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.—Okamoto, R., Li, Y., Noma, K., Hiroi, Y., Liu, P.-Y., Taniguchi, M., Ito, M., Liao, J. K. FHL2 prevents cardiac hypertrophy in mice with cardiac-specific deletion of ROCK2.

Angiotensin II (Ang II) can induce cardiac hypertrophy through effects on cardiomyocytes and the vasculature. The direct myocardial effects of Ang II involve activation of small GTP-binding proteins, such as RhoA (1, 2) and Rac1 (3, 4). Rho-associated coiled-coil containing kinases (ROCKs) are serine/threonine protein kinases that mediate the downstream effects of RhoA on the actin cytoskeleton. Currently, there are 2 ROCK isoforms, ROCK1 and ROCK2 (5–8). Pharmacological inhibition of ROCKs suggests that ROCKs play an important role in the pathogenesis of diverse cardiovascular diseases, such as cerebral and coronary vasospasm, hypertension, cardiac hypertrophy, and ischemia-reperfusion injury (9–11). Because most ROCK inhibitors target the highly homologous amino terminus ATP-dependent kinase domain, they inhibit ROCK1 and ROCK2 at equimolar concentrations (8, 12). Therefore, it has not been possible using a pharmacological approach to delineate the tissue-specific contribution of each ROCK isoform in the pathogenesis of cardiovascular disease. Furthermore, when given in vivo and for prolonged periods of time, ROCK inhibitors could also inhibit other serine/threonine kinases, such as PKC and PKA (8, 12). Thus, the isoform-specific role of ROCKs in cardiovascular disease remains to be determined.

Recently, a cardiac-specific transcriptional cofactor, four-and-a-half LIM-only protein 2 (FHL2) has been shown to be an important negative regulator of cardiac hypertrophy (13–15). FHL2 binds and inhibits extracellular signal-regulated kinase (ERK) in cardiomyocytes (16), thereby preventing ERK-induced cardiac hypertrophy. FHL2 also inhibits serum response factor (SRF)-dependent transcription in a Rho-dependent manner in embryonic stem cells and heart (17). However, the potential upstream regulators of FHL2 in cardiac hypertrophy are not known. In this study, we found that deletion of ROCK2 in cardiomyocytes leads to the up-regulation of FHL2. Using mutant mice with cardiac-specific targeted deletion of ROCK2, we showed that the inhibitory effect of ROCK2 deletion on cardiac hypertrophy is dependent on up-regulation of FHL2.

MATERIALS AND METHODS

Generation of cardiac-specific ROCK2^−/− mice

The conditional targeting vector was constructed to delete a genomic fragment containing exon 3 of the ROCK2 gene by homologous recombination (see Fig. 1A). Two loxP sites flanking exon 3 were introduced. The neomycin resistance (Neo) gene was inserted between exon 3 and the 3′ loxP site. The 5′ homology arm and the 3′ homology arm were inserted upstream of the 5′ loxP site and downstream of 3′ loxP site, respectively. The linearized targeting vector was injected into embryonic stem cells derived from SV/129 mice. Neomycin-resistant clones were screened for homologous recombination by genomic Southern blot. The correctly targeted clones were injected into C57BL/6 blastocysts. Male heterozygous ROCK2^flox/flox mice were bred to C57BL/6 females to obtain heterozygous pups. The α-myosin heavy-chain (αMHC)-Cre transgenic mice (mixed background) were generated as described previously (18) and were backcrossed >10 generations onto C57/BL6 background.

Figure 1.

Decreased cardiac hypertrophy in c-ROCK2^−/− mice. A) Schematic diagram of ROCK2 allele, targeting vector, targeted allele, and knockout allele. Shown are positions for the neomycin cassette (Neo), loxP sites (loxP), and restriction enzyme site for BamHI. B) Southern blot analysis of hearts and tails of WT and c-ROCK2^−/− after BamHI digestion. C) Immunoblots of ROCK1 and ROCK2 in heart endothelial cells and cardiomyocytes of WT and c-ROCK2^−/− mice. D) Immunohistochemical staining of ROCK2 in the left ventricle of WT and c-ROCK2^−/− mice. E) Top panel: photomicrographs of hearts from WT and c-ROCK2^−/− mice at 4 wk after saline or Ang II infusion. Middle panel: hematoxylin and eosin staining of perfused-fixed heart in longitudinal cross sections. Bottom panel: echocardiographical images of hearts from WT and c-ROCK2^−/− mice. Scale bars = 3 mm. F) Wheat-germ agglutinin staining of hearts in WT and ROCK2^−/− mice treated with saline or Ang II (n=8/group). *P < 0.001 vs. Ang II-treated WT; ^†P < 0.01 vs. saline-treated c-ROCK2^−/−; ^‡P < 0.05 vs. Ang II-treated WT. Scale bar = 50 μm. G) Quantitative analysis of protein expression of myosin light-chain ventricular isoform 2 (MLC2v; n=8/group). *P < 0.01 vs. saline-treated WT; ^†P < 0.01 vs. saline-treated c-ROCK2^−/−; ^‡P < 0.05 vs. Ang II-treated WT. H) RNA dot-blot analysis of atrial natriuretic factor (ANF) and βMHC. The mRNA expression of ANF and βMHC was standardized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH; n=12/group). *P < 0.05 vs. saline-treated WT; ^†P < 0.05 vs. Ang II-treated WT. I) ROCK activity as assessed by immunoblotting of anti-pThr⁸⁵³ and anti-pThr⁶⁹⁶ of MBS1. The anti-pThr⁶⁹⁶MBS1 antibody could also recognize Thr⁶⁴⁶ of MBS2 and Thr⁵⁶⁰ of p85. J) Quantification of ROCK activities using anti-pThr⁸⁵³ antibody (n=12/group). *P < 0.01 vs. saline-treated WT mice. ^†P < 0.05 vs. Ang II-treated WT mice.

