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Am J Physiol Heart Circ Physiol 280: H90–H98, 2001. Brief rapid pacing depresses contractile function via Ca2⫹/PKC-dependent signaling in cat ventricular myocytes YONG GAO WANG, WILLIAM J. BENEDICT, JÖRG HÜSER, ALLEN M. SAMAREL, LOTHAR A. BLATTER, AND STEPHEN L. LIPSIUS Department of Physiology, Stritch School of Medicine, Loyola University Chicago and Cardiovascular Institute, Maywood, Illinois 60153 Received 23 May 2000; accepted in final form 15 August 2000 intracellular calcium; excitation-contraction coupling; stunning; tachyarrhythmias; protein kinase C which is considered to be another form of myocardial stunning. The mechanisms by which short periods of tachyarrhythmia elicit myocardial stunning are much less understood than those responsible for chronic tachycardia-induced cardiomyopathy or ischemia-induced myocardial stunning. To date, experimental studies of RP-induced dysfunction have focused primarily on chronically paced in vivo heart preparations in an attempt to understand the complex changes that lead to congestive heart failure. Research into cellular mechanisms have used myocytes isolated from in vivo paced heart preparations (27, 34, 37). This approach, however, makes it difficult to distinguish between the direct effects of RP per se from the secondary in vivo changes that can affect contractile function. For example, in vivo models of chronic RP have documented changes in sympathetic nerve activity, alterations in the renin-angiotensin system and secretions of atrial natriuretic factor, reduced coronary perfusion, and ventricular remodeling (34). The present study indicates that a brief period of RP of ventricular myocytes depresses contractile function at the myofilament level via stimulation of a Ca2⫹/protein kinase C (PKC)-dependent signaling mechanism. These findings may be relevant to the inhibitory effects of paroxysmal ventricular tachyarrhythmia on cardiac contractile function and the development of pacing-induced cardiomyopathy. Portions of this work have been presented in abstract form (2). METHODS (26, 31) and experimentally (1, 8), chronic tachycardia can depress cardiac function and lead to congestive heart failure. Shorter periods (24 h) of rapid pacing (RP) can depress contractile function without overt signs of heart failure (40). These early changes in excitation-contraction (E-C) coupling may be important in the development of myocardial stunning. Classically, myocardial stunning is a reversible depression in contractile function that follows short periods of ischemia (6, 25). However, termination of atrial (20) or ventricular (23, 26) tachyarrhythmias also is followed by a reversible depression in contractile function, CLINICALLY Address for reprint requests and other correspondence: S. L. Lipsius, Loyola Univ. Medical Center, Dept. of Physiology, 2160 S. First Ave., Maywood, IL 60153 (E-mail: [email protected]). H90 Single cell isolation procedure. The methods used to isolate cardiac myocytes have been reported in detail (29). Briefly, adult cats are anesthetized with pentobarbital sodium (80 mg/kg ip). Isolated hearts were mounted on a Langendorff perfusion apparatus for enzymatic (0.07% type II collagenase; Worthington) cell isolation. After enzyme treatment, tissue obtained from the endocardial-midwall region of the left ventricular free wall was cut into small pieces and incubated in fresh enzyme solution. Isolated ventricular myocytes were stored in a solution of HEPES-Tyrode plus 0.1% albumin until use on the same day. 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. Section 1734 solely to indicate this fact. 0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society http://www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on September 11, 2016 Wang, Yong Gao, William J. Benedict, Jörg Hüser, Allen M. Samarel, Lothar A. Blatter, and Stephen L. Lipsius. Brief rapid pacing depresses contractile function via Ca2⫹/PKC-dependent signaling in cat ventricular myocytes. Am J Physiol Heart Circ Physiol 280: H90–H98, 2001.