INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

AMONG A WIDE VARIETY OF protein kinases present in mammalian cells, two multifunctional protein kinases, calcium/phospholipid-dependent protein kinase [protein kinase C (PKC)] and cAMP-dependent protein kinase [protein kinase A (PKA)], are thought to mediate several phosphorylation reactions in the myocardium (7, 9, 37). Both of these protein kinases are known to regulate cation transport, contractile force development, metabolic processes, gene expression, and cellular growth in the heart (9, 37). Molecular cloning studies (9, 24) have indicated that PKC exists as a family of at least 12 distinct isoforms. The conventional PKC isoforms (, , and ) contain a Ca²⁺-binding domain, which accounts for their activation by Ca²⁺. The novel PKC isoforms (, , , and ) lack the putative Ca²⁺-binding domain and do not require Ca²⁺ for maximal enzymatic activation. Atypical PKC isoforms (, , and ) are distinguished from other members of the PKC gene family by the presence of only a single copy of a cysteine-rich motif. Activation of angiotensin II (ANG II) receptors, ₁-adrenergic receptors, and endothelin-1 receptors has been shown to stimulate PKC via Gq-coupled phospholipase C (PLC-) (9, 37, 42). In contrast, PKA is activated by catecholamines through stimulatory G protein-coupled -adrenergic receptors (9, 14).

Previous work from our laboratory (44) has demonstrated increased activities of cardiac PKC and PKA due to congestive heart failure in cardiomyopathic hamsters. Increased cardiac PKC activity has also been shown in pressure-overloaded cardiac hypertrophy in the rat (13) and pressure-overloaded heart failure in guinea pigs (38), as well as in human failing hearts (5). Varying degrees of changes in PKC activities have been observed in cardiac dysfunction due to diabetes (17, 21, 22, 40, 45). Furthermore, transgenic mice with cardiac overexpression of PKC- or PKC- were found to exhibit gross cardiac hypertrophy and diminished ventricular function (39, 43). The PKA activity has also been observed to increase in cardiac hypertrophy due to volume overload in rats (19). Transgenic mice with overexpression of PKA in the heart have been reported to develop dilated cardiomyopathy and reduced cardiac contractility; however, no changes in PKA activity were seen in the failing human heart or in myocardial infarction (MI) (3, 28). Although cardiac hypertrophy, heart failure, and cardiac dysfunction are known to occur as a consequence of MI (1, 11, 30, 36, 41), there is no literature regarding changes in PKC activities in the infarcted heart. Accordingly, this study was undertaken to examine the status of PKC activities during the development of congestive heart failure in a rat model of MI. Some experiments were also carried out to examine whether the changes in PKC in the failing heart are due to corresponding changes in the contents of PKC isozymes.

