molecular and cellular adaptation of muscle in response to e

PHYSIOLOGICAL REVIEWS Vol. 71, No. 2, April 1991

Printed in U.S.A.

Molecular and Cellular Adaptation of Muscle in Response to Exercise: Perspectives of Various Models

FRANK W. BOOTH AND DONALD B. THOMASON

Department of Physiology and Cell Biology, University of Texas Medical School, Houston, Texas; and Department of Physiology and Biophysics, University of Tennessee Medical School, Memphis, Tennessee

I. Physiological Significance ........................................................................... 541 A. Significance of adaptations to environment ..................................................... 541 B. Significance of adaptations to exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 542 C. Causes of fatigue in skeletal muscle during physical exercise .................................. 542

II. Classification of Models Closely Mimicking Human Physical Activity and Models of Increased Contractile Activity That Do Not Mimic Human Exercise .................................... 544

A. Human physical activity ......................................................................... 544 B. Animal models that closely mimic human physical activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544 C. Animal models of increased contractile activity that do not mimic human physical activity . 544 D. Increased contractile activity in tissue cultures of muscle cells ................................ 545 E. Terminology ...................................................................................... 545

III. Response of Cellular Processes in Skeletal Muscle to Single Bout of Exercise .................... 545 A. Glucose uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545 B. Malonyl-coenzyme A ............................................................................. 547 C. Sarcoplasmic reticulum .......................................................................... 547

IV. Adaptation of Skeletal Muscle to Repeated Bouts of Aerobic Exercise ............................ 547 A. Mitochondria ..................................................................................... 547 B. Glycolytic enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553 C. Lactate dehydrogenase .......................................................................... 555 D. Myosin isoform switching ....................................................................... 556 E. Oxygen flux ....................................................................................... 559

V. Adaptation of Skeletal Muscle to Repeated Bouts of Resistance Exercise ......................... 560 A. Human physical activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560 B. Animal models mimicking human heavy-resistance training .................................. 560 C. Adaptations differ in aerobic and strength training ............................................ 561

VI. Hypertrophy in Animal Models Not Mimicking Human Physical Activity ........................ 561 A. Adaptations differ between certain animal models and humans ............................... 561 B. Animal models of stretch-induced hypertrophy ................................................ 562 C. Animal models of compensatory overload-induced hypertrophy ............................... 563

VII. Muscles or Muscle Cells in Culture Do Not Mimic Human Physical Activity ..................... 564 VIII. Regrowth of Atrophied Skeletal Muscle ............................................................ 565

IX. Summary of Inferred Sites for Gene Expression in Those Animal Models That Closely Mimic Human Physical Activity ....................................................................... 566

X. Adaptations That Affect Cardiac Output ........................................................... 566 A. Stroke volume adaptations ...................................................................... 567 B. Chronotropic adaptations ........................................................................ 572

XI. Adaptations That Affect Cardiac and Peripheral Blood Flow ...................................... 573 A. Coronary blood flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573 B. Muscle blood flow ................................................................................ 573

XII. Adaptations That Affect Cardiac Myocyte Metabolism ............................................ 574 A. Substrate metabolism ............................................................................ 574 B. Oxidative phosphorylation ....................................................................... 574

XIII. Conclusion ........................................................................................... 574

I. PHYSIOLOGICAL SIGNIFICANCE for adaptation to changes in the environment likely

A. SigniIcance of Adaptations to Environment carry over to some of the adaptations occurring because of physical training. Prosser (316,317) has written that

The ability of an animal to adapt to repeated bouts cellular, organ, and systemic alterations that favor sur- of physical exercise over a period of weeks such that viva1 of an animal to an environmental change are said exercise capacity is improved is termed physical train- to be adaptive. Physical exercise, like environmental ing. Some of the inherent mechanisms that are crucial change, disrupts the milieu interieur. Fisher (103) has

0031-9333/91 $1.50 Copyright 0 1991 the American Physiological Society 541

542 FRANK W. BOOTH AND DONALD B. THOMASON Volume 71

emphasized that biochemical and physiological adaptations to a changed environment or physiological stimulus fall into two categories based on their duration. Cel- lular, organ, or systemic alterations that occur on the same time scale as a single exercise bout are said to be acute exercise responses. On the other hand, changes in cells, organs, or systems that persist for appreciable periods after or as a consequence of physical training are said to be exercise adaptations. A function of exercise adaptation seems to be to minimize disruption of homeostasis during an exercise bout. It is this better maintenance of the milieu interieur by the exercise adaptations that favors the functional effectiveness of the animal beyond the resting state. Less disruption in homeostasis permits the animal or human to undergo physical work for longer durations at the same absolute power before fatigue. This review considers molecular and cellular responses to exercise that may signal molecular and cellular adaptations during physical training.

B. Signijicance of Adaptations to Exercise

This review is organized to use some of the known causes of fatigue during physical exercise as links between molecular and cellular changes that occur as a result of physical training and the chronic adaptations that are characteristic of physical training. One can speculate that adaptations that improved an animal’s work capacity enhanced its survival. The genetic ability to alter exercise performance through physical training has not been lost along the evolutionary scale. Conse- quently, not all known molecular and cellular changes to exercise are considered here; this is because their function may not yet be recognized to be associated with adaptations that ameliorate fatigue. In addition, this review does not repeat in great detail material that is available in other reviews. There are 11,689 documents for the Medline MESH word “exertion,” Medline’s term for exercise, between 1984-1989, inclusive. To the reader unfamiliar with the causes of fatigue during physical -exertion, enough description has been given (Table 1) to permit understanding of the physiological significance of the molecular and cellular events. A more detailed review on fatigue is available (108). In addition, the reader is referred to earlier reviews that have compre- hensively documented biochemical responses to a single exercise bout and biochemical adaptations of muscle to physical training.

C. Causes of Fatigue in Skeletal Muscle During Physical Exercise

Fatigue is defined as the inability of the animal or human to continue working at a given exercise intensity. Thus a reduction in power output is seen as fatigue (291). This section briefly delineates how various causes of fatigue (Table 1) prevent an animal or a human from being able to continue working at a given rate or level of

activity. Fatigue mechanisms thus cause an individual either to exercise at a lower intensity or to stop exercising altogether. The reader is referred to an excellent review by Gollnick (132) for a more in-depth coverage of energy metabolism during prolonged exercise.

I. Adenosine triphosphate depletion

The interaction of the myosin and actin filaments during muscle contraction is powered by ATP. Through a complex series of molecular events, energy from the binding and enzymatic cleavage of ATP to ADP and Pi powers the formation of the myosin-actin cross bridge, conformational translocation of the opposing filaments, and release of the cross bridge to begin the cycle again. Thus each cycle of myosin and actin cross-bridge formation consumes an ATP molecule. Furthermore, the pul- satile increase in sarcoplasmic free Ca2+ during contraction initiates cross-bridge cycling; additional ATP is consumed in releasing and sequestering Ca2’. New- sholme and Leech (291) speculate that the small decreases in ATP concentration in skeletal muscle during an all-out sprint would diminish myosin adenosinetri- phosphatase (ATPase) activity and hence cross-bridge cycling, in turn preventing continued sprinting at the same pace. Also a decreased ATP concentration would decrease Ca2’ cycling and possibly contribute to fatigue.

2. LowpH

After continuous high-intensity exercise to exhaustion, intramuscular pH can decrease to 6.6-6.3 (171,274, 336). In working muscle, the decrease in muscle power (work per unit of time) at low pH is attributed to proton interference with the catalytic activity of many enzymes (292). Increased free H+ could produce some, or all, of the following metabolic processes. They are inhibition of phosphofructokinase activity (74, 135, ZZO), which would diminish ATP production via glycolysis; decrease of glycogen breakdown by inhibition of phosphorylase kinase and adenylate cyclase activity and by a shift from HPOZ- to H,PO, (59); decrease in maximum tension due to increased free Ca2’ required to obtain the same submaximal tension (86); increase in the constant for. the apparent binding of Ca2’ to troponin (117), thereby attenuating the contractile response (223); and decrease in Ca2+ release from the sarcoplasmic reticulum (289), which would decrease muscle tension. For further discussions on muscle fatigue, see References 108, 153,291,328,336, and 414.

Force production by muscle is also reduced by the formation of the diprotonated form of Pi (H,P0,)(295). Because Pi and H+ both increase in muscle undergoing intense exercise, the shift in equilibrium causes force to decrease.

3. Glycogen depletion

A relationship between carbohydrate depletion and fatigue was demonstrated by Christensen and Hansen

April 1991 EXERCISE TRAINING ADAPTATIONS 543

TABLE 1. Fatigue types and characteristics, causes, adaptations to delay fatigue onset during exercise, biochemical basis of adaptation, and molecular/cellular signals implicated in adaptation

Some Types of Fatigue

Time to Fatigue

Energy From, %

Anaerobic Aerobic Cause of Fatigue

Adaptive Strategies to Delay Fatigue

Biochemical Basis of Adaptation

Molecular and Cellular Responses

Signaling Adaptation

All-out running sprint of 100 m

All-out 1,500-m run

Marathon run of 26 miles

Lifting heavy objects

-10 s 100 0 ATP and CP Deplete less ATP depletion per unit of power

-4 min 35 65 High Pi Low pH

Less reliance on glycolysis

Less increase in muscle and blood lactate concentrations

Carbohydrate sparing

-2h 0 100 Glycogen depletion

Maximal O2 flux

-1 min 100 0 Insufficient mass of skeletal muscle

Increased maximal stroke volume and cardiac output

Increased O2 flux through skeletal muscle

Increase mass of contractile protein in skeletal muscle

ATP resynthesis processes; more efficient use of ATP

Increased mitochondria

Increased mitochondria Decreased glycolytic

enzymes Improved Ca2’ influx Increased blood volume Other?

Increased capillary density

Increased myoglobin concentration

Increased mitochondria 3

?

CAMP? ATP flux? ADP or ATP

levels?

CAMP? ATP level?

ADP or ATP? ?

FGF? CAMP? Other?

?

Fatigue does not derive from a single cause. Thus different human sports have unique sources of fatigue. This table is not inclusive to all types of fatigue. Fatigue is defined as a reduction in power output. CP, creatine phosphate; FGF, fibroblast growth factor. [Adapted from Newsholme (290) and Fitts and Metzger (108).]

in 1939 (61). In this experiment, subjects exercised until exhaustion, at which time the subjects were hypoglyce- mic. Immediately, 200 g of glucose was given orally. Within 15 min of glucose ingestion, the subjective symp- toms of fatigue were gone, blood glucose had increased, and these subjects were able to exercise for an additional hour. It is possible that central and peripheral nerves, which can only oxidize glucose, are a fatiguing tissue.

Later it was shown that the human’s storage of carbohydrate is limited and is equivalent to +,OOO kcal (56,291). In subjects performing aerobic exercise at 70- 100% of aerobic capacity, fatigue occurs at 1-2 h, and it is associated with hypoglycemia (5), depletion of muscle glycogen (4,31,170,355), and depletion of liver glycogen (5,18). The time to exhaustion at these work intensities is altered by dietary manipulation of preexercise concentrations of muscle glycogen; a carbohydrate-poor diet results in the lowest concentrations of muscle glycogen and the shortest time for work to fatigue, whereas a carbohydrate-rich diet is associated with the highest concentration of glycogen and longest work time (31).

Numerous strategies involving regulation of substrate metabolism can be utilized by exercising organ- isms to conserve carbohydrate stores and thus lengthen the exercise duration before exhaustion. Theoretically, such adaptive strategies could enhance survivability.

Two acute responses to conserve glycogen stores during aerobic exercise involve I) a shunting of blood glucose to working skeletal muscle for oxidation and 2) a mobilization of fatty acid from fat depots, their transport to muscle, and subsequent oxidation by muscle mitochondria. In addition, an adaptive increase in mitochondrial density as well as an adaptive decrease in the activity levels of glycolytic rate-limiting enzymes induced by aerobic training has been shown to play an important role in sparing carbohydrate utilization as a fuel during aerobic exercise. Some of these exercise responses and adaptations are discussed in sections III and IV.

4. Limited maximal oxygen flux

Aerobic training increases maximal oxygen flow (VO 2max) (332). To this day, the underlying mechanism(s) accounting for the increase in Voamax (330) remains controversial. Part of the reason for this contro- versy is related to the multiple adaptations that occur in accordance with the increase in Vozmax. Maximal cardiac output, capillary density in skeletal muscle, myoglobin concentration in skeletal muscle, and mitochondrial density of skeletal muscle all increase in response to aerobic training. Each of these factors is thought to contribute to the increase in Vozmax. Many researchers in


the exercise sciences believe that maximal cardiac output limits VOzmax in healthy humans who are 550 yr old when exercise is performed at sea level (182, 332, 333, 340). Thus adaptive increases in the cardiovascular oxygen-delivery system occurs in response to aerobic training. Because this adaptation improves physical performance, it is discussed in sections x and XI. The functional significance of an increase in VOzmax in response to aerobic training appears to be lengthening the duration that a given intensity of aerobic exercise can be sustained until the organism becomes exhausted (182).

5. Insuficient mass of skeletal muscle

The capacity of skeletal muscle to produce force per unit cross-sectional area is not altered after heavy-resistance training (341). Thus an adaptive increase in muscle cross-sectional area would distribute the same absolute load over a larger cross section, decreasing load per unit of cross section.

II. CLASSIFICATION OF MODELS CLOSELY MIMICKING

HUMAN PHYSICAL ACTIVITY AND MODELS OF

INCREASED CONTRACTILE ACTIVITY THAT

DO NOT MIMIC HUMAN EXERCISE

One of the least appreciated necessities in apprais- ing the exercise literature is to relate each exercise model to its proper human sports activity. Because adaptive responses to physical training are specific to the type of exercise, an exercise model must closely or exactly mimic the human sports activity to extrapolate the findings from animals to a specific human sport. We arbitrarily divide models of increased contractile activity into four categories.

A. Human Physical Activity

Examples of human physical activities are jogging, swimming, cycling, resistance exercise, or weight lifting.

B. Animal Models That Closely Mimic Human Physical Activity

The examples of animal models that mimic human physical activities are few. Some are the running of rats on motor-driven treadmills or animal models where heavy-resistance work is accomplished within the period of 1 h with no exercise for the remainder of the day. Rats running on treadmills have a unique order of fiber recruitment (197).

C. Animal Models of Increased Contractile Activity That Do Not Mimic Human Physical Activity

It is universally accepted that adaptations from a given type of increased contractile activity are specific. Extrapolations of adaptations from one type of increased contractile activity to a different type are in- valid in most cases. Therefore it is unlikely that animal models of increased contractile activity that do not mimic closely the recruitment pattern and duration of a human physical activity are valid models of that physical activity. This concept, although academically sound, is not rigorously practiced when data are interpreted in original research articles.

Examples of different adaptational responses between animal models of increased contractile activity that do not mimic a human physical activity are discussed next. For example, the model of swimming of rats in tanks does not mimic human swimming. Colle- giate male swimmers who trained intensely for 6 mo had an 82% greater citrate synthase activity per gram of deltoid muscle (106). Even when rats are “swum” for 6 h/day for 3 mo, only a 25% increase in citrate synthase activity (per gram muscle) in skeletal muscle occurs. Part of the reason is that rats spend considerable time during the exercise bout below the surface of the water doing little continuous repetitive muscular contraction. They just sit on the bottom of the tank for extended periods. In contrast, the pattern of contractile activity in human skeletal muscle is a repetitive cycling of limbs for a continuous duration of many minutes without a period of inactivity at the bottom of the pool. Further- more, the cardiovascular response of rats to swimming is opposite to the response seen in human swimming (14). During swimming, the heart rate and mean arterial pressure in rats decreases (375); in humans, heart rate can increase to near-maximal values during swimming (265). Therefore the swimming rat model does not mimic human swimming.

A second animal model of increased contractile activity that does not mimic a human physical activity is continuous chronic electrical stimulation. First, the recruitment pattern of muscle fibers differs. Continuous electrical stimulation usually recruits all fibers for extended durations of 812, or 24 h/day. Indeed, because of its continuous pattern, Swynghedauw (380) called it “permanent activation of skeletal muscle.” In contrast, human running has an ordered recruitment of muscle fibers [at low intensities of running, type I fibers are preferentially recruited; at higher running intensities or after prolonged submaximal running, type II fibers are also recruited (for review see Ref. 341)]. Second, there is a difference in some of the acute responses that occur with continuous electrical stimulation and running. Skeletal muscles that undergo chronic continuous stimulation exhibit a 300% increase in chloride space throughout the Znd-10th wk of stimulation (167), a progressive increase in intracellular Ca2+ concentration until it is 300% higher at the 2nd wk of stimulation, then a progressive decrease (Ca2’ was determined after dis-


continuing stimulation and anesthetizing the rabbits) (369), and finally a 25% decrease in muscle size after 3 wk of stimulation (425). It is unlikely that chloride space and Ca2’ concentration are altered at the 2nd wk of treadmill running for 2 h/day. Muscle size is unaltered by 2 h/day of running by rats (181). Another difference between chronic stimulation and intermittent running is that ,&adrenergic receptor density increases 320% on the hearts of rats with chronically stimulated skeletal muscle (239) but that ,&adrenergic receptor density is unchanged on hearts from rats who have undergone daily bouts of running on motor-driven treadmills (426). Thus the model of continuous stimulation of skeletal muscle does not mimic the human sport of running.

There is no single regimen for human strength training. However, it is generally agreed that the major lift exercises (press, pulls, and squats) are seldom practiced for >2 days/wk (44). If these lifts are practiced too frequently and too intensely, overtraining invariably results (44). Within a training day, for the purposes of building muscle mass, a large number of sets (5-7) and a moderate number of repetitions (4-7), using moderate amounts of weight (-80% of 1 repetition maximum), are employed (44). Approximately O.l-0.2% of the time within a week is spent training the muscle. Such training produces an enlargement rate of 0.23% /day for the cross-sectional area of elbow flexors during isometric training by humans (201).

Certain animal models for muscle hypertrophy do not mimic the work schedule of human training programs. Although human programs of strength training consist of a low repetition number against a high resistance, some animal models induce hypertrophy of skeletal muscle by a regimen of continuous repetition (24 h/ day) at low resistance. Two such animal models (compensatory overload and continuous stretch) produce a much more rapid enlargement than is produced by human strength training. When muscle’s synergists are disabled by surgical ablation, the remaining muscle enlarges at a rate of 0.86-3.95% /day over a period of weeks in seven different reports (for references see Ref. 259). During the first 5 days postsurgery, muscle enlarges 6.6%/day (294). A second animal model produces an even faster rate of muscle enlargement. Muscle enlarges at a rate of 11% /day during the first 5 days when the muscle is continuously stretched by a weight (249). Both of these models undergo a “permanent activation of skeletal muscle” (for references see Ref. 380). In contrast only 0.1% -0.2%) instead of 100%) of the available time is devoted to increased contractile activity in human strength training. Thus the animal models of compensatory overload and continuous stretch do not mimic the human sport of resistance or strength training.

D. Increased Contractile Activity in Tissue Cultures of Muscle Cells

Either intermittent stretching (411) or electrical stimulation (41) has been used to increase contractile

activity in cultured muscle cells, but neither of these mimics exactly a human physical activity because of the absence of the load of the body or the absence of systemic responses, such as neural, hormonal, and immuno- logical.

E. Terminology

The word exercise is defined as “active: bodily exertion for the sake of restoring the organs and functions to a healthy state or keeping them healthy” (Stedman’s Medical Dictionary, 24th ed.) and as “regular or repeated use of a faculty or bodily organ or bodily exertion for the sake of developing and maintaining physical fitness” (Webster’s New Collegiate Dictionary, 8th ed.). These definitions of exercise can be applied to human physical activities and to most animal models that closely mimic human physical activity, because these activities involve repeated body exertion invoking multiple organ systems-for periods of <2 h/day. On the other hand, it is less than precise to employ the term exercise to animal models of increased contractile activity that do not mimic a human physical activity or to increased contractile activity in tissue cultures of muscle cells. These models often do not include the entire body or evoke the appropriate responses of multiple organ systems or do not have alternative cycles of short exercise bouts with long rest intervals between the repeated bouts.

