INTRODUCTION

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EXERCISE TRAINING-INDUCED improvements in intrinsic cardiac contractile function in normal hearts have been well established (22, 27, 28). When instituted after myocardial infarction (MI), exercise training has also been shown to exert beneficial effects on cardiovascular function in both humans (11, 13) and animals (23, 24). Focusing on cellular mechanisms that may contribute to exercise training-induced enhancements in cardiac function, our laboratory has recently shown in rats that a program of high-intensity sprint training (HIST) instituted shortly after MI was effective in attenuating myocyte hypertrophy, restoring myosin heavy chain isoenzyme distribution toward normal, increasing Na⁺/Ca²⁺ currents (I_Na/Ca), normalizing intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients during excitation-contraction, improving sarcoplasmic reticulum (SR) Ca²⁺ uptake, and enhancing SR Ca²⁺ content (30, 35).

At the level of a single cell, surviving myocytes isolated from hearts post-MI demonstrated either increase (18, 33), decrease (6, 15, 17, 20, 33), or no change (3, 6, 18, 33) in maximal myocyte contraction amplitudes. The discrepancies in results reported by different investigators may relate to differences in animal species, infarct sizes, experimental conditions [temperature, extracellular Ca²⁺ concentration ([Ca²⁺]_o), and pacing frequency], and extent of left ventricular (LV) remodeling post-MI. In a previous study, we characterized contraction abnormalities in myocytes isolated from rat hearts 3 wk post-MI (33). Under conditions that preferentially favored Ca²⁺ efflux over influx (low [Ca²⁺]_o and high pacing frequency), MI myocytes shortened more than those from rats that had received sham operations. Conversely, under conditions that favored Ca²⁺ influx (high [Ca²⁺]_o and low pacing frequency), Sham myocytes shortened to a greater extent than MI myocytes. At intermediate [Ca²⁺]_o and pacing frequencies, differences in steady-state contractile amplitudes between Sham and MI myocytes were no longer significant. These observations were consistent with the hypothesis that both Ca²⁺ influx and efflux pathways were subnormal in MI myocytes and that they contributed to abnormal myocyte contraction. Of the major Ca²⁺ influx and efflux pathways in our 3-wk rat MI model, only I_Na/Ca (36), but not Ca²⁺ current (I_Ca; 32), was depressed in MI myocytes. Because HIST was effective in improving I_Na/Ca and [Ca²⁺]_i dynamics in MI myocytes (30, 35), the present study was undertaken to evaluate whether HIST would restore contractile behavior of MI myocytes toward normal.

	METHODS

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Animal preparation and exercise-training protocol. MI was induced in male Sprague-Dawley rats (~300 g) by techniques previously described in detail (6, 23, 24, 30-36). The 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 (526 ± 13 g, n = 15) continued to walk on the treadmill at low intensity (0° grade, 10 m/min, 10 min/day, Mondays and Thursdays) (Sham-Sed) for another 7-9 wk before death. At 3 wk postoperation, MI rats were randomly assigned to either a sedentary (MI-Sed; 524 ± 12 g, n = 13) or training (MI-HIST; 492 ± 8 g, n = 13) group. Animal weights were measured at time of death. 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, 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.

Myocyte isolation. After 6-8 wk of training (9-11 wk postoperation), cardiac myocytes were isolated separately from the septum and LV free wall by methods previously described in detail (7, 8). Isolated myocytes were seeded onto laminin-coated coverslips in 2 ml of medium 199 (pH 7.4, 95% air-5% CO₂, 37°C) for 2 h before contraction experiments. Myocytes were randomly chosen for experiments from among those that retained rod-shape and sharp cross striations, adhered to cover glass, and showed no membrane blebs.

