INTRODUCTION

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VENTRICLES THAT HAVE SURVIVED myocardial infarction (MI) typically undergo myocardial remodeling. Post-MI remodeling is a complex time-dependent process that involves morphological, molecular biological, neurohumoral, and electrophysiological changes. With the focus on electrophysiological alterations, the two most consistent abnormalities that have been described in association with myocyte hypertrophy are prolongation of action potential duration (APD) (16, 23, 28, 29) and reduction of transient outward current (I_to) density (21, 28, 29). Because I_to plays a major role in early action potential (AP) repolarization, it can indirectly influence the amplitude and duration of L-type Ca²⁺ current (I_Ca) and Na⁺/Ca²⁺ exchange current (I_NaCa) during excitation-contraction.

Exercise training instituted after MI has been shown to improve cardiovascular function in humans (1, 11, 12, 14) and animal models (25, 26). We recently showed that high-intensity sprint training (HIST) implemented shortly after MI was effective in attenuating myocyte hypertrophy, shifting myosin heavy chain isozyme distribution toward that observed in sham myocytes, increasing sarcolemmal Na⁺/Ca²⁺ exchange currents and sarcoplasmic reticulum Ca²⁺ contents, improving intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients and sarcoplasmic reticulum Ca²⁺ uptake, and restoring normal myocyte contractility (34, 35, 39). The present study was undertaken to evaluate whether HIST, an exercise program that we previously showed to increase cardiac output and maximal stroke volume (SV_max) in rats with chronic MI (25) as well as reverse selected cellular maladaptation after MI (34, 35, 39), would shorten the prolonged APD and improve depressed I_to associated with myocyte hypertrophy.

	METHODS

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Animal preparation and exercise-training protocol. Male Sprague-Dawley rats (~250 g) were randomly divided into three groups: sham-sedentary (Sham-Sed), MI-sedentary (MI-Sed), and MI with HIST (MI-HIST). To induce MI, the left main coronary artery of each anesthetized (3% halothane-97% O₂), intubated, and ventilated rat was ligated 3-5 mm distal to its origin from the ascending aorta. In our previous studies (26) employing similar coronary ligation techniques, left ventricular (LV) infarct size, as determined by histological measurement, averaged 36 ± 3%. Sham operation, except that the coronary artery was not ligated, was identical to MI. All surviving rats received rat chow and water ad libitum and were maintained on a 12:12-h light-dark cycle. Two weeks after operation, all rats were introduced to the treadmill (0° grade, 10 m/min, 10 min/day, 5 days/wk) to acclimatize for 1 wk. MI-Sed and Sham-Sed rats continued at the same speed and degree of incline twice per week for another 7-9 wk until they were killed. For the entire training period, the training protocol consisted of five consecutive 1-min running bouts daily, 5 days/wk, and each running bout was interspersed with 1 min of rest. During the 1st wk of training (week 4 after MI), treadmill speed was set at 66 m/min and grade was set at 15°. During the 2nd wk of training, 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 previously showed that this HIST regimen resulted in significant increases in cardiac output and SV_max in post-MI rats (25). 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 after operation), rats were anesthetized with pentobarbital sodium (50 mg/kg body wt ip); their hearts were then excised for myocyte isolation. Myocytes were isolated from the septum and LV free wall portions of the myocardium, as previously described (4-6, 34-39). The infarct scars in MI hearts were excised before the final enzymatic digestion step. Myocytes 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 electrophysiological measurements (6, 37, 38). Patch-clamp studies were performed within 2-10 h of myocyte isolation. As in our previous studies (34-39), only myocytes that retained elongated shape and sharp cross-striations, adhered to cover glass, and showed no membrane blebs were randomly chosen for electrophysiological measurements.

AP measurements. Whole cell patch-clamp experiments were performed at 29°C and as described by Hamill et al. (15) and adapted by us for cardiac myocytes (37-39). Briefly, the Axopatch-1C patch-clamp amplifier (Axon Instruments, Foster City, CA) and pCLAMP 6.4 software were used. Pipettes were pulled from 1.5-mm-OD tubing (Garner Glass, Claremont, CA). After fire polishing and backfilling, pipette resistances were ~0.8-1.5 M. APs from isolated LV myocytes were recorded using current-clamp configuration at 1.5 times threshold stimulus and 4-ms duration. Pipette solution consisted of (in mM) 140 KCl, 4 MgCl₂, 0.06 CaCl₂, 10 HEPES, 5 potassium EGTA, 3.1 Na₂ATP, and 5 Na₂-creatine phosphate, pH 7.1. External solution consisted of (in mM) 132 NaCl, 5.4 KCl, 1.8 CaCl₂, 1.8 MgCl₂, 0.6 NaH₂PO₄, 10 HEPES, and 10 glucose, pH 7.4.

