INTRODUCTION

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EXERCISE TRAINING INSTITUTED after myocardial infarction (MI) has been shown to improve cardiovascular function in both humans (13, 17) and animal models (31, 32). The cellular mechanisms underlying these training-induced improvements in cardiac function post-MI are beginning to be elucidated (52). Focusing on the 3-wk MI rat model, one sees that myocytes surviving MI exhibited cellular hypertrophy (6, 49-51, 53), shift of myosin heavy chain (MHC) isoenzyme distribution from fast to slow isoforms (32, 52), depressed sarcolemmal (SL) Na⁺/Ca²⁺ exchange (11, 53) and Na⁺-K⁺-ATPase activities (10), reduced dihydropyridine-binding sites (9, 49) and -adrenergic responsiveness (48, 49), and decreased sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) 2 expression (47, 51) and sarcoplasmic reticulum (SR) Ca²⁺ contents (53). Functionally, surviving myocytes isolated from infarcted left ventricles (LV) had altered intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients during a twitch (6, 48, 51), depressed SR Ca²⁺ uptake manifested as prolonged half-time (t_1/2) of [Ca²⁺]_i decline (51), and contraction abnormalities (6, 27, 50). It is important to recognize that cardiac hypertrophy models induced by physiological (exercise training) or pathological (pressure overload, postinfarction, rapid pacing, thyrotoxicosis) stimuli, although sharing many similar cellular alterations, have fundamentally distinct functional and phenotypic manifestations. For example, myocyte hypertrophy associated with renovascular hypertension was affected primarily by increases in cell width (45), whereas myocytes isolated from post-MI hearts exhibited predominantly increases in cell length compared with sham controls (6, 35, 50). In addition, in hypertensive cardiac hypertrophy induced in rats by aortic banding, both SL Na⁺-dependent Ca²⁺ uptake and Ca²⁺-ATPase activities were significantly increased (33) rather than decreased as observed in 3- to 4-wk postinfarction rat myocytes (11, 51, 53). Even within the postinfarction model, there are substantial differences in results reported by different investigators for MI myocytes (6, 24, 27, 50). For example, Na⁺/Ca²⁺ exchange has been reported to decrease in the 3- to 4-wk rat MI model (11, 53) but increase in the 8-wk rabbit MI model (26). The discrepancies may relate to differences in species, infarct size, experimental conditions {temperature, extracellular Ca²⁺ concentration ([Ca²⁺]_o), stimulation frequency}, presence or absence of overt LV failure at time of myocyte isolation, myocyte selection (proximal or distal to infarct), and extent of ventricular remodeling after MI (reviewed in Ref. 50). It is important, when comparing studies on MI myocytes reported by different investigators, that the above-listed confounding factors be taken into account.

Some of the MI-induced cellular maladaptations may potentially be reversed by exercise training. For example, in normal hearts, exercise training shifted MHC isoenzyme distribution from slow to fast forms (37), increased SL Na⁺-K⁺-ATPase activities (21), enhanced both Ca²⁺ influx and efflux pathways (29), enhanced calmodulin-stimulated SR Ca²⁺ uptake (25), and improved myocyte contractile performance (29). Indeed, in a recent study, a program of high-intensity sprint training (HIST) instituted shortly after MI was effective in attenuating myocyte hypertrophy, shifting MHC isoenzyme distribution toward that observed in sham myocytes, and increasing SL Na⁺/Ca²⁺ exchange currents and SR Ca²⁺ contents (52).