It is now well known that PKC is activated by ANG II through the PLC--mediated mechanisms in cardiomyocytes (37). Furthermore, ANG II-induced activation of PKC has been demonstrated to result in the stimulation of cardiac gene expression, cell growth, and remodeling of the myocardium (4, 9, 37). Accordingly, the stimulation of the renin-angiotensin system (RAS) is considered to play a critical role in the activation of PKC that regulates the hypertrophic process and cardiac performance (38). Because the RAS is activated in congestive heart failure (12) and treatment of infarcted animals with ANG-converting enzyme (ACE) inhibitors has been shown to produce beneficial effects on heart function and attenuate changes in PLC activities (36, 41), it was planned to test the effect of imidapril, a long-acting ACE inhibitor (41), on PKC activities and PKC isozyme contents in the failing heart. PKA activity and content of the MI-induced failing hearts with or without imidapril treatment were also maintained to test whether changes in PKC are of a specific nature.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Experimental model and hemodynamic assessment. Experiments were conducted in accordance with the "Guide to the Care and Use of Experimental Animals" issued by the Canadian Council of Animal Care, and the protocols were approved by the University of Manitoba Animal Care Committee. MI was induced in male Sprague-Dawley rats (175-200 g) by occlusion of the left coronary artery as described earlier (1, 11, 35, 36). The mortality of rats on coronary occlusion was ~30% within 48 h. The sham control rats were operated in the same way except that the coronary artery was not ligated. These animals were fed rat chow and water ad libitum and then maintained for 1, 2, 4, and 8 wk after the coronary artery ligation before the assessment of cardiac function and biochemical changes. In another series of experiments, some sham and MI rats were divided into untreated and treated groups at 4 wk after the operation. The untreated infarcted animals received distilled water, whereas treated animals were given imidapril hydrochloride dissolved in distilled water at a concentration of 1 mg/ml once a day by gavage at a volume of 1 ml · kg¹ · day¹ for 4 wk. All animals were assessed hemodynamically before death and the heart was dissected out. The left ventricle (LV) and right ventricle (RV) were separated, weighed, frozen in liquid nitrogen, and stored at 70°C. Also, the scar tissue from the infarcted hearts was separated and weighed. The removal of scar from the LV was necessary to obtain the noninfarcted myocardium for analysis, as used in other investigations (1, 11, 35), and to determine whether or not to use the viable LV tissue (including the septum) for biochemical studies. Because the scar weight-to-total LV weight ratio (including septum and scar tissue) was found to exhibit a linear relationship with infarct size, as measured morphometrically (1, 11, 35, 36), the scar weight-to-total LV weight ratio was used as a marker to determine the extent of scar size. It should be pointed out that ~10% of the untreated and treated animals showed small infarct size (scar weight-to-total LV weight ratio <15% corresponding to scar size <30% of the free LV wall). Thus the hemodynamic data from the animals showing small infarct were not included and the cardiac tissue from these animals was discarded. The lung wet weight-to-dry weight ratio (an index of pulmonary congestion) and heart weight-to-body weight ratio (an index of cardiac hypertrophy) were also measured in experimental animals. The heart weight-to-body weight ratio included both ventricles, including infarct. For hemodynamic studies, the animals were anesthetized with an intraperitoneal injection of ketamine (60 mg/kg) and xylazine (10 mg/kg). The LV systolic pressure, LV end-diastolic pressure (LVEDP), heart rate, rate of pressure development (+dP/dt), rate of pressure decay (dP/dt), and mean arterial blood pressure (MAP) were measured in these anesthetized animals according to the procedure described earlier (1, 11, 35, 36).

Preparation of tissue extract for enzyme determination. The preparations of tissue extract for PKC was carried out by the method described earlier (21, 44); all procedures were carried out at 4°C. The ventricular tissue (50 mg) was minced in 1 ml of buffer A (50 mM Tris · HCl, 0.25 M sucrose, 10 mM EGTA, 4 mM EDTA, 20 µg/ml leupeptin, and 200 U/ml aprotinin, pH 7.5) and homogenized (Polytron PT3000, Brinkmann Instruments; Mississauga, Ontario, Canada) at setting 8 for 2 × 30 s and sonicated for 2 × 15 s. In one set of experiments, the homogenate was incubated with 1% Triton X-100 (Sigma; St. Louis, MO) on ice for 60 min to solubilize PKC enzyme, which is bound with subcellular structures. This Triton X-100-treated homogenate was then centrifuged at 100,000 g for 60 min in an ultracentrifuge (model L70, Beckman Instruments), and the supernatant obtained was labeled as the homogenate fraction. In another set of experiments, the homogenate without Triton X-100 treatment was centrifuged at 100,000 g for 60 min to separate the soluble and particulate-bound enzyme. The resulting supernatant was labeled as the cytosolic fraction, whereas the pellet was resuspended in 1 ml of buffer A with 1% Triton X-100 and incubated on ice for 60 min. The resuspended pellet was centrifuged at 100,000 g for 60 min and this supernatant was labeled as the particulate fraction. For preparation of tissue extract for PKA determination, ~50 mg of frozen cardiac tissue were homogenized in 1 ml buffer B at pH 7.4 containing (in mM) 5 histidine-HCl, 0.1 phenylmethylsulfonyl fluoride, 50 KH₂PO₄, 25 NaF, 10 EDTA, 750 KCl, and 0.2 dithiothreitol. After gentle mixing, the homogenate was centrifuged at 100,000 g for 60 min at 4°C and the supernatant was used for analysis of PKA activity and protein level (3, 19).

