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Am J Physiol Heart Circ Physiol 291: H1803-H1813, 2006. First published April 28, 2006; doi:10.1152/ajpheart.01345.2005
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Enhanced calcium mobilization in rat ventricular myocytes during the onset of pressure overload-induced hypertrophy

Beatriz M. R. Carvalho,1,* Rosana A. Bassani,2,* Kleber G. Franchini,3 and José W. M. Bassani1,2

1Departamento de Engenharia Biomédica/Faculdade de Engenharia Elétrica e de Computação, 2Centro de Engenharia Biomédica, and 3Departamento de Clínica Médica/Faculdade de Ciências Médicas Universidade Estadual de Campinas, Campinas, Brazil

Submitted 20 December 2005 ; accepted in final form 19 April 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Early cardiovascular changes evoked by pressure overload (PO) may reveal adaptive strategies that allow immediate survival to the increased hemodynamic load. In this study, systolic and diastolic Ca2+ cycling was analyzed in left ventricular rat myocytes before (day 2, PO-2d group) and after (day 7, PO-7d group) development of hypertrophy subsequent to aortic constriction, as well as in myocytes from time-matched sham-operated rats (sham group). Ca2+ transient amplitude was significantly augmented in the PO-2d group. In the PO-7d group, intracellular Ca2+ concentration ([Ca2+]i) was reduced during diastole, and mechanical twitch relaxation (but not [Ca2+]i decline) was slowed. In PO groups, fractional sarcoplasmic reticulum (SR) Ca2+ release at a twitch, SR Ca2+ content, SR Ca2+ loss during diastole, and SR-dependent integrated Ca2+ flux during twitch relaxation were significantly greater than in sham-operated groups, whereas the relaxation-associated Ca2+ flux carried by the Na+/Ca2+ exchanger was not significantly changed. In the PO-7d group, mRNA levels of cardiac isoforms of SR Ca2+-ATPase (SERCA2a), phospholamban, calsequestrin, ryanodine receptor, and NCX were not significantly altered, but the SERCA2a-to-phospholamban ratio was increased 2.5-fold. Moreover, greater sensitivity to the inotropic effects of the beta-adrenoceptor agonist isoproterenol was observed in the PO-7d group. The results indicate enhanced Ca2+ cycling between SR and cytosol early after PO imposition, even before hypertrophy development. Increase in SR Ca2+ uptake may contribute to enhancement of excitation-contraction coupling (augmented SR Ca2+ content and release) and protection against arrhythmogenesis due to buildup of [Ca2+]i during diastole.

sarcoplasmic reticulum; excitation-contraction coupling; relaxation; beta-adrenergic responsiveness

SUSTAINED INCREASE IN CARDIAC workload leads to ventricular hypertrophic growth, characterized by an increase in cardiomyocyte volume (3). Although compensated hypertrophy may represent an adaptive response that allows the heart to meet the circulatory demand, in the long term, it is usually associated with high morbidity and mortality, often evolving to heart failure, in which altered cell Ca2+ cycling usually accompanies deterioration of contractile function (15, 16, 29, 62).

Sarcoplasmic reticulum (SR) function is of paramount importance for Ca2+ cycling and mechanical function in mammalian cardiac myocytes, not only because most of contraction-activating Ca2+ is released from this organelle, but also because Ca2+ sequestration by the SR Ca2+-ATPase is the main pathway for cytosolic Ca2+ removal, which allows proper relaxation and diastolic function. It is estimated that Ca2+ transport between SR and cytosol comprises 70–90% of total Ca2+ cycling during each beat, whereas most of the remaining Ca2+ fluxes are transsarcolemmal [influx and efflux mainly via L-type Ca2+ channels and Na+/Ca2+ exchanger (NCX), respectively] (6, 16).

A common finding in most studies of animal and human hypertrophied, failing myocardium is depression of the SR contribution to excitation-contraction coupling (ECC) and relaxation due to intrinsic changes in SR function or other mechanisms, such as SR Ca2+ unloading by increased NCX activity (14, 16, 18, 29, 44, 45, 47, 57, 61). Experimental manipulations to increase SR Ca2+-ATPase activity have been successful at rescuing ventricular performance in animal models of heart failure (18, 38, 39 but see Ref. 31), whereas experimental impairment of SR function can precipitate heart failure under pressure overload (PO) (50). On the other hand, up- and downregulation of the NCX and SR Ca2+ pump gene expression, respectively, in association with a trend toward decreased ability of the SR to release and/or take up Ca2+, were reported in nonfailing, chronically hypertrophied myocardium, even though Ca2+ transients, contractions, and ventricular performance in vivo were not markedly depressed (2, 22, 35, 36, 57).