—The purpose of this study is to determine the effects of brief rapid pacing (RP; ⬃200–240 beats/min for ⬃5 min) on contractile function in ventricular myocytes. RP was followed by a sustained inhibition of peak systolic cell shortening (⫺44 ⫾ 4%) that was not due to changes in diastolic cell length, membrane voltage, or L-type Ca2⫹ current (ICa,L). During RP, baseline and peak intracellular Ca2⫹ concentration ([Ca2⫹]i) increased markedly. After RP, Ca2⫹ transients were similar to control. The effects of RP on cell shortening were not prevented by 1 M calpain inhibitor I, 25 M 5 G L-N -(1-iminoethyl)-orthinthine, or 100 M N -monomethylL-arginine. However, RP-induced inhibition of cell shortening was prevented by lowering extracellular [Ca2⫹] (0.5 mM) during RP or exposure to chelerythrine (2–4 M), a protein kinase C (PKC) inhibitor, or LY379196 (30 nM), a selective inhibitor of PKC-. Exposure to phorbol ester (200 nM phorbol 12-myristate 13-acetate) inhibited cell shortening (⫺46 ⫾ 7%). Western blots indicated that cat myocytes express PKC-␣, -␦, and -⑀ as well as PKC-. These findings suggest that brief RP of ventricular myocytes depresses contractility at the myofilament level via Ca2⫹/PKC-dependent signaling. These findings may provide insight into the mechanisms of contractile dysfunction that follow paroxysmal tachyarrhythmias. PACING-INDUCED CONTRACTILE DYSFUNCTION state times (60–90 s), and then held at 300–250 ms (200–240 beats/min) for 5 min. Progressive shortening of the pacing cycle length was used to allow APD to accommodate to the changing cycle length. If electromechanical alternans appeared, the pacing cycle length was increased a few milliseconds to a cycle length at which alternans did not appear. After pacing at the shortest cycle length, we progressively lengthened the pacing cycle length in reverse order back to control (1,000 ms) (see Fig. 1). At least 3 min of pacing at cycle lengths ⱕ 300 ms was required to elicit consistent RP-induced inhibition of cell shortening. Measurements of peak cell shortening were determined as an average of the last five beats at each pacing cycle length using a custom software program (LabView). The RP protocol was always accomplished by stimulated action potentials. Because RP induces sustained changes in cell shortening, in some experiments a single cell could not serve as its own control. In this case, RP-induced inhibition of cell shortening was studied in two groups of cells from the same hearts: control and test group cells. Measurement of intercellular Ca2⫹ concentration. Single ventricular myocytes were loaded with fluorescent Ca2⫹ indicator by exposure to 5 M acetoxymethyl esters (AM) of indo 1 (indo 1-AM; Molecular Probes, Eugene, OR) for 20 min at 20°C. For fluorescence measurements, a coverslip of cells was mounted on the stage of an inverted microscope. Indo-1 fluorescence was excited at 360 nm, and the Ca2⫹-dependent changes of fluorescence emitted from single cells was recorded simultaneously at 405 nm (F405) and 485 nm (F485), respectively, with photomultiplier tubes. Changes of the intracellular Ca2⫹ concentration ([Ca2⫹]i) are expressed as changes in the ratio (R ⫽ F405/F485). Analysis of PKC isoenzyme expression by Western blotting. Acutely isolated cat ventricular myocytes were centrifuged (1,000 g for 10 min) and resuspended in lysis buffer [20 mM Tris 䡠 HCl (pH 7.5) containing 0.5 mM EGTA, 0.5 mM EDTA, 10 mM mercaptoethanol, 0.5% Triton X-100, 1 mM sodium vanadate, 10 g/ml leupeptin, 10 g/ml aprotonin, and 1 mM Pefabloc]. After sonication and repeat centrifugation, we assessed the protein content of the supernatant fraction