J Mol Cell Cardiol 28, 1565–1574 (1996)

Immediate-early Gene Responses toDifferent Cardiac Loads in the EjectingRabbit Left VentricleBryan K. Slinker1, Richard L. Stephens1, Steven A. Fisher2

and Qinglin Yang1

1Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, WashingtonState University and 2Division of Cardiology, Case Western Reserve University, USA

(Received 20 February 1996, accepted in revised form 12 March 1996)

B. K. S, R. L. S, S. A. F Q. Y. Immediate-early Gene Responses to Different CardiacLoads in the Ejecting Rabbit Left Ventricle. Journal of Molecular and Cellular Cardiology (1996) 28, 1565–1574.Clinical and experimental observations in humans and animals have shown that different cardiac adaptationsoccur in response to different types of hemodynamic overload. However, very little is known about how differenthemodynamic loads lead to these different cardiac adaptations. Accordingly, we studied the acute response ofejecting isolated rabbit hearts to independently varied systolic and diastolic mechanical loads at constant coronaryperfusion pressure. We studied the combined effects of low end-diastolic volume (EDV) and low systolic ejectionpressure (Pej ), compared to low EDV and high Pej, high EDV and low Pej, and high EDV and high Pej, on theexpression of c-fos, c-jun, and egr-1. Further, although we did not seek to clarify the role of these immediate-earlygenes in cardiac hypertrophy, we hypothesized that they should not all respond in the same manner to thesedifferent mechanical loads. In these ejecting hearts we found that the expression of these immediate-early genesdid not all respond alike to the different mechanical loads: both c-fos and egr-1 were strongly induced at both 30and 60 min. However, at 30 min only c-fos depended on the level of EDV (P=0.01). Neither c-fos nor egr-1 wasinfluenced by EDV at 60 min. The expression of c-jun was largely insensitive to all loading conditions. Weconclude that EDV, independent of Pej, influences the pattern and time course of expression of some immediately-early genes and that these different immediate-early genes do not respond in parallel to changes in cardiacloading. 1996 Academic Press Limited

K W: Cellular oncogenes; Proto-oncongenes; c-fos; c-jun; egr-1; Cardiac hypertrophy; Isolated heartpreparation.

resultant cardiac adaptations are clearly more com-Introductionplex (Kennedy et al., 1968; Kennedy et al., 1970;Dodge et al., 1973). However, although numerousAltered hemodynamic loading of the heart is a

principal factor controlling cardiac hypertrophy potential pathways for the transduction of alteredmechanical force into signals that control cellular(Morgan and Baker, 1991; Schneider et al., 1991;

Watson, 1991; Komuro and Yazaki, 1993). Studies adaptaion(s) have been identified (Marban and Ko-retsune, 1990; Morgan and Baker, 1991; Schneiderin both humans and animal models of cardiac

disease have shown that different hemodynamic et al., 1991; Neyses and Vetter, 1992; Vandenburgh,1992; Komuro and Yazaki, 1993; Sadoshima andloads result in different types of cardiac hypertrophy.

This is often expressed in terms of the dichotomy Izumo, 1993; Miki et al., 1994), it is not knownhow different mechanical loads lead to differentof pressure v volume overload hypertrophy (Gross-

man, 1980), although the altered loads and the adaptations in the whole heart.

Please address all correspondence to: Bryan K. Slinker, VCAPP Department, Washington State University, Pullmann, WA, 99164-6520, USA.

0022–2828/96/071565+10 $18.00/0 1996 Academic Press Limited1565

B. K. Slinker et al.1566

Many isolated heart studies have shown in- role(s) of these immediate-early genes in cardiachypertrophy, we hypothesized that if they do havecreased protein synthesis or immediate-early gene

expression in response to increased systolic pressure a role, they should not all respond in the samemanner to these four different mechanical loads.(Hjalmarson and Isaksson, 1972; Schreiber et al.,

1975; Takala, 1981; Morgan et al., 1986; Schun-kert et al., 1991; Kolbeck-Ruhmkorff et al., 1993).