Although much is known about the changes in myocyte Ca2+ homeostasis during late stages of PO-induced ventricular hypertrophy and transition to heart failure, little information is available on the acute responses to hemodynamic overload. Some authors reported a change in gene expression of proteins involved in SR Ca2+ transport and augmented SR Ca2+ uptake in ventricular subcellular preparations studied a few days after aortic constriction (2, 4, 42, 43). However, to our knowledge, Ca2+ balance during the onset of PO-induced hypertrophy has not been directly investigated in intact myocytes. A better understanding of early adjustments to overload and the underlying cell mechanisms is important, inasmuch as it may reveal aspects of the adaptive strategy involved in the acute physiological response of the heart to the hemodynamic perturbation. Insight into such a strategy may be valuable in the development of approaches to prevent and treat heart failure.

The aim of this study was to examine systolic and diastolic function in isolated rat left ventricular myocytes 1 wk after PO imposition by aortic constriction. Cells were studied 2 days after aortic banding, when, despite the PO, ventricular hypertrophy is still absent, and on the 7th day, after stable hypertrophic growth has been attained (51). Our findings indicate enhanced SR-cytosol Ca2+ cycling, even before development of hypertrophy, which may be important for cardiac acute adaptation to the increased circulatory load, as well for signaling involved in development of hypertrophy.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Induction of sustained PO. Male Wistar rats (8–12 wk-old) were subjected to constriction of the transverse aorta under anesthesia with ketamine and diazepam (66 and 0.44 mg/kg, respectively, ip), as described elsewhere (59). A clip (0.9-mm aperture) was placed between the brachiocephalic trunk and the left common carotid artery branches. Except for the latter step, the surgical procedure for sham-operated rats was the same. PO and sham-operated rats were studied 2 days (PO-2d and Sh-2d, respectively) and 7 days (PO-7d and Sh-7d, respectively) after surgery, when femoral and carotid arteries were cannulated for blood pressure measurement under pentobarbital sodium anesthesia (50 mg/kg ip). Then the animals were euthanized by pentobarbital overdose, and the hearts were rapidly removed for further experiments. All procedures and care of the rats were approved by the Institutional Committee for Ethics in Animal Research (Proc. N 1070-1).

RT-PCR. After the left ventricles were homogenized in TRIzol, total RNA was isolated and precipitated with isopropanol. cDNA was synthesized in 6-µg aliquots of mRNA with the Superscript II preamplification system (Invitrogen, São Paulo, Brazil), as previously described (40). Taq DNA polymerase was used for cDNA amplification in the presence of specific primers for the cardiac isoforms of SR Ca2+-ATPase (SERCA2a; M. Porter and A. M. Samarel, personal communication), phospholamban (PLB) (27), ryanodine receptor (RyR2) (17), calsequestrin (CSQ) (49), sarcolemmal NCX (NCX1) (48), and beta-actin (40). For each cDNA species, the number of amplification cycles was that necessary for 50% saturation, as determined in preliminary assays. mRNA levels were normalized to that of beta-actin mRNA [which is not affected in this PO model (40)] determined in the same assay.

Isolated ventricular myocytes. The myocytes were enzymatically dissociated as described elsewhere (8). Cells from at least four different hearts were used for each experimental protocol. Myocytes were perfused with modified Tyrode solution at 23°C and field stimulated at 0.5 Hz (unless otherwise stated) with 5-ms voltage pulses.

Unloaded cell shortening was measured with a video edge detector. Cells were loaded with the fluorescent Ca2+ indicator indo 1-AM (5 µM; Molecular Probes, Eugene, OR) for 15 min. Indo 1 was excited at 360 nm, and intracellular free Ca2+ concentration ([Ca2+]i) was estimated from the ratio of emission at 410 nm to emission at 485 nm, which was converted to [Ca2+]i by use of calibration parameters determined in vivo, as previously described (8). The time course of relaxation and [Ca2+]i decline was evaluated by the respective half-time values (t0.5-R and t0.5-Ca).

The SR Ca2+ content was estimated as the peak increase in cytosolic total Ca2+ concentration ([Ca2+]T) evoked by 10 mM caffeine in 0 Na+-0 Ca2+ solution (11), which yields estimates comparable to those obtained by integration of the extracellular Na+-dependent inward current evoked by millimolar caffeine (28). Passive Ca2+-buffering parameters previously determined in ventricular rat myocytes (13) and measured [Ca2+]i were used for [Ca2+]T estimation, with the assumption that intracellular indo 1 concentration was 20 µM. The fraction of the SR Ca2+ content released during a twitch (fractional Ca2+ release) was directly estimated according to Bassani et al. (5, 7). Briefly, after determination of steady-state SR Ca2+ content at 0.5 Hz, electrical stimulation was resumed for 5 min to reload the SR, and the cell was incubated with 5 µM thapsigargin (Calbiochem, San Diego, CA) in 0 Na+-0 Ca2+ solution for 90 s to irreversibly block the SR Ca2+-ATPase. Then Tyrode solution was switched on, a single twitch was evoked, and the remaining SR Ca2+ load was determined. The fractional release thus corresponds to the amount of Ca2+ lost from the SR during the twitch as a percentage of the steady-state SR Ca2+ load. Data from cells in which thapsigargin failed to completely abolish further SR Ca2+ reloading were discarded.