SR Ca²⁺ transport systems occupy important roles in cardiac excitation-contraction coupling. With depolarization, influx of extracellular Ca²⁺ via L-type Ca²⁺ channels and reverse Na⁺/Ca²⁺ exchange (34) triggers SR Ca²⁺ release, with resultant increases in [Ca²⁺]_i and myofilament activation. During diastole, most of the myoplasmic Ca²⁺ is resequestered in SR by SERCA 2, with a small amount extruded by forward Na⁺/Ca²⁺ exchange and, to a minor extent, by SL Ca²⁺-ATPase (51). The SR Ca²⁺ content determines not only the maximal amount of Ca²⁺ available for release but also the gain of SR Ca²⁺ release channels (43). We have previously demonstrated that, in addition to decreased SR filling by Ca²⁺ influx, the lower SR Ca²⁺ content in post-MI myocytes (53) was due to reduced SR Ca²⁺ uptake and SERCA 2 expression, but not to enhanced SR Ca²⁺ leak (51). The present study was undertaken to evaluate whether HIST, an exercise program that we have previously shown to increase cardiac output and maximal stroke volume in rats with chronic MI (31), as well as reverse selected cellular adaptation post-MI (52), would improve SR Ca²⁺ uptake, enhance SERCA 2 expression, and restore [Ca²⁺]_i dynamics toward normal in post-MI myocytes.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animal preparation and exercise-training protocol. Male Sprague-Dawley rats (~300 g) were anesthetized (3% halothane-97% O₂), intubated, and ventilated. The left main coronary artery was ligated 3-5 mm distal to its origin from the ascending aorta. Survivors typically had 36 ± 3% of LV infarcted, according to our laboratory's previous histological measurements (32). Sham operation was identical except that the coronary artery was not occluded. All surviving rats received rat chow and water ad libitum and were maintained on a 12:12-h light-dark cycle. At 2 wk postoperation, all rats were started on the treadmill (0° grade, 10 m/min, 10 min/day, 5 days/wk; Precision Biomedical Systems, State College, PA). At 3 wk postoperation, sham-operated rats (511 ± 10 g; n = 22) continued to walk on the treadmill [0° grade, 10 m/min, 10 min/day, Mondays and Thursdays; sedentary (Sham-Sed)] for another 7-9 wk before death. At 3 wk postoperation, MI rats were randomly assigned to either a training (MI-HIST; 494 ± 7 g, n = 19) or a sedentary (MI-Sed; 513 ± 7 g, n = 22) group. MI-Sed rats participated in the same treadmill-walking program as Sham-Sed rats for 6-8 wk before they were killed. During the first week of training (week 4 post-MI), MI-HIST rats ran five consecutive 1-min bouts daily, 5 days/wk, and each running bout was interspersed with 60 s of rest. Treadmill speed and grade were set at 66 m/min and 15°, respectively. During the second week of training (week 5 post-MI), treadmill speed was progressively increased to 97 m/min. The treadmill grade and speed were then held constant for the remainder of the training period. We have previously shown that HIST was effective in improving cardiac function post-MI, both in vivo (31) and at the cellular level (52). Use of HIST also circumvented potential problems with different degrees of exercise stress produced by endurance training.

LV myocyte isolation. After 6-8 wk of training (9-11 wk postoperation), rats were anesthetized with pentobarbital sodium (35 mg/kg body wt ip), and their hearts were excised for myocyte isolation. Myocytes were isolated separately from the septal and LV free wall as previously described (6, 7). The infarct scars in MI hearts were excised before the final enzymatic digestion step. Myocytes isolated from either the septum or LV free wall were allowed to adhere for 2 h to laminin-coated coverslips in 2 ml of medium 199 (pH 7.4; 95% air-5% CO₂; 37°C) before [Ca²⁺]_i transient measurements (8). Only myocytes that retained rod-shape and sharp cross striations, adhered to the cover glass, and showed no membrane blebs were randomly chosen for experiments.

[Ca²+]_i transient measurements in single myocytes. Freshly isolated myocytes were exposed to 2 µM of fura 2-AM for 15 min at 37°C (6, 8). Fura 2-loaded myocytes mounted in a Dvorak-Stotler chamber were bathed with medium 199 ([Ca²⁺]_o = 5.0 mM; 37°C) buffered with 20 mM HEPES, placed on a temperature-controlled stage (37°C) of a Zeiss IM35 inverted microscope, and field stimulated to contract at 1 Hz between wire electrodes, as previously described (6, 45, 48, 50, 51). Excitation light (360 and 380 nm, ±10-nm band pass; Ionoptix, Milton, MA) was directed to individual myocytes only during data acquisition to minimize photobleaching. Epifluorescence (510 ± 10 nm) collected by a Nikon UV Fluor ×100/1.3 NA oil objective was passed through a pinhole (1.6 mm) and captured by a photomultiplier (Hamamatsu R928-07). Output from the photomultiplier was routed through an amplifier/discriminator (model C609; Thorn EMI, Middlesex, UK) before arrival at a counter/timer board (model C660, Thorn EMI) situated in a PC-compatible computer.