III. RESPONSE OF CELLULAR PROCESSES IN SKELETAL

MUSCLE TO SINGLE BOUT OF EXERCISE

Three important processes are associated with a single exercise bout that could contribute to fatigue. They are glucose uptake, malonyl-CoA concentration, and sarcoplasmic reticulum function.

A. Glucose Uptake

I. Description of response

The acute response to a single bout of aerobic exercise is a shift of glucose uptake to exercising muscle fibers away from most of the other organs (except the brain). This effect is coordinated by a decrease in plasma insulin (162) that is caused by sympathetic inhibition of insulin release from pancreatic ,&cells (326). Thus insulin-stimulated uptake of glucose is diminished in most organs and tissues due to decreases in circulating insulin levels. Superimposed on the reduction in plasma insulin is a preferential shift of blood flow to contracting skeletal muscle away from most other organs, including noncontracting skeletal muscle (332), so that the amount of glucose (concentration X flow rate) presented per unit time to these other organs is dimin-


ished. However, the major factor increasing glucose uptake into contracting skeletal muscle fibers involves an increase in their insulin sensitivity (30, 71, 96, 204, 206, 280, 327). Noncontracting skeletal muscle, lung, and liver respond to acute exercise with no change in insulin sensitivity (206). King et al. (230) have proposed that the key factor accounting for the increased whole body insulin sensitivity observed in trained human subjects is due to the persistent effects of the last bout(s) of exercise as opposed to more long-term adaptations to training. The greatly increased insulin sensitivity of skeletal muscle in the postexercise period likely functions to permit the rapid muscle glycogen resynthesis that occurs in the presence of low plasma insulin levels (145).

2. Glucose transporter response

The mechanism by which a single bout of exercise increases the amount of glucose that is transported into the contracting muscle appears to be due, in part, to a recruitment of glucose transporters to the sarcolemma from the cytosol and/or an increased turnover of glucose transporters within the sarcolemma. In 1965, it was reported that an acute bout of contractile activity increased the rate of glucose uptake into skeletal muscle by increasing the maximal velocity ( Vmax) of transport without significantly altering the Michaelis constant (K,) (183). From these results, the investigators predicted an increase in either the number or the turnover of the glucose transporters within contracting skeletal muscle. This prediction has recently been proven by numerous laboratories. Each of these reports is considered next. The compound cytochalasin B binds specifically to glucose transporters and when radiolabeled provides an index of the number of glucose transporters. With the use of this technology, a twofold increase in D-ghmse- inhibitable cytochalasin B-binding sites in purified plasma membranes from the red gastrocnemius muscle of rats was measured 1 h after a l-h treadmill run (177). However, these investigators showed that the four- to fivefold increase in glucose uptake into skeletal muscle postexercise was proportionately larger than the twofold increase in glucose transporters incorporated into the plasma membrane. Hirshman et al. (177) speculated that an increase in the intrinsic activity of glucose transporters may occur. The results from a second laboratory (88) showed a threefold increase in glucose uptake, a twofold increase in glucose-inhibitable cytochalasin B- binding sites in isolated plasma membranes, and no change in cytochalasin B binding in isolated intracellular membrane fractions in hindlimb skeletal muscle of rats after a 45-min run on the treadmill. Douen et al. (88) interpreted their results as a lack of an exercise-induced decrease in cytochalasin B binding in the intracellular membranes, thereby implying the existence of a second recruitable transporter pool that is not in the isolated intracellular membranes within the muscle fiber. In a third laboratory (373), no change in cytochalasin B binding to isolated sarcolemma was noted when

muscle samples were taken 15 min after the end of a 45-min treadmill run by rats. However, the Vmax of glucose uptake increased 3.2-fold. Sternlicht et al. (373) concluded that the increase in Vmax for glucose uptake was due solely to an increased transport rate of existing glucose transporters in the sarcolemma. In a fourth study (119), the same isolation procedures used in both the second (88) and third (373) studies were employed. Both techniques gave similar results for this group in that a 66% decrease was observed in the ratio of cytochalasin B-binding sites in the intracellular membrane relative to the plasma membrane of rodent gastrocnemius and quadriceps muscles immediately after a 2-h treadmill run. Fushiki et al. (119) interpreted their results as an exercise-induced translocation of glucose transporters to the plasma membrane during the exercise. In a fifth report (141), the plasma membrane glucose transporter number in rodent red gastrocnemius muscle was shown to be elevated 63,77, and 0% immediately, 30 min, and 2 h, respectively, after a l-h run on the treadmill. In the same samples, facilitated D-glucose

transport in plasma membrane vesicles was increased 310, 79, and 0% immediately, 30 min, and 2 h, respectively, after a single exercise bout. Goodyear et al. (141) concluded that the reversal of the exercise-induced increase in transporter intrinsic activity is more rapid than the reversal of the increased transporter number because of the larger decrease in activity in the first 30 min after exercise. In a sixth study, an acute l-h run by rats on a motor-driven treadmill approximately doubled the number of glucose transporters and carrier turnover number in skeletal muscle plasma membrane vesicles (231). The mean affinity constant of the glucose transporter was not altered. Evidence has been presented that the increased insulin sensitivity in skeletal muscle after swimming exercise results from an altered postreceptor step after insulin binding (58). In summary, recent studies conclusively show that the increase in glucose uptake produced by an acute bout of exercise is due to an increased recruitment of glucose transporters from an internal storage pool to the sarcolemma and also due to an increased turnover of glucose transporters within the sarcolemma.

3. Control of glucose transporter response

Protein kinase C translocation during muscle contraction has been suggested to play a regulatory role during contraction, possibly in the activation of glucose transport. Only 2 min of repetitive tetanic contraction caused a maximal translocation of protein kinase C from the cytosol to the particulate fraction (325). The experimental model was 60 200-ms trains of indirect electrical stimulation of rat calf muscle. In a later study it was shown that only 2 min is required for diacylglycerol to increase twofold to its maximal value in calf muscles during indirect stimulation at a similar contraction frequency as that reported in the above study (64). However, the translocation of protein kinase C from the


cytosol to the particulate fraction did not peak until the 10th min of contraction. The uptake of Z-deoxyglucose increased with an even slower time course. Cleland et al. (64) concluded that the production of diacylglycerol may be causal for the translocation of protein kinase C, which, together with an accumulated exposure to Ca2’ during contractile activity, might activate glucose transport.

Other factors may play a role in the exercise-induced stimulation of glucose uptake in contracting skeletal muscle. However, the findings on these factors are sometimes contradictory. Some papers report an increase in insulin receptor number in the plasma membranes of skeletal muscle from chronic aerobically trained rats (36,85,343), whereas other findings suggest no change in insulin receptor number (144,405). Never- theless, a humoral factor is required because in vitro contraction does not enhance the sensitivity of glucose transport to insulin (58). This factor is not insulin, because contractile activity of skeletal muscle in the hind- quarter perfused without insulin increases muscle plasma membrane glucose transport by increasing glucose transporter number and intrinsic activity (142). A correlation of 0.95 existed between the GLUT-4 isoform of the glucose transporter and Z-deoxyglucose uptake in contracting skeletal muscle (166). Fast-twitch oxidative muscle (type IIa) had the highest levels, whereas fast- twitch glycolytic (type IIb) muscle had the lowest GLUT-4 and Z-deoxyglucose uptake. There is no effect of a single bout of exercise on both basal and insulin- stimulated receptor autophosphorylation and on basal and insulin-stimulated exogenous kinase activity in any type of skeletal muscle (405). On the other hand, insulin receptor kinase activity increases in aerobically trained skeletal muscle (85,343). Acute exercise apparently does not stimulate glucose transport via the ,&adrenergic receptor (372). Diacylglycerol and protein kinase C are the most promising findings to date as a part of the signal cascade that is involved in the increased recruitment of glucose transporters into the sarcolemma during acute exercise.

B. Malon yl-Coenx yme A

Malonyl-CoA serves as a regulatory molecule to inhibit fatty acid oxidation. Thirty minutes of treadmill exercise by rats causes a 36% decrease in malonyl-CoA in the gastrocnemius muscle (427). This decrease in malonyl-CoA would decrease its inhibition of carnitine acyltransferase I activity, thereby enhancing fatty acid oxidation. Winder et al. (427) speculated that the significance of a reduction in malonyl-CoA in exercising muscle is a contributing factor to the increase in fatty acid oxidation in muscle that occurs during prolonged submaximal exercise. Thus an acute response during prolonged aerobic exercise is the shift to oxidation of fatty acids, which in turn conserves the limited stores of carbohydrate in the body. A similar adaptive response that

also spares carbohydrate as a fuel for aerobic exercise is presented in section IV.

C. Sarcoplasmic Reticulum

Numerous reports show a decrease in the function of the sarcoplasmic reticulum in fast-twitch muscles I) after prolonged exhaustive exercise @8,54,55,107,352), 2) after high-intensity exercise (55), and 3) during chronic electrical stimulation (165,251). A 50% decrease in both the initial rate and the total capacity of Ca2+ uptake by isolated sarcoplasmic reticulum occurs on the 2nd day of l&h/day indirect stimulation of fast-twitch muscle. The decrease appears to be due to an inactiva- tion of sarcoplasmic reticulum Ca2+-ATPase activity without a change in its protein concentration or in its isoform distribution (251). The 50% decrease in sarcoplasmic reticulum Ca2+ -ATPase activity is apparently causally related, according to Leberer et al. (251), to the 50% reduction in ATP binding, as determined by the binding of fluorescein isothiocyanate (a competitor for the ATP-binding site on the sarcoplasmic reticulum Ca2+-ATPase).

The functional significance of the decrease of sarcoplasmic reticulum Ca2+ -ATPase with different types of muscle contractile activity is debatable. Some believe that it could be the cause of muscle fatigue (28, 54, 55) during acute contractile activity. On the other hand, Le- berer et al. (251) suggested that decreased Ca2’ uptake by the sarcoplasmic reticulum in chronically stimulated fast-twitch muscle could be a factor in the 300% increase in free Ca2+, which was reported by Sreter et al. (369) in continuously stimulated fast-twitch muscle (a time delay existed between ending stimulation, anesthetizing rabbits, and taking muscle samples). Sreter et al. (369) suggested that free Ca2’ is closely connected with changes in gene expression associated with the fast-to- slow fiber transformation (see sect. IvD). Another functional consequence of the decreased uptake of Ca2+ by the sarcoplasmic reticulum is the parallel lengthening of the time to peak tension and the half-relaxation time (165). Whether the ATP cost of muscle work is altered at the time of this change (the 4th day of continuous indirect stimulation of fast-twitch muscle) is unknown.

IV. ADAPTATION OF SKELETAL MUSCLE TO REPEATED

BOUTS OF AEROBIC EXERCISE

Adaptation is defined here as a semipermanent change(s) occurring in the structural and/or functional properties of cells, tissues, and organ systems after weeks of repeated exercise bouts. If daily exercise is dis- continued altogether, the adaptation is maintained for several days before it begins to disappear.

A. Mitochondria

I. Description of adaptation

In 1967, Holloszy (179) reported a twofold increase in the capacitv of skeletal muscle to oxidize pvruvate in


rats that underwent 12 wk of aerobic training by running on a treadmill for 2 h/day. Concomitantly, the activities of the enzymes of the mitochondrial electron transport chain doubled per unit of weight in the same skeletal muscles of the trained rats. In addition, the doubling of protein concentration for cytochrome c, a protein in the electron transport chain, provided evidence that the training adaptation in oxidative capacity involved an increased amount of protein and was not due to an increased catalytic activity of the same amount of enzyme protein. Numerous investigators have verified the adaptive increase in mitochondrial density in aerobically trained skeletal muscle in both rats and in other species, including humans (for references see Ref. 341).

Z. Localization of signal for adaptation

The signal or inducer for the adaptive increase in skeletal muscle mitochondrial density in response to aerobic training appears to be an endogenous rather than a systemic factor. For example, in human subjects, if only a single leg undergoes aerobic training on cycle ergometer (the contralateral leg is noncontracting), mitochondrial density increases only in the trained leg (281). In a study examining a wide spectrum of athletes, succinate dehydrogenase activity (a protein on the inner membrane of the mitochondrion) was found to be highest in those skeletal muscle groups that were engaged directly in the training (134). For example, bicyclists had twice the succinate dehydrogenase activity per gram of skeletal muscle in their legs compared with their arms; conversely, canoeists had 36% greater activity in their arms than in their legs. Moreover, the increase in mitochondrial density in aerobically trained skeletal muscle appears to be independent of certain hormones. Increases in succinate dehydrogenase activity and mitochondrial protein per unit of skeletal muscle weight have been shown to occur in aerobically trained hypophysectomized, thyroidectomized, or dia- betic rats (136). Thus the exercise response that signals the adaptive increase in mitochondrial density likely re- sides within the contracting muscle.

3. Protein synthesis

An increase in mitochondrial protein synthesis, a decrease in mitochondrial protein degradation, or both would be needed to permit the adaptive increase of mitochondrial density in aerobically trained skeletal muscle. A major role for an increase in synthesis rate was deduced from the following results. Similar half-lives for the time course of the increase (training) and the decrease (detraining) of selected mitochondrial proteins between their control and trained steady-state quantities were interpreted as no effect on the protein degradation rate of these mitochondrial proteins by aerobic training (39,385,386); half-life is dictated solely by deg-

radation rate. The mitochondrial markers examined were cytochrome c, citrate synthase activity, and 3-ke- toacid CoA-transferase activity. In this study, rats underwent 2 h of treadmill running every day during training.

+$.. Messenger ribonucleic acid

An increase in the synthesis rates of proteins local- ized in the mitochondria of trained skeletal muscle could occur because of an increase in pretranslation (mRNA quantity), translation (increased utilization of mRNA), or posttranslation (increased assembly) or a combination of these mechanisms. Clearly there is an increase in mRNAs coding for mitochondrial proteins in skeletal muscle undergoing increases in contractile activity (Table 2).

5. Control of protein expression

I) AEROBIC TRAINING. Only a single mRNA, cytochrome c mRNA, has been examined in skeletal muscles of rats trained by treadmill running (Table 2). Simi- lar percentage increases in citrate synthase enzyme activity and cytochrome c mRNA occur in skeletal muscles at the 14th day of training (283). This suggests a pretranslational control mechanism. Citrate synthase activity has been shown to be proportional to the volume fraction of mitochondria within a muscle (45). Thus it can be deduced that a percentage increase in cytochrome c mRNA is likely proportional to the percentage increase in mitochondrial volume fraction at the 14th day of the treadmill-running program.

In skeletal muscles of rats trained to run 2 h/day on a motor-driven treadmill, enzymes for fatty acid oxidation and the respiratory chain increase 100%) while certain tricarboxylic acid cycle enzymes only increase 50% (184, 277) and other tricarboxylic acid cycle enzymes, such as citrate synthase, increase 100% (105). The reason why one tricarboxylic acid cycle enzyme increased by a smaller percentage than fatty acid oxidation and respiratory chain enzymes in treadmill running is unknown. Another study (75) shows the maintenance of a constant proportion of the individual components of the inner mitochondrial membrane constituents during their adaptive increase in skeletal muscle because of aerobic training. We conclude that the proportional increase in mitochondrial components likely implies some role for an assembly control, which is a subcategory of posttranslational control.

In studies where rats were run daily on motor- driven treadmills for different durations, the resultant new steady-state level of mitochondria is directly proportional to the time spent running, up to a specific time duration. Beyond this duration, further increases did not occur. In rats trained 10,30,60, or 120 min/day, the percentage increase in citrate synthase activity and cytochrome c concentration in the gastrocnemius muscle


TABLE 2. Changes in mitochondrial proteins and their mRNAs as a result of increases in contractile activity

Increase in, %

Stimulation Duration Enzyme

Enzyme mRNA for activity enzyme Muscle Stimulated Reference

100 min/day, Citrate synthase 14 days Cytochrome c

12 h/day, 28 days

24 h/day, 3- 5 days

24 h/day, 10 days

24 h/day, 21 days

Animal model mimicking human physical activity: aerobic training by chronic treadmill running

30-41 27-57

Soleus, plantaris, red quadriceps, gastrocnemius

283

Animal model that does not mimic human physical activity: permanent activation of small group of,muscles by chronic indirect electrical stimulation

Citrate synthase 400 700

Citrate synthase Cytochrome oxidase ,&subunit of F,ATPase Citrate synthase Cytochrome oxidase ,&subunit of F,ATPase VIC subunit of

cytochrome oxidase Cytochrome b Citrate synthase Cytochrome oxidase Cytochrome b ,&subunit of F,ATPase VIC subunit of

cytochrome oxidase

178 123

185 213,182 215,154

197,192

112 252

500,557,336 412, 642

500, 654 265,219

220

Extensor digitorum longus

Tibialis anterior, extensor digitorum longus


351

239

239,425


239,425

progressively increased with the time of running each day. The data are (for citrate synthase and cytochrome c, respectively) 15 and 12% (10 min), 57 and 31% (30 min), 87 and 38% (60 min), and 128 and 92% (120 min) greater than control (105). These observations imply that the control mechanism(s) is titrated by exercise duration. Further discussion of this concept is merited. A later study examined the variable of exercise intensity (running speed) in the context of exercise duration on the quantity of the adaptive increase in mitochondrial density in skeletal muscle. Dudley et al. (89) found that it is possible to induce the same increase in cytochrome c concentration in skeletal muscle, as seen with longer exercise times, by employing faster running speeds combined with shorter run times, provided the exercise duration exceeded a minimum threshold value (82). An increase in exercise intensity likely recruits more motor units (416). Thus mitochondrial concentration per gram of whole muscle would be increased more because additional untrained muscle fibers are recruited per unit of time at higher speeds of running.

A minimal duration of daily exercise that is dependent on exercise intensity is necessary to induce a detect- able increase in mitochondrial density (105). Further increases in mitochondrial density are directly related to duration, but there is a maximal duration beyond which further daily bouts of treadmill running do not induce a further increase in mitochondria. These findings imply that the percentage increase in mitochondrial density by contractile activity is titrated by both

exercise duration and intensity (82). Roles for pretranslation, translation, and/or posttranslation events are also demonstrated by another study. Cytochrome c protein synthesis rate and cytochrome c mRNA are 81 and 60%, respectively, of control values in the red quadriceps muscle after 7 days of fixation in a shortened position and are 192 and 126%, respectively, of control values on the 4th day of recovery from the joint fixation (284). Although its mRNA increases during recovery, the increase in cytochrome c protein synthesis rate is much greater. Therefore these data imply an increase in pretranslational, translational, and posttranslational control of cytochrome c protein expression when the contractile activity of an atrophied skeletal muscle increases. Furthermore, the integration of these various control mechanisms may represent a general adaptive response for control of mitochondrial protein expression during aerobic training of muscle.

II) CHRONIC ELECTRICAL STIMULATION. AS with aerobic training, although mRNAs encoding mitochondrial proteins are increased in skeletal muscles undergoing an electrically stimulated increase in contractile activity, this increase in mRNA is not likely to be the sole mechanism inducing an adaptive increase in mitochondrial density in the model of chronic continuous stimulation. In Table 2, cases exist where the percentage increase in mRNA is less than the percentage increase in its protein product. For example, the VIC subunit of cytochrome oxidase mRNA is 220% of the control level, whereas cytochrome oxidase enzyme activity is 412 and


OL 1. * . * 1 * * . * 1 . . . . 1. * . . 1. . . * 1,

0 10 10 30 40 50

Period of stimulation (days)

FIG. 1. Time course of adaptive increases in citrate synthase mRNA (top) and citrate synthase enzyme activity (bottom) during 1% h/day stimulation of fast-twitch muscle. Note discordance between rates of increase for enzyme activity and mRNA. [From Seedorf et al. (35U.l

642% of control values in muscles that have undergone continuous electrical stimulation for 21 days. This and similar observations by Williams et al. (425) led them to state that “this finding suggests that pretranslational regulation alone is insufficient to account fully for the changes in expression of the protein products of the F,-ATPase and cytochrome oxidase subunit VIC genes. Enhanced translational efficiency, accelerated transport of these proteins from their cytoplasmic sites of synthesis across the mitochondrial membranes, or increased stability of the proteins may also be required to support the accelerated mitochondrial biogenesis induced by electrical stimulation” (425).

