INTRODUCTION

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MANY STUDIES CONCERNING functional, metabolic, immunologic, and morphological changes in the heart subjected to acute ischemia have been published in the last two decades (37). More recently, attention has shifted to cellular and molecular changes in viable myocytes recovering from chronic coronary occlusion/ligation. Adaptive changes in myocardium surviving an infarction included progressive desensitization to the positive inotropic effects of extracellular Ca²⁺ (9), side-to-side slippage of myocytes (31), increased transverse chamber diameter and decreased capillary density (30), and increases in collagen concentration and mature cross-linking (24). Known cellular and subcellular changes in surviving myocytes subsequent to infarction included cellular hypertrophy (1, 9, 21, 30, 39, 40), altered contractile protein biochemistry (2, 27), depressed sarcolemmal Na⁺/Ca²⁺ exchange (13, 40) and Na⁺-K⁺-ATPase (12) activities, reduced dihydropyridine binding sites (39) and -adrenergic responsiveness (38, 39), and sustained reduction in high-energy phosphate levels (15). Despite almost universal agreement that cardiac performance was compromised in rat hearts that have recovered from moderate infarction (1, 9, 25, 27, 30), the cellular mechanisms underlying the global myocardial dysfunction remain to be clarified.

With the focus on excitation-contraction abnormalities, alterations in intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients during a twitch (9, 22, 38) and contraction abnormalities (9, 18, 20-22, 25) have been observed in myocytes after myocardial infarction (MI). In addition, abnormalities in individual steps involved in excitation-contraction coupling, such as L-type Ca²⁺ current (21), Na⁺/Ca²⁺ exchange (13, 21, 40), sarcoplasmic reticulum (SR) Ca²⁺ content (40), myosin heavy chain isoform composition (27), and myofilament Ca²⁺ sensitivity (20), have been reported in myocytes isolated from postinfarction hearts. It is not clear how much or how little these identified cellular or subcellular defects contributed to MI myocyte contractile abnormalities, let alone contractile dysfunction, in the postinfarction left ventricle (LV). The present study was undertaken to differentially perturb myocyte Ca²⁺ influx and efflux mechanisms and examine their effects on the differences in myocyte shortening characteristics between Sham-treated animals and animals in which MI was induced. The rationale was that if MI caused myocyte contractile abnormalities primarily by affecting Ca²⁺ influx and/or efflux pathways, then steady-state contractile amplitudes of LV myocytes isolated from Sham-operated and 3-wk-postinfarction rat hearts should respond differently to altered pacing frequency and extracellular Ca²⁺ concentration ([Ca²⁺]_o). In addition, relaxation from caffeine-induced contractures was used to indirectly test the functional significance of reduced Na⁺/Ca²⁺ exchange activity previously observed in MI myocytes (13, 40).

	METHODS

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Animal preparation. Male Sprague-Dawley rats (~300 g) were anesthesized with halothane (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. In our previous studies (27) employing similar techniques, LV infarct size, as determined histologically, averaged 36 ± 3%. Three weeks after MI, survivors (466 ± 6 g, n = 36) and Sham-operated rats (454 ± 8 g, n = 47, P = 0.2325) were anesthesized with pentobarbital sodium (35 mg/kg body wt ip) and their hearts were excised for myocyte isolation. Our previous studies indicate that, 3 wk after MI, myocyte adaptations include cellular hypertrophy as reflected by an ~10% increase in cell length (9) and by a 13-15% increase in whole cell capacitance (39, 40), reduced dihydropyridine binding sites (39), altered [Ca²⁺]_i transients (9, 38), decreased Na⁺/Ca²⁺ exchange currents and SR Ca²⁺ contents (40), and attenuated response to -adrenergic agonists (38, 39).

LV myocyte isolation. Myocytes were isolated from the septal and LV free wall portions of the myocardium, as previously described (9-11, 26); 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 experiments.

