Abstract 4369579: Loss of G Protein-Coupled Receptor Kinase 2 S-Nitrosylation Abolishes Beta 3 Adrenergic Receptor-Mediated Cardioprotection Evidence from Functional and Proteomic Analyses

G Protein-Coupled Receptor Kinase 2 (GRK2) contributes to cardiac dysfunction through β-Adrenoceptor (βAR) desensitization following its upregulation. Inhibition of GRK2 has been shown to improve cardiac function. Nitric oxide (NO), through the formation of S-nitrosothiols (SNOs), serves as an endogenous inhibitor of GRK2 activity. β3-Adrenoceptors (β3ARs), in contrast, resist GRK2 and are upregulated in cardiac pathologies. Activation of β3ARs can confer cardioprotection via NO signaling. We hypothesized that β3AR activation confers cardioprotection through NO-dependent inhibition of GRK2, and that this protection is lost when GRK2 is resistant to S-nitrosylation. Furthermore, we proposed that proteomic profiling would reveal molecular pathways underlying this mechanism. Male C57BL/6J wild-type (WT) and GRK2-C340S knock-in (KI) mice, which carry a Cys340Ser mutation making GRK2 resistant to S-nitrosylation, underwent 40 minutes of left anterior descending coronary artery occlusion followed by 24 hours of reperfusion under isoflurane anesthesia. The β3AR agonist CL 316,243 was given at reperfusion onset. Cardiac function was evaluated by echocardiography (ejection fraction, EF; fractional shortening, FS), and infarct size was assessed by TTC staining. Heart tissues were subjected to mass spectrometry-based quantitative proteomics and pathway analysis. In WT mice, CL 316,243 significantly improved EF and FS compared to vehicle-treated controls. These effects were abolished in GRK2-C340S mice, indicating that GRK2 S-nitrosylation is necessary for β3AR-mediated cardioprotection. WT mice also had significantly smaller infarct size compared to KI mice. Proteomic analysis revealed 397 significantly differentially expressed proteins (137 downregulated, 260 upregulated) in CL-treated KI mice versus WT. Additionally, enriched pathway analysis showed an upregulation of the innate immune response (52-associated proteins) and a downregulation of lipolysis regulation (19-associated proteins). β3AR stimulation protects the heart after ischemia/reperfusion through NO-dependent S-nitrosylation of GRK2, which is essential for this effect. However, proteomic analysis revealed that CL-treated KI mice were enriched in novel pathways, including innate immunity and lipid metabolism, proposing a potential mechanism for the loss of protection and, therefore, implicating the β3AR–NO–GRK2 axis as a promising therapeutic target.

Duke Scholars

Author Rajika Roy Surgery, Cardiovascular and Thoracic Surgery