516 biphasic changes in maximum relaxation rate during...

516

Biphasic Changes in Maximum RelaxationRate during Progressive Hypoxia in

Isometric Kitten Papillary Muscle andIsovolumic Rabbit Ventricle

MARTIN G. ST. JOHN SUTTON, ERIK L. RITMAN, AND NORMAN F. PARADISE

SUMMARY We studied the effects of graded hypoxia on mechanical performance of cardiac ventric-ular muscle by producing controlled, stepwise decreases in partial pressure of oxygen (Po2) in themedium bathing the kitten papillary muscle preparation and in the perfusate of the Langendorff-prepared rabbit heart. For kitten papillary muscle at 30°C and with stimulation rate at 30/min,maximum rate of contraction (+dT/dtmM) and maximum rate of relaxation (—dT/dtm.*) were 184 ± 10inN/mm2 per sec and 162 ± 12 mN/mm2 per sec, respectively, during control conditions with Po2 at 634± 7 mm Hg. Step decreases in Po2 from 634 mm Hg produced decreases in steady state -dT/dtmu, thatwere significantly greater than corresponding decreases in +dT/dtma», except at the lowest Po2. WhenPo2 (mm Hg ± SE) was 411 ± 10, 218 ± 4, and 92 ± 3, steady state +dT/dtmM vs. -dT/dt™, (expressedas % of pre-hypoxia control value ± SE) were: 97 ± 4 vs. 85 ± 7, 76 ± 5 vs. 59 ± 6, and 47 ± 5 vs. 28 ± 4,respectively. When the lowest Po2 of 34 ± 6 mm Hg was achieved, considerable shortening of theduration of the mechanical cycle occurred, and values for +dT/dtm«x and —dT/dtma, (expressed as % ofpre-hypoxia control value ± SE) of 28 ± 7 and 21 ± 7, respectively, were not significantly different.Graded hypoxia similarly affected left ventricular isovolumic pressure developed by the coronaryperfused rabbit heart. In both preparations, changes in relaxation relative to changes in contractionduring progressive hypoxia were biphasic: decreases in maximum relaxation rate were disproportion-ately greater than decreases in maximum contraction rate with intermediate hypoxia, but theproportionality was restored when severe hypoxia produced a decrease in cycle duration.Circ Res 47: 516-524, 1980

SEVERAL studies of ventricular function of intactanimals and humans seem to demonstrate thatmyocardial relaxation is slowed, or impaired, incomparison to myocardial contraction during is-chemia (Barry et al., 1974; Chesebro et al., 1976;McLaurin et al., 1973; St. John Sutton et al., 1978).However, results from studies of the effects of hy-poxia on contraction and relaxation of isolated car-diac muscle preparations seem to differ from theresults obtained from these studies on intact ani-mals. Hypoxia produced by changing the aeratinggas composition from 95% O2-5% CO2 to 95% N2-5%CO2 was associated with a substantial decrease inboth force development and total duration of themechanical cycle of isolated cardiac tissue, but theatmosphere of 95% N2-5% CO2 apparently did notaffect rate or duration of relaxation to any greater

From the Program in Physiology, Northeastern Ohio UniversitiesCollege of Medicine, Rootstown, Ohio and the Department of Physiologyand Biophysics, Mayo Foundation and Mayo Clinic, Rochester, Minne-sota.

Supported in part by the Akron District Chapter of the AmericanHeart Association, National Institutes of Health (NIH) Grant HL04664and by NIH Biomedical Research Development Grant 1406.

Address for reprints: Dr. Norman F. Paradise, Program in Physiology,Northeastern Ohio Universities College of Medicine, Rootstown, Ohio44272.

Received January 24, 1979; accepted for publication May 2, 1980.

extent than rate or duration of contraction (Bing etal., 1976; Nakhjavan et al., 1971; Tyberg et al., 1970;Weisfeldt et al., 1974). There exists the possibility,however, that these on-off changes in oxygen partialpressure (Po2) may mask effects of intermediatedegrees of hypoxia on the contraction-relaxationcycle.

The present study was undertaken to elucidatethe effects of varying degrees of hypoxia on con-traction and relaxation of mammalian ventricularmuscle. The major aim was to determine whethergraded hypoxia affects rates of relaxation differ-ently than rates of contraction, as appears to be thecase for the intact heart during ischemia. Gradedand controlled reductions in Po2 of the superfusatebathing the isolated, isometrically contracting pap-illary muscle were produced in a stepwise fashion,and subsequent changes in force development andrate of force development were recorded. Similarly,isovolumic pressure development was recordedfrom the left ventricle of the Langerdorff-preparedrabbit heart during progressive, stepwise decreasesin perfusate Po2. Analysis of the recorded datapermitted the effects of mild, moderate, and severedegrees of hypoxia on relaxation and contractionprocesses of cardiac ventricular muscle to be com-pared in two different preparations.

