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Circulation. 2007;116:596-605
Published online before print July 23, 2007, doi: 10.1161/CIRCULATIONAHA.106.682773
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(Circulation. 2007;116:596-605.)
© 2007 American Heart Association, Inc.

Arrhythmia/Electrophysiology

Gi{alpha}1-Mediated Cardiac Electrophysiological Remodeling and Arrhythmia in Hypertrophic Cardiomyopathy

Hongmei Ruan, MD, PhD; Scherise Mitchell, PhD; Monika Vainoriene, MD; Qing Lou, BA; Lai-Hua Xie, PhD; Shuxun Ren, MD; Joshua I. Goldhaber, MD; Yibin Wang, PhD

From the Division of Molecular Medicine, Departments of Anesthesiology (H.R., S.M., M.V., Q.L., Y.W.), Physiology (L.-H.X., S.R., J.I.G, Y.W.), and Medicine (J.I.G., Y.W.), Cardiovascular Research Laboratory (J.I.G, Y.W.), and Molecular Biology Institute (Y.W.), David Geffen School of Medicine, University of California at Los Angeles, Los Angeles.

Correspondence to Yibin Wang, PhD, Department of Anesthesiology, Physiology, and Medicine, Cardiovascular Research Laboratory, Molecular Biology Institute, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095. E-mail yibinwang{at}mednet.ucla.edu

Received December 7, 2006; accepted May 29, 2007.


*    Abstract
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 *Abstract
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Background— Cardiac hypertrophy is a major risk factor for arrhythmias and sudden cardiac death. However, the underlying signaling mechanisms involved in the induction of arrhythmia and electrophysiological remodeling in cardiac hypertrophy are unclear.

Methods and Results— Using an inducible gene-switch approach, we achieved tissue-specific and temporally regulated induction of a well-established hypertrophic pathway, the Ras-Raf–mitogen-activated protein kinases pathway, in adult mouse heart. On Ras activation, the transgenic animal developed ventricular hypertrophy and arrhythmias. The development of ventricular arrhythmias was temporally correlated with electrophysiological remodeling in isolated ventricular myocytes, including action potential prolongation, increased sodium-calcium exchanger activity, reduced outward potassium currents, sarcoplasmic reticulum Ca2+ defects, and loss of protein kinase A–dependent phospholamban phosphorylation. From genome-wide expression profiling, we discovered a selective induction of G{alpha} inhibiting subunit 1 (Gi{alpha}1) expression in the Ras transgenic heart. Treatment of transgenic animals with the Gi/o inhibitor pertussis toxin normalized the phospholamban phosphorylation by protein kinase A, reversed the action potential prolongation, and significantly reduced the frequency of cardiac arrhythmias in Ras transgenic animals.

Conclusions— These data suggest that selective induction of G{alpha} inhibiting subunit 1 expression and activity is a novel downstream event in hypertrophic signaling that may be a critical factor leading to cellular electrophysiological remodeling and cardiac arrhythmias in hypertrophic cardiomyopathy.


Key Words: hypertrophy • cardiomyopathy • arrhythmia • electrophysiology • signal transduction


*    Introduction
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 up arrowAbstract
 *Introduction
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Hypertrophic cardiomyopathy is the most common form of heart disease caused by genetic mutations of cardiac proteins, whereas left ventricular hypertrophy can be caused by mechanical overload associated with hypertension and valvular disease. Both may be characterized by myocyte hypertrophy, interstitial fibrosis, and induction of fetal gene expression.1–4 Both the genetic and acquired forms of cardiac hypertrophy are risk factors for sudden cardiac death associated with electrophysiological remodeling and resultant cardiac arrhythmias.5–8 Investigation of the underlying mechanisms of these manifestations has been the main focus of the field in recent years.

Clinical Perspective p 605

Ventricular arrhythmias are the most common cause of sudden cardiac death in hypertrophic cardiomyopathy and other cardiac diseases, including heart failure.9–11 Abnormal conduction and heterogeneous action potential propagation are the main causes of reentrant arrhythmias in ischemic hearts. In contrast, triggered arrhythmias caused by early or delayed afterdepolarizations are thought to be a major cause of ventricular arrhythmias in nonischemic cardiomyopathies.12 Although prolonged action potential duration (APD), repressed outward K+ currents, and increased sodium-calcium exchanger (NCX) activity are commonly observed features of electrophysiological remodeling in hypertrophic and failing hearts, the underlying molecular mechanisms that initiate these changes in the hypertrophic heart remain poorly understood.

