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Circulation. 1997;96:2397-2406

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*Heart Failure

(Circulation. 1997;96:2397-2406.)
© 1997 American Heart Association, Inc.

Articles

Modulation of the Renin-Angiotensin Pathway Through Enzyme Inhibition and Specific Receptor Blockade in Pacing-Induced Heart Failure

II. Effects on Myocyte Contractile Processes

Francis G. Spinale, MD, PhD; Rupak Mukherjee, PhD; Julie P. Iannini, BS; Steve Whitebread, BS; Latha Hebbar, MD; Mark J. Clair, BS; D. Mark Melton, BS; Montgomery H. Cox, BS; Patrick B. Thomas, BS; ; Marc de Gasparo, MD

From the Division of Cardiothoracic Surgery, Medical University of South Carolina, Charleston, and the Pharmaceutical Division, Novartis, Basel, Switzerland (S.W., M. de G.).

Correspondence to Francis G. Spinale, MD, PhD, Cardiothoracic Surgery and Physiology, Medical University of South Carolina, Charleston, SC 29425.


*    Abstract
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 *Abstract
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Background The goal of this study was to determine the effects of ACE inhibition alone, AT1 angiotensin (Ang) II receptor blockade alone, and combined ACEI and AT1 Ang II receptor blockade in a model of congestive heart failure (CHF) on isolated LV myocyte function and fundamental components of the excitation-contraction coupling process.

Methods and Results Pigs were randomly assigned to one of five groups: (1) rapid atrial pacing (240 bpm) for 3 weeks (n=9), (2) concomitant ACEI (benazeprilat, 0.187 mg · kg-1 · d-1) and rapid pacing (n=9), (3) concomitant AT1 Ang II receptor blockade (valsartan, 3 mg/kg/d) and rapid pacing (n=9), (4) concomitant ACEI and AT1 Ang II receptor blockade (benazeprilat/valsartan, 0.05/3 mg · kg-1 · d-1) and rapid pacing (n=9), and (5) sham controls (n=10). LV myocyte shortening velocity was reduced with chronic rapid pacing compared with control (27.2±0.6 versus 58.6±1.2 µm/s, P<.05) and remained reduced with AT1 Ang II receptor blockade and rapid pacing (28.0±0.5 µm/s, P<.05). Myocyte shortening velocity increased with ACEI or combination treatment compared with rapid pacing only (36.9±0.7 and 42.3±0.8 µm/s, respectively, P<.05). Myocyte ß-adrenergic response was reduced by >50% in both the rapid pacing group and the AT1 Ang II blockade group and improved by 25% with ACEI and increased by 54% with combined treatment. Both L-type Ca2+ channel density and the relative abundance of sarcoplasmic reticulum Ca2+ ATPase density were reduced with rapid pacing and returned to control levels in the combined ACEI and AT1 Ang II blockade group.

Conclusions The unique findings of this study were twofold. First, basic defects in specific components of the myocyte excitation-contraction coupling process that occur with CHF are reversible. Second, combined ACEI and AT1 Ang II blockade may provide unique benefits on myocyte contractile processes in the setting of CHF.


Key Words: heart failure • angiotensin • calcium channels • cardiovascular disease • myocardium


*    Introduction
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 up arrowAbstract
 *Introduction
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The development and progression of CHF is accompanied by fundamental defects in myocardial contractile processes.1 2 3 4 5 6 7 8 9 10 11 12 Specifically, diminished myocyte contractility,1 2 3 alterations in myocyte electrophysiology,11 and defects in excitation-contraction coupling processes have all been reported with the development of severe CHF.4 5 6 7 8 9 10 Chronic ACEI has been demonstrated to have favorable effects on LV function and survival in patients with CHF. The institution of ACEI has been shown to improve LV geometry, loading conditions, and neurohormonal status in both humans and animals with developing CHF.13 14 15 16 17 18 19 20 21 However, whether ACEI provides intrinsic protective effects on myocyte contractile processes and whether these effects are primarily mediated by reduced production of Ang II and subsequently diminished AT1 Ang II receptor activity remains unclear. Specific AT1 Ang II receptor antagonists have now been developed and applied in the setting of hypertension and CHF.22 23 24 25 Moreover, combined ACEI and AT1 Ang II receptor blockade has been demonstrated to have additional beneficial effects with respect to systemic blood pressure in the setting of hypertension25 and LV function in a model of CHF.26 Accordingly, the goal of this study was to determine the specific effects of ACEI, AT1 Ang II receptor blockade, and combined therapy in a model of CHF with respect to myocyte contractility and excitation-contraction coupling processes.

Past reports from this laboratory and others have demonstrated that chronic pacing tachycardia in animals causes progressive and time-dependent changes in LV geometry and pump function, neurohormonal system activation, and abnormalities in sarcolemmal transduction systems.26 27 28 29 30 31 32 33 34 35 36 37 More importantly, this laboratory has reported that the development of pacing-induced CHF causes alterations in myocyte contractility, inotropic responsiveness, and electrophysiology.27 28 29 30 31 32 33 37 More recently, we reported that concomitant ACEI with chronic rapid pacing improved indices of myocyte contractility.29 Accordingly, this model of pacing-induced CHF was used to test the central hypothesis that combined ACEI and AT1 Ang II receptor blockade will provide enhanced beneficial effects on myocyte contractile processes compared with either treatment alone.