To obtain a cardiomyocyte-specific deletion of the ROCK2 gene, mice with homozygous ROCK2 conditional floxed allele (ROCK2^flox/flox) were crossed with αMHC transgenic mice, which resulted in the generation of mice with genotypes of αMHC-positive ROCK2^flox/flox (c-ROCK2^−/−) and αMHC-negative ROCK2^flox/flox (control). Male αMHC-positive 8- to 12-wk-old ROCK2^flox/flox mice and their littermates were used in this study. The mice were maintained in the Harvard Medical School animal facilities. The Standing Committee on Animals at Harvard Medical School approved all protocols pertaining to experimentation with animals. Haploinsufficient ROCK2 and ROCK1 mice (ROCK2^+/− and ROCK1^+/−) and FHL2 mice (FHL2^+/−) were generated as described previously (14, 19).

Mouse model of cardiac hypertrophy

Ang II (400 ng/kg/min) or saline was infused in 8-wk-old male c-ROCK2^−/− or their age-matched control littermates [wild-type (WT) or αMHC-Cre mice] with the use of subcutaneously implanted miniosmotic pumps (Alzet model 2004; Durect Corp., Cupertino, CA, USA) for 4 wk, as described previously (20).

Histological analysis

Histological analyses were performed as described in detail in Supplemental Material. Ten sections were examined in each heart, and the results obtained in each group were averaged as described previously (4).

Western blot analysis

The anti-ROCK1(sc-5560), anti-ROCK2(sc-5561 and sc-1852), anti-p-myosin binding subunit 1 (MBS1; MYPT1, Thr⁶⁹⁶, sc-33360), anti-SRF (sc-335, G-20), and anti-connective tissue growth factor (CTGF; sc-14939) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-MBS1 and anti-pMBS1 (Thr⁸⁵³) antibodies were custom-made by Covance (Princeton, NJ, USA); the anti-β-actin polyclonal antibody (RB-9421-PO) was from Lab Vision (Freemont, CA, USA); the anti-cleaved caspase 3(CS 9661), anti-pERK(CS 9101), and ERK(CS 9102) antibodies were from Cell Signaling (Beverly, MA, USA); the anti-FHL2 monoclonal antibody (k-0055) was from MBL (Woods Hole, MA, USA); the anti-c-myc monoclonal antibody (ab9106) was from Abcam (Cambridge, MA, USA); the anti-lamin A/C monoclonal antibody (MAB3211; JoL2) was from Millipore (Bedford, MA, USA); and the anti-muscle RING finger protein 3 (MuRF3) antibody (ab4351) was from Abcam. Bands were visualized with the ECL detection kit (Pierce Biotechnology, Rockford, IL, USA) and quantitated by densitometry (Image 3.1; U.S. National Insitutes of Health, Bethesda, MD, USA).

RNA analysis

RNA dot blot analysis and real-time quantitative RT-PCR were performed as described in detail in Supplemental Material.

ROCK activity assay

Rho-kinase activity was measured and expressed as the ratio of phospho-MBS1/MBS1 as described previously (19). Membranes were immunoblotted with anti-phospho-specific Thr⁸⁵³-MBS1 and anti-MBS1 antibodies.

Cell culture

Cardiomyocytes were isolated from ventricles of control and c-ROCK2^−/− mice as described previously (4). Heart endothelial cells were isolated by 2-step immunoselection with platelet endothelial cell adhesion molecule-1 and intercellular adhesion molecule-2-conjugated magnetic beads as described previously (19).

Immunoprecipitation studies

Immunoprecipitation of SRF from whole heart and cells was performed as described in detail in Supplemental Material.

Electrophoretic mobility shift assay

Nuclear proteins were isolated from heart ventricular samples, and SRF activity was examined as described previously (4, 21).

GST pulldown studies

Endogenous ERK was pulled down from native mouse hearts with GST-FHL2 using a IP buffer containing zinc ion (10 mM HEPES, pH 7.9; 100 mM KCl; 5 mM MgSO₄; 0.5% Nonidet P-40; 50 mM ZnSO₄; and 1 mM DTT), as described in detail in Supplemental Material.

Statistical analysis

All data are expressed as means ± se. Paired data were evaluated by Student's t tests, and a 1-way analysis of variance (ANOVA) was used for multiple comparisons. A value of P < 0.05 was considered statistically significant.