Cardiac hypertrophy can be induced by a number of intracellular signaling pathways, including mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinase/Akt, Gq/protein kinase C, and calmodulin-activated protein kinases, as well as calcineurin.13–18 They have been shown to promote cell growth, fetal gene induction, and other features of cardiac hypertrophy in both cultured myocytes and intact animals. Although numerous transgenic models are already established to investigate the specific role of different hypertrophic signaling pathways in intact animals, the direct impact of a hypertrophic pathway on the electrophysiological properties of ventricular myocytes and their direct contribution to arrhythmogenesis remains unclear. In an earlier study,19 we generated a transgenic mouse model with targeted and chronic activation of the Ras-Raf–MAPK pathway in heart by cardiac expression of a constitutively activated H-Ras-v12 mutant. This transgenic model developed diastolic dysfunction, sarcoplasmic reticulum (SR) Ca2+ defects, and increased early mortality due to apparent sudden cardiac death.19 Thus, it is a useful in vivo model to investigate the potential impact of a hypertrophic pathway on ventricular myocyte electrophysiology and cardiac rhythm. However, because the original study was performed in animals with chronic activation of the Ras pathway, any cellular changes in the Ras-activated cardiomyocytes could have been the direct result of downstream MAPK or other secondary responses, which makes it difficult to distinguish primary mechanisms from epiphenomena. In the present study, we have established a new transgenic model with temporally regulated Ras induction in adult heart. This new model provides us with a powerful system to dissect primary versus secondary effects of a hypertrophic signaling pathway on electrophysiological remodeling and arrhythmias.

By characterizing the cardiac phenotype and cellular function at different time points after Ras induction, we observed immediate induction of cardiomyocyte hypertrophy correlated with MAPK activation. In addition, ventricular arrhythmias were detected after 7 days of transgene induction. The onset of cardiac arrhythmias was temporally correlated with electrophysiological remodeling at the cellular level, including action potential prolongation, decreased total outward K+ currents, SR Ca2+ defects, and loss of protein kinase A (PKA)–dependent phospholamban (PLB) phosphorylation. From genome-wide gene expression profiling, we identified selective induction of G{alpha} inhibiting subunit 1 (Gi{alpha}1) expression, which preceded the onset of arrhythmias, cellular remodeling, and loss of PKA activity. Application of pertussis toxin (PTX), a selective inhibitor of Gi/o, in Ras transgenic animals restored PKA-dependent phosphorylation of PLB, returned APD to normal, and markedly attenuated arrhythmic activity. These results suggest that Gi{alpha}1 induction is a novel downstream event of persistent hypertrophic signaling activation. Enhanced Gi{alpha} activity may play an important role in electrophysiological remodeling and arrhythmogenesis in hypertrophic cardiomyopathy.


*    Methods
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 *Methods
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Animals and Treatment Procedure
Transgenic mouse lines carrying {alpha}MHC-loxp-GFP-loxp-H-RasV12 and {alpha}MHC-MerCreMer have been reported previously.19–21 The genotypes of their offspring were identified by polymerase chain reaction (PCR). All experiments were performed on adult mice (C57black) between 10 and 20 weeks of age. Wild-type animals treated with identical tamoxifen regimens were used as the control group (WT). {alpha}MHC-loxp-GFP-loxp-H-RasV12 and {alpha}MHC-MerCreMer double-transgenic animals were used as the experimental group (Ras). PTX was administered by single intraperitoneal injection at a dose of 30 µg/kg body weight. All mice were generated at the University of California at Los Angeles (UCLA; Los Angeles, Calif) and were treated according to the guidelines and protocols approved by the UCLA Institutional Animal Care and Use Committee (IACUC).

Echocardiography and Electrocardiography
Echocardiography was performed with a VisualSonics Vevo 660 (VisualSonics Inc, Toronto, Canada) equipped with a 35-MHz linear transducer as reported previously.22,23 Two-lead ECG recordings (Indus Instruments, Houston, Tex) were performed on anesthetized mice (by inhalation of oxygenated 1.5% to 2.5% isoflurane, 37°C). The ECG signals were acquired at 1.0 KHz, collected for a total duration of 4 minutes for each mouse. The recordings were stored digitally and analyzed with a self-designed LQ2 program to tally abnormal rhythm–based QRS profile, interval, and duration (Q.L., unpublished data, 2007). The total duration of abnormal rhythms or the total number of abnormal QRS or RR interval distributions were tallied by the LQ2 program with identical criteria applied to all experimental groups. The abnormal rhythm was identified by several features, including reversed axis, missing P waves, bradycardia, or tachycardia.