*    Methods
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 up arrowIntroduction
 *Methods
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Experimental Model and Design
The present study used rapid pacing in pigs, which has been demonstrated to invariably cause functional and neurohormonal characteristics of CHF.26 27 28 30 31 32 33 Atrial pacing leads and modified pacemakers were surgically implanted in 46 weight-matched Yorkshire pigs (Hambone Farms, Reevesville, SC; 20 to 25 kg) as described previously.26 27 28 After recovery from the surgical procedure, the animals were randomly assigned to the following treatment groups: (1) rapid atrial pacing (240 bpm) for 3 weeks (n=9), (2) concomitant ACEI (benazeprilat, 3.75 mg/d) and rapid pacing (n=9), (3) concomitant AT1 Ang II receptor blockade23 (valsartan, 60 mg/d) and rapid pacing (n=9), (4) concomitant ACEI and AT1 Ang II receptor blockade (benazeprilat/valsartan, 1/60 mg/d, respectively) and rapid pacing (n=9), and (5) sham controls (n=10). The drug treatment protocols were begun at the initiation of pacing and continued for the entire 21-day pacing protocol. The dosage selection for monotherapy and combination therapy was based on initial dose-response studies in which appropriate inhibition of the Ang I/II pressor response was achieved without a significant effect on resting blood pressure.26 Through minimization of the effects of drug treatment on resting blood pressure, the potential confounding and differential loading effects of ACEI, AT1 Ang II receptor blockade, or combination treatment could be minimized. After completion of the specific treatment protocol, the animals were anesthetized as described previously,26 27 28 and a sternotomy was performed. The heart was quickly extirpated and placed in a phosphate-buffered ice slush, and the coronary arteries were flushed. The great vessels were removed at the aortic and pulmonary valves. The region of the LV free wall incorporating the circumflex artery (5x5 cm) was excised and prepared for myocyte isolation. The LV apex and midventricular region was cut into 1x1-cm cubes and snap-frozen in liquid nitrogen for subsequent membrane preparation. All animals were treated and cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Research Council, Washington, 1996).

Myocyte Isolation and Contractile Function
LV myocytes were isolated from all the pigs used in this protocol by methods described previously.27 28 29 30 31 32 33 Briefly, the circumflex coronary artery was perfused and recirculated with an oxygenated Krebs solution containing aerobic substrates and collagenase (1 mg/mL, Worthington, type II; 146 U/mg) for 20 minutes. The LV myocardium was then minced into 2-mm sections. The LV tissue was placed in an oxygenated trituration solution containing 400 µmol/L CaCl2 and collagenase and was gently agitated. The supernatant was removed and filtered, and the cells were allowed to settle. The pellet of cells was resuspended in cell culture medium (medium M199, 2 mmol/L Ca2+, Gibco Laboratories). By these methods, a high yield (75±5%) of viable myocytes was obtained, with no difference in the percent yield in any of the treatment groups. Viable myocytes were defined as those that were quiescent in culture, maintained a rod-shaped morphology at physiological Ca2+ concentrations, and excluded trypan blue dye.

Isolated myocyte function was examined as previously reported by this laboratory.27 28 29 30 31 32 33 Briefly, a thermostatically controlled chamber (37°C) containing a volume of 2.5 mL and two stimulating platinum electrodes was used to image the isolated myocytes on an inverted microscope (Axiovert IM35, Zeiss Inc). A x20 long-working-distance Hoffmann modulation contrast objective (Modulation Optics Inc) was used to image the myocytes. Myocyte contractions were elicited by field stimulation of the tissue chamber at 1 Hz (S11, Grass Instruments) with current pulses of 5-ms duration and voltages 10% above contraction threshold. Myocyte contractile performance was examined at a constant stimulation frequency and contraction rate of 1 Hz. Myocyte motion signals were captured and entered through an edge-detector system (Crescent Electronics). The distance between the left and right myocyte edges was converted into a voltage signal, digitized, and entered into a computer (80386; ZBV2526, Zenith Data Systems) for analysis. Parameters computed from the digitized contraction profiles include percent shortening, velocity of shortening, velocity of relengthening, time to peak contraction, time to 50% relaxation, and duration of contraction. Through the use of increased extracellular Ca2+ or ß-adrenergic receptor stimulation, the capacity of the myocyte to respond to an inotropic stimulus can be examined.2 27 28 29 30 The development of CHF in patients and animals has been reported to be associated with abnormalities in inotropic responsiveness.1 2 3 27 28 29 30 31 32 33 36 38 Accordingly, myocyte response to a specific inotropic stimulus was examined in the presence of either 8 mmol/L extracellular Ca2+ or 25 nmol/L isoproterenol [(-)Isoproterenol, Sigma Chemical Co]. The concentration of isoproterenol and Ca2+ used in this study has been demonstrated previously to provide near-maximal contractile response for this myocyte preparation.27 30

