Mechanism of fibrotic cardiomyopathy in mice expressing truncated Rho-associated coiled-coil protein kinase 1

Abstract

We have previously found that in failing human hearts, Rho-associated coiled-coil protein kinase 1 (ROCK1) is processed by caspase-3 into an active isoform, ROCKΔ1. The purpose of the current investigation was to elucidate the pathological consequences of truncated ROCK1 accumulation in the heart, the associated molecular mechanism of ROCKΔ1-mediated cardiac phenotype, and the molecular signaling between Rho kinase activation in cardiomyocytes and extracellular matrix response. We generated transgenic mice expressing ROCKΔ1 in cardiomyocytes to mimic the situation observed in human heart disease, whereas an additional kinase-deficient mouse was generated as a control. The ROCKΔ1 transgenic mice developed fibrotic cardiomyopathy with diastolic dysfunction. Transgenic hearts displayed activated TGFβ1 and NF-κB signaling and a release of a subset of cytokines and were susceptible to angiotensin II stress. Treatment with a Rho kinase inhibitor attenuated the fibrotic phenotype. Cardiac fibroblasts differentiated into myofibroblasts when cocultured with transgenic cardiomyocytes but not with wild-type cardiomyocytes. Inhibitors of Rho kinase as well as TGFβR1 and NF-κB decreased these effects. The serum response factor-dependent TGFβ1 regulation was shown to be responsible for the Rho kinase-mediated activation of TGFβ1 signaling. We conclude that ROCKΔ1 is a novel fibrotic factor. Activation of TGFβ1 and NF-κB signaling contributes to the Rho kinase-mediated pathological fibrosis.

Figure 1.

Generation of transgenic ROCKΔ1 mice along with elevated Rho kinase activity. A) Two transgenic mouse lines expressing intermediate (I) and low (L) levels of ROCKΔ1 protein in the heart were generated and assessed by Western blot with the antibody specific against the N (top panel) and C termini of ROCK1 (middle panel), respectively. Transgene ROCKΔ1 was detected only by anti-ROCK1 N-terminal and not by anti-ROCK1 C-terminal antibody, confirming the truncated ROCKΔ1 expression (without C-terminal domain). Endogenous full-length ROCK1 protein can be detected by both antibodies. B) Relative expression levels of ROCKΔ1 protein over the endogenous ROCK1. No effect on endogenous ROCK1 and ROCK2 protein expression was observed in the transgenic (TG) heart. C) Endogenous ROCK1 mRNA level (low-expression mouse line) was also assessed by qPCR, and the data were pooled from 10 mice/group with analyses for each mouse in triplicate. D, E) Hyperphosphorylation of 2 Rho kinase direct substrates, MLC (D) and MYPT1 (E), was detected in the transgenic heart of the low-expression mouse line. WT, wild type.

Figure 2.

ROCKΔ1 transgenic mice developed interstitial and perivascular fibrotic cardiomyopathy. A) Heart sections were stained with Sirius red. Transgenic (TG) heart showed noticeable collagen deposition (red) in interstitial and perivascular regions compared with the wild-type (WT) heart under physiological (untreated) conditions. B, C) Significant increases in fibrotic and stress factors were detected in the transgenic hearts by qPCR (B) and immunoblot analyses (C). qPCR data were pooled from 10 mice/group with analyses for each mouse in triplicate. D) Quantification of fibrotic areas. E) Even more severe fibrotic ardiomyopathy was observed with Sirius red staining in the mutant heart after ANG II treatment compared with the WT heart. col, collagen. *P < 0.01 vs. WT; **P < 0.01 vs. ANG II-treated WT; ^§P < 0.01 vs. untreated WT; ^ΔP < 0.01 vs. untreated TG.

Figure 3.

A–F) Cardiac fibrosis in the transgenic heart, both untreated (A–C) and treated with ANG II (D–F), was attenuated by the Rho kinase inhibitor Fasudil. Significant decreases in collagen depositions (A, D; Sirius red staining) and fibrotic factors were observed after Fasudil treatment in the transgenic hearts analyzed by qPCR (B, E) and immunoblot analysis (C, F). qPCR data were pooled from 10 mice/group with triplicate analyses for each mouse. G) Quantification of fibrotic area over total analyzed heart area. *P < 0.001 vs. water-treated group; **P < 0.001 vs. ANG II-treated group.

Figure 4.