Cardiomyocyte Isolation
Ventricular cardiomyocytes were enzymatically isolated from WT and Ras mouse hearts as described previously.22,23 Isolated myocytes were stored in a modified Tyrode’s solution containing 1 mmol/L CaCl2 at room temperature until used.22,23

Electrophysiology
Action potentials and ionic currents were measured under current- and voltage-clamp conditions with the whole-cell configuration of the patch-clamp technique at room temperature. The Axopatch-200B amplifier was coupled to a Digidata 1200A personal computer interface and was controlled by pCLAMP 7 software (Molecular Devices/Axon Instruments, Sunnyvale, Calif). We used the pCLAMP 9.2 Clampfit module for data analysis. Fire-polished electrodes had resistances of 1.5 to 2.5 M{Omega}. Action potentials were recorded in response to 1- to 2-ms depolarizing current pulses of 2 to 4 nA delivered at 1 Hz. The bath solution for action potential recording was a modified Tyrode’s solution containing (in mmol/L) 137 NaCl, 5.4 KCl, 0.33 NaH2PO4, 1.0 MgCl2, 10 HEPES, 10 glucose, 1.0 CaCl2 (pH 7.4). The pipette solution contained (in mmol/L) 140 KCl, 10 NaCl, 1 MgCl2, 5 Mg2ATP, 10 HEPES, 2 EGTA (pH 7.2). APD was measured as the time from the upstroke to 50% and 90% repolarization. Tetrodotoxin (10 µmol/L) and nifedipine (20 µmol/L) were added to the bath solution when K+ currents were recorded.23,24 The inward NCX current (INCX) was recorded during the 1.5-second application of 10 mmol/L caffeine with a rapid solution exchanger as described previously.25 The application of caffeine was always preceded by a set of six 100-ms conditioning depolarizations from –40 to 0 mV to ensure a consistent degree of SR Ca2+ loading.25 A holding potential of –40 mV was maintained during caffeine application. For Ca2+ transient observation, myocytes were loaded with the Ca2+ indicator fluo-3 pentapotassium salt (50 µmol/L, Molecular Probes Inc, Eugene, Ore) via the pipette solution, which also contained (in mmol/L) 110 CsCl, 30 TEA-Cl, 10 NaCl, 10 HEPES, 5 Mg2ATP, 0.1 cAMP, 0.05 fluo-3. Fluorescence signals were detected via a Zeiss Pascal 5 laser-scanning confocal microscope operating in line-scan mode (Carl Zeiss, Jena, Germany).22

Western Blot Analysis
Western blots were performed as described previously.19 A total of 25 to {approx}30 µg of total and membrane protein from left ventricles was loaded on 4% to 12% Bis-Tris gel, followed by electric transfer to nitrocellulose membranes. The following primary antibodies were used: anti-Ras (1:1000, Upstate Biotechnology, Lake Placid, NY); anti-PLB phosphor-Ser 16 (1:2000, Cyclacel Limited, Dundee, United Kingdom); anti-PLB phosphor-Thr 17 (1:2000, Badrilla Co, Leeds, United Kingdom); anti-Gi{alpha}1, 2, and 3 (1:500, SC-391, SC-7276, and SC-262, Santa Cruz Biotechnology, Santa Cruz, Calif); anti-PLB (1:1000, Affinity Bioreagents, Golden, Colo); and anti-Na+/Ca2+ exchanger (NCX, 1:1000, a gift from Kenneth D. Philipson’s laboratory, UCLA).

Real-Time Reverse-Transcription PCR
Total RNA was extracted from left ventricle with TRIzol reagent (Invitrogen, Carlsbad, Calif). Five micrograms of RNA was used to reverse transcribe the first-strand cDNA with a Superscript first-strand synthesis kit (Invitrogen). cDNA transcripts were quantified by the iCycler iQ real-time PCR detection system with iQ SYBR Green Supermix (Bio-Rad, Hercules, Calif). GAPDH mRNA were used for normalization. Each reaction was performed in duplicate.

Statistical Analysis
Data are presented as mean±SEM. Means of 2 samples were compared with an unpaired Student t test. Multigroup comparisons were made by ANOVA. P<0.05 was considered statistically significant.