Membrane Preparation and L-Type Ca2+ Receptor Density
Dihydropyridine binding to LV crude membrane preparations was performed to determine the abundance of the L-type Ca2+ channels.4 39 The LV membranes were prepared by techniques described previously.28 38 Briefly, 15 g of LV free wall, from which the epicardial fat had been trimmed away, was placed in 10 volumes of ice-cold buffer containing 250 mmol/L sucrose, 5 mmol/L Tris, and 1 mmol/L EGTA and homogenized. The homogenate was centrifuged at 250g for 10 minutes, the pellet discarded, and the supernatant spun at 50 000g for 15 minutes. The resultant pellet was resuspended with an ice-cold buffer of 50 mmol/L Tris-HCl (pH 7.4). The preparation was recentrifuged and resuspended twice in Tris buffer. To ensure that membrane protein was not lost during centrifugation, supernatants from each step of the membrane isolation procedure were examined for Na+,K+-ATPase activity by assaying for p-nitrophenophosphatase activity.28 Dihydropyridine binding was performed on these crude membrane preparations with [3H]nitrendipine as described previously.4 39 Briefly, membrane preparations (0.08 to 0.1 mg protein/tube) were incubated with 0.25 to 10 nmol/L [3H]nitrendipine in the absence (total binding) or presence (nonspecific binding) of 100 µmol/L unlabeled nifedipine. This concentration of nifedipine has previously been demonstrated to inhibit >95% of the specific [3H]nitrendipine binding.4 The reaction volume for this assay was 250 µL. Samples were incubated in the dark for 60 minutes, after which the reactions were terminated by the addition of 1 mL ice-cold Tris-HCl buffer and vacuum filtration through Whatman GF/C filters. The filters were placed in vials containing 10 mL scintillation fluid, and the radioactivity was counted on a scintillation counter at an efficiency of 39% to 41%. All binding assays were performed in duplicate for each pig, and specific binding was determined by subtraction of nonspecific binding from total binding. Bmax and Kd values were determined by Scatchard analysis with the LIGAND program (Biosoft).

SR Ca2+ ATPase and Phospholamban Abundance
The relative abundances of SR Ca2+-ATPase and phospholamban were examined in LV membrane preparations by standard immunoblotting procedures.8 9 40 41 The samples were thawed on ice and diluted in a sample buffer (10% SDS, 4% sucrose, 0.25 mol/L Tris-HCl, and 0.1% pyronin Y dye, pH 6.8). The samples were then size-fractionated in a Mini Protean II Cell (BioRad) with a discontinuous system of a 4% polyacrylamide stacking and a 10% polyacrylamide separating gel. The gels were run at 15 mA/gel with a running buffer temperature of 10°C until the tracking dye had run off the bottom of the gel. The fractionated proteins were then electrophoretically transferred to polyvinylidene difluoride membranes (BioRad) at a constant voltage of 100 V for 1.5 hours (Trans-Blot Electrophoretic Cell, BioRad). Adequate transfer of the proteins to the membranes was confirmed by a lack of protein staining on the gels by use of a 0.15% Coomassie blue stain (Sigma). The membranes were then incubated for 1 hour at room temperature with 5% BSA/TBS. After washing, the membranes were incubated overnight with a 1:5000 dilution of mouse monoclonal anti–SR Ca2+-ATPase (clone 2A7-A1)40 or 1:1000 mouse monoclonal anti-phospholamban (No 13678, Upstate Biotechnology)41 in a buffer containing TBS, 1% BSA, and 0.1% Tween 20 (25°C and gentle rocking). These dilutions were selected on the basis of preliminary titration experiments. The membranes were washed with this buffer and then incubated with a 1:350 dilution of peroxidase-conjugated anti-mouse IgG (Sigma) for 2 hours at 25°C. After vigorous washing, the membranes were immersed in a solution of 3-amino-9-ethyl carbazole (Sigma). Substitution of the primary antibody with nonimmune mouse serum was used as a negative control in all immunoblotting procedures. The blots were digitized with a Kodak DCS 420 digital camera, which provides high resolution (1500x1000 pixels) and consistent exposure control between scans. The size-fractionated banding pattern was determined by quantitative image analysis (Gel-Pro Analyzer, Media Cybernetics). A 3-pixel-wide profile was constructed along the long axis of each lane and plotted as a two-dimensional array with line intensity on the y axis and molecular weight on the x axis. After correction for background signal, the integrated area corresponding to SR Ca2+-ATPase or phospholamban was computed. Immunoblotting procedures were performed with 1, 5, and 10 µg of LV membrane protein for each sample, and each immunoblot contained samples from each treatment group. The quantitative results were linear with protein, and all measurements were computed relative to control values.

Myocardial Protein Content
To determine whether chronic ACEI, AT1 Ang II receptor blockade, or combination treatment influenced the absolute contractile protein content, MHC and actin were measured in LV myocardial samples. LV myocardial samples (1 g) were homogenized in a 62.5 mmol/L Tris-HCl buffer (1:10 wt/vol) with a tissue grinder (Tissumizer, Tekmar Co). The homogenate was vortexed and diluted into serial dilutions ranging from 1:100 to 1:1000, and protein content was determined by a standardized colorimetric assay (Bio-Rad Protein Assay). The MHC content was determined in these myocardial homogenates by gradient SDS-PAGE.29 31 The samples were initially separated with a 4% stacking gel and were then resolved with a 10% to 13% gradient with a constant current and voltage set at 70 mV. The gels were run for 17 hours at a constant temperature of 12°C to 15°C, stained for 3 hours in a 0.3% Coomassie blue R-250 solution, and destained for an additional 3 hours. The stained gels were then scanned and digitized by the previously described image analysis system and subjected to two-dimensional densitometric analysis. After digital subtraction of background density, the integrated optical density of the bands corresponding to MHC were computed. The integrated optical density values obtained from the two-dimensional quantification were then transformed to actual values by use of purified porcine cardiac MHC and actin standards (1 to 6 µg; Sigma) that had been simultaneously electrophoresed on each gel. All experiments were performed in duplicate, and results were expressed with respect to wet weight of LV myocardium.