Assays of PKC and PKA activities. PKC activities in small samples of nonpurified homogenate, cytosolic, and particulate fractions from the ventricular tissue were measured in the presence of okadaic acid, a highly specific inhibitor of type 1 and type 2A phosphatases (2, 15), by following methods described elsewhere (21, 44). The Ca²⁺-dependent PKC activity was determined with a PKC assay kit (Upstate Biotechnology; Lake Placid, NY) in the reaction buffer C containing (in mM) 20 MOPS, pH 7.2, 25 -glycerol phosphate, 1 sodium orthovanadate, 1 dithiothreitol, and 4 CaCl₂. Substrate cocktail containing 500 µM PKC substrate peptide in buffer C, inhibitor cocktail containing 2 µM PKA inhibitor peptide in buffer C, and lipid activator containing 0.5 mg/ml phosphatidyl serine and 0.05 mg/ml diglyceride in buffer C was used. The Ca²⁺-independent PKC activity was determined in a reaction buffer D containing (in mM) 20 MOPS (pH 7.2), 25 -glycerol phosphate, 1 sodium orthovanadate, 1 dithiothreitol, and 1.25 EGTA. Substrate cocktail (specific for PKC- and - isozymes; Quality Controlled Biochemicals; Hopkinton, MA) containing 500 µM PKC substrate peptide in buffer D, inhibitor cocktail containing 2 µM PKA inhibitor peptide in buffer D, and lipid activator containing 0.5 mg/ml phosphatidyl serine and 0.05 mg/ml diglyceride in buffer C was used. The sequence of the peptide substrate (supplied by Upstate Biotechnology) used for PKC activity assay was QKRPSQRSKYL. The reactions for both Ca²⁺-dependent and Ca²⁺-independent PKC activities were initiated by the addition of [-³²P]ATP (10 µl) and allowed to proceed at 30°C for 10 min. The incorporation of ³²P from -³²P into a synthesized substrate, which is a more specific substrate for PKC than Histone H-1 protein (40, 45), was measured as described elsewhere (21, 44). On the other hand, PKA activity was determined with the use of the PKA assay kit (Upstate Biotechnology). The reaction was initiated by adding [-³²P]ATP (1 part of [-³²P]ATP in 9 parts of kit ATP solution). The PKA activity was assayed as described earlier (44) by measuring the incorporation of ³²P from [-³²P]ATP into the substrate (3, 19).

Analysis of PKC isozyme and PKA protein content. The relative protein content of PKC-, -, -, and - isozymes was obtained by running 10% mini-SDS-PAGE, followed by Western blot analysis with homogenate, cytosolic, and particulate fractions (21, 44). The SDS-PAGE loading buffer contained 0.25 M Tris · HCl (pH 6.8), 8% (wt/vol) SDS, 45% glycerol, 20% -mercaptoethnol, and 0.006% bromophenol blue. The proteins in both fractions separated by SDS-PAGE were electroblotted to Immobilon-P transfer membranes (Millipore; Bedford, MA), which were incubated with polyclonal anti-PKC-, -, -, and - isozyme antibodies (Life Technologies) for 1 h at a concentration of 1:1,000, respectively, and were subsequently incubated with biotinylated anti-rabbit IgG (1:5,000; Amersham) for 40 min and then finally with streptavidin conjugated horseradish peroxidase (1:5,000; Amersham) for 30 min. It should be pointed out that a recombinant standard (Bio-Rad Laboratories; Hercules, CA) was run on SDS-PAGE with each sample to confirm the molecular weight of PKC isozymes. Ponceau staining of the blots was performed to ensure no difference between control and experimental samples with respect to protein loading and protein transfer. The content of PKC isozyme was determined with an imaging densitometer (model GS-670, Bio-Rad) with the Image Analysis Software version 1.0. The relative protein content for each experimental sample was expressed as a percentage of the respective control value (band density of the sham control sample was considered as 100%). The information about the cross-reactivity of PKC isoform antibodies (supplied by Life Technologies) indicated that antibodies directed against PKC- recognize PKC- and to a lesser extent PKC-. Nonetheless, the bands for PKC-, -, and - were distinguished on the basis of molecular weight. Because the antibody against PKC- did not distinguish PKC-I and PKC-II, the band for PKC- was considered to be due to both I and II isoforms.

