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 t...