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Am J Physiol Heart Circ Physiol 274: H1361-H1370, 1998;
0363-6135/98 $5.00
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Vol. 274, Issue 4, H1361-H1370, April 1998

Effects of left ventricular hypertrophy on force and Ca2+ handling in isolated rat myocardium

Lars S. Maier2, Rolf Brandes1, Burkert Pieske2, and Donald M. Bers1

1 Department of Physiology, Loyola University Chicago, Maywood, Illinois 60153; and 2 Medizinische Klinik III, Universität Freiburg, 79106 Freiburg, Germany

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

To study the effect of left ventricular (LV) hypertrophy on force and Ca2+ handling in isolated rat myocardium, LV hypertrophy was induced in rats by banding of the abdominal aorta. After 16 wk, arterial pressure was assessed by catheterization. LV trabeculae were isolated and loaded with indo 1 salt by iontophoretic injection. Isometric force and intracellular free Ca2+ concentration ([Ca2+]i) were measured at stimulation frequencies between 0.25 and 3 Hz and rest intervals between 2 and 240 s. Sarcoplasmic reticulum (SR) Ca2+ content was also investigated using rapid cooling contractures (RCC). Systolic and diastolic pressure as well as heart weight-to-body weight ratios were significantly elevated in banded compared with control animals (167 vs. 117 mmHg, 108 vs. 83 mmHg, and 4.6 vs. 4.0 mg/g, respectively). At high frequencies, twitch relaxation and [Ca2+]i decline rates were significantly slower in banded compared with control rats, and diastolic [Ca2+]i was higher in the banded rat muscles (at 3 Hz, force half-time = 83 vs. 68 ms; time constant of [Ca2+]i decline = 208 vs. 118 ms; and diastolic [Ca2+]i = 505 vs. 353 nM). These differences could not be ascribed to altered Na+/Ca2+ exchange, since twitch relaxation and Ca2+ handling were not different between groups in the presence of caffeine (or cyclopiazonic acid plus ryanodine), where relaxation depends primarily on Na+/Ca2+ exchange. After long rest intervals (>= 120 s), control rats showed a significant rest potentiation of force and Ca2+ transients, whereas banded rats did not. In addition, RCC amplitudes increased with rest in control but were unaltered in banded rats. In summary, pressure-overload hypertrophy was associated with slower twitch relaxation and [Ca2+]i decline but also with blunted rest potentiation of twitches and SR Ca2+ content of LV trabeculae. The decrease in SR Ca2+-ATPase function in banded rats may contribute to the observed diastolic dysfunction associated with pressure-overload hypertrophy.

systemic hypertension; iontophoretic injection; rapid cooling contractures; force-frequency relationship; postrest relationship

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

LEFT VENTRICULAR hypertrophy is an adaptive response to systemic pressure overload and may be a stage in the pathway toward end-stage congestive heart failure (1). There are numerous potential etiologies for development of hypertrophy and failure. Indeed, a wide array of experimental models of hypertrophy exist, and many have implicated altered sarcoplasmic reticulum (SR) Ca2+ management, including downregulation of the SR Ca2+-ATPase (1, 14, 17, 25, 26). Several reports have shown smaller contractions or Ca2+ transients and slower relaxation or intracellular Ca2+ concentration ([Ca2+]i) decline (4, 20, 27, 32), but some have shown no depression of Ca2+ transient amplitude or decline (23, 29). Moreover, different models exhibit different phenotypes at different stages, and the cause of cardiac hypertrophy and how it may lead to heart failure is not yet completely understood.

In recent studies using the same animal model as used here, hypertrophic hearts of hypertensive rats showed a downregulation of SR Ca2+-ATPase at the mRNA and protein level during the progression of left ventricular hypertrophy, suggesting an impaired SR Ca2+ uptake capacity (39). Despite the reduced SR Ca2+-ATPase protein in this model, functional studies in isolated cardiac myocytes investigating Ca2+ fluxes and SR Ca2+ loading did not reveal any significant differences in relaxation or [Ca2+]i decline between normal and hypertrophied myocytes under steady-state conditions (15, 29). Further explanation of this apparent dichotomy was a motivating factor for the present study. One possible explanation raised was that Ca2+ transients and voltage-clamp studies were done on a self-selected population of myocytes that were the best survivors of the cell isolation. Thus the present study uses multicellular preparations. There is also the possibility that the isolated myocytes were not challenged sufficiently from an energetic standpoint to display altered [Ca2+]i dynamics (23), and here we have increased pacing frequency.

