INTRODUCTION

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CARDIAC HYPERTROPHY and failure are accompanied by chronic changes in several sarcolemmal ion transport systems involved in intracellular Ca²⁺ regulation, including Na⁺/Ca²⁺ exchange (30, 43) and Ca²⁺ current (31, 32, 38). However, the extent to which cardiac intracellular pH (pH_i) regulatory systems are affected by cardiac overload is unresolved. Previous studies of pH_i regulation in hypertrophied myocardium have focused on pressure overload-induced cardiac hypertrophy. For example, Na⁺/H⁺ exchange activity was enhanced in papillary muscles from spontaneously hypertensive rats (35), and aortic banding induced activation of mRNA for the NHE-1 isoform of Na⁺/H⁺ exchange in rabbit ventricular muscle (44). However, there is no information available concerning the effects of chronic infarct-induced remodeling on Na⁺/H⁺ exchange. Infarct-induced models of left ventricular dysfunction are useful in terms of their clinical significance. Although recent advances in the treatment of acute myocardial infarction (MI) have decreased overall cardiac mortality, many patients are left with chronic heart damage (22). Thus animal models that simulate postinfarction dysfunction may be of use in the clinical setting.

In cardiac muscle, Na⁺/H⁺ exchange is largely responsible for the recovery of pH_i from intracellular acid loading (20) and is stimulated by ANG II (13, 23, 26). Activation of Na⁺/H⁺ also contributes to the myocardial damage and arrhythmias associated with ischemia-reperfusion (10, 19, 46). The cardiac renin-angiotensin system is activated during cardiac hypertrophy and failure, and enhanced intracardiac production of ANG II may cause adverse effects on ventricular function (15, 29, 37, 39). In this regard, numerous studies have shown that inhibition of angiotensin-converting enzyme attenuates the cardiac dysfunction accompanying MI and heart failure (8, 36, 45).

The major focus of this project was to obtain new insight into the contribution of Na⁺/H⁺ exchange to cellular events related to postinfarction (post-MI) remodeling in spared ventricular myocytes. Because ANG II significantly modulates cardiac Na⁺/H⁺ exchange in normal cells and may contribute to the pathophysiology of heart failure, we examined the effect of ANG II on Na⁺/H⁺ exchange in post-MI ventricular myocytes. We also evaluated the inotropic actions of ANG II on both cell types. All experiments were performed on single ventricular myocytes isolated from adult rabbit hearts using fluorescent indicators to measure pH_i and Ca²⁺ transients. The use of single cells avoids the possible complications associated with ANG II-induced changes in pH_i and intracellular Ca²⁺ concentration ([Ca²⁺]_i) that may occur in noncardiac cells present in multicellular preparations and cell suspensions.

	MATERIALS AND METHODS

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Solutions and myocyte bathing system. Cell bathing solutions were held at 36.5 ± 0.5°C in glass reservoir bottles. Solutions were delivered by gravity from the bottles to the myocyte bath through thermally jacketed gas-impermeable tubing, thereby ensuring that the solution pH and temperature were the same in the bottles and the bath. The 1-ml Plexiglas bath had a clear glass bottom and was mounted on the stage of an inverted microscope (Diaphot, Nikon, Tokyo, Japan). The bottom of the bath was lightly coated with laminin (Collaborative Research, Bedford, MA) to improve myocyte adhesion. Solution flow and depth were held at ~4-6 ml/min and 2 mm, respectively. The temperature of solutions in the bath was 36.5 ± 0.5°C. Complete replacement of a solution in the bath required ~4 s.

Production of myocardial infarction. Rabbits were preanesthetized with acepromazine (16 mg/kg im) and xylazine (3 mg/kg im). Approximately 10-15 min later, they were anesthetized with isoflurane, orally intubated, and ventilated at 40 breaths/min. Under sterile conditions, a left lateral thoracotomy was made in the fifth intercostal space and a 6-0 silk suture was placed around the proximal portion (1-2 mm from the left atrium) of the large branch of the circumflex artery running on the lateral aspect of the heart. Lidocaine HCl (1 mg/kg iv) was administered at the time of coronary artery occlusion, and a second dose (0.5 mg/kg) was given 15 min later. Hearts from the infarcted rabbits showed transmural scars of the lateral wall and apex over ~25% of the total left ventricular circumference.

