Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis

Liver fibrosis is characterized by activation of hepatic stellate cells, which are then involved in synthesis of matrix proteins and in regulating matrix degradation. In the acute phases of liver injury and as liver fibrosis progresses, there is increased expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Among the changes described, striking features include increased expression of gelatinase A (MMP-2) and membrane type 1-MMP (MT(1)-MMP; MMP-14) as well as TIMP-1 and TIMP-2. These molecules and other family members are involved in regulating degradation of both normal and fibrotic liver matrix. This article outlines recent progress in this field and discusses the mechanisms by which MMPs and TIMPs may contribute to the progression and regression of liver fibrosis. Recently described properties of MMPs and TIMPs of relevance to the pathogenesis of liver fibrosis are outlined. The proposal that regression of liver fibrosis is mediated by decreased expression of TIMPs and involves degradation of fibrillar collagens by a combination of MT(1)-MMP and gelatinase A, in addition to interstitial collagenase, is explored.     

Electrical stimulation of cardiomyocytes activates mitochondrial matrix metalloproteinase causing electrical remodeling

Cardiac arrhythmias, instigated by mechanical and electrical remodeling, are associated with activation of extracellular matrix metalloproteinases (MMPs). However, the connection between intracellular MMPs activation and arrhythmogenesis is not well established. Previously, we determined localization of MMP in the mitochondria using confocal microscopy. We tested the hypothesis that electrical pacing induces the activation of mitochondrial MMP (mtMMP) and is associated with myocyte mechanical dysfunction. Myocytes were isolated and field stimulated at 1 and 4 Hz. Myocyte mechanics and calcium transient was studied using Ion-Optix system. Mitochondrial MMP-9 activation was evaluated using zymography. There was a 25% increase in 1 Hz and 40% increase in 4 Hz stimulation. We observed an increase in mtMMP activation with increase in electrical pacing compared to 0 Hz with a significant increase (p < 0.05, n = 3). Field stimulation at 4 Hz decreased cell re-lengthening. The levels of calcium transient were reduced with increase in contraction frequency. We conclude that electrical stimulation activates mtMMP-9 that is associated with myocyte mechanical dysfunction.     

MMP-9 Gene Ablation and TIMP-4 Mitigate PAR-1-Mediated Cardiomyocyte Dysfunction: A Plausible Role of Dicer and miRNA

Although matrix metalloproteinase-9 (MMP‐9) is involved in cardiomyocytes contractility dysfunction, tissue inhibitor of metalloproteinase-4 (TIMP‐4) mitigates the effect of MMP‐9, and proteinase-activated receptor-1 (PAR‐1, a G-protein couple receptor, GPCR) is involved in the signaling cascade of MMP‐9-mediated cardiac dysfunction, the mechanism(s) are unclear. To test the hypothesis that induction of dicer and differential expression of microRNAs (miRNAs) contribute, in part, to the down regulation of sarcoplasmic reticulum calcium ATPase isoform 2a (serca-2a) in MMP-9 and PAR-1-mediated myocytes dysfunction, ventricular cardiomyocytes were isolated from C57BL/6J mice and treated with 3 ng/ml of MMP-9, 12 ng/ml of TIMP-4, and 10 and 100 μM of PAR-1 antagonist with MMP-9. Specific role of MMP-9 was determined by using MMP-9 knock out (MMP-9KO) and their corresponding control (FVB) mice. Ion Optics video-edge detection system and Fura 2-AM loading were used for determining the contractility and calcium release from cardiomyocytes. Quantitative and semi-quantitative PCR were used to determine the expression of dicer, TIMP-4 and serca-2a. miRNA microarrays were used for assessing the expression of different miRNAs between MMP-9KO and FVB cardiomyocytes. The results suggest that MMP‐9 treatment attenuates the voltage‐induced contraction of primary cardiomyocytes while TIMP‐4, an inhibitor of MMP‐9, reverses the inhibition. MMP‐9 treatment is also associated with reduced Ca²⁺ transients. This effect is blocked by a PAR‐1 antagonist, suggesting that PAR‐1 mediates this effect. The effect is not as great at high concentrations (100 μM) perhaps due to mild toxicity. The PAR‐1 antagonist effect did not affect calcium transients unlike TIMP‐4. Interestingly, we show that MMP‐KO myocytes contract more rapidly and release more Ca²⁺ than FVB. The relevant RNA species serca-2a is induced and dicer is inhibited. There is selective inhibition of miR-376b and over-expression of miR-1, miR-26a, miR-30d, and miR-181c in MMP‐9KO that are implicated in regulation of G-PCR and calcium handling.     