Whole cell capacitance measurements. Whole cell patch-clamp recordings were performed at 29°C, as described by Hamill et al. (18) and adapted by our laboratory for cardiac myocytes (49, 51-53). Cell membrane capacitance (C_m) for each cell was measured by applying a small hyperpolarizing pulse (10 mV, 16 ms) and integrating the resulting current change (digitized at 50 kHz, 0.5-kHz filter) over time (49). Leakage currents were subtracted from current traces by standard techniques by using hyperpolarizing pulses (5 mV × 2).

SERCA 2 and calsequestrin immunoblotting. Myocytes were suspended in 10 mM Tris-1 mM EDTA buffer with protease inhibitor cocktail (100:1; Sigma Chemical) and sonicated for 3 × 15 s. Cell homogenates (45 µg/lane) in SDS sample buffer were subjected to electrophoresis in a 7.5% polyacrylamide gel. Proteins from SDS-PAGE were transferred onto Trans-Blot membranes (Bio-Rad, Hercules, CA). A monoclonal antibody (1:500 dilution) against rat SERCA 2 (MA3-919, Affinity Bioreagents, Golden, CO) was used. Rabbit anti-mouse antibody was used as the secondary antibody. For calsequestrin, rabbit anti-calsequestrin antibody (1:1,000; SWant, Bellinzona, Switzerland) was used. Donkey anti-rabbit IgG (1:2,400; Amersham, Buckinghamshire, UK) was used as the secondary antibody. Membranes were detected with the enhanced chemiluminescence-Western blotting system (Amersham). Protein band signal intensities were quantitated by scanning the blots with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Phospholamban immunoblotting. Myocytes were suspended in 8 M urea buffer containing (in mM) 30 HEPES, 5 EDTA, 5 EGTA, 1 phenylmethylsulfonyl fluoride, 50 NaF, 5 Na⁺ pyrophosphate, 0.001 okadiac acid, 0.4 1-(5-isoquinolinesulfonyl)-2-methylpiperazine HCl (H-7, Calbiochem, La Jolla, CA), and 0.05 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62, Calbiochem) and 1% SDS and were sonicated for 3 × 15 s. These conditions were used to maintain the native state of phospholamban during homogenization (4). Cell homogenates (1 µg/lane) were subjected to electrophoresis in a 15% polyacrylamide gel. Proteins from SDS-PAGE were transferred onto Trans-Blot membranes. A monoclonal antibody (1:3,000) against rat phospholamban (MA3-922, Affinity Bioreagents) was used. Sheep anti-mouse antibody (1:4,000; Amersham) was used as the secondary antibody. Membranes were detected with the enhanced chemiluminescence-Western blotting system, and protein band signals were quantitated.

Statistics. All results are expressed as means ± SE. Significance of differences among the means of groups (Sham-Sed, MI-Sed, MI-HIST) was determined by one-way ANOVA. A priori comparisons of means of any two groups (e.g., MI-Sed vs. MI-HIST) were then performed by using F tests as tests of significance. In analyses of a parameter (e.g., systolic [Ca²⁺]_i) as a function of group (Sham-Sed, MI-Sed, MI-HIST) and location (septum, LV free wall), two-way ANOVA was performed to determine significance of difference. A linear model fitted by standard least squares in a commercial software package (JMP version 3.1, SAS Institute, Cary, NC) was used. In all analyses, a P of 0.05 was taken to be statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effects of MI with and without HIST on cell capacitance. Our laboratory has previously demonstrated that HIST was effective in ameliorating cellular hypertrophy observed in post-MI myocytes (52). In the present study, we used C_m, a cell-size indicator, as a marker for the presence of MI and HIST effects. Similar to our laboratory's previous studies (49, 52, 53), C_m in MI-Sed myocytes was significantly (P = 0.005) larger (by 25%) than that in Sham-Sed myocytes (Table 1). There were no differences in C_m measured in myocytes isolated from the septum (distant to infarct) or LV free wall (proximal to infarct). Six to eight weeks of HIST instituted 3 wk post-MI reduced C_m to values similar to those observed in Sham-Sed myocytes (Table 1).

