Beta-adrenergic stimulation of the heart, which results in activation of the cAMP-dependent protein kinase (PKA) signaling pathway, is the main extrinsic regulator of cardiac function. Compartmentalization of PKA via A kinase anchoring proteins (AKAPs) modulates PKA activity in many cell types, however, research into the role of AKAP-mediated PKA anchoring in the heart is in the early stages of study. Thus, the goals of this proposal are: 1) to investigate the role of AKAP-mediated PKA anchoring in adult rat cardiac myocytes and 2) to determine if this anchoring is altered in the spontaneously hypertensive rat (SHR).
In Specific Aim 1, recombinant adenovirus directing expression of a blocking peptide (Ht31) that interrupts AKAP-mediated anchoring of PKA or a non-blocking control peptide (Ht31P) will be generated, identified, purified and titrated for use with adult rat ventricular myocytes.
Under Specific Aim 2, the consequence of disrupting PKA anchoring will be measured at two levels: 1) the phosphorylation state of known PKA substrates under baseline and stimulated conditions will be measured and 2) the physiological effects of PKA-dependent phosphorylation will be studied, specifically the effect on Ca2+ sensitivity o the actomyosin ATPase, Ca2+ cycling, contractility and the location of PKA within the cell. The nature of AKAP/PKA interaction in the SHR and age-matched control Wistar-Kyoto rat (WKY) will be studied in Specific Aim 3. This interaction will be assessed in the SHR via co-immunoprecipitation and surface plasmon resonance studies. The level of each protein will also be measured. These studies will help to clarify the role of AKAP-mediated anchoring of PKA in the heart and identify alterations of PKA anchoring in hypertrophied hearts.
| Russell, Mary A; Lund, Linda M; Haber, Roy et al. (2006) The intermediate filament protein, synemin, is an AKAP in the heart. Arch Biochem Biophys 456:204-15 |
| Fink, M A; Zakhary, D R; Mackey, J A et al. (2001) AKAP-mediated targeting of protein kinase a regulates contractility in cardiac myocytes. Circ Res 88:291-7 |
| Zakhary, D R; Fink, M A; Ruehr, M L et al. (2000) Selectivity and regulation of A-kinase anchoring proteins in the heart. The role of autophosphorylation of the type II regulatory subunit of cAMP-dependent protein kinase. J Biol Chem 275:41389-95 |