RESULTS

Generation of cardiac-specific c-ROCK2^−/−

The lethality observed in ROCK2^−/− mice in utero has precluded investigating the role of global ROCK2 deletion in adult mice (22). Therefore, mutant mice with conditional ROCK2 allele were developed, which could permit deletion of ROCK2 in cardiomyocytes in a tissue-specific manner (Fig. 1A). To achieve cardiomyocyte-specific deletion, we crossed mice containing conditional floxed ROCK2 alleles (exon 3) with αMHC-Cre transgenic mice (23). Cre-mediated excision of ROCK2 allele was observed only in the heart and not in the tail (Fig. 1B). Furthermore, ROCK2 expression was markedly reduced in cardiomyocytes but not in vascular endothelial cells from c-ROCK2^−/− mice (Fig. 1C, D). Using a ROCK2 N-terminal antibody, the expression of the truncated ROCK2 protein containing aa 1–74 (exons 1 and 2) was not observed in the hearts of c-ROCK2^−/− mice (data not shown).

Decreased cardiac hypertrophy in c-ROCK2^−/− mice

There were no differences in basal body weight, blood pressure, left ventricular (LV) volume, and cardiac function in WT littermates and c-ROCK2^−/− mice (Table 1). Furthermore, no anatomical or functional cardiac abnormalities were observed in c-ROCK2^−/− mice for up to 25 wk of age. Despite similar increases in systolic blood pressure by Ang II infusion in both groups, c-ROCK2^−/− mice exhibited less cardiac hypertrophy, reduced end-diastolic wall thickness, and decreased LV mass (Fig. 1E and Table 1). This corresponded to decreased cardiomyocyte cross-sectional areas in the hearts of c-ROCK2^−/− mice compared to those of either WT littermates or αMHC-Cre mice (Fig. 1F). The induction of fetal cardiac genes by Ang II, such as ventricular myosin light chain 2 (MLC2v) and βMHC was also reduced in the hearts of c-ROCK2^−/− mice (Fig. 1G, H). Interestingly, the basal expression of atrial natriuretic factor (ANF) was somewhat higher in the hearts of c-ROCK2^−/− mice than WT mice, but was not induced by Ang II infusion (Fig. 1H).

Table 1.

Hemodynamic and echocardiographic parameters

Parameter	WT		c-ROCK2−/−
	Saline	Ang II	Saline	Ang II
n	12	12	13	12
BW (g)	31.8 ± 1.10	35.0 ± 1.40	33.6 ± 1.10	33.9 ± 1.20
HR (bpm)	615 ± 22	642 ± 10	595 ± 19	636 ± 21
SBP (mmHg)	107 ± 400	121 ± 4**	105 ± 300	121 ± 3†
LVDd (mm)	3.08 ± 0.11	3.03 ± 0.13	3.26 ± 0.14	3.21 ± 0.15
LVDs (mm)	1.81 ± 0.09	1.76 ± 0.12	1.94 ± 0.08	1.87 ± 0.15
IVS (mm)	0.81 ± 0.03	1.11 ± 0.03**	0.79 ± 0.03	0.83 ± 0.03‡
PW (mm)	0.81 ± 0.03	1.11 ± 0.04**	0.77 ± 0.03	0.86 ± 0.03†,‡
FS (%)	41.3 ± 1.20	42.5 ± 2.50	40.1 ± 2.20	42.3 ± 2.70
EF (%)	79.5 ± 1.20	79.9 ± 2.20	77.5 ± 2.10	79.3 ± 2.90
LV mass (mg)	79.4 ± 5.30	126.1 ± 10.6**	83.1 ± 5.70	89.7 ± 5.7†,‡
HW/TL (mg/mm)	7.4 ± 0.3	9.3 ± 0.4**	7.2 ± 0.2	8.0 ± 0.2†,‡

Ang II, angiotensin II; c-ROCK2^−/− mice, cardiomyocyte-specific ROCK2 deletion mice; BW, body weight; HR, heart rate; SBP, systolic blood pressure; LVDd, left ventricular end-diastolic dimension; LVDs, LV end-systolic dimension; IVS, intraventricular septum; PW, posterior wall; FS, fractional shortening; EF, ejection fraction; HW, heart weight; TL, tibial length. Values are expressed as means ± se.

^**P < 0.01 vs. saline-treated WT;

^†P < 0.05 vs. saline-treated c-ROCK2^−/−;

^‡P < 0.05 vs. Ang II-treated WT.

To determine whether deletion of ROCK2 affected overall ROCK activity in the heart, we evaluated ROCK activity in the hearts of WT and c-ROCK2^−/− mice before and after Ang II infusion. Phosphospecific antibodies against conserved phosphorylation sites of ROCK, such as Thr⁸⁵³ and Thr⁶⁹⁶ of MBS1, Thr⁶⁴⁶ of MBS2, and Thr⁵⁶⁰ of p85 were used to assess ROCK activity (24). Both basal and Ang II-stimulated ROCK activities were substantially reduced in c-ROCK2^−/− mice compared to that of WT mice (Fig. 1I, J). These findings indicate that deletion of ROCK2 leads to an overall decrease in basal and Ang II-stimulated ROCK activity in the heart.

