Disruption of protein kinase A localization using a trans-activator of transcription (TAT)-conjugated A-kinase-anchoring peptide reduces cardiac function.
Localization of protein kinase A (PKA) via A-kinase-anchoring proteins (AKAPs) is important for cAMP responsiveness in many cellular systems, and evidence suggests that AKAPs play an important role in cardiac signaling. To test the importance of AKAP-mediated targeting of PKA on cardiac function, we designed a cell-permeable peptide, which we termed trans-activator of transcription (TAT)-AKAD for TAT-conjugated A-kinase-anchoring disruptor, using the PKA binding region of AKAP10 and tested the effects of this peptide in isolated cardiac myocytes and in Langendorff-perfused mouse hearts. We initially validated TAT-AKAD as a PKA localization inhibitor in cardiac myocytes by the use of confocal microscopy and cellular fractionation to show that treatment with the peptide disrupts type I and type II PKA regulatory subunits. Knockdown of PKA activity was demonstrated by decrease in phosphorylation of phospholamban and troponin I after beta-adrenergic stimulation in isolated myocytes. Treatment with TAT-AKAD reduced myocyte shortening and rates of contraction and relaxation. Injection of TAT-AKAD (1 microM), but not scrambled control peptide, into the coronary circulation of isolated perfused hearts rapidly (<1 min) and reversibly decreased heart rate and peak left ventricular developed pressure. TAT-AKAD also had a pronounced effect on developed pressure (-dP/dt), consistent with a delayed relaxation of the heart. The effects of TAT-AKAD on heart rate and contractility persisted in hearts pretreated with isoproterenol. Disruption of PKA localization with TAT-AKAD thus had negative effects on chronotropy, inotropy, and lusitropy, thereby indicating a key role for AKAP-targeted PKA in control of heart rate and contractile function.
Patel
Hemal H
HH
Department of Anesthesiology, University of California, San Diego, La Jolla, California 92093-0654, USA.
Hamuro
Lora L
LL
Chun
Byeong Jo
BJ
Kawaraguchi
Yoshitaka
Y
Quick
Alexander
A
Rebolledo
Brian
B
Pennypacker
Juniper
J
Thurston
Jackie
J
Rodriguez-Pinto
Natalia
N
Self
Christopher
C
Olson
Gary
G
Insel
Paul A
PA
Giles
Wayne R
WR
Taylor
Susan S
SS
Roth
David M
DM
D054758:A Kinase Anchor Proteins / Q000378:metabolism*; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit / Q000378:metabolism; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006321:Heart / Q000187:drug effects* / Q000502:physiology*; D006339:Heart Rate / Q000187:drug effects; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D032342:Muscle Cells / Q000166:cytology / Q000378:metabolism; D009200:Myocardial Contraction / Q000187:drug effects; D010455:Peptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D010477:Perfusion; D010539:Permeability; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D015533:Transcriptional Activation / Q000187:drug effects*
D054758:A Kinase Anchor Proteins; D000318:Adrenergic beta-Agonists; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D010455:Peptides; D047428:Protein Kinase Inhibitors; D017868:Cyclic AMP-Dependent Protein Kinases