The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Previous studies have demonstrated that chronic activation of the Ras pathway in mouse heart leads to cardiac hypertrophy and contractile dysfunction associated with SR Ca2+ cycling defects.19,26 Ho et al27,28 also demonstrated a direct impact of Ras-MAPK activation on neonatal myocyte SR Ca2+ regulation. To establish the direct downstream effect of Ras activation in adult heart, we developed a new transgenic model by breeding the {alpha}MHC-floxed-Ras line19 with the {alpha}MHC-MerCreMer line in which tamoxifen-inducible Cre is targeted in heart.21,29 Tamoxifen treatment for 3 days led to marked induction of the transgene H-Ras-V12 expression in heart (Figure 1A) and downstream MAPK activation.20 At day 4 (1 day after completion of the tamoxifen treatment) and 1 week after induction, significant myocyte hypertrophy was already observed in the Ras transgenic heart on the basis of posterior wall thickness of the left ventricle, heart weight, single-cell capacitance, and fetal cardiac gene expression (Figure 1). These data suggest that MAPK activation and ensuing hypertrophy are the immediate downstream effects of Ras activation in adult hearts.


Figure 1185341
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  		Figure 1. Inducible expression of H-Ras-v12 in adult mouse heart leads to cardiac hypertrophy and severe arrhythmia. A, Immunoblot of Ras protein in animals of different genotypes with or without tamoxifen treatment as indicated. MCM indicates {alpha}MHC-MerCreMer. B through D, Cardiac hypertrophy based on posterior wall thickness determined by echocardiography, heart weight vs body weight (HW/BW), left ventricle weight vs body weight (LV/BW), and capacitance of ventricular myocytes isolated from at least 3 different hearts measured from WT control (open bar) or double-transgenic animals (shade bar) at 4 to 10 days after tamoxifen treatment as specified. n Indicates number of myocytes or animals in each group. E, Autoradiogram of dot-blot analysis of cardiac fetal gene expression profile in Ras transgenic vs WT control samples. BNP indicates brain natriuretic peptide; ANF, atrial natriuretic factor; ß-MHC, ß-myosin heavy chain; SERCA, SR reticular Ca2+ ATPase 2a; and {alpha}-Sk-actin, {alpha}-skeletal actin. *P<0.05, **P<0.01.

In addition to cardiac hypertrophy, transgenic animals also gradually developed severe arrhythmias. Using 2-lead ECG recordings, we observed sinus arrest, idioventricular rhythm, ventricular tachycardia, conduction block, and atrial fibrillation (Figure 2). We quantified arrhythmia activity on the basis of the total duration of abnormal beats within 4 minutes of ECG recording (Figure 2C). Arrhythmia induction in the Ras transgenic animals first reached significant levels at around day 7 after tamoxifen induction and was further increased at day 14 to an average of 158±22 seconds (or 65.8±9.2% of 240 seconds’ total duration) in females and 43±30 seconds (17.9±12.5% of 240 seconds’ total duration) in male transgenic animals (P<0.001 versus day 14 of control males and females, P<0.001 versus day 0 and 5 of Ras males and females). These observations suggest that continuing Ras activation in the adult mouse heart induces progressive arrhythmias, with severity influenced by gender-specific factors. Because female Ras transgenic animals demonstrated a more consistent arrhythmia phenotype, we used only female animals for the subsequent cellular and molecular studies.


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  		Figure 2. Representative ECG recordings of Ras transgenic animals at day 0 (A, before tamoxifen) and day 10 (B, after tamoxifen) of treatment. C, Total duration of abnormal rhythm detected (see Methods) in 4 minutes of recording in WT (Control) and Ras transgenic animals (n=4 each group) on days 5, 7, 9, and 14 after tamoxifen treatment. **P<0.001 Ras vs Control at same time point. Sec indicates seconds.

To understand the cellular basis of the observed arrhythmias in Ras transgenic hearts, we characterized the electrophysiology profile from isolated left ventricular cardiomyocytes of female hearts. As shown in Figure 3A and 3B, the APD in Ras transgenic myocytes (n=8) was normal compared with controls (n=4) at day 4 postinduction, with APD50 at 4.07±0.95 versus 4.07±1.00 ms (P=NS) and APD90 at 17.25±6.7 versus 17.5±3.6 ms (P=NS, Ras versus control). However, at day 8 postinduction, APD was significantly prolonged, with APD50 at 10.9±5.3 versus 4.2±1.6 ms (P<0.001) and APD90 at 36.9±13.9 versus 15.6±4.6 ms (P<0.001, Ras [n=10] versus control [n=10]). Therefore, the onset of arrhythmias in Ras transgenic hearts is temporally correlated with significant electrophysiological remodeling at the cellular level.