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 and placed on a temperature-controlled stage (37°C) of a Zeiss IM35 inverted microscope (11, 36). Measurements of myocyte shortening-relengthening were made between 2 and 6 h after isolation, because overnight culture (18 h) resulted in significant slowing of cell shortening dynamics (36). Fields of myocytes were chosen at random, and myocytes were field stimulated to contract at 0.1-5 Hz between platinum wire electrodes spaced 2 mm apart, as previously described (9, 26, 36). Frequency of contraction was varied via a programmable multichannel stimulator (STIM-6, Ionoptix, Milton, MA). Myocytes imaged by an Olympus DApoUV ×40/1.30 NA oil objective were focused on the high-resolution metal oxide silicon imager of a sequential scanning camera (model TM-640, Pulnix America, Sunnyvale, CA). Output of the video camera was fed into a video edge detector (model VED 103, Crescent Electronics, Sandy, UT). The myocyte image was rotated so that the long axis was horizontal and within the scan field. Proper adjustments of the raster line from which motion was sampled, light level thresholds, and left and right edge excursion limits allowed for optimal analog voltage output from left and right cell edges. Voltage outputs (left, right, and right-left difference) from the video edge detector were digitized on-line by a 12-bit analog-to-digital board (model ADA2710, Real Time Devices, State College, PA) in a personal computer. Data were permanently stored on a 51/4-in. 150-Mbyte Bernoulli disk (Iomega, Roy, UT) and analyzed off-line by custom-written software (Ionoptix). For calibration of edge detector voltage output vs. myocyte length, a high-resolution test target (model 22-8635, Ealing Electro-Optics, Natick, MA) was used.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Isolated myocyte shortening dynamics. A freshly isolated Sham myocyte at 1.8 mM extracellular Ca2+ was stimulated to contract as described in METHODS. Before start of pacing, left (upward deflection) and right (downward deflection) edge detector signals were offset to 0 mV. A: at 0.1 Hz, myocyte returned to its resting cell length after each beat (left and right edge detector signals returned to 0 mV). B: time-expanded view of A from which steady-state contraction amplitudes were calculated. C: at 5.0 Hz, left and right edges of same myocyte clearly did not return to 0 mV after each beat. Rather, at steady-state end diastole, myocyte assumed an end-diastolic cell length that was shorter than resting cell length. D: time-expanded view of C. Systolic shortening ("systolic" in D) is defined as sum of maximal left and right cell edge excursion from resting cell length, i.e., 0 mV. Net shortening is defined as systolic shortening minus steady-state deviation from resting cell length ("diastolic" in D) at end diastole. End-diastolic cell length is calculated from difference between resting cell length and steady-state deviation from resting cell length at end diastole.

Caffeine-induced contracture. Myocytes bathed in 5 mM extracellular Ca²⁺ were paced at 1 Hz. At 200 ms after the 11th beat, a trigger transistor-transistor logic pulse of 4.8-s duration was generated by STIM-6 to initiate caffeine (5 mM) application by puffer (Ionoptix) superfusion, which allowed rapid solution changes around a single cell (34). We previously showed that a 2.4-s pulse with 5 mM caffeine was sufficient to deplete the SR of all releasable Ca²⁺ (40). The caffeine-induced contracture and subsequent relaxation were captured by the video edge detector and stored for off-line analysis.

Statistics. Values are means ± SE. Single between-group comparisons (e.g., cell length, caffeine-induced contracture amplitude, Sham vs. MI) were made by using unpaired Student's t-tests. In experiments in which myocyte shortening was measured as a function of experimental group (Sham vs. MI), pacing frequency, and [Ca²⁺]_o, three-way ANOVA was performed to determine significance of difference. A linear model fitted by standard least squares was used. 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 prior MI on cell length. At 3 wk after MI, LV myocytes isolated from MI hearts averaged 125.0 ± 1.4 µm (n = 170) and were significantly (P < 0.0001) longer than those isolated from Sham hearts (114.4 ± 1.2 µm, n = 206). Our current ~9% increase in cell length was similar to the ~10% increase in cell length (9) reported previously for myocytes at 3 wk after MI. There was no change in cell width in MI myocytes. Our present finding suggests that MI myocytes used in the present series of experiments had undergone a degree of hypertrophy similar to that reported in our previous studies (9, 39, 40).