Myocyte shortening measurements. Myocytes adherent to coverslips were bathed in 0.6 ml of air- and temperature-equilibrated (37°C), HEPES-buffered (20 mM, pH 7.4) medium 199 containing either 0.6 or 5.0 mM [Ca²⁺]_o and were placed on a temperature-controlled stage (37°C) of a Zeiss IM35 inverted microscope (6, 29, 33). Measurements of myocyte contractile activity were made between 2 and 6 h after isolation, because overnight culture (without continuous pacing) resulted in significant slowing of cell-shortening dynamics (29). Fields of myocytes were chosen at random, and myocytes were field stimulated to contract between platinum wire electrodes spaced 2 mm apart, as previously described (6, 22, 29, 33). Stimulus conditions were either 0.6 mM [Ca²⁺]_o and 2 Hz or 5.0 mM [Ca²⁺]_o and 0.1 Hz. For brevity, only the value of [Ca²⁺]_o was explicitly mentioned throughout the text, with the understanding that 0.6 mM [Ca²⁺]_o was always paired with 2 Hz, and 5.0 mM [Ca²⁺]_o with 0.1 Hz. These conditions were chosen to maximize the contractile differences between Sham and MI myocytes (6, 33). It should be noted that, in our rat MI model, there were no contraction amplitude differences between Sham and MI myocytes when paced at [Ca²⁺]_o of 1.8 mM at 0.1-5 Hz (33). Myocytes viewed through an Olympus DApoUV ×40/1.30 numerical aperature oil objective were imaged by a charge-coupled device video camera (Ionoptix, Milton, MA), and myocyte length, width, and motion measurements were acquired by a personal computer with interface and software purchased from Ionoptix. Data were permanently stored on a zip drive (Iomega, Roy, UT) and analyzed off-line by Ionoptix software. For calibration of pixels vs. micrometers, a high-resolution test target (model 22-8635, Ealing Electro-Optics, Natick, MA) was used.

Statistics. All results are expressed as means ± SE. In analyses of a parameter (e.g., contraction amplitude) as a function of group (Sham-Sed, MI-Sed, MI-HIST), location (septum, LV free wall), and [Ca²⁺]_o (0.6, 5.0 mM), three-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, P  0.05 was taken to be statistically significant.

	RESULTS

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Effects of MI ± HIST on surviving cardiac myocyte sizes. For all three groups, there were no differences in cell lengths or cell widths measured in myocytes isolated from the septum (distant to infarct) or LV free wall (proximal to infarct) (insignificant location and group × location interaction effects, Table 1). Two-way ANOVA indicates significant differences in mean cell lengths (P < 0.0001) but not mean cell widths (P = 0.16) among the three groups (Table 1). When cell length data were analyzed without regard to location (composite in Table 1), MI-Sed myocytes were significantly (P < 0.0001) longer (by 14%) than Sham-Sed myocytes. Six to eight weeks of HIST instituted 3 wk post-MI significantly (P < 0.0001) reduced cell length, although not back to values measured in Sham-Sed myocytes (Table 1).

Effects of MI ± HIST on myocyte motion dynamics. Figure 1 shows representative steady-state twitches in Sham-Sed, MI-Sed, and MI-HIST myocytes, from which maximal contraction amplitude (% resting cell length), time required to reach one-half maximal contraction amplitude (time to 1/2 peak), maximal shortening (dL/dt) and relengthening (+dL/dt) velocities, and half time of relaxation (t_1/2) can be calculated. To maximize the differences in contractile behavior between Sham-Sed and MI-Sed myocytes, experimental conditions were chosen to favor either Ca²⁺ influx over efflux pathways (5.0 mM [Ca²⁺]_o, 0.1 Hz) or vice versa (0.6 mM [Ca²⁺]_o, 2 Hz) (22, 33). Data are summarized in Table 2. Across the three groups, there were no differences in any of the measured contraction parameters between myocytes isolated from the septum and those isolated from LV free wall (three-way ANOVA demonstrates lack of location or group × location effects). Therefore, it is reasonable to simplify our data analysis by removing myocyte origin (septum vs. LV free wall) from further consideration.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   High-intensity sprint training (HIST) improves contractile performance in myocytes from rats with induced myocardial infarction (MI). Isolated myocytes were paced (0.1 Hz) to contract at 37°C and extracellular Ca2+ concentration ([Ca2+]o) of 5 mM. Shown are steady-state paced twitches of myocytes from sham-operated (Sham)-sedentary (Sed) (A), MI-Sed (C), and MI-HIST (E) rats and time-expanded views of steady-state twitches from which peak contraction amplitude, time required to reach half maximal contraction amplitude (time to half peak), shortening velocity (dL/dt), relengthening velocity (+dL/dt), and half time of relaxation were measured for Sham-Sed (B), MI-Sed (D), and MI-HIST (F) myocytes. Note that HIST restored peak contraction amplitude in MI myocytes to that observed in Sham-Sed myocytes. Results are summarized in Table 2.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Contraction parameters in Sham-Sed, MI-Sed, and MI-HIST myocytes. Maximal contraction amplitude (max contract ampl), time to 1/2 peak, maximal dL/dt, maximal +dL/dt, and half time of relaxation are measured at 0.6 mM [Ca2+]o and 2 Hz (A, C, E, G, and I) and at 5.0 mM [Ca2+]o and 0.1 Hz (B, D, F, H, and J). Data for all myocytes are presented, without regard to location (septal vs. left ventricular free wall) because there was no location difference in any of the contraction parameters among the 3 groups by 3-way ANOVA. After significant differences were detected by 2-way ANOVA, post hoc analysis was performed to compare Sham-Sed vs. MI-Sed, MI-Sed vs. MI-HIST, and Sham-Sed vs. MI-HIST myocytes. %RCL, percent resting cell length. *P < 0.0002, Sham-Sed vs. MI-Sed; P < 0.03, MI-Sed vs. MI-HIST.

