Repair of the infarcted heart requires TGF-β/Smad3 signaling in cardiac myofibroblasts. However, TGF-β–driven myofibroblast activation needs to be tightly regulated in order to prevent excessive fibrosis and adverse remodeling that may precipitate heart failure. We hypothesized that induction of the inhibitory Smad, Smad7, may restrain infarct myofibroblast activation, and we examined the molecular mechanisms of Smad7 actions. In a mouse model of nonreperfused infarction, Smad3 activation triggered Smad7 synthesis in α-SMA⁺ infarct myofibroblasts, but not in α-SMA^–PDGFRα⁺ fibroblasts. Myofibroblast-specific Smad7 loss increased heart failure–related mortality, worsened dysfunction, and accentuated fibrosis in the infarct border zone and in the papillary muscles. Smad7 attenuated myofibroblast activation and reduced synthesis of structural and matricellular extracellular matrix proteins. Smad7 effects on TGF-β cascades involved deactivation of Smad2/3 and non-Smad pathways, without any effects on TGF-β receptor activity. Unbiased transcriptomic and proteomic analysis identified receptor tyrosine kinase signaling as a major target of Smad7. Smad7 interacted with ErbB2 in a TGF-β–independent manner and restrained ErbB1/ErbB2 activation, suppressing fibroblast expression of fibrogenic proteases, integrins, and CD44. Smad7 induction in myofibroblasts serves as an endogenous TGF-β–induced negative feedback mechanism that inhibits postinfarction fibrosis by restraining Smad-dependent and Smad-independent TGF-β responses, and by suppressing TGF-β–independent fibrogenic actions of ErbB2.

BACKGROUND: Cardiac fibroblast activation contributes to adverse remodeling, fibrosis, and dysfunction in the pressure-overloaded heart. Although early fibroblast TGF-β (transforming growth factor-β)/Smad (small mother against decapentaplegic)-3 activation protects the pressure-overloaded heart by preserving the matrix, sustained TGF-β activation is deleterious, accentuating fibrosis and dysfunction. Thus, endogenous mechanisms that negatively regulate the TGF-β response in fibroblasts may be required to protect from progressive fibrosis and adverse remodeling. We hypothesized that Smad7, an inhibitory Smad that restrains TGF-β signaling, may be induced in the pressure-overloaded myocardium and may regulate fibrosis, remodeling, and dysfunction. METHODS: The effects of myofibroblast-specific Smad7 loss were studied in a mouse model of transverse aortic constriction, using echocardiography, histological analysis, and molecular analysis. Proteomic studies in S7KO (Smad7 knockout) and overexpressing cells were used to identify fibroblast-derived mediators modulated by Smad7. In vitro experiments using cultured cardiac fibroblasts, fibroblasts populating collagen lattices, and isolated macrophages were used to dissect the molecular signals responsible for the effects of Smad7. RESULTS: Following pressure overload, Smad7 was upregulated in cardiac myofibroblasts. TGF-β and angiotensin II stimulated fibroblast Smad7 upregulation via Smad3, whereas GDF15 (growth differentiation factor 15) induced Smad7 through GFRAL (glial cell line-derived neurotrophic factor family receptor α-like). MFS7KO (myofibroblast-specific S7KO) mice had increased mortality, accentuated systolic dysfunction and dilative remodeling, and accelerated diastolic dysfunction in response to transverse aortic constriction. Increased dysfunction in MFS7KO hearts was associated with accentuated fibrosis and increased MMP (matrix metalloproteinase)-2 activity and collagen denaturation. Secretomic analysis showed that Smad7 loss accentuates secretion of structural collagens and matricellular proteins and markedly increases MMP2 secretion. In contrast, Smad7 overexpression reduced MMP2 levels. In fibroblasts populating collagen lattices, the effects of Smad7 on fibroblast-induced collagen denaturation and pad contraction were partly mediated via MMP2 downregulation. Surprisingly, MFS7KO mice also exhibited significant macrophage expansion caused by paracrine actions of Smad7 null fibroblasts that stimulate macrophage proliferation and fibrogenic activation. Macrophage activation involved the combined effects of the fibroblast-derived matricellular proteins CD5L (CD5 antigen-like), SPARC (secreted protein acidic and rich in cysteine), CTGF (connective tissue growth factor), ECM1 (extracellular matrix protein 1), and TGFBI (TGFB induced). CONCLUSIONS: The antifibrotic effects of Smad7 in the pressure-overloaded heart protect from dysfunction and involve not only reduction in collagen deposition but also suppression of MMP2-mediated matrix denaturation and paracrine effects that suppress macrophage activation through inhibition of matricellular proteins.

Background: The effects of TGF‐βs (transforming growth factor‐βs) in failing hearts involve cell‐specific actions mediated through receptor‐regulated Smads or Smad‐independent pathways. The inhibitory Smad, Smad7, is a negative feedback regulator that restrains excessive TGF‐β/receptor‐regulated Smad signaling, while also exerting TGF‐β–independent effects. We hypothesized that cardiomyocyte Smad7 upregulation contributes to the pathogenesis of pressure overload–induced heart failure and investigated the mechanisms underlying its actions. Methods: Cardiomyocyte‐specific Smad7 knockout mice and corresponding Cre+ and Smad7 ^fl/fl controls underwent transverse aortic constriction. Echocardiographic, histological, transcriptomic, and proteomic analyses were performed. RNA sequencing identified candidate pathways modulated by Smad7. Effects of Smad7 loss on TGF‐β and ERBB2 (erb‐b2 receptor tyrosine kinase 2) cascades were examined in vivo and in cardiomyocyte‐like H9c2 cells using western blotting. Results: Smad7 was upregulated in cardiomyocytes and fibroblasts following transverse aortic constriction and in patients with nonischemic cardiomyopathy. Cardiomyocyte‐specific Smad7 knockout mice had no baseline abnormalities but developed worse systolic and diastolic dysfunction and increased cardiomyocyte hypertrophy after pressure overload. Cardiomyocyte Smad7 loss did not alter fibrosis after transverse aortic constriction but increased macrophage infiltration. Cardiomyocyte‐specific Smad7 knockout hearts showed reduced expression of genes associated with cardiac contraction. Bioinformatic analysis identified TGF‐β1, angiotensin, and ERBB2 as candidate upstream regulators mediating the effects of Smad7 loss. In vivo, cardiomyocyte Smad7 inhibited activation of TGF‐β receptor 1 ( TGFBR1)/Smad2/3 and ERBB2 without affecting TGF‐β receptor 2 ( TGFBR2) activity. In the absence of Smad7, pressure‐overloaded cardiomyocytes exhibited increased TGFBR1/Smad3 activation. In H9c2 cells, endogenous Smad7 restrained TGFBR1/Smad3 activation without affecting TGFBR2 or ERBB2 activity. Conclusions: Cardiomyocyte Smad7 protects the pressure‐overloaded heart from dysfunction, regulating genes involved in muscle contraction, likely through direct suppression of TGFBR1/Smad3.