The effect of the Ras homolog gene family (Rho), member A/Rho associated coiled-coil forming protein kinase pathway in atrial fibrosis of type 2 diabetes in rats

Abstract

Diabetes mellitus promotes atrial structural remodeling, thereby producing atrial arrhythmogenicity. Atrial arrhythmia can substantially increase the risk of premature death. The aim of this study was to investigate the role of Ras homolog gene family, member A (RhoA)/Rho associated coiled-coil forming protein kinase (ROCK) in atrial fibrosis in diabetic hearts, and the effects of fasudil hydrochloride hydrate on atrial fibrosis. An eight-week-old male Sprague-Dawley rat model of type 2 diabetes was established using a high-fat diet combined with streptozotocin [30 mg/kg, once, intraperitoneal (i.p.)]. Animals were randomly divided into three groups: Control rats, untreated diabetic rats that received vehicle, and treated diabetic rats that received Rho kinase inhibitor fasudil hydrochloride hydrate (10 mg/kg/day, i.p., for 14 weeks). The morphological features of atrial fibrosis were observed using Masson staining. The mRNA expression levels of RhoA, ROCK1, ROCK2, type-I and type-III procollagen were assessed with quantitative polymerase chain reaction. The protein levels of RhoA, ROCK1 and ROCK2 were evaluated using western blot analysis. The atria of untreated diabetic rats showed evident atrial fibrosis as compared to the control rats; the mRNA expression levels of RhoA, ROCK1, ROCK2, type-I and type-III procollagen were upregulated; and the protein levels of RhoA, ROCK1 and ROCK2 were increased. The treatment with fasudil hydrochloride hydrate significantly reduced atrial fibrosis, mRNA levels of RhoA, ROCK1, ROCK2, type-I and type-III procollagen, and the protein levels of RhoA, ROCK1 and ROCK2. The results suggested that RhoA/ROCK was involved in atrial fibrosis, and that fasudil hydrochloride hydrate ameliorates atrial fibrosis through the RhoA/ROCK pathway in rats with type 2 diabetes.

Keywords: Ras homolog gene family; atrial arrhythmogenicity; atrial fibrosis; fasudil; member A (RhoA)/Rho associated coiled-coil forming protein kinase pathway.

Figure 1

The deposition of collagen in the atrial interstitium in Masson-stained sections of the control, diabetic and diabetic rats treated with fasudil (DM+fas). Heart tissues from the diabetic group show focal regions of fibrosis (blue) in the interstitium (P<0.01 vs. the control and DM+fas groups). Magnification, ×400. The number of rats in each group was six.

Figure 2

mRNA expression levels of (A) RhoA, (B) ROCK1 and (C) ROCK2, in untreated diabetic, control and DM+fas rats are evaluated by quantitative polymerase chain reaction. The intensities of RhoA, ROCK1 and ROCK2 were standardized to that of GAPDH. The number of rats in each group was six. The mRNA expression levels of RhoA, ROCK1 and ROCK2 were significantly increased in diabetic rats and decreased in DM+fas rats. ^*P<0.01 vs. control rats,^**P<0.01 vs. diabetic rats. DM+fas, diabetic treated with fasudil.

Figure 3

The mRNA expression levels of (A) type I and (B) type III procollagen in untreated diabetic, control and DM+fas rats was evaluated by quantitative polymerase chain reaction. The intensities of type I and III procollagen were standardized to that of GAPDH. The number of rats in each group was six. The mRNA expression levels of type I and III procollagen were significantly increased in diabetic rats and decreased in DM+fas rats. ^*P<0.01 vs. control rats,^**P<0.01 vs. diabetic rats. DM+fas, diabetic treated with fasudil.

Figure 4

The protein levels of (A) RhoA, (B) ROCK1 and (C) ROCK2 in control, diabetic and DM+fas rats were evaluated by western blot analyses. The protein levels of RhoA, ROCK1 and ROCK2 were significantly increased in diabetic rats and decreased in DM+fas rats. ^*P<0.05 vs. control rats, ^**P<0.05 vs. diabetic rats. DM+fas, diabetic treated with fasudil.