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Opposing Actions of Fibroblast and Cardiomyocyte Smad3 Signaling in the Infarcted Myocardium.

ABSTRACT: BACKGROUND:Transforming growth factor-?s regulate a wide range of cellular responses by activating Smad-dependent and Smad-independent cascades. In the infarcted heart, Smad3 signaling is activated in both cardiomyocytes and interstitial cells. We hypothesized that cell-specific actions of Smad3 regulate repair and remodeling in the infarcted myocardium. METHODS:To dissect cell-specific Smad3 actions in myocardial infarction, we generated mice with Smad3 loss in activated fibroblasts or cardiomyocytes. Cardiac function was assessed after reperfused or nonreperfused infarction using echocardiography. The effects of cell-specific Smad3 loss on the infarcted heart were studied using histological studies, assessment of protein, and gene expression levels. In vitro, we studied Smad-dependent and Smad-independent actions in isolated cardiac fibroblasts. RESULTS:Mice with fibroblast-specific Smad3 loss had accentuated adverse remodeling after reperfused infarction and exhibited an increased incidence of late rupture after nonreperfused infarction. The consequences of fibroblast-specific Smad3 loss were not a result of effects on acute infarct size but were associated with unrestrained fibroblast proliferation, impaired scar remodeling, reduced fibroblast-derived collagen synthesis, and perturbed alignment of myofibroblast arrays in the infarct. Polarized light microscopy in Sirius red-stained sections demonstrated that the changes in fibroblast morphology were associated with perturbed organization of the collagenous matrix in the infarcted area. In contrast, ?-smooth muscle actin expression by infarct myofibroblasts was not affected by Smad3 loss. Smad3 critically regulated fibroblast function, activating integrin-mediated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-2 (NOX-2) expression. Smad3 loss in cardiomyocytes attenuated remodeling and dysfunction after infarction. Cardiomyocyte-specific Smad3 loss did not affect acute infarct size but was associated with attenuated cardiomyocyte apoptosis in the remodeling myocardium, accompanied by decreased myocardial NOX-2 levels, reduced nitrosative stress, and lower matrix metalloproteinase-2 expression. CONCLUSIONS:In healing myocardial infarction, myofibroblast- and cardiomyocyte-specific activation of Smad3 has contrasting functional outcomes that may involve activation of an integrin/reactive oxygen axis.

SUBMITTER: Kong P

PROVIDER: S-EPMC5812838 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Opposing Actions of Fibroblast and Cardiomyocyte Smad3 Signaling in the Infarcted Myocardium.

Kong Ping P Shinde Arti V AV Su Ya Y Russo Ilaria I Chen Bijun B Saxena Amit A Conway Simon J SJ Graff Jonathan M JM Frangogiannis Nikolaos G NG

Circulation 20171211 7

<h4>Background</h4>Transforming growth factor-βs regulate a wide range of cellular responses by activating Smad-dependent and Smad-independent cascades. In the infarcted heart, Smad3 signaling is activated in both cardiomyocytes and interstitial cells. We hypothesized that cell-specific actions of Smad3 regulate repair and remodeling in the infarcted myocardium.<h4>Methods</h4>To dissect cell-specific Smad3 actions in myocardial infarction, we generated mice with Smad3 loss in activated fibrobla ...[more]

PMID: 29229611

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Project description:BackgroundCellular cardiomyoplasty for myocardial infarction has been developed using various cell types. However, complete differentiation and/or trans-differentiation into cardiomyocytes have never occurred in these transplant studies, whereas functional contributions were reported.Methods and resultsSkeletal muscle interstitium-derived CD34(+)/CD45(-) (Sk-34) cells were purified from green fluorescent protein transgenic mice by flowcytometory. Cardiac differentiation of Sk-34 cells was examined by in vitro clonal culture and co-culture with embryonic cardiomyocytes, and in vivo transplantation into a nude rat myocardial infarction (MI) model (left ventricle). Lower relative expression of cardiomyogenic transcription factors, such as GATA-4, Nkx2-5, Isl-1, Mef2 and Hand2, was seen in clonal cell culture. However, vigorous expression of these factors was seen on co-culture with embryonic cardiomyocytes, together with formation of gap-junctions and synchronous contraction following sphere-like colony formation. At 4 weeks after transplantation of freshly isolated Sk-34 cells, donor cells exhibited typical cardiomyocyte structure with formation of gap-junctions, as well as intercalated discs and desmosomes, between donor and recipient and/or donor and donor cells. Fluorescence in situ hybridization (FISH) analysis detecting the rat and mouse genomic DNA and immunoelectron microscopy using anti-GFP revealed donor-derived cells. Transplanted Sk-34 cells were incorporated into infarcted portions of recipient muscles and contributed to cardiac reconstitution. Significant improvement in left ventricular function, as evaluated by transthoracic echocardiography and micro-tip conductance catheter, was also observed.Conclusions and significanceSkeletal muscle-derived multipotent Sk-34 cells that can give rise to skeletal and smooth muscle cells as reported previously, also give rise to cardiac muscle cells as multi-myogenic stem cells, and thus are a potential source for practical cellular cardiomyoplasty.