Effects of MI with and without HIST on [Ca²+]_i dynamics. Figure 1, A, C, and E, shows paced twitches (1 Hz) of Sham-Sed, MI-Sed, and MI-HIST myocytes, respectively, in medium 199 with 5.0 mM [Ca²⁺]_o at 37°C. The higher than physiological [Ca²⁺]_o was chosen to maximize the differences in [Ca²⁺]_i dynamics and contraction abnormalities in our rat MI model (6, 50, 51). Time-expanded views (Fig. 1, B, D, and F) of steady-state twitches allowed systolic and diastolic [Ca²⁺]_i values to be determined from daily intracellular fura 2 fluorescence calibrations. As shown in Table 2, systolic [Ca²⁺]_i was lowest and diastolic [Ca²⁺]_i was highest in MI-Sed myocytes. Two-way ANOVA (Table 3, top and middle) confirmed a significant group effect (Sham-Sed vs. MI-Sed vs. MI-HIST). Across all three groups, myocytes originating from the septum had higher systolic [Ca²⁺]_i (Table 2, top) but not diastolic [Ca²⁺]_i (Table 2, middle) values than those from the LV free wall, as indicated by significant location effect in Table 3, top, but not in Table 3, middle. Neither systolic nor diastolic [Ca²⁺]_i had significant group × location interaction (Table 3, top and middle), indicating myocyte origin (septum vs. LV free wall) did not affect the inherent differences in [Ca²⁺]_i values among Sham-Sed, MI-Sed, and MI-HIST myocytes.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Intracellular Ca2+ concentration ([Ca2+]i) transients in myocytes from sham-operated sedentary rats (Sham-Sed), rats that underwent myocardial infarction and were kept sedentary (MI-Sed), and rats that underwent myocardial infarction and were subject to high-intensity sprint training (MI-HIST). Fura 2-loaded myocytes were paced (1 Hz) to contract at 37°C and extracellular [Ca2+] of 5 mM. A, C, and E: steady-state paced [Ca2+]i transients of Sham-Sed, MI-Sed, and MI-HIST myocytes, respectively. B, D, and F: time-expanded views of steady-state [Ca2+]i transients from which systolic [Ca2+]i, diastolic [Ca2+]i, and half-time (t1/2) of [Ca2+]i decline were measured for Sham-Sed, MI-Sed, and MI-HIST myocytes, respectively. Note the gradual decline in [Ca2+]i and longer t1/2 in MI-Sed myocyte compared with Sham-Sed or MI-HIST myocytes. Composite results are summarized in Table 2.

Effects of MI with and without HIST on SERCA 2 and calsequestrin abundance in cardiac myocytes. To further elucidate the mechanisms by which HIST normalized t_1/2 of [Ca²⁺]_i decline (and by inference, SR Ca²⁺ uptake) in MI myocytes, we performed immunoblots of isolated cardiac myocyte homogenates probed with antibody to SERCA 2 (Fig. 2). We detected a band of an apparent molecular mass of 90-100 kDa, similar to the SERCA 2 cloned from rat hearts (22). Amounts of SR Ca²⁺-ATPase were higher in Sham-Sed myocytes compared with MI-Sed or MI-HIST myocytes (Table 4). One-way ANOVA indicated that there were significant (P < 0.0001) differences in mean SERCA 2 amounts among the three groups. A priori comparisons of means indicated significant differences in SERCA 2 abundance between Sham-Sed and MI-Sed myocytes (P < 0.001), as previously reported (51). What is unexpected is the finding that HIST resulted in a further significant (P < 0.025) decrease in SERCA 2 in MI myocytes (comparing MI-Sed and MI-HIST).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Immunoblots of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2 and calsequestrin. Proteins in myocyte homogenates (45 µg/lane) were separated by gel electrophoresis and transferred to Trans-Blot membranes, and SERCA 2 and calsequestrin were identified by immunoblotting, as described in METHODS. Note that, compared with Sham-Sed myocytes, MI-Sed myocytes had significant decreases in SERCA 2, whereas MI-HIST myocytes exhibited additional downregulation in SERCA 2 expression. There were no differences in calsequestrin expression among the 3 groups. Composite results are summarized in Table 4. Nos. on left, apparent molecular mass.