MethodsIn contrast, most isolated heart studies have failedto show that diastolic volume overload induces

Isolated rabbit heart preparationchanges in protein synthesis or immediate-earlygenes (Hjalmarson and Isaksson, 1972; Schreiberet al., 1975; Morgan and Baker, 1991; Schunkertet al., 1991). For example, Schunkert et al. (1991)

Isolated heart

Experiments were done in 61 male NZW rabbitsshowed that increased systolic pressure de- [mean BW=3.09±0.27 (..) kg], using a protocolvelopment (or wall stress) in an isovolumically approved by the Institutional Animal Care and Usebeating rat heart preparation increased expression Committee. Animals were pre-anesthetized withof the immediate-early genes c-fos and c-jun. In intramuscular injections of ketamine and xylazinecontrast, when the hearts were arrested so that (35 and 7.5 mg/kg, respectively), and anesthesiasystolic stress was absent, no expression of c-fos or was maintained with either isoflurane (1–2%) orc-jun was found as a function of increasing end- InovarVet (0.044 mg/kg fentanyl and 2.2 mg/kgdiastolic volume (EDV)1, implying that systolic, and droperidol, i.m.). The thorax was opened via anot diastolic, stress (or stretch) was the mechanical median sternotomy. Heparin (1000 units) was in-inducer of immediate-early gene expression. jected into the right atrium, and after 1 min the

However, failure to show that diastolic stress is heart was removed from the chest and placed in aa stimulus for cardiac hypertrophy in these ex- high-potassium buffer solution containing (in m):periments could be due to the difficulty of separating 120 Na+, 36 K+, 143 Cl−, 2.5 Ca2+, 1.1 Mg2+,diastolic, systolic, and coronary pressures in the 12 HCO3

−, 0.4 PO42−, and 11.1 glucose, as well as

intact heart. Most of these studies have been done 2.5 u/l insulin). The aorta was cannulated andin isovolumically beating hearts where systolic wall retrograde perfusion of the coronary arteries wasstress was increased by increasing end-diastolic begun using high-potassium buffer that wasvolume and where muscle did not shorten against a bubbled with a 95% O2–5% CO2 gas mixture (per-load. When traditional working hearts were studied, fusate was not recirculated). Coronary pressure wasEDV could not be controlled independently of sys- kept constant at 90 mmHg. The arrested heart wastolic load. As a result, the effects of the different placed in a temperature-controlled chamber to keepmechanical forces imposed by diastolic and systolic temperature constant at 30°C.loads were impossible to separate. Some in- The left atrium was incised and the chordaevestigators have tried to make this separation by tendinea were cut. A thin empty latex balloon wasarresting the heart so that systolic force is absent secured to the volume-servo system. The heart was(Takala, 1981; Schunkert et al., 1991). However, positioned so that the end of the volume servo-cardiac arrest is an inadequate substitute for dia- system “snapped” into the mitral annulus and thestole because contraction may modulate gene ex- balloon tip was pulled through a puncture in thepression and/or protein synthesis (McDermott and left ventricle (LV) apex. A suture was placed in theMorgan, 1989; Samarel and Engelmann, 1991; atrio-ventricular groove and tightened to secure theVandenburgh, 1992; Kubisch et al., 1993; Sharp heart to the volume-servo system and the balloon inet al., 1993). the LV. Unipolar electrodes were attached to the LV

The purpose of our study, therefore, was to in- apex. Once these preparations were completed, thevestigate the acute response of ejecting isolated perfusate was changed to one containing less potas-rabbit hearts to different mechanical loading con- sium (NaCl was substituted for KCl to bring theditions by independently varying peak systolic ejec- final concentrations of Na+ and K+ to 147 andtion pressure (Pej) and EDV at constant coronary 7.4 m, respectively). The heart was paced at 90–perfusion pressure. We studied the combined effects 100 beats per min.of low EDV and low Pej, compared to low EDV andhigh Pej, high EDV and low Pej, and high EDV and