using a bicinchoninic acid assay (Pierce, Rockford, IL), and 100– 300 g of extracted protein were separated by SDS-PAGE and Western blotting. Separated proteins were probed with specific monoclonal antibodies to PKC-␣, -, -␦, and -⑀ (Transduction Laboratories, Lexington, KY). Protein bands were visualized using an enhanced chemiluminescence method (Amersham, Arlington Heights, IL). Similarly prepared tissue extracts of the rat brain (50 g) and cat brain (50 g) served as positive controls. Fig. 1. Short-term rapid pacing (RP) inhibits contraction of ventricular myocytes. A: starting at a pacing cycle length (CL) of 1,000 ms, CL was progressively decreased (descending), held at 250 ms for 5 min, and then progressively lengthened (ascending) back to control (1,000 ms). After RP, cell shortening was inhibited at each pacing CL. B: original recordings of action potentials (AP) and cell shortening recorded from a myocyte stimulated at 1,000 ms before (control) and after RP. After RP, peak cell shortening was decreased without changes in resting cell length. Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on September 11, 2016 Recording techniques. Ventricular myocytes selected for study were elongated and relaxed and exhibited regular striations and normal action potential configurations when stimulated. Action potentials and ionic currents were recorded in the whole cell configuration (15) using a perforated (nystatin; 150 g/ml)-patch method (17). Cells were superfused at 35 ⫾ 1°C with external solutions containing (in mM) 137 NaCl, 5.4 KCl, 2.0 CaCl2, 1.0 MgCl2, 5 HEPES, and 11 glucose, which were titrated with NaOH to pH 7.35 and bubbled with 100% O2. The internal pipette solution contained (in mM) 120 potassium glutamate, 20 KCl, 1.0 MgCl2, 3 Na2ATP, and 5 HEPES; pH 7.2. Isolation of the L-type Ca2⫹ current (ICa,L) was accomplished by replacing intrapipette K⫹ with cesium (Cs⫹) and adding 5 mM CsCl to external solutions to block K⫹ currents. Access resistance stabilized at 10–15 M⍀ within 5–10 min of forming a gigaseal. Action potentials (bridge) and ionic currents (discontinuous single-electrode voltage clamp) were recorded with an Axoclamp-2A amplifier (Axon Instruments) at a sampling rate of 8–10 kHz. A second oscilloscope was used to monitor the duty cycle to ensure complete settling of the voltage transient between samples. Unloaded cell shortening was monitored using a video-based edge detector (Crescent Electronics), which uses a single-raster line-scanning technique to detect edge motion at one or both ends of a cell. Computer software (pCLAMP version 7) was used to generate voltageclamp protocols as well as acquire and analyze voltage and current signals. Voltage and current traces were sampled by a 12-bit resolution analog-to-digital converter using a Pentium III computer. Data were stored on hard disk and Axotape (Axon Instruments) for later analysis. Computer software (LabView; National Instruments) was used to analyze cell shortening and action potential duration (APD) at 90% repolarization (APD90). In voltage-clamp protocols, ICa,L was activated by depolarizing steps to 0 mV from a holding potential of ⫺40 mV, which inactivates fast Na⫹ current and T-type Ca2⫹ current. ICa,L was measured as the difference between peak and steady-state current without compensation for leak currents. Data are presented as means ⫾ SE. Data obtained from two different groups of cells from the same heart were statistically analyzed using two-tailed unpaired Student’s t-test, with significance at P ⬍ 0.05. Data obtained from a single cell serving as both control and test were analyzed for statistical significance using two-tailed paired Student’s t-test at P ⬍ 0.05. RP protocol. The RP protocol consisted of the following: cells were electrically stimulated through the recording pipette at a cycle length of 1,000 ms (60 beats/min) until APD and cell shortening reached steady state, paced at progressively shorter cycle lengths (700, 500, and 400 ms) for steady- H91 H92 PACING-INDUCED CONTRACTILE DYSFUNCTION Drugs. The drugs used included the following: chelerythrine (Alexis, San Diego, CA), calpain inhibitor I (Calbiochem, La Jolla, CA), L-N5-(1-iminoethyl)-orthinthine (L-NIO) (Alexis, San Diego, CA), NG-monomethyl-L-arginine (LNMMA) (Alexis), and LY-379196 (generously provided by Dr. Chris Vlahos, Eli Lilly Research Laboratories). Generally, cells under study were exposed to drugs for ⬃5 min before the RP protocol was performed unless stated otherwise. RESULTS Figure 1, A and B, shows the effects of electrical pacing on unloaded cell shortening (contraction) of single ventricular myocytes. In Fig. 1A, a myocyte was initially paced at a cycle length of 1,000 ms (60 beats/ min). Cycle length was progressively decreased to 250 ms (240 beats/min), maintained there for 5 min, and then progressively returned to 1,000 ms (see METHODS). After RP at 250 ms for 5 min, peak (systolic) cell shortening was decreased at each pacing cycle length. Diastolic cell length was not significantly different before and after RP. Figure 1B shows an original recording of action potential configuration and cell shortening before and after RP in the same ventricular myocyte. RP-induced inhibition of cell shortening were typically accompanied by a decrease in APD90. In 26 cells, RP decreased APD90 by ⫺9 ⫾ 3% (P ⬍ 0.05). However, RP-induced changes in APD90 were variable among cells. In other words, it was not uncommon for RP to exert little to no effect on APD90 at the same time that it markedly decreased cell shortening. This is reflected in the finding that RP-induced changes in peak cell shortening and APD90 did not show a significant correlation (r ⫽ ⫺0.17; P ⫽ 0.406). Experiments described below will show that RP-induced inhibition of cell shortening was, in fact, independent of membrane voltage. In a total of 43 cells obtained from eight hearts, RP decreased peak cell shortening by ⫺44 ⫾ 4% (P ⬍ 0.001). Fig. 3. L-type Ca2⫹ current (ICa,L) is unchanged after RP. Original recordings of ICa,L before (left) and after RP (right) in the same ventricular myocyte. RP had no discernible effects on holding current, peak Ca2⫹ current amplitude, or time course of inactivation. Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on September 11, 2016 Fig. 2. RP-induced inhibition of contraction is independent of changes in membrane voltage. Cell shortening was elicited by either AP or constant voltage-clamp pulses (clamp) before and after RP in the same cells. After RP, cell shortening was inhibited when stimulated by either AP or clamp pulses, showing that RP-induced inhibition of contraction is not due to changes in membrane potential induced by RP. RP-induced shortening of APD90 could result from activation of ATP-sensitive K⫹ channels, indicating cellular hypoxia. However, exposure to 5 M glibenclamide, a specific inhibitor of ATP-sensitive K⫹ channels (11), failed to prevent RP-induced shortening of APD90 [control ⫺16 ⫾ 7% (n ⫽ 6) vs. glibenclamide ⫺14 ⫾ 6% (n ⫽ 7)]. This result is consistent with reports that RP of unloaded cardiac myocytes does not cause hypoxia (45). RP-induced changes in cell shortening and APD90 may result from a nonspecific timedependent rundown in cell viability. This possibility was examined by stimulating cells at 1,000 ms without RP for the same time period as the RP protocol (⬃15 min). There were no discernable changes in cell shortening or APD90. To determine whether changes in membrane voltage induced by RP (such as shortening of APD) may be responsible for inhibition of cell shortening, we used constant depolarizing voltage-clamp pulses to trigger cell shortening before and then after RP in the same myocytes. During voltage