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Am J Physiol Heart Circ Physiol 276: H53-H62, 1999;
0363-6135/99 $5.00
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Vol. 276, Issue 1, H53-H62, January 1999

Effect of angiotensin-converting enzyme inhibition on protein kinase C and SR proteins in heart failure

Yasuchika Takeishi, Ajit Bhagwat, Nancy A. Ball, Darryl L. Kirkpatrick, Muthu Periasamy, and Richard A. Walsh

Division of Cardiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

We tested the hypothesis that activation of protein kinase C (PKC) isoforms in pressure-overload heart failure was prevented by angiotensin-converting enzyme (ACE) inhibition, resulting in normalization of cardiac sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) 2a and phospholamban protein levels and improvement in intracellular Ca2+ handling. Aortic-banded and control guinea pigs were given ramipril (5 mg · kg-1 · day-1) or placebo for 8 wk. Ramipril-treated banded animals had lower left ventricular (LV) and lung weight, improved survival, increased isovolumic LV mechanics, and improved cardiomyocyte Ca2+ transients compared with placebo-treated banded animals. This was associated with maintenance of SERCA2a and phospholamban protein expression. Translocation of PKC-alpha and -epsilon was increased in placebo-treated banded guinea pigs compared with controls and was attenuated significantly by treatment with ramipril. We conclude that ACE inhibition attenuates PKC translocation and prevents downregulation of Ca2+ cycling protein expression in pressure-overload hypertrophy. This represents a mechanism for the beneficial effects of this therapy on LV function and survival in heart failure.

angiotensin II; hypertrophy; sarcoplasmic reticulum calcium adenosine triphosphatase; phospholamban

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

CONGESTIVE HEART FAILURE (CHF) is a major and growing public health problem with a high mortality rate (16). Myocardial hypertrophy is an adaptive response to hormonal and mechanical stimuli that increase cardiac work (33, 35). Initially, the resultant increased work is compensatory to normalize wall stress and maintain cardiac function. If the stimulus for pathological hypertrophy is sufficiently intense and prolonged, decompensated hypertrophy ensues and ultimately leads to CHF. However, precise mechanisms accounting for the transition from compensated to decompensated hypertrophy have not yet been completely characterized (33, 35).

In cardiac muscle, the sarcoplasmic reticulum (SR) plays an important role in excitation-contraction coupling through the regulation of intracellular free Ca2+ concentration (1). Muscle relaxation is initiated by Ca2+ transport from the cytosol into the SR by the cardiac SR Ca2+ ATPase (SERCA) 2a. The function of SERCA2a is regulated by phospholamban (6). We reported that downregulation of SERCA2a and phospholamban is a marker of the transition from compensated hypertrophy to a decompensated stage of CHF (12). However, the exact signaling pathways affecting downregulation of SERCA2a and phospholamban are poorly understood. In vitro studies using neonatal cardiomyocytes showed that protein kinase C (PKC) activation by phorbol ester decreases SERCA2 mRNA and protein expression that is associated with a reduction of Ca2+ transport by the SR (9, 21, 22). These observations suggested that downregulation of SERCA2 may occur by a PKC-related process that can be attenuated by angiotensin-converting enzyme (ACE) inhibitors.

The activation of the angiotensin II-mediated signal transduction pathway has been implicated in in vitro neonatal cardiomyocyte hypertrophy (24, 37). We recently demonstrated that acute left ventricular (LV) stretch activates PKC, and this activation is attenuated by an angiotensin II type-1 receptor antagonist in the adult guinea pig heart (19). It has also been reported that PKC expression is increased in cardiac hypertrophy induced by pressure overload in rats (7). We have also shown that transgenic Galpha q overexpression in the mouse heart causes PKC activation and a dilated cardiomyopathy with overt heart failure (5a) and that PKC expression is elevated in failed human heart (3). Finally, postnatal cardiac specific overexpression of the PKC-beta 2 isoform in transgenic mice causes a cardiomyopathy with LV hypertrophy (LVH) and in vivo cardiac dysfunction (34). Taken together, these observations suggest that PKC activation plays a critical role in the development of cardiac hypertrophy and heart failure.

ACE inhibitors have been shown to regress LVH in animals (2, 14) and human subjects (30). The prevention trials such as the Cooperative North Scandinavian Enalapril Survival Study (CONSENSUS) and Studies of Left Ventricular Dysfunction (SOLVD) clearly showed that therapy with an ACE inhibitor improves LV ejection fraction and reduces the rate of related hospitalizations in patients with CHF (5, 28). On the basis of these findings, ACE inhibitors have been used increasingly for treatment of CHF. Because angiotensin II plays a central role in the activation of PKC that regulates gene expression, intracellular Ca2+ levels, the hypertrophy process, and contractile state (3, 5a, 7, 17, 19, 34), modulation of PKC activity by ACE inhibition may contribute to the beneficial effects of this pharmacotherapy.