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  		Figure 3. Electrophysiological remodeling in Ras transgenic ventricular myocytes. A and B, 50% and 90% decay of action potential measured from left ventricular myocytes (with number indicated) of WT and Ras animals at day 4 or day 8 after tamoxifen induction. The insets are representative recordings with blue tracing for WT and red tracing for Ras. C through F, Outward K+ currents were measured in ventricular myocytes isolated from WT or Ras transgenic hearts after 8 days of tamoxifen induction as described in Methods. C and D, Representative current recordings under voltage-clamp protocol illustrated in E. F, Current-voltage (I-V) relationship of total outward K+ currents in Ras (n=9) and WT (n=16) myocytes. **P<0.01, Ras vs WT.

Prolongation of APD in cardiomyocytes implies increases in inward currents or decreases in outward currents. Our previous studies found no significant change in inward Ca2+ currents in constitutively active Ras myocytes.19 We therefore examined outward (repolarizing) K+ currents in the Ras ventricular myocytes. As shown in Figure 3, at day 8 after induction, outward K+ current was reduced significantly in Ras transgenic myocytes (Figure 3D) compared with control (Figure 3C). In contrast, K+ current densities in myocytes isolated from day 4 postinduction hearts were completely unchanged (data not shown). These data suggest that a reduced outward K+ current may contribute to APD prolongation in Ras myocytes. We also measured NCX current and SR Ca2+ release in response to caffeine application in fluo-3–loaded myocytes using confocal microscopy and the patch-clamp technique. We found a strong trend toward increased inward peak NCX current density in response to caffeine in Ras transgenic cells compared with controls (Figure 4A, 4B, and 4C; Ras 1.70±0.53 pA/pF versus control 1.22±0.30 pA/pF, P=0.1). However, peak caffeine-induced Ca2+ transients (F/F0; Figure 4A, 4B, and 4D) were markedly reduced in Ras cells, which indicates reduced SR Ca2+ content. Because NCX current is dependent on SR Ca2+ release, this reduction in SR Ca2+ content will lead to an underestimation of NCX activity. To correct for this, we normalized peak NCX current density to the peak corresponding Ca2+ transient.25 Normalized NCX current, a more accurate reflection of absolute NCX activity, was significantly increased in Ras myocytes compared with controls (Figure 4F). We also found that Ca2+ transient decay was significantly slowed in Ras myocytes versus controls (Figure 4A, 4B, and 4E). These data indicate that the increase in arrhythmia activity in Ras transgenic animals is temporally associated with profound cellular electrophysiological changes, including reduced outward K+ currents, increased NCX activity, loss of SR Ca2+, and impaired SR Ca2+ uptake. These changes are known contributing factors to APD prolongation and arrhythmias.12


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  		Figure 4. Ca2+ regulation defects in Ras transgenic ventricular myocytes. Representative tracings of Ca2+ transients (A) and NCX currents (B) after rapid application of caffeine in ventricular myocytes isolated from Ras or WT control hearts at day 8 after tamoxifen induction. C, Average values of peak NCX current densities (INCX). D, Average values of peak Ca2+ transient (F/F0). **P<0.01, Ras vs WT. E, Half-time of Ca2+ transients decay after removal of caffeine. **P<0.01, Ras vs WT. F, Average values of NCX activity as measured from the ratio of peak NCX current density (INCX) against corresponding peak F/F0, **P<0.01, Ras vs WT. G, Representative immunoblot for phosphor-ERK, phospho-ser-16 (PLB-S16-p), total PLB, protein phosphatase 1 (PP1), and NCX with protein extracts prepared from WT or Ras transgenic hearts at different time points of tamoxifen induction as indicated.

Consistent with our cellular physiology data, we found that NCX1 protein expression was significantly increased at day 7 after Ras induction, and the increase persisted for at least 14 days (Figure 4G). There was also a reciprocal decrease in PKA-dependent PLB Ser-16 phosphorylation at and beyond day 7 after Ras induction, whereas PLB phosphatase PP1 protein was modestly increased during the same time period (Figure 4G). This is consistent with the delayed SR Ca2+ uptake observed in Ras ventricular myocytes. However, these changes are unlikely to be direct consequences of Ras-induced MAPK activation, because MAPK activities are already highly induced at an earlier time point (day 4), when cellular physiology remains unaffected. In contrast, when cellular remodeling was detected at later time points (beyond day 8), the MAPK activities in the Ras transgenic hearts had already diminished (Figure 4G). Therefore, unlike a MAPK-dependent hypertrophy, a MAPK-independent pathway is likely responsible for the cellular electrophysiology remodeling observed in the Ras transgenic heart.