I_to measurements. To isolate I_to, pipette solution consisting of (in mM) 135 KCl, 1 CaCl₂, 14 EGTA, 10 HEPES, and 5 MgATP, pH 7.1, was used. ATP (5 mM) was included in the pipette solution to block ATP-sensitive outward K⁺ current. Myocytes were bathed in 0.6 ml of temperature (29°C)- and air-equilibrated external solution containing 0.5 mM CdCl₂ to block I_Ca and I_NaCa. Resting membrane potentials were held at 80 mV. I_to was elicited by depolarizing pulses (5 s, from 40 to 60 mV in 10-mV increments) and acquired at a sampling rate of 5 kHz. Myocyte capacitance (C_m) measurements and leakage current subtraction were performed as described previously (37-39). I_to kinetics were determined by least-squares fitting of a two-exponential function [A₁exp (t/₁) + A₂exp(t/₂)], where t is time, ₁ and ₂ are time constants of the fast and slow I_to components (I_to f and I_to s), respectively, and A₁ and A₂ are peak amplitude of I_to f and I_to s, respectively (16, 17, 28, 29). The data for voltage dependence of steady-state I_to activation were fitted to a Boltzmann distribution of the form
where V is the test activation potential, V_h is the test potential eliciting one-half of maximal current (I_max), and k is the slope factor (22). Similarly, voltage dependence of steady-state inactivation of I_to was determined by the classical voltage prepulse protocol of Tillotson (32), the interpulse duration between the prepulse and test pulse (80 to +60 mV) was set at 3 ms, and the data were fitted to a Boltzmann function of the form
where V is the prepulse potential and I_max, V_h, and k have their usual meanings.

Statistics. Values are means ± SE. Significance of differences among the means of C_m was determined by one-way ANOVA. A priori comparison of means of any two groups (Sham-Sed vs. MI-Sed, MI-Sed vs. MI-HIST, Sham-Sed vs. MI-HIST) was then performed by using F tests as tests of significance. In experiments in which I_to measurements were made as functions of experimental group (Sham-Sed vs. MI-Sed vs. MI-HIST), voltage, and location (septum vs. LV free wall), three-way ANOVA was performed to determine significance of difference. A linear model-fitted standard least squares (JMP version 3.3, SAS Institutes, Cary, NC) was used. For analysis of AP measurements, two-way ANOVA (experimental group and location) was used to determine statistical significance. Post hoc paired comparisons were performed between Sham-Sed and MI-Sed, between MI-Sed and MI-HIST, and between Sham-Sed and MI-HIST. In all analyses, P  0.05 was taken to be statistically significant.

	RESULTS

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Animal weight. At the time of death, the weights of the rats in each group were 505 ± 9 g (n = 16), 509 ± 16 g (n = 16), and 505 ± 12 g (n = 15) for Sham-Sed, MI-Sed, and MI-HIST, respectively. There were no statistically significant differences in body weights among the three groups.

Effects of prior MI ±HIST on C_m. We previously demonstrated that myocyte hypertrophy 3 wk after MI was reflected by a 13-15% increase in C_m (37, 38), an estimate of cell surface area. In the present study, C_m in MI-Sed myocytes 11 wk after MI was 24% larger than that in Sham-Sed myocytes (Table 1; P < 0.01). This suggests that myocyte hypertrophy not only persisted but might have progressed during the 9- to 11-wk post-MI period. MI-induced hypertrophy was significantly (P < 0.01) attenuated by HIST. In addition, there were no significant (P = 0.16) differences between mean C_m values of Sham-Sed and MI-HIST myocytes.