Hemodynamic measurements. To assess the extent of left ventricular dysfunction after MI, intracardiac pressures were recorded in a separate group of rabbits. Rabbits were anesthetized with ketamine (50 mg/kg im) and xylazine (3 mg/kg im). The right carotid artery was isolated by cut down and cannulated with a 1-mm micromanometer-tipped catheter (Millar Instrument, Houston, TX). The catheter was retrogradely passed across the aortic valve under constant pressure monitoring. Left ventricular systolic and end-diastolic pressures were recorded.

Myocyte isolation. Results were obtained from male rabbits (~2.5-4.0 kg) with normal (n = 37) and infarcted hearts (n = 42). The heart was retrogradely perfused via the aorta (60 mmHg pressure) with a Ca²⁺-free solution at 37°C for 5 min containing (in mM) 126.0 NaCl, 4.4 KCl, 22.0 dextrose, 5.0 MgCl₂, 20.0 taurine, 5.0 creatine, 5.0 sodium pyruvate, 1.0 NaH₂PO₄, 24.0 HEPES, and 12.5 NaOH (pH 7.3). This was followed by 15-20 min of recirculation of the same solution (200 ml) containing 1 mg/ml collagenase (class II, Worthington Biochemical, Freehold, NJ), 0.1 mg/ml protease (type XIV, Sigma Chemical), and 0.1 mM CaCl₂. The heart was then perfused for 5 min with the same solution containing no enzymes. All cell isolation solutions were equilibrated with 100% O₂. The left ventricle was separated from the right ventricle and the atria. For normal hearts the free wall of the left ventricle was minced. For infarcted hearts the scar was removed, and then a 2- to 3-mm rim of surviving tissue surrounding the scar was dissected free and minced. We chose to use tissue from the peri-infarcted region to minimize potential heterogeneity in cellular geometry or physiology in different regions of the infarcted heart. The minced tissue from normal and infarcted hearts was gently shaken in the 0.1 mM Ca²⁺ (enzyme-free) solution and strained, and the remaining myocytes were allowed to settle in a storage solution containing 1.0 mM Ca²⁺. The yield of Ca²⁺-tolerant cells using this procedure was >60%. All myocytes used in this study were rod shaped in appearance, had well-defined striations, and did not spontaneously contract. All cells were studied 2-5 h after isolation.

Measurement of cell size. Cell length and width were measured in a large number of myocytes from control and infarcted hearts. To avoid bias, the microscope stage was randomly moved, and all the quiescent rod-shaped cells visible in the field were measured.

Measurement of pH_i, Ca²⁺ transients, and myocyte contractions. pH_i was measured in single myocytes according to previously described procedures (26, 41, 48). Myocytes were equilibrated at 37°C for 10 min in the normal solution containing 13 µM SNARF 1-AM. They were then placed in the bath, and normal solution containing no indicator was continuously directed through the bath. pH_i measurements were begun 30-40 min later. Excitation at 515 ± 5 nm was provided by a 200-W mercury-arc lamp and was directed to the myocyte bath via the objective lens (×40, oil, NA 1.3, Nikon). Myocytes were also illuminated from above with low-intensity blue light (410 ± 20 nm), which had no effects on the SNARF 1 emission signal. Optical signals from a myocyte were collected by the objective lens and sent out the microscope side port to an intensified charge-coupled device (ICCD) television camera (model IC-100, Photon Technology International, S. Brunswick, NJ) and a dichroic mirror (610 nm). The camera detected the cell image, and the mirror directed the fluorescence emission to two photomultiplier tubes equipped with band-pass filters centered at 640 ± 15 and 580 ± 15 nm. An adjustable rectangular window in the side port restricted the optical image and fluorescence to the cell of interest. The ratio of fluorescence emission (640 nm/580 nm) was obtained on-line with an analog divider circuit. Autofluorescence was <1% of the fluorescence signal from SNARF 1-loaded cells. Background fluorescence at each wavelength was measured as the fluorescence from a clean, cell-free area next to the cell under study and was electronically subtracted from the loaded cell signal. All pH_i measurements were performed on resting myocytes, except those in which cell shortening and pH_i were simultaneously recorded.