Relaxation-associated Ca2+ fluxes carried by the SR Ca2+ pump, NCX, and the slow transporters (sarcolemmal Ca2+-ATPase plus mitochondrial Ca2+ uniporter) were calculated as described elsewhere (6, 9) from the [Ca2+]i decline phase of different types of transients: 1) a caffeine-evoked transient in 0 Na+-0 Ca2+ solution (SR Ca2+ accumulation and NCX were inhibited), 2) an electrically evoked twitch after thapsigargin treatment (SR uptake was inhibited), and 3) a control twitch (all transporters were functional). Total Ca2+ flux (time derivative of [Ca2+]T) was considered the sum of the fluxes carried by the transporters that were functional at each type of transient. Empirical kinetic parameters of these transporters were estimated for each cell, with the flux mediated by a given transporter considered to be equal to Vmax/{1 + (Km/[Ca2+]i)n}, where Vmax is the maximal velocity of transport, Km is the [Ca2+]i at which velocity is half-maximal, and n is the Hill coefficient. In this model, we assume that Ca2+ transport by each system depends only on its kinetic parameters and [Ca2+]i. The estimated Ca2+ flux carried by each pathway was integrated over 1 s after the peak of the twitch Ca2+ transient and expressed as micromolar (i.e., micromoles of Ca2+ per liter of nonmitochondrial cell water).

To investigate diastolic SR Ca2+ loss, the amplitude of caffeine-evoked contractures in 0 Na+-0 Ca2+ solution was taken as an index of the SR Ca2+ content before (steady state) and after prolonged diastole (postrest). A steady-state contracture was evoked immediately after interruption of stimulation at 0.5 Hz for 5 min. After caffeine washout followed by electrical stimulation for 5 min, the cells were rested in Ca2+-free solution for 3 min (11) and then exposed again to caffeine.

Concentration-effect curves to isoproterenol (Iso) were determined at 0.5-Hz stimulation during simultaneous measurement of cell shortening and [Ca2+]i. After stabilization of the inotropic response at each Iso concentration, the cells were rested for 1 min, when the frequency of spontaneous contractions was recorded. The maximal responses to the agonist and the negative logarithm of the molar Iso concentration that evoked half-maximal responses (pD2) were obtained by nonlinear curve fitting.

Solutions. The composition of Tyrode solution was as follows (mM): 140 NaCl, 6 KCl, 1.5 MgCl2, 1 CaCl2, 5 HEPES, and 11 glucose (pH 7.4 at 23°C). In the 0 Na+-0 Ca2+ solution, LiCl and EGTA replaced NaCl and CaCl2, respectively. Iso-HCl stock solution contained 1 mM ascorbic acid. Thapsigargin and indo 1 were dissolved in DMSO. Unless otherwise indicated, all reagents were obtained from Sigma Chemical (St. Louis, MO).

Data analysis. Values are means ± SE, unless otherwise indicated. Most comparisons were made by two-way analysis of variance. When, in addition to the type of surgery and postsurgical period, a third factor was involved (i.e., extracellular [Ca2+] or stimulation rate), a three-way analysis of variance followed by Bonferroni's test was used. mRNA data were compared by the Mann-Whitney U test. Percent contributions of transporters to relaxation were transformed to arcsinFormula (where p is the fractional percentage) for normal distribution before statistical analysis, then means and limits of the 95% confidence interval were converted back to percentages. Differences were considered statistically significant at P < 0.05. Prism 4.0 (Graphpad Software, San Diego, CA) was used for nonlinear curve fitting and most of the statistical tests.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Data in Table 1 show that aortic stenosis significantly increased (P < 0.001) diastolic and systolic arterial pressures upstream from the clamp (carotid artery), whereas pressure downstream from the clamp (femoral artery) was comparable to carotid pressure in sham-operated rats. The systolic pressure gradient (~40 mmHg) was stable until 7 days after surgery. Heart rate was similar in all groups.


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  		Table 1. Pressure overload and LV hypertrophy

 
Left ventricular hypertrophy, identified as a significant increase in left ventricular wall mass relative to the right ventricle wall and whole body mass, was absent on the second day, but evident on the seventh day after aortic constriction (Table 1), when a significant increase in myocyte width (P = 0.019 for surgery-time interaction), but not length, was observed, as typical of PO (3). With the assumption that myocyte thickness was unchanged, volume was estimated as ≥30% greater in PO-7d than in Sh-7d myocytes, which could account for most of the left ventricular growth.