In addition, the relative roles that pretranslation, translation, and posttranslation play in increasing mitochondrial density in chronically stimulated skeletal muscle vary during the time course of stimulation. Dur- ing the first 6 days of indirect electrical stimulation, citrate synthase activity increases without any increase in citrate synthase mRNA (351; Fig. 1). Then, from day 7-10 of stimulation, citrate synthase mRNA increased 600-700%. These researchers interpreted these results as an enhanced translation of existing mRNA during the initial increase in citrate synthase activity. In another example, the percentage increase in cytochrome-c oxidase enzyme activity and cytochrome-c oxidase mRNA is similar after days 3-7 of indirect electrical stimulation (10 h/day) (192; Fig. 2). This implies that pretranslational control is solely responsible for the increased enzyme activity with the stimulation protocol. On the other hand, between the time interval of 14-35 days of stimulation, the percentage increase in cy-

tochrome-c oxidase enzyme activity exceeds the percentage increase in its mRNA. This implies the involvement of translational and/or postranslational control in addition to pretranslational control during this later time interval of stimulation. The latter example emphasizes that, for a given mitochondrial protein, different combinations of pretranslation, translation, and posttranslation occur during the time course of its increased expression in response to chronic electrical stimulation.

Despite the complexity of mechanisms contributing to the increased expression of individual mitochondrial proteins in electrically stimulated skeletal muscle, it is clear that their final concentration per gram of muscle is maintained in constant proportion. For example, Reichman et al. (319) found that during chronic electrical stimulation experiments, increases in enzyme activity levels of terminal substrate oxidation (tricarboxylic acid cycle, fatty oxidation, respiratory chain) occur in parallel to maintain a constant proportion of these enzymes. On the other hand, Chi et al. (60) noted that the timing of the changes for individual enzymes in chronically stimulated muscle indicates that more than one kind of signal is operative.

A higher percentage of muscle fibers recruited could be used to explain why the percentage increase in mitochondrial density is greater in chronic electrical stimulation studies than in treadmill run training experiments. Durations of treadmill running for >2 h/day do not further increase mitochondria density in the working skeletal muscles beyond 200% of control values in rats (386). On the other hand, %400-500% increases in respiratory and tricarboxylic acid cycle enzymes per unit of muscle weight occur in muscles that are stimulated either 12 (319) or 24 h/day (425) for 21 days. One possible explanation for why the maximal increase in mitochondrial density is 500% of control in the electrical stimulation studies compared with only 200% of control in muscles from treadmill run rats may be that a

Cytochrome c oxidase Subunit mRNA (% of control)

FIG. 2. Parallel increase in mRNAs for 2 subunits of cytochrome- c oxidase are shown in relation to increase in cytochrome-c oxidase enzyme activity during IO-h/day stimulation of fast-twitch muscle. mRNAs are encoded by different genes. Subunit III is mitochondrially encoded, whereas subunit VIC is nuclear encoded. Also note diver- gence from identity line after 14 days of stimulation. [From Hood and Pette (191).]


greater percentage of muscle fibers per unit mass are activated by electrical stimulation.

In contrast to the observed increase in enzyme activities of aerobic metabolism after indirect electrical stimulation of the tibialis anterior muscle of rats, guinea pigs, and rabbits, 10 h/day of electrical stimulation did not alter the basal levels of aerobic enzymes in mice (358). Mice had high basal levels of aerobic enzymes. This means that transgenic mice may not be applicable to training studies concerned with aerobic enzymes.

6. Heme expression

The rate-limiting enzyme regulating heme synthesis is &aminolevulinic acid synthase. The activity of this enzyme in the red portion of the vastus lateralis muscle in rats is doubled 17 h after a 4,000-m run on a treadmill (185). At the same time postexercise, no change in cytochrome c protein concentration occurs. This observation implies that upregulation of heme synthesis is an early regulatory event mediated by .muscle contraction.

7. Factors regulating molecular changes

In view of the above observations, three potential regulators of the increase in mitochondrial density by aerobic exercise and by chronic stimulation are discussed. These include adenosine 3’,5’-cyclic monophosphate (CAMP), hypoxia, and creatine phosphate.

I) ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE AND

AEROBIC TRAINING. Kraus et al. (239) recently hypothe- sized that an elevated level of CAMP within skeletal muscle during its chronic continuous stimulation is the signal for the increase in mRNAs transcribing proteins of mitochondria. This section considers the evidence as to whether this hypothesis for the nonphysiological model of human exercise supports a similar hypothesis for aerobic training. Immediately after a single run on a motor-driven treadmill lasting either 5, 10, or 30 min, CAMP concentration doubles in the red and in the white quadriceps muscles (128). If an increase in CAMP is the sole factor inducing the increased mitochondrial density in aerobically trained skeletal muscle, then mitochondria should be increased after weeks of training at these durations. However, rats that undergo 13 wk of running for 10 min daily on a motor-driven treadmill have no significant change in mitochondrial density, whereas rats that run 30 min/day for 13 wk have a 31 and 57% increase in cytochrome c concentration and citrate synthase activity per gram of muscle, respectively, in the gastrocnemius muscle (105). Duration of the doubling of CAMP concentration in skeletal muscle after either a IO- or a 30-min run is unknown. However, in the heart, a single 60-min run on a motor-driven treadmill results in CAMP being increased for a 24-h period after the run (306). Thus the postexercise duration of CAMP increases in skeletal muscle requires documentation.

The remaining results related to a potential role for

CAMP as the signal causing mitochondrial proliferation in aerobically trained skeletal muscle are data related to ,&adrenergic receptors. Because ,&adrenergic receptor stimulation by agonists increases CAMP concentrations, whereas ,8-adrenergic receptor blockage by antagonists decreases CAMP, the strategy of numerous studies in animals and humans has been to use pharmacological manipulation of ,&adrenergic receptor activation in vivo to monitor resultant changes in mitochondrial density of skeletal muscle. Two-hour daily infusions of the syn- thetic catechol dobutamine into human subjects during 3 wk of bedrest increased citrate synthase activity in their vastus lateralis muscle but caused no significant change in succinate dehydrogenase and cytochrome oxidase activities (376). Conversely, the normally observed increase in citrate synthase, cytochrome oxidase, ,&hydroxyacyl-CoA dehydrogenase, malate dehydrogenase, and alanine aminotransferase activities found in aerobically trained skeletal muscle of rats is almost com- pletely blocked by a dosage of propranolol that decreases exercise heart rate by 25% but not by the &-selective blocker atenolol (211). Ji et al. (211) concluded that “&-adrenergic mechanisms play an essential role in the training-induced enzymatic adaptation in skeletal muscle.” Although chronic P-blockade does not prevent an adaptive increase in mitochondrial enzymes in the vastus lateralis muscle of humans after 8 wk of bicycle training, the increase is not as great as occurs in the placebo group. In this study, succinate dehydrogenase, cytochrome-c oxidase, and ,8-hydroxyacyl-CoA dehydrogenase activities do not increase as much compared with placebo, whereas citrate synthase activities are unaf- fected by the ,&blockade during training (377). ,&Adren- ergic receptor density is increased in skeletal muscle as a result of aerobic training (423). A correlation of 0.63, or a probability of 36%) was obtained between succinate dehydrogenase activity and ,8-adrenergic receptor density in the gastrocnemius muscles of rats examined from control, swim-trained, and run-trained groups (423). Thus many reports involving training studies suggest a connection between an increase in either ,&adrenergic receptor density or an increase in CAMP levels and an increase in mitochondria of skeletal muscle. How- ever, some reports do not support this association.

A number of studies report that the ,8-adrenergic receptor does not play a role in the exercise-induced adaptation of mitochondrial enzymes. After 6 wk of daily injections of L-epinephrine in sedentary rats, hearts hypertrophy 11%, but respiratory capacity, citrate synthase and succinate dehydrogenase activities, and cytochrome c concentrations of skeletal muscles do not change (101). Chronic ,&adrenergic blockade does not prevent the exercise-induced increase in enzymes of skeletal muscle in rats (216). Ji et al. (211) speculated that the failure of this study to observe a role for ,8-adrenergic receptors in the exercise-induced increase in mitochondrial density was due to an insufficient ,&blockade. The adaptive increase in citrate synthase, succinate dehydrogenase, cytochrome-c oxidase, and P-hydroxyacyl- CoA dehydrogenase activities by swim training of rats

552 FRANK W.BOOTH AND DONALD B.THOMASON Volume 71

is not prevented by adrenodemedulation and/or sympa- thectomy (169). The potential role of CAMP as a signal for mitochondrial biogenesis in aerobically trained muscle requires more experimentation.

II) ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE AND CHRONIC STIMULATION. Kraus et al. (239) interpreted their data demonstrating an increase in CAMP during electrical stimulation as a correlation between the increase in CAMP and/or ,&adrenergic receptor density and the increase in mRNAs for mitochondrial proteins. An examination of their data reveals that the percent- ages of increase in CAMP and mRNA are not always in proportion to each other. The F,-ATPase mRNA is significantly increased 85% in skeletal muscle after 3-5 days of chronic stimulation, but CAMP is unchanged after 3 days of stimulation and unreported after 5 days (239). The F,-ATPase mRNA remains doubled from the 3rd to the 10th day of stimulation, but CAMP triples in concentration in the same time period of stimulation. Finally, F,-ATPase mRNA remains doubled from the 10th to 21st day of stimulation while CAMP concentration in stimulated muscle decreases from 308 to 154% of control. Further testing of the hypothesis, as suggested by the originators of the hypothesis, is merited.

III) ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE AND TISSUE CULTURE. In primary Cultures of rat muscle cells, agents that increase intracellular concentrations of CAMP have been consistently shown to increase the levels of two enzymes of oxidative metabolism, fumarase and malate dehydrogenase (250). In another study, only NADH cytochrome-c reductase activity and glucose oxidation are consistently increased by drugs that elevate CAMP in myotube cultures derived from satellite cells (112). Other mitochondrial enzymes are not consistently increased by drugs that increase CAMP in the same study. Data from another report (349) suggest that the cascade of increased free Ca2+, increased prostaglandin synthesis, and increased CAMP lead to an increase in creatine kinase activity in fusing myoblasts. Indometh- acin inhibits both the increase in CAMP and creatine kinase under the above conditions. As cautioned, findings in tissue culture must be replicated in whole animals during exercise to validly conclude that a response in culture occurs in the animal during physical exercise.

IV)HYPOXIAANDAEROBICTRAINING. Increasedac- tivities of succinic oxidase, 3-hydroxyacyl-CoA dehydrogenase, citrate synthase, and cytochrome-c oxidase in skeletal muscle have been reported in humans with peripheral arterial insufficiency that causes intermittent claudification (53, 187, 189). These investigators remarked that the similarity of their findings with changes after aerobic training suggest a “common trigger mechanism” (187). Holm et al. (189) later suggested that “the restricted supply of oxygen to the muscle tissue might induce increased activity of the mitochondrial enzyme succinic oxidase” in the condition of intermittent claudification (188). The same investigators observed that successful revascularization reduces the increase in succinic oxidase activities of skeletal muscle back toward control values (189). Another experimental

model has been employed. In human subjects, both legs were separately trained on a single-leg ergometer. Ci- trate synthase activity and myoglobin concentration increase more in skeletal muscle from the leg that trained under hypobaric conditions (PO, = 572 Torr) than from the contralateral leg that trained under normobaric conditions (388). Terrados et al. (388) suggested that a lowered PO,, rather than a difference in substrate flux (which they infer to be the same because work intensities were the same), is the causal factor for greater increases in citrate synthase and myoglobin under hypobaric conditions. However, Saltin and Gollnick (341) cited extensive circumstantial evidence against the hypothesis that tissue hypoxia is the initial stimulator for the exercise-induced increase in mitochondrial density in aerobically trained skeletal muscle. Further recent support against a role for hypoxia as a signal inducing mitochondrial biogenesis is the report that strenuous exercise in the expedition to Mount Everest and Lhotse resulted in a decrease in the enzyme activities of citric acid cycle and respiratory chain and an increase in glycolytic enzyme activities (196). The data from hypoxia studies cited above do not provide a definitive conclusion.

V)HIGH-ENERGYPHOSPHATELEVELSANDAEROBIC TRAINING. There is recent evidence that depletion of tissue ATP and creatine phosphate may serve as a stimulus to induce an increase in mitochondrial density in skeletal muscle (244,356). Rats that ingest a diet of 1% ,&guanidinopropionic acid for 6 wk have a 90% decrease in creatine phosphate and a 50% decrease in ATP in skeletal muscle (104). After 6-10 wk of this diet, an increase in the activities of citrate synthase, Z-oxoglutar- ate dehydrogenase, and 3-hydroxyacyl-CoA dehydrogenase occurs in some of the fast-twitch muscles but not in slow-twitch muscle (356). Cytochrome c mRNA increases 60 and 67% in the white quadriceps and soleus muscles, respectively, when rats are fed a 1% ,B-guanidinopropionic acid diet for 22 days (244). This observation infers that decreased ATP and creatine phosphate concentrations in skeletal muscle could play a contributing role in upregulating mitochondrial density during aerobic training. However, the red quadriceps muscle demonstrated no change in cytochrome c mRNA quantity when fed 1% ,&guanidinopropionic acid for 22 days. Thus any role of high-energy phosphate depletion in in- ducting mitochondrial biogenesis involves more than a pretranslational step.

VI) HIGH-ENERGY PHOSPHATE LEVELS AND CHRONIC STIMULATION. In the same model, continuous electrical stimulation of fast-twitch muscle (which increases CAMP) produces a 50% decrease in creatine phosphate after 8 days; however, no apparent change occurs within the first 30 h (168). All of the increase in citrate synthase mRNA occurs between the 6th and 10th day of chronic electrical stimulation (12 h/day) of fast- twitch muscle (351), which is the time period associated with the decrease in creatine phosphate. In summary, data exist to support a hypothesis that a decrease in the


high-energy status of fast-twitch skeletal muscle can induce mitochondrial biogenesis.

VII)HIGH-ENERGY PHOSPHATES AND SPRINTING. Because sprinting (anaerobic exercise) decreases high- energy phosphates without an increase in mitochondrial density, an additional factor other than high-energy phosphates must be involved in mitochondrial biogenesis. The additional factor is duration. Thirty minutes, but not 10 min, of endurance running was necessary to invoke a significant increase in mitochondrial density in skeletal muscle (105). All-out sprint exercise has a duration of <l min. Thus the duration of reduced high-energy phosphate is not long enough to signal mitochondrial biogenesis in a single bout of sprint exercise. A combination of duration would be required with the decrease in high-energy phosphates to produce on increase in mitochondrial density, if high-energy phosphates are the inducing signal for increased mitochondria.

8. How adaptation alters fatigue

Some functional adaptations that could result from an increase in mitochondrial density were suggested by Holloszy in 1973 (180). Holloszy reasoned that changes in ADP and/or ATP concentrations from their homeostatic levels would be only one-half as much in skeletal muscle with twice the mitochondria; after aerobic training both trained and untrained muscles consume equal quantities of oxygen and produce similar amounts of ATP from their mitochondria while working at the same absolute power. Moreover, Holloszy suggested that to produce the same amount of ATP per unit of muscle, muscle with twice the mitochondrial density would need only one-half of the change in ADP and Pi levels to produce one-half the ATP amount per unit of mitochondrial weight. The hypothesis that ADP and ATP concentrations would need to be disrupted less from their homeostatic levels to obtain the same absolute ATP production in muscle has been verified. The ATP levels in human skeletal muscle decrease less when exercising at the same absolute power after training compared with before training in humans (342). Two additional studies verified and extended these findings (65, 92). Both found that the concentration of ATP decreases less and that the concentrations of Pi, ADP, and ammonia increase less at the same oxygen uptake in rat skeletal muscle with high mitochondrial content. The important metabolic consequence of the smaller disruption in these compounds is the predicted lower substrate fluxes through creatine kinase, adenylate kinase, AMP deaminase, and glycolysis as a result of the adaptive increase in mitochondria in aerobically trained skeletal muscle (65,92). These adaptive alterations in metabolic flux would diminish the increase in lactic acid formation and carbohydrate depletion, thus delaying the onset of fatigue due to glycogen depletion. Because H,PO, decreases force production in skeletal muscle (295), an adaptive increase in mitochondria results in less of an

increase in Pi, and thus of H,PO,, resulting in a lessened decrease in force production at the same power after aerobic training. In addition, the increase in mitochondria of aerobically trained skeletal muscle provides another adaptive function for sparing carbohydrate as a fuel for muscle contraction. Enzymes involved in the activation (cytosolic), transfer to intramitochondrial site (cytosolic and mitochondrial), and ,&oxidation (mitochondrial) of free fatty acids are increased in aerobically trained skeletal muscle (279). These enzyme adaptations correspond to trained skeletal muscle having an increased capacity to oxidize free fatty acids (279). Hol- loszy and Coyle (182) concluded that “the glycogen- sparing effect of increased fat oxidation probably plays a major role in endurance that occurs with training.” Sparing carbohydrate oxidation during prolonged aerobic work delays fatigue.

B. Glycolytic Enzymes

I. Description of adaptation

I) AEROBIC TRAINING. A decreased glycolytic flux in skeletal muscle occurs at the same exercise intensity after aerobic training compared with before training (170). This adaptation occurs during aerobic training because of a decrease in the maximal activity of glycolytic enzymes per unit of skeletal muscle weight and because of a decrease in allosteric factors that activate phosphofructokinase activity at a given absolute workload.

The decrease in glycolytic enzymes after aerobic training appears to be limited to fast-twitch red muscles. After 12 wk of treadmill running by rats for daily durations of 2 h/day, several glycolytic enzymes decreased ~20% in fast red skeletal muscle and increased 18-35% in slow red skeletal muscle (21); however, there was no change in glycolytic enzymes in fast white skeletal muscle. In this particular study, exercise intensity may not have been great enough to recruit the fast white skeletal muscle. Baldwin et al. (20) noted among various rat skeletal muscles a high correlation between the activities of phosphofructokinase and actomyosin ATP- ase, indicative of the greater reliance of fast white muscle on glycolysis.

II) CHRONIC STIMULATION. Greater percentage decreases in glycolytic enzyme activities have been observed during continuous (24 h/day) electrical stimulation experiments, probably because the daily duration of contraction was longer than in the Z-h daily running protocol. During continuous stimulation of a fast-twitch muscle comprised of red and white regions, aldolase activity is reduced to 66 and 92% of control after 10 days of stimulation and is further reduced to 26 and 41% of control after 21 days of stimulation in the red and white regions, respectively (239,425). During the first 2 wk of chronic stimulation of the fast-twitch red and white tibialis anterior muscle. no change in the maximal activ-


ity of phosphofructokinase and in the concentration of fructose 1,6-diphosphate occurred, but both decrease 80% during the subsequent 4 wk of stimulation. By the 5th wk of stimulation, both are at levels found in the control slow-twitch soleus muscle (167).

2. SigniJicance enzymes

of adaptive decrease in glycolytic

A theoretical analysis of the metabolic significance of changes in enzyme activities was presented by Goll- nick and Saltin (137). The model predicts that for glycolytic flux to be maintained at the same rate in aerobically trained muscles with decreased glycolytic enzymes, substrate concentrations would have to be higher in the trained muscles. This has not been shown empirically. Similar concentrations of glucose 6-phosphate were measured before and after training in the vastus lateralis muscle of ll- to 13-yr-old boys at rest, at 500 m/min, at 750 m/min, and at maximal exercise on a bicycle (95). The following logical deductions can be made. Glucose 6-phosphate levels reflect fructose 6- phosphate levels (the substrate for phosphofructokinase, the rate-limiting enzyme in glycolysis). If true, then the substrate-driven reaction velocity of phosphofructokinase in aerobically trained skeletal muscle would be decreased in fast-twitch muscles after training at the same absolute power, since the maximal activity of phosphofructokinase per gram of muscle decreased without any change in glucose 6-phosphate levels. No information exists on the effects of training on fructose 2,6-diphosphate concentrations in skeletal muscle, although its concentration decreases in the liver during exercise (84,429). Other modulatory factors of phosphofructokinase are considered next.

