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 2

Deletion of ROCK2 in cardiac fibroblasts attenuates angiotensin II–induced (Ang II–induced) cardiac hypertrophy, fibrosis, and diastolic dysfunction.

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Deletion of ROCK2 in cardiac fibroblasts attenuates angiotensin II–induc...
(A) Representative photomicrographs and H&E-stained sagittal sections of hearts from fibroblast-specific ROCK2-deficient (ROCK2Postn–/–) and littermate control (ROCK2flox/flox) mice at 4 wk after saline or Ang II infusion. Scale bars: 3 mm. (B and C) Quantitative analysis of the ratios of heart weight to body weight and to tibial length from ROCK2Postn–/– and ROCK2flox/flox mice treated with saline or Ang II (n = 10 each). (D and E) Representative sections from hearts immunostained with wheat germ agglutinin and quantification of cardiomyocyte cross-sectional area in ROCK2Postn–/– and ROCK2flox/flox mice treated with saline or Ang II (n = 5 each). Scale bars: 25 μm. (F and G) Representative sections from hearts stained with Picrosirius red and quantification of interstitial fibrosis area in ROCK2Postn–/– and ROCK2flox/flox mice treated with saline or Ang II (n = 5 each). Scale bars: 50 μm. (H) Representative echocardiographic M-mode images of left ventricles from ROCK2Postn–/– and ROCK2flox/flox mice treated with saline or Ang II (n = 10 each). (I) Representative echocardiographic images of mitral inflow pattern to evaluate diastolic dysfunction measured by transmitral Doppler velocity ratio of early-to-atrial wave (E/A ratio) (n = 10 each). **P < 0.01 vs. saline-treated each genotype. #P < 0.05, ##P < 0.01 vs. Ang II-treated ROCK2flox/flox mice. Data are expressed as mean ± SEM. P values were calculated using one-way ANOVA with Tukey’s HSD test. (J) Comparison of ROCK activity (phosphorylated myosin-binding subunits [p-MBS]/total MBS [t-MBS]) in HFpEF patients (n = 10) with age- and sex-matched controls (n = 10). Data are expressed as mean ± SEM. P value was calculated using paired t test. (K) Relationship between diastolic dysfunction grades and leukocyte ROCK activity (p-MBS/t-MBS) in 19 patients with normal or mild (grades 0 and 1) and moderate to severe (grades 2 and 3) diastolic dysfunction. Mean ROCK activity is higher in patients with moderate to severe compared with normal to mild diastolic dysfunction (1.34 ± 0.31 vs. 0.69 ± 0.34). Data are expressed as mean ± SD. P value was calculated using paired t test.