Roles And Mechanisms Of TGFβ-Smad2/3 Signaling Pathways In Cardiac Remodeling After Myocardial Infarction In Mice

Background: Acute myocardial infarction(AMI)is the major mortality world wide.Post-MI cardiac remodeling is critically associated with myofibroblast transdifferentiation.Durng the process of cardiac repair and remodeling,transforming growth factor(TGF-βs)is one of the most important cytokines to regulate fibroblast responses,by activating Smad2 or Smad3 signaling,or via Smad-independent pathways.We have previously demonstrated that fibroblast-specific Smad3 is critically implicated in repair of the infarcted heart;Cardiomyocyte Smad3 signaling accentuates cardiac remodeling.However,the role of fibroblast and cardiomyocyte Smad2 in homeostasis and myocardial infarction remains unknown.Objectives: This study investigates the distinct roles of cell specific Smad2 and Smad3 signaling in homeostasis and myocardial infarction,and explores the mechanisms responsible for the distinct effects of Smad2 and Smad3 in the infarct myofibroblasts.Methods: Both in vitro and in vivo approaches are used to compare the distinct effects of cell specific Smad2 and Smad3 in homeostasis and post-MI cardiac remodeling.In vivo,we use Cre-loxP system to generate mice with loss of Smad2 or Smad3 in fibroblast(Col1a2-Cre),myocyte(aMHC-Cre),and activated myofibroblasts(Periostin-Cre)to examine the effects of cell specific loss of Smad2/3 on baseline cardiac geometry and function;a mouse model of permanent myocardial infarction was used to examine the distinct role of myofibroblasts specific Smad2 and Smad3 loss(FS2KO and FS3O)in cardiac repair and remodeling,and the effects on myofibroblast activation,as well as topographical organization of the fibroblasts-based scar.In vitro,we use siRNA knockdown experiment to explore the actions of Smad2 and Smad3 signaling on integrin expression,extracellular matrix(ECM)synthesis,planar cell polarity(PCP)signaling pathway essential for fibroblast alignment.Results: Compared to wild type(WT)mice,cardiac fibroblasts from global Smad3 knockout(S3KO)mice express markedly increased matrix metalloproteinase-8(MMP-8)when stimulated with TGF-β1;Moreover,TGF-β1 significantly promoted secretion of MMP-8 and its active form in supernatant of S3 KO fibroblasts.In normal heart,WB shows evident baseline activation of pSmad2,but not pSmad3.In vitro,Smad2 and Smad3 knockdown play various roles in expression of adhesion molecules and extracellular matrix(ECM)genes.In vivo,tamoxifen induced fibroblasts specific Smad2 and Smad3 loss have no significant effects on homeostasis,cardiac baseline geometry and function.In contrast to cardiomyocyte specific Smad3 loss(CMS3KO),male CMS2 KO developed eccentric hypertrophy,systolic dysfunction and dilative remodeling at the age of 6 months old;cardiac dysfunction in female CMS2 KO mice is delayed at 9 months old.From Western blot,Smad2 expression shows 90% reduction in male CMS2 KO cardiomyocyte,while only 50% reduction in female CMS2 KO mcie,compared to Smad2 fl/fl.In a mouse model of myocardial infarction,Smad2 and Smad3 activation in infarct myofibroblasts peaked 7 days after coronary occlusion.In vitro,TGF β1,β2 and β3,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 FS3 KO mice,FS2 KO 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,FS2 KO and Smad2 fl/fl mice had comparable adverse remodeling.Although both FS3 KO and FS2 KO animals had increased myofibroblast density in the infarct,only FS3 KO mice exhibited impaired scar organization,associated with perturbed alignment of infarct myofibroblasts in the infarct area and border zone.In vitro,Smad3 but not Smad2 knockdown downmodulated fibroblast α2 and α5 integrin expression.Moreover,Smad3 knockdown reduced expression of the GTPase RhoA,whereas Smad2 knockdown markedly increased fibroblast RhoA levels.Besides,the core PCP pathway involves the Frizzled(Fz)family of multipass transmembrane proteins.Analysis of frizzled gene expression in isolated cardiac fibroblasts with S2 KD or S3 KD are divergent.It did not reveal consistent effects that could explain the in vivo observations.Conclusions: Cardiomyocyte specific Smad2 signaling is important to maintain baseline cardiac geometry and function.In contrast,cardiomyocyte specific Smad3 is critically involved in post-MI cardiac remodeling.In non-reperfused MI,fibroblasts-specific Smad3 signaling protect the infarcted heart from adverse remodeling,playing a critical role in activation and topographical organization of fibroblast-based scar,while fibroblast specific Smad2 loss does not disrupt scar organization,and is associated with a modest and transient reduction in adverse remodeling.Smad3-dependent integrin expression may be important for fibroblast activation,whereas RhoA may transduce planar cell polarity pathway signals,essential for fibroblast alignment.In conclusion,cardiomyocyte specific Smad2 signaling is associated with homeostasis;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.