The Rho-associated coiled-coil containing kinases, ROCK1 and ROCK2, are important regulators of cell shape, migration, and proliferation through effects on the actin cytoskeleton. However, it is not known whether ROCK2 plays an important role in the development of cardiac hypertrophy. To determine whether the loss of ROCK2 could prevent cardiac hypertrophy, cardiomyocyte-specific ROCK2-null (c-ROCK2(-/-)) were generated using conditional ROCK2(flox/flox) mice and α-myosin heavy-chain promoter-driven Cre recombinase transgenic mice. Cardiac hypertrophy was induced by Ang II infusion (400 ng/kg/min, 28 d) or transverse aortic constriction (TAC). Under basal conditions, hemodynamic parameters, cardiac anatomy, and function of c-ROCK2(-/-) mice were comparable to wild-type (WT) mice. However, following Ang II infusion or TAC, c-ROCK2(-/-) mice exhibited a substantially smaller increase in heart-to-body weight ratio, left ventricular mass, myocyte cross-sectional area, hypertrophy-related fetal gene expression, intraventricular fibrosis, cardiac apoptosis, and oxidative stress compared to control mice. Deletion of ROCK2 in cardiomyocytes leads to increased expression of four-and-a-half LIM-only protein-2 (FHL2) and FHL2-mediated inhibition of serum response factor (SRF) and extracellular signal-regulated mitogen-activated protein kinase (ERK). Knockdown of FHL2 expression in ROCK2-deficient cardiomyocytes or placing ROCK2-haploinsufficient (ROCK2(+/-)) mice on FHL2(+/-)-haploinsufficient background restored the hypertrophic response to Ang II. These results indicate that cardiomyocyte ROCK2 is essential for the development of cardiac hypertrophy and that up-regulation of FHL2 may contribute to the antihypertrophic phenotype that is observed in cardiac-specific ROCK2-deficient mice.

Background: Phosphorylation of the regulatory light chain of myosin (MLC) has roles in cardiac function. In vitro, myosin phosphatase target subunit 2 (MYPT2) is a strongly suspected regulatory subunit of cardiac myosin phosphatase (MP), but there is no in-vivo evidence regarding the functions of MYPT2 in the heart. Methods and Results:Transgenic mice (Tg) overexpressing MYPT2 were generated using the α-MHC promoter. Tg hearts showed an increased expression of MYPT2 and concomitant increase of the endogenous catalytic subunit of type 1 phosphatase (PP1cδ), resulting in an increase of the MP holoenzyme. The level of phosphorylation of ventricular MLC was reduced. The pCa-tension relationship, using β-escin permeabilized fibers, revealed decreased Ca 2+ sensitization of contraction in the Tg heart. LV enlargement with associated impairment of function was observed in the Tg heart and ultrastructural examination showed cardiomyocyte degeneration. Conclusions:Overexpression of MYPT2 and the increase in PP1cδ resulted in an increase of the MP holoenzyme and a decrease in the level of MLC phosphorylation. The latter induced Ca 2+ desensitization of contraction and decreased LV contractility, resulting in LV enlargement. Thus, MYPT2 is truly the regulatory subunit of cardiac MP in-vivo and plays a significant role in modulating cardiac function. (Circ J 2010; 74: 120 - 128)

Background & AimsCardiac myosin light chain kinase (cMLCK) plays an obligatory role in maintaining the phosphorylation levels of regulatory myosin light chain (MLC2), which is thought to be crucial for regulation of cardiac function. To test this hypothesis, the role played by ventricular MLC2 (MLC2v) phosphorylation was investigated in the phenylephrine-induced increase in twitch tension using the naturally-occurring mouse strain, C57BL/6N, in which cMLCK is down regulated.Methods and ResultsBy Western blot and nanoLC-MS/MS analysis, cMLCKs with molecular mass of 61-kDa (cMLCK-2) and/or 86-kDa were identified in mice heart. Among various mouse strains, C57BL/6N expressed cMLCK-2 alone and the closest relative strain C57BL/6J expressed both cMLCKs. The levels of MLC2v phosphorylation was significantly lower in C57BL/6N than in C57BL/6J. The papillary muscle twitch tension induced by electrical field stimulation was smaller in C57BL/6N than C57BL/6J. Phenylephrine had no effect on MLC2v phosphorylation in either strains but increased the twitch tension more potently in C57BL/6J than in C57BL/6N. Calyculin A increased papillary muscle MLC2v phosphorylation to a similar extent in both strains but increased the phenylephrine-induced inotropic response only in C57BL/6N. There was a significant positive correlation between the phenylephrine-induced inotropic response and the levels of MLC2v phosphorylation within ranges of 15–30%.ConclusionsWe identified a new isoform of cMLCK with a molecular mass of 61kDa(cMLCK-2) in mouse heart. In the C57BL/6N strain, only cMLCK-2 was expressed and the basal MLC2v phosphorylation levels and the phenylephrine-induced inotropic response were both smaller. We suggest that a lower phenylephrine-induced inotropic response may be caused by the lower basal MLC2v phosphorylation levels in this strain.

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