Effects of prior MI ±HIST on AP. We recorded AP in myocytes isolated from the septum and LV free wall (Fig. 1). Two-way ANOVA (group and location) indicated no statistically significant differences in resting membrane potential and AP amplitude among Sham-Sed, MI-Sed, and MI-HIST myocytes isolated from septum or LV free wall (Table 2). AP amplitudes, however, were significantly (P = 0.007) higher in LV free wall myocytes than in septal myocytes across all three groups. In addition, APD at 50% repolarization (APD₅₀) was significantly (P = 0.0015) longer in myocytes isolated from septum than in myocytes isolated from LV free wall, but no such differences were detected in APD at 90% repolarization (APD₉₀). APD₅₀ and APD₉₀ were longest in MI-Sed myocytes (Table 2). Two-way ANOVA confirmed a significant (P = 0.0001) group effect (Sham-Sed vs. MI-Sed vs. MI-HIST), but the lack of significant group × location interaction effect indicated that myocyte origin (septum vs. LV free wall) did not affect the inherent differences in APD₅₀ and APD₉₀ values among Sham-Sed, MI-Sed, and MI-HIST myocytes.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   High-intensity sprint training (HIST) restores action potential (AP) duration (APD) in myocytes subjected to myocardial infarction (MI) toward normal. APs in myocytes isolated from the septum (A) and left ventricular (LV) free wall (B) from sham-operated-sedentary (Sham-Sed), MI-Sed, and MI-HIST groups were measured as described in METHODS. Data are summarized in Table 2.

Effects of prior MI ±HIST on I_to amplitudes and decay. I_to was acquired from septal and LV free wall myocytes. After reaching peak amplitude, I_to decayed with apparently fast (I_to f) and slow (I_to s) phases. Decay of I_to was fitted by two exponentials: A₁ and ₁ for I_to f and A₂ and ₂ for I_to s (see METHODS). Figure 2 shows the A₁ and A₂ voltage-current density relationship of I_to for Sham-Sed, MI-HIST, and MI-Sed myocytes isolated from the septum (A and C) and LV free wall (B and D). Three-way ANOVA indicates significant group (P = 0.0001), voltage (P = 0.0001), and group × location × voltage interaction (P < 0.0004) effects for peak current densities for I_to f (A₁) and I_to s (A₂). Post hoc analysis indicates that I_to f (A₁) and I_to s (A₂) were significantly (P = 0.0001) lower in MI-Sed than in Sham-Sed myocytes. HIST was able to restore I_to in MI myocytes toward normal, although not to the same levels observed in Sham-Sed myocytes (Fig. 2; P < 0.007, Sham-Sed vs. MI-HIST).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Current-voltage relationships of transient outward current (Ito) in Sham-Sed, MI-Sed, and MI-HIST myocytes isolated from septum (A and C) and LV free wall (B and D). Peak amplitudes of fast (Ito f, A and B) and slow (Ito s, C and D) components of Ito at each test potential were measured from current traces. Values are means ± SE. Error bars are not shown if they fall within boundaries of symbol. A: Sham-Sed (n = 11), MI-Sed (n = 13), and MI-HIST (n = 16) myocytes. B: Sham-Sed (n = 9), MI-Sed (n = 11), and MI-HIST (n = 8) myocytes.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Kinetics of Ito inactivation. Ito traces were fitted by a 2-exponential function (see METHODS). Time constants for Ito f and Ito s (1 and 2, respectively) are shown as a function of test voltage. A and C: septum. B and D: LV free wall.

Effects of prior MI ±HIST on steady-state inactivation and activation parameters of I_to. To further evaluate voltage-dependent properties of I_to in Sham-Sed, MI-Sed, and MI-HIST myocytes, steady-state inactivation of I_to was determined using the classical two-pulse protocol (32). In this series of experiments, we did not separately evaluate myocytes isolated from septum and LV free wall. Figure 4 shows the sigmoidal relationships between the extent of inactivation (f = I/I_max) and prepulse potential for the three groups of myocytes (Sham-Sed, MI-Sed, and MI-HIST) for I_to f (A, C, and E) and I_to s (B, D, and F). Least-square fitted values for V_h and k are shown in Table 3. There are no statistically significant differences in steady-state inactivation parameters of I_to f and I_to s among the three groups.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Steady-state activation and inactivation of Ito in Sham-Sed, MI-Sed, and MI-HIST myocytes. Voltage dependence of activation (squares) and inactivation (circles) of Ito f (A, C, and E) and Ito s (B, D, and F) in Sham-Sed (A and B), MI-Sed (C and D), and MI-HIST (E and F) myocytes at 1.8 mM extracellular Ca2+ and 29°C are shown. Steady-state activation data were derived from experiments depicted in Fig. 2. Steady-state inactivation of Ito was evaluated by a classic double-pulse experiment (see METHODS). Values are means ± SE. Error bars are not shown if they fall within boundaries of symbol. Data are summarized in Tables 3 and 4. I, current.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Time course of recovery from inactivation of Ito f and Ito s in Sham-Sed, MI-Sed, and MI-HIST myocytes. Two identical pulses [from 80 to +60 mV, 600-ms duration for Ito f (A) and 2,500-ms duration for Ito s (B)] separated by interpulse intervals of increasing duration were delivered to the myocytes. The initial pulse should maximally activate Ito (Imax). The current (I) during the 2nd pulse was measured. I/Imax reflects recovery of Ito from maximal channel activation and is plotted for Ito f (A) and Ito s (B) as a function of interpulse intervals. Data were fitted to a cumulative gamma function and analyzed as previously described (24). Results are summarized in Table 5. , MI-HIST; +, MI-Sed; , Sham-Sed.