Studies of hypertrophic human hearts are primarily limited to end-stage failing human myocardium in which a downregulation of SR Ca2+ pump on the mRNA and protein level has been reported (22, 30). The enlarged and failing human heart exhibits a negative force- and [Ca2+]i-frequency relationship (vs. positive in nonfailing) and also shows blunting of postrest potentiation compared with the nonfailing heart (34, 36, 38). Study of force-frequency relationships and postrest twitch behavior can be useful in understanding alterations in Ca2+ handling in the heart, and we have used this strategy here. It is possible that depressed and slowed Ca2+ transients are more uniformly characteristic of heart failure than of hypertrophy (especially during the compensated stage in which systolic pressure remains elevated; see Ref. 17).

Rat ventricle typically exhibits a negative force-frequency relationship, paralleled by a decrease in SR Ca2+ content [measured using rapid cooling contractures (RCC); see Refs. 8 and 42]. In nonfailing human myocardium (as in rabbit), a positive force-frequency relationship is accompanied by increased SR Ca2+ content (8, 37). However, failing human myocardium shows a negative force-frequency relation, and SR Ca2+ content fails to increase at higher frequencies. Additionally, postrest decay of twitch force in normal rabbit and failing human myocardium is associated with a parallel decline in SR Ca2+ content, in contrast to the rest potentiation and increased SR Ca2+ content in normal rat and nonfailing human myocardium (8, 37).

The main aims of the present study were to use multicellular preparations from control and hypertrophic hearts to evaluate 1) whether SR Ca2+-ATPase function is altered during relaxation in these muscle preparations (as found biochemically), 2) whether there is a more negative force-frequency relationship and less postrest potentiation in the banded vs. control rats, and 3) whether changes in SR Ca2+ load parallel the changes in twitch force and Ca2+ transients. For this purpose, we microinjected indo 1 free acid into isolated rat trabeculae to measure twitch force and Ca2+ transients simultaneously. In addition, we investigated SR Ca2+ content using RCCs in these trabeculae, a technique that can be used to estimate SR Ca2+ load (7, 13).

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Experimental animals and hemodynamic measurements. The procedure used for banding the abdominal aorta has been previously described (15, 16, 29, 39). Briefly, young male Sprague-Dawley rats (150-200 g body wt) were randomly divided into banded, sham operated, and unoperated animals. After anesthetization with ketamine (60-90 mg/kg im) and xylazine (1-2 mg/kg im) and dissection of the abdominal aorta, suprarenal coarctation was produced in the banded group. Sham rats were operated on without application of the suprarenal band. Some control rats were not operated on at all. Sixteen weeks after surgery, all rats were anesthetized again (as above), and hemodynamic measurements and body weight were determined. A 3-Fr micromanometer-tipped catheter (MMI-Gaeltec, Hackensack, NJ) was introduced via the right carotid artery to measure systemic blood pressure at the aortic arch.

Muscle preparation and solutions. After hemodynamic measurements, the hearts were excised and washed with a modified Krebs-Henseleit buffer (KHB) containing (in mM) 108 NaCl, 6 KCl, 24 NaHCO3, 1.2 MgCl2, 2 CaCl2, 4 glucose, 10 pyruvate, and 20 IU/l insulin, continuously bubbled with 95% O2 and 5% CO2 to bring pH to 7.4. After washing, hearts were perfused retrograde through the aorta with KHB containing 30 mM 2,3-butanedione monoxime (BDM), which arrested contraction and cleared blood from the ventricles before weighing. This cardioplegic solution has been shown to protect the myocardium from a high rate of energy utilization and especially from cutting injury (33). The left ventricle was opened carefully along the septum, and thin trabeculae were removed, including a piece of the surrounding free wall (carnal) at both ends.

After dissection, the trabecula was mounted in a muscle chamber described by Brandes and Bers (10). One end of the trabecula was held by a wire basket that was glued onto a piezoresistive force transducer (AE875; Aksjeselskapet Micro-Elektronikk, Horten, Norway) held by a micromanipulator. The other end was hooked on a stainless steel pin attached to another micromanipulator. After mounting, the trabecula was kept in KHB solution with BDM for 5-10 min before switching to standard KHB superfusate (15 ml/min at room temperature, 25°C). After washout of BDM, the trabecula was stimulated at the default 0.5 Hz with platinum field electrodes (2-ms pulses at 1.2 times threshold).

Isometric force and fluorescence (see below) signals were recorded and stored in a computer. The muscles were allowed to equilibrate for 30 min after being gradually stretched to the length that produced maximum isometric force. To inhibit SR function during part of the experiment, 20 mM caffeine was added to the KHB. After caffeine was washed out, a second approach to block SR function was used. This entailed superfusion with KHB containing a mixture of 50 µM cyclopiazonic acid (CPA) and 1 µM ryanodine.