Enhanced Ca2+ mobilization during ECC. Figure 1 depicts representative twitch Ca2+ transients and contractions recorded from PO and sham myocytes at steady-state stimulation (0.5 Hz). Mean contraction and Ca2+ transient amplitude ({Delta}[Ca2+]i), as well as t0.5-R and t0.5-Ca, are shown in Table 2. Although differences in peak shortening did not attain statistical significance, {Delta}[Ca2+]i was increased by 20% in PO-2d (P < 0.01 for surgery vs. time interaction), but not in PO-7d, cells. A significant interaction (P < 0.01) was also observed for diastolic [Ca2+]i, which was decreased in hypertrophied myocytes, but not in PO-2d cells.


Figure 1
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  		Fig. 1. Twitch in myocytes from pressure-overloaded (PO) ventricles. A: representative steady-state Ca2+ transients {intracellular Ca2+ concentration, ([Ca2+]i)} at 0.5 Hz in rat ventricular myocytes at days 2 and 7 after aortic constriction (PO-2d and PO-7d) or sham surgery (Sh-2d and Sh-7d). B: superimposed traces of cell shortening [percentage of resting cell length (RCL)]. C: superimposed Ca2+ transients in PO and Sh myocytes. Dashed lines, traces from Sh groups.

 

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  		Table 2. Ca2+ transient, contraction, and Ca2+ fluxes during a twitch

 
Greater t0.5-R values (Table 2, Fig. 1B) were observed only in hypertrophied cells (P < 0.001, surgery-time interaction), even though t0.5-Ca was unaffected, as seen in hypertrophied myocytes from spontaneously hypertensive rats (SHR) (54). Thus prolonged relaxation in PO-7d cells was probably due to changes in the Ca2+-myofilament interaction and/or cell mechanical properties [e.g., upregulation of beta-myosin heavy chain and greater cytoskeletal stiffness and viscosity caused by increased microtubule density (24, 58)], rather than slower cytosolic Ca2+ removal.

The PO vs. sham differences observed in Ca2+ transients at 0.5 Hz were maintained over a broad range of stimulation rates (0.1–2 Hz). Although diastolic [Ca2+]i increased with frequency (P < 0.001), it was consistently lower in PO-7d cells (P < 0.001; Fig. 2A). Ca2+ transient amplitude showed a small, but significant (P < 0.05), rate-dependent increase that was more evident in PO-2d cells (P < 0.01; Fig. 2B). The [Ca2+]i decline during relaxation was accelerated by increasing rate (P < 0.001), as typical of rodent myocytes and possibly due to enhancement of SR Ca2+ uptake associated with Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity (34, 62). This response was similar in all groups (Fig. 2C). The differences between groups during changes in stimulation rate were maintained when extracellular [Ca2+] ([Ca2+]o) was decreased to 0.5 mM. Additionally, the positive inotropic effect of increasing [Ca2+]o from 0.5 to 2 mM was comparable among groups (not shown).


Figure 2
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  		Fig. 2. Effect of stimulation rate on Ca2+ transients: diastolic [Ca2+]i (Dia [Ca2+]i, A), transient amplitude ({Delta}[Ca2+]i, B), and half time for [Ca2+]i decline (t0.5-Ca, C) at different stimulation rates in Sh-2d, PO-2d, Sh-7d, and PO-7d myocytes. Values are means ± SE (n = 6).

 
Fractional SR Ca2+ release during a twitch was enhanced in PO compared with sham cells (P < 0.01 for surgery only; Fig. 3A), independently of the postsurgery period (P = 0.462 for surgery-time interaction). A similar pattern of change was observed for the SR Ca2+ content, which was increased in PO cells (P < 0.01 for surgery only, P = 0.848 for surgery-time interaction; Fig. 3B). Thus, considering both changes, one can conclude that the amount of Ca2+ released from the SR during a twitch is markedly increased in the early phases of hypertrophy development.


Figure 3
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  		Fig. 3. Enhanced sarcoplasmic reticulum (SR)-cytosol Ca2+ cycling in early PO. A: fractional SR Ca2+ release during a twitch [n = 6 (Sh groups) and n = 7 and 5 (PO-2d and PO-7d, respectively)]. B: SR Ca2+ content in ventricular myocytes from PO [PO-2d (n = 47) and PO-7d (n = 45)] and sham groups [Sh-2d (n = 65) and Sh-7d (n = 62)]. C: integrated Ca2+ fluxes mediated by SR Ca2+-ATPase (SR) and Na+/Ca2+ exchanger (NCX) during twitch relaxation calculated from average [Ca2+]i values and transporter kinetic parameters in each group.