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GRADED HYPOXIA AND MYOCARDIAL RELAXATION/St. John Suttonetal. 517

MethodsPreparationPapillary Muscles

Following the removal of hearts from chloroform-anesthetized kittens (450-2200 g), the right ventri-cle was opened rapidly and submerged in oxygen-ated physiologic salt solution. Papillary muscleswere carefully excised and arranged to contractisometrically by securing the severed tuft of theventricular insertion in a spring-loaded Perspexclamp and tying the tendinous end of the muscledirectly to a glass rod which was connected to aglass extension of an RCA 5734 mechano-electronictransducer (manufacturer's specification for fre-quency response, 12,000 hertz). The muscle wasbathed in physiologic salt solution of the followingcomposition (mmol/liter): Na+, 135; K+, 5.0; Ca2+,2.0; Mg2+, 1.0; Cl", 98; HC(V, 24; HPO4

=, 1.0; SO4=,

1.0; CH3COO", 20; glucose, 10. An internal circula-tion within the bathing chamber (Blinks, 1965) wascreated by bubbling gases through the solution.

During an initial 2- to 3-hour equilibration, mus-cle length was increased until force developmentupon stimulation was maximal. Thereafter, musclelength was kept constant. Muscles were stimulatedat 20/min during the equilibration period by uni-directional pulses applied through punctate elec-trodes (Blinks, 1965). Stimulus duration was 2 msecand stimulus strength was slightly above threshold(range: 1.0-1.5 V).

Rabbit HeartsAfter heparinization (300 U/kg) of thoracotom-

ized male rabbits (2-3 kg) under Nembutal anes-thesia (40 mg/kg, iv), the aorta was cannulated andthe heart excised from the chest cavity. A smallincision in the left atrium was made to permit thepassage of a fluid-filled balloon through the mitralvalve into the left ventricle to record isovolumicpressures. Perfusate with a composition identical tothat employed for the studies on papillary musclewas delivered to the hearts at a constant rate of 35ml/min (Gilson peristaltic pump, #HP16). After a1-hour equilibration period, the atrioventricularnode was crushed and electrical pacing of the rightventricle was initiated to maintain heart rate con-stant during the subsequent experimental proce-dures.

Procedures

Papillary MusclesThe bathing solution was aerated with combi-

nations of gases issuing from two pressurized cyl-inders, one containing 95% O2-5% CO2 and the other95% N2-5% CO2. During the 2- to 3-hour equilibra-tion period, only the 95% O2-5% CO2 gas mixturewas employed. Oxygen partial pressure achieved insuperfusate during aeration with 95% O2-5% CO2

was approximately 635 mm Hg. Thereafter, theoxygen tension in the bathing medium was reducedin stepwise decrements by adjusting the relativeproportion of O2 and N2 bubbling through the cham-ber. In three experiments, each 10-minute stepwisereduction in oxygen tension was followed by a re-turn to control conditions for 15 minutes by restor-ing aeration with 95%.O2-5% CO2. The last stepwisereduction in oxygen tension produced nearly com-plete anoxia as a consequence of aeration with the95% N2-5% CO2 gas mixture only. Oxygen partialpressure in superfusate during aeration with thisoxygen-deficient gas mixture was approximately 30mm Hg. In a second series of 28 experiments, thegraded reductions in oxygen tension were producedat 10-minute intervals without intervening periodsof reoxygenation. In a third series of six experi-ments, mechanical function was studied for pro-longed periods during exposure to 95% O2-5% CO2only. In a fourth series of four experiments, oxygentension was reduced to approximately one-third ofcontrol in a single step and subsequent changes inmechanical function were studied for 70 minutes. Ina fifth series of four experiments, the effects onmechanical function of seven step decreases in ox-ygen tension to approximately one-third of controlwith six intervening periods of reoxygenation wereevaluated.

The effect of decreasing oxygen tension on pap-illary muscle mechanical performance was studiedat several stimulation rates (range: 20-60/min) withtemperature at 30 ± 0.1 °C. Oxygen tension and pHwere measured using a Radiometer CopenhagenBMS3 Mkll microsystem and a PHM73 pH/gasmonitor. Solution pH of 7.45 remained constantthroughout all experimental procedures and wasindependent of the composition of the aeratinggases. After a change in composition of the aeratinggases, equilibration of the gases with the bathingsolution occurred within approximately 75 seconds.

Tension (T) developed by the muscle and therate of change of tension (dT/dt), obtained by elec-tronic differentiation of the tension signal, wererecorded continuously on paper at 2.5 mm/min(Electronics for Medicine, Inc.). The frequency re-sponse of this recording system was flat for frequen-cies up to 100 hertz. Additionally, photographicrecords of tension traces were obtained from aTektronix 7613 storage oscilloscope. Measured pa-rameters included: (1) peak developed tension (PT),(2) time-to-peak tension (TTP), (3) maximum rateof contraction (-t-dT/dtw,), (4) maximum rate ofrelaxation (—dT/dtmax), and (5) one-half relaxationtime (V2RT), the time during which tension fell fromits peak to a value midway between peak and rest-ing levels.

