Fibroblast deletion of ROCK2 attenuates cardiac hypertrophy, fibrosis, and diastolic dysfunction
Toru Shimizu, Nikhil Narang, Phetcharat Chen, Brian Yu, Maura Knapp, Jyothi Janardanan, John Blair, James K. Liao
Toru Shimizu, Nikhil Narang, Phetcharat Chen, Brian Yu, Maura Knapp, Jyothi Janardanan, John Blair, James K. Liao
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Research Article Cardiology

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

Abstract

Although left ventricular (LV) diastolic dysfunction is often associated with hypertension, little is known regarding its underlying pathophysiological mechanism. Here, we show that the actin cytoskeletal regulator, Rho-associated coiled-coil containing kinase-2 (ROCK2), is a critical mediator of LV diastolic dysfunction. In response to angiotensin II (Ang II), mutant mice with fibroblast-specific deletion of ROCK2 (ROCK2Postn–/–) developed less LV wall thickness and fibrosis, along with improved isovolumetric relaxation. This corresponded with decreased connective tissue growth factor (CTGF) and fibroblast growth factor–2 (FGF2) expression in the hearts of ROCK2Postn–/– mice. Indeed, knockdown of ROCK2 in cardiac fibroblasts leads to decreased expression of CTGF and secretion of FGF2, and cardiomyocytes incubated with conditioned media from ROCK2-knockdown cardiac fibroblasts exhibited less hypertrophic response. In contrast, mutant mice with elevated fibroblast ROCK activity exhibited enhanced Ang II–stimulated cardiac hypertrophy and fibrosis. Clinically, higher leukocyte ROCK2 activity was observed in patients with diastolic dysfunction compared with age- and sex-matched controls, and correlated with higher grades of diastolic dysfunction by echocardiography. These findings indicate that fibroblast ROCK2 is necessary to cause cardiac hypertrophy and fibrosis through the induction CTGF and FGF2, and they suggest that targeting ROCK2 may have therapeutic benefits in patients with LV diastolic dysfunction.

Authors

Toru Shimizu, Nikhil Narang, Phetcharat Chen, Brian Yu, Maura Knapp, Jyothi Janardanan, John Blair, James K. Liao

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Figure 9

Inhibition of ROCK2 decreases extracellular release of FGF2 from rat neonatal cardiac fibroblasts (RNCFs).

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Inhibition of ROCK2 decreases extracellular release of FGF2 from rat neo...
(A) Detection of CTGF and FGF2 protein expression in the mixture of supernatant and eluted extracellular matrix of RNCFs, stimulated by 10 ng/ml TGF-β1 for 24 hours, with or without 2.5 μM Y27632. Ponceau S staining of the membrane shows equal loading. (B) ELISA quantification of FGF2 concentration in eluted extracellular matrix of RNCFs, stimulated by TGF-β1 with or without Y27632 (n = 6–9, each in triplicate). **P < 0.01 vs. vehicle-stimulated RNCFs. ##P < 0.01 vs. TGF-β1-stimulated RNCFs without Y27632. (C) Detection of CTGF and FGF2 protein expression in the mixture of supernatant and eluted extracellular matrix of TGF-β1–stimulated RNCFs, transfected with control, ROCK1, or ROCK2 siRNA. (D) ELISA quantification FGF2 concentration in eluted extracellular matrix of TGF-β1–stimulated RNCFs, transfected with control, ROCK1, or ROCK2 siRNA (n = 5–8, each in triplicate). **P < 0.01 vs. vehicle-stimulated RNCFs transfected with each siRNA. ##P < 0.01 vs. TGF-β1–stimulated RNCFs transfected with control siRNA. †P < 0.05 vs. TGF-β1–stimulated RNCFs transfected with ROCK1 siRNA. Data are expressed as mean ± SEM. (E and F) Representative fluorescent images and quantification of cellular hypertrophy of rat H9C2 cardiomyocytes stained with sarcomeric α-actinin (green), in response to 0–100 ng/ml FGF2 for 24 hours. Nuclei are stained with DAPI (blue). (n = 30–50 each). Scale bars: 25 μm. (G–I) Quantification of RT-PCR analysis of hypertrophic markers of Acta1 (encoding skeletal muscle α-actin), Myh7 (β-myosin heavy chain), and Nppa (atrial natriuretic factor) in H9C2 cells stimulated by FGF2 (n = 3–4 each). *P < 0.05, **P < 0.01 vs. vehicle-stimulated H9C2 cells. Data are expressed as mean ± SEM. P values were calculated using one-way ANOVA with Tukey’s HSD test.