Determination of intracellular buffering power and net flux of acid equivalents. To determine net H⁺ flux (J_H⁺) it is necessary to measure intracellular buffering power. For experiments performed in the absence of CO₂ and HCO₃, we assumed that HCO₃ buffering was zero and that total intracellular buffering power (B_T) was equal to intrinsic buffering power (B_i). B_i was experimentally estimated using step reductions in NH₄Cl. To accurately estimate B_i it was necessary to eliminate transsarcolemmal flux of acid equivalents via Na⁺-HCO₃ cotransport, Cl/HCO₃ exchange, and Na⁺/H⁺ exchange. Thus all myocyte bathing solutions used for B_i measurements contained no added HCO₃, CO₂, or Na⁺ and were buffered with HEPES. To minimize Ca²⁺ accumulation via reverse Na⁺/Ca²⁺ exchange, the solutions also contained no added Ca²⁺. pH_i was allowed to stabilize in Na⁺-free solution before application of NH₄Cl. B_i (mM/pH) was calculated as previously described (20, 48) where pH_i is the measured change in pH_i and [NH⁺₄]_i is the change in intracellular concentration of NH⁺₄ calculated as follows where [NH₄]_o is the total extracellular concentration of NH₄Cl and pH_o is extracellular pH (7.4), with the dissociation constant (pK) taken as 9.5. The relationship between B_i and pH_i was determined over the pH_i range of ~6.5-7.4 in normal and infarcted hearts. The best-fit equation of the data (B_i vs. pH_i) was used to estimate B_i at any given pH_i in other cells.

Data acquisition. Analog signals (fluorescence ratio and cell shortening) were simultaneously recorded digitally with a computer using AxoScope and AxoTape software (Axon Instruments, Foster City, CA). pH_i signals were filtered at 1 Hz and digitized at a rate of 2 Hz. Cell shortening and fluo 3 signals were filtered at 500 Hz and digitized at 1 kHz.

Statistical analysis. Values are means ± SE. Statistical analysis was performed using paired and unpaired Student's t-test. P < 0.05 was considered significant.

	RESULTS

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Dimensions of normal and post-MI remodeled myocytes. The mean length and width of normal myocytes were 132.2 ± 3.1 and 29.4 ± 0.7 µm, respectively (301 cells from 4 hearts). The corresponding values from post-MI cells were 142.9 ± 1.3 and 30.4 ± 0.4 µm, respectively (498 cells from 7 hearts). Cell length is significantly different in the two groups (P < 0.01, unpaired t-test). These changes in cell size suggest a predominant volume overload, which is typical of the infarcted ventricle (6). This characteristic pattern of cellular hypertrophy confirms that the single cells used in this project were subjected to the pathological processes that involve the surviving myocardium after transmural infarction. Hemodynamic measurements are summarized in Table 1.

B_i in post-MI remodeled myocytes. To quantitatively assess J_H⁺ via Na⁺/H⁺ exchange, it is necessary to determine B_i. It is also important to directly measure B_i, since it may be changed by cardiac hypertrophy. All B_i measurements were performed in Na⁺-free solution containing no added CO₂ or HCO₃. An example of a B_i determination in a post-MI cell is shown in Fig. 1A, and the results from normal and post-MI cells are summarized in Fig. 1B. The B_i-pH_i relationship is given by B_i = 143.1  17.2pH_i for normal cells (35 measurements in 15 cells) and B_i = 76.7  8.1pH_i for post-MI cells (27 measurements in 13 cells). There was no significant difference in B_i between the two cell types.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Measurement of intrinsic buffer capacity (Bi) in rabbit ventricular myocytes. A: Bi determination in a spared myocyte after myocardial infarction (post-MI). B: summary of relationship between Bi and intracellular pH (pHi) in normal () and post-MI myocytes (). There was no significant difference in Bi between groups.