Volume-servo systemhigh Pej, on the expression of the immediate-earlygenes c-fos, c-jun, and egr-1. In addition, although Details of the volume-servo system have been pub-

lished previously (Campbell et al., 1992). The systemwe did not seek to address the potential causal

Immediate-early Genes and Cardiac Mechanical Loads 1567

is composed of a linear motor, a piston-cylinder series. In another experimental series, two othergroups were subjected to a mechanical load for 60device, a linear variable displacement transducer

(LVDT), an analog position controller, and a high min: both groups were designated high Pej groups,one with low EDV (n=7) and one with high EDVcurrent amplifier. The piston-cylinder device was a

modified 5 ml glass syringe. The piston barrel was (n=7). These two groups comprise the 60 min ex-perimental series. EDV was set by adding volume toconnected to one end of the motor shaft. The flared

end of the cylinder was attached to the LV balloon the intraventricular balloon with a 1-cc syringe andthe volume-servo system was used to control Pej.and placed in the mitral annulus. A 5F catheter-

tip pressure transducer (Millar, Houston, TX, USA)was introduced into the center of the balloon via

Protocola side port. The position of the piston, and thusrelative LV volume, was measured by the LVDT A heart was mounted on the volume-servo system(Trans-Tek, Inc.). Piston position was controlled by as described above. The group assignment (withinone of two analog proportional-integral-differential each of the two experimental series) was chosen at(PID) compensators, one for volume and one for random and both the parameters of the volume-pressure. Volume and pressure command signals servo system and the EDV were adjusted ac-were fed continuously from the computer control cordingly. When the heart was beating stably atsystem to the volume-servo system as the reference the desired settings of Pej and EDV, the 30 or 60input to the PID compensators. The output voltage min experimental period was started. At the end offrom the two PID compensators was switched to this period pacing was stopped, volume was quicklyexert either volume or pressure control as required removed from the LV, and the heart was quicklyfor the contraction mode. placed in 4°C buffer. LV/septal and right ventricle

myocardium were then frozen separately in liquidnitrogen and stored at −80°C until processed.

Computer control system

Likewise, details of the computer control systemControl groupshave been published previously (Campbell et al.,

1992). A computer system with an analog-to-digital To rule out non-specific effects of heart isolation,converter (Scientific Solutions, Lab Master DMA we included control groups of three hearts each.operating at 2 KHz) continuously sampled LV vol- For the 30-min experimental series these groupsume and pressure. Digital-to-analog converters were: (C1) hearts removed from anesthetized rabbitswere used to send the volume and pressure com- as soon as the thorax could be opened; (C2) heartsmands to their respective PID compensators. Two taken to the stage of isolation from the rabbit;different modes of pressure control were utilized. In (C3) hearts taken to the stage of mounting on thethree of the hearts in each of the four experimental volume-servo system and switchover from high K+

groups, a constant pressure command signal was to low K+ perfusion buffer; and (C4) hearts isolateddelivered to “pressure-clamp” the ejecting LV at a and mounted to the volume-servo system, but main-fixed level. In the remaining hearts the load was tained unloaded (i.e., empty) and arrested for 30provided by a Windkessel model that, when solved min. For the 60-min experimental series a singlein real time, simulated an arterial load on the LV control group, (C5), included hearts isolated and(Kirkpatrick et al., 1991). mounted to the volume-servo system, but main-

tained unloaded and arrested for 60 min.