To identify the molecular nature of Ras-induced cellular remodeling, we performed genome-wide cDNA microarray analysis using mRNA samples prepared from transgenic hearts at day 4 (early) and day 14 (late) time points after induction of Ras (Figure 5). We have recently reported a global analysis of the same microarray data.20 Among the genes that are significantly changed at both early and later time points of Ras activation, we found a marked induction of the PTX-sensitive Gi{alpha}1 gene in Ras transgenic hearts compared with controls.20 This was unexpected, because the Gi{alpha}1 gene is reported to be selectively expressed in brain and kidney, whereas Gi{alpha}2 and Gi{alpha}3 are more abundant in normal heart.30,31 However, we confirmed by real-time quantitative reverse-transcription PCR that Gi{alpha}1 mRNA was indeed induced by {approx}20-fold in Ras transgenic hearts at day 14 postinduction (Figure 5A). By immunoblot using anti-Gi{alpha}1 antibodies, we further demonstrated that Gi{alpha}1 was also highly induced in Ras transgenic hearts at the protein level compared with its undetectable level in WT controls (Figure 5B). In contrast, Gi{alpha}2/3, G{alpha}o1, and G{alpha}s mRNA were not significantly altered in the transgenic heart (Figure 5C). Furthermore, immunoblots did not detect significant increases in Gia2 and Gia3 proteins in the Ras transgenic hearts compared with controls (Data Supplement, Figure I). These data suggest that Ras activation in adult heart led to selective induction of Gi{alpha}1 expression.


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  		Figure 5. Gi{alpha}1 induction in Ras transgenic heart. A, Relative mRNA levels of Gi{alpha}1 measured by quantitative reverse-transcription PCR at different time points as indicated. *P<0.05,**P<0.01, Ras vs WT. B, Gi{alpha}1 protein measured by immunoblot from membrane protein preparations of WT control and transgenic Ras hearts 14 days after tamoxifen induction. C, Relative mRNA levels of Gi{alpha}2, Gi{alpha}3, G{alpha}o1, and G{alpha}s measured by quantitative reverse-transcription PCR from 3 samples of each group normalized against GAPDH at different time points after tamoxifen induction.

Because Gi{alpha}1 is known to function as an endogenous ß-adrenergic signal desensitizer via negative regulation of adenylyl cyclase activity and subsequent PKA inactivation,31–33 our finding raises the possibility that enhanced Gi{alpha}1 inhibits PKA-dependent downstream signaling in heart, as manifested in hypophosphorylation of PLB. To test this possibility, we administered a single injection of the selective Gi/o inhibitor PTX on day 10 after tamoxifen induction in both WT and Ras transgenic animals (Figure 6A). PTX treatment did not affect transgene H-Ras-V12 expression at day 13 after tamoxifen induction (3 days after PTX treatment; data not shown) or PKA-dependent PLB-S16 phosphorylation in WT control hearts (Figure 6C). However, PTX treatment restored PLB-S16 phosphorylation in Ras transgenic heart to normal levels (Figure 6C). Similarly, the APD measured from PTX-treated Ras transgenic cardiomyocytes (n=13) was also reduced to the same levels as WT controls (APD50 at 5.85±1.62 and APD90: at 13.77±2.97 ms, P=NS versus WT control; Figure 7A and 7B). Longitudinal observation from the same group of animals throughout the experimental protocol showed that the frequency of abnormal heart rhythms was markedly reduced from an average of 29.1% at day 10 (pre-PTX treatment) to an average of 1.2% at day 13 (3 days after PTX; n=4; Figure 7C and 7D). Therefore, PTX treatment reversed the loss of PKA signaling, normalized electrophysiological properties, and attenuated arrhythmias at the molecular, cellular, and whole-animal levels. These results strongly support an important role of elevated Gi{alpha} signaling in arrhythmia induction and molecular/cellular remodeling in response to persistent hypertrophic growth signaling in heart.


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  		Figure 6. Effect of PTX treatment on PKA-mediated PLB phosphorylation. A, Treatment protocol for Ras activation by tamoxifen and Gi{alpha} inhibition by PTX in WT and Ras transgenic animals. B through D, Immunoblot and quantification for PKA-dependent Ser-16 phosphorylation (pSer16-PLB) and calmodulin-activated protein kinase–dependent Thr-17 phosphorylation (pThr17-PLB) and total PLB in left ventricular tissues prepared from 3 samples of each different treatment group of WT and Ras transgenic hearts, as indicated.