	DISCUSSION

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The most consistent electrophysiological abnormality in association with cardiac hypertrophy is prolongation of APD (16, 23, 28, 29). Focusing on the rat post-MI model, Thollon et al. (30) were the first to report prolongation of APD in post-MI rat ventricular myocytes. Prolongation of APD was subsequently reaffirmed in 3- to 4-wk (28) and 16-wk (29) post-MI rat myocytes, 8-wk post-MI rabbit myocytes (20), and 5-day post-MI canine myocytes (21). Our present results on APD prolongation in post-MI rat myocytes (Fig. 1, Table 2) are in agreement with findings previously reported by other investigators (28-30). Prolongation of APD might be an important compensatory mechanism to increase Ca²⁺ influx in post-MI myocytes during the AP. In 3-wk post-MI rat myocytes, we previously demonstrated that I_NaCa (38), but not I_Ca (37), was depressed. By prolonging the plateau phase of the AP (Fig. 1), more Ca²⁺ may enter via reverse Na⁺/Ca²⁺ exchange, thus partially compensating for the depressed I_NaCa in 3-wk MI myocytes. Theoretically, additional Ca²⁺ influx in post-MI myocytes might also occur via I_Ca during the prolonged plateau of the AP. This is less likely in view of the fact that there were no differences in inactivation time constants in I_Ca between Sham and MI myocytes (37) and that the time constants for Ca²⁺-induced inactivation of I_Ca (32) were of much smaller amplitude (6-8 ms) (37) than the APD (60-120 ms; Table 2). On the other hand, prolongation of APD may be one of the mechanisms for development of ventricular tachyarrhythmias in the hypertrophied ventricle (28). Viewed in this context, APD prolongation in post-MI myocytes may be regarded as a maladaptation with potentially fatal consequences. Thus the first major finding of our present study is that HIST instituted shortly after MI was able to significantly shorten the prolonged APD (Fig. 1, Table 2). In addition, the beneficial effects of HIST were applicable to myocytes proximal (LV free wall) and distal (septum) to the infarct.

I_to is a critical determinant in the early stage of AP repolarization and, thus, affects APD. The results of Qin et al. (28) indicate that the prolonged APD of post-MI rat ventricular myocytes could be explained by decreases in I_to f and I_to s. With few exceptions (19), I_to density has been reported to be lower in hypertrophied myocytes. This is especially the case in hypertrophied myocytes isolated from post-MI hearts (21, 28, 29). Our present findings that the amplitudes of I_to f and I_to s were lower in MI-Sed myocytes than in Sham-Sed myocytes (Fig. 2) are consistent with previous reports by other investigators (21, 28, 29). Thus the second major finding of our study is that HIST was able to restore I_to (both fast and slow) in MI myocytes toward normal (Fig. 2). This important finding may underlie the ionic basis of shortened APD in post-MI myocytes by HIST.

There are many explanations for the observed reduction in I_to and its restoration toward normal by HIST in the post-MI myocytes. At the level of a single myocyte, modulation of I_to may be mediated by changes of molecular conformation, pH, ionic milieu, glucose oxidation, channel phosphorylation, and channel protein expression. Conformation changes of the I_to channel molecule appear unlikely to be the mechanism by which HIST enhanced I_to in post-MI myocytes, because kinetics of I_to inactivation (Fig. 3), steady-state activation and inactivation parameters (Fig. 4), and time-dependent recovery from inactivation (Fig. 5) were similar among Sham-Sed, MI-Sed, and MI-HIST myocytes. Modulation of pH and ionic compositions is also an unlikely mechanism by which HIST improved I_to in post-MI myocytes in vivo, since the beneficial effects of HIST on I_to were observed when the intracellular environment was controlled by dialysis with the pipette solution under whole cell patch-clamp conditions.