Experimantal designAnalyses

Experimental groupsCardiac mechanics data

Six experimental groups were studied. Four of thesegroups were subjected to a mechanical load for 30 Data records, each consisting of five beats of LV

pressure and volume sampled at 250 Hz, weremin. Two of these groups were designated as lowEDV groups, one with low Pej (n=9) and one collected every 10 min throughout the 30 or 60

min experimental period. The variables of interestwith high Pej (n=8). The other two groups weredesignated as high EDV groups, again, one with were extracted (using software developed in our

laboratory) from each of the five beats in the recordlow Pej (n=7) and one with high Pej (n=8). Thesefour groups comprise the 30 min experimental taken two-thirds of the way into the experimental

B. K. Slinker et al.1568

period: 20 min for the 30-min series and 40 min coding region from pcfos-3 (American Type CultureCollection), subcloned into the vector pGEM4z, isfor the 60-min series. The average of these five

beats was taken as the value for that heart. Spe- linearized with AVA II and transcribed as above;the 0.9kb Pstl fragment of the humancifically, end-diastolic pressure (Ped) was determined

as the value of pressure when LV dP/dt first exceeded c-jun coding region from pEHJ-2 (kindly providedby Peter Vogt), subcloned into pGEM4z, is linearized10% of peak positive dP/dt; stroke volume (SV) was

determined as the difference between EDV and the with Acc l and transcribed as above; pTRI-/Egr-1-rat (Ambion) is transcribed as described above.end-systolic volume, which was determined as the

value of volume signal just prior to refilling; ejection Reactions, using approximately 10 lg total RNA,are performed according to the Ambion Hybspeedfraction (EF) was calculated as SV/EDV; Pej was

determined as the peak value of LV pressure; and RPA instruction manual, with slight modifications.RNase digestions are performed at 37°C for 30 minheart rate (HR) was determined from the interbeat

interval. In addition, simple wall stresses were cal- using a 1:1000 dilution of the RNase A and RNaseT1 mixture supplied with the kit. RNase digestedculated. For example, relative end-diastolic stress

(EDr) was calculated as solutions were electrophoresed on 5% poly-acrylamide/8 urea gels and visualized on film.

(1)EDr=Ped · (EDV)1/3

Films were scanned with a Molecular DynamicsPersonal Densitometer. After accounting for back-This relative stress is derived assuming sphericalground, the optical density (OD) of the GAPDH andgeometry. Wall stress, r, is given byimmediate-early gene of interest in each lane were

(2) recorded and the result for that heart was expressedr=Pr2h as ratio of these two OD values. Results in the 30

min series were normalized so that the immediate-early gene/GAPDH OD ratio of the average value

where P is pressure, r is radius, and h is wallfrom all four control groups (C1–C4) equalled 1.0.

thickness. Assuming constant h, the factor 2h isResults in the 60-min series were normalized so

constant and can be dropped. Volume is pro-that the immediate-early gene/GAPDH OD ratio of

portional to r3, and so V1/3 can be substituted for r,the single control group equalled 1.0.

resulting in a relative wall stress

(3)rrel=P · V1/3

Statistics

Substituting a specific pressure, e.g., Ped, and vol- Data were summarized as mean±.. unless other-ume, e.g., EDV, results in a relative wall stress for wise noted. The 30 min experimental series wasa specific event in the cardiac cycle (e.g., for end designed such that the analysis of the pattern ofdiastole as given by Equation 1). Similar sub- immediate-early gene expression in the four ex-stitutions were done to calculate end-systolic stress perimental cardiac loading groups was done using(ESr). Peak-systolic stress (Peakr) was determined two-way ANOVA (level of EDV is one factor andas the maximal value calculated according to Equa- level of Pej is the second factor). Because the 60-tion 3, for the period between end diastole and end min experimental series had only two loadingsystole. groups, these were compared using an unpaired t-

test. The mean OD of the four control groups (C1–C4) in the 30-min experimental series were com-

RNase protection assaypared using one-way ANOVA.