clamp, each cell was held at its respective resting membrane voltage (⫺75 to ⫺80 mV) and stepped to ⫹10 mV for 200 ms at 1 Hz. Cell shortening was measured before and after RP when triggered by either action potentials or voltage-clamp pulses at 1 Hz. The graph in Fig. 2 summarizes the results obtained in a total of eight cells obtained from two hearts. Generally, the amplitude of cell shortening under control conditions was smaller, although not significantly (P ⫽ 0.5), when triggered by a voltageclamp pulse than by an action potential. Nevertheless, RP inhibited cell shortening to a similar extent when triggered by action potentials (⫺52 ⫾ 8%) or by depolarizing voltage-clamp pulses (⫺48 ⫾ 10%). These findings indicate that potential changes in membrane voltage induced by RP are not responsible for the inhibition of contractility induced by RP. ICa,L plays a critical role in triggering cell shortening and may influence APD90. We therefore used voltage clamp to determine whether RP inhibited ICa,L. ICa,L was measured by voltage clamping the same cell before and then after imposing the RP protocol. As shown in Fig. 3, in cells obtained from three hearts, RP had no PACING-INDUCED CONTRACTILE DYSFUNCTION H93 Fig. 4. Intracellular Ca2⫹ concentration ([Ca2⫹]i) measurements recorded from a ventricular myocyte before, during, and after RP. Top: stimulated AP. Bottom: [Ca2⫹]i transients. During RP, baseline and peak [Ca2⫹]i increased. After RP, baseline and peak [Ca2⫹]i returned to control values. Vm, membrane voltage; F405 and F480, fluorescence at 405 and 480 nm, respectively. [Ca2⫹]i is significantly elevated, and, after RP, SR Ca2⫹ release and reuptake are not significantly changed. In addition, neither during nor after RP did we observe any signs that cells were Ca2⫹ overloaded or damaged. In other words, RP never induced Ca2⫹-mediated afterdepolarizations, Ca2⫹ waves, or spontaneous Ca2⫹ transients. Also, cells did not exhibit blebbing or become granular as a result of RP. RP-induced increases in Ca2⫹ influx could, in principle, activate a number of Ca2⫹-mediated signaling pathways that may underlie RP-induced inhibition of cell shortening. Therefore, we tested the role of Ca2⫹ influx by lowering the extracellular Ca2⫹ concentration ([Ca]o) from 2 to 0.5 mM during the RP protocol. Measurements of cell shortening were obtained in two groups of cells (control and low [Ca]o) from the same two hearts before and after RP when the [Ca]o ⫽ 2 mM. In control cells, RP elicited a typical decrease in cell shortening (3.6 ⫾ 0.8 vs. 1.8 ⫾ 0.4 m; ⫺48%) (n ⫽ 5). Figure 5, A–C, shows selected recordings of action potentials and cell shortening before, during, and after RP, when the [Ca]o was reduced to 0.5 mM during RP. Comparing cell shortening before and after RP, it is Fig. 5. RP-induced inhibition of contraction is dependent on Ca2⫹ influx. AP and cell shortenings recorded before (A), during (B), and after (C) RP at a CL of 300 ms. During RP, the extracellular calcium concentration ([Ca]o) was reduced from 2 to 0.5 mM. Lowering [Ca]o during RP prevented RP-induced inhibition of cell shortening. Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on September 11, 2016 affect on peak ICa,L density (control 8.9 ⫾ 1.8 vs. after RP 9.0 ⫾ 1.9 pA/pF; n ⫽ 13) or the rapid (1) and slow (2) time constant of ICa,L inactivation (control 1 ⫽ 5 ⫾ 0.8 and 2 ⫽ 44 ⫾ 3 ms vs. after RP 1 ⫽ 6 ⫾ 0.8 and 2 ⫽ 45 ⫾ 5 ms; n ⫽ 4). A key step in cardiac E-C coupling is Ca2⫹ release from the sarcoplasmic reticulum (SR). Therefore, fluorescent microscopy and the Ca2⫹-sensitive dye indo 1-AM were used to determine whether RP altered the handling of intracellular Ca2⫹. Figure 4 shows typical measurements of [Ca2⫹]i obtained before, during, and after RP. This cell was rapidly paced at 225 beats/min for 6 min. During RP, baseline and peak [Ca2⫹]i were