Another potential mechanism for I_to enhancement in post-MI myocytes by HIST is modulation of phosphorylation state of the I_to channel molecule (8, 27). Because exercise training has been shown to increase adenylate cyclase activity (3), it is reasonable to hypothesize that HIST improves I_to density in post-MI hearts by enhancing channel phosphorylation, possibly through increased adenylate cyclase activity.

In rat myocardium, Kv1.4 and Kv4.2 are candidate channel clones for the cardiac I_to current (2, 10), although in terms of mRNA expression, function, and pharmacology, the Kv4.2 channel much more closely resembles rat and human I_to than does Kv1.4 (9). In addition, protein expression of the Kv1.4 channel was not detected in rat hearts (2). In myocytes isolated from 3-wk MI rats, Gidh-Jain et al. (13) reported that Kv4.2, Kv1.4, and Kv2.1 (putative I_to s channel) mRNAs, as well as Kv4.2 and Kv2.1 protein levels, were significantly decreased compared with controls. HIST may improve I_to density by simply ameliorating myocyte hypertrophy (reduction in C_m; Table 1) without changing I_to channel expression, thereby increasing the ratio of I_to channel number to unit membrane surface area, by enhancing transcription and translation of Kv4.2 and Kv2.1 channels, or by reducing degradation/turnover of these two protein molecules.

Finally, there are a number of important limitations to this study. The first limitation concerns the intensity of our sprint training program. Initial studies utilizing chronic endurance training of low to moderate intensity (26) did not result in improvement in LV pump function in post-MI rats. By contrast, when HIST was instituted shortly after MI was induced in the rat, cardiac output and SV_max increased compared with MI sedentary rats (25). Indeed, 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 a substantial increase in maximal cardiac output, without worsening hemodynamic status or causing further myocardial damage (11). In another clinical study involving 29 patients with prior MI, improvement in cardiac function, at rest and during exercise, was noted only in the high-intensity training group (1). The second limitation relates to the fact that we did not examine the effects of HIST on APD and I_to in Sham myocytes. Exercise training has been known to prolong the duration of ventricular AP plateau in adult rats (31), whereas the ionic basis for the prolongation is not known. Although it is of considerable interest to examine potential changes in APD and I_to density in normal myocytes by HIST, our previous (34, 35, 39) and present focus continues to be the elucidation of cellular mechanisms by which HIST improved global myocardial contractile function in post-MI rats (25). It is interesting to point out that the "dual" effects of exercise training on cardiac APD, physiological prolongation in normal myocytes (31) and reversal of pathological prolongation in post-MI myocytes (Fig. 1, Table 2), are reminiscent of the dual effects of exercise training on cardiac myocyte size: physiological hypertrophy in normal myocytes (24) and regression of pathological hypertrophy in post-MI myocytes (34, 35, 39) (Table 1). The third limitation concerns the well-documented regional variation of I_to in the heart of several species, including rats (7) and humans (18, 33). Although we separately measured I_to in LV myocytes isolated from the septum (distant to infarct) and LV free wall (close to infarct) and demonstrated that AP amplitude was significantly lower and APD₅₀ significantly longer in septal myocytes (Table 2), we did not further separate myocytes on the basis of their epi- or endocardial origins. Because it is known that transmural heterogeneity to AP morphology and I_to properties exists (2, 18), some of the quantitative changes in I_to density reported in the present study may theoretically be explained by inadvertent "selection" of subepicardial myocytes (8 times higher Kv4.2 expression than subendocardial myocytes) (2) in one group (e.g., Sham-Sed myocytes). We believe that this is unlikely, inasmuch as the same selection (e.g., more subepicardial myocytes in Sham-Sed group vs. more subendocardial myocytes in MI-Sed myocytes) would have to consistently occur in septal and LV free wall populations. Thus, although our data involving increased density of I_to f and I_to s in MI-HIST myocytes (compared with MI-Sed myocytes) represented pooled results for all myocytes across the septum or LV free wall, our primary conclusion that HIST shortened APD in post-MI myocytes by increasing I_to density remains valid.

In summary, we have shown that HIST decreased whole cell capacitance and shortened the pathological prolongation of APD in myocytes from rat hearts with a moderate-sized LV infarct. We hypothesize that normalization of APD in post-MI myocytes by HIST was likely mediated by restoration of the depressed I_to density toward normal.