Effects of MI with and without HIST on phospholamban expression in cardiac myocytes. One possible explanation for the apparent discrepancy that HIST improved SR Ca²⁺ uptake (shorter t_1/2 of [Ca²⁺]_i decline) but decreased SERCA 2 abundance in MI myocytes (Fig. 2) is that HIST enhanced SR Ca²⁺ transport by affecting phospholamban expression and/or phosphorylation state (41). To approach this, we performed immunoblots for phospholamban in isolated myocyte homogenates (Fig. 3). We detected a band of apparent molecular mass of 25-30 kDa, corresponding to the pentameric form of phospholamban (42). There were no quantitative differences in phospholamban between Sham-Sed and MI-Sed myocytes (Table 4). By contrast, MI-HIST myocytes contained significantly less phospholamban compared with either Sham-Sed (P < 0.01) or MI-Sed (P < 0.01) myocytes (Table 4).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Immunoblots of phospholamban. Freshly isolated myocytes were homogenized in 8 M urea buffer containing both protein kinase and phosphatase inhibitors (see METHODS). Proteins in myocyte homogenates (1 µg/lane) were loaded onto a 15% polyacrylamide gel, separated by gel electrophoresis, and transferred. Phospholamban pentamers were identified by immunoblotting (see METHODS). There were no differences in phospholamban protein levels between Sham-Sed and MI-Sed myocytes, but MI-HIST myocytes had significant decreases in phospholamban expression. Composite results are summarized in Table 4. Nos. on left, apparent molecular mass.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

There are several methodological issues that bear discussion before the interpretation of our data. The first concerns the intensity of our sprint training program, especially instituted so shortly post-MI. We chose the HIST regimen because, within a reasonable period (6 wk) of training, it effected a statistically significant maximal stroke volume increase in post-MI hearts (31), whereas 8-10 wk of moderate endurance running only resulted in a trend (P = 0.08) in cardiac output improvement (32). At the cellular level, our laboratory has previously shown that HIST attenuated post-MI cellular hypertrophy and restored the reduced Na⁺/Ca²⁺ exchange current and SR Ca²⁺ content in MI-Sed myocytes toward normal (52). By contrast, endurance running was shown to have little to no effect on L-type Ca²⁺ current (28) and Na⁺/Ca²⁺ exchange activity (23), although the training paradigm was effective in promoting a modest (5-9%) myocyte hypertrophy in normal rats (28, 29). Choice of HIST also circumvents potential problems with different degrees of exercise stress produced by endurance training. In addition, our laboratory's previous (31, 52) and present experiments clearly demonstrated that HIST did not cause harmful or even fatal effects that may occur when rats with moderate-size LV infarcts were subjected to a strenuous exercise program. In this light, it is interesting to note that more recent clinical studies indicate that high-intensity exercise training in men with reduced LV function after MI or coronary artery bypass graft resulted in substantial increases in maximal cardiac output, without worsening hemodynamic status or causing further myocardial damage (12). In another clinical study involving 29 patients with prior MI, improvement in cardiac function, both at rest and during exercise, was noted only in the high-intensity training group (1).

The second methodological issue involves studying separately myocytes isolated from the septum (spared and distant from infarct) and the LV free wall (close to infarct). The rationale is that surviving myocytes close to the infarct may be subjected to greater hemodynamic stress during ventricular remodeling and may thus exhibit different cellular adaptations from those distant from the infarct (36). For example, in rats with infarct sizes (38%) similar to those in present study, Olivetti et al. (35) reported at 4 wk post-MI larger increases in cell volume (43 vs. 20%) and cell length (25 vs. 13%) in myocytes isolated from the LV free wall compared with the septum. By contrast, in rat hearts studied 1 wk post-MI, Lefroy et al. (24) reported that there were no significant differences in cell dimensions between the myocytes isolated close to the infarct and those isolated distant from the infarct; although the basal contraction amplitude was reduced in myocytes from the infarcted region compared with the noninfarcted region. In our present study, we did not detect significant differences in C_m (an index of cell surface area and thus cell size) between myocytes isolated from septum and those from LV free wall in the three groups studied (lack of location effects, Table 1). Systolic [Ca²⁺]_i and t_1/2 of [Ca²⁺]_i decline, however, were consistently different between myocytes isolated from septum and those from LV free wall across the three groups (significant location effect, Table 3). The absence of significant group × location interaction effects (Table 3), however, indicates that myocyte origin (septum vs. LV free wall) did not affect the inherent differences in [Ca²⁺]_i dynamics among Sham-Sed, MI-Sed, and MI-HIST myocytes. Therefore, it is reasonable to discuss our data without regard to location differences.