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  		Figure 7. Normalized electrophysiology and attenuated arrhythmia by PTX in Ras transgenic animals. A, APD at 50% (APD50) and B, APD at 90% (APD90) measured from ventricular myocytes of different experimental groups isolated at the end of the treatment protocol shown in Figure 6. C, Representative ECG tracing and D, total number of abnormal QRS incidents during 4 minutes of recording from 4 individual Ras transgenic animals at the 3 time points of the treatment protocol indicated. Sec indicates seconds.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
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Cardiac hypertrophy is a major risk factor for cardiac arrhythmias and sudden cardiac death, yet the specific mechanisms whereby hypertrophic signaling results in electrical remodeling and arrhythmias are unknown. In this report, we took advantage of an inducible transgenic system to achieve an efficient, targeted, and temporally regulated activation of the Ras pathway in adult heart so that we could dissect the downstream effects of this well-established prohypertrophic signaling pathway on cardiac rhythm and cellular physiology. Molecular, cellular, and whole-animal analyses showed that within 4 days of H-Ras-v12 transgene induction in adult mouse heart, there was an increase in downstream MAPK activity and cardiomyocyte hypertrophy. Transgenic animals developed significant cardiac arrhythmias within 1 week of induction, which temporally coincided with profound electrophysiological changes in ventricular myocytes. Notably, APD was markedly prolonged, outward K+ currents were suppressed, and NCX activity and protein were increased. In addition to these electrophysiological changes, systolic SR Ca2+ release was reduced, and diastolic Ca2+ uptake was delayed (Figure 4). These cellular changes were closely associated with impairment of PKA signaling as reflected in PLB hypophosphorylation. Therefore, the present in vivo study suggests that Ras-induced MAPK activation has a direct and immediate effect on myocyte hypertrophy. In contrast, the cellular electrophysiological remodeling and intracellular Ca2+ cycling defects are most likely secondary consequences of prolonged Ras activation. From comprehensive gene expression profiling in Ras transgenic hearts, we found a significant induction of Gi{alpha}1 expression in response to Ras activation at both early and later time points. The functional significance of the enhanced Gi{alpha}1 signaling in electrophysiological remodeling and arrhythmogenesis was further demonstrated by the normalization of APD and significant attenuation of arrhythmias in Ras transgenic animals after PTX treatment. In summary, the present study provides the first in vivo evidence for 2 distinct downstream effects of Ras activation in heart: an early MAPK-dependent cardiac hypertrophy and delayed Gi{alpha}1-mediated electrophysiological remodeling and arrhythmias. Interestingly, extracellular signal-regulated kinase activation was only observed in day 4 samples, which suggests an endogenous negative-feedback mechanism that limits the duration of downstream extracellular signal-regulated kinase activity in spite of sustained Ras activation in adult mouse heart.

Gi{alpha} proteins couple to muscarinic receptors and ß2-receptors to inhibit adenylyl cyclase activity.30,34 Enhanced Gi{alpha} signaling through targeted expression of Gi-coupled receptor in heart was reported to induce pathological remodeling and ventricular arrhythmias,35 as well as contractile dysfunction,36 in transgenic mice. Increased expression of Gi{alpha} proteins was also observed in human diseased heart and in numerous animal models of heart failure.37–42 These data suggest that elevated Gi{alpha} signaling and negative regulation of PKA in heart is a common feature associated with the onset of heart failure. However, most of the studies focused on the Gi{alpha}2 and Gi{alpha}3 subunits. A selective induction of Gi{alpha}1 expression in a model of hypertrophic cardiomyopathy has not been reported or investigated. In the present report, we not only demonstrated the induction of Gi{alpha}1 at both mRNA and protein levels but also showed that PTX treatment effectively reversed the electrophysiological remodeling and attenuated the arrhythmias. Therefore, the observed Gi{alpha}1 induction is likely an important contributor to electrophysiological abnormalities and arrhythmias in hypertrophic hearts. However, the absolute amount of Gi{alpha}1 protein induction relative to Gi{alpha}2 or Gi{alpha}3 protein levels in heart is unknown. Although PTX is a selective inhibitor for all isotypes of Gi and Go, the specific contribution of Gi{alpha}1 induction will need to be investigated with other approaches in the future. Notably, genetic inactivation of Gi{alpha}2 or Gi{alpha}3, the predominant isotypes expressed in heart, does not affect cardiac function or ß-adrenergic receptor sensitivity,31 which suggests redundant activity in normal myocardium. On the other hand, genetic inactivation of Gi{alpha}1 has a major impact on adenylyl cyclase activity in hippocampus neurons.30 A concomitant induction of other G proteins, such as G{alpha}s, can potentially alter the final outcome in PKA signaling and function. Future investigation for the global and local PKA activity in Ras transgenic hearts is needed. Although the present report suggests a Gi{alpha}1-mediated mechanism in cardiac arrhythmia induction during the hypertrophic response, the underlying molecular and cellular basis remains unclear and could be quite complicated. In fact, Grimm et al43 have reported that PTX treatment in normal rats under ß-adrenergic stimulation actually increased the propensity of cardiac arrhythmias. It is possible that enhanced Gi{alpha} activity in hypertrophic cardiomyopathic heart or loss of Gi{alpha} activity after ß-adrenergic receptor stimulation has the same effect of promoting triggered arrhythmias via separate mechanisms, 1 of which involves Gi{alpha}-mediated inhibition of SR Ca2+ ATPase activity for SR Ca2+ uptake, with the other involving PKA-dependent RyR phosphorylation in SR Ca2+ leakage. Indeed, increasing SR Ca2+ ATPase activity and reducing RyR-mediated Ca2+ leakage have had a similar impact on attenuating cardiac arrhythmias in failing hearts.44–46 This speculation will need to be tested experimentally.

