Abstract 18646: The Dynamic Relationship Between GRK2 and NOS/NO Critically Determines Cardiac Ischemia/Reperfusion Injury
Background and Aims: G protein-coupled receptor kinase 2 (GRK2) and endothelial nitric oxide synthase (eNOS) play key roles in cardiac signaling and function. Increased GRK2, seen in injured myocardium, causes increased cell death and injury after ischemia while reciprocally, eNOS/NO exerts protection. A link between these two systems occurs as GRK2 can be regulated via S-nitrosylation (SNO) at Cys340, which inhibits its activity. Alternatively, increased GRK2 activity inhibits NO in the heart. The aim of this study was to determine whether there is a dynamic interaction between cardiac GRK2 and eNOS/NO.
Methods: We investigated a direct interaction between GRK2 and eNOS in cultured myocytes and hearts. Cardiac-specific transgenic mice overexpressing GRK2 (Tg-GRK2) and mice with expression of the GRK2 inhibitor βARKct (Tg-βARKct) were used to create hybrid mice with concurrent modulation of eNOS. Finally, we created novel knock-in mice where all endogenous GRK2 was a mutant form (Cys340Ser) resistant to SNO regulation. All mice were characterized basally and subjected to ischemia/reperfusion (I/R) injury and infarct size and cardiac phenotypes determined.
Results: 1). Co-immunoprecipitation discovered an interaction between GRK2 and eNOS in cardiac tissue, which was significantly increased in the ischemic heart. 2). TgGRK2/TgeNOS hybrid mice showed significantly reduced infarct size and improved function post I/R compared to Tg-GRK2 mice. Protection offered by βARKct was reversed in mice without eNOS. 3). SNO-GRK2 was increased by isoproterenol and GSNO in WT mice but was not observed in GRK2-C340S mice. 4). Interestingly, GRK2-C340S mice exhibited significantly larger injury post I/R compared to WT mice with decreased cardiac dysfunction. GSNO treatment reduced infarct size in WT mice but had no effects in GRK2-C340S mice. Also, pre-treatment with the NOS inhibitor L-NAME increased infarct size in WT mice but not in GRK2-C340S mice.
Conclusions: Our data demonstrates a unique relationship between GRK2 and NO signaling in the heart, which has direct implications for mechanisms of cardiac injury. This includes the demonstration that GRK2 is a nodal pro-death kinase after I/R injury and its SNO is a requirement for NO-mediated cardioprotection.
- © 2012 by American Heart Association, Inc.