prominently elevated compared with control, resulting in an increase in mean [Ca2⫹]i. After RP baseline, [Ca2⫹]i and the peak Ca2⫹ transient amplitude returned to control values. Moreover, there was no change in diastolic [Ca2⫹]i or the time course of relaxation of the Ca2⫹ transient, suggesting that SR Ca2⫹ uptake was unchanged. In five cells obtained from two hearts, during RP baseline, [Ca2⫹]i increased from 0.33 ⫾ 0.03 (control) to 0.41 ⫾ 0.03 (⫹24%; P ⬍ 0.05). These results indicate that, during RP, time-averaged H94 PACING-INDUCED CONTRACTILE DYSFUNCTION Fig. 6. Inhibition of protein kinase C (PKC)- abolishes RP-induced inhibition of contraction. A: control recordings of AP and cell shortening before and after RP. RP markedly inhibited cell shortening. B: recordings of AP and cell shortening from another myocyte in the presence of LY-379196, a selective inhibitor of PKC-. RP-induced inhibition of cell shortening was abolished. tory (43, 44) have shown that both L-NIO and L-NMMA inhibit NO-mediated processes in cat atrial myocytes. In cells from the same three hearts, L-NIO (control ⫺47 ⫾ 10% vs. L-NIO ⫺48 ⫾ 8%; n ⫽ 11) or L-NMMA (control ⫺69 ⫾ 22% vs. L-NMMA ⫺60 ⫾ 21%; n ⫽ 6) both failed to prevent RP-induced decreases in cell shortening, suggesting that NO is not mediating the inhibitory effects of RP on contractility. Activation of PKC is an important Ca2⫹-dependent signaling mechanism, which is known to be associated with negative inotropic effects in the heart (38, 41, 42). We therefore examined the role of PKC signaling by performing the RP protocol in two groups of cells from the same four hearts in the absence and presence of 2–4 M chelerythrine, a specific nonselective PKC inhibitor (16). In the test cell group, chelerythrine alone had no significant effect on peak cell shortening. When compared with control cells, however, chelerythrine significantly attenuated RP-induced decreases in cell shortening (control ⫺59 ⫾ 7%, n ⫽ 8, vs. chelerythrine ⫺14 ⫾ 3%, n ⫽ 9) (P ⬍ 0.001). In the transgenic mouse, overexpression of PKC- decreases myofilament Ca2⫹ responsiveness and contractility, possibly via phosphorylation of troponin I (38). Therefore, to assess more specifically the role of the Ca2⫹-dependent isoenzyme PKC-, we tested the effects of 30 nM LY-379196, a selective inhibitor of PKC- (33). At 30 nM, the specificity of LY-379196 for inhibition of PKC- is more than an order of magnitude greater than for inhibition of other PKC isoenzymes (33). Figure 6, A and B, shows original recordings of action potentials and cell shortenings obtained from two different myocytes. In a control cell (Fig. 6A), RP markedly decreased cell shortening (⫺81%) and slightly shortened APD90 (⫺7%). In the test cell group, LY-379196 alone elicited a small increase in basal cell shortening (⫹18 ⫾ 9%; n ⫽ 7). In a cell exposed to Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on September 11, 2016 evident that RP failed to decrease cell shortening (4.7 ⫾ 0.6 vs. 4.6 ⫾ 0.7 m; ⫺3 ⫾ 4%) (n ⫽ 6). In addition, measurements of cell shortening during RP in normal [Ca]o (2.9 ⫾ 0.5 m) and low [Ca]o (1.3 ⫾ 0.4 m) (P ⬍ 0.05) indicate that cell shortening was significantly smaller in cells paced in low [Ca]o. This is consistent with a reduced Ca2⫹ influx during RP in low [Ca]o. Moreover, the records (Fig. 5B) show that lowering [Ca]o did not interfere with excitation at high frequencies of stimulation. These results, in conjunction with those in Fig. 4, indicate that RP-induced increases in Ca2⫹ influx and subsequent elevation of [Ca2⫹]i is an essential factor in the mechanism by which RP inhibits cell shortening. In ischemic myocardial stunning, elevation of [Ca2⫹]i is thought to activate the Ca2⫹-sensitive protease calpain, resulting in degradation of contractile myofibrillar protein (9, 13). To determine whether a similar mechanism