AKAP-dependent regulation of Cardiac SR Ca handling

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Project Summary/ Abstract Beta-adrenergic receptor (β-AR) activation affects cardiac excitation-contraction coupling (ECC) through coordinated increases in sarcoplasmic reticulum (SR) Ca uptake by SERCA and release via ryanodine receptor (RyR) channels. Reduced SR Ca uptake and sensitized SR Ca release contribute to reduced SR Ca content in heart failure (HF), and are key therapeutic targets in HF. However, there are key gaps in our fundamental understanding of ß-AR regulation of these SR Ca handling processes in cardiac myocytes, which our team is well- poised to address. SR Ca uptake is basally inhibited by phospholamban (PLB), an effect that is relieved by PLB phosphorylation by PKA. A quantitative mechanistic question is how a small number of PKA molecules in the cell can rapidly phosphorylate the far more numerous (>200- fold) and widely distributed PLB, to uniformly accelerate [Ca]i decline and SR Ca load upon ß-AR activation. It is unlikely that dedicated anchoring of PKA near each small cluster of PLB molecules would suffice from a quantitative standpoint. New data suggest that PLB phosphorylation can decrease PLB affinity of A-kinase anchoring protein, AKAP7. We will test the hypothesis that upon ß-AR activation, the AKAP7 that is bound to un-phosphorylated PLB phosphorylates nearby PLB molecules, but then detaches, only to bind again quickly to a neighboring area of unphosphorylated PLBs (i.e. “hovering” along the SR, phosphorylating sequential PLB clusters). This new paradigm, which differs from the standard, accepted 1:1 relationship between other AKAPs and their targets, may be critical to rapid adrenergic fight-or- flight response. Aim 1 will test how AKAP7 mobility is influenced by PLB binding and phosphorylation to expedite adrenergic cardiac lusitropy. Aim 2 will test consequences of disrupting the AKAP7-PLB interaction with respect to spatiotemporal spread of SERCA activation in single myocytes. Aim 3 will test for AKAP7-CaMKII interaction and its potential effects on PLB & RyR. This work will provide novel and clear mechanistic data regarding the fundamental mechanism of rapid synchronous adrenergic activation throughout the myocyte and heart. This may present a novel type of AKAP function, where targeted translocation of the AKAP amplifies signaling to very large numbers of target sites.