	DISCUSSION

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There are major differences among the various MI models utilized by various investigators to study excitation-contraction abnormalities in surviving myocytes, depending on the animal species, infarct sizes, experimental conditions, and time of recovery from acute MI (2, 3, 6, 15-18, 20, 25, 30-36). This wide spectrum of changes was also observed in failing human hearts, in which at least two different phenotypes existed at both ends of the spectrum: systolic dysfunction alone vs. systolic and diastolic dysfunction (14). To facilitate discussion of the mechanisms by which HIST may improve MI myocyte contractile performance, it would be fruitful to summarize known excitation-contraction abnormalities in our rat MI model. Compared with Sham myocytes, in MI myocytes' action potential duration was prolonged (37), I_Ca characteristics were unchanged (32), I_Na/Ca was depressed (36) although there were no detectable changes in Na⁺/Ca²⁺ exchange protein levels (35), SR Ca²⁺ uptake was lower concomitant with lower levels of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2; Ref. 34), SR Ca²⁺ content was reduced (36), myosin heavy chain isoenzyme shifted from V₁ to V₃ pattern (24, 35), and contractile performance was impaired (6, 33). It should be noted that many of our findings in the rat MI model have been independently corroborated by the work of Afzal and Dhalla (2). It should also be pointed out that the phenotypic manifestations (decreased SERCA2 but no change in Na⁺/Ca²⁺ exchange protein levels) of our rat MI model are consistent with those observed in group III failing human hearts as defined by Hasenfuss et al. (14), in which both systolic and diastolic dysfunction were present (in our case, lower contraction amplitude and longer t_1/2, Fig. 2, B and J).

Compared with Sham-Sed myocytes, at high [Ca²⁺]_o and low pacing frequency, MI-Sed myocytes contracted ~30% less. In addition, there was significant prolongation of contraction, both in terms of time to 1/2 peak and half time of relaxation. In 6-wk MI rats, the time to peak tension measured in the noninfarcted LV posterior papillary muscle was prolonged compared with that in sham-operated rats (19). The prolonged time to 1/2 peak might be due to continued transsarcolemmal Ca²⁺ influx (via I_Ca and reverse I_Na/Ca) because of prolonged action potential duration, prolonged SR Ca²⁺ release, depressed SR Ca²⁺ uptake, or reduced Ca²⁺ extrusion by forward Na⁺/Ca²⁺ exchange. The prolonged action potential duration in MI-Sed myocytes (18, 37) might be a mechanism to allow more Ca²⁺ entry via reverse Na⁺/Ca²⁺ exchange during the plateau phase of the action potential. This interpretation received indirect support in that, when pacing frequency was increased to 2 Hz with proportional shortening of action potential duration, the time to 1/2 peak was no longer different between Sham-Sed and MI-Sed myocytes. This simplistic interpretation must be viewed in the context that during a cardiac action potential, there are complicated interactions between I_Ca and I_Na/Ca and that it is extremely difficult to fully characterize these two (and other) currents during an action potential. A prolonged t_1/2 may be explained by reduced SR Ca²⁺ uptake, decreased Ca²⁺ extrusion by forward Na⁺/Ca²⁺ exchange, and lower myosin ATPase activity in MI-Sed myocytes. It is interesting to note the important role Na⁺/Ca²⁺ exchange played in both contraction and relaxation in diseased myocytes, as has been recently demonstrated in failing human myocytes (9, 12, 14).