 
Increased SR Ca2+ uptake during relaxation. Available evidence indicates that, at steady-state activity, Ca2+ fluxes during ECC and relaxation are fairly balanced across the sarcolemma and the SR membrane (16, 60). Thus one would expect that enhanced systolic SR Ca2+ release in PO myocytes would be accompanied by increased SR Ca2+ uptake during relaxation. To investigate this possibility, we estimated the individual Ca2+ fluxes and the relative contributions of the SR and NCX to the total integrated Ca2+ flux associated with twitch relaxation. SR-mediated Ca2+ flux was increased in PO compared with sham myocytes (P = 0.044 for surgery only, P = 0.782 for surgery-time interaction), whereas neither the NCX-mediated flux nor the relative contributions of SR and NCX to cytosolic Ca2+ removal was significantly changed (Table 2, Fig. 3C). Ca2+ mobilization during a twitch (estimated as total integrated flux in Table 2, i.e., sum of fluxes carried by SR, NCX, and slow transporters) was greater in PO cells (P = 0.031 for surgery only, P = 0.715 for surgery-time interaction). Therefore, the increase in Ca2+ cycling between SR and cytosol during ECC and relaxation demonstrates enhanced SR function before and during development of ventricular hypertrophy.

The functional changes in Ca2+ cycling in hypertrophied myocytes were not accompanied by a significant alteration in NCX1, RyR2, CSQ, PLB, and SERCA2a mRNA levels relative to beta-actin mRNA, although there was a trend toward an increase in SERCA2a and NCX mRNA abundance in PO-7d cells (0.10 > P > 0.05). Unchanged message levels of SERCA2 and RyR 5 days after aortic banding have also been reported by Anger et al. (2). However, the SERCA2a-to-PLB mRNA ratio was ~2.5-fold greater in PO-7d than in Sh-7d cells (P < 0.05; Fig. 4), which is consistent with the functional data that indicate increased SR Ca2+ uptake.


Figure 4
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  		Fig. 4. Increased SR Ca2+-ATPase (SERCA2a)-to-phospholamban (PLB) mRNA ratio in PO. Left ventricular levels of mRNA species that code for proteins involved in Ca2+ cycling: SR Ca2+ release channel (RyR2), calsequestrin (CSQ), phospholamban (PLB), SR Ca2+-ATPase (SERCA2a), and Na+-Ca2+ exchanger (NCX). mRNA levels were normalized to that of beta-actin determined in the same preparation. Points represent data from individual hearts: Sh-7d ({circ}) and PO-7d ({triangleup}). Horizontal lines, median for each group. *P = 0.036 (Mann-Whitney U test).

 
Increased diastolic SR Ca2+ loss. Increased SR Ca2+ load leads to greater SR Ca2+ release during systole (7, 52, 60) and diastole (10, 53). Our present results point out a greater amount of Ca2+ stored in the SR and released during systole in myocytes from PO rats. To ascertain whether diastolic SR Ca2+ release was also affected, we analyzed SR Ca2+ depletion at rest, during which cells were perfused with Ca2+-free medium. In this condition, stimulation of NCX-mediated Ca2+ efflux unmasks the time-dependent SR Ca2+ loss in the rat ventricle, which is otherwise not apparent because of reuptake of the leaked Ca2+ (11). SR Ca2+ loss was assessed by the ratio of the amplitude of postrest to steady-state caffeine contractures. The analysis of variance revealed that this ratio was lower in PO than in sham cells (P = 0.01 for surgery alone, P = 0.151 for surgery-time interaction; Fig. 5, A and B), which indicates greater diastolic SR Ca2+ leak.


Figure 5
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  		Fig. 5. Greater SR Ca2+ loss in early PO. A: typical cell shortening of myocytes from Sh-2d (dashed traces) and PO-2d (solid traces) rats during caffeine-evoked contractures after steady-state stimulation (Cf1) and 3 min of rest in Ca2+-free solution (Cf2). B: Cf2-to-Cf1 amplitude ratio. Values are means ± SE [n = 10 (Sh-2d and PO-2d), 8 (Sh-7d), and 9 (PO-7d)]. C: rate of spontaneous contractions at rest as a function of extracellular Ca2+ concentration ([Ca2+]o). Values are means ± SE; n = 11 (Sh-2d and PO-2d), 14 (Sh-7d), and 12 (PO-7d).

 
Because Ca2+ overload and greater diastolic SR Ca2+ release may underlie spontaneous activity in the myocardium (10, 16, 37), we also analyzed the rate of spontaneous contractions at different [Ca2+]o. As shown in Fig. 5C, spontaneous activity during rest was enhanced by an increase in [Ca2+]o (P < 0.001), and, independently of the influence of [Ca2+]o, the rate of spontaneous contractions was significantly greater in PO than in sham cells (P < 0.01 for surgery alone, P = 0.525 for surgery-time interaction).