may operate in the contractile dysfunction induced by RP, we performed the RP protocol in cells exposed to calpain inhibitor I, a cell-permeant inhibitor of calpain (30). The experiments were performed by either exposing cells to 1 M calpain inhibitor I acutely (5–10 min) or by incubating cells in the inhibitor for up to 1 h before the RP protocol was initiated. Because similar results were obtained with the two methods, the data have been pooled. Calpain inhibitor I failed to affect RP-induced inhibition of cell shortening (control ⫺37 ⫾ 11% vs. calpain inhibitor I ⫺39 ⫾ 7%; n ⫽ 6 cells from 2 hearts). In cardiac myocytes, constitutive nitric oxide (NO) synthase activity is stimulated by RP-induced elevation of [Ca2⫹]i (21), and NO can inhibit cardiac contractility (5, 21). To assess the potential role of NO, we performed two series of experiments to test the effects of 25 M L-NIO and 100 M L-NMMA, both inhibitors of NO synthase (28). Previous work from this labora- PACING-INDUCED CONTRACTILE DYSFUNCTION H95 LY-379196 (Fig. 6B), RP-induced inhibition of cell shortening was abolished, whereas APD90 still shortened (⫺13%). In cells obtained from the same two hearts, LY-379196 blocked RP-induced inhibition of cell shortening (control ⫺69 ⫾ 11%, n ⫽ 5, vs. LY379196 ⫺9 ⫾ 4%, n ⫽ 7) (P ⬍ 0.001). To further evaluate the role of PKC activation, we directly stimulated phorbol ester-sensitive PKC isoenzymes by exposure to 0.2 M phorbol 12-myristate 13-acetate (PMA) for 5 min. PMA consistently and significantly decreased peak cell shortening (⫺46 ⫾ 7%; P ⬍ 0.01; n ⫽ 5) much the same as RP (data not shown). Exactly which PKC isoenzymes are expressed in the heart is somewhat controversial (39), and feline cardiac PKC isoenzyme expression has not been previously evaluated. Therefore, cellular extracts of cat ventricular myocytes were probed with monoclonal antibodies specific for PKC-␣, -, -␦, and -⑀ , the major PKC isozymes reported to be present in ventricular myocytes of several different species, including humans (37). As shown in Fig. 7, PKC-␣ (A), PKC-␦ (C), and PKC-⑀ (D) were all detected in cell extracts of freshly isolated myocytes obtained from four hearts. In addition, as shown in Fig. 7B, an 80-kDa band was detected, which comigrated with PKC- isolated from both the cat and rat brain. These results are consistent with the presence of PKC- (as well as PKC-␣, -␦, and -⑀) in adult cat ventricular myocytes. DISCUSSION The main finding of the present study is that RP of ventricular myocytes for just a few minutes elicits depression of systolic contractile function via activation of a Ca2⫹/PKC-dependent signaling mechanism. Moreover, the depression in contractile function that follows brief RP cannot be explained by alteration in the basic handling of intracellular Ca2⫹ during E-C coupling. More specifically, RP-induced inhibition of contraction was not associated with changes in 1) the trigger for SR Ca2⫹ release, i.e., Ca2⫹ influx via ICa,L; 2) SR Ca2⫹ release as determined from peak intracellular Ca2⫹ transient amplitude; or 3) reuptake of SR Ca2⫹, as determined from relaxation of the intracellular Ca2⫹ transient. Baseline levels of [Ca2⫹]i also were unchanged after RP, indicating that diastolic [Ca2⫹]i was unaffected. This is consistent with our finding that RP did not significantly affect diastolic cell length. Although RP generally shortened APD90, there was no significant correlation between changes in APD90 and peak contraction. In addition, the use of voltage-clamp pulses to elicit contraction also directly demonstrated that RP-induced inhibition of contraction is independent of RP-induced changes in action potential configuration. These results lead to the conclusion that RPinduced inhibition of contractile function occurs at the level of the contractile myofilaments. In contrast to the present