Adenylyl cyclase type 6 (AC6) has favorable effects on the failing heart. However, AC6 has two shortcomings that, if resolved, would provide an optimal therapeutic transgene. First, AC6's large size prevents its expression in adeno-associated virus (AAV) vectors, which would enable longer-term expression. Second, AC6's propensity to increase intracellular levels of cAMP is a potential impediment. Recent data from our laboratory indicate that the beneficial effects of AC6, in large measure, are independent of cAMP generation. The elimination of the amino terminus and the two transmembrane domains (M1 &M2) of AC and subsequent fusing the two cytoplasmic domains (C1 &C2) with an 8 amino acid linker yields a C1C2 protein. C1C2 has an intact catalytic domain, but is disengaged from membrane-associated ?-adrenergic receptors (?AR), and therefore less responsive to ?AR stimulation. Our preliminary data indicate that increased expression of C1C2 in cardiac myocytes mimics the beneficial effects of AC6 on Akt activation and ATF3 expression, has favorable effects on cell survival, and improved function of the failing heart. Moreover, C1C2 is sufficiently small to be inserted in an AAV vector with a regulated expression cassette, and will be better suited for targeting to specific microdomains owing to its smaller size. Hypothesis C1C2 expression - independently ?-adrenergic receptor stimulation - will have beneficial effects on the failing heart. The following 5 Aims will examine the mechanism of action and effects of C1C2 on heart function.
Aim 1 To determine the mechanisms by which C1C2 has beneficial effects on cardiac myocytes.
Aim2 To determine if cardiac-directed expression of the optimal C1C2 construct will have favorable effects on normal heart function and have minimal deleterious effects even after prolonged high-level cardiac expression.
Aim 3 To determine if cardiac-directed expression of C1C2 will improve function of the failing heart.
Aim 4 To determine whether cardiac gene transfer of an AAV vector enabling regulated expression of an optimal C1C2 can safely improve function of the failing mouse heart.
Aim 5 To determine whether cardiac gene transfer of an AAV vector enabling regulated expression of an optimal C1C2 can safely improve function of the failing pig heart, perform biodistribution and toxicology studies, and file an IND for initiation of a clinical trial.
This Project focuses upon the discovery and potential use of new therapeutic genes for the treatment of clinical congestive heart failure so its relevance to the mission of the NHLBI and NIH is clear.
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