Abstract P2132: The Raf Kinase Inhibitor Protein Protects Mitochondria From Ischemic Stress

Abstract

Background: The Raf kinase inhibitor protein (RKIP) increases cardiac inotropy without detrimental remodeling and even protects from the development of heart failure in response to chronic pressure overload or catecholamines. These RKIP effects are mediated via simultaneous and persistent activation of cardiac β1- and β2-adrenergic receptors (βAR). Hypothesis: Since an intact mitochondrial function is central for the energy supply and cardiomyocyte health, which are parameters that had been proven to be positively influenced by RKIP in the heart, we here aimed to analyze the impact of RKIP and βAR signaling on mitochondria. Methods and Results: Electron microscopy, respiration measurements and confocal analysis of the mitochondrial membrane potential (mtMP) revealed that the mitochondria of RKIP knockout mice are of smaller size and abnormal shape, show a reduced ADP-stimulated respiration and a hyperpolarized mtMP compared to WT mitochondria. And, expression levels of several respiratory proteins were reduced. In contrast, mitochondrial content in RKIP-tg mouse hearts was enhanced, mitochondrial function maintained, and expression of proteins involved in respiratory complexes and mitochondria-related transcription (e.g. Tfam, NRF-1) was enhanced. To evaluate the stress resistance of RKIP-tg hearts, we subjected isolated cardiomyocytes or hearts of RKIP-tg or WT, respectively, to ischemia/reperfusion (I/R) stress. I/R effectively decreased the mtMP in WT cardiomyocytes and impaired ADP-stimulated respiration of isolated WT mitochondria after ex vivo I/R. In contrast, RKIP-tg mitochondria were protected from mtMP collapse and impaired respiration after I/R. To understand this protective RKIP-mediated effect and whether βAR are involved, we analyzed mtMP in neonatal cardiomyocytes of β2AR knockout mice, upon application of different βAR ligands, pertussis toxin and inhibitors of the PI3K or AKT. These experiments suggest a RKIP-mediated mitochondrial protection via β2AR/Gi/PI3Kα/pAKT signaling. Discussion: Our study shows that RKIP-βAR signaling protects mitochondria from I/R stress suggesting βAR modulation as a potential therapeutic strategy to protect the myocardium in ischemic conditions.