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 (AC VI ) 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 AC VI expression. We therefore conducted a series of experiments focused on how gene transcription is influenced by AC VI in cultured neonatal rat cardiac myocytes, with a particular focus on genes that might influence cardiac function. We found that overexpression of AC VI down-regulated mRNA and protein expression of phospholamban, an inhibitor of the sarcoplasmic reticulum Ca 2؉ -ATPase. We determined that the cAMPresponsive-like element in the phospholamban (PLB) promoter was critical for down-regulation by AC VI . Overexpression of AC VI did not alter the expression of CREB, CREM, ATF1, ATF2, or ATF4 proteins. In contrast, overexpression of AC VI increased expression of ATF3 protein, a suppressor of transcription. Following AC VI 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 AC VI has effects on gene transcription that are not directly dependent on cAMP generation. Adenylyl cyclase (AC)1 is the effector molecule in the ␤-adrenergic receptor-G-protein-AC signaling pathway in cardiac myocytes and other cells. Previous studies showed that the amount of AC sets a limit on the ability of cardiac myocytes to generate cAMP (1), and cardiac-directed expression of AC type VI (AC VI ) has pronounced favorable effects on cardiovascular function in normal and failing hearts (2-6).The mechanisms explaining these favorable effects of AC VI are not precisely known. The most direct explanation is that the benefits stem from increased intracellular levels of cAMP; this explanation is contrary to current dogma in heart failure which asserts that inotropic agents that increase cAMP are bad for the heart. Indeed, pharmacological agents that stimulate the ␤-adrenergic receptor or decrease the breakdown of cAMP increase cardiac function but do not appear to prolong life (7-9). In contrast, AC VI , a dominant AC isoform in mammalian cardiac myocytes, improves global cardiac function, attenuates myocardial hypertrophy, and increases survival in murine cardiomyopathy (3, 4). However, when cardiac-directed ␤-adrenergic receptor expression is used to treat this same model, life is shortened (10), underscoring the marked differences evoked by these signaling elements, both of which are associated w...

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