Chronic administration of hexarelin attenuates cardiac fibrosis in the spontaneously hypertensive rat

Cardiac fibrosis is a hallmark of heart disease and plays a vital role in cardiac remodeling during heart diseases, including hypertensive heart disease. Hexarelin is one of a series of synthetic growth hormone secretagogues (GHSs) possessing a variety of cardiovascular effects via action on GHS receptors (GHS-Rs). However, the role of hexarelin in cardiac fibrosis in vivo has not yet been investigated. In the present study, spontaneously hypertensive rats (SHRs) were treated with hexarelin alone or in combination with a GHS-R antagonist for 5 wk from an age of 16 wk. Hexarelin treatment significantly reduced cardiac fibrosis in SHRs by decreasing interstitial and perivascular myocardial collagen deposition and myocardial hydroxyproline content and reducing mRNA and protein expression of collagen I and III in SHR hearts. Hexarelin treatment also increased matrix metalloproteinase (MMP)-2 and MMP-9 activities and decreased myocardial mRNA expression of tissue inhibitor of metalloproteinase (TIMP)-1 in SHRs. In addition, hexarelin treatment significantly attenuated left ventricular (LV) hypertrophy, LV diastolic dysfunction, and high blood pressure in SHRs. The effect of hexarelin on cardiac fibrosis, blood pressure, and cardiac function was mediated by its receptor, GHS-R, since a selective GHS-R antagonist abolished these effects and expression of GHS-Rs was upregulated by hexarelin treatment. In summary, our data demonstrate that hexarelin reduces cardiac fibrosis in SHRs, perhaps by decreasing collagen synthesis and accelerating collagen degradation via regulation of MMPs/TIMP. Hexarelin-reduced systolic blood pressure may also contribute to this reduced cardiac fibrosis in SHRs. The present findings provided novel insights and underscore the therapeutic potential of hexarelin as an antifibrotic agent for the treatment of cardiac fibrosis.     

Regulation of myocardial matrix metalloproteinase expression and activity by cardiac fibroblasts

Cardiac fibroblasts (CF) play a key role in orchestrating the structural remodeling of the myocardium in response to injury or stress, in part through direct regulation of extracellular matrix (ECM) turnover. The matrix metalloproteinases (MMPs) are a family of over 25 zinc-dependent proteases that together have the capacity to degrade all the protein components of the ECM. Fibroblasts are a major source of several MMPs in the heart, thereby representing a viable therapeutic target for regulating ECM turnover in cardiac pathologies characterized by adverse remodeling, such as myocardial infarction, cardiomyopathy, hypertension and heart failure. This review summarizes current knowledge on the identity and regulation of MMPs expressed by CF and discusses future directions for reducing adverse myocardial remodeling by modulating the expression and/or activity of CF-derived MMPs.     

Matrix metalloproteinase-14 is a mechanically regulated activator of secreted MMPs and invasion

Matrix metalloproteinases (MMPs) are extracellular matrix (ECM) degrading enzymes and have complex and specific regulation networks. This includes activation interactions, where one MMP family member activates another. ECM degradation and MMP activation can be initiated by several different stimuli including changes in ECM mechanical properties or intracellular contractility. These mechanical stimuli are known enhancers of metastatic potential. MMP-14 facilitates local ECM degradation and is well known as a major mediator of cell migration, angiogenesis and invasion. Recently, function blocking antibodies have been developed to specifically block MMP-14, providing a useful tool for research as well as therapeutic applications. Here we utilize a selective MMP-14 function blocking antibody to delineate the role of MMP-14 as an activator of other MMPs in response to changes in cellular contractility and ECM stiffness. Inhibition using function blocking antibodies reveals that MMP-14 activates soluble MMPs like MMP-2 and -9 under various mechanical stimuli in the pancreatic cancer cell line, Panc-1. In addition, inhibition of MMP-14 abates Panc-1 cell extension into 3D gels to levels seen with non-specific pan-MMP inhibitors at higher concentrations. This strengthens the case for MMP function blocking antibodies as more potent and specific MMP inhibition therapeutics.     