3. Mitochondrial adaptations produce allosteric modijications to phosphofructokinase

A decreased glycolytic flux in aerobically trained skeletal muscle working at the same power is also produced by a decreased allosteric activation of phosphofructokinase. As discussed in section IVAN, at a given absolute work rate or power output the homeostatic disruption of high-energy phosphates is less because of the adaptive increase in mitochondrial density in aerobically trained skeletal muscle (65, 92). Because a decrease in ATP and corresponding increases in ADP and Pi activate phosphofructokinase activity (292), smaller changes in the concentration of these nucleotides at a given absolute work rate after training would produce less activation of phosphofructokinase so that glycolytic flux is less, the rate of carbohydrate depletion .is reduced, and time for work until exhaustion is prolonged. Furthermore, the adaptive increase in mitochondria also diminishes the amount of increase in another acti- vator of phosphofructokinase activity, ammonia (NH,). Muscle with a high density of mitochondria shows a

smaller increase in AMP than muscle with a low density of mitochondria during exercise at the same absolute work intensity (65,92). An increase in AMP concentration results in an increased flux through AMP deaminase, the first reaction of the purine nucleotide cycle. This enzyme catalyzes the deamination of AMP to IMP and NH, (255), primarily in fast-twitch red muscle (91). At the same absolute work rate, NH, increases less in muscle with a high mitochondrial density, thus activat- ing glycolytic flux to smaller degree than in untrained skeletal muscle.

Also associated with the adaptive increase in mitochondrial density in aerobically trained skeletal muscle are increases in enzymes of ,&oxidation and the capacity to oxidize fatty acids (279). The potential metabolic significance of increased fatty acid oxidation on glycolytic flux is given by the following experiment. When plasma free fatty acids are increased experimentally in untrained rats during treadmill running, glycogen depletion decreases in fast and slow red skeletal muscle and citrate concentrations increase (323). In a later experiment performed in situ to avoid the 5-min delay that occurs when the animal stops running and the muscle sample is obtained, citrate and glycogen concentrations are higher in stimulated muscle perfused with fatty acids than in the hindlimb perfused without fatty acids (322). These investigators concluded that the higher citrate level in the muscle perfused with fatty acids inhib- ited phosphofructokinase (322). A similar conclusion has been made from the results of a human study. A fat-rich diet, which causes an inhibition of glycolysis in skeletal muscle both at rest and at the 5th min of bicy- cling, is associated with a higher citrate concentration in the quadriceps muscle (208). Jansson and Kaijser (208) concluded that inhibition of glycolysis may be mediated by the inhibition of phosphofructokinase by an increased muscle citrate concentration. Thus both positive and negative allosteric modulators of phosphofructokinase are altered in such a quantitative manner so as to activate phosphofructokinase less in aerobically trained skeletal muscle undergoing the same absolute work rate as an untrained muscle. The decreased glycolytic flux that occurs during submaximal aerobic exercise serves to conserve the limited stores of carbohydrate in the body and thus extends the time of work until fatigue.

4. Messenger ribonucleic acid alterations

I) AEROBIC TRAINING. No data are known to exist. II)CHRONICSTIMULATION. Apretranslationalmech-

anism appears to be the major, if not the exclusive, control site for the decrease in glycolytic enzymes noted in chronically stimulated skeletal muscle. Aldolase A mRNA is 27% of control after 10 days of indirect electrical stimulation of fast-twitch muscle (239), whereas aldolase enzyme activity is 66 and 92% of control activity after the same duration of stimulation in two different experiments (239,425). Thus a large decrease in al-


dolase A mRNA level precedes a decrease in its enzyme activity1

5. Factors regulating molecular changes

1)CHRONIC STIMULATION. Provision ofpropranolol, a ,&adrenergic antagonist, to rabbits prevents a decrease in aldolase A activity and its mRNA after 10 days of electrical stimulation of skeletal muscle for 24 h/day. Propranolol inhibits some of the decrease in aldolase A mRNA (55% of control with propranolol vs. 29% of control without propranolol) after 21 days of stimulation (239). Propranolol does not affect the increase in CAMP that occurs in the continuously stimulated muscles. Kraus et al. (239) concluded that the decrease in aldolase A mRNA in chronically stimulated skeletal muscle is not a consequence of direct regulation by CAMP.

II) TISSUE CULTURE. Increasing CAMP in cultured muscle cells did not decrease the activity of glycolytic enzymes (112, 250), as found in chronic stimulation of skeletal muscle. However, the addition of caffeine to cultured myotubes causes a decrease in the activities of phosphofructokinase, pyruvate kinase, and lactate dehydrogenase (112). These results were interpreted by Freerksen et al. (112) to be a result of a prolonged increase in cytosolic Ca” acting to trigger events that result in the downregulation of glycolytic enzymes in skeletal muscle. This idea, if held to be true, must include a duration factor, because the adaptive response of phosphofructokinase to high-resistance strength training is variable. Maximal activity of phosphofructokinase in strength-trained skeletal muscle has been reported to not change (155, 194), to increase (67, 240), and to decrease (390). Because cytosolic free Ca2’ levels increase in strength-trained muscles, which show no consistent adaptive change in phosphofructokinase activity, another explanation possibly is needed. It may be that the minimal duration of the increase of free cytosolic Ca2’ level in response to the strength-training stimulus is not long enough to downregulate the pretranslational control of glycolytic enzymes. In addition to increasing cytosolic free Ca2+, caffeine also increases CAMP and mechanical activity of the cell. Muscle cultures exposed to caffeine not only have a downregulation of the certain enzymes involved in glycolytic flux, but the activities of some proteins of the mitochondrial electron transport chain (NADH cytochrome-c reductase and succinic cytochrome-c reductase) increase (112). However, increased CAMP without increased free Ca2’ does not decrease glycolytic enzymes (112).

C’. Lactate Dehydrogenase

1. Description of adaptation and aerobic and strength training

Daily running exercise affects total lactate dehydrogenase activitv differentlv in various fiber tapes. After

aerobic training, total lactate dehydrogenase activity decreases in the gastrocnemius muscle of rats (138,277) and humans (8, 222), decreases in fast-twitch fibers of rats (21) and humans (9), increases in the slow soleus muscle of rats (21), decreases in slow-twitch fibers of the vastus lateralis muscle of humans (9), and increases in the rat heart (138,212). Total activity of lactate dehydrogenase is either unchanged, (222, 390), increased (240), or decreased in muscle biopsies taken from strength-trained muscle of humans (9). These reports thus indicate that although aerobic training decreases total lactate dehydrogenase activity in fast-twitch muscle, strength training has no consistent effect on this enzyme.

In addition to a decrease in total activity, isoforms of lactate dehydrogenase in skeletal muscle shift because of aerobic training (the isoform terminology used in the original reference is retained here). The relative activity of lactate dehydrogenase isoforms 1 and 2, expressed as a percentage of total lactate dehydrogenase activity, is greater in aerobically trained subjects than in nonconditioned subjects (222). In a related study, the Vmax and K, of lactate dehydrogenase increases and decreases, respectively, when lactate is used as the substrate in a homogenate of skeletal muscle of rats that had undergone 10 wk of treadmill running for 1 h/day (212). Also the Vmax of this enzyme decreases when pyruvate is used as the substrate in these rat muscle homoge- nates (212). The shift in enzyme isoform is an adaptation that decreases pyruvate transfer to lactate while increasing lactate conversion to pyruvate in aerobically trained skeletal muscle of rats. In another report, a greater activity of lactate dehydrogenase isozyme 1 is observed in the slow-twitch fibers of endurance-trained humans than in untrained individuals (9). Chronic stimulation of fast-twitch muscle also increases the heart isoform (isoform 1) of lactate dehydrogenase (359).

2. Messenger ribonucleic acid alterations

I) AEROBIC TRAINING. No data are known to exist. II)CHRONICSTIMULATION. Apretranslationalmech-

anism appears to be the exclusive control for the switch in lactate dehydrogenase isoforms that results from increased contractile activity. In the study of Seedoff et al. (351), a similar time course for the changes in activities and mRNAs for lactate dehydrogenase isoforms were reported. A parallel increase in the protein subunit of the heart isoform of lactate dehydrogenase and its mRNA occurs during chronic stimulation (12 h/day) of rabbit extensor digitorum longus muscle. Likewise, proportional decreases in the protein subunit and mRNA of the muscle isoform of lactate dehydrogenase occurs during chronic stimulation (351). More of the H, isoform of lactate dehydrogenase protein exists in slow than in fast muscle (191).


Concurrent with a decrease in glycolysis in aerobi- calls trained muscle is another adaptation that also


tends to lower lactate levels. The function of the training-induced decrease in the total activity of lactate dehydrogenase and in a shift to the heart isoform of this enzyme in skeletal muscle is to lessen the increase in lactate concentration muscle at a given level of absolute power. Indeed this was demonstrated by Karlsson et al. (221) in 1972. They observed smaller increases in lactate concentration in the skeletal muscles of humans after training when exercised at the same absolute power as before training. The consequence of smaller increases in muscle lactate during work would be an adaptive lengthening of time before the onset of fatigue. The pK, of lactic acid is 3.7. Increases in lactic acid would lower pH in muscle. Further information on the functional role of lactic acid during exercise can be found in Brooks (43).

D. Myosin Isofbrm Switching

1. Human physical activity

Normal ambulatory activity by humans maintains the expression of the slow myosin heavy-chain isoform by fibers at some genetically set limit. Further physical training results only in very small percentage shifts from fast to slow myosin heavy chain.

Five studies have demonstrated histochemically a decrease in the percentage of type IIb fibers in skeletal muscle of humans in response to long-term aerobic exercise training (7, 25, 195, 203, 357). However, the fiber type replacing the histochemical type IIb is variable. A significant increase in slow type I fiber percentage was noted in two of the above reports (195,357) where training lasted 6 and 15 wk, a significant increase in fast type IIa fibers with no change in type I fiber percentage was reported in two studies (7,203) where training duration was 8 and 24 wk, and no significant change in either type I or IIa fiber percentage was found in an 8-wk training study (25). An early study, which only examined the percentage of slow type I fibers, found no change from their pretraining level after a 5-mo aerobic training program (133). When type I fiber percentage has been shown to increase by training, the increases were from 50 to 56% (195) and from 41 to 47% (357). Thus small or no changes in slow type I percentage occur in aerobically trained human muscle when analyzed by histochemical procedures on muscle biopsies.

Histochemistry classifies a fiber into broad categories of fast or slow. However, in the rat soleus muscle, single muscle fibers have been shown to have both fast and slow myosin heavy chains (120). Two populations of fibers were observed. One type was identified histochemically as slow and failed to react against fast myosin antibodies (120). This population consisted entirely of slow myosin heavy chains (321). However, the second population of fibers in the rat soleus muscle was identified histochemically as fast but has both fast and slow mvosin heavv chains (120. 321). Thus when fast fibers

have small percentage shifts in myosin heavy-chain isoforms from fast to slow, they remain fast fibers by histochemical analysis. We speculate that this explanation could account for the variable observations in human skeletal muscle as to whether aerobic training will shift the myosin heavy chain from the fast to the slow isoform.

With the advent of the analysis of isoform type within single muscle fibers, unequivocal evidence from humans now exists for the shift from fast to slow in aerobically trained skeletal muscle. In one of the human studies, no significant increase in type I fiber percentage was detected with histochemistry; however, a shift was noted within individual fast IIA fibers from exclusive expression of fast myofibrillar protein isoforms toward a mixed pattern of fast and slow isoforms, as determined by one-dimensional electrophoresis of proteins from fragments of the same fiber type (25). Other reports support the idea that aerobic training increases the percentage of “hybrid” fibers containing both fast and slow isoforms. By histochemistry, 13% of the type II fibers are converted into a fiber type demonstrating intermediate myofibrillar ATPase activity in the triceps brachi muscle in men and women after a 36-day ski event over 800 km (345). Isolated fibers demonstrating the histochemical category of intermediate myofibrillar ATPase activity were shown, using immunohistochemical analysis, to have a coexistence of slow and fast isoforms of myosin heavy chains and of troponins C, I, and T (345). These findings are consistent with the reduced VmLlx of individual type II fibers of the human deltoid muscle after 6 mo of intense swimming training (106). A decreased Vmax in single type II fibers would be caused by an increased percentage of the slow myosin heavy- chain isoform (321). Taken together, these findings from humans suggest that small quantities of isoform switching from fast to slow contractile proteins occur after aerobic training.

A decrease from 57 to 48% of the fibers identified as slow-twitch fibers, after application of a myofibrillar stain, occurred after a sprint training program (207). Training consisted of “all-out” sprints for either 15 or 30 s on a mechanically braked bicycle ergometer by humans. Training was performed 2-3 days/wk, with the sprint number progressively increasing from 4 to 12 each day during the 4- to 6-wk program. Four other sprint training experiments did not produce an increase in the percentage of fast-twitch fibers in the trained muscle (for references see Ref. 207).

2. Chronic stimulation

The type of switching of myosin isoforms that occurs during chronic stimulation does not represent the type of adaptation occurring in the sports training of normal humans. Although there is a subtle change in myosin isoforms when normal ambulatory humans un- dertake a program of physical training, a massive shift in myosin isoforms occurs when skeletal muscle is invol-


untarily recruited continuously for either 12 or 24 h/day for several weeks. Chronic indirect electrical stimulation (24 h/day) of the rabbit tibialis anterior muscle results in a synchronous switching from fast protein isoforms of myosin to slow protein isoforms. For the myosin light chains, switching of protein isoforms begins during the 2nd wk and is largely completed by the 7th wk of chronic stimulation (46). In the same muscles, a downregulation of the fast type IIb myosin heavy chain starts at 2 wk, but the appearance of slow myosin heavy-chain protein does not occur until the 4th wk of chronic stimulation (46,267). A similar experiment published in the same year found that the switch of myosin light chains from fast to slow protein isoforms is more delayed. The switch in myosin light chains occurs during the 8th-13th wk of chronic indirect stimulation (24 h/day) in the rabbit tibialis anterior muscle (350). If stimulation is only 12 h/day, smaller changes in protein quantities of certain myosin light chains occur with no change in myosin light-chain 3f (350). In contrast to the 2-mo delay in switch of protein from fast to slow myosin light chain, the activity of myosin light-chain kinase decreases by 50% after only 1 day when stimulating the rabbit tibialis anterior muscle for 24 h/day (237). The potential significance of decreased myosin light-chain phosphorylation would be a shift of the pCa-tension relationship toward lower pCa values and an attenuation of isometric force (110).

In 1981, Kwong and Vrbova (243) reported that fast-twitch muscle from small vertebrates (rats) has less switching to slow-type muscle during chronic IO Hz stimulation than does fast-twitch muscle from larger animals (rabbits and cats). They observed that despite the prolonged stimulation, the twitch duration of fast muscles in rats is changed little. They remarked that this observation differs from the findings obtained earlier for rabbits and cats, which show that a slowing of contraction speed is achieved by 10 Hz stimulation for a similar duration. Furthermore, they commented that it appeared that the synthesis of contractile proteins of the fast type is favored in small mammals. Likewise in the heart, Baldwin (14) indicates that the V, (fast) isozyme of cardiac myosin is greater in rodents than in larger animals.

Support for this concept has been made by the analysis of myosin protein isoform composition. Although major increases in the protein isoforms of slow myosin light-chains 1s and 2s occur in rabbit fast-twitch muscle during the 3rd-7th wk of continuous stimulation (24 h/ day) (46), less extensive changes are reported in rats. After 6 wk of chronic stimulation (IO h/day) of rat fast- twitch muscle, changes in myosin are restricted to fast isoforms. Myosin light-chain 3f and fast myosin heavy- chain IIb decrease while myosin light-chain If and fast myosin heavy-chain IIa increase (234). Only small amounts of slow myosin heavy chain appear in rat skeletal muscle stimulated 10 h/day for 28 days (233).

Pluskal and Sreter (314) concluded that the close correlation between changes in mRNA and protein levels of mvosin light chains. as thev switch from fast to

slow isoforms during the 3rd to 7th wk of continuous indirect electrical stimulation of rabbit fast-twitch muscle, implies a pretranslational control mechanism. Heilig and Pette (164) also noted a switch from fast to slow myosin light-chain mRNAs in rabbit fast-twitch muscle after 28 days of indirect electrical stimulation (12 h/day). They suggested that the induced transformation of myosin light-chain pattern was due to a switch in gene transcription. The initiation of the switch in myosin light-chain isoform mRNAs precedes initiation of the switch in myosin heavy-chain isoform mRNA in the rabbit skeletal muscle.

A different time course of switching of myosin light-chain isoform occurs in fast-twitch muscle in rats than that described for rabbits. Although a nearly complete switch from fast to slow myosin light-chain mRNA occurs after -4 wk of indirect electrical stimulation in the rabbit (164,314), minimal switching occurs in rats whose muscles were stimulated for a similar duration (232, 233). An additional difference exists between the two animal species. Although control of switching appears to be pretranslational in rabbits (164, 314), a more complex control is apparent in rats, as discussed next.

The control mechanism altering the protein isoforms of myosin light chains during chronic stimulation of rat fast-twitch muscle seems to be specific to the isoform. Although myosin light-chain isoform 2s protein is increased, no change in either its synthesis rate or mRNA is seen during chronic stimulation of fast-twitch muscle (233). Kirschbaum et al. (233) suggested that protein degradation of myosin light-chain 2s is reduced. An earlier study from the same laboratory (22) found no change in slow myosin light chains during chronic stimulation (IO h/day for 56 days) of rat fast-twitch muscle. In addition, the percentage decrease in the protein quantity of myosin light-chain 3f is more than the percentage decrease in its mRNA. Kirschbaum et al. (233) stated that “this might indicate an increased turnover of LC3f or the existence of additional posttranscrip- tional regulations of LC3f expression.” On the other hand, the percentage increases in the protein synthesis rates, protein quantity, and mRNA quantity are similar for myosin light-chains If and Is, in chronically stimulated fast muscles of the rat. Thus a pretranslational control is evident for these light-chain isoforms. In summary, each myosin light-chain isoform exhibits different control sites (pretranslation, translation, and posttranslation) during isoform switching produced by chronic stimulation in rat fast-twitch muscle.

Switching in myosin heavy-chain isoforms also differs between rabbits and rats for chronically stimulated fast-twitch muscle. Whereas slow myosin heavy- chain protein is markedly increased between 21 and 28 days of continuous electrical stimulation of rabbit muscle (46), no slow myosin heavy-chain protein is apparent in 56-day stimulated fast-twitch muscle of rats (232).

In rats, myosin heavy-chain mRNA switching is restricted to fast isoforms during indirect electrical stimulation (10 h/dav) of fast-twitch muscle. A progressive


decrease of fast myosin heavy-chain IIb mRNA to nearly undetectable levels occurs in the first 7 days of stimulation of rat fast muscle (234). Fast myosin heavy- chain IIa mRNA is markedly increased between the 4th and 7th days of stimulation in the same rat muscle (234). Thus a switch from the fast isoform of myosin heavy chain found in low oxidative fast fibers (type IIb) to that found in high oxidative fast fibers (type IIa) occurs early and before any switches in the mRNAs for myosin light chains in stimulated fast muscle of rats. Only minor increases in slow myosin heavy-chain I mRNA were observed from the 4th to 8th wk of stimulation of rat fast muscle (234).

The rapid decrease in fast myosin heavy-chain IIb mRNA in chronically stimulated fast muscle in the rat has been confirmed in rabbits. In the chronically stimulated (24 h/day) tibialis anterior muscle of the rabbit, fast myosin heavy-chain IIb mRNA per gram of muscle is 43,36, and 8% of the contralateral control at the 4th, lOth, and Zlst day, respectively (47). In the same rabbits, fast myosin heavy-chain IIb mRNA per gram of extensor digitorum longus muscle is 50,19, and 33% of contralateral control at the 4th, lOth, and Zlst day, respectively, of continuous indirect electrical stimulation (47). Thus, in both rats and rabbits, fast myosin heavy- chain IIb mRNA rapidly decreases during continuous excitation of fast muscle.

Correlations between the time courses of changes in protein and their mRNA quantities have also been made in rat skeletal muscle. An increase in fast myosin heavy-chain IIa mRNA occurs between the 2nd and 4th days of chronic stimulation of rat fast-twitch muscle (234). However, in the words of Kirschbaum et al. (234), “a remarkable delay” existed before fast myosin heavy- chain IIa protein increased. These investigators suggested that “regulatory steps might exist between transcription and translation in heavy chain expression for fast myosin heavy chain IIa.”