Effects of pacing frequency on myocyte contraction. At low pacing frequency of 0.1 Hz, the myocyte returned to its resting cell length after each beat. This is indicated by the left (upward) and right (downward) edge detector signals returning to 0 mV (both video edge detector signals were offset to 0 mV before beginning of stimulation) after each beat (Fig. 1, A and B). At 5 Hz, however, it is clear that left and right edge detector signals did not return to their baseline of 0 mV after each beat (Fig. 1, C and D). This suggests that, at moderate-to-high pacing frequencies, myocytes initiated each contraction from an "end-diastolic" cell length that was significantly shorter than the resting cell length. One consequence of this phenomenon is that, at high pacing frequencies, cell shortening amplitudes calculated from resting cell position ("systolic" in Fig. 1D) would overestimate the true myocyte contraction amplitude by the difference between resting and end-diastolic cell lengths ("diastolic" in Fig. 1D). The relationship between systolic and net shortening amplitudes at various pacing frequencies is shown for Sham myocytes in Fig. 2. On cessation of pacing at 5 Hz and at 1.8 mM extracellular Ca²⁺, myocytes usually exhibited aftercontractions (Fig. 1D), suggesting transient high intracellular Ca²⁺ loading. Two observations indicate that pacing myocytes at high frequencies and physiological [Ca²⁺]_o did not cause irreversible injury. First, after pacing, myocytes uniformly relaxed from their end-diastolic cell lengths to resting cell lengths, albeit slowly (Fig. 1D). Second, after undergoing a period of high-frequency contractions, myocytes could again be paced to contract after a brief period of rest.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Relationship between systolic and net shortening and steady-state diastolic position. Definitions are as in Fig. 1D. Data are for Sham myocytes incubated at 1.8 mM extracellular Ca2+. Means ± SE for systolic and net shortening and steady-state deviation from resting cell length at end diastole are shown at 0.1 (n = 90), 1.0 (n = 44), 2.0 (n = 43), and 5.0 Hz (n = 31). Error bars are not shown if they fall within boundaries of symbol.

Effects of prior MI on myocyte shortening. Under a variety of experimental conditions, MI myocytes tended to shorten to an extent that was different from Sham myocytes (Table 1). This general observation is supported by the existence of a significant ANOVA group (Sham vs. MI) effect (Table 2). Across both experimental groups, myocytes tended to shorten more as [Ca²⁺]_o was progressively increased (Table 1; significant [Ca²⁺]_o effect, Table 2). Increasing pacing frequency tended to reduce net cell shortening amplitudes in Sham and MI myocytes (Table 1; significant frequency effect, Table 2). The significant group × [Ca²⁺]_o interaction (Table 2) indicates that the magnitude and/or direction of the effect of [Ca²⁺]_o on cell shortening was different across experimental groups (Sham vs. MI). Inspection of data in Table 1 indicates that, at low [Ca²⁺]_o (0.6 mM), MI myocytes tended to shorten more than Sham myocytes, with the difference in shortening amplitudes greater at higher pacing frequencies (>0.1 Hz). By contrast, at high [Ca²⁺]_o (5.0 mM), Sham myocytes tended to contract more than MI myocytes, with the difference in shortening amplitudes largest at low pacing frequencies (0.1 Hz). This between-group difference in shortening amplitudes was no longer apparent at intermediate [Ca²⁺]_o (1.8 and 3.0 mM).

Effects of prior MI on fractional resting cell length at end diastole. At pacing frequencies 1 Hz, myocytes did not return to resting cell length after each beat (Fig. 1). Rather, they started the next beat from the end-diastolic cell length, which was uniformly shorter than resting cell length. This is most likely due to the fact that, at pacing frequencies 1 Hz, end-diastolic [Ca²⁺]_i levels were significantly higher than resting [Ca²⁺]_i levels measured in quiescent myocytes (38). Table 3 summarizes the relationship between fractional resting cell length at end diastole and pacing frequency at 1.8 and 5.0 mM extracellular Ca²⁺. Three-way ANOVA demonstrated significant frequency effect only (Table 4), indicating that increasing the pacing frequency resulted in shorter end-diastolic cell lengths. Given the insignificant three-way (group × [Ca²⁺]_o × frequency) interaction, to increase statistical power it is appropriate to remove the three-way interaction from analysis (28). Reanalysis of data for the fractional resting cell length at end diastole with the assumption of only two-way interaction indicated significant [Ca²⁺]_o and frequency main effects (Table 4). Neither analysis suggested a significant Sham vs. MI difference. To summarize, increasing pacing frequency or [Ca²⁺]_o resulted in shorter end-diastolic cell length in Sham and MI myocytes. There was no difference in fractional resting cell lengths at end diastole between Sham and MI myocytes. However, because the absolute resting cell lengths of Sham myocytes were ~10% shorter than those of MI myocytes, the absolute end-diastolic length of MI myocytes was still ~10% longer than that of sham myocytes.

