Inhibition of cardiac myofibroblast formation and collagen synthesis by activation and overexpression of adenylyl cyclase
adrenomedullin; angiotensin-ii; cardiac fibroblast; cyclic AMP; extracellular-matrix; failure; fibrosis; gene-expression; growth-factor-beta; heart; lung fibroblasts; modulation; necrosis-factor-alpha; phenotypic; rat ventricular myocytes; Science & Technology - Other Topics; smooth muscle actin
Transformation of fibroblasts to myofibroblasts, characterized by expression of alpha-smooth muscle actin (alpha-SMA) and production of extracellular matrix (ECM) components, is a key event in connective tissue remodeling. Approaches to inhibit this transformation are needed in tissues, such as the heart, where excessive ECM production by cardiac fibroblasts (CFs) causes fibrosis, myocardial stiffening, and cardiac dysfunction. We tested whether adenylyl cyclase (AC) activation (increased cAMP levels) modulates the transformation of adult rat CF to myofibroblasts, as assessed by immunofluorescent microscopy, immunoblotting, and collagen synthesis. A 24-h incubation of CF with TGF-beta or angiotensin II increased alpha-SMA expression, which was inhibited by the AC agonist forskolin and a cAMP analog that activates protein kinase A. Treatment with forskolin blunted serum-, TGF-beta-, and angiotensin II-stimulated collagen synthesis. CFs engineered to overexpress type 6 AC had enhanced forskolin-promoted cAMP formation, greater inhibition by forskolin of TGF-beta-stimulated alpha-SMA expression, and a decrease in the EC50 of forskolin to reduce serum-stimulated collagen synthesis. The AC stimulatory agonist adrenomedullin inhibited collagen synthesis in CF that overexpressed AC6 but not in controls. Thus, AC stimulation blunts collagen synthesis and, in parallel, the transformation of adult rat CF to myofibroblasts. AC overexpression enhances these effects, "uncovering" an inhibition by adrenomedullin. These findings implicate cAMP as an inhibitor of ECM formation by means of blockade of the transformation of CF to myofibroblasts and suggest that increasing AC expression, thereby enhancing cAMP generation through stimulation of receptors expressed on CF, could provide a means to attenuate and prevent cardiac fibrosis and its sequelae.
Swaney J S; Roth D M; Olson E R; Naugle J E; Meszaros J G; Insel P A
Proceedings of the National Academy of Sciences of the United States of America
2005
2005-01
Journal Article
<a href="http://doi.org/10.1073/pnas.0408704102" target="_blank" rel="noreferrer noopener">10.1073/pnas.0408704102</a>
Angiotensin II enhances adenylyl cyclase signaling via Ca2+/calmodulin - G(q-)G(s) cross-talk regulates collagen production in cardiac fibroblasts
a(2b) receptors; adenosine inhibits collagen; Biochemistry & Molecular Biology; caveolae; Heart failure; in-vivo; nitric-oxide; pathway; protein-synthesis; smooth-muscle cells; ventricular myocytes
Cardiac fibroblasts regulate formation of extracellular matrix in the heart, playing key roles in cardiac remodeling and hypertrophy. In this study, we sought to characterize cross-talk between G(q) and G(s) signaling pathways and its impact on modulating collagen synthesis by cardiac fibroblasts. Angiotensin II (ANG II) activates cell proliferation and collagen synthesis but also potentiates cyclic AMP ( cAMP) production stimulated by beta-adrenergic receptors (beta-AR). The potentiation of beta-AR-stimulated cAMP production by ANG II is reduced by phospholipase C inhibition and enhanced by overexpression of G(q). Ionomycin and thapsigargin increased intracellular Ca2+ levels and potentiated isoproterenol- and forskolin-stimulated cAMP production, whereas chelation of Ca2+ with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid/AM inhibited such potentiation. Inhibitors of tyrosine kinases, protein kinase C, or Gbetagamma did not alter this cross-talk. Immunoblot analyses showed prominent expression of adenylyl cyclase 3 (AC3), a Ca2+-activated isoform, along with AC2, AC4, AC5, AC6, and AC7. Of those isoforms, only AC3 and AC5/6 proteins were detected in caveolin-rich fractions. Overexpression of AC6 increased betaAR-stimulated cAMP accumulation but did not alter the size of the ANG II potentiation, suggesting that the cross-talk is AC isoform-specific. Isoproterenol-mediated inhibition of serum-stimulated collagen synthesis increased from 31 to 48% in the presence of ANG II, indicating that betaAR-regulated collagen synthesis increased in the presence of ANG II. These data indicate that ANG II potentiates cAMP formation via Ca2+-dependent activation of AC activity, which in turn attenuates collagen synthesis and demonstrates one functional consequence of crosstalk between G(q) and G(s) signaling pathways in cardiac fibroblasts.
Ostrom R S; Naugle J E; Hase M; Gregorian C; Swaney J S; Insel P A; Brunton L L; Meszaros J G
Journal of Biological Chemistry
2003
2003-07
Journal Article
<a href="http://doi.org/10.1074/jbc.M212659200" target="_blank" rel="noreferrer noopener">10.1074/jbc.M212659200</a>