Osteoprotegerin differentially regulates protease expression in osteoclast cultures

Cysteine proteases and matrix metalloproteinases (MMPs) are important factors in the degradation of organic matrix components of bone. Osteoprotegerin (OPG) is an osteoblast-secreted decoy receptor that inhibits osteoclast differentiation and activation. This study investigated the direct effects of human OPG on cathepsin K, MMP-9, MMP-2, and tissue inhibitors of metalloproteinases (TIMP1 and TIMP2) expressed by purified rabbit osteoclasts. The expression of two osteoclast markers, namely tartrate-resistant acid phosphatase (TRAP) and cathepsin K, was inhibited by 100 ng/mL hOPG, whereas MMP-9 expression was enhanced. Gelatinase activities were measured using a zymographic assay, and hOPG was shown to enhance both pro-MMP-9 and MMP-2 activities. Concomitantly, TIMP1 expression was greatly stimulated by hOPG, whereas TIMP2 mRNA levels were not modulated. Overall, these results show that hOPG regulates the proteases produced by purified osteoclasts differentially, producing a marked inhibitory effect on the expression of cathepsin K, the main enzyme involved in bone resorption.     

Ataxia telangiectasia mutated kinase plays a protective role in ��-adrenergic receptor-stimulated cardiac myocyte apoptosis and myocardial remodeling

β-Adrenergic receptor (β-AR) stimulation induces cardiac myocyte apoptosis and plays an important role in myocardial remodeling. Here we investigated expression of various apoptosis-related genes affected by β-AR stimulation, and examined first time the role of ataxia telangiectasia mutated kinase (ATM) in cardiac myocyte apoptosis and myocardial remodeling following β-AR stimulation. cDNA array analysis of 96 apoptosis-related genes indicated that β-AR stimulation increases expression of ATM in the heart. In vitro, RT-PCR confirmed increased ATM expression in adult cardiac myocytes in response to β-AR stimulation. Analysis of left ventricular structural and functional remodeling of the heart in wild-type (WT) and ATM heterozygous knockout mice (hKO) 28 days after ISO-infusion showed increased heart weight to body weight ratio in both groups. M-mode echocardiography showed increased percent fractional shortening (%FS) and ejection fraction (EF%) in both groups 28 days post ISO-infusion. Interestingly, the increase in %FS and EF% was significantly lower in the hKO-ISO group. Cardiac fibrosis and myocyte apoptosis were higher in hKO mice at baseline and ISO-infusion increased fibrosis and apoptosis to a greater extent in hKO-ISO hearts. ISO-infusion increased phosphorylation of p53 (Serine-15) and expression of p53 and Bax to a similar extent in both groups. hKO-Sham and hKO-ISO hearts exhibited reduced intact β1 integrin levels. MMP-2 protein levels were significantly higher, while TIMP-2 protein levels were lower in hKO-ISO hearts. MMP-9 protein levels were increased in WT-ISO, not in hKO hearts. In conclusion, ATM plays a protective role in cardiac remodeling in response to β-AR stimulation.     

Early induction of matrix metalloproteinase-9 transduces signaling in human heart end stage failure

Extracellular matrix (ECM) turnover is regulated by matrix metalloproteinases (MMPs) and plays an important role in cardiac remodeling. Previous studies from our lab demonstrated an increase in gelatinolytic-MMP-2 and -9 activities in endocardial tissue from ischemic cardiomyopathic (ICM) and idiopathic dilated cardiomyopathic (DCM) hearts. The signaling mechanism responsible for the left ventricular (LV) remodeling, however, is unclear. Administration of cardiac specific inhibitor of metalloproteinase (CIMP) prevented the activation of MMP-2 and -9 in ailing to failing myocardium. Activation of MMP-2 and -9 leads to induction of proteinase activated receptor-1 (PAR-1). We hypothesize that the early induction of MMP-9 is a key regulator for modulating intracellular signaling through activation of PAR and various downstream events which are implicated in development of cardiac fibrosis in an extracellular receptor mediated kinase-1 (ERK-1) and focal adhesion kinase (FAK) dependent manner. To test this hypothesis, explanted human heart tissues from ICM and DCM patients were obtained at the time of orthotopic cardiac transplants. Quantitative analysis of MMP-2 and -9 gelatinolytic activities was made by real-time quantitative zymography. Gel phosphorylation staining for PAR-1 showed a significant increase in ICM hearts. Western blot and RT-PCR analysis and in-situ labeling, showed significant increased expression of PAR-1, ERK-1and FAK in ICM and DCM. These observations suggest that the enhanced expression and potentially increased activity of LV myocardial MMP-9 triggers the signal cascade instigating cardiac remodeling. This early mechanism for the initiation of LV remodeling appears to have a role in end-stage human heart failure.     