Our study is the first to demonstrate that a program of exercise training instituted shortly after recovery from acute MI was effective in improving cardiac contractile performance at the level of a single myocyte. Concerns about the clinical appropriateness and safety of high-intensity exercise training post-MI have been previously addressed, both in humans (1, 10) and animal models (23, 30, 35). Focusing on the rat MI model, we have previously demonstrated that 6-8 wk of HIST effected significant increases in cardiac output and maximal stroke volume in post-MI hearts (23). Although systemic factors (central cardioregulatory and peripheral vascular adaptations, and so forth) likely contributed to HIST-induced improvements in cardiac performance in post-MI rats, cellular adaptations to HIST may be equally important in improved myocardial function. Indeed, HIST has been shown to exert beneficial effects on multiple cellular events involved in excitation-contraction coupling in post-MI myocytes, including but not limited to restitution of normal isoenzyme distribution of contractile proteins, restoring normal action potential duration, enhancing I_Na/Ca, normalizing [Ca²⁺]_i transients during a twitch, improving SR Ca²⁺ uptake, and increasing SR Ca²⁺ contents (30, 35, 37).

In our present study, we did not detect significant differences in cell lengths, cell widths, and contraction parameters between myocytes isolated from septum and those from LV free wall in the three groups studied (lack of location effect; see Tables 1 and 2). In a previous study from our laboratory, whole cell capacitance measurements also failed to support myocyte origin (septum vs. LV free wall) as an important determinant in capacitance values in MI myocytes (30). Our findings are in agreement with those of Lefroy et al. (15) but not with those of Olivetti et al. (25). In any event, lack of location and group × location interaction effects in all contraction parameters examined makes it reasonable to discuss our present data without reference to location differences.

The observation that MI-Sed myocytes were ~14% longer than Sham-Sed myocytes but cell widths were similar between the two groups (Table 1) was in agreement with our previous report (6). Other investigators also reported that increases in cell lengths were larger than increases in cell widths in post-MI myocytes (3, 18, 20, 25), a characteristic consistent with volume-overload hypertrophy (4). The first major finding is that HIST was effective in attenuating cellular hypertrophy in MI-Sed myocytes, primarily by decreasing cell lengths with no effects on cell widths (Table 1). It is interesting to speculate that, by directly modulating cell hypertrophy in postinfarction myocytes, HIST may minimize ventricular dilation post-MI. Indeed, 6 wk of graduated swim training attenuated LV dilation by ~30% in post-MI rat hearts (26). It is also interesting to note the "dual" effects of exercise training on cardiac myocyte size: physiologic hypertrophy in normal myocytes (21, 22) and regression of pathologic hypertrophy in post-MI myocytes (30, 35; Table 1).

The second major finding is that HIST was effective in restoring normal contraction amplitude in post-MI myocytes stimulated at 5.0 mM [Ca²⁺]_o and 0.1 Hz. Under these experimental conditions, HIST has been shown to increase I_Na/Ca, systolic [Ca²⁺]_i, SR Ca²⁺ uptake, and SR Ca²⁺ content in post-MI myocytes (30, 35). Positive influences by HIST on all these cellular mechanisms involved in excitation-contraction coupling may enhance contractile performance in post-MI myocytes. On the other hand, at 0.6 mM [Ca²⁺]_o and 2 Hz, HIST did not attenuate the "supranormal" contraction amplitude observed in MI-Sed myocytes (Fig. 2A). This may be due to the fact that the major Ca²⁺ efflux pathway, I_Na/Ca, was not restored to normal levels by HIST (35); that Na⁺/Ca²⁺ exchange assumed a more important role in contraction and relaxation in diseased myocytes (12, 14); that HIST normalized the isoenzyme distribution patterns (and, by inference, contractile efficiency) of contractile proteins (23, 35); or that exercise training increased the Ca²⁺ sensitivity of myofilaments (22), which could be subnormal in MI myocytes (16), although the last point is controversial (3).

The time to reach one-half maximal contraction amplitude was prolonged in MI-Sed myocytes (Fig. 2D), similar to the findings in surviving myocytes isolated from healing canine infarcted hearts (14) and 6-wk MI rats (19). HIST was effective in reducing time-to-1/2 peak in MI myocytes, probably by shortening action potential duration (37) and enhancing SR Ca²⁺ uptake (30) and sarcolemmal Ca²⁺ extrusion by Na⁺/Ca²⁺ exchange (35). In this light, it should be pointed out that improvement in I_Ca by HIST in MI myocytes is an unlikely mechanism for reducing time to 1/2 peak shortening. Our laboratory has previously shown that the intrinsic properties of I_Ca were not different between Sham and MI myocytes (32) and that endurance training (albeit not HIST) did not modify I_Ca characteristics in running rats (21).