3. Decreased weight bearing

The removal of normal ambulatory activity results in large shifts of the myosin isoforms in humans and rats (397). The percentage of type I fibers in the vastus lateralis muscle decreases from 54 to 43% after 6 wk of limited mobility in a movable cast brace after knee surgery to eight athletes (152). In a human case study, the recovery of type I fibers after atrophy was documented. The percentage of type I fibers in a cross-country skier at the time of knee surgery was 81%) was 58% 6 wk after limb immobilization from surgery, and was 86% after intense training in the time period from 2-6 mo postsurgery (152).

Removal of the weight-bearing function of the slow soleus muscle of the rat produces rapid and large changes in the content and percent composition of slow myosin (399). Approximately 84% of slow myosin protein is lost from the soleus muscle by the 28th day of its nonweight bearing (399). After onlv 7-8 davs of non-

weight bearing by the slow soleus muscle of the rat, total myofibrillar protein content and total slow heavy- chain protein content are decreased by 26 and 30%, respectively (399). Because myofibrillar protein synthesis rate is decreased by 59% during most of the 7-day period of nonweight bearing (396), it is likely that a reduction in synthesis rate plays an important role in the downregulation of slow myosin protein expression. The lack of any significant change in slow myosin heavy-chain mRNA in the 7-day unwei .ghted soleus muscle suggests that a decrease in translation of this mRNA plays the major role in decreasing synthesis of slow myosin heavy-chain protein (396). In addition, a numerical mod- eling analysis indicates that the degradation rate of myofibrillar protein in the slow soleus muscle begins to increase on the 3rd-4th day of its unweighting (396). Thus a decrease in myofibrillar protein synthesis initiates protein loss. After 1 wk of nonweight bearing, both the decrease in myofibrillar protein synthesis and the increase in myofibrillar protein degradation rate in the soleus muscle maintain the continued loss of protein. After 1 mo of nonweight bearing, the new smaller myofibrillar protein mass in the soleus is maintained exclu- sively by the decreased myofibrillar protein synthesis rate. Therefore both translational and posttranslational control mechanisms, although differing in time and magnitude, play roles in the approach to a new steady-state level of protein expression in the nonweight-bearing soleus muscle. A pretranslational control mechanism also becomes active, although to a lesser extent, as evidenced by the de nova synthesis of additional myosin isoforms (399) and the expression of their mRNAs (396).

If a slow soleus muscle is permitted 28 days of recovery from 56 days of nonweight bearing, slow myosin protein increases from 2.1 to 11.2 mg/pair soleus muscles (399). Normal controls have 13.4 mg slow myosin/ pair soleus muscles. Thus, in contrast to fast muscle of the rat where slow myosin gene expression is resistant to change by continuous electrical stimulation, slow myosin gene expression in slow muscle changes greatly in response to weight-bearing exercise. Furthermore, if adjunct treadmill-running exercise is provided during or after the nonweight bearing, fast isoforms of myosin are upregulated in addition to the recovery of slow myosin content (398). These data, taken collectively, indicate the complexity of the control mechanisms by which normal contractile function can influence the quantity and quality of protein expression.

4. Factors regulating myosin isoform switching

A switch from fast to slow myosin isoforms has been shown to occur after chronic administration of either a &-adrenergic antagonist or a chemical that decreases muscle ATP and creatine phosphate. Treatment of rats for 8 wk with the &-antagonist butoxamine causes a 13% decrease in the cross-sectional area for tvee II fibers without affecting tvpe I fiber size in the


soleus muscle (440). In addition, the percentage of type I fibers increases from 68 to 83% of total fibers in the soleus muscle and from 2 to 6% in the extensor digitorum longus muscle. Interestingly, chronic administration of the &-adrenergic agonist clenbuterol has the opposite effect. Type I fiber percentage decreases from 88 to 63% of the total fibers in the rat soleus muscle (440). These data imply that increased CAMP would decrease the percentage of type I fibers. If true, this observation would conflict with the finding of increased CAMP and increased type I fibers in chronically stimulated fast- twitch muscle (239). Thus a change in a single regulatory signal is insufficient to explain a change in contractile protein isoform by contractile activity.

A chronic decrease in ATP and creatine phosphate in rats fed a 1% ,&guanidinopropionic acid diet results in a predominantly slow muscle becoming pure slow muscle but with no apparent alteration of fast isoforms in fast muscle (356). With histochemical methods, the proportion of type I fibers in the soleus muscle changes from 81 to 100% after 6-10 wk of the diet (356). After 7 wk of a 2% ,&guanidinopropionic acid diet, fast myosin isoform 3 protein decreases 60% in the mouse extensor digitorum longus muscle (276). The contractile properties of the soleus muscle exhibit a significant slowing, whereas no change in contractile characteristics was noted in the plantaris of the same rats after 10 wk of a 1% ,&guanidinopropionic acid diet (311). Because 6 wk of a 1% ,&guanidinopropianic acid diet decreases creatine phosphate 90% and ATP 50% in skeletal muscle (104), one interpretation of the above studies is that a reduction in creatine phosphate and ATP plays some role in signaling an isoform switch from fast to slow myosin in skeletal muscle. Likely this interpretation needs direct confirmation at the level of the control of gene transcription. If high-energy phosphates play a role in signaling myosin isoform composition, then weight bearing must have an interactive control function. When weight bearing is decreased, the phosphocreatine level decreases and type I fiber percentage decreases in the soleus muscle of nonweight-bearing limbs (146,397) and of immobilized limbs (38,262). As stated, we speculate that multiple signals or factors from altered contractile activity interact to change gene expression.


A switch to slow myosin heavy chain would reduce the oxygen cost of work. The energy cost per unit force per cross-sectional area is greater in fast than in slow muscle (242). For a brief tetanus of t9 s, the energy cost of the mouse fast-twitch extensor digitorum longus muscle is 2.9 times that of the mouse soleus muscle, which contains an equal mixture of fast and slow fibers (73). After 9 s of tetanus, the fast muscle has only 1.5 times the normalized energy cost of that of the mouse soleus muscle (for reviews see Refs. 242, 318). A similar event occurs in the heart. Slow (V,) myosin is more eco-

nomical than is fast (V,) myosin (6). Thus the functional role of the adaptive switching of myosin isoform from a fast isoform with a high ATPase to a slow isoform with a lower ATPase is that energy costs per unit of force would be less after training. The reputed energy conservation by the adaptive conversion to slow myosin in skeletal muscle is not reflected in the overall oxygen uptake, which is unchanged at the same absolute workload in humans after training (332). One explanation is either that the percentage shift to slow myosin is very small in humans or that the mass of muscle that responds by this mechanism is small.

E. Oxygen Flux

I. Capillaries

This topic has been reviewed extensively by Saltin and Gollnick (341). In brief, skeletal muscles in both humans and animals adapt to aerobic training by an increase in the number of their capillaries (341). Indeed, training-induced increases in whole body maximal aerobic power are comparable to increases in the ratio of capillaries per muscle fiber (341). Nevertheless, the physiological significance of this adaptation is debatable. Saltin and Gollnick (341) conclude that the capil- larization of skeletal muscle is not limiting to whole body maximal aerobic power in humans. Furthermore, they speculate on the physiological role for the adaptation. Diffusion distances for gases and substrates, espe- cially free fatty acids, would be reduced in the aerobically trained skeletal muscle (341). Saltin and Gollnick (341) suggest that it is the decreased diffusion distance that is the function of the increased capillarity. Re- cently Rota et al. (330) concluded that their data were in accord with the notion that maximal aerobic power was the balance between convective oxygen delivery by the blood and its subsequent diffusive movement to myofibrillar mitochondria through the sarcoplasm.

Increased concentrations of fibroblast growth factor were noted in skeletal muscle after 21 days of continuous electrical stimulation (286). Because fibroblast growth factor induces capillary proliferation (111) and because continuous electrical stimulation also results in capillary proliferation (68), it is possible that fibroblast growth factor links increased contractile activity to angiogenesis in skeletal muscle. Whatever the signal that links contractile activity with angiogenesis, the response is rapid. As little as 4 days of electrical stimulation (8 h/day) can evoke a 20% increase in capillary-to- muscle fiber ratio (68). However, no significant increase in fibroblast growth factor was noted after 3 days of electrical stimulation (286).

2. Myoglobin

An adaptive increase in myoglobin concentration in response to run training occurs in the skeletal muscles

of rats (308) but not of humans (209,378). Svendenhag et B. Animal Models Mimicking Human Heavy- al. (378) showed that humans who trained at 75% of Resistance Training their maximal aerobic power had no change in myoglobin concentration of skeletal muscle while indicators of Animal models that closely resemble some, but not respiratory capacity in the same muscle increased 62- all, aspects of heavy-resistance training as performed 82%. However, in rabbits, an -X-fold increase in myo- by humans have been developed. Some of these models globin mRNA content occurs in fast-twitch muscles are described next. Mice and hamsters were trained to after 21 days of continuous indirect electrical stimula- obtain food by pulling and holding a weighted food bas- tion (409). These authors concluded that pretransla- ket (129-131). Although muscle fiber cross-sectional tional mechanisms were important in establishing of area increased, muscle size was unchanged. Cats were this adaptation (409), since myoglobin protein increased conditioned to lift weights with their right forelimb only twofold in rat fast-twitch muscle that was chroni- against a heavy resistance to receive a food reward (139, cally stimulated for 21 days. Thus myoglobin concentra- 140). Both an increase in muscle fiber diameter and in tion in skeletal muscle is not altered by human physical muscle size occurred. Muscle enlarged at a rate of activity but is increased by pretranslational mecha- O.O7%/day over 34 wk of training, which was similar to nisms in continuously stimulated animal muscle (224). the growth rate found in humans who underwent heavy- The role of protein degradation in these responses is resistance training. unknown. Another animal model employed to mimic human

In a recent review, Wittenberg and Wittenberg resistance training is to electrically contract the skele- (431) discuss the function of myoglobin. Myoglobin- tal muscles of anesthesized rats against a resistance us- facilitated oxygen diffusion mediates a large part of the ing a training paradigm similar to the human (432). In total oxygen flux through the sarcoplasm. In view of its these studies calf muscles are contracted against a function, myoglobin concentrations are not limiting to heavy resistance, resulting in plantar flexion. Exercise maximal aerobic power in humans, since aerobic power bouts are performed in sets of six repetitions/set (4 or 32 can increase in the absence of an increase in myoglobin sets/day), 2 days/wk (432). This training paradiam is concentration (378). No explanation is available for the very similar to the repetition pattern employed in hu- significance of the adaptive increase in myoglobin con- man resistance training. An inherent aspect of this centration in skeletal muscles of animals that have un- model, the simultaneous recruitment of all muscle dergone aerobic training by phasic treadmill running. fibers within a muscle (as opposed to an orderly recruit-

ment of fibers during voluntary contractions by humans), produced a serendipitous finding. Both the ankle

V. ADAPTATION OF SKELETAL MUSCLE TO REPEATED

BOUTS OF RESISTANCE EXERCISE

extensors and flexors in the same limb are induced to contract. However, the gastrocnemius muscle (ankle extensor) shortens while the antagonist tibialis anterior muscle (ankle flexor) lengthens during active cross-

A. Human Physical Activity bridge formation. The response of these two fast-twitch muscles differs depending on the type of training: concentric (shortening while contracting) or eccentric

Human regimens of heavy-resistance training gen- (lengthening while contracting). Although apparent erally consist of intermittent bouts of low-frequency overtraining inhibits hypertrophy of the concentrically repetitions (3-10 sets of 6-8 repetitions/set) with high trained gastrocnemius muscle, the same number of ex- loads (67-75% of maximal voluntary contraction) and ercise repetitions produces significant hypertrophy of long recovery periods between training bouts (2-3 days the eccentrically trained tibialis anterior muscle. A of rest between workout days) (259). Studies in humans milder resistance training program results in similar have been performed to observe the rates of skeletal hypertrophy of the eccentric and concentric contracted muscle enlargement during heavy-resistance training. muscle (432). For example, muscle cross-sectional area of elbow flex- In addition to muscle size, protein synthesis rates ors was increased 8% after 8 wk (O.l4%/day increase) and skeletal cu-actin mRNA were determined in the ani- and was increased 23% after 100 days (023%/day in- mal model of concentric and eccentric contraction crease) (201). In other studies of progressive high-inten- within the same limb. Results from these studies indi- sity resistance training by humans, arm circumference cated that alterations in translational and posttransla- increased 11% after 5 mo (O.O?%/day increase) (262) tional control mechanisms may be as important, or and thigh girth was 3% greater after 6 mo (O.OZ%/day more important, than pretranslational control in effect- increase) (154). In the latter study, areas of fast-twitch ing the new size of the muscle (433, 434). For example, fibers were increased by 27%, whereas no change in the after a single bout of either 192 concentric or eccentric cross-sectional area of slow-twitch fibers occurred. contractions, both mixed and myofibrillar protein syn- Thus in studies of heavy-resistance training in humans, thesis rates increase 50-60% at 12-17 h and at 36- to muscle enlargement is relatively slow, averaging 41-h postexercise (433, 434). However, skeletal cu-actin NO.1 % /day. mRNA and cytochrome c mRNA are not altered at these



times (433, 434). An increase in translation of protein can be inferred from such data. In addition, the increase in protein synthesis rates is not necessarily associated with muscle hypertrophy if the initial exercise bout was repeated twice weekly for 10 wk. The muscle trained by concentric contraction shows no hypertrophy. Because an earlier experiment, which employed milder resistance training, resulted in 18% hypertrophy of the concentrically contracting gastrocnemius muscle (432), it was speculated that the heavier resistance, the increased daily repetitions, or both may cause comparable increases in both protein degradation and synthesis rates in the latter report (in which no hypertrophy of the concentrically trained gastrocnemius muscle occurred) (433). If true, a posttranslational control mechanism is present in concentric resistance training.

In contrast to the minimal effects of hypertrophy with concentric contraction, the eccentrically trained tibialis anterior muscle not only hypertrophies but enlarges more when daily contractions are increased from 24 to 19.2 or when plantar flexion occurs against a greater load. If expressed for the whole tibialis anterior muscle, increases of 41% in skeletal a-actin mRNA, of 38% in total RNA, and of 28% in protein (0.4%/day increase) exist after the lo-wk program of heavy-resistance eccentric training (434). These changes are even more remarkable when the duration of training is calculated as a percentage of the lo-wk period. Only 0.1% of available time was actually spent in training eccentrically. This implies that the cellular and molecular signals eliciting from heavy-resistance eccentric training are more dependent on the mechanical load than on the contractile duration. Moreover, the -40% increase in the muscle’s content of skeletal a-actin mRNA and in total RNA imply that repeated applications of low-repetition, high-resistance eccentric exercise is sufficient to upregulate pretranslational control mechanisms. On the other hand, the heavy-resistance concentric training program does not increase skeletal a-actin mRNA (434).

The differential responses to concentric and eccentric resistance training in the rat model of human training emphasize that multiple control sites (pretranslational, translational, and posttranslational) can be elic- ited during training. Future interpretations of mechanisms of muscle hypertrophy in animal models should only be related to the human sport that it mimics, since various types of resistance training in rats give different combinations of pretranslational, translational, and posttranslational control.

C. Adaptations Difer in Aerobic and Strength Training

Programs of aerobic training and of heavy-resistance training by humans result in different adaptive changes in the structure and function of skeletal muscle, some of which are contrasted next. (Aerobic training is the involvement of large masses of skeletal muscle in

rhythmic exercise of low resistance so that whole body 0, uptake is increased many fold over the resting level.) Although hypertrophy of skeletal muscle occurs after the onset of heavy-resistance training by humans (154, 155), aerobic training does not produce hypertrophy of skeletal muscle (181). Indeed, if humans combine heavy- resistance training and aerobic training, strength development by skeletal muscle is usually less rapid than with heavy-resistance training only (90, 173, 338, 339). The decreased strength development could be due to diminished recruitment of muscle fibers by central com- mand (339). Although heavy-resistance training by humans increases the cross-sectional area of fast-twitch fibers (391), the percentage relative area of a muscle that is composed of fast-twitch fibers decreases in aerobically trained skeletal muscle (133). Although heavy- resistance training decreases the mitochondrial volume in human skeletal muscle (257, 260, 261), aerobic training increases mitochondrial volume in human skeletal muscle (193). Likewise, although mitochondrial enzyme activity per gram of skeletal muscle is unchanged (346) or decreased (390, 391) by heavy-resistance training by humans, it increases with aerobic training (341). Heavy- resistance training by humans either decreases, or has no effect, on capillary density in hypertrophied skeletal muscle (389), but aerobic training increases the capillary per muscle fiber ratio, capillary density, and the number of capillaries around a given muscle fiber (341). These findings lead to the conclusion that molecular and cellular responses initiating adaptations are likely different for heavy-resistance training versus aerobic training, since the two result in different patterns of protein expression in skeletal muscle of humans.

VI. HYPERTROPHY IN ANIMAL MODELS NOT MIMICKING

HUMAN PHYSICAL ACTIVITY

A. Adaptations Difler Between Certain Animal Models and Humans

Some of the adaptive protein expression occurring in animal models of skeletal muscle hypertrophy are not similar to the adaptations that occur in human skeletal muscle after heavy-resistanc.e training. Thus these animal models are classified as not mimicking the human sport of resistance training. For example, significant hypertrophy of skeletal muscle does not occur for 2 mo in humans undergoing heavy-resistance training (401), but significant hypertrophy of skeletal muscle occurs within days of the onset of continuous stretch and of continuous overload of rat or chicken muscles (123,249). Mitochondrial volume density decreases in human skeletal muscle during heavy-resistance training (257, 260, 261), but mitochondrial enzymes rapidly increase in the stretched hypertrophying fast-twitch muscle of the chicken wing (97,186). In addition, the percentage of the total cross-sectional area that is composed of fast-


twitch fibers increases in human skeletal muscle after chronic heavy-resistance training (389,390), but the percentage of the muscle cross section that is composed of fast-twitch fibers decreases in the animal models of continuous stretch and of continuous overload (147, 199, 210). Increases in fiber diameter of both fast- and slow- twitch fibers often occurs in human skeletal muscle after heavy-resistance training (261). Magnesium-stimulated myofibrillar ATPase activity decreases in human skeletal muscle after 6 mo of heavy-resistance training (390), so it is possible that slow myosin isoforms increase in fast-twitch muscle with 6 mo of heavy-resistance training. A definite shift from fast to slow myosin heavy chain occurs in continuous stretch (186) and in continuous overload (406).

B. Animal Models of Stretch-Induced Hypertrophy

I. Description

Those models that chronically stretch a muscle produce a muscle hypertrophy that occurs much more rapidly than that produced by a human undergoing a program of heavy-resistance training. “Stretch” can be produced by any one of numerous models, some of which give different adaptive changes in the hypertrophying muscle. In 1944, Thomsen and Luco (400) reported that when the ankle joint of a cat was immobilized in dorsi- flexion for 14 days, there was an increase in the weight of the stretched soleus muscle and a decrease in the weight of the shortened tibialis anterior muscle. Con- versely, fixation of the joint in plantar-flexion reversed the effects, i.e., the soleus muscle atrophied and the tibialis anterior muscle hypertrophied. These authors interpreted their results to suggest that the degree of tension, or stretch, to which the muscle was exposed during joint fixation determined the adaptive new size of the muscle. In later work it was determined that muscle enlargement occurs by the addition of sarcomeres in series (421) without any increase in muscle fiber diameter (368).

In 1953, transient hypertrophy of the denervated side of the diaphragm was reported (365). This finding was attributed to chronic, periodic stretching of the denervated fibers by contractions of the still functional and periodically contracting contralateral hemidiaphragm (149,365). Transient increases both in muscle size and in total RNA were noted. Even though after 7 days of denervation the denervated side of the diaphragm was 55% larger with 74% more RNA, by day 40 of denervation the denervated muscle size had regressed to control values and RNA was only 50% of control (40). Increases in protein synthesis rate of 41,94,43, and 33% have been reported for the denervated hemidiaphragm at 1,3,5, and IO days, respectively (408). Protein degradation rates are increased by 55% on day 1 and by -156% on days 3-7 on the denervated side of the diaphragm. Thus although this model further emphasizes a

role of stretch in the induction of muscle hypertrophy, its shortcoming is the transient nature, since hypertrophy in human skeletal is not transient during heavy- resistance training programs.