Data Analysis
Indices of LV myocyte function were compared by ANOVA. For the myocyte function studies, an ANOVA using a randomized block split-plot design was used. The treatment effects were pacing and drug therapy. Each pig was considered a complete block. Thus, the numbers of myocytes studied from each animal were considered repeated observations within each block. If the ANOVA revealed significant differences, pairwise tests of individual group means were compared by use of Bonferroni probabilities. All statistical procedures were performed with the BMDP statistical software package (BMDP Statistical Software Inc). Results are presented as mean±SEM. Values of P<.05 were considered to be statistically significant.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
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All the pigs that were entered into the study completed their respective treatment protocols, and myocytes were successfully harvested from all animals. Significant LV dysfunction and neurohormonal system activation occurred after 3 weeks of chronic rapid pacing. The specific effects on LV function and geometry and neurohormonal status for the pigs included in this study were the focus of a previous report.26

Myocyte Contractility and Inotropic Response
Resting myocyte length for the different groups is shown in Fig 1Down. Myocyte resting length was measured in more than 500 isolated myocytes from each group and formed a gaussian distribution. In the rapid pacing group, myocyte length significantly increased compared with the control group (166±2 versus 138±1 µm, respectively, P<.05). In the concomitant ACEI and rapid pacing group, myocyte length was reduced from rapid pacing–only values (147±1 µm, P<.05) but remained higher than controls (P<.05). In the AT1 Ang II receptor blockade and rapid pacing group, myocyte length was similar to rapid pacing–only values (161±1 µm). In the combined ACEI and AT1 Ang II blockade group, myocyte length was reduced from rapid pacing–only values (151±1 µm, P<.05) and was similar to ACEI-alone values. Indices of isolated myocyte contractile function under basal conditions in the control group, in the chronic rapid pacing group, and in the three different drug treatment groups are shown in Table 1Down. Steady-state myocyte contractile function was significantly reduced in the rapid pacing–only group compared with control values. For example, myocyte percent and velocity of shortening were reduced by 50% from control values. In the concomitant ACEI and rapid pacing group, myocyte contractile function was significantly improved from rapid pacing–only values. Specifically, myocyte velocity of shortening was 35% higher in the ACEI group than rapid pacing–only values. Although myocyte function was improved with concomitant ACEI during rapid pacing, myocyte contractile function remained lower than control values. In the concomitant AT1 Ang II blockade and rapid pacing group, myocyte contractile performance was similar to rapid pacing–only values. In the combined ACEI and AT1 Ang II blockade group, myocyte function was significantly improved from rapid pacing–only values. For example, with combined ACEI and AT1 Ang II blockade, myocyte velocity of shortening was 51% higher than rapid pacing–only values. Myocyte contractile function with combined ACEI and AT1 Ang II blockade and rapid pacing remained lower than control values. There was no significant difference in steady-state myocyte contractile function between the ACEI-alone group and the combined ACEI and AT1 Ang II blockade group.



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  		Figure 1. Frequency distribution for myocyte resting length in control, chronic rapid pacing, rapid pacing and ACEI, rapid pacing and AT1 Ang II blockade, and combined ACEI and AT1 Ang II blockade with rapid pacing (n=>500 myocytes/group). Sample for each group approximated gaussian distribution. Isolated myocyte resting length increased with chronic rapid pacing compared with control value (P<.05). With rapid pacing and ACEI, myocyte length was reduced from rapid pacing alone (P<.05) but remained higher than control values (P<.05). In AT1 Ang II receptor blockade group, resting length was unchanged from rapid pacing–only values. With combined AT1 Ang II receptor blockade and rapid pacing, resting length was similar to ACEI-only values. Absolute mean values for myocyte lengths are given in text.


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  		Table 1. Isolated Myocyte Contractile Function With Rapid Pacing Heart Failure: Effects of ACE Inhibition and AT1 Ang II Receptor Blockade During the Progression of Heart Failure

Inotropic responsiveness of isolated myocytes was examined in the presence of either 25 nmol/L isoproterenol or 8 mmol/L extracellular Ca2+, and the results from this series of studies are shown in Table 1Up. ß-Receptor stimulation with isoproterenol increased myocyte function from basal values in all groups. In the presence of isoproterenol, myocyte function remained significantly lower in all rapid pacing groups than in the control group. However, in the ACEI and rapid pacing group, myocyte function was higher after ß-receptor stimulation than rapid pacing–only values. In the AT1 Ang II receptor blockade group, myocyte ß-adrenergic response was unchanged from rapid pacing–only values. In the combined ACEI and AT1 Ang II blockade group, myocyte ß-adrenergic response was significantly higher than rapid pacing–only values. Moreover, in the combined ACEI and AT1 Ang II blockade group, myocyte function after ß-receptor stimulation was 25% higher than ACEI-alone values. With increased extracellular Ca2+, myocyte contractile function was significantly lower in all rapid pacing groups than control values. In the ACEI group, myocyte function with increased Ca2+ was significantly higher than rapid pacing–only values. In the AT1 Ang II receptor blockade group, myocyte Ca2+ response was similar to rapid pacing–only values. In the combined ACEI and AT1 Ang II blockade group, myocyte Ca2+ response was increased by >40% from both rapid pacing values and ACEI-only values. Because baseline myocyte function was different in the various treatment groups, myocyte contractile response to inotropic stimulation can be difficult to interpret. Accordingly, the absolute change in myocyte velocity of shortening after the addition of isoproterenol or Ca2+ was computed for each individual myocyte. The results from this analysis of >2000 myocytes are presented in Fig 2Down. The absolute change in myocyte velocity of shortening after ß-receptor stimulation was reduced by 50% in the rapid pacing–only group. In the ACEI and rapid pacing group, the absolute change in myocyte velocity of shortening was higher after ß-receptor stimulation than rapid pacing–only values. In the AT1 Ang II receptor blockade and rapid pacing group, the absolute change in myocyte velocity of shortening with ß-receptor stimulation was unchanged from rapid pacing–only values. In the combined ACEI and AT1 Ang II blockade group, the absolute change in myocyte velocity of shortening after ß-adrenergic response was higher than rapid pacing–only values and from ACEI-alone values. The absolute change in myocyte velocity of shortening with increased Ca2+ was higher in the combined ACEI and AT1 Ang II blockade group than in control and all other rapid pacing groups.



