FHL2 prevents cardiac hypertrophy in mice with cardiac‐specific deletion of ROCK2

Abstract

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.

Fibroblast deletion of ROCK2 attenuates cardiac hypertrophy, fibrosis, and diastolic dysfunction

“…The underlying mechanism is due, in part, to ROCK2-dependent expression of FGF2 and CTGF in fibroblasts, which acts in a paracrine manner to elicit cardiomyocyte hypertrophy and cardiac fibrosis. These findings are consistent with previous animal studies showing that nonselective ROCK inhibitors can prevent the development of cardiac hypertrophy and fibrosis, and that ROCK2 rather than ROCK1 is the primary mediator of cardiac hypertrophy (10,15,27,28).…”

“…Similar findings were observed with hypertrophic markers such as atrial natriuretic factor (ANF), skeletal muscle α-actin (α-SK), and β-MHC; fibrotic markers of collagen I and fibronectin 1; and a central mediator in the pathogenesis of cardiac remodeling, transforming growth factor (TGF-β1) ( Figure 5, F-I, and Supplemental Figure 3, A-E). We have previously shown that ROCK2 in cardiomyocytes enhances apoptosis and MAPK pathway in the development of Ang II-induced cardiac remodeling, through activation of cleaved caspase-3 and extracellular signal-regulated kinase (ERK), respectively (15). Similarly, Ang II-enhanced apoptosis was reduced in heart tissues of ROCK2…”

“…To obtain a fibroblast-specific constitutively active ROCK gene, the caROCK flox/flox mice were crossed with Postn-Cre transgenic mice, which resulted in the generation of mice with genotypes of Postn-Cre Ang II Infusion. Eight-to 10-week-old ROCK2 Postn-/-and caROCK Postn-/-mice, and their age-matched littermate control mice, were implanted with mini-osmotic pumps (ALZET model 2004; Durect Corp) containing Ang II (A9525; Sigma-Aldrich; 1,000 ng/kg/min) or vehicle (saline) for 4 weeks to induce cardiac fibrosis and hypertrophy, as previously described (15). Briefly, a 1.0-cm vertical midscapular skin incision was made in mice anesthetized with isoflurane, followed by a creation of a 3.5-cm deep pocket, where a miniosmotic pump was inserted.…”

“…Four weeks after the implantation of the miniosmotic pump, blood pressure and heart rate were measured by noninvasive tail-cuff method (BP-2000; Visitech Systems Inc.) on a preheated 37°C plate to dilate the tail artery, as previously described (15). The average of no less than 10 successive measurements for each mouse was taken as the individual blood pressure and heart rate values.…”

“…Transthoracic echocardiography was performed with a high-resolution microimaging system equipped with a 30-MHz transducer (Vevo2100; VisualSonics Inc.), as previously described (15). After isoflurane inhalation, heart rate was adjusted to 450-550 beats/min in each group by adjusting the concentration of isoflurane.…”