Some earlier shortcomings were remedied by the model of stretching the slow-twitch chicken anterior latissimus dorsi (ALD) muscle. Feng et al. (102) first showed that, on its denervation, the ALD muscle undergoes permanent hypertrophy. Later it was deduced that the weight of the denervated wing is the causal factor for the hypertrophy. Supporting this deduction is the observation that attaching additional weights to the denervated wing produces a more rapid and greater hypertrophy than produced by denervation alone (364). These investigators concluded that stretch is the stimulus for growth. Furthermore, they observed the same effect in the innervated ALD muscle. It is also clear that the biochemical adaptations that occur in chronically stretched skeletal muscle are specific to the initial fiber type of the muscle. It must be further emphasized that stretch is not the only stimulus that can generate enlargement of muscle. As mentioned, active mechanical forces also induce muscle enlargement.

2. Adaptive changes in protein expression

Adaptive increases in the mass of the chicken slow- twitch ALD muscle are associated with a switch to a slower myosin isoform, without any change in mitochondrial density (186). With growth of the ALD muscle as the normal chicken grows older, there is a develop- mentally regulated gene switch; the expression of the slow myosin 1 heavy-chain gene is repressed while the slow myosin 2 heavy-chain gene expression is induced. By the attachment of a weight to the wing of 5-wk-old chickens, the switch from slow myosin 1 to slow myosin 2 heavy chain is accelerated (227). The appearance of slow myosin 2 during development is closely correlated with the slowing of the maximal velocity of shortening (320).

Continuous stretch of fast-twitch muscle produces different types of adaptations than those described for the effects on continuous stretch on the ALD muscle. Some of the adaptations in continuously stretched fast muscle are as follows. The percentage increase and the duration of the increase in protein synthesis rate are smaller in continuously stretched fast than slow muscle (249). Mitochondrial density increases in continuously stretched fast muscle (148). Activity of citrate synthase increases 100% and oxidation of succinate increases 174% in fast-twitch muscle that is stretched for 5 wk (186). The same muscles double in mass for this duration of stretching. The activity of &aminolevulinate synthase, the rate-limiting enzyme for heme synthesis, precedes the increase in the activity of cytochrome-c oxidase, an enzyme containing heme in continuously stretched fast-twitch muscle. After 3 days of stretch, no change in cytochrome-c oxidase activity is detected, whereas a 150% increase in &aminolevulinate synthase


activitv and a 60% increase in the mRNA for b-aminolevulinate synthase occurs (97). Essig et al. (97) suggested that both pretranslation and either translation or posttranslation effects, or both, increase b-aminolevulinate synthase activity during stretch of a fast-twitch muscle.

3. Protein synthesis and degradation

When a weight is chronically attached to the wing of a chicken, a 140% increase in protein content occurs in the slow-twitch ALD muscle (249). The increase in muscle protein synthesis rate exceeds the increase in muscle protein degradation rate. As shown in Figure 3, 20% of the increase in the protein synthesis rate ac- counts for net muscle growth while 80% of the increased synthesis contributes to an increased turnover of proteins. Increased protein degradation would be classified as a posttranslational control. An increase in protein translation occurs on day I followed by an increase in RNA quantity on day 3 so that the ratio of protein synthesis per unit of RNA returns to normal at this time (249). Because -80% of RNA is rRNA, the assumption was made that measurements of RNA approximated rRNA quantity. Therefore an increase in the pretranslational control occurs by day 3 of the stretch. When the ALD muscle is stretched, a large increase in the subsar- colemmal concentration of myosin heavy-chain mRNA occurs in the region of these rapidly growing fibers (94). Deoxyribonucleic acid quantity doubles in the hypertrophying ALD muscle, and Laurent et al. (249) deduced that this is likely satellite cell proliferation and incorporation into muscle fibers. This deduction has been confirmed with three observations in the 1st wk of the chronically stretched ALD muscle (226). First, there is an increase in close contact between satellite cells and the sarcolemma. Second, the volume and surface area of

-4Or

10 20 30 40 50 60

t Days of Hypertrophy wemht add-&d

FIG. 3. Calculated percentage rate of protein synthesis as function of time after initiation of constant stimulus (weight added) for hypertrophy of fast-twitch muscle. During growth period, increase in protein synthesis rate over control (normal replacement) is shown. Majority of increase in protein synthesis does not contribute to growth but is involved in remodeling (wastage). [From Millward w-5).1

the satellite cells triples. Third, satellite cell number increases.

The myotendinous junction of a continuously stretched fast-twitch muscle in rabbits is a site of accumulation of slow myosin heavy-chain mRNA (83). Dix and Eisenberg (83) speculate that increased slow myosin heavy-chain mRNA at the myotendinous junction would contribute to an increase in regional protein synthesis and myofibril assembly at this site (83). A large cytoplasmic space that was devoid of myofibrils formed at the myotendinous junction. The sequence of myofibril assembly in this model was found to be that actin filaments attached in part to vinculin on the tendon and sarcolemma. Thick contractile filaments join the thin filaments. Z-bodies then assemble with the thick and think filaments setting the sarcomeric register (83).

The above observations have been extended by Gregory et al. (143) in a report on the synthesis rates of individual contractile proteins and mRNA levels during overload hypertrophy of the chicken ALD muscle. Total protein synthesis rates double at 24 h of overload with no change in polyadenylated mRNA quantity. An increase in translation is inferred. Actin protein synthesis rates double at 24 h and triple at 72 h of overload. In contrast the protein synthesis rates of slow myosin 1 and of slow myosin 2 decrease at 24 h and then increase at 72 h of overload. Because slow myosin 2 replaces slow myosin 1, a selective degradation of slow myosin 1 was inferred. In addition, the relative quantity of slow myosin 1 mRNA decreases even though its absolute protein synthesis rate appears unchanged. These data support the concept that the response of synthesis rates of individual proteins differ among proteins. If true, measurement of changes in the synthesis rate of mixed proteins may be misleading for certain specific proteins.

C. Animal Models of Compensatory Overload-Induced Hypertrophy

1. Protein synthesis and degradation

Another model used for studying adaptive changes to increased usage is compensatory hypertrophy of skeletal muscle. The workload of selected muscles are increased by either tenotomy of synergistic muscles or ablation of some of the synergistic muscle (12,19,123). An increase in protein synthesis rate has been shown to account for net protein gain in skeletal muscles undergoing compensatory hypertrophy (124,158). Excess protein is likely being synthesized because the fractional rate of protein degradation increases 73% in fast-twitch muscle during the 3rd-7th day of its compensatory hypertrophy (272). Most of the increase is blocked by fenbufen, a prostaglandin inhibitor (272).

2. Ribonucleic acid and deoxyribonucleic acid

Ribonucleic acid concentration increases in skeletal muscles undergoing compensatory hypertrophy (124,

564 FRANK W. BOOTH AND DONALD B. THOMASON Volume ?I

158). This increase in RNA appears to be essential to the muscle growth, because treatment of rats with actino- mycin D prevents the compensatory hypertrophy (125, 127). An increased synthesis of RNA in vivo is detect- able within 24 h after initiating the compensatory hypertrophy (363). An increase in RNA polymerase quantity in isolated nuclei in vitro occurs on the 3rd day, but not the 1st or 2nd day, of compensatory hypertrophy (363). Sobel and Kaufman (363) interpret their observations to mean that an increase in the activity of RNA polymerase occurs in vivo on day 1, whereas the quantity of RNA polymerase increases by day 3 (363). Fur- thermore, they suggest that RNA polymerase I (rRNA) and II (mRNA) both increase on day 3. However, it was later demonstrated that proliferating capillaries and fi- broblasts are the major site of the new RNA synthesis in skeletal muscle undergoing compensatory hypertrophy (205). Furthermore, this increase was found to occur only in the distal portion of the hypertrophying soleus muscle. A 30% increase in DNA per whole muscle has been observed on the 4th day of compensatory hypertrophy (158). Some of this DNA increase must be from satellite cell proliferation, as the number of satellite cells increases from 5.8 to 16.6% in the 7th day of compensatory hypertrophy by soleus muscle (159).

3. Myosin isoforms

Tsika et al. (406) showed an increase of fast IIa myosin heavy-chain protein along with the increase in slow myosin heavy-chain protein during continuous overload hypertrophy of the plantaris muscle. Func- tional overload of the plantaris muscle of rats, which is caused by removal of synergist muscles, results in the slow myosin isoform increasing from 4.3 to 18.3% of myosin after 6 wk of the overload (406). Tsika et al. (406) interpreted their results to suggest that the slow myosin isoform can be expressed in increased quantities in fast- twitch muscle if the weight-bearing function is trans- ferred from the slow soleus muscle to the fast plantaris muscle. Thus mechanical stress has a significant impact on the expression of slow myosin in rat skeletal muscle; this conclusion is in agreement with the results from decreased weight-bearing experiments (sect. IVD3).

4. Messenger ribonucleic acids

Extensive evidence exists that myosin isoform expression is shifted from fast to slow during compensatory overload, particularly in fast muscles (15, 19, 147, 199,406). For the most part, a parallel increase occurs in slow myosin heavy-chain mRNA, ,&slow myosin heavy- chain protein, and the percentage of slow-twitch fibers in the plantaris muscle during O-31 days of compensatory hypertrophy (70). A similar parallelism was reported in a study from another group. The ,&slow myosin heavy chain mRNA and its protein increase 235 and 130%, respectively, at the 4th wk of compensatory hy-

pertrophy of the plantaris muscle, whereas increases of 310 and 450%) respectively, occur at the 11th wk of plantaris muscle hypertrophy (310). In the same muscle, there is an approximate parallel increase of 50% in fast IIa myosin heavy-chain mRNA and its protein and an -50% decrease both in fast IIb myosin heavy-chain mRNA and its protein. The report attributed the increase in fast IIa myosin as an intermediate step in the conversion of fast to slow myosin (310). Changes in ,& slow myosin heavy chain mRNA do not occur in the first 7 days of compensatory hypertrophy in the plantaris but increase at day IO (70). The increase in mRNA seems to precede the increase in ,&slow myosin heavy-chain protein (70). Thus pretranslational control appears to be the exclusive control of isoform switching of myosin heavy chain during compensatory hypertrophy in the rat.

Likewise, the decrease in phosphorylase expression in the plantaris muscle during compensatory hypertrophy appears to be totally a result of pretranslational control, because both phosphorylase mRNA and enzyme activity decrease in parallel (70). However, in contrast to the more delayed onset of myosin heavy-chain isoform switching, the decrease in phosphorylase mRNA is complete by the 2nd day of compensatory hypertrophy of the plantaris muscle (70). Crerar et al. (70) concluded that these two genes, ,&slow myosin heavy chain and phosphorylase, are “discoordinately regulated” in this model of hypertrophy.

5. Shortcomings

A number of the responses and adaptations of muscle during compensatory hypertrophy are unusual. The initial 40% increase in the wet weight of the plantaris muscle is due, in part, to edema, connective tissue proliferation, and cells involved in tissue repair (12,198,225). Both edema and leukocyte invasion peak at the 4th day of compensatory hypertrophy of the plantaris muscle. Thereafter, protein accumulation and increases in muscle wet weight parallel each other. Crerar et al. (70) speculated that a transient 70% decrease in ,&slow myosin heavy-chain mRNA on the 2nd day of compensatory hypertrophy is related to “surgical trauma.” During chronic compensatory hypertrophy of the plantaris muscle, a decrease in maximum isometric tetanic tension per unit of cross-sectional area occurs (334). Thus, as stated by Gutmann et al. (150) in 1971, compensatory hypertrophy of skeletal muscle represents a reaction to functional overload that is of doubtful adaptational value because of the decrease in maximum isometric tension per unit of cross-sectional area of the hypertrophied muscle.

VII. MUSCLES OR MUSCLE CELLS IN CULTURE DO NOT

MIMIC HUMAN PHYSICAL ACTIVITY

Numerous reports exist in which organ or tissue culture was employed for the purpose of determining


the chemical linkage between stretch and muscle hypertrophy. An excellent critical review of this area was recently published by Vandenburgh (411). Only selected topics are repeated here. The reader is referred to that review for a more comprehensive treatment.

Shortcomings of most organ culture systems are that skeletal muscles in vitro are in a negative nitrogen balance (118,411) and that organ cultures are not viable long enough to produce hypertrophy. For example, during incubation of the rat diaphragm in unsupplemented Krebs Ringer buffer, the net rate of protein degradation is Z-Z.5 times greater than the rate of protein synthesis (118). It is possible that mechanisms controlling protein synthesis and degradation differ from in vivo mechanisms when such large differences in protein balance exist. Therefore effects of stretch may be acting though different mechanisms than present in vivo. However, under selected experimental conditions, nitrogen balance can be made to zero in the incubated fast-twitch epitrochlearis muscle (374).

In contrast to whole muscles, Vandenburgh (411) indicated that primary cultures of skeletal muscle cells provide an advantage over whole muscle cultures, because cultures of muscle cells can be maintained in a condition of positive nitrogen balance for weeks. More- over, mechanical stretching of embryonic chicken skeletal myotubes while they are cultured leads to an increased accumulation of total protein and myosin heavy chains (412). A shortcoming of cultured muscle cells is that often they do not express slow myosin or adult isoforms. Also cultured cells cannot mimic the mechanical loading existent in human sport, nor do they experience the hormonal and neural environment of muscle in human physical activity.

Stretch of either cultured whole muscle or muscle cells results in an increased rate of protein synthesis (23,41,48,126,300,301,412). Cultured skeletal myotubes respond to passive stretch by a 60-70% increase in the Vmax of the sodium pump (413). No increase in the number of sodium pumps was detected. Ouabain prevents the stretch-induced increase in protein synthesis (413). Vandenburgh and Kaufman (413) suggested that sodium pump activation may be involved in the stretch-induced cell growth of cultured cells. On the other hand, tetrodotoxin inhibits voltage-dependent sodium channels and spontaneous activity but does not prevent the increases in either the protein synthesis rate or in protein accumulation that occurs when muscle cells are stretched in culture (412).

It has been suggested that stretch of whole muscles increases prostaglandin synthesis by the muscle, which in turn increases muscle protein synthesis (361). Prosta- glandin secretion into the culture medium increases in mechanically stimulated muscle cells (163). Both indo- methacin and meclofenamic acid, which are prostaglandin synthesis inhibitors, decrease the rate of protein synthesis in intermittently stretched muscles in vitro (361). An earlier report from the same laboratory indicated that muscles incubated under control conditions have a protein synthesis rate that is 22% of those found in vivo.

Another prostaglandin synthesis inhibitor, fenbufen, does not prevent compensatory hypertrophy in rats but lessens the increase in protein synthesis rates at the 7th day of hypertrophy (272). No role was found for prostaglandins as a causal factor in the acute stimulation of protein synthesis in the heart by hypertensive aortic pressures or insulin in vivo (361).

Although one report found that the calcium iono- phore A23187 increases protein synthesis rates in incubated skeletal muscles (219), two later studies have not observed an effect (331,361). Thus increased sarcoplasmic free Ca2’ may not link increased contractile activity to increases in protein synthesis in skeletal muscle. On the other hand, increased free Ca2’ has been shown to increase protein degradation in skeletal muscle in vitro (219). It is clear that muscle hypertrophy is associated with an increased protein degradation (249). A potential consequence of increased free Ca2’ is membrane dam- age. Vitamin E has been shown to inhibit the efflux of creatine kinase from A23187-treated skeletal muscle in vitro (313). Phoenix et al. (313) suggested that vitamin E inhibits the muscle sarcolemmal changes induced by intracellular Ca2+ overload, which, in the absence of vitamin E, causes intracellular enzyme efflux (313).

VIII. REGROWTH OF ATROPHIED SKELETAL MUSCLE

Multiple sites controlling gene expression are invoked in the early period of recovery from muscle atrophy, before demonstrable muscle enlargement. Protein content of fast-twitch muscle decreases 27% during 7 days of limb fixation in a shortened position in rats (407). During the initial 4 days of recovery from the ‘I-day immobilization, muscle weight does not increase (37). Nevertheless, actin protein synthesis rate, which is 33% of the control level at the 7th day of hindlimb immobilization, returns to the control value on the 2nd day of recovery and is three times higher than control in the fast-twitch muscle on the 4th recovery day after limb immobilization in rats (285). During the recovery, rats were permitted cage activity. The skeletal cw-actin mRNA is 53% of control at the 7th day of limb immobilization, and its increase during the first 2 recovery days parallels the increase in actin synthesis rate; this suggests that pretranslational mechanisms are the cause of the initial increase in actin protein synthesis rate in fast-twitch muscle recovering from atrophy (285). How- ever, further increases in actin synthesis from the 2nd- 4th day appear to be under translational control, since actin protein synthesis rate is 300% of control on the 4th recovery day, but skeletal cu-actin mRNA is only 128% of control (285). Because muscle protein mass is unchanged, this suggests an increased remodeling (increased protein degradation). Thus in the initial phases of recovery as fast-twitch muscle prepares to regrow from atrophy, pretranslational, translational, and posttranslational mechanisms for skeletal cu-actin gene expression are invoked.

The recovery of cytochrome c gene expression from

566 FRANK W. BOOTH AND DONALD B. THOMASON

7 days of limb immobilization has a somewhat different time course (284). During the first 2 days of recovery, cytochrome c protein synthesis rate and cytochrome c mRNA were maintained at the low levels found at the start of recovery from muscle atrophy caused by 7 days of hindlimb immobilization in rats (284). However, at the 4th recovery day, cytochrome c protein synthesis rate is 192% of control while cytochrome c mRNA is 126% of control. Because the percentage increase in cytochrome c protein synthesis is greater than the percentage increase in cytochrome c mRNA, it is likely that both pretranslational and translational mechanisms are invoked in as muscle begins to recover from atrophy.

IX. SUMMARY OF INFERRED SITES FOR GENE

EXPRESSION IN THOSE ANIMAL MODELS THAT

CLOSELY MIMIC HUMAN PHYSICAL ACTIVITY

To make a general conclusion about the potential sites of gene expression in skeletal muscle where altered muscle usage produces a change in protein quantity, three different proteins, the quantities of which are altered in four animal models of various human physical activities are shown in Table 3.

The composite of the data leads to the next conclusion. Multiple sites (pretranslational, translational, and posttranslational) are inferred to be evoked as protein quantities adapt to new steady-state levels because of chronic changes in muscle usage. Such an analysis suggests that the control of gene expression in skeletal muscle during chronic changes in human physical activity is very complex. It is likely that most, if not all, of the following are altered: gene transcription, mRNA stability, protein translation, protein assembly, and protein degradation. If true, this means that efforts to delineate all mechanisms by which human physical activity produces adaptive changes in protein quantity will require more research time than envisioned a decade ago.

Volume 71

X. ADAPTATIONS THAT AFFECT CARDIAC OUTPUT

The molecular and cellular adaptations of the cardiovascular system to exercise training are considered, as they ultimately affect the capacity to deliver oxygen and nutrients. The data must be viewed with the bias that the adaptations, if important, affect the economy of delivery. This is an underlying premise of adaptation. Therefore the adaptations are considered as they affect the minute work of the heart within the entire cardiovascular system: heart minute work = heart rate x

stroke volume X pressure. To provide a more efficient delivery of oxygen and nutrients during exercise, the exercise training adaptations of the cardiovascular system are viewed with the inclination that pressure increases are minimized in favor of adaptations that provide an enhanced stroke volume. This supposition is based on the tenet that changing the work of the heart by changing volume is energetically more efficient than by changing pressure (98). Although the adaptation of the cardiovascular system to exercise training has been extensively reviewed (35, 348), significant advances have been made in the tools and concepts with which to approach the molecular and cellular mechanisms of adaptation.