Effects of prior MI on caffeine-induced contractures. After steady-state twitch contraction was achieved, application of 5 mM caffeine to a Sham myocyte at end diastole caused a large contracture due to SR Ca²⁺ release and then relaxation to a shorter resting cell length in the continued presence of caffeine (Fig. 3A). The incomplete relaxation (Fig. 3B) is thought to be due to increased myofilament sensitivity to Ca²⁺ by caffeine (32). Relaxation in the continued presence of caffeine was due to cellular Ca²⁺ efflux mechanisms, since SR Ca²⁺ accumulation was inhibited by caffeine (4, 40). Caffeine-induced contracture amplitudes in MI myocytes were 29.52 ± 1.75% of resting cell length (n = 12) and significantly (P < 0.03) smaller than those in Sham myocytes (35.03 ± 1.62% of resting cell length, n = 13). More importantly, the half time of relaxation from caffeine-induced contracture was significantly (P < 0.02) longer in MI myocytes (1.542 ± 0.149 s, n = 12; Fig. 3, C and D) than in Sham myocytes (1.098 ± 0.097 s, n = 13; Fig. 3, A and B).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Caffeine-induced contractures in Sham myocytes and myocytes in which myocardial infarction (MI) was induced. For clarity of presentation, only differential (right edge  left edge) voltage signal is shown. A: after 11 twitches (1 Hz, 37°C, 5.0 mM extracellular Ca2+) to ensure steady-state sarcoplasmic reticulum Ca2+ load, caffeine (5 mM) was "puffed" onto a sham myocyte to induce contracture and myocyte relaxation was followed until contracture was complete. Note first negative treppe phenomenon characteristic of rat myocytes. Caffeine induced a large myocyte contracture that slowly relaxed toward a shorter resting cell length secondary to increased myofilament Ca2+ sensitivity by caffeine (32). At low (5 mM) caffeine concentrations, rapid transient phase of contraction largely reflects changes in intracellular Ca2+ concentration (32). Relaxation in continued presence of caffeine was due to Ca2+ efflux mechanisms, since sarcoplasmic reticulum Ca2+ accumulation was inhibited (40). B: time-expanded view of caffeine-induced contracture in Sham myocyte shown in A. C: caffeine-induced contracture in an MI myocyte under same experimental conditions. D: time-expanded view of caffeine-induced contracture in MI myocyte shown in C. Relaxation was more prolonged in MI myocyte.

	DISCUSSION

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The major determinants of cardiac myocyte contractile behavior are 1) Ca²⁺ influx, 2) Ca²⁺ efflux and sequestration, 3) SR Ca²⁺ content, 4) myofilament Ca²⁺ sensitivity, and 5) percent myosin heavy chain isoform. Previous studies by our group (9, 27, 38-40) and others (12, 13, 18, 20-22, 25) have identified individual steps in excitation-contraction coupling that were altered in surviving myocytes after MI. However, it is difficult to unequivocally establish that a particular altered Ca²⁺ homeostastic pathway is an important causative factor in decreased contraction in MI myocytes. To approach this, in the present study we designed experimental conditions to manipulate Ca²⁺ fluxes and examined the effects on the difference between Sham and MI myocyte contractile behavior. This is possible because, in the myocardium, a transient imbalance between cellular Ca²⁺ influx and efflux could be created by changing pacing frequency and [Ca²⁺]_o (6, 26). This imbalance produced an alteration in cellular Ca²⁺ load that was manifest primarily as a change in SR Ca²⁺ content (6). Because SR Ca²⁺ content is a major determinant of the amount of SR Ca²⁺ released per twitch, alterations in SR Ca²⁺ content would be observed as changes in contraction amplitude (6).

In rat myocardium, high pacing frequency and low [Ca²⁺]_o favored Ca²⁺ efflux pathways (forward Na⁺/Ca²⁺ exchange, sarcolemmal Ca²⁺-ATPase), whereas low pacing frequency and high [Ca²⁺]_o favored Ca²⁺ influx pathways [Ca²⁺ current (I_Ca) and reverse Na⁺/Ca²⁺ exchange current (I_NaCa)] (6, 26). If defects in Ca²⁺ extrusion are important pathogenetic factors of abnormal contractile behavior after MI, in the absence of other changes (e.g., myofilament Ca²⁺ sensitivity) (1, 2, 22), one would expect that, under conditions designed to favor Ca²⁺ efflux, maximal twitch amplitude measured in MI myocytes would be greater than that in Sham myocytes. The difference in twitch amplitudes between Sham and MI myocytes would narrow as conditions changed to favor Ca²⁺ influx. Conversely, if defects in Ca²⁺ influx pathways constituted the major determinant of abnormal contractile behavior in MI myocytes, then one would expect MI myocytes to contract less under conditions favoring Ca²⁺ influx, with the differences in twitch amplitude between MI and Sham myocytes narrowing/disappearing under conditions favoring Ca²⁺ efflux.