At the end of each experiment, muscle lengthand weight were measured. Cross-sectional area(wr2) and diameter (2r) of each muscle were com-puted assuming a cylindrical shape and a specific


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518 CIRCULATION RESEARCH VOL. 47, No. 4, OCTOBER 1980

gravity of unity. Peak developed tensions were nor-malized by muscle cross-sectional areas and wereexpressed in units of milli-Newtons per mm2 (mN/mm2). Mean diameter (mm ± SE) for all musclesstudied was 0.84 ± 0.03.

Rabbit HeartsImmediately prior to its arrival at the heart,

perfusate was passed through a membrane oxygen-ator (Travenol Laboratories, Inc., #5M0321) whereequilibration with gases was achieved. Step de-creases in perfusate Po2 were achieved in five heartsby adjusting the proportion of O2 and N2 passingthrough this gas exchange system. Progressive stepdecreases in P02 were produced without interveningperiods of reoxygenation and each decrease in Po2was maintained for 15 minutes instead of 10 min-utes. Po2 of arterial perfusate was not measuredduring the course of each experiment because theremoval of samples of arterial perfusate for analysiswould have necessitated a temporary reduction ofperfusate flow to the heart. However, at the termi-nation of each experiment, the heart was removedfrom the arterial line and the experimental protocolwas repeated so that the arterial perfusate Po2'scould be measured. Isovolumic pressure develop-ment of the left ventricle was recorded (GouldRecorder, #2600) throughout the experimental pro-cedures with temperature at 30°C. The frequencyresponse of the catheter-transducer (Gould Sta-tham P23)-recording system was evaluated by pro-ducing full scale (50-mm) sinusodial pressure fluc-

tuations with a Multifunction Pressure Generator(model MPG-30, Millar Instrument, Inc.) Changesin recorded pulse amplitude were within 5% forfrequencies up to 13 hertz.

Statistical AnalysesAnalysis of variance for single-factor experiments

having repeated measures and for two-factor exper-iments having repeated measures were employed,and Newman-Keuls tests were used to assess thestatistical significance of differences between indi-vidual pairs of means (Winer, 1971).

Results

Effects of Graded Hypoxia on MechanicalFunction of Papillary Muscle

The changes in mechanical function of the iso-metric papillary muscle following a step decrease inPo2 are illustrated in Figure 1. Mechanical functionof this muscle stabilized within 10 minutes of thestep decrease in Po2. The transient increases in PT,-1-dT/dtmax, and —dT/dtmax occurring immediatelyafter the decrease in Po2 and shown in Figure 1were not always observed.

Photo-oscillographic recordings of steady statebeats following step decreases in P02 with interven-ing periods of reoxygenation are shown in Figure 2for selected procedures from a single experiment.There is a direct relationship between PT and Po2,with greater decreases in PT being associated withlarger step decreases in Po2. It is apparent from the

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180

160

140

100

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8 10

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MINUTESFIGURE 1 Transient changes in papillary muscle performance following a step decrease in Po2- A decrease in Po2

from 650 to 146 mm Hg (shown at time zero) produced a transient increase and subsequent decline in peak developedtension (PT), maximum rate of contraction (+dT/dtTnaj), and maximum rate of relaxation (—dT/dtmaJ. Note theproportionately greater effect of this level of hypoxia on —dT/dtmal than on either PT or +dT/dtmsiI. Arrows labeledTrace 1, Trace 2, and Trace 3 designate times at which photographic traces of beats 1, 2, and 3, respectively, of insetwere recorded. Temperature was 30°C and stimulation rate was 50/min. Muscle diameter was 0.66 mm.


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GRADED HYPOXIA AND MYOCARDIAL RELAXATION^. John Sutton et al. 519

P02 ControlP02 Test

FIGURE 2 Effect on tension twitches of progressive re-ductions in oxygen tension with intervening 15-minuteperiods of reoxygenation. Upper traces are steady statecontrol beats recorded at the end of 15-minute periods ofreoxygenation with 95% O2-0% N2-5% CO?.. Lower tracesare steady state test beats recorded 10 minutes aftereach step decrease in Po2. Partial pressures of oxygen(mm Hg) measured during the control and test periods(Po2 control/Po 2 test) are reported to the right of eachpanel. Peak developed tension (mN/mm2) of controlbeats (upper traces) vs. test beats (lower traces) were 56.2vs. 48.6 (panel A), 55.7 vs. 42.7 (panel B), 54.8 vs. 28.6(panel C), and 54.3 vs. 21.6 (panel D). Experimentalconditions: temperature, 30° C; stimulation rate, 30/min;muscle, diameter, 0.93 mm.

data in Figure 2 that decreases in TTP were asso-ciated with the decreases in Po2. However, the totalduration of the contraction-relaxation cycle wasaffected minimally by the changes in Po2 shown inthis figure. Furthermore, the effects of 10-minuteintervals of hypoxia, whether mild, moderate, orsevere, are nearly completely reversible, as indi-cated by the restoration of tension development(upper trace in each panel of Figure 2) during each15-minute period of reoxygenation with the 95% O2-0% N2-5% CO2 gas mixture. The same sets ofchanges in mechanical function were observedwhen progressive hypoxia was produced withoutintervening periods of reoxygenation (compare Fig-ure 2 with inset in Figure 5). The effects of gradedhypoxia on mechanical function were independentof the protocol employed. Therefore, most of theexperiments performed in this study followed theprotocol for producing progressive hypoxia withoutintervening periods of reoxygenation.