Matrix metalloproteinase expression in cardiac myocytes following myocardial infarction in the rabbit

Myocardial infarction (MI), leads to cardiac remodeling, thinning of the ventricle wall, ventricular dilation, and heart failure, and is a leading cause of death. Interactions between the contractile elements of the cardiac myocytes and the extracellular matrix (ECM) help maintain myocyte alignment required for the structural and functional integrity of the heart. Following MI, reorganization of the ECM and the myocytes occurs, contributing to loss of heart function. In certain pathological circumstances, the ECM is modulated such that the structure of the tissue becomes damaged. The matrix metalloproteinases (MMPs) are a family of enzymes that degrade molecules of the ECM. The present experiments were performed to define the time-course, isozyme subtypes, and cellular source of increased MMP expression that occurs following MI in an experimental rabbit model. Heart tissue samples from infarcted and sham animals were analyzed over a time-course of 1-14 days. By zymography, it was demonstrated that, unlike the sham controls, MMP-9 expression was induced within 24 hours following MI. MMP-3 expression, also absent in sham controls, was induced 2 days after MI. MMP-2 expression was detected in both the sham and infarcted samples and was modestly up-regulated following MI. Tissue inhibitor of metalloproteinase-1 (TIMP-1) expression was evaluated and shown to be down-regulated following MI, inverse of MMP-9 and MMP-3 expression. Further, MMP-9 and MMP-3 expression was detected by immunohistochemistry in myocytes within the infarct. Additional studies were conducted in which cultured rat cardiac myocytes were exposed to a hypoxic environment (2% O2) for 24 hours and the media analyzed for MMP expression. MMP-9 and MMP-3 were induced following exposure to hypoxia. It is speculated that the net increase in proteolytic activity by myocytes is a contributing factor leading to myocyte misalignment and slippage. Additional studies with a MMP inhibitor would elucidate this hypothesis.     

Peroxynitrite and myocardial contractility: in vivo versus in vitro effects

Generation of peroxynitrite (ONOO-) as a result of altered redox balance has been shown to affect cardiac function; however, inconsistencies in the data exist, particularly for myocardial contractility. The hypothesis that the cardiac impact of ONOO- formation depends on its site of generation, intravascular or intramyocardial, was examined. Cardiac contractility was assessed by pressure-volume analysis to delineate vascular versus cardiac changes on direct infusion of ONOO- into the right atria of conscious dogs both with normal cardiac function and in heart failure. Additionally, ONOO- was administered to isolated murine cardiomyocytes to mimic in situ cardiac generation. When infused in vivo, ONOO- had little impact on inotropy but led to systemic arterial dilation, likely as a result of rapid decomposition to NO2- and NO3-. In contrast, infused ONOO- was long lived enough to abolish beta-adrenergic (dobutamine)-stimulated contractility/relaxation, most likely through catecholamine oxidation to aminochrome. When administered to isolated murine cardiomyocytes, ONOO- induced a rapid reduction in sarcomere shortening and whole cell calcium transients, although neither decomposed ONOO- or NaNO2 had any effect. Thus, systemic generation of ONOO- is unlikely to have primary cardiac effects, but may modulate cardiac contractile reserve, via blunted beta-adrenergic stimulation, and vascular tone, as a result of generation of NO2- and NO3-. However, myocyte generation of ONOO- may impair contractile function by directly altering Ca2+ handling. These data demonstrate that the site of generation within the cardiovascular system largely dictates the ability of ONOO- to directly or indirectly modulate cardiac pump function.     

Plasma concentrations of matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-1 and osteopontin reflect severity of heart failure in DOCA-salt hypertensive rat