To determine whether ROCK2 could also mediate cardiac hypertrophy in response to transaortic constriction (TAC), we performed TAC procedure in WT and c-ROCK2^−/− mice. The hearts of these mice developed cardiac hypertrophy as exhibited by increased heart and lung weights-to-tibial length ratios, reduced end-diastolic wall thickness, and decreased LV mass (Supplemental Fig. S1A, B and Supplemental Table S1). Comparable increases were observed in αMHC-Cre mice compared to WT mice, indicating lack of Cre toxicity in cardiomyocytes of αMHC-Cre mice (data not shown). In contrast, c-ROCK2^−/− mice subjected to TAC developed substantially less cardiac hypertrophy. This corresponded to decreased cardiomyocyte cross-sectional areas in the hearts of c-ROCK2^−/− mice compared to that of WT mice (Supplemental Fig. S1C). These findings suggest that ROCK2 is also critical for the cardiac hypertrophic response to TAC.

Up-regulation of FHL2 in c-ROCK2^−/− mice

FHL2 has recently been found to be an antihypertrophic factor through its negative interactions with SRF and ERK (16). To determine whether deletion of ROCK2 could affect FHL2 expression, we measured protein expression of FHL2 in WT and c-ROCK2^−/− mice. In the hearts of WT mice, FHL2 expression was decreased after stimulation with Ang II (Fig. 2A), suggesting that down-regulation of FHL2 by Ang II may mediate some of the hypertrophic effects of Ang II. In the hearts of c-ROCK2^−/− mice, basal FHL2 protein level was higher compared to that of WT mice and was not decreased by Ang II infusion. The increased basal expression of FHL2 and the lack of FHL2 down-regulation by Ang II infusion, therefore, may prevent the development of cardiac hypertrophy in c-ROCK2^−/− mice. Indeed, FHL2 was recently found to form an autoinhibitory feedback loop in response to RhoA activation (17). Interestingly, compared to WT mice, MuRF3 expression was decreased in the hearts of c-ROCK2 mice (Fig. 2B), suggesting that the up-regulation of FHL2 in c-ROCK2^−/− mice may also be partly attributed to the decrease of MuRF3.

Figure 2.

FHL2 mediates the downstream effects of ROCK2 on cardiac hypertrophy. A) Immunoblot showing expression of FHL2 and β-actin in WT and c-ROCK2^−/− mice treated with saline or Ang II (n=12/group). *P < 0.05 vs. saline-treated WT; ^†P < 0.05 vs. Ang II-treated WT. B) Immunoblot showing expression of MuRF3 in WT and c-ROCK2^−/− mice (n=6/group). *P < 0.05 vs. WT mice. C) Cardiomyocyte hypertrophy as assessed by leucine uptake in rat neonatal cardiomyocytes transfected with control (−), ROCK2 siRNA, and/or FHL2 siRNA with or without Ang II (100 nM) or isoproterenol (1 μM) stimulation (n=4). *P < 0.05 vs. control cells treated with Ang II or isoproterenol; ^†P < 0.05 vs. control cells transfected with ROCK2 siRNA treated with Ang II or isoproterenol. D) ANF expression in cardiomyocytes transfected with control (−), ROCK2 siRNA, or FHL2 siRNA, and treated with Ang II (100 nM) (n=4). *P < 0.05 vs. control cells treated with Ang II; ^†P < 0.05 vs. cells transfected with ROCK2 siRNA treated with Ang II.

To determine whether FHL2 could play an important role as a downstream mediator of ROCK2 in cardiac hypertrophy, we knocked down ROCK2 and FHL2 using specific siRNAs and evaluated hypertrophic responses to Ang II in rat neonatal cardiomyocytes. The knockdown of ROCK2 leads to decreased Ang II- and isoproterenol-induced leucine uptake (Fig. 2C). This inhibitory effect of ROCK2 knockdown was rescued, in part, by concomitant knockdown of FHL2. Furthermore FHL2 knockdown alone increased the hypertrophic response to Ang II and isoproterenol. These findings correlated with changes in ANF expression (Fig. 2D), indicating that the up-regulation of FHL2 may mediate some of the antihypertrophic effects observed in c-ROCK2^−/− mice. Interestingly, FHL2 is degraded in the heart by MuRF3, a member of the RING-finger E3 ubiquitin ligases, which are expressed in striated muscle and regulate protein levels via ubiquitin-mediated proteasome degradation (25).

Inhibition of SRF/ERK by FHL2 in c-ROCK2^−/− mice

Next, we investigated whether deletion of ROCK2 affects SRF and ERK signaling pathways. Transfection of SRF cDNA increased serum response element (SRE) promoter activity by >10-fold, which was reduced by >50% with cotranfection of FHL2 cDNA (Fig. 3A). Similar findings were observed with SRF-induced SM22 and ANF promoter activities, which were reduced by 20-30% with FHL2 overexpression. Cotransfection with ROCK2 but not ROCK1 cDNA completely prevented the inhibitory effects of FHL2 on SRF-induced SRE promoter activity. These results suggest that ROCK2 can negatively regulate FHL2's inhibitory effects on hypertrophic gene transcription. Because FHL2 is also known to inhibit SRF activity through its physical interaction with SRF (17), we examined whether inhibition of ROCK by the ROCK inhibitor, fasudil, could alter the interaction of FHL2 with SRF. Treatment of rat aortic smooth muscle cells (RSMCs) and C2C12 myoblasts with fasudil increased the interaction of FHL2 with SRF (Supplemental Fig. S2A), suggesting that ROCK2 antagonizes FHL2 function, in part, by preventing FHL2 interaction with and inhibition of SRF. Indeed, compared to WT cardiomyocytes, there was substantial increase in FHL2 interaction with SRF in cardiomyocytes from c-ROCK2^−/− mice, and this interaction was substantially increased after stimulation with Ang II (Fig. 3B). Although FHL2 expression and FHL2-SRF interaction are increased in c-ROCK2^−/− mice, basal SRF activity in c-ROCK2^−/− mice was not different compared to that of WT mice (Fig. 3C). Perhaps this is one reason why c-ROCK2^−/− mice do not show any baseline phenotype. Another possibility is that other signaling pathways may compensate, such as myocardin and/or actin cytoskeletal regulation of SRF in c-ROCK2^−/− mice. Because Ang II-stimulated SRE/SRF activation is decreased in cardiomyocytes from c-ROCK2^−/− mice, these results suggest that ROCK2 may regulate SRE/SRF activation and cardiac hypertrophy by preventing FHL2-SRF interaction.