Another important finding of the present study is that HIST shortened t_1/2, which was prolonged in MI-Sed myocytes (Fig. 2J). Longer t_1/2 in MI-Sed myocytes, compared with Sham-Sed myocytes, was expected in view of previous findings that SR Ca²⁺ uptake (2, 34), SR CaATPase expression (34), and Na⁺/Ca²⁺ exchange activity (36) were reduced in MI rat myocytes. Improvement in SR Ca²⁺ uptake (30) and forward Na⁺/Ca²⁺ exchange (35) in MI-Sed myocytes by HIST may contribute to the reduction of t_1/2. This simple interpretation must be tampered by the fact that, in rabbit, rat (5), and canine (17) cardiac myocytes, relaxation was complete well before the end of the [Ca²⁺]_i transient. For example, in rat myocytes stimulated at 1.0 mM [Ca²⁺]_o and 22°C, t_1/2 from a twitch was 0.08 ± 0.01 s, whereas the time constant of [Ca²⁺]_i transient decline was 0.194 ± 0.010 s (5). Under our experimental conditions of 5.0 mM [Ca²⁺]_o and 37°C, t_1/2 from a twitch was 120 ± 3 ms (Fig. 2J) but half time of [Ca²⁺]_i decline was 197 ± 5 ms (30). The fact that myocyte relaxation was complete well ahead of [Ca²⁺]_i transient suggests that mechanisms in addition to enhanced SR Ca²⁺ uptake and forward Na⁺/Ca²⁺ exchange contribute to improved myocyte mechanical relaxation.

Finally, there are a number of important limitations to this study. The first is that, compared with Sham myocytes, surviving myocytes isolated from our rat MI model did not exhibit significant contraction amplitude differences when stimulated at "physiological" [Ca²⁺]_o of 1.1-1.9 mM and at 0.1-5 Hz (6, 33). Absence of significant differences in twitch amplitudes between Sham and rat MI myocytes was also reported by Anand et al. (3). However, contraction abnormalities in our rat MI myocytes could clearly be demonstrated under high [Ca²⁺]_o and low pacing frequencies and also at low [Ca²⁺]_o and high pacing frequencies (33; Fig. 2). We interpreted the complex pattern of contraction abnormalities to be consistent with the notion that Ca²⁺ influx and efflux pathways were reduced in MI myocytes. In view of this, together with our laboratory's previous observation that HIST improved Na⁺/Ca²⁺ exchange activity (which affects both Ca²⁺ influx and efflux) in MI myocytes (35), in the present study we purposefully examined the potential beneficial effects of HIST on MI myocyte contraction under conditions that favored Ca²⁺ influx over efflux (high [Ca²⁺]_o and low pacing frequency) and vice versa (low [Ca²⁺]_o and high pacing frequency). The second limitation relates to the fact that we did not examine the effects of HIST on Sham myocytes. Although it is of considerable interest to delineate potential changes in excitation-contraction coupling in normal myocytes by HIST, previous (30, 35) and present focus by our laboratory continues to be the elucidation of cellular mechanisms by which HIST improved global myocardial contractile function in post-MI rats (23). The third limitation concerns the fact that in the study of contraction abnormalities of single myocytes isolated from MI hearts, it is difficult to ascertain for each individual myocyte how close to or far away from the infarcted area it was. Theoretically, surviving myocytes close to the infarct may be subjected to greater hemodynamic stress during recovery and may thus exhibit different cellular adaptation from those distant from the infarct. As a first approach, we separately examined myocytes isolated from the septum (spared and distant from infarct) and the LV free wall (close to infarct). There were no differences in myocyte dimensions and contraction parameters between myocytes isolated from septum and those from LV free wall in the three groups studied (Tables 1 and 2). The fourth limitation is that there were no direct measurements of [Ca²⁺]_i transients in myocytes from the three experimental groups. In a previous study (30), our laboratory has shown that, at 5.0 mM [Ca²⁺]_o, HIST was effective in restoring the depressed systolic [Ca²⁺]_i and lowering the elevated diastolic [Ca²⁺]_i of MI-Sed myocytes toward those observed in Sham-Sed myocytes. Thus the observed improvements in contraction amplitudes by HIST in the present study may be related to its beneficial effects on Ca²⁺ homeostasis in MI myocytes.

In summary, we have demonstrated that HIST attenuated cellular hypertrophy post-MI, primarily by reduction in cell lengths with little effect on cell widths. In our rat MI model, which exhibited both systolic and diastolic dysfunction, HIST was effective in improving many of contraction parameters measured in MI myocytes in the unloaded state. We hypothesize that enhanced Na⁺/Ca²⁺ exchange activity, reduced action potential duration, and improved SR Ca²⁺ uptake may be some of the more important mechanisms responsible for improved contractile performance by HIST in post-MI myocytes.