Role of S-Nitrosylation on Beta-Adrenergic Signaling in Cardiac Injury and Repair

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SUMMARY: During the development of heart failure (HF), especially after ischemic injury, derangements in myocardial β- adrenergic receptor (βAR) signaling contribute centrally to pathogenesis, including signal uncoupling and receptor desensitization leading to myocyte death and contractility defects. Waning of signaling through cardiac βARs and other G protein-coupled receptors (GPCRs) is classically regulated via receptor phosphorylation and internalization mediated by GPCR kinases (GRKs) and β-arrestins (β-Arrs). This is significant since the activity of GRK2, which is elevated in myocardium after injury/stress, is pathologic in HF and its inhibition is therapeutic. Through a long-standing collaboration, the Koch and Stamler laboratories have found that GPCRs are regulated by nitric oxide (NO), through S-nitrosylation of cysteine to form protein S-nitrosothiol (SNO), including profound regulation of GRK2 and of β-Arr2. Since cardiac GPCRs, including all three βARs, can activate NO synthase (NOS) enzymes, there is a need to discover how this can promote SNO-mediated cardioprotection, especially downstream of β2- and β3ARs. Our prior work has shown that NO from endothelial NOS (eNOS) inhibits GRK2 by S-nitrosylation at Cys340. Loss of SNO-based regulation in GRK2-C340S mutant knock-in (KI) mice leads to un-checked and enhanced GRK2 activity, and to increased ischemic injury, and to dysfunction during aging. Our labs have also shown that neuronal/inducible NOS (n/iNOS) activity can regulate β-Arr2 through SNO-Cys253 to maintain physiological βAR signaling in the heart. The loss of this SNO-β-Arr2 regulation in β-Arr2-C253S KI mice leads to increased βAR desensitization and HF. Additionally, the Stamler lab recently discovered that β2AR is S-nitrosylated at Cys265 and that this modification regulates β2AR desensitization. Together these data suggest tightly integrated regulation of βAR/GPCR function via receptor-stimulated S-nitrosylation, which plays a central but largely unappreciated role in controlling myocardial function. Our data provides novel insight into consequences of the nitroso-redox imbalance in failing heart. The Central Hypothesis of this Multi-PI proposal is that cardiac βAR signaling and desensitization via GRK2 and β-Arr2 are regulated by S-nitrosylation and that nitroso-redox stress can be understood in terms of altered SNO of receptor, GRK and β-Arr to significantly impair the heart’s response to injury. Specific Aims are: [1] To determine whether GRK2 inhibition via S-nitrosylation plays a mechanistic role in selective βAR responses during cardiac ischemic injury; [2] To determine if β2AR is S-nitrosylated in the ischemic heart and whether this impacts injury and repair; [3] To determine if regulation of β-Arr2 by S-nitrosylation tunes βAR responses during cardiac dysfunction after injury and is integrated with β2AR and GRK2 SNO regulation. Successful completion of these studies will illuminate the role of S-nitrosylation in the integrated adrenergic response to cardiac injury and repair. These fundamental discoveries will reveal new insights into the regulation of cardiac function in health and disease by defining therapeutic interventions to promote cardioprotection, and to serve as a paradigm for signaling systems in other organs and diseases.