Increased inotropic sensitivity to beta-adrenoceptor stimulation. Plasma catecholamine levels rise soon after the onset of hypertrophy in aorta-constricted rats (1), and myocardial responsiveness to beta-adrenoceptor activation may be altered at this early stage. Concentration-effect curves to Iso inotropic effects (increase in Ca2+ transient and contraction amplitude) were shifted to the left in PO-7d myocytes (Fig. 6, A and B), with a significant increase in Iso pD2 (P = 0.016 and 0.021 for surgery-time interaction for shortening and [Ca2+]i responses, respectively; Table 3), whereas the maximal inotropic responses to Iso were not significantly altered. When Iso facilitation of spontaneous activity during rest was examined, a significant increase in maximal response was observed in the PO compared with the sham myocytes (P = 0.038 for surgery alone, P = 0.357 for surgery-time interaction; Fig. 6C). The pD2 values for this response were comparable in all groups (P = 0.624 for surgery; Table 3).


Figure 6
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  		Fig. 6. Greater responsiveness to isoproterenol (Iso) in hypertrophied myocytes: inotropic responses to Iso {increase in twitch cell shortening (A) and concurrent Ca2+ transient amplitude ({Delta}[Ca2+]i), B} in ventricular myocytes from sham-operated and PO rats. Responses are expressed as percentage of respective maximal response (Max). C: Iso-induced increase in spontaneous activity during rest. Curve parameters are presented in Table 3.

 

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  		Table 3. Inotropic response to isoproterenol

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main finding of the present study was enhanced SR-cytosol Ca2+ cycling in rat left ventricular myocytes before and at the onset of PO-induced hypertrophic growth. This is, to our knowledge, the first report in which Ca2+ fluxes during ECC and relaxation were examined in intact myocytes early in the development of hypertrophy.

Aortic constriction is a maneuver commonly used to induce PO and ventricular hypertrophy. The resulting increase in blood pressure in the proximal aorta is evident a few minutes after the constriction (47, 59), whereas days are required for development of hypertrophy. Cardiac function may be depressed initially, but it gradually recovers or even improves with the development of hypertrophy (41–43). Increased intracellular Ca2+ cycling was also described in hypertrophied myocytes from nonfailing SHR hearts (54). The presence of a similar change during the onset of left ventricular hypertrophy induced by stepwise imposition of PO suggests that enhanced ventricular Ca2+ cycling might be a response to increased hemodynamic load, rather than a characteristic of that particular genetic model of hypertension.

Our results show enhanced ECC during the first week after aortic constriction. Inotropic and lusitropic responses to changes in stimulation rate and [Ca2+]o in cells from PO and sham groups were qualitatively comparable, which indicates preservation of contractile reserve, in contrast to the blunted response to rapid pacing in failing, hypertrophied myocardium (30) and apparently reduced ECC efficiency at low [Ca2+]o in chronic, compensated, PO-induced hypertrophy (36).

Before development of hypertrophy (PO-2d group), twitch Ca2+ transient amplitude was greater, apparently because of markedly increased fractional SR Ca2+ release. Higher peak [Ca2+]i in this group would favor SR Ca2+ uptake during relaxation as a result of a substrate concentration-dependent increase in enzyme velocity. Accordingly, the estimated Ca2+ flux mediated by the SR Ca2+-ATPase during relaxation was also greater, which demonstrates enhanced SR-cytosol Ca2+ cycling. Despite the indication of greater Ca2+ fluxes between SR and cytosol also in PO-7d myocytes, twitch Ca2+ transient amplitude was not increased. Stronger Ca2+ buffering due to greater abundance of myofibrillar proteins (3) would not be a likely explanation, inasmuch as changes in Ca2+ buffering capacity do not seem to accompany ventricular hypertrophy (44). Nevertheless, systolic [Ca2+]i variation might be attenuated because of the small, but significant, decrease in diastolic [Ca2+]i in PO-7d cells. Lower [Ca2+]i implies that less Ca2+ would be bound to buffers during diastole, and the greater availability of Ca2+ binding sites might increase the efficiency of Ca2+ buffering during systole, even if buffering capacity remained unchanged. On the basis of Ca2+ buffering data previously obtained in ventricular rat myocytes (13), we estimate that the 15% decrease in diastolic [Ca2+]i in PO-7d cells would allow additional buffering of ~7–10 µM total Ca2+ during systole without a change in the free Ca2+ transient amplitude. Additionally, enhanced SR Ca2+ uptake might also limit a possible increase in the Ca2+ transient due to increased SR Ca2+ release (5, 6).