The present study was designed to test the hypothesis that activation of PKC isoforms in pressure-overload heart failure was prevented by ACE inhibition associated with normalization of SERCA2a and phospholamban protein levels and improvement in intracellular Ca2+ handling.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Preparation of animals. Subtotal descending thoracic aortic banding was performed in adult male Charles River guinea pigs (250-300 g) as described previously (12). After anesthesia with pentobarbital sodium (25 mg/kg ip), the descending thoracic aorta was exposed through an intercostal incision. A uniform degree of constriction around the descending thoracic aorta was produced by tying a 2-0 surgical silk ligature tightly around a 6-mm length of hypodermic tubing with an external diameter of 1.24 mm. The tubing was then withdrawn from the ligature, and the chest incision was surgically closed. Sham-operated control animals underwent the same operation, but the aorta was not banded. Aortic-banded animals and sham-operated control animals were housed and fed under identical conditions. One day after surgery, the banded and sham-operated animals were randomized to receive either ramipril (5 mg · kg-1 · day-1) or vehicle. Ramipril was administered orally for a total duration of 8 wk by a tuberculin syringe after being dissolved in orange juice. All surgery was performed by the same investigator, and <10% operative mortality was observed in the banded animals. During the 8-wk treatment period, guinea pigs were monitored daily for determination of survival rate.

Heart perfusion. After 8 wk of treatment with ramipril or vehicle, the guinea pigs were anesthetized with intraperitoneal ketamine (54 mg/kg), acepromazine (1.8 mg/kg), and xylazine (10.9 mg/kg) and heparinized. Hearts were quickly excised and perfused by the Langendorff method with a modified Krebs-Henseleit buffer containing (mM) 113.8 NaCl, 4.7 KCl, 1.1 MgSO4, 0.12 KH2PO4, 23.6 NaHCO3, 2.5 CaCl2, 6.0 mannitol, and 11.0 glucose. The solution was saturated with 95% O2-5% CO2 (pH 7.4) at 37°C. A saline-filled latex balloon attached to a 3-F micromanometer catheter (Millar Instruments) was inserted into the LV through the mitral valve for pressure measurements (12). The balloon was inflated to achieve initial minimum diastolic pressure of 10 mmHg and was kept isovolumic during the perfusion. Heart rate and aortic and LV pressure were continuously monitored on a Gould MK200 multichannel recorder interfaced to an IBM computer. Analog signals were digitized on-line at a sampling frequency of 1,000 Hz, and hemodynamic parameters were derived by custom-designed software. Ten to fifteen beats were averaged for each condition, and premature contractions were excluded from the analysis. The maximum rate of isovolumic pressure development (+dP/dt) was calculated and used as an index of LV contractility. The minimum rate of pressure development (-dP/dt) was measured to assess changes in the rate of isovolumic relaxation. In addition, the time to peak pressure (TPP) and time to 50% isovolumic relaxation (RT1/2) were also quantified. These values were normalized by developed pressure (DP) as TPPc (TPP/DP × 10-1) and RT1/2,c (RT1/2/DP × 10-1). The coronary flow rate was adjusted to 10 ml · min-1 · g net heart wt-1 and was kept constant throughout the experiment.

Preparation of isolated LV cardiomyocytes. LV myocytes were isolated from the hearts of guinea pigs as previously described (11, 32). Briefly, the heart was rapidly excised and placed in a dish of oxygenated Ca2+-free Joklik's modified buffer pH 7.2 (GIBCO BRL). The aorta was cannulated with a 16-gauge needle, flushed briefly with buffer, and mounted onto a perfusion apparatus. The right ventricular outflow tract was excised, and the coronary arteries were perfused at 10 ml/min first with Ca2+-free Joklik's buffer for 4 min followed by Joklik's buffer containing 25 µM Ca2+, 90 U/ml collagenase I, 90 U/ml collagenase II (Worthington Biochemical), 1% albumin, and 2% donor calf serum, pH 7.2. The perfusion temperature was maintained at 37°C, and all buffers were continuously bubbled with 95% O2-5% CO2. After 15-20 min of perfusion, the heart was removed from the perfusion apparatus and transferred to a watch glass containing low-Ca2+ Joklik's buffer supplemented with 25 µM Ca2+ and 2% donor calf serum. The LV was isolated, minced, and gently pipetted into a 20-ml conical tube containing 10 ml of the buffer. The tissue was agitated to release loosened cells into the solution, which were then allowed to settle. Supernatant containing the isolated cells was immediately transferred to a new 50-ml conical tube. Isolation of the cells was repeated four times, and the cell supernatants were pooled. The pooled supernatant was centrifuged at 500 rpm for 1 min, and the cell pellet was resuspended in 20 ml of low-Ca2+ Joklik's buffer. After the cells were allowed to settle for 15 min, they were resuspended in physiological buffer (in mM: 132 NaCl, 4.8 KCl, 1.2 MgCl2 · 6H2O, 5 glucose, and 10 HEPES; pH 7.2) supplemented with 2.5 mM Ca2+.