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  		Figure 2. Absolute change in myocyte velocity of shortening in controls, after 3 weeks of chronic rapid pacing, and after concomitant ACEI, AT1 Ang II receptor blockade (AT1 block), or a combination of both with rapid pacing (ACEI/AT1 block). After ß-receptor stimulation with 25 nmol/L isoproterenol, absolute change in myocyte velocity of shortening was significantly reduced in rapid pacing–only group compared with control group. In ACEI and rapid pacing group, absolute change in myocyte velocity of shortening was higher than rapid pacing–only values. In AT1 Ang II receptor blockade group, absolute change in myocyte velocity of shortening after ß-receptor stimulation was unchanged from rapid pacing–only values. In combined ACEI and AT1 Ang II blockade group, absolute change in myocyte velocity of shortening was higher than rapid pacing–only and ACEI-only values. With increased extracellular Ca2+ (8 mmol/L), absolute change in myocyte velocity of shortening was increased in ACEI and AT1 Ang II blockade group. Indices of contractile function at baseline and after inotropic stimulation are summarized in Table 1Up. *P<.05 vs control, +P<.05 vs rapid pacing only, §P<.05 vs ACEI and rapid pacing.

Myocyte Contractile Protein Content and L-Type Ca2+ Receptor Density
LV myocardial content for MHC and actin for the control group and for each treatment group are summarized in Table 2Down. The biochemical content for MHC and actin were not different in any of the treatment groups from control values. The relative L-type Ca2+ density of LV membrane preparations was determined through radiolabeled binding experiments using nitrendipine.4 39 Specific binding of [3H]nitrendipine to all membrane preparations was saturable, and representative binding curves for the different treatment groups are presented in Fig 3Down. Bmax and Kd were determined from these binding assays, and these results are shown in Table 2Down. In the chronic rapid pacing group, Bmax was reduced by 36% compared with control values. In the ACEI group, Bmax was increased from rapid pacing–only values (P=.08). In the AT1 Ang II receptor blockade group, Bmax remained significantly lower than control values and was similar to rapid pacing–only values. In the combined treatment group, Bmax was normalized. The relative binding affinity for nitrendipine remained unchanged from control values in all treatment groups.


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  		Table 2. LV Myocyte Contractile Protein, L-Type Ca2+ Channel, and SR Characterization With Rapid Pacing Heart Failure: Effects of ACEI, AT1 Ang II Receptor Blockade, or combined ACEI and AT1 Ang II Receptor Blockade During the Progression of Heart Failure



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  		Figure 3. Specific binding of L-type Ca2+ channel ligand nitrendipine to LV sarcolemmal preparations obtained from control membrane preparations, after chronic rapid pacing, and chronic rapid pacing with either concomitant ACEI, AT1 Ang II receptor blockade (AT1 block), or combined ACEI and AT1 Ang II receptor blockade (ACEI/AT1 block). Specific binding was saturable in all sarcolemmal preparations. Scatchard analysis was performed to determine Bmax and Kd for nitrendipine binding; results from this analysis are presented in Table 2Up. In rapid pacing group and monotherapy treatment groups, Bmax was decreased from control values. Nitrendipine binding in combined ACEI and AT1 Ang II receptor blockade group was similar to control values.

Abundance of SR Ca2+-ATPase and Phospholamban
A representative immunoblot for SR Ca2+-ATPase and for phospholamban is shown in Fig 4Down. A strong signal was detected for all LV membrane preparations at the 105-kD region and is consistent with the positive signal for SR Ca2+-ATPase obtained with this antiserum.40 For phospholamban, a doublet staining pattern was observed at the 29- to 27-kDa region, which is consistent with the pentameric form of this protein.9 40 41 Substitution of nonimmune mouse sera or deletion of the primary antibody in the immunoblotting procedure abolished these signals. Membrane preparations with identical protein concentrations were analyzed from each treatment group and the relative abundances of SR Ca2+-ATPase and phospholamban determined with respect to the control signal (Table 2Up). In the chronic rapid pacing group, the relative abundance of SR Ca2+-ATPase was reduced from control levels. In both monotherapy treatment groups, the relative abundance of SR Ca2+-ATPase was similarly reduced from control values. However, in the combined ACEI and AT1 Ang II blockade treatment group, SR Ca2+-ATPase levels were normalized. The relative abundance of phospholamban was unchanged with chronic rapid pacing or in any of the treatment groups.