Indo 1 measurements and [Ca2+]i calibration. A glass electrode was filled with a filtered H2O solution of 2-4 mM indo 1 free acid (Molecular Probes, Eugene, OR) and backfilled with 0.2 M KCl. The electrodes typically had a resistance of 150-200 MOmega and a tip diameter of <0.1 µm, determined by measuring minimum electrode air pressure needed to force a bubble through the tip (31). A single cell of the trabecula was impaled, using a membrane potential of -40 mV or more negative as the criteria. Indo 1 was iontophoretically injected into the cell by applying a negative current of 5-10 nA (3). To measure resistance and membrane potential, and also to inject indo 1, a Microprobe System was used (M707A; World Precision Instruments, Sarasota, FL). After injection (using 3-4 sites), the trabecula was stimulated at 1 Hz for 30-60 min to promote diffusion of indo 1 through the gap junctions to other cells. No measurements were done until the muscle fluorescence was homogeneous (3), as determined by visual inspection through the microscope and minimal motion artifact in the indo 1 ratio. Wier et al. (44) recently reported that iontophoretic loading of fluo 3 was not completely uniform from cell to cell using confocal microscopy. Although homogeneous loading is ideal, some undetected heterogeneity of cellular indo 1 concentration would be unlikely to affect our results (partly due to the ratiometric nature of indo 1 measurements). Loading with indo 1 caused a two- to threefold increase in fluorescence vs. background (at 385 nm and diastole).

Indo 1 fluorescence was excited at 350 nm, and emission was detected at 385 nm (F385) and 456 nm (F456). From this, the commonly used indo 1 ratio (R) was calculated after subtraction of the background fluorescence at each wavelength. Because the background fluorescence intensities slowly decreased with time (2, 10), this time dependency was measured in separate control experiments (n = 3). After an initial ~100 min of illumination (at 350 nm), the backgrounds decayed at a constant rate of ~0.1%/min. Thus, as the experiments progressed, smaller background intensities were used to calculate R. Changes in pacing frequency also alter background fluorescence (which is partially due to NADH; see Ref. 11), and attempts were therefore made to additionally correct for these acute changes. However, these changes in NADH background fluorescence were much smaller than the changes in indo 1 fluorescence, and correction for such acute background changes did not alter R significantly. Thus, no correction for phasic changes in background fluorescence was used.

Conversion of R values to [Ca2+]i was performed as previously described by Brandes et al. (12). Briefly, the relationship between F385 and F456 was first determined to obtain the slope, defined as the gain adjuster (bH). To reduce motion artifacts during determination of bH, 10-20 mM BDM was added to nearly abolish contraction (while only partially inhibiting [Ca2+]i transients). With the use of bH, the minimum and maximum values of R (Rmin and Rmax, respectively) can be calculated
R<SUB>min</SUB> = R<SUB>max</SUB> × (S<SUB>385</SUB>/S<SUB>456</SUB>) (1)
R<SUB>max</SUB> = (1 − S<SUB>456</SUB>)/[(S<SUB>385</SUB> − 1) × bH] (2)
where S385 and S456 are calculated by dividing the indo 1 fluorescence intensity in the absence of Ca2+ by that at saturating [Ca2+] (at 385 and 456 nm, respectively). These values were previously determined in protein solutions as S385 = 0.051 and S456 = 2.27 (12).

After Rmin and Rmax were determined, [Ca2+]i was calculated according to (19)
[Ca<SUP>2+</SUP>]<SUB>i</SUB> = <IT>K</IT><SUB>d</SUB> × S<SUB>456</SUB> × (R − R<SUB>min</SUB>)/(R<SUB>max</SUB> − R) (3)
where the Ca2+ dissociation constant of indo 1 (Kd) was taken as 256 nM. Note that a different Kd value simply scales [Ca2+]i linearly and would not alter the relative comparison of [Ca2+]i described in the present study.

RCC. Some muscles were used for investigating SR Ca2+ content using RCCs essentially as previously described (7, 13). For these experiments, left ventricular trabeculae were isolated and mounted in a chamber with one end connected to a fixed pin and the other to a force transducer (using fine silk suture). Bath temperature was measured on a strip chart recorder. RCCs were elicited by a rapid switch to low temperature superfusate via solenoid valves at the chamber inlet, which allowed rapid bath cooling from 25 to 1°C. The cold solution was maintained by a refrigerated circulating bath that cooled the solution and also surrounded the tubing connected to the muscle bath. Muscles were not stimulated during the cooling period. With this setup, the surface of a muscle can be cooled to <5°C in <300 ms and the core of a muscle with a diameter of 400 µm in <2 s (7, 13).