We typically had a yield of viable myocytes of ~80% for both banded and sham-operated guinea pigs. Viable cells were carefully selected on the basis of standard morphological criteria used by our laboratory (5a, 11, 32, 34).

Measurement of intracellular calcium. Cytosolic free Ca2+ was measured by ratio imaging of fura 2 fluorescence as described previously (11, 32). In brief, isolated cardiomyocytes were loaded with fura 2 by incubation of a 1-ml suspension for 30 min at 37°C with fura 2 for a final concentration of 7 µM in low-Ca2+ Joklik's buffer. Fura-loaded myocytes were allowed to settle, and the pellet formed was resuspended in the physiological buffer described in Preparation of isolated LV cardiomyocytes. The fura-loaded cells were placed in a perfusion chamber on the stage of a microscope (Olympus IMT-2) and constantly superfused with oxygenated physiological buffer at room temperature. The imaging of the cells was acquired through a charge-coupled device (Model GP-CD60 Panasonic) and viewed on a monitor (PVM-122 Sony). Two platinum electrodes placed in the bathing fluid were connected to a Grass S9 stimulator and used to stimulate the myocytes with pulses of 2-ms duration at frequencies of 0.25, 0.5, and 1.0 Hz (15, 30, and 60 beats/min, respectively). Myocytes were paced for 20 s at each of the stimulation rates, and pacing was continuous through stimulation rate changes. Cytosolic free Ca2+ was measured by ratio imaging of 340- to 380-nm fluorescence of fura 2 (emission wave length 510 nm) using a PTI Delta Scan-1 dual-beam spectrophotofluorometer [Photon Technology International (PTI)] coupled to an Olympus IMT-2 with UV transparent optics. Signals were transferred to a Pentium P60 computer and analyzed by Felix (PTI) software. The baseline, the amplitude, and the times for 50 and 80% decay (T50 and T80) of the intracellular Ca2+ signal were measured.

Measurement of cardiomyocyte mechanical properties. One-half of the isolated cells from each heart were used for Ca2+ kinetic studies, and the other half were used for mechanical studies. Isolated cardiomyocytes were placed in a perfusion chamber on the stage of a microscope (Olympus IMT-2) and constantly superfused with oxygenated physiological buffer at room temperature. The imaging of the cells was acquired through a charge-coupled device (model GP-CD60 Panasonic), viewed on a monitor (PVM-122 Sony), and recorded on a videotape as described previously (11, 32). Cells were stimulated to contract using a similar protocol to that in the Ca2+ studies. Myocyte contractile parameters measured were percent shortening, rate of shortening, and rate of relengthening as determined from videotaped images using a dedicated video motion edge detector (Crescent Electronics) and recorded on a Gould MK200A chart recorder. Cardiomyocyte dimensions measured from the videotaped images were compared with a calibration micrometer on the microscope stage.

For measurements of intracellular Ca2+ and mechanical properties, five to eight cardiomyocytes were selected from each animal at random and analyzed. The cells used for the separate Ca2+ studies and mechanical studies were from the same animals. Statistical analyses were performed on the basis of the number of animals rather than the number of cells in each group.

Separation of membrane and cytosolic fractions for PKC localization. Membrane and cytosolic fractions of detergent-extracted PKC were prepared as previously described (19). Briefly, LV tissue was homogenized in lysis buffer containing (mM) 25 Tris · HCl, 5 EGTA, 2 EDTA, 100 NaF, 0.02 leupeptin, 0.01 E64, 0.12 pepstatin, 0.2 phenylmethylsulfonyl fluoride, and 5 dithiothreitol. An 800 g crude particulate fraction was discarded, and the supernatant was centrifuged at 100,000 g for 60 min. The pellet constituted the membrane-particulate fraction, and the supernatant was the cytosolic fraction. The particulate fraction was resuspended in homogenizing buffer containing 0.5% Triton X-100 and centrifuged at 100,000 g for 60 min, and the resulting detergent-treated supernatant was the membrane fraction.

Western blot analysis. Subcellular localization of PKC isoforms was examined by quantitative immunoblotting (19). Equal amounts of cytosolic and membranous protein extracts (8 µg) for each group were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk overnight at 4°C and incubated overnight with PKC isoform-specific primary antibodies (Santa Cruz) at 4°C. To ensure the specificity of immunoreactive proteins, transferred membranes were incubated with primary antibodies in the presence and absence of the corresponding blocking peptide (Santa Cruz).

