Tissue fibrosis is a major cause of organ dysfunction during chronic diseases and aging. A critical step in this process is transforming growth factor ␤1 (TGF-␤1)-mediated transformation of fibroblasts into myofibroblasts, cells capable of synthesizing extracellular matrix. Here, we show that SIRT3 controls transformation of fibroblasts into myofibroblasts via suppressing the profibrotic TGF-␤1 signaling. We found that Sirt3 knockout (KO) mice with age develop tissue fibrosis of multiple organs, including heart, liver, kidney, and lungs but not whole-body SIRT3-overexpressing mice. SIRT3 deficiency caused induction of TGF-␤1 expression and hyperacetylation of glycogen synthase kinase 3␤ (GSK3␤) at residue K15, which negatively regulated GSK3␤ activity to phosphorylate the substrates Smad3 and ␤-catenin. Reduced phosphorylation led to stabilization and activation of these transcription factors regulating expression of the profibrotic genes. SIRT3 deacetylated and activated GSK3␤ and thereby blocked TGF-␤1 signaling and tissue fibrosis. These data reveal a new role of SIRT3 to negatively regulate aging-associated tissue fibrosis and discloses a novel phosphorylation-independent mechanism controlling the catalytic activity of GSK3␤.

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Background: miR-378 is a newly discovered cardiomyocyte-enriched miRNA. Results: By targeting Grb-2, miR-378 blocks activation of the hypertrophic signaling cascade and gene expression. Its deficiency contributes to the development of hypertrophy in a Ras activity-dependent manner. Conclusion: miR-378 is a negative regulator of cardiac hypertrophy. Significance: Cellular restoration of miR-378 will be beneficial in preventing adverse cardiac remodeling.Understanding the regulation of cardiomyocyte growth is crucial for the management of adverse ventricular remodeling and heart failure. MicroRNA-378 (miR-378) is a newly described member of the cardiac-enriched miRNAs, which is expressed only in cardiac myocytes and not in cardiac fibroblasts. We have previously shown that miR-378 regulates cardiac growth during the postnatal period by direct targeting of IGF1R (Knezevic, I., Patel, A., Sundaresan, N. R., Gupta, M. P., Solaro, R. J., Nagalingam, R. S., and Gupta, M. (2012) J. Biol. Chem. 287, 12913-12926). Here, we report that miR-378 is an endogenous negative regulator of cardiac hypertrophy, and its levels are down-regulated during hypertrophic growth of the heart and during heart failure. In primary cultures of cardiomyocytes, overexpression of miR-378 blocked phenylephrine (PE)-stimulated Ras activity and also prevented activation of two major growth-promoting signaling pathways, PI3K-AKT and Raf1-MEK1-ERK1/2, acting downstream of Ras signaling. Overexpression of miR-378 suppressed PE-induced phosphorylation of S6 ribosomal kinase, pERK1/2, pAKT, pGSK-3␤, and nuclear accumulation of NFAT. There was also suppression of the fetal gene program that was induced by PE. Experiments carried out to delineate the mechanism behind the suppression of Ras, led us to identify Grb2, an upstream component of Ras signaling, as a bona fide direct target of miR-378-mediated regulation. Deficiency of miR-378 alone was sufficient to induce fetal gene expression, which was prevented by knocking down Grb2 expression and blocking Ras activation, thus suggesting that miR-378 interferes with Ras activation by targeting Grb2. Our study demonstrates that miR-378 is an endogenous negative regulator of Ras signaling and cardiac hypertrophy and its deficiency contributes to the development of cardiac hypertrophy.Cardiac hypertrophy is an adaptive response of myocytes to increased workload that often develops as a consequence of hypertension or valvular heart diseases (1, 2). Because adult cardiomyocytes are unable to divide, they respond to growth stimuli by increasing their cell size, a process known as hypertrophy. During hypertrophy, myocytes not only grow in size but also add sarcomeres and induce the expression of a group of genes, which are usually expressed during fetal heart development. These changes are initially considered as compensatory to manage the increased workload on the heart; however, prolonged hypertrophy leads to pathological ventricular remodeling, which is an established precursor for heart failure. In recent years,...

This article has been withdrawn by the authors. Lanes 1 and 2 of the actin immunoblot in Fig. 3B were reused in lanes 3 and 4 of the actin immunoblot in Fig. 7C. The ANF immunoblot in Fig. 3B was reused as ANF in Fig. 9F. Control lanes 1 and 2 of the ANF immunoblot in Fig. 8C were duplicated. The Grb2 and GAPDH immunoblots in Fig. 9C were inappropriately manipulated.

Background: MicroRNA-378, a cardiomyocyte-specific miRNA, is down-regulated during heart failure. Results: miR-378 inhibition induced TGF␤1 expression, which correlated with the activity of c-Fos, c-Jun, and Ras. Conditioned media of miR-378-depleted myocytes induced fibroblast activation by utilizing TGF␤1-dependent paracrine mechanisms. Conclusion: miR-378 is a negative regulator of TGF␤1 and cardiac fibrosis. Significance: miR-378 offers therapeutic potential for the management of heart failure.