Increase in SR Ca2+ load and the trigger for SR Ca2+ release (i.e., Ca2+ current) may augment fractional Ca2+ release (7, 52). Although some authors have found greater Ca2+ influx during a longer action potential in PO (14), shortening or no change in action potential duration has also been reported in this condition (26). In our present experiments, a major alteration in Ca2+ influx seems unlikely for the following reason: if we consider that Ca2+ efflux during relaxation (mostly via NCX) matches systolic Ca2+ influx at steady-state stimulation (16, 60), the lack of increase in the estimated NCX-mediated Ca2+ flux during twitch relaxation indicates that systolic Ca2+ influx should be comparable in PO and sham cells. Previous estimates of Ca2+ efflux via NCX with the present approach have shown agreement with direct measurement of Ca2+ influx by Ca2+ current integration (6, 16, 21). Thus it is likely that an increase in SR Ca2+ content is the main mechanism responsible for enhanced fractional SR Ca2+ release in PO myocytes. Greater SR Ca2+ loading in other models of ventricular hypertrophy has been attributed to an increase in NCX-mediated Ca2+ influx during the action potential (19, 55), rather than enhanced SR function. In our model, however, experimental findings did not suggest major changes in total Ca2+ influx and/or NCX function.

Additional RyR regulation by endogenous signaling molecules might further contribute to the increase in fractional SR Ca2+ release in PO cells. Interestingly, larger Ca2+ transient and spark amplitude, without concomitant changes in L-type Ca2+ current and SR Ca2+ content, were observed in myocytes from SHR with compensated hypertrophy and attributed to greater efficacy of the trigger signal to elicit SR Ca2+ release (54). Increased sensitivity to Ca2+-releasing agonists in RyR isolated from PO hearts has also been described (32). Activation of Ca2+-calmodulin-dependent enzymes has been considered an important step in hypertrophy signaling (34, 62). Ventricular CaMKII {delta}-isoform expression and activity are increased as soon as 1 day after aortic banding and remain high for 7 days after banding (20, 62). This enzyme has been shown to positively modulate Ca2+ release from the SR (34, 61, 62). It is tempting to speculate whether the CaMKII enzyme would play a role in the stimulation of SR Ca2+ release in the early phase of PO.

Increased SERCA2 expression and in vitro activity have been described during the onset of mild hypertrophy due to aortic stenosis (4, 42, 43). These changes were implicated in the enhancement of left ventricular function, which may accompany the development of compensated hypertrophy (41–43). Here we present experimental evidence of greater SR Ca2+ uptake in intact PO-7d cells, namely, increased SR-mediated Ca2+ transport during relaxation, despite unchanged Ca2+ transient amplitude. The SERCA-to-PLB ratio has been considered the main determinant of SR Ca2+-ATPase activity (18). Our finding of an increased SERCA2a-to-PLB mRNA ratio in the PO-7d group, although not informative in terms of actual protein levels, is in agreement with functional data that indicate greater SR Ca2+ uptake in this group. Enhanced SR Ca2+ pump function at the onset of hypertrophy might be the mechanism underlying lower diastolic [Ca2+]i in PO-7d myocytes. Ito et al. (30) observed no change in the SERCA2a-to-PLB mRNA ratio 1 mo after aortic ligation, when hypertrophy was still compensated. Thus an increase in this ratio may represent an early, transient adaptive response to PO.

An intriguing observation is that hypertrophied myocytes were supersensitive to the inotropic effects of the beta-adrenoceptor agonist Iso. Diminished beta-adrenoceptor density and responsiveness are common findings during chronic cardiac hypertrophy associated with hypertension (25). However, in early PO, beta-adrenoceptor density apparently remains unaltered (1). In our study, supersensitivity to Iso was observed 1 wk after aortic ligation, when increases in plasma norepinephrine concentration and ventricular epinephrine content have been reported (1, 56). Inasmuch as supersensitivity to Iso and epinephrine due to augmented efficiency of beta2-adrenoceptor signaling was reported during adaptation to stress (12), it is possible that a similar change might account for the greater beta-adrenergic responsiveness in hypertrophied myocytes. Although further studies are necessary to investigate this possibility, the present findings indicate increased inotropic reserve at the onset of hypertrophy, which is in sharp contrast to the impairment of beta-adrenergic cascade signaling in the failing heart (25).

Spontaneous contractions are thought to arise from greater diastolic Ca2+ release from an overloaded SR, which may give rise to delayed afterdepolarizations and arrhythmia due to the generation of depolarizing membrane current by electrogenic Ca2+ extrusion via NCX (10, 37, 45). The higher rate of spontaneous contractions and SR Ca2+ loss during rest in both PO groups may be associated with a greater propensity to arrhythmia during the PO acute phase, especially under conditions that favor Ca2+ overload, such as increased [Ca2+]o and beta-adrenoceptor stimulation. In the case of Iso-induced enhancement of spontaneous activity, an increase in the apparent efficacy (but not potency) of the agonist was observed in both PO groups, probably due to greater diastolic SR Ca2+ leak. However, in the PO-7d group, in which the pD2 values for Iso inotropic effects were increased, the ratio of the concentration required for 50% of maximal proarrhythmic and inotropic effects to that required for inotropic effects was markedly greater than in the other groups (~18 vs. 4). As a result, considerable enhancement of contractility might be achieved at levels of beta-adrenergic mediators that are not high enough to be arrhythmogenic.