To compare the relative protein levels of SERCA2a and phospholamban, a similar amount of whole LV homogenate (2 µg) for each group was separated by 15% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (12). Membranes were blocked with 5% nonfat dry milk for 2 h and reacted with primary antibodies for phospholamban (Affinity Bioreagents), SERCA2a, and cardiac actin (Sigma) at room temperature for 1 h.

Blots were then incubated for 1-2 h with secondary antibody (horseradish peroxidase conjugated, KPL Laboratories) and visualized by enhanced chemiluminescence (Amersham Life Science). The degree of labeling was quantified by a computer program (NIH) and expressed in relative scan units. The scan units of signals were linear in the range of 2-12 µg homogenate protein loaded onto the gel lanes for PKC and 1-10 µg for SERCA2a and phospholamban.

Statistical analysis. Data are presented as means ± SE. Reported data were analyzed by analysis of variance followed by Student-Newman-Keuls test. If data were not normally distributed or failed equal variance tests after log10 transformations, they were analyzed by nonparametric statistics. Survival curves were calculated according to the Kaplan-Meier actuarial method and compared by the log-rank test. Values with P < 0.05 were considered as statistically significant.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Effect of ACE inhibition on LVH and pulmonary congestion. Banded and sham-operated animals were treated with ramipril or vehicle for 8 wk after surgery and killed for analysis. In banded guinea pigs treated with vehicle, the degree of LVH assessed by LV weight and a LV weight-to-body weight ratio were significantly higher than sham-operated guinea pigs (Table 1). The degree of pulmonary congestion defined by lung weight and a lung weight-to-body weight ratio were significantly higher in banded guinea pigs treated with vehicle than in sham-operated guinea pigs. Banded guinea pigs treated with ramipril had significantly lower LV weight-to-body weight ratios (3.37 ± 0.05 vs. 3.82 ± 0.18, P < 0.01) and lung weight-to-body weight ratios (6.37 ± 0.51 vs. 8.49 ± 1.26, P < 0.05) than banded guinea pigs treated with vehicle. However, treatment with ramipril did not reverse the degree of LVH to the normal levels of sham-operated animals. These findings suggested that, with the dose used in the present study (5 mg · kg-1 · day-1), ramipril attenuated the degree of LVH but did not prevent completely this pathological response in this pressure-overload model of decompensated hypertrophy.

                              
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  		Table 1.   Left ventricular weight and lung weight

ACE inhibition and survival. Kaplan-Meier survival curves show that the attenuation of LVH and pulmonary congestion was associated with improved survival in ramipril-treated banded guinea pigs (Fig. 1). After enrollment, 7 of 19 guinea pigs (36%) in the vehicle-treated banded group died, whereas 1 of 18 (5%) in the ramipril-treated banded group died over the 8-wk period. These results demonstrated that treatment with ramipril significantly reduced mortality (P < 0.05). The mortality over 8 wk in sham-operated guinea pigs was similar to that of the ramipril-treated banded group.


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  		Fig. 1.   Kaplan-Meier survival curves for sham-vehicle (SV), sham-drug (SD), band-vehicle (BV), and band-drug (BD) groups. Actuarial survival of banded guinea pigs administered ramipril was significantly improved relative to banded guinea pigs given vehicle alone.

Isolated heart function. To examine the functional consequences of ACE inhibition, the isolated heart of each animal was perfused in a Langendorff apparatus. The developed LV pressure (109 ± 10 vs. 82 ± 7 mmHg, P < 0.05), isovolumic parameters of LV contractility (+dP/dt: 1,934 ± 174 vs. 1,408 ± 131 mmHg/s, P < 0.05), and speed of relaxation (-dP/dt: 1,700 ± 137 vs. 1,248 ± 138 mmHg/s, P < 0.05) were significantly increased in the ramipril-treated banded group compared with the vehicle-treated banded group (Table 2). There was no significant difference in isovolumic LV function between the ramipril-treated banded group and sham-operated groups.

                              
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  		Table 2.   Isolated isovolumic left ventricular performance

Isolated cardiomyocyte mechanics and calcium transients. Figure 2 shows representative analog recordings of LV cardiomyocyte mechanics and intracellular Ca2+ signals from banded and sham-operated guinea pigs treated with ramipril or vehicle. Banded guinea pigs treated with vehicle showed less cardiomyocyte contraction and lower amplitude of intracellular Ca2+ signal compared with sham-operated guinea pigs. Treatment with ramipril improved cardiomyocyte contraction and amplitude of the Ca2+ signals.


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  		Fig. 2.   Representative analog recordings of cardiomyocyte mechanics (% shortening, A) and Ca2+ transients (B) of cells isolated from guinea pigs of SV, SD, BV, and BD groups. Intracellular Ca2+ was obtained by ratio imaging of fura 2. Banded guinea pig treated with vehicle shows decreased cardiomyocyte contraction and lower amplitude of Ca2+ signals compared with sham-operated guinea pigs. These changes are reversed by treatment with ramipril.

