| The majority of the nonmyocytes, cardiac fibroblasts (CFs), occupied60～70%of the total cell number of the heart. CFs are found throughout the cardiac tissue, surrounding myocytes and bridging the myocardial interstitial components. CFs exhibited a wide range of functions, including extracellular matrix (ECM) turnover regulation, cytokine synthesis and secretion, mechanical and electrical signal transduction, and angiogenesis modulation. Therefore, CFs play a critical role in maintaining normal cardiac function, as well as in cardiac remodeling during pathological conditions.Cardiac remodeling is the common outcome of various kinds of cardiac disease, which is frequently associated with myocardial fibrosis (MF). CFs are the primary cell type responsible for MF. In response to pathological stimuli, CFs undergo a phenotypic transformation to become cardiac myofibroblasts (CMFs). CMFs are highly proliferative and migrative, and remodel the cardiac interstitium by increasing synthesis and secretion of ECM components and matrix-degrading metalloproteinases (MMPs). To stimulate the remodeling process further, they secrete increased amounts of growth factors and cytokines, which can in turn act on various kinds of cardiac cells by the autocrine and paracrine effects. Although these changes serve initially as an important reparative wound healing response, in the longer term, they become maladaptive and lead to abnormal myocardial stiffness and ultimately, ventricular dysfunction and heart failure (HF). Therefore, it has a great potential to developing antifibrotic and cardioprotective drugs that using CFs as targets.Pirfenidone (5-methyl-l-phenyl-2-[1H]-pyridone) is a novel, small molecule antifibrotic drug. Previously, it has been reported that pirfenidone could attenuate cardiac fibrosis, improve cardiac function and reverse cardiac remodeling in several different models of cardiac fibrosis. CFs are at the heart of myocardial fibrosing, and play a pivotal role in cardiac remodeling. However, although a serial of animal studies on cardiac fibrosis have consistently shown that pirfenidone exhibited significant antifibrotic effects, and CFs may be its main targets, no published information is available regarding the biologic effects of pirfenidone on isolated CFs, and the underlying cellular and molecular mechanisms of the potential effects of this drug. Therefore, this study was aimed to investigate the effects of pirfenidone on CF functions that are important in the cardiac remodeling process such as proliferation, phenotype transformation, collagen contraction, migration and cytokine secretion, and explore possible cellular and molecular mechanisms that underlies its potential antifibrotic effects.In this study, we found that pirfenidone significantly inhibited the proliferation of cultured CFs, lowered the ratio of Ki67positive staining cells, in a non-cytotoxic way. Pirfenidone also inhibited the phenotype transformation of CFs to CMFs, evidenced by the lowered expression of α-smooth muscle actin (a-SMA). Moreover, the contractility and migratory ability of CFs were also significantly impeded. Besides, pirfenidone reduced the synthesis and secretion of MMP-9and increased that of TIMP-1, i.e., decreased the ratio of MMP-9/TIMP-1in CFs, MMP-9/TIMP-1ratio has been shown to be upregulated during cardiac remodeling and its balance is important in the regulation of ECM turnover and cardiac fibroblast migration. In addition, pirfenidone decreased the synthesis and secretion of TGF-β1but enhanced that of IL-10in CFs, which may serve as another aspect of its antifibrotic and cardioprotective effects.This study identified the direct effects of pirfenidone on CFs, and revealed several new cellular and molecular mechanisms that underlies its antifibrotic effects, which may be helpful for the future application of pirfenidone in the treatment of cardiac remodeling and MF. During recent years, with the development of genomics and proteomics, there have been many great breakthroughs in the screening and identification of cardiac remodeling and myocardial fibrosis (MF) related genes. It has been reported that the gene, TGF-β stimulated clone-22(TSC-22), was upregulated in several cardiopathy and MF models, and it is assumed to be involved in the MF process. In consistent with previous reports, we also found the upregulation of TSC-22in the fibrotic cardiac tissue of heart failure (HF) patients in our preliminary study, while the dynamic expression pattern of TSC-22after cardiac injury, and furthermore, its inducing factors and biologic functions in CFs, the primary cell type responsible for cardiac fibrosis, are still not clear. This study was aimed to investigate the dynamic expression pattern of TSC-22protein during the process from myocardial infarction (MI) to HF, explore the possible inducing factors of TSC-22and its function role in CFs.Our in vivo results showed that, The protein expression of TSC-22in rats'infarcted myocardium exhibited a dynamic pattern after MI, and its level was significantly elevated1day and7days after MI, while decreased4weeks after MI. In vitro experiment results showed that TGF-β1induced the expression of TSC-22in CFs, mediated by TGF-β1type I receptor, and the subcellular location was also changed after the treatment of TGF-β1, mainly expressed in the nuclear. At the same time, we also found that the expression of TSC-22was significantly elevated when CFs reached the condition of contact inhibition. By transfecting CFs with TSC-22specific small interfering RNA (siRNA), knocking down the expression of TSC-22, the expression of a-smooth muscle actin (α-SMA) in TGF-β1induced CFs was significantly decreased, which implies that TSC-22plays a positive role in the process of TGF-β1induced CFs-cadiac mycfibroblasts (CMFs) transition. This study may help to understand the molecular basis of cardiac remodeling, and provide new antifibrotic targets for the therapy of MF.
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