Project description:Coronary artery disease is a severe ischemic condition characterized by the reduction of blood flow in the arteries of the heart that results in the dysfunction and death of cardiac tissue. Despite research over several decades on how to reduce long-term complications and promote angiogenesis in the infarct, the medical field has yet to define effective treatments for inducing revascularization in the ischemic tissue. With this work, we have developed functional biomaterials for the controlled release of immunomodulatory cytokines to direct immune cell fate for controlling wound healing in the ischemic myocardium. The reparative effects of colony-stimulating factor (CSF-1), and anti-inflammatory interleukins 4/6/13 (IL4/6/13) have been evaluated in vitro and in a predictive in vivo model of ischemia (the skin flap model) to optimize a new immunomodulatory biomaterial that we use for treating infarcted rat hearts. Alginate hydrogels have been produced by internal gelation with calcium carbonate (CaCO3) as carriers for the immunomodulatory cues, and their stability, degradation, rheological properties and release kinetics have been evaluated in vitro. CD14 positive human peripheral blood monocytes treated with the immunomodulatory biomaterials show polarization into pro-healing macrophage phenotypes. Unloaded and CSF-1/IL4 loaded alginate gel formulations have been implanted in skin flap ischemic wounds to test the safety and efficacy of the delivery system in vivo. Faster wound healing is observed with the new therapeutic treatment, compared to the wounds treated with the unloaded controls at day 14. The optimized therapy has been evaluated in a rat model of myocardial infarct (ischemia/reperfusion). Macrophage polarization toward healing phenotypes and global cardiac function measured with echocardiography and immunohistochemistry at 4 and 15 days demonstrate the therapeutic potential of the proposed immunomodulatory treatment in a clinically relevant infarct model.

Project description:TGF-?s regulate fibroblast responses, by activating Smad2 or Smad3 signaling, or via Smad-independent pathways. We have previously demonstrated that myofibroblast-specific Smad3 is critically implicated in repair of the infarcted heart. However, the role of fibroblast Smad2 in myocardial infarction remains unknown. This study investigates the role of myofibroblast-specific Smad2 signaling in myocardial infarction, and explores the mechanisms responsible for the distinct effects of Smad2 and Smad3. In a mouse model of non-reperfused myocardial infarction, Smad2 activation in infarct myofibroblasts peaked 7?days after coronary occlusion. In vitro, TGF-?1, -?2 and -?3, but not angiotensin 2 and bone morphogenetic proteins-2, -4 and -7, activated fibroblast Smad2. Myofibroblast-specific Smad2 and Smad3 knockout mice (FS2KO, FS3KO) and corresponding control littermates underwent non-reperfused infarction. In contrast to the increase in rupture rates and adverse remodeling in FS3KO mice, FS2KO animals had mortality comparable to Smad2 fl/fl controls, and exhibited a modest but transient improvement in dysfunction after 7?days of coronary occlusion. At the 28?day timepoint, FS2KO and Smad2 fl/fl mice had comparable adverse remodeling. Although both FS3KO and FS2KO animals had increased myofibroblast density in the infarct, only FS3KO mice exhibited impaired scar organization, associated with perturbed alignment of infarct myofibroblasts. In vitro, Smad3 but not Smad2 knockdown downmodulated fibroblast ?2 and ?5 integrin expression. Moreover, Smad3 knockdown reduced expression of the GTPase RhoA, whereas Smad2 knockdown markedly increased fibroblast RhoA levels. Smad3-dependent integrin expression may be important for fibroblast activation, whereas RhoA may transduce planar cell polarity pathway signals, essential for fibroblast alignment. Myofibroblast-specific Smad3, but not Smad2 is required for formation of aligned myofibroblast arrays in the infarct. The distinct in vivo effects of myofibroblast Smad2 and Smad3 may involve Smad3-dependent integrin synthesis, and contrasting effects of Smad2 and Smad3 on RhoA expression.

Project description:The present study evaluated the reversal of diabetes-mediated impairment of angiogenesis in a myocardial infarction model of type 1 diabetic rats by intramyocardial administration of an adenoviral vector encoding thioredoxin-1 (Ad.Trx1). Various studies have linked diabetes-mediated impairment of angiogenesis to dysfunctional antioxidant systems in which thioredoxin-1 plays a central role.Ad.Trx1 was administered intramyocardially in nondiabetic and diabetic rats immediately after myocardial infarction. Ad.LacZ was similarly administered to the respective control groups. The hearts were excised for molecular and immunohistochemical analysis at predetermined time points. Myocardial function was measured by echocardiography 30 days after the intervention. The Ad.Trx1-administered group exhibited reduced fibrosis, oxidative stress, and cardiomyocyte and endothelial cell apoptosis compared with the diabetic myocardial infarction group, along with increased capillary and arteriolar density. Western blot and immunohistochemical analysis demonstrated myocardial overexpression of thioredoxin-1, heme oxygenase-1, vascular endothelial growth factor, and p38 mitogen-activated protein kinase-beta, as well as decreased phosphorylated JNK and p38 mitogen-activated protein kinase-alpha, in the Ad.Trx1-treated diabetic group. Conversely, we observed a significant reduction in the expression of vascular endothelial growth factor in nondiabetic and diabetic animals treated with tin protoporphyrin (SnPP, a heme oxygenase-1 enzyme inhibitor), even after Ad.Trx1 therapy. Echocardiographic analysis after 4 weeks of myocardial infarction revealed significant improvement in myocardial functional parameters such as ejection fraction, fractional shortening, and E/A ratio in the Ad.Trx1-administered group compared with the diabetic myocardial infarction group.This study demonstrates for the first time that impairment of angiogenesis and myocardial dysfunction can be regulated by Ad.Trx1 gene therapy in streptozotocin-induced diabetic rats subjected to infarction.

Dataset Information

Opposing Actions of Fibroblast and Cardiomyocyte Smad3 Signaling in the Infarcted Myocardium.

Publications

Opposing Actions of Fibroblast and Cardiomyocyte Smad3 Signaling in the Infarcted Myocardium.

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