The group data for mechanical properties of isolated LV cardiomyocytes are shown in Table 3. Cardiomyocyte percent shortening in the vehicle-treated banded group was significantly reduced compared with the sham vehicle group at each stimulation rate of 15, 30 and 60 beats/min (P < 0.05). Cardiomyocyte function in banded guinea pigs was improved by treatment with ramipril, but these changes were not statistically significant.

                              
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  		Table 3.   Mechanical properties of left ventricular cardiomyocytes

The group data for intracellular Ca2+ kinetics of isolated LV cardiomyocytes are shown in Table 4. The vehicle-treated banded group showed significantly lower amplitude at 30 beats/min (P < 0.05) and longer T80 and T50 at 15, 30, and 60 beats/min (P < 0.05) compared with the sham vehicle group. T80 and T50 were significantly shorter in the ramipril-treated banded group compared with the vehicle-treated banded group at 15, 30, and 60 beats/min (P < 0.05).

                              
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  		Table 4.   Calcium transients from isolated left ventricular cardiomyocytes

Protein levels of phospholamban and SERCA2a. To examine the effects of ACE inhibition on SERCA2a and phospholamban protein expression, quantitative immunoblotting was performed. As shown in Fig. 3 protein levels of SERCA2a and phospholamban decreased in banded guinea pigs treated with vehicle, and treatment with ramipril prevented downregulation of SERCA2a and phospholamban in banded guinea pigs. The protein level of actin was unchanged among the four groups. In the group data (Fig. 4), there was a 23% increase of SERCA2a and a 21% increase of phospholamban in the ramipril-treated banded group compared with the vehicle-treated banded group (P < 0.05). The SERCA2a-to-phospholamban ratio was not different in the banded group, because both SERCA2a and phospholamban protein levels were decreased in banded animals treated with vehicle and increased by treatment with ramipril.


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  		Fig. 3.   Representative immunoblots of sarcoplasmic reticulum Ca2+ ATPase (SERCA2a), actin, and phospholamban. A similar amount of whole left ventricular homogenate (2 µg) from guinea pigs from SV, SD, BV, and BD groups was subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. Membrane was cut into 3 pieces and probed separately with antibodies specific to SERCA2a, actin, and phospholamban. Protein levels of SERCA2a and phospholamban were decreased in banded guinea pig treated with vehicle compared with sham groups. Treatment with ramipril prevented downregulation of SERCA2a and phospholamban levels in banded guinea pig. Protein level of actin was not different among the 4 animals. Positions of molecular mass markers (kilodaltons) are indicated on right.


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  		Fig. 4.   Group data for SERCA2a (A) and phospholamban (B) protein expression. Left ventricular homogenates from 4 SV and 4 SD animals were pooled. Data for BV and BD groups were obtained from 6 animals/group. Protein levels of SERCA2a and phospholamban were reduced in banded animals treated with vehicle compared with sham-operated controls. Treatment with an angiotensin-converting enzyme (ACE) inhibitor prevented downregulation of SERCA2a and phospholamban protein levels. * P < 0.05 vs. BV group.

Subcellular distribution of PKC isoforms. At least 11 PKC isoforms are presently identified and perhaps have played different roles in cell signaling. Because PKC isoform diversity was reported between species and/or developmental stages (20, 23, 29), we examined Western blotting of seven PKC isoforms: alpha , beta 1, beta 2, delta , epsilon , gamma , and zeta . Among them, the LV of adult guinea pig expressed five PKC isoforms, alpha , beta 2, epsilon , gamma , and zeta , whereas no significant immunoreactivity was detected for beta 1- and delta -isoforms (data not shown). In subsequent experiments, significant translocation of PKC-alpha and -epsilon was observed in aortic-banded guinea pigs.

