Abstract
IL-6 has been shown to play a major role in collagen up-regulation process during cardiac hypertrophy, although the precise mechanism is still not known. In this study we have analyzed the mechanism by which IL-6 modulates cardiac hypertrophy. For the in vitro model, IL-6-treated cultured cardiac fibroblasts were used, whereas the in vivo cardiac hypertrophy model was generated by renal artery ligation in adult male Wistar rats (Rattus norvegicus). During induction of hypertrophy, increased phosphorylation of STAT1, STAT3, MAPK, and ERK proteins was observed both in vitro and in vivo. Treatment of fibroblasts with specific inhibitors for STAT1 (fludarabine, 50 μM), STAT3 (S31-201, 10 μM), p38 MAPK (SB203580, 10 μM), and ERK1/2 (U0126, 10 μM) resulted in down-regulation of IL-6-induced phosphorylation of specific proteins; however, only S31-201 and SB203580 inhibited collagen biosynthesis. In ligated rats in vivo, only STAT3 inhibitors resulted in significant decrease in collagen synthesis and hypertrophy markers such as atrial natriuretic factor and β-myosin heavy chain. In addition, decreased heart weight to body weight ratio and improved cardiac function as measured by echocardiography was evident in animals treated with STAT3 inhibitor or siRNA. Compared with IL-6 neutralization, more pronounced down-regulation of collagen synthesis and regression of hypertrophy was observed with STAT3 inhibition, suggesting that STAT3 is the major downstream signaling molecule and a potential therapeutic target for cardiac hypertrophy.
Highlights
Involvement of various neurohumoral and growth factors has been illustrated in the development of cardiac fibrosis both in vitro and in vivo [3, 8]
Western blotting showed a significant decrease in IL-6 levels in rat ventricular tissue after IL-6 neutralization together with a significant decrease in expression of hypertrophy marker genes as revealed by RT-PCR (Fig. 1B), suggesting that increased circulating IL-6 after renal artery ligation was responsible for development of cardiac hypertrophy in rats
Isolated cardiac fibroblasts treated with IL-6 or AngII was used to generate hypertrophy in vitro
Summary
Reverse Transcription PCR (RT-PCR) and Real Time RT-PCR— Total RNA from cells (24 h treatment) and tissues (on the 15th day after surgery) was isolated using TRIzol reagent (Invitrogen) following manufacturer’s instructions. Estimation of Total Collagen by Hydroxyproline Assay—Hydroxyproline assay was performed to measure total collagen content both in renal artery-ligated ventricular tissue (on the 15th day after surgery) as well as in fibroblast culture supernatant (24 h treatment) [25]. STAT3 siRNA for in vivo delivery (In vivo Direct siRNA; s129047; Ambion) was dissolved in RNase free 1ϫ sterile PBS and was directly injected to the ventricles of ligated animals (n ϭ 10) following the manufacturer’s instructions at a concentration of 10 nmol from the 1st day of ligation until the 14th day [22] Another group of renal artery-ligated rats was treated with nonspecific control siRNA (NSsiRNA) (4404020; Ambion).
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