HEPES-buffered solution: effect of post-MI remodeling on Na⁺/H⁺ exchange and steady-state pH_i. It seems possible that post-MI remodeling may affect expression and/or the ion transport capacity of Na⁺/H⁺ exchange. To test the latter hypothesis, we activated Na⁺/H⁺ exchange by inducing rapid intracellular acidosis using NH₄Cl prepulses in normal and post-MI cells. In the absence of CO₂-HCO₃, pH_i recovery from intracellular acidosis in normal ventricular myocytes is mediated by forward Na⁺/H⁺ exchange. The results are summarized in Fig. 2, in which J_H⁺ is expressed as a function of pH_i during recovery from acid loading. Although the mean values of J_H⁺ in post-MI myocytes were less than normal, the differences at each pH_i did not achieve statistical significance (P = 0.09-0.31, unpaired t-test). Similarly, the mean steady-state pH_i in post-MI myocytes (7.09 ± 0.02, n = 29) was not significantly different from that in normal myocytes (7.05 ± 0.02, n = 23).

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Summary of relationship between pHi and net H+ efflux (JH+) via Na+/H+ exchange in normal () and post-MI () myocytes. JH+ was determined during pHi recovery from intracellular acidosis after a 10 mM NH4Cl prepulse. All solutions were buffered with HEPES. n = 4-10. There was no significant difference in JH+ between groups at each pHi (unpaired t-test).

HEPES-buffered solution: effect of ANG II on Na⁺/H⁺ exchange and steady-state pH_i in post-MI remodeled myocytes. Hypertrophied and failing hearts have an altered neurohormonal responsiveness (4) and an activated renin-angiotensin system (15, 37, 39). We previously showed that ANG II stimulates Na⁺/H⁺ exchange in normal cardiac cells (23, 26). In the present experiments we examined the effects of ANG II (100 nM) on Na⁺/H⁺ exchange in spared post-MI myocytes. Normal and post-MI myocytes displayed an ANG II-induced increase in the rate of recovery from acid loading that is indicative of stimulated Na⁺/H⁺ exchange (Fig. 3). However, in the post-MI myocytes the stimulatory effect was suppressed. The results are summarized in Fig. 4, with J_H⁺ expressed as a function of pH_i. In contrast to post-MI myocytes, ANG II significantly increased J_H⁺ in normal myocytes at pH_i 6.65, 6.70, and 6.75. This difference in ANG II responsiveness is further emphasized by comparing J_H⁺ in the two groups at pH_i 6.85. In the presence of ANG II, J_H⁺ was significantly less (unpaired t-test, P < 0.05) in post-MI myocytes (2.46 ± 0.84 mM/min, n = 7) than in normal cells (4.70 ± 0.69 mM/min, n = 8).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Effect of 100 nM ANG II on pHi recovery from intracellular acidosis in normal and spared post-MI myocytes. All solutions were buffered with HEPES. Stimulatory effect of ANG II on pHi recovery from intracellular acidosis is blunted in post-MI myocyte.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Summary of effect of 100 nM ANG II on relationship between JH+ via Na+/H+ exchange and pHi in normal (A; , control; , ANG II) and spared post-MI myocytes (B; , control; , ANG II). Experimental protocol is shown in Fig. 3. Each myocyte served as its own control for ANG II application. n = 3-7. * Significantly increased compared with control (P < 0.05, paired t-test).