Therefore, the effect of ACE inhibition on the subcellular localization of these two PKC isoforms was examined using isoform-specific antibodies. Figure 5A represents Coomassie blue staining of an SDS-polyacrylamide gel. This figure demonstrates that the same amounts of membranous and cytosolic proteins from each of the four groups were loaded onto each gel lane. To ensure equivalent quantitative transfer of proteins, the nitrocellulose membrane was stained with Ponceau S (Fig. 5B). Representative immunoblots of PKC-alpha and -epsilon are shown in Fig. 6. The membrane-associated immunoreactivity of PKC-alpha and -epsilon was markedly increased in banded guinea pigs treated with vehicle compared with sham-operated groups. These increases in immunoreactivity of membrane fractions were attenuated by treatment with ramipril. The cytosol-associated immunoreactivity of PKC-alpha was unchanged, but that of PKC-epsilon was markedly increased in the banded groups. The immunoreactivity was specific to PKC-alpha and -epsilon , because it was blocked by competing peptides. The group data for PKC immunoblots are summarized in Table 5. The membrane-associated immunoreactivity of PKC-alpha and -epsilon was significantly increased in banded guinea pigs treated with vehicle compared with sham-operated groups (alpha : 139.5 ± 5.5 vs. 185.7 ± 10.7 scan unit, P < 0.01, epsilon : 147.2 ± 9.1 vs. 219.3 ± 2.5 scan unit, P < 0.01). These increases of PKC-alpha , but not -epsilon , were attenuated significantly by treatment with ramipril (alpha : 152.0 ± 4.1 scan unit, P < 0.05, epsilon : 194.0 ± 1.9 scan unit, P = not significant). As shown in Fig. 7, the membrane-to-cytosol ratios of PKC-alpha and -epsilon were significantly reduced in banded guinea pigs treated with ramipril compared with those treated with vehicle (alpha : 1.09 ± 0.04 vs. 0.74 ± 0.03, P < 0.01, epsilon : 1.16 ± 0.04 vs. 0.89 ± 0.03, P < 0.01). ACE inhibition with ramipril attenuated the translocation of PKC-alpha and -epsilon isoforms in pressure-overload heart failure produced by aortic banding.


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  		Fig. 5.   Coomassie blue staining of an SDS-polyacrylamide gel (A) and Ponceau S staining of transferred nitrocellulose membrane (B). Similar amounts of membranous and cytosolic protein extracts were subjected to SDS-polyacrylamide gel electrophoresis for each animal. Positions of molecular mass markers (kilodaltons) are indicated on left.


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  		Fig. 6.   Representative immunoblots of protein kinase C (PKC)-alpha (A) and -epsilon (B). Membranous and cytosolic protein extracts (8 µg) for each animal were subjected to SDS-polyacrylamide gel electrophoresis. Immunoblot analysis was performed with antibodies specific to PKC-alpha and -epsilon that had not (-) and had (+) been incubated with corresponding blocking peptide. Membrane-associated immunoreactivity of PKC-alpha and -epsilon was increased in banded guinea pig treated with vehicle, and these changes were attenuated in banded guinea pig treated with ramipril. Cytosol-associated immunoreactivity of PKC-alpha was unchanged among 4 animals, but PKC-epsilon showed markedly increased immunoreactivity of cytosol in banded guinea pigs. Immunoreactivity of each band was specific to PKC-alpha and -epsilon , because it was blocked by corresponding blocking peptide. Positions of molecular mass markers (kilodaltons) are indicated on right.

                              
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  		Table 5.   Subcellular distribution of protein kinase isoforms


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  		Fig. 7.   Comparison of membrane/cytosol ratios of PKC-alpha (A) and -epsilon (B) obtained from 6 guinea pigs in each group. Membrane-to-cytosol ratios of PKC-alpha and -epsilon were significantly reduced by ACE inhibition. * P < 0.01 vs. SV, SD, and BD.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The results of the present study demonstrate that in this model of decompensated pressure-overload hypertrophy, ACE inhibition caused attenuation of LVH, improved survival, enhanced isolated heart function, and produced improvement in cardiomyocyte Ca2+ transients. These functional changes were associated with attenuation of PKC translocation and related prevention of downregulation of Ca2+ cycling proteins.

Isolated heart and cardiomyocyte function in LVH and CHF. Banding the ascending or descending aorta is a common procedure used to create animal models of compensated and decompensated LVH. Decreased LV norepinephrine content, diminished maximal LV isometric pressure-generating capacity, and variable histological evidence of fibrosis are typically observed in such banded animals (27). In isolated papillary muscles from guinea pigs with abdominal aortic banding, markedly diminished maximal tension development is observed (13). At the isolated cardiomyocyte level, it has been shown that both the velocity and percentage of myocyte shortening are significantly decreased in hypertrophied guinea pig cells (26). In the present study, decreases of developed LV pressure, +dP/dt, and -dP/dt in Langendorff-perfused isovolumic hearts, decreased myocyte shortening, and pulmonary congestion from banded guinea pigs indicated contractile depression typical of decompensated pressure-overload hypertrophy.