Data analysis. Data were expressed as means ± SE. The differences among different groups were evaluated statistically by one-way ANOVA, followed by the Newman-Keuls test. A P value <0.05 was taken to represent a significant difference.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Time course changes in general characteristics, hemodynamics, and cardiac PKC activities in experimental rats. Coronary occlusion in rats resulted in extensive LV infarction and the noninfarcted cardiac muscle underwent significant hypertrophy at 1, 2, 4, and 8 wk after the operation. These changes are reflected by the presence of a large scar (average infarct size varied from 38 to 42% of the LV free wall corresponding to the average values in the range of 19-21% for scar weight-to-total LV weight ratio in different groups) and increased ratio of heart weight-to-body weight ratio at all time points (Table 1). These values for infarct size in animals used in this study are comparable to those reported by others (20). The RV weight and viable LV weight were increased at 2, 4, and 8 wk after MI was induced compared with the respective sham control animals. There was a significant increase in the wet weight-to-dry weight ratio of the lungs in 4- and 8-wk infarcted animals, indicating the presence of pulmonary edema (Table 1). An increase in LVEDP and a decrease in both +dP/dt_max and dP/dt_max were observed in 1-, 2-, 4-, and 8-wk MI animals (Table 1). These results are consistent with earlier observations in this experimental model (1, 11, 35), which have indicated that the experimental animals at 4 and 8 wk after the coronary occlusion are at early and moderate stages of congestive heart failure, respectively. The Ca²⁺-dependent PKC activity increased by 53, 79, 47, and 159% in the LV homogenate, whereas the Ca²⁺-independent PKC activity was elevated by 47, 88, 30, and 201% in the LV homogenate at 1, 2, 4, and 8 wk of MI compared with control values, respectively (Table 1). On the other hand, the Ca²⁺-dependent PKC activity in the RV homogenate was increased by 54, 24, 34, and 97% and the Ca²⁺-independent PKC activity was augmented by 47, 24, 29, and 238% of control values at 1, 2, 4, and 8 wk of MI, respectively.

Effect of imidapril treatment on general characteristics, hemodynamics, and cardiac PKC activities in experimental rats. The beneficial effects of imidapril treatment on heart function and cardiac PKC activities were tested by treating the 4-wk experimental animals with imidapril for 4 wk. The results in Table 2 indicate cardiac hypertrophy, lung congestion, elevated LVEDP, depressed +dP/dt, and dP/dt as well as increased Ca²⁺-dependent and Ca²⁺-independent PKC activities in both LV and RV homogenates in the 8-wk infarcted animals. All of these changes were partially normalized by treatment with imidapril. Treatment of sham control animals with imidapril did not show any significant effect on any of these parameters (Table 2). The values for LV systolic pressure and MAP in the untreated and treated groups were not different from each other. Furthermore, the average scar weight-to-total LV weight ratio in the untreated and treated animals were 20.3 ± 0.08 and 22.8 ± 1.1; these values correspond to infarct size of ~41 and 46% of the free LV wall area and were not different (P > 0.05) from each other.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Protein kinase C (PKC) activities in cytosolic (C) and particulate (P) fractions in left (LV) and right ventricles (RV) from sham control (sham), imidapril-treated sham (sham + Imp), myocardial infarcted (MI), and imidapril-treated MI (MI + Imp) rats 8 wk after operation. A: Ca2+-dependent PKC activity in cytosolic fraction; B: Ca2+-dependent PKC activity in particulate fraction; C: Ca2+-independent PKC activity in cytosolic fraction; D: Ca2+-independent PKC activity in particulate fraction. Values are means ± SE for 6 experiments in each group. *P < 0.05, significantly different from sham control; #P < 0.05, significantly different from MI.