Total RNA was extracted from the frozen myocardialsamples using the Single-Step RNA Isolation pro-tocol (Chomczynski and Sacci, 1987) with slight Resultsmodifications. All RNA samples were stored aspellets at −80°C in 2 ll 3 sodium acetate (pH The four different loading condition assignments

produced the desired range of cardiac mechanics5.2) and 20 ll 100% ethanol until used for electro-phoresis. Templates were prepared as follows: variables (Tables 1 and 2). Representative samples

from the RNase protection assays, illustrating thepTRI-glyceraldehyde-3-phosphate dehydrogenase(GAPDH)-rat (Ambion) is transcribed using T7 RNA response of c-fos, c-jun, egr-1, and GAPDH in two

hearts from each experimental loading group inpolymerase and [a-32P]-CTP according to the Am-bion Maxiscript manual, with slight modifications; the 30 min experimental series (as well as control

groups C1 and C3) are shown in Figure 1.the 1.5-kb EcoRI/Sstl fragment of the mouse c-fos

Immediate-early Genes and Cardiac Mechanical Loads 1569

Table 1 Average values of cardiac mechanics variables for the four experimental groups ofthe 30-min series

Group

LowEDV LowEDV HighEDV HighEDVVariable LowPej HighPej LowPej HighPej

EDV (ml) 0.91±.21 0.93±.16 2.06±.34 1.88±.22Pej (mmHg) 9.3±7.3 5.3±8.8 11.9±7.8 15.4±13.2EDr (mmHg/mm) 9.6±8.0 5.1±8.5 15.0±9.2 18.6±15.9SV (ml) 0.68±.29 0.14±.08 1.47±.39 0.12±05EF (%) 65±12 15±8 71±11 6±3Pej (mmHg) 60±11 103±9 64±13 111±11Peakr (mmHg/mm) 51±2 99±13 74±14 135±13ESr (mmHg/mm) 32±7 94±12 45±9 133±12HR (beats/min−1 ) 105±16 99±20 91±19 99±19

Table 2 Average values of cardiac mechanics variablesfor the two experimental groups of the 60-min series

Group

LowEDV HighEDVVariable HighPej HighPej

EDV (ml) 1.08±.43 2.21±.10Ped (mmHg) 2.1±1.4 9.7±5.5EDr (mmHg/mm) 2.3±1.6 12.6±7.2SV (ml) 0.11±.05 0.09±.03EF (%) 9±3 4±1Pej (mmHg) 117±11 131±10Peakr (mmHg/mm) 123±8 169±15ESr (mmHg/mm) 119±9 167±14HR (beats/min−1 ) 88±2 87±2

GAPDHc-fos

GAPDH

egr-1

GAPDH

c-jun

Figure 1 Portions of RNase protection assay films show-ing the response of c-fos, egr-1, and c-jun in two leftventricles in each of the four experimental groups, andin a control group, all from the 30-min experimentalResponse to 30 min of mechanical loading (Fig. 2)series. The leftmost pair of lanes in each panel is from acontrol group (C3 for c-fos and C1 for egr-1 and c-jun).

Both c-fos and egr-1 were induced by 30 min of the The second pair (counting from the left) is from the lowmechanical loads. In contrast, c-jun did not respond EDV: low Pej group; the third pair is from the low EDV:

high Pej group; the fourth pair is from the high EDV: lowstrongly to the mechanical loads (< two-fold in-Pej group; and the fifth pair (i.e., the rightmost pair) iscrease).from the high EDV: high Pej group.Both an inspection of the data plots showing the

average responses of these immediate-early genesin the four experimental groups (Fig. 2) and theresults of the two-way ANOVAs suggest that theresponse of c-fos was different than the responses respectively). The ANOVAs for each of these im-of egr-1 and c-jun. The expression of c-fos varied mediate-early genes showed no significant inter-significantly with the level of EDV (P=0.01) but action between EDV and Pej.not with the level of Pej (P=0.16). Specifically, theexpression of c-fos at either level of Pej was lowerwith high EDV than with low EDV. In contrast, therelative expression of both egr-1 and c-jun among Response to 60 min of mechanical loading (Fig. 3)the four loading groups did not vary significantlywith either the level of EDV (P=0.86 and P= Comparison of the low EDV v high EDV group (both

high Pej ) at 60 min (Fig. 3) showed no significant0.90, respectively) or Pej (P=0.38 and P=0.51,

