Abstract 2100: Beneficial Effects of Adenylyl Cyclase 6 (AC6) Gene Transfer Persist in a Catalytically Inactive AC6 Mutant With Impaired cAMP Generating Capacity
Background. Cardiac-directed expression of AC6 has pronounced favorable effects on cardiac function not apparently solely linked with cAMP production. To determine rigorously whether cAMP generation is required for the beneficial effects of AC6, we generated a catalytically inactive AC6 mutant (AC6mut) that has markedly diminished cAMP generating capacity compared to normal AC6.
Methods & Results. We replaced aspartic acid with alanine at position 426 in the C1 loop of AC6. Gene transfer of AC6mut (adenovirus-mediated) in adult rat cardiac myocytes showed impaired cAMP generation (Figure⇓, left) despite identical cellular distribution vs normal AC6 transgene. Despite marked reduction in cAMP generation, AC6mut gene transfer increased Akt phosphorylation and activity and suppressed PHLPP2 (an Akt-specific phosphatase) activity similarly to that observed with AC6 gene transfer. Moreover, AC6mut gene transfer increased Ca2+ transients in cardiac myocytes after isoproterenol stimulation similarly to AC6 gene transfer (Figure⇓, right). Finally, as compared with AC6 gene transfer, AC6mut gene transfer reduced apoptosis (p<0.05) induced by isoproterenol (1 μM, 24h) in cultured cardiac myocytes.
Conclusion. The beneficial effects of AC6 gene transfer persist in a catalytically inactive AC6mut, indicating that salutary effects (Akt activation, increased Ca2+ handling) occur in the absence of increased cAMP generation. These findings, together with the added benefits of AC6mut vis-à-vis reduced apoptosis, indicate that AC6mut may be a suitable candidate gene for heart failure therapy.
This research has received full or partial funding support from the American Heart Association, Western States Affiliate (California, Nevada & Utah).