Our first finding that HIST ameliorated the 26% cellular hypertrophy observed in post-MI myocytes (Table 1) confirmed our laboratory's previous report that HIST was effective in attenuating increases in cell length and C_m in post-MI myocytes (52). Reducing myocyte hypertrophy post-MI by HIST may positively impact on ventricular remodeling and minimize development of dilated cardiomyopathy. The decrease in C_m by HIST in post-MI myocytes in the present study can be used as a "cellular marker" that HIST was similarly effective in modulating post-MI myocyte maladaptations, as in our laboratory's previous studies (52).

Studies with SR membrane fractions (2, 16) and intact myocytes (51) have demonstrated decreased SR Ca²⁺ uptake but not increased SR Ca²⁺ leak (51) in post-MI myocytes. Decreased SR Ca²⁺ uptake affected both the rate of decline of the [Ca²⁺]_i transient and thus myocyte relaxation during diastole, as well as SR-releasable Ca²⁺ content and thus the peak systolic [Ca²⁺]_i and force of contraction. Thus a major finding of the present study is that HIST restored t_1/2 of [Ca²⁺]_i decline and, by inference, SR Ca²⁺ uptake (51) in post-MI myocytes to normal (Tables 2 and 3). Given that there was no difference in L-type Ca²⁺ currents between Sham and MI myocytes (49) and assuming no change in SR Ca²⁺ release channels, the significantly higher systolic [Ca²⁺]_i in MI-HIST myocytes compared with MI-Sed myocytes (Tables 2 and 3) is consistent with the notion that SR Ca²⁺ contents were higher in MI-HIST myocytes. Indeed, direct electrophysiological measurements have shown that HIST was effective in increasing SR Ca²⁺ contents in post-MI myocytes (52).

In our rat MI model, SERCA 2 protein levels decreased (51) but Na⁺/Ca²⁺ exchange protein levels were unchanged (52). These observations are similar to the group III failing human hearts defined by Hasenfuss et al. (20) in which there was disturbed diastolic function as evidenced by a progressive increase in end-diastolic force as pacing frequency was increased. Because there is a highly positive correlation between end-diastolic [Ca²⁺]_i levels and diastolic relaxation abnormalities (39), it is not unexpected that diastolic [Ca²⁺]_i in MI-Sed myocytes was significantly higher than that in Sham-Sed myocytes (Tables 2 and 3; Ref. 48). HIST, by improving both SR Ca²⁺ uptake (present study) and SL Na⁺/Ca²⁺ exchange (52) in post-MI myocytes, resulted in significant lowering of diastolic [Ca²⁺]_i in MI-HIST compared with MI-Sed myocytes (Tables 2 and 3). A surprise finding is that diastolic [Ca²⁺]_i was significantly lower in MI-HIST myocytes compared with Sham-Sed myocytes. One trivial explanation is that the "significant" difference is artifactual and has no physical meaning. A more likely explanation is that, unlike normal myocytes, Na⁺/Ca²⁺ exchange may play a more dominant role in contraction and relaxation in MI myocytes, as has been recently demonstrated in myocytes isolated from end-stage human hearts (14). HIST, by enhancing both Na⁺/Ca²⁺ exchange (52) and SR Ca²⁺ uptake in the abnormal milieu of MI myocytes, may result in further lowering of diastolic [Ca²⁺]_i in MI-HIST myocytes compared with Sham-Sed myocytes. A third possibility is that HIST increased sarcoplasmic Ca²⁺ buffering capacity and/or Ca²⁺ binding to SR and SL. This type of training adaptation would be consistent with both slightly lower systolic (not significant, P = 0.07) and diastolic (significant, P = 0.003) [Ca²⁺]_i in MI-HIST myocytes compared with Sham-Sed myocytes. In this light, it is interesting that Tibbits et al. (44) have demonstrated that Ca²⁺ binding sites in papillary muscle preparations from exercise-trained rats increased by 63% compared with sedentary controls. Penpargkul et al. (38) also reported enhanced binding of Ca²⁺ by cardiac SR of hearts from exercise-trained rats. A fourth speculative possibility to account for lower diastolic [Ca²⁺]_i in MI-HIST myocytes is that exercise training enhanced SL ATP-dependent Ca²⁺ extrusion (40) and/or mitochondrial metabolism (3), thus effectively lowering the "set point" for Ca²⁺ regulation (5) in the trained myocyte. It should be emphasized that Ca²⁺ homeostasis in the cardiac myocyte is very complicated, and it is very difficult to draw unambiguous conclusions unless all possible Ca²⁺ homeostatic pathways and Ca²⁺ sinks are simultaneously evaluated in the three experimental groups.