B. K. Slinker et al.1570

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Figure 2 Relative mRNA OD ratios (immediate-early/ Figure 3 Comparison of responses in the 30- and 60-min experimental series. [The data for the 30-min seriesGAPDH) for the three immediate-early genes, normalized

so that the average value of the four 30-min series control (open bars) are identical to those of the same loadinggroup shown in Fig. 2, and are redrawn here for com-groups=1.0, in each of the four different groups of the

30 min experimental series. The values are mean±.. parison.] For the 60-min group (hatched bars), the rel-ative mRNA OD ratios (immediate-early gene/GAPDH)for n=9 LV samples for the low EDV: low Pej group, n=

8 LV samples for the low EDV: high Pej group, n=7 for for the three immediate-early genes were normalized sothat the average value of the single control group (C5)=the high EDV: low Pej group, and n=8 for the high EDV:

high Pej group. The horizontal dashed line is at a value 1.0. The values for the two 60-min conditions aremean±.. for n=7 LV samples in the low EDV: high Pejof 1.0 (i.e., level of expression in control hearts).group (n=6 for c-jun) and n=7 LV samples for the highEDV: high Pej group. The horizontal dashed line is at avalue of 1.0.

differences for c-fos (P=0.85), egr-1 (P=0.42), orc-jun (P=0.40). Thus, the difference in c-fos ex- than at 30 min, indicating that the time course of

its expression is delayed somewhat relative to thepression that was observed between low EDV andhigh EDV at 30 min is no longer apparent at 60 expression of c-fos. However, c-jun was not strongly

induced (still < two-fold increase).min. egr-1 expression is much higher at 60 min

Immediate-early Genes and Cardiac Mechanical Loads 1571

Table 3 Immediate-early gene expression (immediate-early gene OD/GAPDH OD) in the control groups

Group P value∗

C1 C2 C3 C4 C5

c-fos 0.05±0.02 0 0 0.12±0.15§ 0.38±.014 0.34egr-1 0.03±0.03 0.01±0.02 0.03±0.04 0.46±0.67† 0.22±0.12 0.43c-jun 0.99±0.07 1.40±0.31 0.75±0.61 1.18‡ 0.54±0.13 0.29

∗ From the ANOVA comparing the four control groups from the 30-min experimental series (groups C1–C4).§ 1 of the 3 samples had a value of 0.29. The other two samples had values of 0.02 and 0.05.† One of the three samples had a value of 1.23. The other two samples had values of 0.05 and 0.09.‡ Two of the three samples were lost due to technical error, so there is only one value to report for this group.

Control groups not shown an independent effect of EDV or stresson immediate-early gene expression or protein syn-

Low or undetectable expression of c-fos and egr-1 thesis in isolated hearts (Hjalmarson and Isaksson,was observed for all control groups in the 30-min 1972; Schreiber et al., 1975; Morgan et al., 1986;series (C1–C4 in Table 3). In contrast, c-jun was Schunkert et al., 1991). In addition, our findingexpressed in all of the control groups. There was that c-jun is relatively insensitive to Pej group as-no significant difference among the C1–C4 group signment in these ejecting rabbit hearts, is differentmeans for any of the immediate-early genes (Table than shown by others in isovolumically beating rat3), and thus these control groups were pooled to hearts, where it has been shown to be induced withobtain a single average value for normalization. high systolic loads (Schunkert et al., 1991). On theSimilar values were observed in the 60-min series other hand, our c-jun results are not particularlycontrol group (C5 in Table 3). surprising in one respect. Compared to c-fos, c-jun

is thought to be present at low levels in manytissues, including cardiac muscle (Vogt et al., 1990;Lin et al., 1993; Woodgett et al., 1995), and othersDiscussionhave shown detectable c-jun mRNA in LV myo-cardium under control, or baseline, conditionsThe different systolic and diastolic mechanical loads(Reiss et al., 1993; Knoll et al., 1994).we studied in these ejecting hearts differently in-