Experimental protocol. Force-frequency relations were tested by stepwise changes in stimulation rate from a baseline frequency of 0.25 to either 0.5, 1, 2, or 3 Hz. With the use of these frequencies, SR Ca2+ content was also assessed by RCCs induced 2 s after interruption of steady-state stimulation. To investigate the influence of rest, steady-state stimulation at 0.5 Hz was interrupted for rest intervals of 2-240 s. The first twitch after resumption of stimulation (postrest twitch) was compared with the last steady-state twitch. Similarly, RCCs induced after different rest intervals were compared with the RCC induced 2 s after interruption of 0.5-Hz stimulation (i.e., at the time when the next steady-state twitch would have occurred). SR function was blocked by adding either 20 mM caffeine or a combination of 50 µM CPA and 1 µM ryanodine to the KHB.

Statistics. All data are expressed as means ± SE unless otherwise mentioned. Statistical analysis was performed with Student's t-test (paired or unpaired) or two-way analysis of variance followed by Student-Newman-Keuls test as appropriate. Statistical significance was taken as P <=  0.05. Determination of the time constant of [Ca2+]i decline (tau Ca) of [Ca2+]i transients was done by fitting the declining phase to a monoexponential curve. Mechanical relaxation was analyzed by determining the half-time (t1/2) of relaxation, since the data were not well described by a single exponential.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Hemodynamic measurements. Table 1 summarizes values obtained from hemodynamic measurements for seven banded and five control rats (n = 3 sham operated and n = 2 unoperated). These results confirmed the hypertensive and hypertrophic status of the banded rats, as reported previously with this same hypertrophic model (15, 39). Most importantly, systolic and diastolic systemic pressures were significantly increased in the group of banded rats by 43 and 30%, respectively (systolic pressure of 166.5 ± 6.4 mmHg for banded and 116.5 ± 4.9 mmHg for control rats; diastolic pressure of 107.5 ± 2.7 mmHg for banded and 82.6 ± 3.7 mmHg for control rats). Mean arterial pressure was also increased by 36% in banded compared with control rats (127.2 ± 3.9 mmHg in banded and 93.9 ± 4.1 mmHg in control rats). The ratios of heart weight to body weight of the banded rats were also significantly increased compared with control rats (4.6 ± 0.2 vs. 4.0 ± 0.1 mg/g), with no difference in body weight (482.4 ± 11.3 vs. 477.0 ± 7.5 g). There were no differences in cardiac or hemodynamic parameters between sham-operated and unoperated control animals.

                              
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  		Table 1.   Heart weight and arterial pressure

In Fig. 1A, the values for systolic and diastolic systemic pressure of both groups are presented. The correlation of systolic and diastolic pressure was highly significant (P < 0.0001, regression line r = 0.98). In addition, mean values, including error bars for each group, are plotted. There was no overlap between the normotensive control and hypertensive banded rats.


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  		Fig. 1.   Correlations of hemodynamic measurements and heart weight-to-body weight ratio. A: correlation between diastolic and systolic systemic pressure for individual animals. B: correlation between the ratio of heart weight to body weight and systolic systemic pressure. Results from 7 banded rats (black-triangle), 3 sham-operated rats (open circle ), and two unoperated control rats (square ) are shown. Regression lines (solid lines) are plotted for the data with 95% confidence bands (broken lines), and P values and r values are shown. Average results for control and banded groups are shown as crossed SE bars.

Figure 1B shows systolic systemic pressure and heart weight/body weight. The correlation was highly significant (P < 0.002, regression line r = 0.82). In addition, mean values, including error bars for each group, are plotted. The degree of hypertension gave better discrimination between the groups (i.e., there was some overlap with respect to heart weight/body weight). Nevertheless, the banded group was differentiated as both hypertrophic and hypertensive compared with the control group.

Force-frequency relationship. Figure 2, A and B, shows representative isometric twitches and Ca2+ transients, respectively, in an indo 1-loaded trabecula from a banded rat. Stimulation frequency was increased from 0.25 to 2 Hz. As can be seen, when stimulation frequency was increased, isometric twitch tension decreased (from 29.8 to 26.9 mN/mm2) as did Ca2+ transient amplitude (from 1,230 to 1,146 nM).


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  		Fig. 2.   Changes in isometric force (A) and Ca2+ transients (B) with increasing stimulation frequency from 0.25 to 2 Hz. This trabecula is from a banded rat. [Ca2+]i, intracellular Ca2+ concentration.