The effects of graded hypoxia on mechanicalfunction of seven papillary muscles (mean equiva-lent diameter, 0.84 ± 0.07 mm) stimulated at 30/min and with temperature at 30°C are summarized

in Figures 3-6. The modulating effects of Po2 onmaximum rate of relaxation and maximum rate ofcontraction are shown in Figure 3. Progressive de-creases in Po2 produced proportionately greaterdecreases in steady state — dT/dtma* as compared to+dT/dtmax except at the lowest Po2.

In contrast to the disproportionate effects ofgraded hypoxia on -f-dT/dtmax and — dT/dtmax, pro-gressive decreases in Po2 produced nearly propor-tional changes in steady state PT and +dT/dtmaxover the entire range of Po2's studied (Fig. 4). Figure5 shows the effects of progressive, step reductionsin Po2 on both steady state peak developed tensionand time-to-peak tension (TTP). Superimposedtracings of steady state beats recorded from a singlemuscle are shown in the inset of Figure 5. It isapparent from this set of tracings that decreases inPo2 did not affect the total duration of the contrac-tion-relaxation cycle until measured Po2 reached 29mm Hg. At this lowest Po2, there was a considerableshortening in the total duration of the mechanicalcycle (lowest trace in inset of Figure 5). This obser-vation is representative of all of the experimentalobservations of this study. The duration of themechanical cycle decreased substantially only whenan atmosphere of 95% N2-5% CO2 was used to aeratethe bathing medium.

The data in Figure 6 show that steady state one-half relaxation time (V&RT) was unaltered bychanges in Po2 between 634 ± 7 mm Hg (V&RT =236 ± 30 msec) and 92 ± 3 mm Hg (V2RT = 240 ±21 msec). However, there was a significant decreasein '/2RT when Po2 was 34 ± 6 mm Hg. This decreasewas related to the considerably shortened durationof the mechanical cycle associated with the lowestachievable Po2.

Data from experiments performed to assess thestability of the preparation in the absence of de-creases in Po2 are reported in row 1 of Table 1. The6% increase in tension development after 70 minutesindicated that the changes in mechanical functionof the papillary fibers during the 70 minutes ofprogressive hypoxia (Figs. 3-6) were attributable tothe decreases in Po2 only, and not to a spontaneousdeterioration of the preparation. Changes in PTfollowing the onset of a sustained, constant level ofhypoxia and repetitive bouts of hypoxia to the samelevel with intervening periods of reoxygenation areshown in rows 2 and 3, respectively, of Table 1.Overall, papillary muscle function remained rela-tively stable under these experimental conditions.In contrast, decreases in PT during progressive stepdecreases in Po2 (Figs. 4 and 5) were considerablylarger. Data in Figure 4 show that PT was 87 ± 6,73 ± 6, and 54 ± 6% of pre-hypoxia control whenPo2 was 317 ± 8, 218 ± 4, and 131 ± 3 mm Hg,respectively. Thus, the decreases in mechanicalfunction reported in Figures 3-6 can be attributedalmost exclusively to the decreases in Po2 and notto long-term, cumulative effects of hypoxia.


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*P < 0.05Mean ± SE

100

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600 400 200

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FIGURE 3 Effects of stepwise decreases in Po2 on maximum rate of contraction and maximum rate of relaxation ofisometric papillary muscle. Decreases in P02 were produced without intervening periods of reoxygenation. Mean steadystate values (expressed as % of pre-hypoxia control ± SE) of maximum rate of contraction, +dT/dtma%, and maximumrate of relaxation, —dT/dtmal, are plotted against mean P02. Standard errors of mean values of P02 are shown by thehorizontal bars. During control conditions with mean Po2 of 634 ± 7 mm Hg, mean +dT/dtmal and mean —dT/dtmta

were 184 ± 10 and 162 ± 12 mN/mm2 per sec, respectively. Decreases in Po2 produced significant decreases in both+dT/dtmal. and —dT/dtmal (P < 0.001). Furthermore, decreases in —dT/dtmBX were significantly greater than decreasesin +dT/dtma% (P < 0.001), and the interaction between Po2 and ±dT/dtmaI was significant (P < 0.001). Mean values of+dT/dtmBI designated by asterisks (*) are significantly greater than corresponding mean values of —dT/dtma% (P <0.01). These data show that decreases in —dT/dtmaz are larger than corresponding decreases in +dT/dtmal atintermediate Po2's but not at the lowest achievable Po2.