The cardiovascular system is extremely responsive to functional demand on a beat-to-beat basis, often maintaining function by drawing on one mechanism to compensate for another that has been compromised. This intrinsic responsiveness often makes it difficult to definitively single out one factor or mechanism responsible for a particular adaptation. For example, during adrenergic ,&receptor blockade in humans, maximal cardiac output is maintained, despite a reduced heart rate, by an increase in stroke volume (32). Another example occurs during exhaustive exercise. Cardiovascular drift, i.e., a progressive decrease of systolic pressure and stroke volume, is compensated by increasing heart rate, resulting in no net change in work (278). With this com- pensating ability as a potential complication to inter- pretion, the cellular and molecular adaptations to exer-

TABLE 3. Inferred or directly measured sites of altered gene expression in skeletal muscle of adult rats

Activity Model Protein Pretranslational Translational Posttranslational

Nonweight bearing

Recovery from atrophy

Run training

Resistance training

Actin

Myosin heavy chain

Actin

Cytochrome c

Actin

Cytochrome c

Actin

Cytochrome c

rRNA

X X X

X X X

X X X

cl

e X X X

X

cl X X

X X X

X X X

cl

El X

X, Site is affected; x II

, inferred as first site of temporal regulation.


cise training that affect cardiac output are considered next. They fall into the two broad categories of stroke volume and chronotropic adaptations.

A. Stroke Volume Adaptations

The stroke volume adaptations to exercise training can be further subdivided into those that alter stroke volume by the Frank-Starling mechanism and those that alter ejection volume through changes in contractility (inotropic adaptations). In the untrained individual exercising at relatively low work loads, the Frank-Star- ling mechanism is the most important factor for maintaining cardiac output; at near maximal work loads, where end-diastolic volume cannot be further increased, an increase in ejection fraction is necessary for an increase in cardiac output (175). These mechanisms for providing sufficient cardiac output to maintain a given work load are modified and enhanced as a result of training. For example, in sprint-trained rats, cardiac output can be maintained at a greater level than in the sedentary animal despite a lower or comparable heart rate due to an increased stroke volume (121,176). Next, some of the mechanisms whereby exercise training alters the Frank-Starling and inotropic mechanisms for. maintaining cardiac output are explored.

0 Right Atrial Pressure

B

vasodilation

cardiac function

I. End-diastolic volume: Frank-Starling mechanism


1)ADAPTATIONSTHATAUGMENTVENOUSRETURN ORINCREASE CENTRALBLOODVOLUME. A)Homeostatic mechanisms. Exercise training increases the plasma and blood volumes in humans as a result of adjustments in fluid conservation (69,115,161,298). The expansion of blood volume serves to increase mean circulatory filling pressure (228) either directly or by autoinfusion during exercise, thus augmenting cardiac output by the Frank- Starling mechanism (Fig. 4A). We examine some of the

3 a 5

C

vasodilation enhanced contractility

0 0 m

cardiac function

mechanisms involved in fluid conservation as reflected in those hormones responsible for electrolyte and water balance.

Despite the increase in blood volume and the lower mean arterial pressure that are nominally found in en-

0

,


durance-trained subjects, at rest atria1 natriuretic pep- FIG. 4. At given level of exertion, cardiac output is equilibrium of

tide (ANP), plasma renin activity (PRA), antidiuretic vascular function and cardiac function. A: as result of training, in-

hormone (ADH or vasopressin), and aldosterone levels creased blood volume in endurance-trained subjects would shift equilibrium point on cardiac function curve to greater potential cardiac

A

trained cardiac function

are not different from the untrained individual (115, output. This response is similar to effect of transfusion. B: given in- 116,415). Therefore resting levels of these factors do not creased blood volume in trained individual, vasodilation (decreased

explain the increase in blood volume. To determine if peripheral resistance) would augment venous return and shift equilib-

there is a difference in the ability of trained and un- rium point further up cardiac function curve. C: in addition to effects

trained subjects to handle a water load, Freund et al. of increased blood volume and decreased peripheral resistance in trained individual, an increase in cardiac contractility would further

(115) examined the plasma levels of these hormones increase cardiac output.

after water ingestion. They found that water ingestion does not differentially affect ANP, PRA, or aldosterone in the endurance-trained subjects (runners) compared creases, whereas the plasma ADH decrease of the with untrained control subjects. However, the plasma trained individuals is blunted (115). The fact that there ADH level in the untrained individuals significantly de- is no difference in the change in plasma volume and


there is a similar decrease in plasma osmolality in the trained and untrained subjects in response to an in- gested water load (115) indicates a decrease in sensitivity of the osmoreceptors in trained individuals. This is in contrast to a previous study that indicated an increase in plasma ADH levels because, in response to water load or whole body immersion, endurance-trained individuals (runners and swimmers) exhibit a greater ADH excretion (63) despite an apparent decrease in the glomerular filtration rate in trained individual [as esti- mated from creatinine clearance (115)]. Thus there appears to be some uncertainty about the response of ADH as a possible mediator of the increased plasma volume in athletes. To add to this uncertainty is the observation that during exercise, despite the lack of a difference in plasma ADH levels between trained and untrained individuals exercising at the same relative work load or at maximum work load, plasma ADH levels are lower than in the trained individual at the same absolute work load (66, 415). This adaptation in ADH response would lead to an increase in free-water clearance in the trained individuals. In further contrast is the observation that trained subjects exhibit a decreased plasma osmolality, perhaps as a result of increased sensitivity of osmoreceptors (114). This finding is clearly in conflict with the apparently blunted osmoresponse to water challenge just mentioned. On the basis of these data, we can only conclude that the ADH response and osmoreceptor adaptation to exercise training remains an open question.

Exercise itself stimulates the renin-angiotensin-aldosterone axis, setting into motion a means by which fluid conservation can occur. However, aerobic training has no differential effect on the renin-angiotensin-aldosterone axis response to exercise in endurance-. trained versus untrained individuals, except that trained individuals exhibit an attenuated PRA level at 90 % vo, max (256). Although a diminished PRA level relative to control would apparently work against fluid conservation, adaptation to the training apparently also occurs at the level of the adrenal glomerulosa cell because, during the exercise, the increased aldosterone level is not correlated with its known regulatory substances (256). On the other hand, Hespel et al. (172) found a slight nonsignificant depression of the renin- angiotensin-aldosterone axis with endurance training but observed that the decrease in these substances was negatively correlated with the increase in work capacity after training. Therefore the greatest depression of the renin-angiotensin-aldosterone axis occurs in those individuals on whom training has the greatest effect (172).

An exciting development within the past decade with regard to fluid homeostasis has been the character- ization of ANP. The effects of ANP are somewhat mixed and even counterintuitive with regard to exercise train-, ing adaptation. It is worthwhile to briefly review the putative mechanisms by which ANP secretion is regulated. The causative factors involved in ANP release are primarily related to changes in atria1 pressure. Mea- sures that increase central blood volume increase ANP release (87). whereas a decreased blood volume does not

change ANP levels (178). Increasing the mean arterial pressure appears to be a stimulus for ANP release (293, 335), although the suggestion has been made that heart rate may also be influential (337). However, cardiac pac- ing in dogs does not change ANP levels, whereas increased atria1 pressure does cause ANP release (417). The actual cellular mechanism by that atria1 cells are stimulated to secrete ANP in response to changes in pressure or stretch has yet to be fully defined. Prevent- ing cultured rat atria1 myocytes from contracting reduces the secretion of ANP in a manner that apparently is not dependent on calcium; nevertheless, ANP secretion is stimulated by phorbol ester, indicative of a protein kinase C-mediated stimulus (200). Increased sym- pathoadrenal activity may also contribute to ANP secretion (392) as discussed below.

How then could a factor known to induce natriuresis contribute to an overall improvement of cardiovascular function after exercise training? In addition to the natriuretic effect of the peptide, infusion of physiological doses of ANP into a normal individual causes arterial vasodilation and a decreased total peripheral vascular resistance (34,49). In untrained individuals this results in decreased cardiac output as a result of decreased venous filling pressure (122), but in the trained individual with sufficient vascular reserve, this would have the net effect of shifting toward greater cardiac output on the cardiac function curve. Atria1 natriuretic peptide also inhibits the secretion of aldosterone, although at plasma concentrations that may exceed those normally found (3). One possible mechanism for this response is a differential effect of ANP on the voltage-sensitive Ca2+ channels of the glomerulosa cells, inhibiting the Ca2’ current derived from T-type channels (258). These non- natriuretic consequences of ANP action may play the more important role in the exercise training adaptation, because ANP levels do not indicate a blunted natriuresis. Resting levels of ANP are similar in both trained and untrained individuals, although glomerular filtration rate is lower in the endurance-trained subject (115), perhaps diminishing the potential for natriuresis. Fur- thermore, ANP levels increase with various exercise regimen (116), but ANP levels increase more during exercise in subjects receiving ,&adrenoreceptor blockade (393). This suggests an influence of sympathetic drive (which is depressed in trained individuals) on the inhibition of ANP release. Thus, although it is difficult to see how natriuresis as a result of ANP release could augment venous return, the secondary effects of ANP may be more important to the trained individual. This is cer- tainly an area that deserves further research.

B) Vascular tone. Although the concept of the “muscle pump” as an important factor in augmenting flow to the venous side of the circulatory system has been un- derstood for over 40 years (315), little attention has been given to potential adaptations that can occur at the level of the arteriolar musculature to augment flow to exercising muscle. Such adaptations could serve to enhance the muscle pump in a manner analogous to the enhancement of cardiac output bv an increased venous return.


Several lines of evidence suggest that exercise training adaptations in vascular tone do occur, although the mechanisms for the adaptation remain to be explained. Using microspheres, Armstrong and Laughlin (IO) demonstrated that blood flow to exercising endurance- trained muscle is greater than in control animals. The fact that this muscle is working and acts as a muscle pump would augment venous return (245). Further evidence of an adaptation in arteriolar tone with exercise training is the decreased total peripheral vascular resistance observed during recovery from exercise in trained subjects (76). Central to the mechanism of the adaptation that affects arteriolar tone is the training-induced attenuation of central baroreflex function. In treadmill- trained rabbits, experimentally induced inhibition of cardiac afferent nerve activity results in a normal renal sympathetic nerve response to changes in pressure, ameliorating the attenuated response observed after training (77, 78). Exercise training in rabbits also enhances blood flow to the renal and mesenteric arteries through an increased inhibitory effect of the cardiac vagal afferents on the exercise-induced increase in vascular resistance of these arteries, as determined by blockade of cardiac efferent nerve activity (79). Thus baroreceptor function apparently adapts with exercise training (151) such that greater blood flow is maintained in nonworking organs, a phenomenon that is one of the hallmarks of endurance exercise training (332). This attenuated baroreceptor response may also contribute to the increased plasma volume by decreasing the renal vascular response, i.e., attenuated sympathetic nerve activity to increased blood pressure during the exercise bout itself. In addition to a central mechanism for maintaining blood flow to the working muscle and thus augmenting venous return, adaptation at the arteriolar level also occurs, as indicated by the increased capacity for maximal vasodilation [i.e., a greater maximal conductance (271, 353, 362)]. All of these data indicate a training-induced enhancement of venous return by effectively increasing venous filling pressure.

II)INFLUENCEOFEXERCISEBRADYCARDIAONFILL- ING TIME. One of the most striking effects of exercises training is the resulting bradycardia. The physiological significance of the bradycardia on cardiovascular adaptations to exercise is apparent under the conditions of ,&adrenergic receptor blockade. In untrained humans given ,&adrenergic blockers while exercising at intensities requiring maximum oxygen consumption, cardiac output is maintained at control levels (despite a slower heart rate) by increasing stroke volume (32). The slower heart rate allows a longer filling time and a greater end-diastolic volume, resulting in a greater stroke volume. The exact mechanism responsible for training bradycardia remains elusive. Treadmill training of rats produces a bradycardia that does not depend on ,& adrenergic mechanisms, either systemically or cen- trally, because receptor blockade with either the general ,&blocker propranolol or the cardioselective ,&blocker metoprolol does not prevent the training-induced bradycardia (296). This observation is consistent with the

data from swim-trained rats. ,&Adrenergic receptor density on the myocardium of these animals does not change, suggesting that the site of action is not ,&adrenergic receptors (426). However, there is a decreased myocardial cholinergic and a-adrenergic receptor density in swim-trained rats, but the significance of these changes is unknown (426). The mechanism of exercise training-induced bradycardia is discussed more fully in section XB.

2. Ejection fraction (size and contractility)

In addition to the changes in cardiac output that occur as a result of changes in stroke volume by the Frank-Starling mechanism, it is possible that the fraction of blood ejected during each stroke can be changed by adaptations in contractility and functional mass of the ventricles. These adaptations, because they occur at the cellular level, are often difficult to detect functionally. For example, in pigs trained by treadmill running for 10 wk, no adaptations were observed in one study where contractility was assessed by measurement of ventricular dimensions and left ventricular change in pressure over time (420). On the other hand, in humans, stroke volume is greater at all levels of upright exercise as a result of training (382). This may be a unique but necessary adaptation in humans because, despite an increase in venous filling pressure, end-diastolic volume decreases and thus an increase in stroke volume is needed to maintain cardiac output during upright exercise (175). Therefore, as discussed next, assessment of function and the correlation of function with molecular and cellular adaptations is not straightforward, reflect- ing the remarkable adaptability and compensatory nature of the cardiovascular system.

I)ADAPTATIONOFHEARTSIZE. Althoughitisgener- ally accepted that the increased functional demand placed on the heart by endurance exercise training will produce cardiac hypertrophy, exercise training models that produce changes in cardiac mass are not always as consistent as would be expected when addressing the mechanisms of the changes (160). In part, the difficulty arises from the desire to project what is observed in an experimental animal to the human condition, which are two entirely different hemodynamic situations. In addition, there appears to be a strong genetic component to cardiac function, as illustrated in dogs where, compar- ing racing greyhounds and mongrels, cardiac size does not correlate well with cardiac functional parameters (307). Furthermore, different types of exercise appear to have different effects, i.e., endurance training in humans has a tendency to produce hypertrophy (normalized for lean body mass), whereas resistance training does not produce such a hypertrophy (relative to lean body mass) despite the apparently similar left ventricular contractility (80, 229, 254, 410). Therefore, adaptation at the cellular and molecular level, despite its proba- ble importance for functional adaptation, may not al- wavs be reflected functionallv.


One of the more popular models for inducing cardiac hypertrophy is that of increased myocardial wall stress as a result of pressure overload (increased afterload). As discussed below, this may not be an appropriate model for studying exercise-induced cardiac hypertrophy, but it serves as an example of some of the points to be considered in adaptation of cardiac protein expression. Early in the onset of pressure overload there is an increased formation of ribosomes (62) and increased protein synthesis rate in a manner that is apparently mediated by a CAMP-protein kinase mechanism (419, 436). Mechanical stretching of S49 mouse lymphoma cells directly stimulates adenylate cyclase (418). Furthermore, the period of exposure need only be brief to produce such changes (436). In young rats, pressure overload causes an increased c-rnyc mRNA expression in both atria and the left ventricle (287). Although expression of the cellular oncogenes is generally considered to be indicative of a growth “program,” the lack of expression of r-fos in response to the pressure-overload model reflects a lack of mitotic activity in the hypertrophy process (287). The link between cellular mechanisms involved in pathological hypertrophy (pressure overload) and normal growth comes from the observation that in the normal growth process of the pig heart the left ventricle maintains a greater rate of ribosome formation than the low-pressure right ventricle (57). This suggests, therefore, that at least some of the fea- tures of the control of protein expression in the pressure-overloaded heart are similar to those involved in exercise-induced cardiac hypertrophy.

However, induction of pressure overload may not be the mechanism for exercise-induced cardiac hypertrophy, because the effect of swim training on myosin isoforms, ATPase activity, and functional indexes are the same in both normal and spontaneously hypertensive rats (354). Also, the head-down tilt caused in the rodent tail-traction model of hindlimb nonweight bearing, a posture that should produce a transient volume overload and increased hydrostatic afterload, causes a decrease in myocardial total mixed protein synthesis rate within just a few hours (396). It is interesting that despite this decrease in protein synthesis rate, no cardiac atrophy occurs (indicating a concomitant decrease in protein degradation) (399). Nonetheless, when treadmill-running exercise is given adjunct to the nonweight- bearing hindlimb, significant cardiac hypertrophy does occur (398). Therefore, although there may be specific cellular and molecular changes as a result of increased pressure, as a cellular system the adaptations may not’ be expressed as a functional adaptation.

A mechanism for exercise-induced cardiac hypertrophy that is not mediated by wall stress must also be considered based on the following evidence. Isoproter- onol is known to cause cardiac hypertrophy (383) without changing functional capacity (17). The fact that a receptor-mediated mechanism may be involved is suggested by the observation that in male rats, ,&adrenergic receptor blockade can prevent a treadmill training- induced cardiac hypertrophy (213). However, it must be

remembered that, with swim training, myocardial ,& adrenergic receptor density does not change (426), indicating there is not the dogmatic up- or downregulation of these receptors that might be expected from a receptor-mediated stimulation. A role for glucocorticoids is also suggested, because the binding capacity for glucocorticoids was increased in the heart of female rats that were swim trained and exhibited cardiac hypertrophy (174). In these same rats there is a decrease in plasma corticosterone levels and no change in androgens. How- ever, there is an additive effect of treadmill exercise and cortisol acetate treatment on cardiac hypertrophy in female rats (241). In these animals the cortisol treatment causes a decreased corticosteroid and androgen binding capacity in the myocardium that was not modulated by the exercise.

Therefore, perhaps not surprisingly, the mechanisms of cardiac protein expression that are influenced by exercise apparently involve complex events. We noted that several signal transduction mechanisms may be active and that the sight of action of these signals may be at several control points, i.e., transcriptional, translational, and posttranslational.

II)ADAPTATIONOFMYOFIBRILLARPROTEIN. Adapta- tions in cardiac myofibrillar protein expression are functionally important, because the contractile machinery modulates calcium sensitivity and transduction of chemical energy into mechanical work (13,24, 190,299, 395). As with cardiac hypertrophy, some of the adaptations observed are dependent on the exercise model employed.

From a pedagogic point of view, the shifts that occur in myosin isoform expression as a result of exercise training in rats appear to be compensatory for the progressively decreased pCa required for activation of the myofibrils (decreased contractility) during an acute bout of exhaustive exercise (27, 29), that is, toward a more energetically active isoform. In swim-trained rats there is either a shift in the relative distribution of the myosin isoforms toward the V, species (269,299,381) or no change in the isoform profile (51). However, in both normotensive and hypertensive rats given swim training in one study, despite the hypertrophy, no change was observed in isoform profile (all V,) or Ca2+-activated ATPase activity, but an increase in stroke and cardiac performance indexes was noted (354). In another study, a functional improvement of hypertensive rats with swim training was also observed, although this time it was accompanied by a shift in the myosin isoform profile (344). Functional improvement with swim training has also been observed accompanied by a decreased expression of the V1 myosin isoform (51). These results are in contrast to those of Pagani and Solar0 (299) in whose study swimming exercise caused an increase in the Ca2+-stimulated ATPase in the trained animals, with no change in M$+-stimulated ATPase, as well as a shift toward the V, myosin isoform. It is interesting to note that in the latter study, hypothyroid animals receiving the swim training did not exhibit the shift in myosin isoform profile toward the V1 isoform. We must there-


fore conclude that the relationship between improved cardiac performance and changes in myosin isoform profile is at best tenuous and oversimplified.

Treadmill-running exercise has a considerably less striking effect on myosin isoform profile than does swim training. As noted by Baldwin (14) and Baldwin et al. (16), even in species with a large potential for myosin isoform shifts toward the V, isoform, there is little evidence to suggest the occurrence of such a shift. Further- more, the age-induced shift in myosin isoforms from V1 to a more equal disposition of all three isoforms in the rat myocardium is not affected by treadmill training (100). Thus it appears as if the control of cardiac myosin isoform expression during treadmill training is under a stringent control to prevent shifts in profile. Indeed this control may be independent of contractile function, as indicated by the data on trained neonatal rats, where the shifts in isoform expression during a 9-wk treadmill training program are exaggerated in sympathectomized animals (283). Furthermore, the metabolic state of rats also controls myosin isoform expression, as indicated by the shift in myosin isoform profile that can be induced by modifying dietary carbohydrate (282). However, an infarct-induced shift in rat myosin isoform profile from the V, isoform to the V, and V, isoforms is partially reversed by treadmill training but does not result in any alteration in maximum cardiac output or stroke volume (288). Covalent modification of the cardiac contractile proteins may also be an adaptation that enhances cardiac output. Phosphorylation of myosin light chains has been correlated with positive inotropic effects in rats (238). Furthermore, myosin light-chain phosphate content correlates with the double product (heart rate X pressure), a factor in minute work (109). The idea that this mechanism may play an important role in the trained individual is based on the observation that, in treadmill-trained rats, there is an increased Ca2+-stimulated myofibril ATPase and an increase in catecholamine-stimulated myosin light-chain phosphorylation (324), a response correlated with positive inotropic effects (238; Fig. 4C).