The increase in frequency also altered the t1/2 of twitch relaxation and tau Ca. Figure 3A shows the relation between t1/2 of relaxation for the twitch and the stimulation frequency when stimulation rate was increased stepwise from 0.25 Hz. In trabeculae from both groups, t1/2 relaxation time decreased monotonically with increases in stimulation frequency (P < 0.05). In control rats, t1/2 relaxation time decreased from 174 ± 18 ms at 0.25 Hz to 68.0 ± 1.8 ms at 3 Hz. In contrast, banded rats showed a decrease from 222 ± 17 to 83.4 ± 4.0 ms. In addition, at a stimulation rate of 3 Hz, trabeculae from banded rats showed a significantly higher t1/2 relaxation time of the isometric twitch.


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  		Fig. 3.   Frequency dependence of twitch force relaxation half-time (t1/2, A), time constant of [Ca2+]i decline (tau Ca, B), twitch amplitude (C), and rapid cooling contracture (RCC) amplitude (D) when raising stimulation frequency. Average values from 7 trabeculae from banded rats (bullet ) and 5 trabeculae from control rats (open circle ); bars show SE. * Significant difference (P < 0.05) compared with value at 0.25 Hz. # Significant difference between control and banded at the same frequency.

Figure 3B shows the relation between tau Ca and stimulus frequency. As frequency increased in control trabeculae, tau Ca decreased progressively from 211 ± 38 ms at 0.25 Hz to 118 ± 8 ms at 3 Hz (P < 0.05). In contrast, myocardium from banded rats showed a decrease in tau Ca from 278 ± 35 ms at 0.25 Hz to 211 ± 25 ms at 1 Hz, without further decline at 2 and 3 Hz. At 2 and 3 Hz, tau Ca was significantly smaller in control compared with that in banded rats.

Figure 3C indicates the relation between isometric twitch amplitude and stimulation frequency. Myocardium of both groups showed a decline of isometric twitch force with increasing stimulation frequency (negative force-frequency relationship; P < 0.05). Compared with the developed force at 0.25 Hz, twitch force at 3 Hz was 36.8 ± 6.3% in banded and 46.2 ± 13.8% in the control group. There was no significant difference between the force-frequency relationship of control and hypertrophic myocardium. Figure 3D shows that SR Ca2+ content, assessed by RCCs, did not change significantly as a function of frequency in the control group (at 3 Hz, RCC amplitude was 88.3 ± 11.3% of the value at 0.25 Hz). A decline in twitch force without a decrease in SR Ca2+ content might imply a depression of excitation-contraction (E-C) coupling at high frequency. In the banded group, the negative force-frequency relationship was accompanied by a significant decrease in RCC amplitude at all frequencies compared with that at 0.25 Hz (e.g., 69.2 ± 4.9% at 3 Hz). There may thus be two factors contributing to the strong depression of twitches in the banded rats at higher frequency (depression of E-C coupling and reduced SR Ca2+ content).

Table 2 summarizes the measured parameters of isometric twitch force and Ca2+ transients. At 0.5 Hz, there were no significant differences between control and banded rats. In contrast, at higher stimulation frequency, e.g., 3 Hz, there was a significant increase in t1/2 of twitch relaxation and tau Ca decline in banded compared with control rats. In addition, diastolic [Ca2+]i was significantly higher in banded compared with control rats (505 ± 55 vs. 353 ± 36 nM).

                              
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  		Table 2.   Contraction and Ca2+ transient parameters

To evaluate whether altered function of Na+/Ca2+ exchange contributes to the changes in relaxation behavior between banded and control rats, net SR Ca2+ uptake was inhibited by inclusion of 20 mM caffeine while measuring steady-state twitch contractions at 0.5 Hz. Table 2 shows that the t1/2 of relaxation and tau Ca of [Ca2+]i decline were greatly increased by caffeine. This is consistent with conclusions of Bassani et al. (5) that >90% of Ca2+ removal in rat ventricular myocytes is normally attributed to SR Ca2+-ATPase. They found that relaxation and [Ca2+]i decline were greatly slowed when SR Ca2+ transport was inhibited, thereby causing the Na+/Ca2+ exchange to be the main mode of Ca2+ removal from the cytosol. Table 2 also shows that, in the presence of caffeine, there were no differences between control and banded rats for measured parameters, in either twitch force or [Ca2+]i. Because caffeine also affects other systems (e.g., sensitizing the myofilaments to Ca2+ and inhibiting phosphodiesterases), SR function was alternatively blocked using a combination of CPA (50 µM) and ryanodine (1 µM). The results were similar to the experiments with caffeine, and no difference between the groups was detectable (see Table 2).