Calcium transients and calcium cycling proteins in LVH and CHF. It has been reported that in myocytes from hypertrophied and failing guinea pig hearts, the peak amplitude of Ca2+ transients is depressed (26). Myocardial tissue from patients with end-stage heart failure has demonstrated abnormal prolonged Ca2+ transients and impaired ability to restore diastolic Ca2+ to normal low levels (8). We previously showed that SR Ca2+ transport is decreased in descending thoracic aortic-banded guinea pigs with heart failure and is accompanied by reduced levels of Ca2+ cycling proteins (12). Decreased cellular uptake of Ca2+ into the SR was also shown in phorbol ester-induced hypertrophy of neonatal rat cardiac myocytes (9, 21, 22). This decrease in Ca2+ transport by the SR observed in CHF may be a result of decreased abundance of cardiomyocyte Ca2+ cycling proteins (1). In the present study, the reduced cardiomyocyte function in banded guinea pigs was paralleled by the decreased amplitude and prolongation of Ca2+ transients. These changes may be interpreted as being caused by diminished SR Ca2+ release and resequestration resulting from depressed protein levels of SERCA2a and phospholamban. Phospholamban in its dephosphorylated state inhibits the SR Ca2+ ATPase (6). Phosphorylation of this protein by cAMP-dependent or Ca2+/calmodulin-dependent protein kinases relieves this inhibition. In genetically engineered mice the stoichiometry between the SR Ca2+ ATPase and phospholamban was shown to be a major determinant of myocardial contractility (11). In the present study and previous studies from our laboratory, both Ca2+ cycling proteins were depressed in heart failure, and the baseline phosphorylation status of phospholamban did not differ between decompensated hypertrophy and normal hearts (12). It is possible that as yet unidentified changes in the biophysical environment of the SR membrane account for the depressed cardiac SR Ca2+ ATPase function in addition to the relative levels of SERCA2a and phospholamban in conventional animal models of heart failure and human clinical heart failure. Taken together, these findings indicate that impaired cardiomyocyte and isolated heart function are, at least in part, related to changes in intracellular Ca2+ handling. However, the complex regulation of Ca2+ transport and its relevance to heart failure are still not fully clarified.

Effect of ACE inhibition on LV performance, survival, and Ca2+ cycling protein levels. The local renin-angiotensin system plays an important role in the development of pressure-overload hypertrophy and heart failure (19, 24, 37), and ACE inhibition may cause regression of clinical and experimental LVH (2, 14, 30). In isolated heart experiments using rats with pressure-overload hypertrophy, LV systolic developed pressure relative to perfusate Ca2+ concentration was significantly higher in banded rats with ACE inhibition compared with those without treatment (36). In the present study isolated, perfused hearts were also examined in the absence of confounding factors of systemic neurohormonal activation and pericardial constraint and under conditions of constant coronary flow, normothermia, and physiologically paced heart rate. The results demonstrated that decreased LV developed pressure, +dP/dt, and -dP/dt were improved by treatment with ramipril. The improved LV chamber performance was accompanied by parallel functional effects of ACE inhibition on Ca2+ signals. ACE inhibition improved survival in banded guinea pigs with heart failure. Studies in patients with chronic heart failure suggest that the beneficial effects of ACE inhibition on heart function and survival are attributed to a decrease in peripheral vascular resistance and antagonism of neurohormonal activation (5, 28). However, in this animal model of pressure-overload hypertrophy, ACE inhibition was unlikely to improve heart function or survival by peripheral vasodilation, because fixed aortic banding prevented significant drug-related unloading of the heart (4). Postmortem analysis of guinea pigs that died during the study revealed pulmonary congestion and pleural and pericardial effusion, which suggested that death was directly related to advanced cardiac failure. We hypothesize that ACE inhibition might delay the transition of hypertrophy to cardiac failure by mechanisms that are at least in part intrinsic to the cardiomyocyte (4, 36).

The exact mechanism(s) whereby ACE inhibition prevents the downregulation of SERCA2a and phospholamban protein levels in decompensated pressure-overload hypertrophy are not currently understood. The decreased expression of SERCA2a and phospholamban protein levels in LVH and heart failure are known to be caused by a decrease in steady-state levels of their respective mRNAs (1). Although both transcriptional and posttranscriptional mechanisms are postulated, the exact signaling pathways affecting SERCA2a and phospholamban expression are poorly understood. It has been shown that PKC activation by phorbol ester can produce a decrease in SERCA2 expression accompanied by a decrease in SR Ca2+ transport in neonatal cardiomyocytes (9, 21, 22). Taken together, these studies suggest that downregulation of SERCA2 may occur by a PKC-related process that can be attenuated by ACE inhibitors.

Translocation of PKC isoforms and CHF. PKC has been implicated as the intracellular mediator of several neurotransmitters, growth factors, and tumor promoters through multiple signal transduction pathways (17). We recently demonstrated that in the adult guinea pig heart, acute LV dilatation produces stretch-mediated activation of phospholipase C that results in inositol phosphate hydrolysis and PKC-epsilon activation (19). Schunkert et al. (25) reported that angiotensin II type-1 receptor and PKC activation were involved in angiotensin II-mediated stimulation of protein synthesis in isolated rat heart. It was also shown that transgenic cardiac-specific Galpha q overexpression results in a dilated cardiomyopathy, PKC activation, and overt heart failure (5a). These findings strongly implicated overreactivity of an angiotensin II-mediated cell signaling pathway in the pathogenesis of cardiac hypertrophy and heart failure.