HEPES buffered solution: effect of the AT₁-receptor antagonist losartan on ANG II-induced stimulation of Na⁺/H⁺ exchange. To identify the receptor subtype involved in the ANG II-induced changes in J_H⁺, we used losartan, a specific nonpeptide blocker of AT₁ receptors (47). As shown in Fig. 5 (normal myocyte), ANG II elicited a fall in steady-state pH_i when applied in the presence of 1 µM losartan. The mean decrease in steady-state pH_i in normal myocytes was 0.011 ± 0.001 (n = 6) after 5 min in ANG II, which is significantly different (P < 0.05, unpaired t-test) from the mean increase in steady-state pH_i (0.032 ± 0.014, n = 8) that occurs when ANG II is applied to normal myocytes in the absence of losartan.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   AT1-receptor inhibitor losartan blocks stimulatory effect of 100 nM ANG II on pHi recovery from intracellular acidosis (A, normal myocyte). B: overlay of pHi recoveries shown in A. All solutions were buffered with HEPES.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Summary of effect of AT1-receptor blocker losartan (1 µM) on relationship between JH+ and pHi in normal (A; , losartan; , losartan + 100 nM ANG II) and post-MI myocytes (B; , losartan; , losartan + 100 nM ANG II). Experimental protocol is shown in Fig. 5. Each cell served as its own control for ANG II application. n = 3-6. ANG II had no significant effect on JH+ in presence of losartan in normal and post-MI myocytes.

HCO₃-buffered solution: effect of ANG II on pH_i recovery from acid loading. We also studied pH_i recovery from 10 mM NH₄Cl prepulses under more physiological conditions using an HCO₃-CO₂ buffer system. In this setting all pH_i regulatory systems are intact and J_H⁺ reflects J_H⁺ via Na⁺/H⁺ exchange and HCO₃ influx via Na⁺-HCO₃ cotransport (20). The results are summarized in Fig. 7. In normal myocytes, ANG II elicited significant (P < 0.05, paired t-test) increases in J_H⁺ at pH_i 6.95, 6.90, and 6.85. In contrast, no significant changes occurred in post-MI myocytes. The mean steady-state pH_i of normal myocytes in HCO₃-buffered solution without ANG II (7.03 ± 0.02, n = 5) was not significantly different from that in post-MI myocytes (7.02 ± 0.02, n = 10).

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Summary of effect of 100 nM ANG II on relationship between JH+ and pHi in normal (A; , control; , ANG II) and post-MI myocytes (B; , control; , ANG II) bathed in an HCO3-CO2 buffer system. Each myocyte served as its own control for ANG II application. n = 3-7. ANG II did not significantly increase JH+ in post-MI myocytes. * Significantly increased compared with post-MI myocytes (P < 0.05, paired t-test).

HEPES-buffered solution: effect of ANG II on pH_i and myocyte shortening during pacing. We and others previously showed that the positive inotropic action of ANG II in normal rabbit ventricular myocytes is mediated, in part, by stimulation of Na⁺/H⁺ exchange (3, 16, 26). To examine the acute inotropic effects of ANG II (100 nM) on post-MI myocytes, we simultaneously measured pH_i and cell shortening in field-stimulated myocytes (cycle length = 3 s). Cell shortening in post-MI myocytes displayed reduced responsiveness to ANG II compared with normal cells (Fig. 8). The accompanying changes in pH_i also differed in the two cell types, with alkalosis in the normal cell and acidosis in the post-MI cells. The mean increase in cell shortening measured 5 min after ANG II application was 91.5 ± 21.4% (n = 8) in normal myocytes compared with 30.0 ± 11.9% in post-MI myocytes (n = 6, P < 0.05, unpaired t-test). The corresponding changes in pH_i in the beating myocytes were 0.06 ± 0.02 and 0.04 ± 0.02 in normal and post-MI myocytes, respectively (P < 0.01, unpaired t-test). In addition to demonstrating that post-MI hypertrophy is associated with a diminished inotropic sensitivity to ANG II, these results also show that the positive inotropic action of ANG II can occur without an accompanying increase in pH_i.

View larger version (38K): [in this window] [in a new window]   Fig. 8.   Effect of ANG II on simultaneously measured cell shortening and pHi in normal and post-MI myocytes paced at a cycle length of 3 s. All solutions were buffered with HEPES. ANG II increased cell shortening and pHi in normal myocyte. In contrast, ANG II caused pHi to fall in post-MI myocyte, and positive inotropic effect was blunted.