Figure 3.

Inhibition of SRF and ERK by FHL2 in ROCK-inhibited cells and c-ROCK2^−/− mice. A) Cos7 cells transfected with promoter luciferase constructs containing serum response element (SRE) were cotransfected with SRF, FHL2, ROCK2, and/or ROCK1 cDNA. Values represent fold activation compared to vector control-transfected cells (n=4). *P < 0.05 vs. control cells transfected with luciferase constructs and SRF cDNAs; ^†P < 0.05 vs. cells overexpressing SRF and FHL2. B) Coimmunoprecipitation of FHL2 and SRF in the hearts of WT and c-ROCK2^−/− mice treated with saline or Ang II for 4 wk; n = 6–10. *P < 0.01 vs. WT without Ang II treatment; ^†P < 0.001 vs. Ang II-treated WT; ^‡P < 0.05 vs. saline-treated c-ROCK2^−/−. C) Electrophoretic mobility shift assay showing SRF activation in hearts of WT and c-ROCK2^−/− mice treated with saline or Ang II (n=6/group). NS, nonspecific band; free, free or nonbinding probe. D) Immunoblot showing pERK and total ERK in WT and c-ROCK2^−/− mice treated with saline or Ang II (n=8). *P < 0.05 vs. saline-treated WT; ^†P < 0.05 vs. Ang II-treated WT.

Because FHL2 could also inhibit ERK signaling by preventing activated ERK from being retained in the nucleus, we evaluated the effect of ROCK on Ang II-induced nuclear translocation of phosphorylated ERK in NIH3T3 fibroblasts. Stimulation with Ang II led to increased ERK phosphorylation in the nucleus, which was inhibited by overexpression of FHL2 (Supplemental Fig. S2B). The inhibitory effect of FHL2 on ERK phosphorylation could be reversed by cooverexpression of constitutive-active ROCK2 (CA-ROCK2). Cooverexpression of a dominant-negative ROCK2 (DN-ROCK2) mutant, however, did not affect ERK phosphorylation beyond that observed with FHL2 overexpression alone, suggesting that FHL2 mediates most of the inhibitory downstream effects of ROCK2 inhibition on ERK phosphorylation in the nucleus. Indeed, the phosphorylation of ERK was attenuated in ROCK2-deficient cardiomyocytes (Fig. 3D). This corresponded with inhibition of ERK activity, as determined by decreased phosphorylation of p90 ribosomal S6 kinase (p90RSK; data not shown). The activities of other hypertrophic signaling molecules, such as JNK, p38MAPK, protein kinase B (Akt), and phosphatase and tensin homologue on chromosome 10 (PTEN) were not different between the hearts of WT and c-ROCK2^−/− mice, with or without Ang II treatment (data not shown). Interestingly, a yeast 2-hybrid screen using ROCK1 and ROCK2 as bait showed that only ROCK2 associated with FHL2 (Supplemental Fig. S3). However, FHL2 was weakly phosphorylated by ROCK2. Nevertheless, these results indicate that FHL2 is a novel downstream regulator of ROCK2 signaling through its inhibitory interactions with SRF and ERK.

Decreased cardiac fibrosis and apoptosis in c-ROCK2^−/− mice

Cardiac hypertrophy is marked by increased fibrosis and cardiomyocyte apoptosis. To determine whether cardiac fibrosis is altered in c-ROCK2^−/− mice, we assessed the degree of cardiac fibrosis with Sirius-red staining and CTGF expression in response to Ang II. In the hearts of c-ROCK2^−/− mice, there was less cardiac fibrosis and smaller induction of CTGF following Ang II infusion compared to that of WT mice (Fig. 4A, B). In addition, the activation of cleaved caspase-3 was greatly reduced in the hearts of c-ROCK2^−/− mice (Fig. 4C). We have previously shown that increased Rac1-dependent NADPH oxidase activity and myocardial oxidative stress are required for Ang II-induced cardiac hypertrophy (4). To determine whether the reduced Ang II-induced cardiac hypertrophy in c-ROCK2^−/− mice is associated with decreased myocardial oxidative stress, we measured NADPH oxidase activity in the hearts of WT and c-ROCK2^−/− mice. Although basal NADPH oxidase activities in the hearts of WT and c-ROCK2^−/− mice were not different, compared to hearts from WT mice, NADPH oxidase activity was reduced in the hearts of c-ROCK2^−/− mice that were treated with angiotensin (Fig. 4D). Cotreatment of diphenyleneiodonium (DPI) inhibited Ang II-induced NADPH oxidase activity in both WT and c-ROCK2^−/− mice. These findings suggest that ROCK2 mediates cardiac hypertrophy and fibrosis and cardiomyocyte apoptosis in response to Ang II by increasing NADPH oxidase activity and myocardial oxidative stress.