Effects of Graded Hypoxia on MechanicalFunction of Rabbit Left Ventricle

The tracings in Figure 7 illustrate the effects ofstep decreases in perfusate Po2 on isovolumic pres-sure developed by the left ventricle of the rabbit

heart. Decreases in perfusate Po2 produced de-creases in both peak left ventricular pressure de-velopment and time-to-peak pressure development.Substantial shortening of the duration of the me-chanical cycle occurred only at the lowest Po2 stud-ied. Data from five experiments are summarized in

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FIGURE 4 Comparison of tension development and rate of tension development during stepwise reductions in PO2-Mean steady state values of peak developed tension (PT) and maximum rate of contraction (+dT/dtma%) are expressedas % of pre-hypoxia control at each P02 level studied. During control conditions with P02 at 634 ± 7 mm Hg, PT was46.7 ± 3.2 mN/mm2 and +dT/dtmiI was 184 ±10 mN/mm2 per sec. Standard errors of mean values of P02 are notshown, but are the same as in Figure 3.


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GRADED HYPOXIA AND MYOCARDIAL RELAXATION/S*. John Sutton et al. 521

40

20

Peak Tension

Time-to-Peak Tension

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6204883882982171389829

600

400rn

200

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600 400 200

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FIGURE 5 Effects of step decreases in P02 on peak developed tension (PT) and time-to-peak tension development(TTP). TTP decreased only moderately with step decreases in Po2 between 634 ± 7 and 218 ± 4 mm Hg, but thereafterthe decreases were pronounced. Inset: Superimposed tracings of steady state beats illustrating relation between tensiondevelopment and Po2. Po2's associated with tracings are listed to the right. Upper trace was recorded with Po2 at 620mm Hg, and progressively lower traces were recorded at correspondingly lower Po2's. Data shown in the inset wereobtained from a muscle with diameter of 0.88 mm.

Table 2. Decreases in developed pressure (row b)and time-to-peak pressure development (row d)during graded hypoxia were similar to the decreasesin PT and TTP observed in the papillary muscleduring graded hypoxia (compare rows b and d ofTable 2 with Figure 5). Changes in +dP/dtmax and—dP/dtmax are reported in rows e and f. Progressivehypoxia produced significant decreases in both4-dP/dUax and -dP/dtmax (P < 0.001). In addition,decreases in —dP/dtmax were significantly greaterthan decreases in +dP/dtmax (P < 0.05) and theinteraction between Po2 and ±dP/dtmax was signifi-cant (P < 0.001). Differences between correspond-ing values of -l-dP/dtmax and -dP/dtmax were statis-tically significant when Po2 (mm Hg) was 313 ± 10

r

FIGURE 6 Effect of stepwise reductions in Po2 on one-half relaxation time (lART). xkRT of 162 ± 7 msecachieved at the lowest Po2of34±6 mm Hg was signifi-cantly less than mean values of lhRT obtained at eachof the other Po2's (P < 0.01). No other pairs of meanvalues were found to be statistically significantly differ-ent. Therefore severe hypoxia, but not intermediate hy-poxia, produced a significant shortening of V2RT.

(column 4, row g), 220 ± 7 (column 5, row g) and135 ± 6 (column 6, row g), but not statisticallysignificant at the lowest Po2 (column 7, row g).Thus, —dP/dtmax was depressed to a greater extentthan +dP/dtmax by intermediate degrees of hypoxia,but the proportionality between —dP/dtmax and-t-dP/dtmax was restored during severe hypoxia.Comparison of the data shown in Figure 7 andTable 2 with the data shown in Figures 3-6 dem-onstrates that step decreases in perfusate Po2 pro-duced effects on mechanical performance of theisolated, coronary perfused rabbit heart which re-sembled closely the effects of decreases in superfus-ate P02 on mechanical performance of the isolated,superfused papillary fiber.

DiscussionStep reductions in superfusate Po2 from approx-

imately 650 to 75 mm Hg were associated withseveral characteristic alterations in the mechanicalfunction of the kitten papillary fiber. A decrease insteady state PT accompanied each step decrease inPo2. TTP also decreased with step decreases in Po2,although the duration of the contraction-relaxationcycle remained nearly invariant in this Po2 range.Thus, since cycle duration remained constant butTTP decreased, there was a decrease in the dura-tion of the upstroke, or contraction phase, and aconcomitant increase in the duration of the down-stroke, or relaxation phase, of the cycle. The dis-proportionately greater effects of decreases in Po2on -dP/dtmax than on -1-dP/dtmax (Fig. 3) are areflection of this set of changes in mechanical func-tion. These findings were observed consistently in


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TABLE 1 Stability of Isolated, Superfused Papillary Muscle Preparation

Peak tension development (% of control) at

Condition Omin lOmin 40 min 70 min

1. Constant Po2 of 632 ± 5 mm Hg 100 102 ± 1 105 ± 1 106 ± 2

2. Sustained decrease in Po2 from 100 78 ± 8 73 ± 10 71 ± 9636 ± 8 to 226 ± 8 mm Hg(n= 4)

3. Ten-minute episodes of hypoxia 100 84 ± 6 81 ± 6 73 ± 7(Po2 = 232 + 4 mm Hg) withintervening 15-minute periods ofreoxygenation (Po2 = 626 ± 10mm Hg) (re = 4)