In experimental diabetic rats produced by streptozotocin injection, activation of PKC-beta 2 (10) and PKC-epsilon (15) was observed in the heart. It was demonstrated that these changes in PKC distribution were prevented by the normalization of blood glucose with insulin (10, 15) or by a specific angiotensin II type 1-receptor antagonist (15).

It would be ideal to use cardiomyocytes for the evaluation of isoform specific translocation rather than whole LV homogenates, because contamination by vascular or interstitial tissue might affect the result (23). However, the LV tissue was carefully separated from the atria, great vessels, and right ventricle in the present study. Thus the majority of LV homogenate used for immunoblotting in the present study was considered to be cardiomyocytes. PKC isoform diversity was reported between species and/or developmental stages (20, 23, 29). It was reported that PKC-alpha , -beta , and -zeta isoforms did not exist in adult rat heart (23). However, in the present study these three isoforms existed, and PKC-delta , one of the major isoforms of rat cardiomyocytes, did not demonstrate immunoreactivity in guinea pig heart.

Presently, at least 11 isoforms of PKC have been identified in vivo. PKC isoform expression is regulated differently during development (23, 29). It has also been reported that PKC isoforms are differentially responsive to neurohormones (20). Isoform-specific activation of PKC has been found in myocardial hypertrophy and failure. The levels of PKC-beta and -epsilon isoforms are increased during development of cardiac hypertrophy induced by pressure overload in adult rats (7). We have examined explanted hearts of patients diagnosed with idiopathic dilated cardiomyopathy or ischemic cardiomyopathy and have found increases of PKC-alpha and -beta expression and unchanged PKC-epsilon expression in failed human hearts (3). In the present study, PKC isoforms alpha , beta 2, gamma , epsilon , and zeta  were found in adult guinea pig hearts, and enhanced translocation of PKC-alpha and -epsilon was observed in the failing heart produced by chronic pressure overload. Acute mechanical stretch has been reported to mediate PKC-epsilon , but not PKC-alpha , activation (19). These findings suggest that responses of PKC isoforms to distinct pathological stimuli are differentially regulated.

Activation of PKC modulates gene expression, intracellular Ca2+ levels, the hypertrophy process, and contractile state through phosphorylation of its substrates (3, 5a, 7, 17, 19, 34). We recently showed that transgenic mice with cardiac-specific overexpression of PKC-beta 2 have depressed cardiomyocyte contractility mediated by enhanced in vivo phosphorylation of cardiac troponin I (32). Therefore, enhanced PKC activity may depress myocardial contractility by multiple mechanisms (18). We reported that acute mechanical stretch-induced translocation of PKC-epsilon was partially blocked by angiotensin II type 1-receptor antagonist. In the present study, translocation of PKC-alpha and -epsilon isoforms in failing heart produced by chronic pressure overload was attenuated by treatment with an ACE inhibitor. Furthermore, this change in PKC distribution was accompanied by functional improvement in intracellular Ca2+ handling and cardiac contractility at the isolated heart level. Because PKC activation by phorbol ester decreases SERCA2 mRNA and protein expression (9, 21), PKC inhibition might contribute to the prevention of downregulation of SERCA2a protein level observed in the present study. To our knowledge, this is the first report showing that activation of PKC in hypertrophied or failed heart induced by chronic pressure overload may be attenuated by chronic ACE inhibition. Furthermore, we demonstrate that downregulation of SERCA2a in heart failure may occur in vivo by a PKC-related process that can be prevented by ACE inhibition, possibly by the attenuation of PKC translocation. Angiotensin II is only one of the hormones that mediate Galpha q stimulation with resultant activation of PKC. Endothelin and alpha -adrenergic agonists such as phenylephrine and prostaglandin F2alpha each also activate this cell signaling pathway by binding to their cognate seven transmembrane spanning receptors. We propose that more complete inhibition of this pathway at the receptor, G protein, or PKC isoform level would augment the beneficial effects of ACE inhibition presented here.

We conclude that attenuation of PKC translocation and improvement in Ca2+ cycling protein levels with resultant amelioration of intracellular Ca2+ handling might contribute to the improvement in survival, cardiac morphometry, and contractile function produced by ACE inhibition in pressure-overload heart failure. These findings support the concept that angiotensin II-mediated PKC activation plays a critical role in the transition from compensated hypertrophy to heart failure and provides insight into an additional favorable mechanism of ACE inhibition in the pharmacotherapy of this pathological process.

    ACKNOWLEDGEMENTS

This study was supported in part by Specialized Center of Research in Heart Failure Grant P50 HL-52318 from the National Heart, Lung, and Blood Institute and a grant from the Japanese Heart Foundation.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: R. A. Walsh, Division of Cardiology, Univ. of Cincinnati College of Medicine, 231 Bethesda Ave., Rm. 3354, Cincinnati, OH 45267-0542.

Received 7 April 1998; accepted in final form 24 August 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Heart Circ Physiol 276(1):H53-H62
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