In the rat MI model, depressed SR Ca²⁺ uptake (2, 51) was generally associated with decreased SERCA 2 mRNA (47) and protein levels (47, 51), although Yue et al. (46) recently reported no changes in both SERCA 2 mRNA and protein levels in rats 1 day to 6 wk post-MI. Our present results that both SR Ca²⁺ uptake function (Fig. 1, Table 2) and SERCA 2 expression (Fig. 2, Table 4) were subnormal in MI-Sed myocytes confirmed our previous results (51) and were in agreement with the results of Zarain-Herzberg et al. (47) and the results in group III failing human hearts of Hasenfuss et al. (20). What is totally unexpected is the finding that, despite improvement in SR Ca²⁺ uptake in MI myocytes by HIST (Fig. 1, Table 2), SERCA 2 expression was further attenuated in MI-HIST myocytes (Fig. 2, Table 4). This observation indicates that improvement in SR Ca²⁺ uptake post-MI by HIST was not due simply to increased SERCA 2 expression.

The activity of SR Ca²⁺-ATPase is modulated through its interaction with phospholamban, a pentamer composed of five identical 6-kDa monomers (42). Dephosphorylated phospholamban binds SERCA 2 and inhibits Ca²⁺ transport activity, primarily by decreasing affinity for Ca²⁺ but also probably by decreasing V_max (41). Phosphorylation of phospholamban blocks the interaction between phospholamban and SERCA 2 and relieves this inhibition. Given the additional decrease in SERCA 2 in MI-HIST myocytes (compared with MI-Sed myocytes), the improvement in SR Ca²⁺ uptake by HIST post-MI could be explained by the reduction in phospholamban expression and/or increases in its phosphorylation state. If one assumes that the stoichiometry of phospholamban to SERCA 2 is a determinant in the level of SR Ca²⁺-ATPase inhibition, then the large decrease in SERCA 2 (73.6%) relative to the decrease in phospholamban (25.2%) in MI-HIST myocytes, compared with Sham-Sed myocytes, suggests that mechanisms other than parallel decreases in phospholamban expression by HIST must be involved to explain the similar SR Ca²⁺ uptake activity between Sham-Sed and MI-HIST myocytes. A likely mechanism is increased phosphorylation of phospholamban by HIST in post-MI myocytes. Although we did not directly address phospholamban phosphorylation in this study, it is interesting to note that exercise training has been shown to increase adenylate cyclase and protein kinase C activities in normal hearts (for review, see Ref. 30).

In summary, we have shown that HIST attenuated cellular hypertrophy and improved [Ca²⁺]_i dynamics and SR Ca²⁺ uptake in myocytes from rat hearts with a moderate-size LV infarct. Improvement in SR Ca²⁺ uptake by HIST post-MI was not mediated by increased SERCA 2 expression, but rather by downregulation of phospholamban expression and possibly increases in its phosphorylation state. We hypothesize that, in post-MI rats, improvements in intracellular Ca²⁺ homeostasis by HIST form the cellular basis of enhancement of cardiac performance.