Control values for peak tension development (mN/mm2 ± SE) for conditions 1, 2, and 3 were 40.5 ± 4.2, 51.2 ±6.3, and 40.2 ± 8.4, respectively. Values reported in row 3 were measured after the first (10-minute), fourth (40-minute), and seventh (70-minute) step decrease in Po2. Tension development during the reoxygenation periodimmediately preceding the seventh step decrease in Po2 (70 minutes, row 3) of 39.5 ± 7.8 mN/mm2 was notstatistically significantly different than tension development immediately preceding the first step decrease in Po2(0 min) of 40.2 ± 8.4 mN/mm2. Experimental conditions: stimulation rate, 30/min; temperature, 30°C; mean musclediameters (mm ± SE): (1) 0.88 ± 0.13, (2) 0.90 ± 0.11, and (3) 0.74 ± 0.06.

both the kitten papillary fiber preparation and inthe coronary perfused rabbit heart. These dataindicate that, compared to contraction, myocardialrelaxation processes are apparently impaired to agreater extent with moderate reductions in oxygensupply.

Under conditions produced by aeration of thesuperfusate or perfusate with 95% N2-5% CO2 (mea-sured Po2 of approximately 30 mm Hg), the dura-tion of the contraction-relaxation cycle shortenedconsiderably in both kitten papillary muscle andcoronary perfused rabbit heart preparations. Under

150

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120/min, 30° C

PO2628

400 msec

FIGURE 7 Relation between perfusate Po2 and steadystate isovolumic pressure developed by the left ventricleof rabbit heart. Peak steady state pressure development(systolic pressure minus diastolic pressure) during con-trol with perfusate Po2 at 628 mm Hg was 106 mm Hg(upper trace) and with perfusate Po2 at 36 mm Hg was19 mm Hg (lower trace). Heart rate was maintainedconstant throughout the experimental procedures byelectrical stimulation of the right ventricle. Not shownby these superimposed tracings are increases in diastolicpressure of 4 and 11 mm Hg when P02 was 140 and 36mm Hg (lower two traces), respectively.

these conditions of lowest Po2, V2RT of kitten pap-illary muscle twitches decreased significantly andvalues of +dT/dtmax and —dT/dtmax, when com-pared to their respective control values, were notstatistically significantly different (Fig. 3). Changesin +dP/dtmax and —dP/dtmax following the onset ofaeration of rabbit heart perfusate with 95% N2-5%CO2 (rows e-g in column 7 of Table 2) were similarto the corresponding changes in +dT/dt,nax and—dT/dtmax recorded from kitten papillary muscle.Severe hypoxia did not appear to have the disparateeffects on contraction and relaxation processes thatwere observed when tissues were studied at slightlyhigher Po2's. Thus, the effects of aeration with 95%N2-5% CO2 on mechanical function of two differentheart muscle preparations at 30°C shown in thisstudy and in previous studies at about 30° C (Binget al., 1976; Nakhjavan et al., 1971; Parmley andSonnenblick, 1969; Tyberg et al., 1970; Weisfeldt etal., 1974) are not representative of the sets ofchanges occurring in mechanical performance ofthe myocardium in response to decrements in P02ranging between approximately 650 and 75 mm Hg.However, the pattern of myocardial relaxation fol-lowing onset of severe hypoxia may be temperaturedependent. Frist and coworkers (1978) studied thekitten papillary fiber stimulated at a rate of 12/minand found that a step change in aeration from 95%O2-5% CO2 to 95% N2-5% CO2 produced a shorteningof V6RT at 29°C, which is similar to the findings ofthe present study, but a prolongation of M>RT wasobserved at 38°C. There exists the possibility thatintermediate degrees of hypoxia affect contractionand relaxation processes in the normothermic rangedifferently than at 30°C.

Delivery of oxygen to the cells of the cylindricallyshaped papillary muscle occurs by diffusion fromthe external solution. According to the formulationof Hill (1928), total tension development by thepapillary muscle under a given set of experimental


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GRADED HYPOXIA AND MYOCARDIAL RELAXATION/St. John Sutton et al. 523

TABLE 2 Effect of Step Decreases in Perfusate Oxygen Partial Pressure (P02) on Mechanical Function of RabbitLeft Ventricle (30° C, 120/min, n = 5)

a.

b.

c.

d.

e.

f.

g-

P02 (mm Hg)Developed pressure(mm Hg)Diastolic pressure(mm Hg)TTP (msec)+dP/dU» (%)-dP/dU« (%)Statistical significance(row e vs. row f)

1

627 ± 14110 + 5

9 ± 1

179 + 7100*

lOOf

2

528 ± 13

100 ± 6

8± 1

179 + 689 ±689 ±3

NS

3

414 ± 1085 ± 7

8± 1

173 ±577 ±573 ±5

NS

4

313 ± 1068±5

8± 1

162 ±566 ±555 ±2

P < 0.01

5

220 + 750±5

10 + 2

151 ±753 ±338±3

P < 0.01

6

135 ±634 ±4

13 ±4

130 ±641 ±426 ±2

P < 0.01

7

31 +20 +

20 ±

111 ±25 +19 +NS

8

3

7

7

4

2

NS = not significant.• Mean pre-hypoxia control value was 1132 + 105 mm Hg/sec.f Mean pre-hypoxia control value was 657 ± 25 mm Hg/sec.