Postrest behavior of control and banded rats. Figure 4, A and B, shows the effects of rest after 0.5-Hz steady-state pacing on twitches and RCCs in trabeculae from control and banded rats, respectively. Figure 4, left, shows steady-state twitches and RCCs (i.e., RCCs induced 2 s after the last steady-state twitch at 0.5 Hz). Figure 4, right, shows twitches and RCCs induced after separate 120-s rests after termination of steady-state 0.5-Hz stimulation. The prominent rewarming spike at the end of each RCC is due to the sudden increase in myofilament Ca2+ sensitivity when the muscle is abruptly rewarmed while [Ca2+]i is still high (8, 21). As can be seen, the control isometric twitch tension increases after the 2-min rest interval (from 36.9 to 59.1 mN). In parallel, the control, postrest RCC also increases from 24.3 to 38.2 mN. In a trabecula from a banded rat (Fig. 4B), twitch amplitude was only slightly increased after 120-s rest (from 37.9 to 42.7 mN), and the RCC amplitude was decreased (from 18.1 to 16.8 mN).


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  		Fig. 4.   Steady-state and postrest twitches and RCCs from control (A) and banded (B) rats. Steady-state twitches (left) were at 0.5 Hz, and the RCC was activated 2 s after the last steady-state twitch. Right: postrest twitches were the first twitch after a 2-min rest period. Postrest RCCs are obtained after reequilibrating the muscle at 0.5 Hz and then a second 2-min rest. Note that RCC amplitudes were measured when force reached a maximum during the cooling contracture, but before the rewarming spike (which is attributed to a rapid increase in myofilament Ca2+ sensitivity upon rewarming).

Figure 5A shows the twitch force amplitude of the first postrest twitch (relative to the steady-state 0.5-Hz twitch) as a function of the duration of the rest interval. In myocardium from control rats, isometric twitch tension of the first postrest twitch increased continuously with increasing rest periods. Postrest potentiation of tension was maximum at long rest intervals (increased to 174 ± 10% at 240 s, P < 0.05). In contrast, in banded rat myocardium, twitch tension of the first postrest beat increased at short rest intervals (increase to 143 ± 15% at 10-s rest interval, P < 0.05) but then continuously declined at longer rest intervals to 120 ± 14% at 240 s (not significant compared with the steady-state 0.5-Hz value).


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  		Fig. 5.   Rest-dependent changes in the amplitude of twitch force (A), [Ca2+]i transient (B), and RCC (C). All values are normalized to the value obtained at the steady-state interpulse interval of 2 s (0.5 Hz). Mean values from 7 trabeculae from banded rats (bullet ) and 5 trabeculae from control rats (open circle ); bars show SE. * Significant difference (P < 0.05) compared with the value at 0.5 Hz (A and B) or 2-s rest (C). # Significant difference between control and banded at the same rest interval.

Figure 5B shows that, in control muscles, postrest [Ca2+]i transient amplitudes increased with rest periods (to 197 ± 35% at 240 s, P < 0.05). Trabeculae from banded rats showed increased Ca2+ transient amplitudes at short rest intervals, but the increase was blunted at longer rest intervals and at 240 s was not significantly different from the steady-state value (138 ± 21%).

Figure 5C shows the RCC amplitude (relative to the steady-state RCC at 2 s) as a function of rest duration. In trabeculae from control rats, the amplitude of the postrest RCC continuously increased with increasing rest intervals (155 ± 15% at 240 s, P < 0.05). In contrast, in trabeculae from banded rats, amplitudes of the postrest RCC did not change at short rest periods (102 ± 6% at 30-s rest interval, not significant) and declined at longer rest intervals to 83.0 ± 8.6% at 240 s. The increase in RCC in control rats at all rest intervals is significantly different from the decline in banded rats.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study is the first report combining the use of indo 1 salt to measure [Ca2+]i and RCC in multicellular preparations from the left ventricle of hypertrophic rat myocardium. The main findings of the present study are that, in banded vs. control rats, relaxation of twitch force and [Ca2+]i decline are impaired at higher stimulation frequencies in combination with higher diastolic Ca2+ concentration. In addition, rest intervals led to less rest potentiation of twitch force, Ca2+ transient, and SR Ca2+ content (based on RCCs) in muscles from banded rats.