Figure 4.

Decreased cardiac fibrosis, cardiomyocyte apoptosis, and myocardial oxidative stress in c-ROCK2^−/− mice. A) Hearts were isolated from WT and c-ROCK2^−/− mice receiving either saline of Ang II infusion for 4 wk and stained with Sirius red. Representative photographic images are shown; n = 10/group. B) Immunoblot showing the expression of CTGF in hearts from WT and c-ROCK2^−/− mice; n = 10/group. *P < 0.05 vs. WT without Ang II treatment; ^†P < 0.05 vs. c-ROCK2^−/− without Ang II treatment; ^‡P < 0.05 vs. Ang II-treated WT. C) Effect of saline or Ang II treatment on apoptosis in the hearts of WT and c-ROCK2^−/− mice as determined by cleaved caspase-3. n = 8/group. *P < 0.05 vs. WT without Ang II treatment; ^†P < 0.05 vs. c-ROCK2^−/− without Ang II treatment; ^‡P < 0.05 vs. Ang II-treated WT. D) NADPH oxidase activity in the hearts of WT and c-ROCK2^−/− mice with or without Ang II treatment, as determined by lucigenin chemiluminescence assay in the presence or absence of diphenyleneiodonium (DPI, 10 μM). n = 8/group. *P < 0.05 vs. WT mice without Ang II treatment. †P < 0.05 vs. c-ROCK2^−/− mice without Ang II treatment, ‡P < 0.05 vs. Ang II-treated WT mice.

Rescue of ROCK2^+/− cardiac phenotype by genetic deletion of FHL2

To determine whether FHL2 could mediate some of the antihypertrophic effects observed in c-ROCK2^−/− mice, we generated haploinsufficient ROCK2^+/− mice on FHL2^+/− background. The haploinsufficient FHL2^+/− mice have ∼50% protein level of FHL2 in the heart compared to WT littermates (Fig. 5A). Similar to c-ROCK2^−/− mice, haploinsufficient ROCK2^+/− mice have increased FHL2 expression in the heart compared to WT littermates. However, the ROCK2^+/−FHL2^+/− double-mutant mice have lower FHL2 expression in the heart compared to ROCK2^+/− mice, i.e., comparable to the level of FHL2 expression in FHL2^+/− mice (Fig. 5A). Similar to c-ROCK2^−/− mice, ROCK2^+/− mice showed decreased cardiac hypertrophy compared to WT mice after the treatment with Ang II (Fig. 5B, C). This corresponded to decreased cardiomyocytes cross-sectional areas (Fig. 5D) and attenuated expression of MLC2v (Fig. 5E) in the hearts of ROCK2^+/− mice compared to that of WT mice. In contrast, FHL2^+/− mice showed augmented hypertrophic response to Ang II compared to WT mice. The decreased cardiac hypertrophy observed in ROCK2^+/− mice in response to Ang II was completely “rescued” in the FHL2^+/−ROCK2^+/− double-mutant mice (Fig. 5B–E). This corresponded with reversal of SRF and ERK activity in FHL2^+/−ROCK2^+/− mice compared to ROCK2^+/− mice (Fig. 5F, G).

Figure 5.

Rescue of ROCK2^+/− cardiac phenotype with genetic deletion of FHL2. A) Immunoblot showing FHL2 and β-actin in hearts of WT, ROCK2^+/−, FHL2^+/−, and FHL2^+/−ROCK2^+/− mice. B) Representative photomicrographs of hearts from WT, ROCK2^+/−, FHL2^+/−, and FHL2^+/−ROCK2^+/− mice at 4 wk after saline or Ang II infusion. Scale bar = 3 mm. C) Ratios of heart weight to tibial length in WT, ROCK2^+/−, FHL2^+/−, and FHL2^+/−ROCK2^+/− mice treated with saline or Ang II (n=5–8/group). *P < 0.01 vs. saline-treated WT; ^†P < 0.05 vs. Ang II-treated WT; ^‡P < 0.05 vs. Ang II-treated ROCK2^+/−. D) Assessment of myocardial cross-sectional area by wheat-germ agglutinin staining of hearts in WT, ROCK2^+/−, FHL2^+/−, and FHL2^+/−ROCK2^+/− mice treated with saline or Ang II (n=5/group). *P < 0.01 vs. saline-treated WT ; ^†P < 0.05 vs. Ang II-treated WT; ^‡P < 0.05 vs. Ang II-treated ROCK2^+/−. Scale bar = 50 μm. E) Quantitative analysis of protein expression of MLC2v (n=5/group). *P < 0.01 vs. saline-treated WT; ^†P < 0.05 vs. Ang II-treated WT; ^‡P < 0.05 vs. Ang II-treated ROCK2^+/−. F) Top panel: representative electrophoretic mobility shift assay showing SRF activation in hearts of WT, ROCK2^+/−, FHL2^+/− and FHL2^+/−ROCK2^+/− mice treated with saline or Ang II. Bottom panel: quantification of SRF activity (n=5/group). *P < 0.01 vs. saline-treated WT; ^†P < 0.05 vs. Ang II-treated WT. ‡P < 0.05 vs. Ang II-treated ROCK2^+/− mice. G) Immunoblot showing pERK and total ERK in WT, ROCK2^+/−, FHL2^+/−, and FHL2^+/−ROCK2^+/− mice treated with saline or Ang II (n=5/group). *P < 0.05 vs. Ang II-treated WT; ^†P < 0.05 vs. Ang II-treated WT; ^‡P < 0.05 vs. Ang II-treated ROCK2^+/−.