Relative protein content of PKC isozymes in infarcted and imidapril-treated infarcted animals. The relative protein contents of PKC-, -, -, and - isozymes in homogenate, cytosolic, and particulate fractions of the LV and RV from 8-wk sham, imidapril-treated sham, infarcted, and imidapril-treated infarcted animals were determined with the use of Western blot analysis. The typical bands representing PKC-, -, -, and - isozymes in these fractions of rat hearts are shown in Figs. 2-4. Polyclonal antibodies to PKC isozymes detected proteins at 76 kDa for -isozyme, 77 kDa for -isozyme, 83 kDa for -isozyme, and 67 kDa for -isozymes. There was a nonspecific band at ~80 kDa below the bands for -isozyme in Figs. 2-4; however, because its identity is unknown, this band was not included in the densitometric analysis. In LV homogenate, the relative protein contents of the PKC-, -, -, and - isozymes were significantly elevated, whereas in the RV homogenate, only PKC-, -, and - isozymes were increased in the infarcted animals (Fig. 2). The relative protein contents for PKC-, -, -, and - isozymes were also increased in LV cytosolic and particulate fractions in the infarcted animals except PKC- isozyme content in the cytosolic fraction was unaltered (Fig. 3). On the other hand, in PKC-, -, and - isozymes, unlike the PKC- isozyme, the contents were increased in the RV cytosolic and particulate fractions from infarcted animals, except that no changes were seen in the RV particulate fraction (Fig. 4). The MI-induced increases in PKC isozymes in both LV and RV cytosolic and particulate fractions were attenuated by imidapril treatment, which showed no effect on the sham control animals (Figs. 2-4).

View larger version (57K): [in this window] [in a new window]   Fig. 2.   Typical immunoblots (top) and analysis of results (A-D) for the relative protein contents of cardiac PKC isoforms in homogenate fraction in LV and RV from sham, sham + Imp, MI, and MI + Imp rats at 8 wk after surgery. Values are means ± SE of 6 experiments in each group. *P < 0.05, significantly different from sham control; #P < 0.05, significantly different from MI.

View larger version (51K): [in this window] [in a new window]   Fig. 3.   Typical immunoblots (top) and analysis of results (A-D) for the relative protein contents of cardiac PKC isoforms (, , , and ) in cytosolic and particulate fractions in LV from sham, sham + Imp, MI, and MI + Imp rats 8 wk after surgery. Values are means ± SE of 6 experiments in each group. *P < 0.05, significantly different from sham control; #P < 0.05, significantly different from MI.

View larger version (52K): [in this window] [in a new window]   Fig. 4.   Typical immunoblots (top) and analysis of results (A-D) for the relative protein contents of cardiac PKC isoforms (, , , and ) in cytosolic and particulate fractions in the RV from sham, sham + Imp, MI, and MI + Imp rats 8 wk after operation. Values are means ± SE of 6 experiments in each group. *P < 0.05, significantly different from control; #P < 0.05, significantly different from MI.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Immunoblots of standard and different samples (lanes A-E) (top) and analysis of results (bottom) for the absolute levels of PKC isoforms ( and ) in the LV and RV of control rats.

PKA activity and relative protein content. The activity and relative protein content of PKA were examined in homogenates from the LV and RV of sham, imidapril-treated sham, untreated infracted, and imidapril-treated infarcted animals. The results showed that there were no significant changes in LV and RV PKA activity and protein content in the infarcted animals (Fig. 6). Imidapril treatment did not affect PKA activity and protein content in the sham or infarcted animals compared with values from untreated animals.