The major new finding of the current study is that MI myocytes could be shown to contract more or less than Sham myocytes, depending on experimental conditions. This phenomenon has not been demonstrated in previous studies (1, 9, 18, 20, 21, 25). Under conditions favoring Ca²⁺ efflux (0.6 mM extracellular Ca²⁺ concentration and 5 Hz), MI myocytes shortened to a greater extent than Sham myocytes (Table 1). Conversely, under conditions favoring Ca²⁺ influx (5 mM extracellular Ca²⁺ and 0.1 Hz), MI myocytes shortened less than Sham myocytes (Table 1). The differences in twitch amplitudes between Sham and MI myocytes narrowed and became insignificant as experimental conditions receded from the two extreme conditions. With the assumption of no change in myofilament Ca²⁺ sensitivity between Sham and MI myocytes (1, 2, 22), this complex pattern of contractile responses is consistent with the hypothesis that alterations in Ca²⁺ influx and efflux pathways contributed to abnormal contractile behavior in MI myocytes. Of the two major regulated Ca²⁺ influx pathways in cardiac ventricular myocytes, I_Ca (21) and reverse I_NaCa (40) have been reported to be lower in MI myocytes. Similarly, Na⁺/Ca²⁺ exchange, the major myocyte Ca²⁺ efflux mechanism (3, 4), was found to be decreased in MI myocytes (13, 40). The results of our current study thus conferred functional significance to the observations that I_Ca (21) and I_NaCa (40) were depressed in MI myocytes.

In light of recent appreciation of the potential roles of reverse I_NaCa in mediating Ca²⁺ entry during depolarization, loading the SR with Ca²⁺, and subsequently triggering SR Ca²⁺ release (16, 19, 29), it is interesting to propose that depressed Na⁺/Ca²⁺ exchange activities (13, 40) may simultaneously lower Ca²⁺ influx and efflux in rat myocytes 3 wk after MI. Although Bassani et al. (3) reported that, at 22°C, Na⁺/Ca²⁺ exchange accounted for only 7% of Ca²⁺ transport during a twitch in rat myocytes, thus raising questions as to the physiological significance of alterations in Na⁺/Ca²⁺ exchange in MI myocytes, it has to be recalled that the activity of Na⁺/Ca²⁺ exchange is extremely temperature sensitive (14, 19, 29, 35). For example, Vornanen et al. (35) clearly demonstrated that SR Ca²⁺ release triggered by Na⁺/Ca²⁺ exchange was almost absent at 23°C but increased with depolarization at 37°C. Indeed, 20% of the decline in [Ca²⁺]_i during depolarization in rat cardiac myocytes at 31°C was attributed to forward Na⁺/Ca²⁺ exchange (8).

Another functional manifestation of depressed Na⁺/Ca²⁺ exchange activity in myocytes 3 wk after MI may be the significantly longer half time of relaxation from caffeine-induced contractures (Fig. 3; RESULTS). Relaxation under continued caffeine presence (SR Ca²⁺ accumulation inhibited) is largely mediated by forward Na⁺/Ca²⁺ exchange with minor contributions by sarcolemmal Ca²⁺-ATPase and mitochondrial Ca²⁺ uniporter (3, 4, 7). With the assumption of small differences in resting membrane potential [3 mV (21)] and similar intracellular Na⁺ concentration, reduction in Na⁺/Ca²⁺ exchange activity would be expected to result in slower Ca²⁺ extrusion and decreased rate of myofilament relaxation during caffeine-induced contracture in MI myocytes. The amplitude of caffeine-induced contracture reflects not only the SR Ca²⁺ content and myofilament Ca²⁺ sensitivity but also the rate of Ca²⁺ extrusion from the sarcoplasm (4). If it is assumed that the sensitivities of myofilaments to Ca²⁺ (1, 2, 22) and caffeine were not changed 3 wk after MI, and, despite a slower rate of Ca²⁺ efflux by depressed Na⁺/Ca²⁺ exchange (13, 40), the amplitude of caffeine-induced contracture was still significantly lower in myocytes 3 wk after MI. This is consistent with the notion that SR Ca²⁺ content was lower in MI myocytes. We previously showed by electrophysiological measurements that SR Ca²⁺ content was indeed reduced in myocytes 3 wk after MI (40).