Furthermore, to determine whether cardiac fibrosis is altered in FHL2^+/−ROCK2^+/− mice, we assessed the degree of cardiac fibrosis with Sirius-red staining in response to Ang II. Similar to c-ROCK2^−/− mice, ROCK2^+/− mice showed decreased cardiac fibrosis compared to WT mice after the treatment with Ang II (Fig. 6A). This corresponded to decreased expression of CTGF and cleaved caspase-3 (Fig. 6B, C) and decreased NADPH oxidase activity (Fig. 6D) in the hearts of ROCK2^+/− mice compared to that of WT mice. In contrast, FHL2^+/− mice showed augmented fibrotic response to Ang II compared to WT mice. The decreased cardiac fibrosis, apoptosis, and oxidative stress observed in ROCK2^+/− mice in response to Ang II was completely rescued in the FHL2^+/−ROCK2^+/− double-mutant mice (Fig. 6B–D). These in vivo findings strongly suggest that the up-regulation of FHL2 due to ROCK2 deficiency may mediate some of the antihypertrophic phenotype observed in c-ROCK2^−/− mice.

Figure 6.

Rescue of ROCK2^+/− cardiac phenotype with genetic deletion of FHL2 in cardiac fibrosis, cardiomyocyte apoptosis, and myocardial oxidative stress. A) Hearts were isolated from WT, ROCK2^+/−, FHL2^+/− and ROCK2^+/− FHL2^+/− mice receiving either saline or Ang II infusion for 4 wk and stained with Sirius red. Representative photographic images are shown; n = 5–8/group. B) Immunoblot showing the expression of CTGF in hearts from WT, ROCK2^+/−, FHL2^+/−, and ROCK2^+/− FHL2^+/− mice; n = 5/group. *P < 0.05 vs. WT mice without Ang II treatment; ^†P < 0.05 vs. ROCK2^+/− mice without Ang II treatment; ^‡P < 0.05 vs. Ang II-treated WT. C) Effect of saline or Ang II treatment on apoptosis in the hearts of WT, ROCK2^+/−, FHL2^+/−, and ROCK2^+/− FHL2^+/− mice as determined by cleaved caspase-3; n = 5/group. *P < 0.05 vs. WT mice without Ang II treatment; ^†P < 0.05 vs. ROCK2^+/− mice without Ang II treatment; ^‡P < 0.05 vs. Ang II-treated WT. D) NADPH oxidase activity in the hearts of WT, ROCK2^+/−, FHL2^+/−, and ROCK2^+/− FHL2^+/− mice with or without Ang II treatment as determined by lucigenin chemiluminescence assay in the presence or absence of DPI (10 μM); n = 5/group. *P < 0.05 vs. WT mice without Ang II treatment; ^†P < 0.05 vs. ROCK2^+/− mice without Ang II treatment; ^‡P < 0.05 vs. Ang II-treated WT.

DISCUSSION

Studies using ROCK inhibitors have shown that ROCK signaling plays an important role in the pathogenesis of cardiac hypertrophy and ventricular remodeling (26–28). However, the mechanism by which ROCK mediates cardiac hypertrophy is not known. Surprisingly, ROCK1-deficient mice develop cardiac hypertrophy in response to TAC or Ang II stimulation similar to that of WT mice (20, 29). These results suggest that ROCK2 may be an important mediator of the hypertrophic process. Indeed, we found that cardiac-specific ROCK2-deficient mice exhibited decreased myocardial hypertrophic response to Ang II and TAC. The mechanism is due, in part, to the up-regulation of FHL2 in the hearts of cardiac-specific ROCK2-deficient mice, and the subsequent inhibition of SRF and ERK by FHL2. Although some studies have suggested a protective role of ERK in cardiac hypertrophy, other studies suggest that the activation of ERK leads to the development of cardiac hypertrophy. Indeed, knockdown or deletion of FHL2 reversed the antihypertrophic phenotype observed in ROCK2-deficient mice. These findings suggest that ROCK2 negatively regulates FHL2 in mediating cardiac hypertrophy.

Interestingly, hearts from c-ROCK2^−/− mice at 25 wk of age exhibited normal thickness, size, and function when compared to WT mice. Thus, it is possible that baseline heart thickness and size are not affected by FHL2, whereas physiological or pathophysiological cardiac hypertrophy, which activates signaling pathways such as SRF and ERK, is blocked by FHL2 up-regulation. Indeed, ROCK1-deficient mice, in contrast to ROCK2-deficient mice, do not exhibit FHL2 up-regulation (data not shown), and developed similar hypertrophic response to agonist infusion and TAC (29, 30). Furthermore, they showed similar ERK activation as WT mice in a model of cardiac hypertrophy and dysfunction induced by Gq transgene (31). These findings indicate that ROCK1 and ROCK2 have distinct roles in mediating cardiac hypertrophy.