View larger version (42K): [in this window] [in a new window]   Fig. 6.   Cardiac protein kinase A (PKA) activities and relative protein content in the LV and RV from sham, sham + Imp, MI, and MI + Imp rats 8 wk after operation. Values are means ± SE of 6 experiments. Bottom, typical immunoblots for PKA protein are shown. No change was found in these groups.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study, we have observed an increase in both Ca²⁺-dependent and Ca²⁺-independent PKC activities in the viable LV as well as LV dysfunction in 1-, 2-, 4-, and 8-wk infarcted animals. Because the activation of PKC by phorbol esters has been shown to exert a negative inotropic effect on the heart due to phosphorylation of troponin I and T and subsequent inhibition of myofibrillar ATPase activity (25), it is possible that the sustained increase in PKC activity may be involved in depressing the LV function on inducing MI. Stimulation of PKC by phorbol esters has also been shown to produce changes in cytosolic Ca²⁺ and negative inotropic effect in cardiomyocytes (6). Although the activation of PKC by phorbol esters has been reported to decrease cardiac sarcoplasmic reticulum Ca²⁺ transport (31) and can explain the depression in LV +dP/dt and dP/dt in the infarcted hearts, the mechanism of decrease in sarcoplasmic reticular Ca²⁺ uptake by the activation of PKC are not clear. Nonetheless, increased PKC activities and cardiac dysfunction have also been observed in diabetic animals (17, 21, 22). Furthermore, attenuation of both increased PKC activities and LV dysfunction in the infarcted animals was found to occur on treatment with imidapril, which has been reported to produce beneficial effects as a consequence of ACE inhibition in this model of heart failure (41). Increased PKC activities have also been observed in failing human hearts (5) as well as in different experimental models of heart failure (32, 38, 44). Thus, in view of such observations, it appears that a sustained increase in PKC activities may be involved in the genesis of contractile dysfunction in heart failure. This suggestion is further supported by the fact that overexpression of PKC isozymes resulted in diminished heart function in transgenic mice (43). Whereas the attenuation of increased PKC activity and depressed cardiac function by treatment of MI animals with imidapril can be explained on the basis of suppression of the RAS, imidapril treatment was found to exert no effect in the control animals. Such results may indicate that both cardiac function and PKC isozymes in normal physiological conditions may not be under the influence of the RAS.

The increased PKC activities in the LV homogenate in infarcted animals does not appear to be due to translocation of cytosolic enzyme to the particulate compartment of the cell because the PKC activities in both LV cytosolic and particulate fractions were increased due to MI. Such an increase in PKC activities in LV is likely to be due to increase in the expression of PKC-, -, -, and - isozymes in the myocardium because the relative contents of these isozymes (except cytosolic PKC- content) were increased in LV homogenate, cytosolic, and particulate fractions on inducing MI. Furthermore, treatment of infarcted animals with imidapril not only partially prevented the increase in LV PKC activities, it also had a similar effect on the isozyme contents in the LV homogenate, cytosolic, and particulate fractions. Whether the observed increase in LV PKC isozymes is due to translational or transcriptional changes in the myocardium remains to be investigated. However, it should be pointed out that PKC- isozyme, a predominant isoform in cardiomyocytes (34), has been shown to be associated with sarcomeres on activation (10) and be responsible for the phosphorylation of troponin I (22, 25). On the other hand, PKC- isoforms have been reported to stimulate the promoter of -myosin heavy chain in the myocardium (18). Accordingly, it seems possible that the depressed LV function in the infarcted heart may be due to increases in both PKC- and - isozyme contents. Because the role of PKC- and - isoforms in altering the function of any subcellular organelle or metabolic site in the myocardium has not been well established, it is difficult to speculate on the exact functional significance of increased PKC- and - isozymes in failing LV from the infarcted animals. However, the translocation of PKC- and - has been reported to occur in failing hearts due to aortic banding in guinea pigs and this change was attenuated by treatment with ramipril, an ACE inhibitor (38).

