Gβγ subunits colocalize with RNA polymerase II and regulate transcription in cardiac fibroblasts

Gβγ subunits mediate many different signaling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear Gβγ signaling, we investigated the functional role of Gβγ signaling in the regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. We identified a novel, negative, regulatory role for the Gβ1γ dimer in the fibrotic response. Depletion of Gβ1 led to derepression of the fibrotic response at the mRNA and protein levels under basal conditions and an enhanced fibrotic response after sustained stimulation of the angiotensin II type I receptor. Our genome-wide chromatin immunoprecipitation experiments revealed that Gβ1 colocalized and interacted with RNA polymerase II on fibrotic genes in an angiotensin II-dependent manner. Additionally, blocking transcription with inhibitors of Cdk9 prevented association of Gβγ with transcription complexes. Together, our findings suggest that Gβ1γ is a novel transcriptional regulator of the fibrotic response that may act to restrict fibrosis to conditions of sustained fibrotic signaling. Our work expands the role for Gβγ signaling in cardiac fibrosis and may have broad implications for the role of nuclear Gβγ signaling in other cell types. Gβγ subunits mediate many different signaling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear Gβγ signaling, we investigated the functional role of Gβγ signaling in the regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. We identified a novel, negative, regulatory role for the Gβ1γ dimer in the fibrotic response. Depletion of Gβ1 led to derepression of the fibrotic response at the mRNA and protein levels under basal conditions and an enhanced fibrotic response after sustained stimulation of the angiotensin II type I receptor. Our genome-wide chromatin immunoprecipitation experiments revealed that Gβ1 colocalized and interacted with RNA polymerase II on fibrotic genes in an angiotensin II-dependent manner. Additionally, blocking transcription with inhibitors of Cdk9 prevented association of Gβγ with transcription complexes. Together, our findings suggest that Gβ1γ is a novel transcriptional regulator of the fibrotic response that may act to restrict fibrosis to conditions of sustained fibrotic signaling. Our work expands the role for Gβγ signaling in cardiac fibrosis and may have broad implications for the role of nuclear Gβγ signaling in other cell types. In recent years, our understanding of the role of paracrine interactions between cardiomyocytes and cardiac fibroblasts in modulating the response to cardiac damage has expanded dramatically. Cardiac fibroblasts, in particular, respond dynamically following damage to the myocardium, characterized by differentiation into myofibroblasts, increased proliferation and migration to areas of damage (1Travers J.G. Kamal F.A. Robbins J. Yutzey K.E. Blaxall B.C. Cardiac fibrosis: the fibroblast awakens.Circ. Res. 2016; 118: 1021-1040Crossref PubMed Scopus (921) Google Scholar, 2Fu X. Khalil H. Kanisicak O. Boyer J.G. Vagnozzi R.J. Maliken B.D. et al.Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart.J. Clin. Invest. 2018; 128: 2127-2143Crossref PubMed Scopus (346) Google Scholar, 3Dobaczewski M. Bujak M. Li N. Gonzalez-Quesada C. Mendoza L.H. Wang X.F. et al.Smad3 signaling critically regulates fibroblast phenotype and function in healing myocardial infarction.Circ. Res. 2010; 107: 418-428Crossref PubMed Scopus (290) Google Scholar). This fibrotic response is modulated by a number of cytokines including the renin-angiotensin system, acting predominantly through the peptide ligand angiotensin II (Ang II, (4Murphy A.M. Wong A.L. Bezuhly M. Modulation of angiotensin II signaling in the prevention of fibrosis.Fibrogenesis Tissue Repair. 2015; 8: 7Crossref PubMed Scopus (105) Google Scholar, 5Kawano H. Do Y.S. Kawano Y. Starnes V. Barr M. Law R.E. et al.Angiotensin II has multiple profibrotic effects in human cardiac fibroblasts.Circulation. 2000; 101: 1130-1137Crossref PubMed Scopus (280) Google Scholar)). Ang II drives changes in fibroblast function both directly through G protein signaling and indirectly by increasing expression of other profibrotic growth factors such as transforming growth factor β1 (TGF-β1) (6Campbell S.E. Katwa L.C. Angiotensin II stimulated expression of transforming growth factor-beta1 in cardiac fibroblasts and myofibroblasts.J. Mol. Cell. 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Involvement of reactive oxygen species in angiotensin II-induced endothelin-1 gene expression in rat cardiac fibroblasts.J. Am. Coll. Cardiol. 2003; 42: 1845-1854Crossref PubMed Scopus (122) Google Scholar, 10Ahmed M.S. Øie E. Vinge L.E. Yndestad A. Øystein Andersen G. Andersson Y. et al.Connective tissue growth factor—a novel mediator of angiotensin II-stimulated cardiac fibroblast activation in heart failure in rats.J. Mol. Cell. Cardiol. 2004; 36: 393-404Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). These signaling events create a feedforward loop, amplifying the fibrotic response from the initial area of damage to more distal regions of the heart (11Ma Z.G. Yuan Y.P. Wu H.M. Zhang X. Tang Q.Z. Cardiac fibrosis: new insights into the pathogenesis.Int. J. Biol. Sci. 2018; 14: 1645-1657Crossref PubMed Scopus (185) Google Scholar). While the fibrotic response initially aids in wound healing, a prolonged, activated fibrotic response worsens adverse cardiac remodeling, and accelerates progression to heart failure (1Travers J.G. Kamal F.A. Robbins J. Yutzey K.E. Blaxall B.C. Cardiac fibrosis: the fibroblast awakens.Circ. Res. 2016; 118: 1021-1040Crossref PubMed Scopus (921) Google Scholar, 12Weber K.T. Sun Y. Bhattacharya S.K. Ahokas R.A. Gerling I.C. Myofibroblast-mediated mechanisms of pathological remodelling of the heart.Nat. Rev. Cardiol. 2013; PubMed Scopus Google Scholar). of the fibrotic response adverse cardiac X. Khalil H. Kanisicak O. Boyer J.G. Vagnozzi R.J. Maliken B.D. et al.Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart.J. Clin. Invest. 2018; 128: 2127-2143Crossref PubMed Scopus (346) Google Scholar, K.T. J. the cardiac to myocardial fibrosis in heart 2016; Scopus Google Scholar). 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