The matrix metalloproteinases (MMPs) and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) play a key role in extracellular matrix maintenance and are altered in the failing heart, both in experimental models and in chronic end-stage heart failure in humans. As the common diagnostic markers of heart failure, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) primarily reflect increased pressure loading, determination of soluble, heart-derived MMPs and TIMPs in plasma, as well as the determination of the emerging fibrosis marker osteopontin (OPN) might be valuable tools for detecting heart fibrosis. In this study the effect of spironolactone treatment on plasma MMP-2, TIMP-1 and OPN levels was assessed in a heart failure animal model. Unilaterally nephrectomized Sprague Dawley rats received subcutaneous injection of 100 mg deoxycorticosterone acetate (DOCA) once a week and 1% (w/v) NaCl in drinking water. Blood pressure was monitored weekly and blood samples were collected after 1, 2 and 4 weeks. After 6 weeks, left ventricular contractility (LVC) and heart weight-to-body weight ratio (HW/BW) were assessed. DOCA treatment increased plasma MMP-2, TIMP-1 and OPN concentrations. Alterations of plasma marker levels were correlated with changes of HW/BW and paralleled impaired LVC. Furthermore, beneficial effects of spironolactone treatment were observed. In DOCA-salt hypertensive rats, plasma concentrations of MMP-2, TIMP-1 and OPN reflected heart failure associated with haemodynamic, functional and morphological changes. Based on these findings, it appears reasonable to use plasma markers of fibrosis to monitor the development of heart failure.     

Reversion inducing cysteine rich protein with Kazal motifs and cardiovascular diseases: The RECKlessness of adverse remodeling

The Reversion Inducing Cysteine Rich Protein With Kazal Motifs (RECK) is a glycosylphosphatidylinositol (GPI) anchored membrane-bound regulator of matrix metalloproteinases (MMPs). It is expressed throughout the body and plays a role in extracellular matrix (ECM) homeostasis and inflammation. In initial studies, RECK expression was found to be downregulated in various invasive cancers and associated with poor prognostic outcome. Restoring RECK, however, has been shown to reverse the metastatic phenotype. Downregulation of RECK expression is also reported in non-malignant diseases, such as periodontal disease, renal fibrosis, and myocardial fibrosis. As such, RECK induction has therapeutic potential in several chronic diseases. Mechanistically, RECK negatively regulates various matrixins involved in cell migration, proliferation, and adverse remodeling by targeting the expression and/or activation of multiple MMPs, A Disintegrin And Metalloproteinase Domain-Containing Proteins (ADAMs), and A Disintegrin And Metalloproteinase With Thrombospondin Motifs (ADAMTS). Outside of its role in remodeling, RECK has also been reported to exert anti-inflammatory effects. In cardiac diseases, for example, it has been shown to counteract several downstream effectors of Angiotensin II (Ang-II) that play a role in adverse cardiac and vascular remodeling, such as Interleukin-6 (IL-6)/IL-6 receptor (IL-6R)/glycoprotein 130 (IL-6 signal transducer) signaling and Epidermal Growth Factor Receptor (EGFR) transactivation. This review article focuses on the current understanding of the multifunctional effects of RECK and how its downregulation may contribute to adverse cardiovascular remodeling.     

BMP-2 regulates cardiomyocyte contractility in a phosphatidylinositol 3 kinase-dependent manner

Bone morphogenetic protein-2 (BMP-2) regulates development of heart during vertebrate embryogenesis. In vitro BMP-2 induces differentiation of precardiac cells into mature cardiomyocytes by inducing the expression of cardiac-specific genes. However, the role of BMP-2 and its signaling in other cardiac functions have not been studied. We examined the action of phosphatidylinositol (PI) 3 kinase in isolated adult rat cardiomyocytes. Incubation of rat ventricular cardiomyocytes with BMP-2 increased the PI 3 kinase activity. Ly294002, a pharmacological inhibitor of PI 3 kinase, blocked BMP-2-induced PI 3 kinase activity completely. To investigate the contractility of isolated cardiomyocytes, fractional shortening was examined. BMP-2 significantly increased the percent fractional shortening of the cardiomyocytes. Inhibition of PI 3 kinase activity completely abolished this action of BMP-2. These data indicate that PI 3 kinase regulates BMP-2-induced myocyte contractility. To further confirm this observation, we used adenovirus-mediated gene transfer to express a constitutively active myristoylated catalytic subunit of PI 3 kinase in rat cardiomyocytes. Infection of cardiomyocytes with the adenovirus vector increased the expression of constitutively active PI 3 kinase within 24 h. Expression of constitutively active PI 3 kinase significantly increased cardiomyocyte contractility. Together, these data show for the first time that the growth and differentiation factor, BMP-2, stimulates cardiomyocyte contractility. Also we provide the first evidence that BMP-2-induced PI 3 kinase activity regulates this cardiomyocyte function.     