results, chronically paced hearts, with (1, 8) or without (40) signs of heart failure, exhibit contractile dysfunction, which is associated with significant alterations in [Ca2⫹]i regulation and E-C coupling. At the cellular level, ventricular myocytes isolated from hearts chronically paced in vivo exhibit more positive resting membrane potentials, triangular action potential configurations, prolonged APD, decreased ICa,L density, and alterations in cytoarchitecture; all of which are thought to contribute to contractile dysfunction (34). In contrast, various models of myocardial stunning (10, 12, 13, 32) exhibit impaired contractile function that is not associated with changes in basic E-C coupling mechanisms. For example, in ischemic myocardial stunning, ICa,L density and SR Ca2⫹ release and reuptake are unchanged, whereas the responsiveness to Ca2⫹ is attenuated, indicating that contractility is affected at the myofilament level. Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on September 11, 2016 Fig. 7. PKC isoenzyme expression in cat ventricular myocytes. Detergentextracted cellular proteins from isolated cat ventricular myocytes (100– 300 g) and cat brain (50 g) and rat brain (50 g) were separated by SDSPAGE and Western blotting. The resulting blot was probed with mouse monoclonal antibodies generated against human PKC-␣ (A), PKC- (B), PKC-␦ (C), and PKC-⑀ (D). Bands were detected by an enhanced chemiluminescence protocol. The position of molecular weight standards is depicted to the left of each blot. H96 PACING-INDUCED CONTRACTILE DYSFUNCTION With regard to our initial explanation, increased expression of PKC- is consistently associated with the contractile dysfunction of cardiomyopathic and failing hearts. For instance, in streptozotocin-induced myopathic hearts, the expression of PKC- is preferentially increased over other PKC isoenzymes (18). In failing human hearts, expression of PKC- was significantly increased compared with nonfailing hearts (3). Moreover, blocking PKC- with LY-333531, a selective PKC- blocker and close analog of LY-379196, indicated that PKC- contributed the greatest amount to the total increase in PKC activity in failing hearts. Transgenic expression of PKC- in adult mice hearts caused mild and progressive ventricular hypertrophy (4, 42). In addition, troponin I is a substrate for PKC phosphorylation, and phosphorylation of troponin I decreases myofilament Ca2⫹ responsiveness (19, 41). In transgenic mouse hearts, the specific overexpression of PKC- decreases myofilament Ca2⫹ responsiveness and contractility, presumably via phosphorylation of troponin I (38). The present results therefore suggest that by raising [Ca2⫹]i, RP activates a Ca2⫹/PKC-dependent signaling mechanism, possibly via PKC-, which subsequently depresses myofilament Ca2⫹ responsiveness. Moreover, the early stimulation of PKC signaling by RP may set in motion further downstream signaling mechanisms that lead to the pathological changes characteristic of chronic tachycardia-induced cardiomyopathy. Further studies, however, involving direct measurements of PKC activation and/or translocation as well as protein phosphorylation of troponin I will be required to prove our hypothesis. We thank Rachel Gulling and Alan Furguson for expert technical assistance with this study and Dr. Chris Vlahos, Eli Lilly and Company Research Laboratories, for generously providing compound LY-379196. Support was provided by the National Heart, Lung, and Blood Institute Grants HL-27652 (to S. L. Lipsius), HL-34328 and HL63711 (to A. M. Samarel), and HL-51941 and HL-62231 (to L. A. Blatter), the American Heart Association National Center (to L. A. Blatter), and the Deutsche Forschungsgemeinschaft (to J. Hüser). L. A. Blatter is an Established Investigator of the American Heart Association. 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