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  		Figure 4. Representative immunoblots for SR Ca2+-ATPase and phospholamban for control membrane preparations (CON), after chronic rapid pacing (RP), and chronic rapid pacing with either concomitant ACEI (ACEI/RP), AT1 Ang II receptor blockade (ATBLK/RP), or combined ACEI and AT1 Ang II receptor blockade (ATBLK/ACEI/RP). Myocardial membrane preparations were loaded from all treatment groups on same gel at equivalent protein concentrations. Positive signals for SR Ca2+-ATPase and phospholamban were consistent for these proteins and antisera.40 41 Signal was digitized and quantified with respect to controls; this analysis is summarized in Table 2Up.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
It has been well established that with the development of severe CHF, a decline in LV myocardial contractility occurs.1 2 3 9 12 Although this is an area of active research, the specific cellular and molecular processes that contribute to the reduced LV contractility with CHF most likely include abnormalities in Ca2+ regulation and excitation-contraction coupling.1 2 3 4 5 6 7 8 9 10 11 12 ACEI has been demonstrated to provide beneficial effects in the setting of CHF.13 14 15 16 17 18 19 20 21 However, whether ACEI provides favorable effects on myocyte contractile processes and whether these potential effects are mediated solely by the AT1 Ang II receptor remains unclear. Accordingly, the first objective of this project was to define whether the effects of monotherapy by ACEI or AT1 Ang II receptor blockade would provide similar and beneficial effects on myocyte contractility and specific components of the excitation-contraction coupling process with developing CHF. A second objective was to determine the potential beneficial effects of combined ACEI and AT1 Ang II receptor blockade on myocyte contractile processes in the setting of CHF. The present study used a model of pacing-induced CHF in which chronic ACEI, AT1 Ang II receptor blockade, or a combination of both treatments was instituted with the initiation of chronic rapid pacing. Chronic rapid pacing and concomitant ACEI significantly improved myocyte contractility and inotropic responsiveness. Chronic rapid pacing and AT1 Ang II receptor blockade did not confer similar protective effects with respect to myocyte contractility. Moreover, combined ACEI and AT1 Ang II blockade provided significant beneficial effects on LV myocyte inotropic response and components of the excitation-contraction coupling process that were greater than that obtained by ACEI alone. Thus, the unique findings of this study were twofold. First, basic defects in specific components of the myocyte excitation-contraction coupling process that occur in this model of CHF are preventable. Second, combined ACEI and AT1 Ang II blockade may provide further beneficial effects on myocyte contractile processes in the setting of CHF.

Myocyte Function and Geometry
Initial studies have demonstrated that AT1 Ang II receptor blockade can be safely instituted in patients with CHF.22 Moreover, combined ACEI and AT1 Ang II receptor blockade has been demonstrated to provide additional effects with respect to lowering systemic blood pressure.25 In a recently completed study,26 the effects of chronic monotherapy with ACEI, AT1 Ang II receptor blockade, or combination therapy were examined with respect to LV function and systemic hemodynamics in the setting of pacing-induced CHF. These past in vivo studies demonstrated that ACEI during chronic rapid pacing improved LV fractional shortening but that monotherapy AT1 Ang II receptor blockade did not. Furthermore, combined ACEI and AT1 Ang II receptor blockade improved indices of LV pump function to a greater degree than either monotherapy alone. However, monotherapy and combination therapy had significant and differential effects on LV preload, afterload, heart rate, and neurohormonal status.26 Thus, whether the changes in LV pump function observed in these past in vivo studies were due to differential effects on myocyte contractile processes remained unclear. Accordingly, the present study examined contractile performance in a large number of isolated myocytes from each treatment group in which extracellular loading and neurohormonal conditions were held constant. Chronic rapid pacing caused myocyte contractile dysfunction, which was improved with concomitant ACEI. Concomitant AT1 Ang II receptor blockade did not provide similar protective effects on myocyte contractile function. Finally, combined ACEI/AT1 Ang II improved the capacity of the myocyte to respond to an inotropic stimulus to a greater extent than that obtained by ACEI alone. Thus, a contributory mechanism for the improved LV pump function after monotherapy with either ACEI or combination therapy during chronic rapid pacing is increased myocyte contractility. The development of pacing-induced CHF was associated with increased myocyte length. The increased myocyte length parallels the significant LV dilation and myocardial remodeling that occurs in this CHF process.27 29 31 In the present study, ACEI or combined ACEI and AT1 Ang II during chronic rapid pacing reduced resting myocyte length. This reduction in myocyte length with either ACEI or combination treatment has been demonstrated previously to be associated with an attenuation in the degree of LV dilation that occurs with chronic rapid pacing.26 29 These findings suggest that a cellular mechanism for the reduction in LV dilation with ACEI during chronic rapid pacing was due to a reduction in myocyte length. However, in the present study, monotherapy with AT1 Ang II receptor blockade during chronic rapid pacing did not decrease myocyte length from pacing CHF values. Furthermore, concomitant AT1 Ang II receptor blockade during chronic rapid pacing did not reduce the degree of LV dilation that invariably occurs with pacing CHF.26 These results suggest that the modulation of LV myocyte function and geometry by ACEI or combination therapy in this model of CHF may not be solely due to the prevention of AT1 Ang II receptor activation. Likely contributory factors for the effects of ACEI and combined treatment on myocyte function and geometry in this model of CHF include potentiation of bradykinin as well as influences on the activity of alternative enzyme systems. These contributory factors have been discussed in greater detail in our recent in vivo study regarding ACEI and combination therapy.26 Nevertheless, it is clear from the present study that a cellular mechanism for the improved LV function observed either with ACEI or through combined ACEI and AT1 Ang II receptor blockade during chronic rapid pacing was improved myocyte contractile performance.