SRF is a potent inducer of cardiac hypertrophy, since cardiac-specific overexpression of SRF caused hypertrophic cardiomyopathy in mouse (21), and tamoxifen-inducible cardiac-specific SRF KO mice develop dilated cardiomyopathy in the adult (32). SRF is also an important regulator of cytoskeletal proteins and transcription factors in cardiomyocytes, smooth muscle, and skeletal muscle cells (33). In cardiac and smooth muscle cells, SRF regulates their differentiation and proliferation. Thus, it is likely that SRF affects the cytoskeletal reorganization that is required for the hypertrophic process. Interestingly, RhoA has been shown to regulate SRF-mediated gene expression though the depletion of globular (G) actin during filamentous (F) actin polymerization (34, 35). At least two Rho-dependent pathways mediate a reduction in G-actin levels. One pathway involves members of the forming family of cytoskeleton proteins (diaphanous; mDia) and the other pathway is through ROCK (36). Thus, in addition to FHL2, ROCK2 could also regulate SRF activity through changes in the actin cytoskeleton. RhoA could also regulate SRF indirectly through serum response cofactors, such as myocardin and myocardin-related transcription cofactors (37, 38). Furthermore, there are other LIM proteins, such as cysteine-rich protein 1 and 2 (CRP1 and CRP2), which could associate with SRF in the nucleus to direct SRF-mediated gene expression (39).

FHL2 is a multifunctional protein and works both as a transcriptional coactivator and a corepressor depending on the circumstances (15). FHL2 has been shown to transduce RhoA signals from the cell membrane into the nucleus in a ROCK-dependent manner (40). In cardiomyocytes, FHL2 functions predominantly as an antihypertrophic factor by preferentially interacting with the activated or phosphorylated form of ERK2 (16). FHL2-deficient mice exhibit exaggerated cardiac hypertrophy in response to β-adrenergic stimulation (13). Consistent with these findings, we found that ERK-p90RSK pathway was down-regulated and cardiac hypertrophy was attenuated in c-ROCK2-deficient mice. In addition, we found that FHL2 can physically bind and inhibit the activity of SRF. This is consistent with previous studies showing an autoregulatory inhibitory feedback mechanism of Rho-SRF signaling in embryonic stem cells and embryonic hearts (17). Our findings indicate that FHL2 is negatively regulated by ROCK2 and leads to the loss of inhibitory interaction between FHL2 and SRF. Alternatively, the Rho-ROCK signaling pathway could also regulate SRF via promoting myocardin-SRF downstream effects.

Although most studies on ROCK have been focused on the actin cytoskeleton, little is known regarding the role of ROCK in regulating gene and protein expression. Several recent studies suggest that ROCK can bind and regulate transcription factors, such as Fork-head in human rhabdomyocarcoma (FKHR) in striated muscles (41). Interestingly, in our yeast 2-hybrid screen, ROCK2 was found to associate with several transcription factors and their cofactors, including FHL2, which was not observed with ROCK1. These results suggest the possibility that ROCK2 might play a greater role in transcriptional regulation than ROCK1 through direct protein-protein interactions. Although constitutive-active ROCK blocked FHL2-mediated inhibition of SRE promoter activity and nuclear translocation of phosphorylated ERK, FHL2 is only weakly phosphorylated by ROCK2 in vitro (data not shown). Thus, it does not appear that the phosphorylation of FHL2 by ROCK2 is the primary downstream mechanism that leads to Ang II-induced cardiac hypertrophy. Rather, the deletion or loss of ROCK2 leads to an up-regulation of FHL2 by unknown mechanisms. A potential mechanism for the up-regulation of FHL2 may be due to decreased expression of MuRF3 in c-ROCK2^−/− mice. MuRF3 regulates protein degradation via ubiquitin-proteasome pathway (42, 43) and has recently been found to mediate degradation of filamin C and FHL2 in the heart (25). Thus, it is possible that the decrease in MuRF3 in c-ROCK2^−/− mice could mediate the up-regulation of FHL2. It remains to be determined, however, whether the decreased MuRF3 expression observed in c-ROCK2^−/− mice is a secondary or indirect effect of ROCK2 deficiency.

In addition to being localized in the nucleus and cytosol, FHL2 is present in the Z disk of cardiac sarcomere as one of its components that tether several protein kinases to titin, such as ERK (16), creatine kinase, adenylate kinase, and phosphofructokinase (44). Therefore, we cannot exclude the possibility that increased levels of FHL2 in c-ROCK2^−/− mice may be playing a direct role on sarcomere function. However, we found that FHL2 and ROCK2 were colocalized mostly in the perinuclear region and not in the sarcomere bundle (data not shown).

In summary, our findings suggest that ROCK2 may be an important therapeutic target for preventing the development of cardiac hypertrophy. However, further studies are required to determine how ROCK2 deletion leads to the up-regulation of FHL2 and whether ROCK2 plays an important role in the transition from compensated cardiac hypertrophy to heart failure.