AKAP-mediated targeting of protein kinase a regulates contractility in cardiac myocytes.
Compartmentalization of cAMP-dependent protein kinase A (PKA) by A-kinase anchoring proteins (AKAPs) targets PKA to distinct subcellular locations in many cell types. However, the question of whether AKAP-mediated PKA anchoring in the heart regulates cardiac contractile function has not been addressed. We disrupted AKAP-mediated PKA anchoring in cardiac myocytes by introducing, via adenovirus-mediated gene transfer, Ht31, a peptide that binds the PKA regulatory subunit type II (RII) with high affinity. This peptide competes with endogenous AKAPs for RII binding. Ht31P (a proline-substituted derivative), which does not bind RII, was used as a negative control. We then investigated the effects of Ht31 expression on RII distribution, Ca(2+) cycling, cell shortening, and PKA-dependent substrate phosphorylation. By confocal microscopy, we showed redistribution of RII from the perinuclear region and from periodic transverse striations in Ht31P-expressing cells to a diffuse cytosolic localization in Ht31-expressing cells. In the presence of 10 nmol/L isoproterenol, Ht31-expressing myocytes displayed an increased rate and amplitude of cell shortening and relaxation compared with control cells (uninfected and Ht31P-expressing myocytes); with isoproterenol stimulation we observed decreased time to 90% decline in Ca(2+) but no significant difference between Ht31-expressing and control cells in the rate of Ca(2+) cycling or amplitude of the Ca(2+) transient. The increase in PKA-dependent phosphorylation of troponin I and myosin binding protein C on isoproterenol stimulation was significantly reduced in Ht31-expressing cells compared with controls. Our results demonstrate that, in response to beta-adrenergic stimulation, cardiomyocyte function and substrate phosphorylation by PKA is regulated by targeting of PKA by AKAPs.
Fink
M A
MA
Department of Molecular Cardiology, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Zakhary
D R
DR
Mackey
J A
JA
Desnoyer
R W
RW
Apperson-Hansen
C
C
Damron
D S
DS
Bond
M
M
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000256:Adenoviridae / Q000235:genetics; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004274:DNA, Recombinant; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D018613:Microscopy, Confocal; D009200:Myocardial Contraction / Q000187:drug effects / Q000502:physiology*; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity; D014162:Transfection; D016276:Ventricular Function
D054758:A Kinase Anchor Proteins; C487775:AKAP5 protein, human; D048868:Adaptor Proteins, Signal Transducing; D000318:Adrenergic beta-Agonists; C415703:Akap5 protein, rat; C484293:Akap6 protein, rat; D002352:Carrier Proteins; D004274:DNA, Recombinant; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D007545:Isoproterenol; D002118:Calcium