Myocyte Inotropic Capacity
Pacing-induced CHF caused diminished myocyte inotropic responsiveness after ß-adrenergic receptor stimulation or in the presence of increased extracellular Ca2+. The blunted ß-adrenergic response with pacing-induced CHF is consistent with past reports from this laboratory and others29 30 32 36 and is similar to that observed in patients with severe CHF.38 Likely contributory factors for the diminished myocyte ß-adrenergic response with pacing-induced CHF are downregulation of ß-receptors, alterations in the ß-receptor transduction pathway, and diminished cAMP production.29 30 36 These alterations in ß-adrenergic responsiveness and transduction with the development of CHF have been postulated to be due to chronically elevated catecholamine levels.38 In the present study, concomitant ACEI during chronic rapid pacing improved myocyte ß-adrenergic responsiveness. Our previous in vivo studies demonstrated that ACEI with chronic rapid pacing reduced circulating catecholamine levels.26 Furthermore, this laboratory has demonstrated previously that ACEI with chronic rapid pacing improved ß-adrenergic receptor density and cAMP production from pacing CHF values.29 Unlike ACEI, however, AT1 Ang II receptor blockade with chronic rapid pacing did not reduce plasma norepinephrine content from pacing CHF values.26 The present study demonstrated that AT1 Ang II receptor blockade with chronic rapid pacing did not improve myocyte ß-adrenergic response from pacing CHF values. These findings provide additional evidence to suggest that the mechanism for the improved myocyte ß-adrenergic response with chronic ACEI is modulation of plasma norepinephrine levels rather than inhibition of myocardial Ang II formation and subsequent AT1 Ang II receptor activation. In the present study, combined ACE and AT1 Ang II receptor blockade with chronic rapid pacing improved myocyte ß-adrenergic response over that observed with ACEI alone. There are two likely contributory mechanisms for the enhanced myocyte ß-adrenergic response with combined therapy in this model of CHF. First, the degree of systemic neurohormonal activation was reduced with concomitant combined ACEI and AT1 Ang II receptor blockade with rapid pacing and thereby played a protective role with respect to sarcolemmal transduction systems.26 Specifically, in addition to reducing plasma catecholamine levels, dual therapy during chronic rapid pacing reduced plasma endothelin levels over pacing CHF and monotherapy values.26 Second, combined treatment during chronic rapid pacing improved the fundamental capacity of the myocyte to respond to an inotropic stimulus. Likely contributory mechanisms for the beneficial effects of combined ACEI and AT1 Ang II receptor blockade on the capacity of the myocyte to respond to an inotropic stimulus are presented below.

Components of the Myocyte Excitation-Contraction Coupling Process
Influx of Ca2+ through the L-type Ca2+ channels is necessary for the initiation of myocyte contraction.42 Moreover, phosphorylation of the L-type Ca2+ channels secondary to ß-adrenergic receptor activation is one mechanism by which ß-adrenergic stimulation increases myocyte contractility.43 Thus, changes in L-type Ca2+ density will have important effects on overall contractility and ß-adrenergic response. In an earlier report, Lew and colleagues44 determined that there was an approximate 1:1 concordance between the number of dihydropyridine binding sites and functional L-type Ca2+ channels. In the present study, the development of pacing-induced CHF resulted in reduced L-type Ca2+ channel abundance, which is consistent with past clinical and experimental reports.5 39 For example, Takahashi and colleagues5 demonstrated a 48% reduction in L-type Ca2+ channel abundance in patients with CHF. It has been reported previously that an {approx}40% reduction in peak L-type Ca2+ current occurred with the development of pacing-induced CHF.32 In the present study, concomitant ACEI and AT1 Ang II receptor blockade with chronic rapid pacing normalized L-type Ca2+ channel abundance. Monotherapy with either ACEI or AT1 Ang II receptor blockade with rapid pacing did not increase L-type Ca2+ channel abundance to that achieved with dual therapy. Combined ACEI and AT1 Ang II receptor blockade with chronic rapid pacing improved myocyte ß-adrenergic responsiveness to a greater degree than monotherapy values. A potential mechanism for the improvement in myocyte ß-adrenergic responsiveness in the combined ACEI and AT1 Ang II receptor blockade group was normalization of L-type Ca2+ channel abundance and function.