conditions may be considered to be the sum of theforce developed by the fraction of cells contractinganaerobically within the core of the muscle and theforce developed by the fraction of cells which areoxygenated adequately and contracting aerobicallyperipherally. To test the extent to which this two-compartment model might be applicable to theinterpretation of the data of the present study, theeffects of progressive decreases in Po2 on mechani-cal function of the left ventricle of the capillaryperfused rabbit heart were investigated. P02 mod-ulation of left ventricular isovolumic pressure de-velopment was found to be similar to the P02 mod-ulation of papillary muscle isometric tension devel-opment. Since diffusion distances for oxygen in thecapillary perfused heart preparation presumablycorrespond to intercapillary distances and ordinar-ily are considerably snorter than distances for oxy-gen diffusion in the isolated papillary muscle, it isunlikely that the decline in force development ofthe papillary muscle results simply from changes inthe relative contribution of an anaerobic set of cellsand an aerobic set of cells. The two-compartmentmodel emerging from Hill's formulation (Hill, 1928)may not, therefore, completely explain the modu-lating effects of Po2 changes on papillary musclefunction that were observed in the present study.This conclusion is in harmony with data reportedby Frezza and Bing (1976) which showed that forcedevelopment by the rat papillary muscle was mod-ulated by changes in Po2 between 550 and 450 mmHg, even though there was no evidence for theexistance of an anaerobic core within the fiber inthis range of values of P02.

Mean diameter of all papillary muscles used inthe present study was 0.84 ± 0.03 mm. Most of themuscles with diameters less than 0.84 mm did notexhibit decreases in PT, +dT/dtmax, or -dT/dtma*following the first step decrease in Po2 from about650 to 550 mm Hg. Therefore, these thinner muscleswere likely to have been oxygenated adequately inthe control state during aeration with 95% O2-5%CO2 gas mixture. Muscles with diameters exceeding

0.84 mm generally exhibited decreases in PT, +dT/dtmax, and —dT/dWax after the first step decrease inP02. The decrease in mechanical function of thesethicker muscles associated with the first step de-crease in P02 suggests that they may not have beenoxygenated adequately during aeration with 95%02-5% CO2. However, the responses of these thickermuscles to progressive hypoxia were the same asthe responses of the thinner muscles, viz., there wasa progressive shortening of the duration of contrac-tion phase and a simultaneous lengthening of theduration of relaxation phase. Thus, although ananaerobic core may have been present in some ofthe thicker muscles under control conditions, itspresence did not appear to affect the mechanicalresponses to step reductions in P02, except that thechanges in mechanical function occurred at higherPo2's in the thicker muscles, compared to the thin-ner muscles.

The effects of graded hypoxia on mechanicalfunction of cardiac ventricular muscle observed inthe present study may be related, in part, to de-creases in the duration of the action potential(McDonald and MacLoed, 1971) with subsequentdecreases in the levels of calcium stored at releasesites within the cardiac cell (Wood et al., 1969).Additionally, altered calcium transport by sarco-plasmic reticulum (Lee et al., 1975) may explainpartially the observed mechanical responses tograded hypoxia. Since, however, the sets of bio-chemical changes associated with varying degreesof hypoxia are complex, these and other possibleexplanations remain speculative.

AcknowledgmentsWe greatly appreciate the advice and assistance of Dr. George

S. Malindzak, Jr., Scott Shorten, and Fred M. Wolf during thecourse of this study.

ReferencesBarry WH, Brooker JZ, Alderman EL, Harrison DC (1974)

Changes in diastolic stiffness and tone of the left ventricleduring angina pectoris. Circulation 49: 255-263

Bing OHL, Brooks WW, Messer JV (1976) Prolongation of


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tension on reoxygenation following myocardial hypoxia: apossible role for mitochondria in muscle relaxation. J Mol CellCardiol 8: 205-215

Blinks JR (1965) Convenient apparatus for recording contrac-tions of isolated heart muscle. J Appl Physiol 20: 755-757

Chesebro JH, Ritman EL, Frye RL, Smith HC, Connolly DC,Rutherford, BD, Davis GD, Danielson GK, Pluth JR, Barn-horst DA, Wallace RB (1976) Videometric analysis of regionalleft ventricular function before and after aortocoronary arterybypass surgery: Correlation of peak rate of myocardial wallthickening with late postoperative graft flows. J Clin Invest58: 1339-1347

Frezza WA, Bing OHL (1976) PO2-modulated performance ofcardiac muscle. Am J Physiol 231: 1620-1624

Frist WH, Palacios I, Powell WJ Jr (1978) Effect of hypoxia onmyocardial relaxation in isometric cat papillary muscle. J ClinInvest 61: 1218-1224

