Abstract 208: Mitochondrial-targeted Grk2 Increases Superoxide Formation And Impairs Cardiomyocyte Respiratory Reserve Capacity

Abstract

β-adrenergic receptors (βARs) are powerful regulators of cardiovascular function and are impaired in heart failure (HF). Signal transduction of βARs is canonically shut down by phosphorylation via G protein-coupled receptor kinase 2 (GRK2) and the subsequent binding of β-arrestins. This process of receptor desensitization is enhanced in HF via the up-regulation of GRK2 and contributes to disease progression. We have recently reported non-canonical actions of GRK2, which contribute to the development of HF independent of βAR desensitization. We have previously shown that GRK2 can act as a pro-death kinase in cardiomyocytes bytranslocating to mitochondria and activating mitochondria permeability transition. This study was designed to gain more understanding of the mitochondrial function of GRK2. We isolated adult cardiomyocytes from cardiac-specific transgenic mice overexpressing GRK2 at levels found in human HF (TgGRK2), and examined superoxide production using the redox sensitive reporter MitoSox Red. Confocal imaging revealed a 4.6 fold increase in superoxide levels in cardiomyocytes overexpressing Grk2 as compared to non-transgenic (NLC) cardiomyocytes (corrected total cell fluorescence 11.59±1.06, TgGRK2 (n= 3 hearts, 88 cells) vs 2.54±0.02 NLC (n=3 hearts, 52 cells), (p<0.001). This indicates that the chronic elevation of GRK2 induces mitochondrial oxidative stress priming the myocyte for enhanced injury. To further explore the mitochondrial actions of GRK2 and consequences of redox stress we examined oxidative phosphorylation by performing oxygen consumption measurements in neonatal rat ventricular myocytes overexpressing GRK2 or GFP-expressing control myocytes. Seahorse analysis showed that cells overexpressing GRK2 have a significant decrease in spare respiratory capacity indicating that cells with elevated GRK2 levels have an impaired capacity to generated ATP during times of stress. Further studies with mutants that limit GRK2 kinase activity or mitochondrial localization demonstrate that mitochondrial GRK2 may be a significant contributor to cellular dysfunction as seen in heart failure.