HEPES-buffered solution: effect of ANG II on the Ca²⁺ transient and myocyte shortening during pacing. To determine whether the ANG II-induced changes in pH_i and contraction described above (Fig. 8) were accompanied by alterations in the Ca²⁺ transient, we repeated the pacing protocol in fluo 3-loaded cells under identical conditions. In both cell types, ANG II increased the amplitude of the Ca²⁺ transient and the simultaneously measured cell shortening (Fig. 9). The results are summarized in Fig. 10 and demonstrate that the ANG II-induced increase in both parameters was significantly less in post-MI myocytes.

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Effect of 100 nM ANG II on simultaneously measured Ca2+ transients [fluo 3 fluorescence (F) in arbitrary units (au)] and shortening in normal ( A) and post-MI (B) myocytes paced at a cycle length of 3 s. All solutions were buffered with HEPES. Exposure to ANG II for 5 min increased both parameters in both cell types.

View larger version (10K): [in this window] [in a new window]   Fig. 10.   Summary of effect of 100 nM ANG II on simultaneously measured Ca2+ transients (open bars) and cell shortening (filled bars) in normal (n = 5) and post-MI (n = 7) myocytes paced at a cycle length of 3 s. ANG II-induced changes in cell shortening and amplitude of Ca2+ transient are expressed relative to their values in control solution. All solutions were buffered with HEPES. ANG II-induced increases in both parameters were significantly attenuated in post-MI myocytes: * P < 0.025, cell shortening; ** P <0.01, Ca2+ transient (unpaired t-test).

	DISCUSSION

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In this study we examined Na⁺/H⁺ exchange activity, Ca²⁺ transients, and cell shortening in rabbit ventricular myocytes isolated from normal and chronically (8-12 wk) infarcted left ventricles. Experiments were performed at pH_o 7.4 in solutions that contained no added CO₂ or HCO₃ (HEPES buffered) or were buffered with 18.0 mM HCO₃-5.0% CO₂. The post-MI myocytes were isolated from the peri-infarcted region of the left ventricles. Given the high incidence of heart failure induced by ischemic heart disease, this model has clinical relevance.

Our results demonstrate for the first time that 1) post-MI hypertrophy is not associated with changes in B_i (Fig. 1), 2) post-MI hypertrophy does not significantly affect the ability of Na⁺/H⁺ exchange to mediate pH_i recovery from intracellular acidosis (Fig. 2), 3) the stimulatory effect of ANG II on Na⁺/H⁺ exchange is significantly reduced in post-MI myocytes (Figs. 3 and 4), 4) in HCO₃-buffered solutions, ANG II does not significantly stimulate pH_i recovery from acidosis in post-MI cells (Fig. 7), 5) the AT₁ receptor mediates the stimulatory action of ANG II on Na⁺/H⁺ exchange in post-MI myocytes (Fig. 6), and 6) in HEPES-buffered solution, post-MI myocytes have a reduced positive inotropic response to ANG II (Figs. 8-10) that correlates with a blunted increase in the amplitude of the Ca²⁺ transient (Figs. 9 and 10).

The percentage of the left ventricle affected by coronary artery ligation in our experiments varied from 20 to 30% of the total mass of the ventricle. Myocytes from post-MI rabbits showed a predominant increase in cell length, suggesting that chronic infarction caused changes in myocyte structure similar to chronic volume overload. Similar changes have been noted in a rat model of post-MI heart failure (33). The main hemodynamic effects of infarction induced by coronary artery ligation in the rabbit are increased left ventricular end-diastolic pressure, slowed left ventricular relaxation, and decreased rate of left ventricular pressure development (25). These effects are also very similar to those reported for the more extensively characterized rat model of postinfarction heart failure (22).

Despite their increased size, post-MI myocytes did not display significant changes in their ability to buffer intracellular H⁺ in the absence of CO₂-HCO₃. Pressure overload hypertrophy in rat (17) and ferret (11) ventricular muscle is also not accompanied by changes in B_i. Similarly, normal B_i values are reported for ventricular muscle from spontaneously hypertensive rats (35). Interestingly, these results contrast with exercise-induced hypertrophy, in which B_i is significantly increased (9).