Numerous physiological and pathological events, from organ development to cancer and fibrosis, are characterized by an epithelial-to-mesenchymal transition (EMT), whereby adherent epithelial cells convert to migratory mesenchymal cells. During cardiac development, proepicardial organ epithelial cells undergo EMT to generate fibroblasts. Subsequent stress or damage induces further phenotype conversion of fibroblasts to myofibroblasts, causing fibrosis via synthesis of an excessive extracellular matrix. We have previously shown that the transcription factor scleraxis is both sufficient and necessary for the conversion of cardiac fibroblasts to myofibroblasts and found that scleraxis knockout reduced cardiac fibroblast numbers by 50%, possibly via EMT attenuation. Scleraxis induced expression of the EMT transcriptional regulators Twist1 and Snai1 via an unknown mechanism. Here, we report that scleraxis binds to E-box consensus sequences within the Twist1 and Snai1 promoters to transactivate these genes directly. Scleraxis upregulates expression of both genes in A549 epithelial cells and in cardiac myofibroblasts. Transforming growth factor-β induces EMT, fibrosis, and scleraxis expression, and we found that transforming growth factor-β-mediated upregulation of Twist1 and Snai1 completely depends on the presence of scleraxis. Snai1 knockdown upregulated the epithelial marker E-cadherin; however, this effect was lost after scleraxis overexpression, suggesting that scleraxis may repress E-cadherin expression. Together, these results indicate that scleraxis can regulate EMT via direct transactivation of the Twist1 and Snai1 genes. Given the role of scleraxis in also driving the myofibroblast phenotype, scleraxis appears to be a critical controller of fibroblast genesis and fate in the myocardium and thus may play key roles in wound healing and fibrosis. NEW & NOTEWORTHY The molecular mechanism by which the transcription factor scleraxis mediates Twist1 and Snai1 gene expression was determined. These results reveal a novel means of transcriptional regulation of epithelial-to-mesenchymal transition and demonstrate that transforming growth factor-β-mediated epithelial-to-mesenchymal transition is dependent on scleraxis, providing a potential target for controlling this process.

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-The phenotype conversion of fibroblasts to myofibroblasts plays a key role in the pathogenesis of cardiac fibrosis. Numerous triggers of this conversion process have been identified, including plating of cells on solid substrates, cytokines such as transforming growth factor-␤, and mechanical stretch; however, the underlying mechanisms remain incompletely defined. Recent studies from our laboratory revealed that the transcription factor scleraxis is a key regulator of cardiac fibroblast phenotype and extracellular matrix expression. Here we report that mechanical stretch induces type I collagen expression and morphological changes indicative of cardiac myofibroblast conversion, as well as scleraxis expression via activation of the scleraxis promoter. Scleraxis causes phenotypic changes similar to stretch, and the effect of stretch is attenuated in scleraxis null cells. Scleraxis was also sufficient to upregulate expression of vinculin and F-actin, to induce stress fiber and focal adhesion formation, and to attenuate both cell migration and proliferation, further evidence of scleraxis-mediated regulation of fibroblast to myofibroblast conversion. Together, these data confirm that scleraxis is sufficient to promote the myofibroblast phenotype and is a required effector of stretch-mediated conversion. Scleraxis may thus represent a potential target for the development of novel antifibrotic therapies aimed at inhibiting myofibroblast formation. transcription factor; cardiac fibroblast; stretch; migration; proliferation MYOFIBROBLASTS, AS THE ACTIVATED form of fibroblasts, are major mediators of tissue fibrosis in the heart, lungs, dermis, kidneys, and gastrointestinal tract (17,26,29,36,42). Excess deposition of fibrillar collagens in the extracellular matrix (ECM) of these tissues imparts increased stiffness and reduced organ function. Cardiac fibrosis entails a poor prognosis, negatively impacting both systolic and diastolic function and eventually leading to heart failure and death (43). There currently exists no treatment for the arrest or reversal of cardiac fibrosis (38). However, alteration of the myofibroblast phenotype may provide a novel means for the treatment and even reversal of fibrotic lesions in various tissue types (20,21,23,26,34,37,49).In response to myocardial injury, the release of damage factors [such as profibrotic transforming growth factor-␤ (TGF-␤)] and mechanical strain resulting from the loss of ECM integrity induce activation of cardiac fibroblasts and subsequent conversion to the myofibroblast phenotype (11,14,22,25,32). Cardiac myofibroblasts are characterized by hypersynthesis of fibrillar collagens type I and III, increased expression of ␣-smooth muscle actin (␣-SMA), increased adhesions and cell size, reduced proliferation and migration, and the formation of stress fibers (12,18,39,44,46). The morphological and functional changes that cardiac fibroblasts undergo during their conversion to myofibroblasts are critical to the wound healing process following myocardial injury. The loca...