b-adrenergic receptors (bARs) are one of the powerful regulators of cardiac function. bAR downregulation (loss of surface receptors) and desensitization (impaired G-protein coupling) are hallmarks of a failing heart. Desensitization is initiated by phosphorylation of bARs by bAR kinase 1 (bARK1 or GRK2) in response to catecholamines resulting in reduced cardiac responses. b-arrestin binds phosphorylated bARs facilitating their endocytosis (downregulation). Phosphorylated bARs are resensitized by dephosphorylation mediated through protein phosphatase 2A (PP2A) in the endosomes and are recycled back to the plasma membrane as nave receptor ready for activation. Contrary to the belief that resensitization is a process that maintains homeostasis, our mechanistic studies in the current RO1 have identified that PI3Kg inhibits PP2A activity, thereby negatively regulating bAR resensitization. We have shown that PI3Kg phosphorylates endogenous inhibitor of PP2A (I2PP2A) on serine 9 & 93 (S9, 93) that robustly binds to PP2A, inhibiting PP2A activity and blocking bAR resensitization, effectively determining that the PI3Kg-I2PP2A-PP2A pathway regulates bAR resensitization. Although we have identified the pathway that regulates bAR resensitization, it is not known whether this pathway is altered and contributes to cardiac hypertrophy and heart failure. Therefore, the goal of the renewal is to determine whether the resensitization pathway is altered in conditions of heart failure and test whether targeting components of resensitization using genetically altered mice could provide beneficial remodeling upon stress. We have generated transgenic mice with cardiac overexpression of wild type I2PP2A (Wt I2PP2A Tg) or I2PP2A mutants S9, 93A (I2PP2A S9, 93A Tg, mimicking dephospho state) or S9, 93D (I2PP2A S9, 93D Tg, phospho state). Our preliminary studies show that subjecting Wt I2PP2A Tg mice or I2PP2A S9, 93D Tg mice to cardiac stress results in accelerated cardiac dysfunction which is markedly ameliorated in I2PP2A S9, 93A Tg mice suggesting a critical role for bAR resensitization in deleterious remodeling. Additionally, we also observed non-classical Gbg-independent PI3Kg recruitment to bARs in response to pro-inflammatory cytokine TNFa which may inhibit PP2A activity leading to loss in bAR resensitization suggesting that TNFa could regulate bAR function by altering the PI3Kg-I2PP2A-PP2A axis. Based on these preliminary studies, we hypothesize that bAR resensitization is inhibited by the PI3Kg- I2PP2A-PP2A pathway upon cardiac stress and releasing the inhibition on bAR resensitization by targeting I2PP2A could preserve bAR function and prevent deleterious cardiac remodeling. We propose the following specific aims to address the hypothesis:
Specific aim 1 : To determine whether regulating cardiac bAR resensitization pathway prevents deleterious cardiac remodeling in response to cardiac stress, Specific aim 2: To identify the molecular basis of PI3Kg mediated I2PP2A-PP2A interaction and its role in bAR resensitization, Specific aim 3: To investigate the molecular mechanisms mediating agonist independent non-canonical recruitment of PI3Kg and inhibition of bAR resensitization in response to TNFa. Determining the role of PI3Kg-I2PP2A-PP2A axis in regulating bAR resensitization in response to cardiac stress will provide comprehensive mechanistic understanding of bAR dysfunction leading to identification of novel therapeutic strategies for heart failure by targeting bAR resensitization pathway.
b-adrenergic receptors (bARs) are powerful regulators of cardiac function and their dysfunction is a hallmark of heart failure. Desensitization, i.e. the inability of bARs to transmit signals into the myocytes, is thought to be the major underlying cause for heart failure. In this context, the mechanisms that allow the receptors to become rejuvenated and competent to transmit signals, i.e., resensitization, are not well understood in heart failure. Consistently, all the current mainstream drugs for heart failure target the bAR desensitization pathway and none target the resensitization. Thus, our current studies on bAR resensitization pathways in heart failure will provide potentially novel therapeutic targets that may have beneficial effects.
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