Disruption of ROCK1 gene attenuates cardiac dilation and improves contractile function in pathological cardiac hypertrophy

Abstract

The development of left ventricular cardiomyocyte hypertrophy in response to increased hemodynamic load and neurohormonal stress is initially a compensatory response. However, persistent stress eventually leads to dilated heart failure, which is a common cause of heart failure in human hypertensive and valvular heart disease. We have recently reported that Rho-associated coiled-coil containing protein kinase 1 (ROCK1) homozygous knockout mice exhibited reduced cardiac fibrosis and cardiomyocyte apoptosis, while displaying a preserved compensatory hypertrophic response to pressure overload. In this study, we have tested the effects of ROCK1 deficiency on cardiac hypertrophy, dilation, and dysfunction. We have shown that ROCK1 deletion attenuated left ventricular dilation and contractile dysfunction, but not hypertrophy, in a transgenic model of Gαq overexpression-induced hypertrophy which represents a well-characterized and highly relevant genetic mouse model of pathological hypertrophy. Although the development of cardiomyocyte hypertrophy was not affected, ROCK1 deletion in Gαq mice resulted in a concentric hypertrophic phenotype associated with reduced induction of hypertrophic markers indicating that ROCK1 deletion could favorably modify hypertrophy without inhibiting it. Furthermore, ROCK1 deletion also improved contractile response to β-adrenergic stimulation in Gαq transgenic mice. Consistent with this observation, ROCK1 deletion prevented down-regulation of type V/VI adenylyl cyclase expression, which is associated with the impaired β-adrenergic signaling in Gαq mice. The present study establishes for the first time a role for ROCK1 in cardiac dilation and contractile dysfunction.

Introduction

The development of postnatal cardiomyocyte hypertrophy, also named pathological hypertrophy, is the common response when heart endures increased hemodynamic load (as with chronic hypertension or valvular stenosis), myocardial injury, and neurohormonal stress. Pathological hypertrophy is characterized by increases in myocyte size and protein content, and a change in gene expression profiles from adult to a “fetal-like” program [1], [2]. Although this adaptation is initially a compensatory response, persistent stress eventually leads to a decompensate congestive heart failure, which is characterized by chamber dilation and myocardial dysfunction. Due to the high mortality of heart failure, there is a growing interest in identifying the signaling mechanisms underlying the development of cardiac hypertrophy and the transition to heart failure.

The Gq class of heterotrimeric G proteins is one important signal transducer that is responsible for the development of cardiac hypertrophy and subsequent cardiac decompensation. This class of proteins couple membrane receptors for neurohumoral factors (i.e., α1-adrenergic agonists, angiotensin II, endothelin, and prostaglandin F2α) in response to the cardiac hypertrophic response. Previous studies demonstrated that Gq signaling is necessary for pressure-overload induced cardiac hypertrophy [3], [4]. Transgenic overexpression of the α subunit of Gq in the myocardium is a well-characterized in vivo pathological hypertrophy model which recapitulates many cellular, molecular, and functional characteristics of pressure overload-induced hypertrophy including left ventricular dilation, depressed ventricular contractility at baseline and in response to β-adrenergic receptor stimulation, diminished basal and agonist stimulated adenylyl cyclase activity, and activation of a fetal cardiac gene program [5], [6]. This transgenic mouse model offers a means of studying the signaling mechanisms in myocardium responsible for cardiac dilation and contractile dysfunction in hypertrophic but not in failing phase. A number of studies have delineated several potential mechanisms by which Gαq overexpression leads to cardiomyocyte hypertrophy, cardiac dilation, and dysfunction. These mechanisms include increased expression and activity of protein kinase C (PKC)α [7], decreased adenylyl cyclase expression and catalytic activity [8], [9], and increased activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) kinase kinase 1 (MEKK1)/c-Jun N-terminal kinase (JNK) signaling pathway [10].

Rho-associated coiled-coil containing protein kinase (ROCK) is a downstream mediator of RhoA GTPase, and is believed to play a critical role in mediating the effects of RhoA on stress fiber formation, smooth muscle contraction, cell adhesion, membrane ruffling, and cell motility. Recent in vivo studies using ROCK inhibitors, Y27632 and fasudil, suggest a role of ROCK in mediating cardiac hypertrophy and remodeling [11], [12], [13], [14]. These pharmacological inhibitors are not able to distinguish between the two isoforms of the family, ROCK1 and ROCK2 [15], [16], [17]. We have recently shown that ROCK1 homozygous deficient mice developed cardiac myocyte hypertrophy in response to pressure overload induced by transverse aortic constriction, indicating that ROCK1 is not critical for the development of cardiac hypertrophy [18]. Interestingly, these mice exhibited significantly reduced interstitial fibrosis [18] and cardiomyocyte apoptosis [19]. This observation is consistent with another recent study using ROCK1 haploinsufficient mice which did not show decreased hypertrophy but decreased perivascular fibrosis induced by angiotensin II [20].

A number of the previous in vitro studies performed in cultured rat neonatal cardiomyocytes have shown that RhoA/ROCK pathway cooperated with Gαq pathway for the induction of cardiomyocyte hypertrophy by G protein-coupled receptor agonists [21], [22], [23], [24]. However, the in vivo interaction between these pathways has not been demonstrated. The combination of the well-characterized transgenic Gαq cardiac hypertrophy model with the ROCK1 deficient mice provides an opportunity to examine the potential in vivo interaction between ROCK1 and Gαq pathways and to test effects of ROCK1 deletion on the development of dilated cardiomyopathy induced by intrinsic cardiac effects in Gαq overexpression transgenic mice.