G Protein-Coupled Receptor Kinase 2 (GRK2) is a culprit in the loss of cardiac contractile function in heart failure due to β-Adrenoceptor (AR) desensitization after its upregulation. Indeed, its inhibition has been demonstrated to improve cardiac function and increased GRK2 in the heart leads to larger injury after an ischemic insult. Nitric oxide (NO) via S-nitrosothiol (SNO) at residue Cys340 is a reported endogenous inhibitor of GRK2 activity. ß3ARs, on the other hand, are known to be resistant to desensitization by GRK2 and they are upregulated in cardiac pathologies. Activation of ß3ARs can be cardioprotective via NO signalling. Thus, in the present study, we aimed to investigate the interaction between ß3ARs and GRK2 through NO signaling to determine if ß3AR cardioprotection can occur via NO-mediated GRK2 inhibition. We used wild type C57BL/6 mice (WT), global β3AR knockout (KO) mice and GRK2-C340S knockin mice, which harbor a point mutation that changes Cys340 with a serine, meaning all endogenous GRK2 globally cannot be inhibited via NO-mediated S-nitrosylation. We exposed WT, GRK2-C340s KI and β3AR KO mice to ischemia/reperfusion (I/R) injury (40 min ischemia followed by 24 hrs reperfusion). We found that WT mice had significantly diminished cardiac function evaluated by echocardiography and Millar Catheterization, and this was rescued by treating these mice with CL316,243 (a selective β3AR agonist) at the time of reperfusion. On the other hand, GRK2-C340S KI mice did worse after I/R injury compared to WT mice and CL316,243 did not rescue this dysfunction as it did in WT mice. As expected, β3AR KO mice had worsened cardiac function compared to WT mice and CL316,243 had no functional benefit. Infarct size measurements after I/R revealed that β3AR KO mice had larger infarcts than WT mice supporting β3ARs as being protective. Indeed, CL316,243 induced robust cardioprotection in WT mice, reducing infarct size. GRK2-C340S mice had larger infarcts than WT mice and CL316,243 failed to offer any cardioprotection. Thus, β3AR-mediated cardioprotection clearly involves inhibition of GRK2 as part of its therapeutic mechanism and this appears to involve NO-mediated S-nitrosylation.
Cloning, Characterization, and Expression of a G-Protein-Coupled Receptor fromLymnaea stagnalisand Identification of a Leucokinin-Like Peptide, PSFHSWSamide, as Its Endogenous Ligand