The results from the present study demonstrated that the defects in the capacity of the myocyte to respond to an inotropic stimulus with pacing CHF are not simply due to alterations in sarcolemmal receptor systems but rather are fundamental defects in myocyte inotropic response to exogenous Ca2+. In studies of human myocardium with end-stage CHF, abnormalities in Ca2+ homeostasis have been identified.1 3 8 12 33 45 For example, Pieske and colleagues1 demonstrated that Ca2+ uptake by the SR was reduced with CHF and was associated with diminished myocardial force generation. Furthermore, clinical studies have reported a reduction in the expression and abundance of SR Ca2+-ATPase with the development of severe CHF.9 10 The present study demonstrated a reduction in the relative abundance of SR Ca2+-ATPase with the development of pacing-induced CHF. Interestingly, the relative abundance of a regulatory protein associated with SR Ca2+-ATPase, phospholamban, was unchanged with pacing-induced CHF. These findings would suggest that significant alterations in the stoichiometric relation between SR Ca2+-ATPase and phospholamban have occurred in this CHF process. The SR Ca2+-ATPase is the fundamental mechanism by which Ca2+ is transported from the myocyte cytosolic compartment to the SR and therefore directly influences myocyte contractile properties. In the dephosphorylated state, phospholamban inhibits Ca2+ uptake by the SR Ca2+-ATPase.43 Phosphorylation of phospholamban through a cAMP-dependent mechanism relieves the inhibitory influence on the SR Ca2+-ATPase and thereby increases uptake of Ca2+ into the SR.43 We reported previously that steady-state cAMP levels are reduced with pacing-induced CHF.29 30 Taken together, these findings would suggest that the reduced SR Ca2+- ATPase abundance with no change in phospholamban content that occurred with pacing CHF diminished the capacity of the myocyte to resequester Ca2+ within the SR. In a past report, this laboratory has demonstrated increased resting intracellular Ca2+ levels within pacing CHF myocytes and that these alterations in Ca2+ homeostasis were associated with a negative velocity of shortening-frequency response.33 Therefore, in the present study, the diminished myocyte inotropic response to increased extracellular Ca2+ with pacing CHF was most likely due to an exacerbation of existing defects in Ca2+ homeostatic processes. In the present study, combined ACEI and AT1 Ang II receptor blockade during chronic rapid pacing improved myocyte contractile function with increased extracellular Ca2+. These findings would suggest that this combined treatment with pacing-induced CHF significantly improved Ca2+ homeostatic processes compared with monotherapy with either ACEI or AT1 Ang II receptor blockade. Additional evidence to support this possibility is that combined treatment with chronic rapid pacing prevented the reduction in SR Ca2+-ATPase abundance. These findings would suggest that combined ACEI and AT1 Ang II receptor blockade may provide particular benefit in the setting of severe CHF in which abnormalities in Ca2+ regulatory mechanisms such as SR Ca2+-ATPase abundance and function have been identified.1 2 3 9 10 12

Study Limitations
Ang II receptors have been identified in a number of cell types within the LV myocardium, including myocytes.46 47 48 49 50 However, increased concentrations of Ang II have not uniformly resulted in a myocardial contractile response.50 51 52 53 54 In rodent myocardium, increased concentrations of Ang II have been demonstrated to increase contractile frequency and influence lusitropy.50 54 In isolated human LV myocytes, concentrations of up to 10 µmol/L of Ang II failed to influence contractile behavior.53 In preliminary studies performed in our laboratory, Ang II in concentrations of 1 µmol/L did not significantly influence contractile function in normal porcine myocytes. Therefore, in the present study, myocyte contractile function was not examined in the presence of Ang II. In a recent study, Cheng et al52 reported that 1 µmol/L Ang II had a negative inotropic effect in a canine myocyte preparation after chronic rapid pacing. Thus, the possibility exists that chronic ACEI, AT1 Ang II receptor blockade, or combination treatment with pacing-induced CHF may influence myocyte contractile response to Ang II. In light of the findings from the present study and the past report by Cheng and colleagues, this issue warrants further investigation. In past reports, it has been demonstrated that changes in steady-state isolated myocyte contractile function directly reflect changes in the intrinsic capacity of the LV myocardium to function against a given load.55 Thus, although the isolated myocyte function studies described in the present study were performed under equivalent unloaded conditions, it is likely that these findings can be translated to intrinsic myocardial contractile capacity. Another important consideration is that this isolated myocyte system differs from in vivo preparations in which capillary diffusion distances are affected by coronary artery disease, hypertrophy, and nonuniform maintenance and control of temperature. The limitations of the isolated myocyte system must be recognized, and extrapolation of the results from these in vitro studies to in vivo conditions should be performed with caution.

Summary
In a model of pacing-induced CHF that causes functional and neurohormonal changes similar to those of the clinical spectrum of CHF,17 the present study demonstrated that specific AT1 Ang II receptor blockade did not provide protective effects similar to those of ACEI with respect to myocyte contractile processes. However, combined ACEI and AT1 Ang II receptor blockade provided additional beneficial effects on the capacity of the myocyte to respond to an inotropic stimulus. Contributory mechanisms for the protective effects of combined treatment on myocyte contractility include improved L-type Ca2+ receptor and SR Ca2+-ATPase density. Thus, dual therapy with both ACEI and AT1 Ang II receptor blockade may provide enhanced beneficial effects on myocyte contractile performance in the setting of CHF.


*    Selected Abbreviations and Acronyms
 
ACEI = ACE inhibition
Ang II = angiotensin II
Bmax = maximal binding
CHF = congestive heart failure
LV = left ventricular
MHC = myosin heavy chain
SR = sarcoplasmic reticulum
TBS = Tris-buffered saline


*    Acknowledgments
 
This study was supported by National Institutes of Health grant HL-45024 (Dr Spinale), a Basic Research Grant from Novartis (Dr Spinale), an American Heart Association Grant-in-Aid (Dr Spinale), and an AHA Medical Student Fellowship Award (Mark Melton). Dr Spinale is an Established Investigator of the American Heart Association. The authors wish to express their appreciation to Charles Basler and Jennifer Hendrick for their excellent technical assistance in this project.

Received February 3, 1997; revision received April 10, 1997; accepted April 18, 1997.


*    References
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