Adenylyl cyclase type VI gene transfer reduces phospholamban expression in cardiac myocytes via activating transcription factor 3

Abstract

Cardiac-directed expression of adenylyl cyclase type VI (ACVI) increases stimulated cAMP production, improves heart function, and increases survival in cardiomyopathy. In contrast, pharmacological agents that increase intracellular levels of cAMP have detrimental effects on cardiac function and survival. We wondered whether effects that are independent of cAMP might be responsible for these salutary outcomes associated with ACVI expression. We therefore conducted a series of experiments focused on how gene transcription is influenced by ACVI in cultured neonatal rat cardiac myocytes, with a particular focus on genes that might influence cardiac function. We found that overespression of ACVI down-regulated inRNA and protein expression of phospholamban, an inhibitor of the sarcoplasmic reticuluna Ca2+-ATPaSe. We determined that the cAMP-responsive-like element in the phospholamban (PLB) promoter was critical for down-regulation by ACVI. Overexpression of ACVI did not alter the expression of CREB, CKEM, ATF1, ATF2, or ATF4 proteins. In contrast, overexpression of ACVI increased expression of ATF3 protein, a suppressor of transcription. Following ACy1 gene transfer, when cardiac myocytes were stimulated with isoproterenol or NKH477, a water-soluble forskolin analog that directly stimulates AC, expression of ATF3 protein was increased even more, which correlated with reduced expression of PLB. We then showed that AC VI-induced ATF3 protein binds to the cAMP-responsive-like element on the PLB promoter and that overexpression. of ATF3 in cardiac myocytes inhibits PLB promoter activity. These findings indicate that ACVI has effects on gene transcription that are not directly dependent on cAMP generation.