The J_H⁺-pH_i relationship for Na⁺/H⁺ exchange in normal myocytes was not significantly different from that of post-MI myocytes (Fig. 2). This contrasts with recent findings that the activity and gene expression of ventricular Na⁺/H⁺ exchange are increased in spontaneously hypertensive rats (35) and pressure-overloaded rabbit hearts (44), respectively. The molecular basis for this difference is unclear. However, chronic pressure and volume overload are known to induce morphologically different forms of myocardial hypertrophy (1) and to elicit distinctly different molecular phenotypes and patterns of peptide growth factor induction (5).

We previously showed that ANG II stimulates Na⁺/H⁺ exchange in normal rabbit atrial (23) and ventricular (26) myocytes. Although the exact mechanism of this effect is unresolved, it seems likely that protein kinase C activation and phosphorylation of the transporter are involved (26). In the present study we demonstrate that the stimulatory action of ANG II on Na⁺/H⁺ exchange is attenuated in post-MI myocytes. A suppressed responsiveness of Na⁺/H⁺ exchange to ANG II and endothelin 1 has also recently been observed in rat ventricular myocytes subjected to pressure-induced hypertrophy (17). This similarity suggests that, despite their differences (1, 5), pressure- and volume-induced left ventricular hypertrophies may share similar mechanisms for inducing impaired sensitivity to vasoactive peptides.

Acid load recovery in HCO₃-buffered solutions is mediated by Na⁺/H⁺ exchange and Na⁺-HCO₃ cotransport (20). In adult rat and guinea pig ventricular myocytes, the relative contribution of Na⁺-HCO₃ cotransport to this process is ~33% (21) and 40% (20), respectively. Values for rabbit ventricular muscle are apparently not available. However, a comparison of our normal J_H⁺ values (without ANG II) in the absence (Fig. 4) and presence of HCO₃ (Fig. 7) suggests that Na⁺-HCO₃ may contribute ~30% at pH_i 6.75. The acid loader, Na⁺-independent Cl/HCO₃ exchange, mediates HCO₃ efflux at pH_i above ~6.7 and thus does not contribute to pH_i recovery from intracellular acidosis (20, 48). In post-MI myocytes equilibrated in HCO₃-buffered solution we found that acid load recovery was not significantly stimulated by ANG II (Fig. 7). Although our results do not reveal the mechanism of this lack of response, a suppressed responsiveness of Na⁺/H⁺ exchange to the peptide is very likely involved. However, we cannot exclude possible contributions by Na⁺-HCO₃ cotransport and Na⁺-independent Cl/HCO₃ exchange, since ANG II is reported to stimulate both transporters in normal adult ventricular muscle (6, 13). Further work is required to characterize the effects of chronic infarct-induced remodeling on these HCO₃-dependent transporters and their responsiveness to vasoactive peptides.

Our results do not reveal the mechanism of reduced sensitivity of Na⁺/H⁺ exchange to ANG II in post-MI myocytes. However, there are several possibilities, including 1) reduction in the density and/or sensitivity of ANG II receptors, 2) suppression of the intracellular signaling pathway leading to protein kinase C activation, and 3) reduction in Na⁺/H⁺ exchanger density and/or the H⁺ sensitivity of the transporter's proton modifier site.

The extent to which mechanism 3 contributes to our findings requires further investigation. However, previous studies of ANG II receptors and intracellular signaling pathways provide possible explanations for our findings. Our losartan experiments indicate that the AT₁ receptor mediates the stimulatory effect of ANG II on Na⁺/H⁺ exchange, in accord with previous findings in normal perfused ferret hearts (13). AT₁-receptor density is reported to increase in rat ventricular myocytes after a relatively brief period (1 wk) of coronary artery ligation (28). In contrast, AT₁-receptor density is decreased in hypertrophied rat ventricle subjected to 8-9 wk of pressure overload (24). Similarly, AT₁ receptor density (2) and expression (14) are reduced in human heart failure. AT₂ density (2) or expression (14) was unchanged. Although the rabbits used in our experiments (8-12 wk post-MI) had not progressed to overt left ventricular failure, these earlier studies raise the possibility that a similar reduction in AT₁-receptor density and expression may contribute, in part, to the decreased ANG II responsiveness we observed. Regarding intracellular signaling, Ito et al. (17) recently reported that the stimulatory effect of phorbol esters on Na⁺/H⁺ exchange was markedly suppressed in hypertrophied rat ventricle, suggesting that the coupling between protein kinase C activation and Na⁺/H⁺ exchange was impaired. A similar mechanism may also be operational in the post-MI myocytes.

