Disruption of Protein Kinase A Localization Using a Trans-activator of Transcription (TAT)-conjugated A-kinase-anchoring Peptide Reduces Cardiac Function

Abstract

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 ␤-adrenergic stimulation in isolated myocytes. Treatment with TAT-AKAD reduced myocyte shortening and rates of contraction and relaxation. Injection of TAT-AKAD (1 M), 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.

A-kinase anchoring proteins: scaffolding proteins in the heart

“…In line with these findings, treatment of Langendorff-perfused mouse hearts with transactivator of transcription-conjugated anchoring disruptor peptides containing the PKA-binding region of AKAP10 strongly reduced the phosphorylation of cardiac PKA substrates, heart rate, and left ventricular developed pressure in response to isoproterenol stimulation (88). Collectively, these findings provide solid evidence for the importance of AKAPs in the compartmentalization of PKA and the regulation of cardiac function.…”

“…Several studies have shown that the disruption of PKA anchoring in cardiomyocytes and in intact hearts inhibits PKAmediated phosphorylation of cTnI and cMyBP-C (15,26,75,88). Although these results demonstrated that cTnI and cMyBP-C phosphorylation requires AKAP-mediated PKA anchoring, the identity of the AKAP(s) mediating this function is still unknown.…”

Cardiac Troponin T, a Sarcomeric AKAP, Tethers Protein Kinase A at the Myofilaments

“…Studies over the past decade have illuminated our understanding of their non-redundant biological role, usually through sequestering each PKA type to distinct intracellular signaling compartments. Recent studies employing various imaging techniques show overlapping staining patterns at the cardiac sarcomere for both PKA-RI and PKA-RII (11,45). The striated staining is largely abolished in the presence of peptide disruptors.…”

Targeting Protein–Protein Interactions Within The Cyclic AMP Signaling System As A Therapeutic Strategy for Cardiovascular Disease

“…Cellpermeable peptides that displace PKA from AKAPs have helped delineate AKAP function in Tcells [105], pancreatic b cells [106], adrenocortical cells [105], cardiac myocytes [107,108], hippocampal neurons [109] and renal principal cells (reviewed in [110]). …”