Indo 1 as a Ca2+ indicator in rat trabeculae. Our results demonstrate that indo 1 loaded by iontophoretic microinjection can be used to measure [Ca2+]i in left ventricular rat cardiac trabeculae as shown for fura 2 (3). The success of the microinjection and the following measurements of [Ca2+]i required micropipettes small enough to minimize damage and depolarization. In most of the experiments, two to four impalements were sufficient to obtain homogeneous distribution of indo 1 throughout the trabeculae. After loading, indo 1 fluorescence could often be measured for several hours, allowing numerous interventions. However, the rate of indicator loss in trabeculae was sometimes relatively fast, especially when small branches were cut during isolation or mounting. The correction made for the slow decline in background autofluorescence also helped to maintain steady-state twitch [Ca2+]i transients at relatively constant levels. This was especially important, as the indo 1 gradually leaked from the preparation (or was photobleached) over the course of long protocols.

Hypertension, hypertrophy, and SR Ca2+ transport. The hemodynamic measurements indicate that the hypertensive state of the banded rats is similar to previous reports using this model (15, 29, 39). The increase in heart weight/body weight (15%) was somewhat smaller than the ~25% increase that we have found in previous rounds of banding, despite using the same approach. It should also be noted that weighing the entire heart rather than the left ventricle underestimates the degree of left ventricular hypertrophy. In previous studies, the 25% increase in heart weight/body weight corresponded to a 43% increase in left ventricular weight/body weight. Thus, although the degree of hypertrophy here was modest, the banded rats were significantly hypertrophic and hypertensive.

In this same experimental model, Qi et al. (39) found significant reductions in SERCA2 expression at the level of mRNA, protein, and SR Ca2+ uptake rate in left ventricular homogenates from rats banded for 16 wk. Isolated myocytes also exhibited significant cellular hypertrophy, but Ca2+ current, SR Ca2+ load, and Na+/Ca2+ exchange were all increased in direct proportion with cell size (15). Indeed, during relaxation in isolated left ventricular myocytes, there was no decrease in the rate of [Ca2+]i decline attributed to either altered SR Ca2+-ATPase or Na+/Ca2+ exchange (29). This was unexpected, given the nearly 50% reduction of SERCA2. Indeed, numerous others have reported reduced SERCA2 expression in pressure overload ventricular hypertrophy (1, 14, 17, 25, 26), and several reports have shown smaller amplitude and slower decline of the cellular [Ca2+]i transient or contraction (4, 18, 20, 27, 32).

One possible explanation raised by Delbridge et al. (15) was that there might be an intrinsic selection of well-compensated hypertrophic cells by the isolation procedure, which were then studied by voltage-clamp or Ca2+ transients. This possibility partly motivated the present study of multicellular preparations, which would avoid the problem of cell self-selection during enzymatic cell isolation.

With multicellular trabeculae in the present study, we found slower force relaxation and [Ca2+]i decline in banded rats during steady-state twitches at higher frequencies. This might be consistent with the possibility above that the isolated myocytes previously studied were somewhat autoselected. That is, well-compensated myocytes (with respect to SR Ca2+ transport) may have preferentially survived to be studied, whereas the multicellular trabeculae here may include both SR compensated and decompensated cells. However, the isolated ventricular myocytes were also not under any mechanical load (in contrast to traeculae here). It is possible that the differences are relatively subtle and are made more apparent at higher work levels (23; in our case, achieved at higher frequency). In addition, as hypertrophy progresses from well compensated to decompensated and failure, these changes in relaxation and [Ca2+]i may become more apparent.

Systolic function and E-C coupling. There was no reduction in either developed force or the amplitude of [Ca2+]i transients in the banded rats (see Table 2). In isolated left ventricular myocytes in this same model, there was also no decrease in Ca2+ transient amplitude in banded rats (29). Unloaded myocyte shortening was, however, lower in cells from banded rats, despite the equivalent Ca2+ transients (29). Because we did not detect any reduction in isometric force in the present study, it is possible that there is greater mechanical resistance to unloaded shortening in myocytes from the banded rat but unaltered Ca2+ concentration dependence of isometric force (24, 28). Thus there is no overt evidence of systolic dysfunction at this stage (although that could change in failure).

In this hypertrophic model, McCall et al. (29) also found that E-C coupling in single left ventricular myocytes in this model was not altered at normal extracellular Ca2+ concentration ([Ca2+]o), based on comparable contraction for a given Ca2+ current trigger and SR Ca2+ load. However, when [Ca2+]o was reduced to 0.5 mM, E-C coupling was strongly depressed in banded vs. control rats. It was speculated that the SR Ca2+ release channels are less sensitive to a given Ca2+ current trigger in the banded rat. Under normal conditions, there still may be sufficient trigger, but when the Ca2+ current trigger is reduced by lowering [Ca2+]o, the trigger may no longer be sufficient in banded rats. Similar depression of E-C coupling was recently reported by Gómez et al. (18) in genetic strains of hypertrophic and failing rat hearts (SH-HF and Dahl salt sensitive). In that study, the depression of E-C coupling was apparent without [Ca2+]o reduction but could be largely restored by increasing Ca2+ current amplitude. Thus there may be a subtle defect in E-C coupling during compensated hypertrophy that becomes more apparent in the transition to heart failure.