The temporally dissociated onsets of hypertrophy and cellular electrophysiological remodeling may have important implications for the pathogenesis of hypertrophic cardiomyopathy. At early stages of Ras activation, growth signals lead to compensated hypertrophy without triggering cellular remodeling. When a growth signal is persistently activated, as in the case of genetic mutations, chronic overload or injury and secondary changes can lead to impaired SR Ca2+ cycling and electrophysiological remodeling, which eventually contribute to the transition into heart failure and the genesis of arrhythmias. Induction of Gi{alpha}1 activity may represent a novel mechanism underlying this critical transition and disease progression. Further studies will be needed to determine whether Gi{alpha}1 induction is both necessary and sufficient to induce cardiac arrhythmia and ventricular electrophysiology remodeling.

The present data also raise several interesting questions. Although our results demonstrate that Gi{alpha}1 induction at both the mRNA and protein level is a major downstream event of Ras signaling, the molecular mechanism that mediates Gi{alpha}1-dependent regulation on PKA signaling and electrophysiological remodeling is still unknown and requires further investigation. Other than membrane K+ currents, the impact of Gi{alpha}1 on SR Ca2+ cycling may also play a role in the development of arrhythmias.47 The induction of Gi{alpha}1 expression in other forms of heart failure will also need to be studied. Nevertheless, Gi{alpha}1 induction most likely represents a single (although critical) element of pathological changes during hypertrophy. Other molecular changes, including changes in KChIP expression (data not shown) may also contribute to the pathogenesis of cardiac arrhythmias. Clearly, further studies in a transgenic model of temporally regulated hypertrophy and arrhythmia such as we have reported here will provide an important system for shedding new light on the molecular and cellular mechanisms of this complex disease.


*    Acknowledgments
 
The authors wish to thank Haiying Pu for her excellent technical assistance and Drs Enrico Stefani and James Weiss for their critical discussion and support.

Sources of Funding

The present study is supported in part by the Laubisch Foundation at the University of California at Los Angeles and grants from the National Institutes of Health (Drs Wang and Goldhaber). Dr Ruan is a recipient of a postdoctoral fellowship award from the Great Western Affiliate of the American Heart Association, and Dr Wang is an Established Investigator of the American Heart Association.

Disclosures

None.


*    References
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CLINICAL PERSPECTIVE

Cardiac arrhythmia is the leading cause of premature death in hypertrophic cardiomyopathy patients, and cardiac hypertrophy is also a major risk factor for sudden cardiac death. Despite their close relationship in clinical manifestations, it is unclear how cardiac hypertrophy leads to cellular remodeling that triggers arrhythmia. In this report, we used sophisticated molecular genetic approaches to activate a potent hypertrophic signaling pathway in adult mouse heart in a temporally regulated fashion. From extensive cellular and molecular studies, we established the time line from hypertrophic stimulation to cellular remodeling and the subsequent onset of arrhythmia. Furthermore, by combining cellular physiology and genomic analysis, we identified a novel signaling mechanism in the development of arrhythmia during cardiac hypertrophy that involves the selective induction of G{alpha} inhibiting subunit 1 and the subsequent loss of protein kinase A activity. This finding offers a thus far unrecognized mechanism for the development of cardiac arrhythmia during hypertrophic response. Identification of G{alpha} inhibiting subunit 1 as a potential mediator in cardiac arrhythmia is potentially important in the future development of effective therapies to prevent and treat sudden cardiac death associated with hypertrophic cardiomyopathy.


*    Footnotes
 
The online-only Data Supplement, consisting of a figure, is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.682773/DC1.




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Circulation, September 18, 2007; 116(12): 1413 - 1423.
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