Abstract 1276: Adrenomedullin Regulates Cardiac Fibroblasts via 2 Different Pathways: Stimulation of Migration through Epac and Attenuation of Transformation of Fibroblasts to Myofibroblasts through PKA
Background Migration and transformation of fibroblasts to myofibroblasts (the latter characterized by expression of α-smooth muscle actin (αSMA)) are key events in connective tissue remodeling after myocardial infarction. We have demonstrated that adrenomedullin (AM) stimulated adenylyl cyclase (AC) activity inhibits transforming growth factor β (TGFβ) -stimulated αSMA expression and collagen synthesis in cardiac fibroblasts (CF) overexpressing AC Type 6 (PNAS 2005).
Hypothesis We hypothesize that AM not only regulates transformation of fibroblasts to myofibroblasts but also CF migration, an essential remodeling process during wound healing.
Methods We examined effects of AM and adenovirus-mediated gene transfer of AC6 (Adv.AC6) on rat CF migration using the Boyden chamber method and assessed the role of Epac and protein kinase A (PKA), both mediators of cAMP action, by using cAMP analogs selective to PKA or Epac. Expression of AC and Epac isoforms were determined by quantitative RT-PCR and αSMA expression was examined by immunoblot analysis.
Results AC6 mRNA was most highly expressed among those (AC 3–9) identified in CF (22.8+ − 3 fold, p<0.001 n=4 relative to AC4). TGFβ (10ng/ml) decreased AC6 mRNA (72+/− 0.05%, p< 0.001, n=4) but AM (1uM) increased AC6 and Epac 2 mRNA (1.4+/− 0.09 and 2.4+/− 0.17 fold, p< 0.05, n=4). TGFβ did not effect CF migration However, angiotensin II (100nM) and endothelin (100nM), which are potent profibrotic factors, inhibited migration. Surprisingly, AM and Adv.AC6 promoted CF migration (4.5+/− 0.2 and 5.8+/− 0.8 fold, p< 0.001). pCPT-cAMP (100 μM), a cAMP analog selective for PKA, inhibited CF migration (60+/− 0.1%, p < 0.05, n=4) However, 8-cCPT-2′-O-Me-cAMP (50μM), selective for Epac, enhanced cell migration (4.9+/− 0.2 fold, p< 0.001, n= 4). Although pCPT-cAMP decreased αSMA expression (95+/− 2%, p<0.001, n=4), 8-cCPT-2′-O-Me-cAMP did not change αSMA expression.
Conclusion AM promotes CF migration through Epac and attenuates transformation of fibroblasts to myofibroblasts through the PKA pathway. The varying ability of agonists that raise cAMP to activate these two pathways likely contributes to heterogeneity in response of CF and to regulate connective tissue remodeling in the heart.