ROCKΔ1 facilitated actin assembly and promoted SRF activation in cardiomyocytes. A, B) Immunoblot displayed ROCKΔ1 (TG-ROCKΔ1) protein expression by transient transfection in cardiomyocytes with the ROCKΔ1 expression plasmid (A), which led to an obvious increase in the Rho kinase activity shown by MYPT1 hyperphosphorylation (B). C) A significant increase in the ratio between polymerized F-actin (in a pellet) and unpolymerized G-actin (in a supernatant) was detected by ROCKΔ1 transfection. D) Immunoblot densitometry was summarized from 2 repeated experiments. E) Same cohort of cardiomyocytes showed enhanced SRF activity in the EMSA assay. F) Densitometry was summarized from E. S, supernatant; P, pellet.

Figure 5.

Up-regulation of TGFβ and SRF by ROCKΔ1. A, B) Enhanced SRF binding to the c-fos SRE was assessed by EMSA assay in the transgenic (TG) heart (A), and the assay intensity was quantified from 6 mice/group (B). C–F) qPCR analysis of TGFβ1 and SRF transcripts. Significant increases in TGFβ1 (C, D) and SRF (E, F) mRNA levels were observed in C2C12 myoblasts transfected with the ROCKΔ1 expression plasmid (C, E), and both increases were diminished by the SRF deficiency induced by the siRNA specific for SRF or by a dominant negative SRF-N (D, F). qPCR data were pooled from 3 repeated experiments in each group with triplicate analyses for each time. Δ1, ROCKΔ1; ROCKΔ1KD, mutant ROCKΔ1 with kinase deficiency; SRF-N, N-terminal SRF (a dominant negative SRF isoform); a.u., arbitrary unit. *P < 0.01 vs. control; ^ΔP < 0.01 vs. wild type (WT).

Figure 6.

Analysis of the TGFβ1 promoter region revealed TGFβ1 as a direct target gene of SRF. A, B) SRF binding to the SRE site in the TGFβ1 promoter region was demonstrated by the EMSA assay in vitro (A) and verified by the ChIP assay with sequencing assessment in vivo (B). C) Addition of SRF activated luciferase activity driven by TGFβ1 promoter, and mutation of the SRE (solid gray bar) diminished the SRF-mediated reporter activity. Data represent the average of 3 experiments with triplicate measurements in every experiment. D) A significant decrease in the SRF protein expression was achieved in the cardiac-specific SRF-knockout heart. E) In parallel with the decline in the SRF expression level, repressed TGFβ1 transcript was detected in the SRF-null heart. qPCR data represent the average of 10 mice/group.

Figure 7.

Activation of TGFβ1 and proinflammatory NF-κB signaling in the transgenic animals. Marked increases in TGFβ1 (A) and IL-1β (E) serum concentrations in transgenic mice were observed compared with the wild-type (WT) mice. Western blots revealed hyperphosphorylated Smad2 (B) and p65 and up-regulated p65 protein expression (D) in transgenic (TG) hearts, and proinflammatory factors were analyzed by qPCR (C). Data were pooled from 10 mice/group with triplicate analyses for each mouse. *P < 0.01 vs. WT.

Figure 8.

Activation of cardiac fibroblasts and inflammatory response in the transgenic heart, and attenuation of the fibrotic response by Rho kinase, TGFβ, and NF-κB signaling inhibitors. A–D) Visualization of α-SMA and macrophage marker F4/80 expression in left ventricles by immunostaining showed that both factors were up-regulated in the transgenic (TG) heart (A, B) compared with the wild type (WT; C, D). E–H) α-SMA expression was also induced in the WT CFs when cocultured with transgenic CMs (E, H) but not with the WT cardiomyocytes (F, G). I–L) Induction of α-SMA was attenuated by treatment with inhibitors Y27632 (I), SB431542 (J), CAPE (K), or both SB431542 and CAPE (L), respectively.

Figure 9.

Proposed mechanism of ROCKΔ1-mediated fibrotic program. In cardiomyocytes, active ROCK1 facilitates actin assembly and potentiates SRF activity by promoting an entry of the SRF accessory factor MAL into the nucleus to form the SRF-MAL complex. SRF binds to the TGFβ1 promoter/enhancer, thus directing the TGFβ1 expression. Meanwhile, Rho kinase activates the NF-κB signaling and induces a subset of cytokines indicative of the profibrotic and inflammatory program. Activation of both signaling pathways initiates a fibrotic response in cardiac fibroblasts and promotes fibrotic remodeling. PKD1, protein kinase D1.