We observed that high EDV delayed the ex-fluenced the expression of the immediate-earlypression of c-fos. The literature provides little in-genes c-fos, c-jun, and egr-1. c-jun was only weaklyformation about this response—only one otherinducible in this isolated rabbit heart preparation

(< two-fold over control). In contrast, there was study has attempted to separate the effects of dia-strong induction of both c-fos and egr-1. In this stolic and systolic loads on immediate-early generespect our results are similar to other reports of expression in a beating and ejecting heart. Kolbeck-discordant responses of c-fos and c-jun to cardiac Ruhmkorff et al. (1993) used a working Langendorffischemia (Brand et al., 1992) and a- v b-adrenergic rat heart preparation to study the time course ofstimulation (Brand et al., 1993). The time course the effect of increased atrial pressure (changingof egr-1 expression was unaffected by either Pej from 8 to 16 cmH2O) at a constant ejection afterloador EDV. In contrast, however, the time course of (80 cmH2O) on c-fos and c-myc expression. Thisexpression of c-fos was delayed when EDV was high maneuver doubled c-fos expression. However, incompared to when EDV was low. This can be seen that study, unlike ours, there was no significantby the significantly lower level of c-fos message at expression of c-fos under their condition of low30 min in the high EDV groups, irrespective of Pej. mechanical loads, and there was no expression ofHowever, by 60 min there is no difference in c-fos c-fos at 60 min under any conditions. There areexpression with EDV. many differences between our preparation and ex-

perimental design and theirs, including species,temperature, controlled heart rate in our study vuncontrolled in their study, and coronary perfusionImmediate-early gene response to diastolic and systoliccontrolled independently of mechanical loads andloadscardiac output in our study, but not in their study.Further, we were able to exert precise control overUnlike our result showing an effect of EDV on the

expression of c-fos, previous studies have generally LV volume and pressure in our study, whereas in

B. K. Slinker et al.1572

working Langendorff hearts there is no direct con- different types of cardiac hypertrophy, a necessary,though not sufficient, condition for any putativetrol over LV volume and pressure. In fact, no pre-

vious study of immediate-early gene expression in signal is that different responses can be dem-onstrated with different mechanical loads. Our res-the working Langendorff heart preparation has

even reported the values of volume and pressure ults do not directly address any causal connectionbetween mechanical loads and hypertrophy via anyin the LV.of these three immediate-early genes. However, ourresults do show that the three immediate-earlygenes do not respond alike to the different in-Immediate-early genes and mechanical signal

transduction in cardiac hypertrophy terventions: c-jun has a very different response pat-tern than c-fos and egr-1 in that it is not strongly

Our purpose was to determine whether different induced above baseline under any of our studyconditions. Furthermore, c-fos and egr-1 have dif-mechanical loads placed on the heart would pro-

duce different cellular responses. For this purpose, ferent responses: the time course of c-fos expressionis influenced by EDV, but the time course of egr-1we studied the response of three immediate-early

genes to the different loads. This is a practical expression is not.choice: we needed a cellular endpoint that couldbe measured relatively easily in the time frameavailable when using an isolated heart (2–4 h). Critique of the experiment

There is considerable evidence that immediate-early genes play a role in cardiac hypertrophy This experiment addressed issues that could not be

studied in vivo because the mechanical loads wouldand, accordingly, they are reasonable markers of aresponse for the purpose of this experiment. Mech- not be sufficiently separable. Futher, our servo-

controlled isolated heart preparation has significantanical forces (e.g., stretch in cell culture, increasedpressure in isolated heart preparations, and aortic advantages over both working and non-working

Langendorff preparations and, accordingly, this isbanding in vivo), as well as numerous growth factorsthat are thought to play a role in cardiac hyper- the first isolated heart study of mechanical signal

transduction in hypertrophy in which adequatetrophy (e.g., angiotensin II and endothelin-1), tran-siently increase the expression of a variety of independent control was exerted over the mech-

anical loading variables. It is also the first study toimmediate-early genes. [This literature has beenreviewed in several recent publications (Marban use an isolated heart from a species other than rat.