Targeted deletion of MMP-9 attenuates myocardial contractile dysfunction in heart failure

Chronic volume overload (VO) on the left ventricle (LV) augments redox stress and activates matrix metalloproteinase (MMP) which causes the endocardial endothelial-myocyte (EM) disconnection leading to myocardial contractile dysfunction. VO-induced MMP-9 activation impairs cardiac functions, in part by endothelial endocardial apoptosis, but the role of MMP-9 on EM functions remains obscure. We conjecture that chronic VO activates MMP-9 and causes EM uncoupling. Arteriovenous fistula (AVF) was created in genetically identical wild type (WT) mice (FVB/NJ) and MMP-9 knockout mice (MMP-9KO, FVB.Cg-MMP9(tm1Tvu)/J). Sham-operated mice were used as controls. Before experimentation the phenotype analysis of MMP-9KO mice was carried out. In-gel-gelatin zymography for MMP-9 activation was performed on LV homogenates. The EM functions were determined on LV rings using tissue myobath. We report a decrease in MMP-9 activity in left ventricular myocardial extracts in MMP-9 deficient mice after AVF. The responses to drugs affecting cardiac functions (acetylcholine (Ach), nitroprusside and bradykinin) were attenuated in AVF mice suggesting the impairment of EM coupling. Interestingly, the EM functions were restored in the MMP-9 deficient mice after AVF. We suggest a direct cause-and-effect relationship between MMP-9 activation and EM uncoupling in LV myocardium after chronic VO and the possible involvement of MMP-9 in myocardial contractile performance.     

Cardiac extracellular matrix tenascin-C deposition during fibronectin degradation

Tenascin-C (TN-C) might aggravate left ventricular remodeling after myocardial infarction (MI). Our previous study demonstrated that ventricular remodeling after MI is linked with the degradation of fibronectin (FN). The aim of the present study was to determine whether cardiac extracellular matrix TN-C deposition after MI requires FN degradation. We found that treatment with angiotensin (ANG) II significantly down-regulated FN while remarkably up-regulated TN-C in co-cultured cardiomyocytes and fibroblasts. Inhibitors of matrix metalloproteinase (MMP)-2, MMP-3 or MMP-9 significantly attenuated ANG II-induced loss of FN and obviously blunted ANG II-induced re-expression of TN-C in co-cultured cells. Moreover, FN fragments dose-dependently induced the deposition of TN-C. In addition, MI induced a significant reduction of FN protein expression and a marked elevation of TN-C expression level at day 7 after MI compared with the sham group. The present findings suggest that cardiac TN-C matrix deposition after MI is induced by FN degradation, which is dependent on the activation of MMPs. These findings might contribute to gain mechanistic insights into the regulation of TN-C formation after MI.     

MiR-34a/miR-93 target c-Ski to modulate the proliferaton of rat cardiac fibroblasts and extracellular matrix deposition in vivo and in vitro

Cardiac fibrosis is associated with diverse heart diseases. In response to different pathological irritants, cardiac fibroblasts may be induced to proliferate and differentiate into cardiac myofibroblasts, thus contributing to cardiac fibrosis. TGF-β signaling is implicated in the development of heart failure through the induction of cardiac fibrosis. C-Ski, an inhibitory regulator of TGF-β signaling, has been reported to suppress TGF-β1-induced human cardiac fibroblasts' proliferation and ECM protein increase; however, the underlying molecular mechanism needs further investigation. In the present study, we demonstrated that c-Ski could ameliorate isoproterenol (ISO)-induced rat myocardial fibrosis model and TGF-β1-induced primary rat cardiac fibroblasts' proliferation, as well as extracellular matrix (ECM) deposition. The protein level of c-Ski was dramatically decreased in cardiac fibrosis and TGF-β1-stimulated primary rat cardiac fibroblasts. In recent decades, a family of small non-coding RNA, namely miRNAs, has been reported to regulate gene expression by interacting with diverse mRNAs and inducing either translational suppression or mRNA degradation. Herein, we selected miR-34a and miR-93 as candidate miRNAs that might target to regulate c-Ski expression. After confirming that miR-34a/miR-93 targeted c-Ski to inhibit its expression, we also revealed that miR-34a/miR-93 affected TGF-β1-induced fibroblasts' proliferation and ECM deposition through c-Ski. Taken together, we demonstrated a miR-34a/miR-93-c-Ski axis which modulates TGF-β1- and ISO-induced cardiac fibrosis in vitro and in vivo; targeting the inhibitory factors of c-Ski to rescue its expression may be a promising strategy for the treatment of cardiac fibrosis.