Hill AV (1928) Diffusion of oxygen and lactic acid throughtissues. Proc R Soc Lond [Biol] 104: 39-96

Lee SL, Balasubramanian V, Dhalla, NS (1976) Excitation-con-traction coupling in heart. XIX. Effect of hypoxia on calciumtransport by subcellular particles in the isolated perfused ratheart. Can J Physiol Pharmacol 54: 49-58

McDonald TF, MacLeod DP (1971) Anoxia-recovery cycle inventricular muscle: action potential duration, contractility andATP content. Pfluegers Arch 325: 305-322

McLaurin LP, Rolett EL, Grossman W (1973) Impaired leftventricular relaxation during pacing-induced ischemia. Am JCardiol 32: 751-757

Nakhjavan FK, Parameswaran R, Lu CY, Srinivasan NV, Gold-berg H (1971) Effects of hypoxia, reoxygenation, and temper-ature on cat papillary muscle. Am J Physiol 220: 1289-1293

Parmley WW, Sonnenblick EH (1969) Relation between me-chanics of contraction and relaxation in mammalian cardiacmuscle. Am J Physiol 216: 1084-1091

St. John Sutton MG, Frye RL, Smith HC, Chesebro JH, RitmanEL (1978) Relation between left coronary artery stenosis andregional left ventricular function. Circulation 58: 491-496

Tyberg JV, Yeatman LA, Parmley WW, Urschel CW, Sonnen-blick EH (1970) Effects of hypoxia on mechanics of cardiaccontraction. Am J Physiol 218: 1780-1788

Weisfeldt ML, Armstrong P, Scully HE, Sanders CA, DaggettWM (1974) Incomplete relaxation between beats after myo-cardial hypoxia and ischemia. J Clin Invest 53: 1626-1636

Winer, BJ (1971) Statistical Principles in Experimental Design,ed 2. New York, McGraw-Hill, pp 261-273 and 514-532

Wood EH, Heppner RL, Weidmann S (1969) Inotropic effects ofelectric currents. I. Positive and negative effects of constantelectric currents or current pulses applied during cardiac ac-tion potentials. II. Hypothesis: calcium movements, excitation-contraction coupling and inotropic effects. Circ Res 24: 409-445

Angiotensin II Increases Electrical Couplingin Mammalian Ventricular Myocardium

KENT HERMSMEYER

SUMMARY Electrical measurements of current flow in ventricular myocardium immersed in siliconeoil showed that angiotensin II increases the cell-to-cell spread of current within seconds. The increasesin current spread and conduction velocity occur without any changes in resting membrane potential ormaximum rate of rise of the action potential. The concentration range was 10 nM to 10 /IM, with an ED50

of 100 nM for angiotensin exposures lasting about 10 seconds. The largest effects were an apparentdecrease in resistance through the cellular pathway to 50% of control and a 40% increase in conductionvelocity, which returned to control in about 15 minutes. Continuous or repeated exposure to angiotensincaused desensitization to appear. These effects were found with or without denervation by 6-hydrox-ydopamine and /?-adrenergic blockade by 1 J»M propranolol in calf, pig, sheep, and rabbit ventricularmyocardium. Therefore, angiotensin appears to increase electrical conduction rapidly and directly incardiac muscle by decreasing resistance through the cellular pathway. Circ Res 47: 524-529, 1980

THE EFFECT of angiotensin II on the mammalianventricular myocardium is to increase maximumtension development (Koch-Weser, 1964). This pos-itive inotropic effect is direct, rather than resultingfrom norepinephrine release (Fowler and Holmes,1964; Koch-Weser, 1965). Unlike norepinephrine,

From the Physiologisches Institut, Universitat Bern, Bern, Switzer-land.

Address for reprints: Dr. K. Hermsmeyer, Department of Pharmacol-ogy, University of Iowa BSB, Iowa City, Iowa 52242.

Supported by Grant HL 16328 and Research Career DevelopmentAward HL00O73 from the National Institutes of Health, by the RocheResearch Foundation for Scientific Exchange and Biomedical Collabora-tion with Switzerland, and by the Schweizerische Stiftung fur Kardiologie.

Received December 17, 1979; accepted for publication May 8,1980.

angiotensin II does not induce arrhythmias, sug-gesting differences in the mechanism of action(Koch-Weser, 1964). There is evidence to suggestthat angiotensin II can cause increased contractionand a prolongation of the cardiac action potentialby enhancement of calcium influx under certainconditions (Freer et al., 1976).

This report suggests another direct action ofangiotensin (II) on cardiac muscle. It has beenfound (unpublished observations) that angiotensinimproves the synchrony of contraction of largesheets of interconnected myocardial cells in tissueculture. The synchrony was increased by a decreasein coupling resistance that averaged 2-fold at its


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M G Sutton, E L Ritman and N F Paradisepapillary muscle and isovolumic rabbit ventricle.

Biphasic changes in maximum relaxation rate during progressive hypoxia in isometric kitten

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1980 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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