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
View: Text | PDF
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

×

Figure 10

Attenuated hypertrophic response of rat H9C2 cardiomyocytes to conditioned medium from ROCK2-silenced rat neonatal cardiac fibroblasts (RNCFs).

Options: View larger image (or click on image) Download as PowerPoint
Attenuated hypertrophic response of rat H9C2 cardiomyocytes to condition...
(A and B) Representative fluorescent images and quantification of cellular hypertrophy of H9C2 cells stained with sarcomeric α-actinin (green) and DAPI (nuclei, blue). H9C2 cells were incubated for 24 hours with conditioned medium from 24-hour 10 ng/ml TGF-β1–stimulated RNCFs with or without 2.5 μM Y27632 (n = 30–50 each). Scale bars: 25μm. Before coculture experiments, TGF-β1 neutralizing antibody was added to the conditioned medium to block the hypertrophic effects of TGF-β1. Y27632 was also almost removed by ultrafiltration before coculture. (C–E) 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 from coculture experiments with TGF-β1–stimulated RNCFs with or without Y27632 (n = 4–5 each). **P < 0.01 vs. H9C2 cells cocultured with vehicle-stimulated RNCFs. #P < 0.05, ##P < 0.01 vs. H9C2 cells cocultured with TGF-β1–stimulated RNCFs without Y27632. (F and G) Representative fluorescent images and quantification of cellular hypertrophy of H9C2 cells, incubated with conditioned medium from TGF-β1–stimulated RNCFs transfected control, ROCK1, or ROCK2 siRNA (n = 30–40 each). Scale bars: 25 μm. TGF-β1 neutralizing antibody was added to the conditioned medium before coculture. (H–J) Quantification of RT-PCR analysis of hypertrophic markers of Acta1, Myh7, and Nppa in H9C2 cells from coculture experiments with TGF-β1–stimulated RNCFs transfected with control, ROCK1, or ROCK2 siRNA (n = 4–5 each). **P < 0.01 vs. H9C2 cells cocultured with vehicle-stimulated RNCFs transfected with control siRNA. ##P < 0.01 vs. H9C2 cells cocultured with TGF-β1–stimulated RNCFs transfected with control siRNA. ††P < 0.01 vs. H9C2 cells cocultured with TGF-β1–stimulated RNCFs transfected with ROCK1 siRNA. Data are expressed as mean ± SEM. P values were calculated using one-way ANOVA with Tukey’s HSD test.