It is conceivable that enhanced diastolic SR Ca2+ loss and systolic fractional release may stem from common mechanisms, such as increased SR Ca2+ content (7, 10, 53) and, possibly, RyR modulation by other signaling molecules, such as CaMKII, which may stimulate diastolic and systolic SR Ca2+ release (61, 62). In addition to SR Ca2+ overload, enhanced NCX function can be a predisposing factor for myocardial spontaneous activity. Accordingly, enhanced susceptibility to arrhythmia has been attributed to SR Ca2+ overload and/or augmented arrhythmogenic currents in experimental models of ventricular hypertrophy and heart failure in which NCX function is upregulated (37, 45, 55). In the present study, however, there was no indication of significant NCX upregulation. This, associated with enhanced SR Ca2+ uptake, may attenuate the potentially arrhythmogenic effects of increased diastolic SR Ca2+ release (23).

The present study presents some limitations, such as the measurement of unloaded cell shortening vs. developed force under mechanical load and the temperature at which the experiments were carried out (23°C), which precluded the use of physiological stimulation rates. However, Puglisi et al. (46) demonstrated that the relative balance among Ca2+ transporters in ventricular myocytes is similar at 25°C and 35°C, although Ca2+ transients and contractions differ in amplitude and time course. Another limitation is that mRNA levels were used to investigate gene expression of proteins involved in Ca2+ transport. It has been shown that changes in mRNA levels may or may not be paralleled by changes in abundance and/or function of the respective protein (33, 36). Nevertheless, the functional changes observed in the PO-7d group (i.e., greater contribution of SR Ca2+ uptake during twitch relaxation and lower diastolic [Ca2+]i), in which a greater SERCA2a-to-PLB mRNA ratio was detected, are in agreement with parallelism of message and protein levels.

In summary, our results show that aortic ligation evokes a myocardial response characterized by enhanced SR-cytosol Ca2+ cycling, even before the onset of hypertrophy. This very early response may be important for maintenance of cardiac output and also may be implicated in the activation of Ca2+-dependent pathways involved in development of hypertrophic growth (14–16, 34, 62), although at the cost of greater propensity to arrhythmia because of augmented diastolic SR Ca2+ release. These changes are likely to be exacerbated in vivo because of stretch-dependent effects on Ca2+ homeostasis exerted by the high wall stress. At the onset of hypertrophy, greater SR Ca2+ uptake (possibly due to higher SERCA2a-to-PLB expression ratio) may help preserve systolic and diastolic functions by maintaining the SR Ca2+ load at a high level and decreasing diastolic [Ca2+]i, respectively, while diminishing net SR Ca2+ loss. At this stage, the inotropic response to sympathetic mediators is likely to be enhanced because of greater cell sensitivity to beta-adrenoceptor stimulation. This scenario is the opposite of that reported in heart failure, when SR Ca2+ pump activity and SR Ca2+ content are depressed, net diastolic SR Ca2+ loss is augmented, and beta-adrenergic responses are attenuated (14, 16, 18, 25, 29, 30, 44, 45, 57, 61). Some of these alterations are present during chronic, compensated hypertrophy (25, 35, 36, 57). Thus it would be plausible to assume that the early changes in Ca2+ homeostasis evoked by PO might represent acute adaptive adjustments of myocardial function to cope with the circulatory overload. However, this short-term adaptive response may eventually degenerate into maladaptive changes and deterioration of cardiac function if the circulatory stress persists, because an increase in Ca2+ mobilization may precede cardiovascular decompensation in some models of hypertrophy (14, 54, 55). It remains to be established whether chronically enhanced Ca2+ cycling might contribute to further development of maladaptation.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnólogico (Brazil) Grant 147459/1999-0 and Fundação de Amparo à Pesquisa do Estade de São Paulo Grant 01/11698-1.


   ACKNOWLEDGMENTS
 
We are grateful to Thais H. Theizen and Elizângela S. Oliveira for excellent technical support and to Drs. Michael Porter and Allen M. Samarel for helpful suggestions.

The present results have been presented in part at the 49th Annual Meeting of the Biophysical Society, Long Beach, CA, February 2005 and published in abstract form (Biophys J 88, Suppl 1: 1556, 2005).


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. Bassani, Centro de Engenharia Biomédica, Universidade Estadual de Campinas, Caixa Postal 6040, 13084-971 Campinas, SP, Brazil (e-mail: rosana{at}ceb.unicamp.br)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* B. M. R. Carvalho and R. A. Bassani contributed equally to this study. Back


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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