Our observed differences in contraction amplitudes (expressed as percentage of resting cell lengths) between Sham and MI myocytes (1-3%) are in the same range previously reported by Litwin and Bridge (<2%) (21), LeFroy et al. (1%) (18), Meggs et al. (4%) (25), and Cheung et al. (6%) (9). However, there are also substantial differences in results reported by different investigators for MI myocytes. For example, in LV myocytes isolated from rats 1 wk after recovery from MI, Meggs et al. and LeFroy et al. reported a reduction in maximal extent of cell shortening (44 and 20% decrease, respectively) under experimental conditions of 1-1.2 mM [Ca²⁺]_o, 30-32°C, and pacing at 0.5-1.0 Hz. By contrast, when studied 3-4 wk after MI, at 1-2 mM [Ca²⁺]_o, 30-37°C, and pacing from 0.2 to 5 Hz, neither we (present study and Ref. 9) nor Anand et al. (1) observed any significant differences in twitch amplitudes between Sham and MI myocytes. The effects of increasing [Ca²⁺]_o on MI myocyte contraction are also controversial. At 5 mM extracellular Ca²⁺, our previous (9) and present results (Table 1) indicate significant differences in maximal extent of cell shortening between Sham myocytes and myocytes 3 wk after MI. Anand et al. (1) and LeFroy et al. (18), however, did not observe statistically significant differences in mean maximal contraction amplitude between sham and MI myocytes stimulated in the presence of increasing [Ca²⁺]_o. The discrepancies in results reported by different investigators may relate to the following confounding factors: 1) species, i.e., rabbit (21) vs. rat (1, 9, 18, 20, 25); 2) infarct size, i.e., small (<25% of LV) (21) vs. moderate (36%) (9, 27) vs. large (>50%) (25); 3) overt LV failure at time of myocyte isolation, i.e., present (1, 25) vs. absent (21); 4) LV myocyte selection, i.e., no selection (9, 20, 25; present study) vs. adjacent to infarct (18) vs. remote from infarct (1); 5) temperature, i.e., 30°C (1, 21, 25) vs. 32°C (18) vs. 37°C (1, 9; present study); 6) [Ca²⁺]_o, i.e., 1.2 mM (25) vs. 2.7 mM (21) vs. range of [Ca²⁺]_o (1, 9, 18; present study); 7) pacing frequency, i.e., 0.2 Hz (9) vs. 0.5 Hz (1, 18) vs. 1 Hz (25) vs. range of frequencies (1, 21; present study); and 8) recovery time after MI, i.e., 1 wk (18, 25) vs. 3 wk (9; present study) vs. 1-6 wk (1) vs. 8 wk (21). The last point bears emphasis, inasmuch as the post-MI ventricle undergoes progressive remodeling (33), so that differences observable 1 wk after MI may no longer be apparent 3 wk after MI. For example, Li et al. (20) recently reported a significant rightward shift of the tension-pCa relationship in skinned myocytes 1 wk after MI, indicating decreased myofilament Ca²⁺ sensitivity. In an earlier report by the same group (2), however, no significant difference in Ca²⁺ sensitivity of myofibrillar ATPase activities was found between control LV and LV that were subjected to severe left coronary artery constriction for 12-13 wk and suffered significant structural damage. Anand et al. (1) did not observe any shift in the [Ca²⁺]_i-cell shortening relationship in myocytes isolated for 1-6 wk after MI. Likewise, Litwin and Morgan (22) did not observe any differences in pCa-tension relationships between LV posterior papillary muscles dissected from Sham and 6-wk MI rats. It is important when comparing studies on MI myocytes reported by different investigators that the above-listed confounding factors be taken into account.

In summary, myocytes isolated from LV 3 wk after MI were longer and shortened less than Sham myocytes under high [Ca²⁺]_o and low pacing frequencies and more under low [Ca²⁺]_o and high pacing frequencies. We interpreted the complex pattern of contraction abnormalities consistent with the notion that Ca²⁺ influx and efflux pathways were reduced in MI myocytes. We hypothesize that abnormalities in Na⁺/Ca²⁺ exchange may partly account for the contractile dysfunction in MI myocytes.