We also found that the stimulatory effects of ANG II on contraction and the Ca²⁺ transient were suppressed in post-MI myocytes bathed in HEPES-buffered solution (Figs. 8-10). Recent work suggests that the positive inotropic action of ANG II in normal rabbit ventricular myocytes is mediated, in part, by stimulation of Na⁺/H⁺ exchange (16, 26). The resulting intracellular alkalosis may increase L-type Ca²⁺ current (18) and myofilament Ca²⁺ sensitivity (16). Our finding of reduced ANG II-induced activation of Na⁺/H⁺ exchange may account, in part, for the suppressed contractile response of post-MI myocytes to ANG II. However, factors other than intracellular alkalosis (e.g., intracellular Na⁺ accumulation) must be involved, since, in contrast to normal cells in which pH_i and cell shortening were increased by ANG II, in post-MI myocytes shortening increased but pH_i fell (Fig. 8). A dissociation between pH_i and contractility during exposure to ANG II has also been reported for normal paced cat papillary muscles and was attributed to activation of Cl/HCO₃ exchange (27). Intracellular acidosis reduces ventricular contactility (34, 41) and thus by itself cannot account for the positive inotropic effect of ANG II we observed in post-MI myocytes. However, we previously showed in normal ventricular myocytes bathed in HEPES-buffered solution (no added CO₂ or HCO₃) that ANG II has a dual effect on pH_i (26): one mechanism acts to stimulate forward Na⁺/H⁺ exchange and increase pH_i; the other, which appears to be metabolic in origin, decreases pH_i. The final effect of ANG II on steady-state pH_i depends on the relative magnitudes of acid extrusion via Na⁺/H⁺ exchange and acid production via the metabolic pathway. Thus the action of ANG II to decrease steady-state pH_i in post-MI myocytes bathed in HEPES-buffered solution may reflect predominance of the acid production pathway. However, the accompanying activation of Na⁺/H⁺ exchange would increase intracellular Na⁺ and, because of Na⁺/Ca²⁺ exchange, would raise cytosolic Ca²⁺ and thus increase Ca²⁺ loading of the sarcoplasmic reticulum. The resulting increase in sarcoplasmic reticulum Ca²⁺ release would act to increase cell shortening, albeit to a smaller extent in post-MI myocytes. Consistent with this hypothesis is our finding that ANG II increased the amplitude of the Ca²⁺ transient in normal and post-MI myocytes (Figs. 9 and 10).

We did not examine the effect of ANG II on contraction and the Ca²⁺ transient in an HCO₃-CO₂ buffer system. However, the suppressed stimulatory effect of ANG II on Na⁺/H⁺ exchange in post-MI myocytes will also be present in this system and by itself would act to reduce ANG II-induced increases in both parameters. The lack of a significant stimulatory effect of ANG II on J_H⁺ in post-MI myocytes in an HCO₃-CO₂ buffer supports this hypothesis.

The physiological significance of a reduced ANG II sensitivity of Na⁺/H⁺ exchange, the Ca²⁺ transient, and contractility in spared post-MI myocytes is unclear. However, suppression of the positive inotropic action of ANG II may help preserve intracellular ATP. Similarly, suppression of ANG II-induced increases in Na⁺/H⁺ exchange and the Ca²⁺ transient may help attenuate the myocardial damage and arrhythmias associated with intracellular Ca²⁺ overload during ischemia-reperfusion (19).