Force-frequency relationship. The reduction in twitch amplitude with increasing frequency in rat ventricle has been explained by either a reduction of SR Ca2+ content or a decreased fractional SR Ca2+ release due to a refractoriness of E-C coupling (8, 9). This negative force-frequency relationship was not altered in the banded group (Fig. 3C), nor was there a significant change in SR Ca2+ load in control with frequency. The decline in twitch force without a decrease in SR Ca2+ content in control would be consistent with a refractoriness of E-C coupling at high frequency. In the banded group, there was also a reduction in SR Ca2+ content (69% at 3 Hz) that might contribute to the slightly stronger depression of twitch force at 3 Hz in banded (37%) compared with 46% in the control group. The more rapid relaxation and [Ca2+]i decline with increasing frequency observed in both control and banded groups is well known (40), depends on acceleration of transport of Ca2+ by the SR, and appears to depend on Ca2+/calmodulin-dependent protein kinase (6). It is not obvious why the difference between control and banded groups is only significant at the higher frequencies. We suspect that this is because of lower variance in the t1/2 of relaxation and tau Ca at higher frequency or because of the higher work load at higher frequency (see above). For example, the ratio of mean t1/2 values in banded vs. control was always between 1.21 and 1.29 at all frequencies. In addition, other groups have found slowed relaxation at lower frequencies using various hypertrophic animal models (4, 20, 27, 32, 35, 43).

Postrest twitches. Control rats exhibited roughly parallel rest-dependent increases of the amplitudes of twitch force, Ca2+ transients, and RCC (Fig. 5). The rest-dependent increase in SR Ca2+ content may, in part, explain the enhanced twitch force and Ca2+ transients. On the other hand, rest potentiation of twitches in normal rat ventricular myocytes can also occur without any increase in SR Ca2+ content (9). This may be the result of a relatively slow recovery of E-C coupling and is the converse of the refractoriness that builds up at higher frequency. The banded rats seem to lack the rest-dependent increase of SR Ca2+ while still showing a moderate degree of rest potentiation of twitches. Stauffer et al. (42) also recently reported reduced SR Ca2+ content in myocytes from hypertensive rats based on RCCs.

The increase in SR content during rest in control rats has been attributed to a net Ca2+ influx via Na+/Ca2+ exchange during rest in rat myocardium due to a relatively high intracellular Na+ concentration (41). If less Ca2+ enters during rest in the cells isolated from banded rats, that could explain the observed results in Fig. 5. Although a slightly lower intracellular Na+ concentration in the cells from banded rats (even by 1 mM) could explain this in principle, there is no evidence indicating that this is the case. The time course of [Ca2+]i decline in the presence of caffeine or CPA plus ryanodine was not different between banded and control groups. From this, we infer that hypertrophy was not accompanied by a great increase in the ability of Na+/Ca2+ exchange to extrude Ca2+ from the cell. This conclusion is consistent with isolated myocyte data showing unaltered [Ca2+]i decline and Na+/Ca2+ exchange current during caffeine-induced contractures and no reduction in Na+-dependent 45Ca2+ fluxes in sarcolemmal vesicles (15, 29). Even without a change in Na+/Ca2+ exchange, the functional impact of this system during rest can still be changed in the banded rats. This is because any reduction in SR Ca2+-ATPase in banded vs. control rats would compete less favorably with an unchanged Na+/Ca2+ exchanger, causing less maintained SR Ca2+ load during rest.

    ACKNOWLEDGEMENTS

We thank Drs. Ming Qi, Kenneth S. Ginsburg, and Allen M. Samarel for assistance in performing animal surgeries and hemodynamic measurements. Furthermore, the excellent technical assistance of Steve Scaglione was greatly appreciated. We thank Dr. Gerd Hasenfuss, Medizinische Klinik III der Universität Freiburg, Germany, for stimulating discussion.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-52478 (to D. M. Bers) and HL-57562 (to R. Brandes) and by grants from the Deutsche Forschungsgemeinschaft and the Boehringer Ingelheim Fonds (to L. S. Maier and B. Pieske).

Address for reprint requests: D. M. Bers, Dept. of Physiology, Loyola Univ. Chicago, 2160 South First Ave., Maywood, IL 60153.

Received 18 July 1997; accepted in final form 30 December 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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