PKC isozymes are known to serve in the signal transduction mechanism and thus play a crucial role in the development of cardiac hypertrophy (23, 29, 34, 39). Transfection of cardiomyocytes with constitutively active PKC was demonstrated to activate genes for atrial natriuretic factor and -myosin heavy chain, which are associated with cardiac hypertrophy (8, 18, 26). Previous studies (13) have shown that the expression of both PKC- and - isozymes is increased in cardiac hypertrophy induced in rats by aortic banding. Furthermore, mechanical stretch has been reported to increase PKC-, but not PKC-, and induce cardiac hypertrophy (27). It is thus possible that cardiac hypertrophy observed in the LV due to MI may be caused by an increase in the content of PKC isozymes including content of PKC-. This view is consistent with the observations that treatment of infarcted animals with imidapril was found to not only reduce the extent of LV hypertrophy but also the level of LV PKC isozymes.

Whereas the Ca²⁺-dependent and Ca²⁺-independent PKC activities were increased in both LV and RV homogenates on inducing MI, some differences between LV and RV were apparent with respect to changes in PKC isozyme contents. For example, the infarcted LV showed an increase in PKC- content in homogenate, cytosolic, and particulate fractions, but no such changes were seen in the RV. Furthermore, unlike RV, no increase in cytosolic PKC- content was detected in the LV. On the other hand, no changes in PKC- and - isozyme contents were seen in the RV, whereas the contents of these isoforms were increased in the LV after MI. Such differential changes in the LV and RV in the infarcted heart indicate that PKC isozymes in different regions of the heart may be regulated differentially. Differences in the behavior of LV and RV with respect to changes in the sarcoplasmic reticular Ca²⁺ pump as well as adenylyl cyclase activities have also been reported during the development of congestive heart failure due to MI (1, 35). Nonetheless, the increase in PKC activities as well as PKC isozymes in both LV and RV in the infarcted animals may be of some specific nature because neither the PKA activities nor PKA protein contents were altered in both LV and RV on inducing MI. Although activation of PKA has been shown to represent a growth promoting signal (19, 33), our data are in agreement with other reports that PKA activity had no relation to the development of cardiac hypertrophy due to MI or pressure overload (28). Furthermore, unlike PKC, no change in PKA activity was observed in the failing human heart (3).

Taken together, the data in this study are consistent with the view that the increased PKC activities in the hypertrophied and failing heart subsequent to MI are due to increased expression of PKC isozymes and that the sustained increase in PKC activity may be involved in cardiac dysfunction on occluding the coronary artery. However, it should be recognized that the observed changes in cardiac function in the MI-induced heart failure may not be entirely due to cardiomyocyte-specific adaptation in PKC signaling because the contribution of alterations in PKC activity from other cell types such as fibroblasts (16) cannot be excluded. Furthermore, despite the association of increased PKC activity and cardiac dysfunction in the failing heart, the exact significance of the observed changes in PKC isozymes in heart failure due to MI remains to be established by the use of PKC inhibitors in this experimental model. The partial prevention of changes in cardiac PKC isozymes and cardiac dysfunction in heart failure due to MI by the ACE inhibitor imidapril indicates that mechanisms other than those mediated by increased formation of ANG II in heart failure may also be implicated in the genesis of cardiac dysfunction. Although ACE inhibition is generally considered to confer beneficial effects on the failing heart by reducing afterload, we did not observe any changes in the MAP in the MI animals on treatment with imidapril for a period of 4 wk. Also, this study does not provide any information regarding the cause-and-effect relationship between changes in heart function and PKC isozyme expression. Accordingly, the exact mechanisms responsible for the observed increase in PKC isozymes during the development of heart failure due to MI as well as for the partial prevention of PKC activities in the failing hearts on treatment with imidapril require further studies.