In general terms, the inotropic training adaptation of the cardiac myofibrillar protein, if indeed one occurs, is toward an energetically more active condition. An example of this is evident in the shift in myosin isoforms toward the V, isoform. Furthermore, covalent modification of these proteins appears to also be concerted toward providing a more responsive system to inotropic stimulation. An example of this effect is enhanced phosphorylation of the phosphorylatable light chain of myosin in treadmill-trained rats without a difference in CAMP levels compared with control animals (324) and changes in contractility associated with activation of CAMP pathways are correlated with the proportion of the V, isoform (430).

III) ADAPTATION OF THE PLASMA MEMBRANES. A) Sarcolemma. Treadmill exercise training induces a lengthened plateau phase of the cardiac action potential, indicative of increased Ca2’ flux across the sarco- lemma1 membrane caused, at least in part, by an in-

creased number of low-affinity sarcolemmal Ca2’-binding sites (402). Part of the increased Ca2+-binding capacity may be a result of the increase in phosphati- dylserine content of the sarcolemmal membrane (404), although the possibility of an effect of the altered lipid composition on the L-type Ca2+ channel has not been investigated. In support of this possible effect of altered lipid composition on the Ca2’ channel is the evidence that an 11-wk training program of rats decreased the K, of the Na+-Ca2’ exchanger in sarcolemmal vesicles from 36.1 to 15.7 PM with no alteration of Vmax (403). This electrogenic exchanger is sensitive to changes in plasmalemmal lipid composition (312). As noted, the lipid profile of the sarcolemmal membrane from the hearts of trained animals does change, and this could account for the change in K, of the Na+-Ca2+ exchanger as well as possible alteration in the L-channels. Re- cently, ANP was shown to stimulate the L-type channel in bovine glomerulosa cells, leaving open the possibility of an endocrine modulation of this channel as a consequence of exercise training (258). Furthermore, ANP can alter the selectivity of the cardiac sodium channel such that it can conduct Ca2’ (366). Another possible effect of exercise training on the sarcolemmal Ca2’ channel is suggested from the observation that the level of the stimulatory guanine nucleotide binding protein (G,) increases in the hearts of treadmill-trained pigs (157); this protein has been shown to activate the Ca2’ channels from bovine sarcolemma (202). Together, these factors could serve to increase Ca2+ flux across the sarcolemma as an adaptation to exercise training.

Although it would not directly affect contractility, hyperkalemia would eventually compromise cardiac contraction (and a few other vital processes). Therefore it is interesting to note that swim training of rats increases the K+-dependent 3-0-methylfluorescein phosphatase activity in the myocardium (indicative of ATPase activity) as well as the number of [3H]ouabain-binding sites in skeletal muscle (236). In humans, the level of expression of the Na+-K+-ATPase in myocardial biopsies correlates with ejection fraction measured in the same individual (235). These data provide evidence that both central and systemic adaptations in the expression of Na+-K+-ATPase in response to exercise training occur, possibly to minimize the potential for hyperkalemia.

B) Sarcoplasmic reticulum. As the primary store of intracellular Ca2+, the sarcoplasmic reticulum (SR) provides an important, albeit difficult, organelle to study the regulation of cardiac contractility. Swim training in rats produces an adaptation in the SR that provides a greater sequestering capacity (268, 309), perhaps providing a more rapid cycling of Ca2+ during the cardiac cycle. This should facilitate relaxation, which would be important for coronary perfusion at a rapid heart rate. The exact mechanism for such an adaptation is not apparent, however, because despite the increased SR Ca2+ binding and uptake there is no difference between swim-trained and control rats in SR Ca2’-ATPase activity (268). This may be an age-dependent effect, however, since senescent male rats show a marked improvement


in papillary muscle contractile function with treadmill training accompanied by an increase in the rate of Ca2’ uptake by the SR (384). In contrast, no change is observed in the cardiac SR Ca2+ uptake of treadmill- trained dogs who were not senescent (367). In addition, an increase in intracellular Ca2+ concentration will increase Ca2’ release from the SR before the Ca2+-induced Ca2’ release phenomenon (99). Therefore the aforemen- tioned adaptations in the sarcolemmal membrane that enhance sarcolemmal Ca2+ flux would also produce an effect at the SR.

IV) ADAPTATIONS IN CATECHOLAMINE SENSITIVITY. The effect of circulating catecholamines on myocardial contractility is a well-documented phenomenon. There- fore the decrease in resting plasma catecholamine levels resulting from exercise training, with the more dra- matic reductions occurring in endurance-trained athletes as opposed to resistance-trained athletes (214, 428), may impact on myocardial contractile adaptations. These should be reflected at the receptor or postreceptor level. One problem with assessing adrenergic receptor changes is the inability to obtain tissue samples from humans. However, an important observation is that the variation in lymphocyte adrenergic receptors may be indicative of variation in other tissues (1,42).

This observation has allowed exercise training adaptation of adrenergic receptors to be studied in humans using blood samples.

In humans, a decreased ,&adrenergic receptor density on lymphocytes in endurance-trained individuals has been observed in one study, but this decrease in density is absent in resistance-trained athletes (214, 215). This observation contrasts with the lack of change in lymphocyte ,&adrenergic receptor density previously observed with running exercise training in humans seen by Williams et al. (424) and the increase observed by Lehmann et al. (252). However, lymphocyte receptors are not indicative of regional variation in receptor density, as indicated by a decrease in the right atria1 ,& adrenergic receptor density in treadmill-trained pigs but no change in left ventricular density (157). Of note is the lower cu-adrenergic receptor density on platelets of weight lifters and perhaps a greater receptor sensitivity in endurance-trained athletes (214,215). This increased sensitivity could be the mechanism by which the circulating catecholamine levels are diminished as a result of presynaptic feedback inhibition. However, resistance- trained subjects apparently have a greater cY-adrenergic receptor density on lymphocytes (252).

At the postreceptor level there appear to be exercise-induced adaptations that augment the response to catecholamines. In trained cats there is an increased responsiveness of adenylate cyclase to catecholamines (435). There is also an increased sensitivity to catecholamines of the papillary muscle of swim-trained rats as manifested in the isoproteronol-induced increased isometric tension and the change in tension per unit of time relative to control tissue (381). In this study they found that an increased affinity of the receptors, and not receptor number. was responsible for the increased

sensitivity. However, in humans, isoproteronol-stimulated CAMP production in lymphocytes was diminished with endurance training but not with resistance training, without an apparent change in sensitivity (214,215). Nonetheless, in treadmill-trained pigs the decreased or unchanged ,8-adrenergic receptor density presented an increased sensitivity of heart rate to isoproteronol(l57). This may be a result of increased levels of G, (157). In this respect the treadmill training differs from pressure overload where there is an increased ,&adrenergic receptor density and decreased adenylate cyclase activity and G, levels (253). There may also be an increased sensitivity of myosin light-chain kinase to CAMP stimulation as indicated by the increased rate and extent of catecholamine-stimulated myosin light-chain phosphorylation in the hearts of treadmill-trained rats despite the lack of a difference in CAMP levels between trained and control hearts (324). However, this observation has not been corroborated in treadmill-trained rats by the recent data of Fitzsimons et al. (110).

On the whole, exercise training apparently produces an increased sensitivity to catecholamines in the heart, either at the level of the receptor or by a postreceptor mechanism. However, regional differences in receptor-mediated catecholamine sensitivity also occur. Therefore it is not too surprising that left ventricular function and catecholamine levels are not strongly correlated, making plasma catecholamine levels a poor indicator of inotropic effects in dogs (439) and inotropic and chronotropic effects in humans (379).

B. Chronotropic Adaptations

As mentioned in section XA~II, exercise training- induced bradycardia has significant functional consequences for the heart. The mechanism by which this bradycardia develops is not clear, however. Exercise bradycardia occurs only in endurance-trained subjects (252) during ergometric tests, indicating an increased vagal tone in these subjects because decreased plasma levels of catecholamine are observed in both resistance- trained and endurance-trained subjects. However, in the context of chronotropic adaptation, adrenergic receptor sensitivity is not altered with training, as evidenced by the lack of change in the responsiveness of heart rate to catecholamine infusion (422). Therefore the decreased circulating levels of catecholamines may nonetheless be important in modulating intrinsic heart rate. Because the sympathetic and parasympathetic mechanisms for controlling heart rate are opposed, Ra- ven and co-workers (360) have defined the concept of “autonomic balance” for the relative influence of parasympathetic and sympathetic tone. Trained subjects have an autonomic balance that is shifted toward greater parasympathetic influence (360).

Despite this conceptualization, the exact cellular mechanism for exercise bradycardia remains to be elu- cidated. Very probably there is a central nervous mechanism for the autonomic balance. but cellular adapta-


tions in the sinoatrial node may also occur. For example, acetylcholine inhibits the hyperpolarization-activated (pacemaker) current in the sinoatrial node cells at concentrations much lower than required to activate the potassium channels previously thought to control pacemaker activity in these cells (81). This means that only a slight increase in vagal tone is required to slow heart rate. In addition, the mechanism of acetylcholine action is to inhibit adenylate cyclase activity (82, 3.29), poten- tially altering pacemaker activity through a second messenger action. However, such a process would be slow on a beat-to-beat basis, and recent evidence has shown that the pacemaker currents are readily modulated by direct interaction of the G proteins with the channel (437). Therefore, given the adaptations that can occur at the receptor and postreceptor level, a large change in vagal tone would not necessarily be required to produce exercise bradycardia in trained subjects. The possibility of cellular adaptation is supported by the data from myocardial-infarcted rats in whom the diminished maximum heart rate can be reversed by treadmill training (288).

XI. ADAPTATIONS THAT AFFECT CARDIAC AND

PERIPHERAL BLOOD FLOW

A. Coronary Blood Flow

The coronary vascular system adapts at the cellular level to changes in functional demand that result from exercise training. There is an increase in coronary ar- tery size and capillary number with training (394) that suggests an angiogenesis. In swim-trained rats a 15- 18% increase in coronary vascular reserve occurs more rapidly than does hypertrophy (50), indicating a more complex mechanism than simple growth; in treadmill- trained miniature swine, coronary vascular reserve increases 22% and the capillary exchange capacity (as determined by the permeability-surface product) increases 51% (247). As a result of the increase in capillary density, there is a decreased diffusion distance for oxygen to the working cells (52). An exception to the increase in capillary density may occur in swim-trained rats where a decreased capillary density has been observed (113). However, in these animals capillary volume remains constant and is accounted for by an increased capillary width (113), which manifests itself functionally as an increase in coronary blood flow in swim-trained rats (269). It is interesting to note that in severely hypertensive rats there is a decreased capillary density in the heart with endurance training despite an improved functional capacity, which is in support of our previous contention that a pressure-overload model is not a model for exercise-induced cardiac hypertrophy (270). This decreased capillary density may account for the decreased coronary blood flow that is manifest in hypertensive rats even after swim training (269).

B. Muscle Blood Flow

Although total hindlimb blood flow does not change with treadmill training in rats, flow is greater to the trained muscle and the visceral organs during exercise (10, 332). This indicates an adaptation in the arterial tone specific to the trained muscle as well as a reflex adaptation.

Receptor-mediated control of muscle arterial tone has some effect but cannot explain all of the exercising muscle-specific adaptations that are observed. For example, ,&adrenergic receptor blockade of rats running at low speed decreases flow to all muscles, but at high speeds ,&blockade has no effect (246). Also, muscarinic receptor adaptations apparently do not play a role in the shift of blood flow to muscle during either the preantici- patory phase or during slow locomotor activity because atropine does not alter the flow (11). Furthermore, the decreased resting blood pressure observed in hypertensive trained rats is not associated with decreased arterial reactivity to norepinephrine (93). Nonetheless, there is an increased sensitivity to infused catecholamines in vasodilator and systolic pressor responses in humans, as indicated by decreased diastolic pressure and increased systolic pressure (379). As a consequence of these mixed responses, other factors must be considered that may be involved in the decrease arterial tone. Hyperpolarization of arterial smooth muscle can cause vasodilation, and several hormones may act by a mechanism that directly (vasoactive intestinal peptide) or indi- rectly (acetylcholine through endothelium-mediated factor release) activates an ATP-sensitive potassium channel, causing hyperpolarization (370). Exercise adaptation in the arterial smooth muscle may alter the sensitivity or action of these channels. Such an increased sensitivity to external vasodilators may be the reason for decreased peripheral resistance in trained subjects during recovery from exercise (76).

Reflex vasoconstriction may play a role in maintaining flow to exercising muscle as well as the increased visceral flow observed as a result of exercise training. In barodenervated rabbits there is a lack of diversion of blood flow toward exercising muscle that is manifest as a large decrease in mean arterial pressure at the onset of exercise, a decrease in maximum coronary flow at rest and exercise, and no change in kidney blood flow or muscle blood flow (156). A desensitization of this baroreflex with training would contribute to the maintenance of flow to the visceral organs (26) as well as contribute to the attenuation of the response of renal sympathetic nervous activity, which may play a role in training-induced hypervolemia (78).

Blood flow to exercise-trained muscle is also augmented by an increased capacity for flow (248,264,353), which may, at least in part, be a result of an increased capillarity (266). The increased capacity for flow is dependent on the training intensity, as apparently are increases in the capillary filtration (either intrinsic or due to increased capillarity) (248,353). Significantly, the increase in total vascular conductance that results from

574 FRANK W. BOOTH AND DONALD B. THOMASON VolunLe 71

training is a result of decreases of the same magnitude in both pre- and postcapillary resistance (353), an adaptation that not only augments flow (Fig. 4B) but also does not alter the balance of fluid movement between capillary and interstitium.

XII. ADAPTATIONS THAT AFFECT CARDIAC MYOCYTE

METABOLISM

Despite the fact that endurance-trained individuals exhibit a decreased heart rate-pressure product and thus have a decreased minute work (76), adaptations occur in cardiac muscle metabolism to apparently make the work more efficient and better sustained.

A. Substrate Metabolism

One of the more striking responses of the myocardial biochemistry to exercise is the twofold increase in CAMP levels in the myocardium for 24 h after exercise, despite an increased phosphodiesterase level (306). This response to an acute bout of exercise is the same for trained and nontrained rats (304,305). ,&Adrenergic receptor blockade or adrenalectomy inhibits the increase in CAMP concentration (302). Another adrenergic receptor-dependent event appears to be the treadmill training-induced increase in myocardial hexokinase activity, which is blocked by ,&adrenergic antagonists (213). The large increase and maintenance of CAMP levels in the myocardium is interesting when it is considered that, for catechol-induced lipolysis, the activation of triacylglycerol lipase by CAMP appears to be mediated by protein kinase C (303). Therefore the adaptation in the responsiveness of adenylate cyclase discussed would be manifest as an increase in lipolysis, providing a more efficient utilization of energy.

In addition to these lipolytic adaptations, there is an increase in glycogen content in swim-trained rats at rest (347), cardiac glucose uptake is enhanced in swim- trained rats, and there is a dissipation of the endocar- dium-epicardium glucose uptake gradient in swim- and run-trained rats (217,218). These adaptations occur in- dependently of the actual cardiac work load or the availability of other substrates and thus are apparently a result of an enhancement at the level of the glucose transporter (218). It is interesting to note that, in skeletal muscle, the contraction-induced translocation of protein kinase C precedes an enhanced glucose uptake (64); perhaps a similar mechanism for enhanced glucose transport in cardiac muscle occurs as a result of the increased CAMP levels observed by Palmer and co- workers (303-305).

Swim training of rats increases the M, isoform of lactate dehydrogenase in the myocardium from 28 to 33% (438), consistent with the decreased Km of lactate dehydrogenase for lactate observed in the hearts of running exercise-trained rats (212). Consequently, the potential for the greater utilization of lactate by the

trained heart would contribute to the smaller increases in plasma lactate and smaller decreases in plasma pH during exercise (180), providing a means for extending the length of exercise time at maximal levels.

B. Oxidative Phosphorylation

As determined by 31P-nuclear magnetic resonance, an increase in myocardial oxygen consumption may occur with only small changes in phosphate metabolites but may be stimulated instead by Ca2+, a phenomenon termed “stimulus-response-metabolism coupling” (273). By the same mechanism as the contractility adaptations that occur because of enhanced Ca2+ availability, oxidative phosphorylation in trained cardiac muscle may be stimulated. However, in treadmill-trained rats, cardiac mitochondria exhibit a decreased retention of Ca2+ and fewer transport sites (367). Despite the apparent lack of an increase in mitochondrial protein in the hearts of treadmill-trained rats (213) and the lack of an increase in myocardial mitochondrial density (136,297), ubiquinone and cytochrome c are increased in concentration with endurance training (33).

However, different training protocols may have differential effects, as suggested by the increased mitochondrial-to-myofibril volume density in swim-trained rats (113). A reversal of the decreased mitochondrial-to- myofibril volume ratio in hypertensive rat myocardium is observed after treadmill training, perhaps by the same mechanism (72). The senescence-associated decrease in cytochrome c concentration in the heart was reversed by a 4-rnoj program of treadmill running started at age 21 mo in rats (371). Interestingly, &aminolevulinic acid synthase activity increases with an acute bout of exercise in the untrained animal but not in the trained animal (2). These data indicate that whatever stimulatory effect exercise training has on mitochondrial expression occurs rapidly and then returns to a steady state as training progresses.

XIII. CONCLUSION

We outlined many of the molecular and cellular adaptations that occur in skeletal muscle and the cardiovascular system as a result of exercise training. In addition, some of the molecular and cellular adaptations occurring in response to models of increased contractile activity that do not mimic human sports are given. An underlying theme is the concept that adaptability serves to provide less disruption of the milieu interieur, minimize fatigue, enhance performance, and improve the economy of energy expenditure during exercise. From the point of view of classic evolutionary theory, the genetic trait of adaptability is maintained.

With aerobic training there is a shift in the trained skeletal muscle to greater reliance on oxidative metabolism to provide energy, although at a diminished oxygen flux per mitochondrion. Furthermore, the contractile


machinery of the trained muscle adapts to utilize energy more efficiently. With resistance training, the primary adaptation is the distribution of the load across a greater muscle mass.

The cardiovascular system adapts to exercise training by minimizing the energy cost of the work. To provide the necessary work, increases in pressure work and heart rate are minimized in favor of augmented stroke volume. To this end adaptations in both inotropic function and blood flow occur. Furthermore, cardiac myocyte metabolism adapts to the demands of training to provide a more efficient and better sustained energy SUPPlY l

Physiologists have just begun to describe and inte- grate the many factors that comprise the “exercise- training response.” These adaptations function to provide a less taxing, and more enjoyable, response to the physical demands of exercise. Furthermore, to define the mechanisms underlying these adaptations to physical activity requires the synthesis of knowledge from multiple disciplines (systems physiology, adaptive physiology, biochemistry, cell biology, molecular biology, integrative physiology, biophysics) to explain how the un- anesthetized human can survive high levels of physical stress.

We gratefully thank Lawana Norris for the care with which the manuscript was prepared. We also appreciate the many helpful comments and suggestions given by Drs. G. Ste- phen Morris, George Taffet, Charlotte Tate, Adrian Sheldon, and the unknown reviewers. We also thank Drs. Phil Gollnick, John Holloszy, and Charles Tipton, whose teaching and research fostered the field of exercise biochemistry.

This work was supported by National Institutes of Health Grant AR-19393 (to F. W. Booth) and National Aero- nautics and Space Administration Grant NAGW’i’O (to D. B. ‘l’homason).

Present address of D. B. Thomason: Dept. of Physiology, Univ. of Tennessee Medical School, Memphis, TN 38163.

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