In spite of these advantages, there are limitationsand Koretsune, 1990; Morgan and Baker, 1991;Neyses and Vetter, 1992; Komuro and Yazaki, 1993; to this study. What we have called “high pressure”

for our loading groups are peak systolic pressuresSchneider et al., 1993).] In addition, the proteinproducts of immediate-early genes have been shown of 100–140 mmHg (in contrast to “low pressures”

of 60–70 mmHg). These are, indeed, high pressuresto regulate cardiac-specific genes that have alteredexpression in most models of cardiac hypertrophy: in the context of this preparation (Tobias et al., in

press), but not particularly high in vivo. Thus,for example, a-myosin heavy chain (Gupta et al.,1991), atrial natriuretic factor (Kovacic-Milivojevic although we show that different mechanical loads,

including high loads in the context of the isolatedand Gardner, 1993), and skeletal a-actin (Bishopricet al., 1992). Furthermore, the increased protein heart, lead to different responses, whether any of

our loading conditions represents an abnormalsynthesis observed in response to angiotensin IIand endothelin-1 stimulation is associated with mechanical load in vivo is unclear.

In contrast to the results of Kolbeck-Ruhmkorffincreased egr-1 message (Neyses et al., 1991, 1993),and can be blocked by anti-sense egr-1 (Neyses et et al. (1993), who observed the lowest c-fos ex-

pression in the condition of lowest preload andal., 1991). Finally, over expression of c-myc hasbeen shown to modulate cardiac hypertrophy in afterload, we found that our low EDV: low Pej group

had strong c-fos expression. Thus, in our approachresponse to triiodothyronine (Robbins and Swain,1992). the lowest loading condition did not represent a

“baseline” of low expression that would serve as aBeyond our practical interest in these genes asmarkers, however, a secondary hypothesis that we reference point for comparison of the responses of

the other loading groups. Therefore, we abandonedwished to address was that these immediate-earlygenes would not all respond alike to the different our initial plan to normalize results to a value of

1.0 for the low EDV: low Pej group and, instead,mechanical loading conditions we imposed. Giventhat different kinds of mechanical loads result in normalized against the control groups.

Immediate-early Genes and Cardiac Mechanical Loads 1573

angiographic methods in the evaluation of vascularFinally, we did not pinpoint the cellular of theheart disease. Prog Cardivoasc Dis 16: 1–23.message we measured from these whole heart

G W, 1980. Cardiac Hypertrophy; Useful ad-homogenates. We assume, that the myocytes are aptation or pathological process? Am J Med 68: 576–the source of the bulk of the measured message, as 584.shown by Schunkert et al., (1991). Nor have we G MP, G M, Z R, S VP, 1991. Egr-1,

a serum-inducible zinc finger protein, regulates tran-any way of determining in our analyses whetherscription of the rat cardiac a-myosin heavy chain gene.the effects we observe are the result of simpleJ Biol Chem 266: 12813–12816.transcriptional changes, or whether post-tran-

H A, I O, 1972. In vitro load and ratscription processing is important. heart metabolism; I. Effect on protein synthesis. Acta

In summary, we studied the effect of different Physiol Scand 86: 126–144.K JW, T RD, B JR, D HT, 1968.combinations of EDV and Pej (i.e., diastolic v systolic

Quantitative angiocardiography III: relationships of leftloads) in ejecting, isolated rabbit hearts. We showedventricular pressure, volume, and mass in aortic valvethat there are differences in the patterns and timedisease. Circulation 38: 838–845.

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valve disease. Circulation 41: 817–824.high EDV delays the expression of c-fos.K RD, C KB, B D, T H, 1991.

Method for studying arterial wave transmission effectsof left ventricular function. Am J Physiol 260: H1003–

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