In the heart, the Raf kinase inhibitor protein (RKIP) acts as a regulator of β-adrenergic receptor (βAR) signaling: cardiac RKIP overexpression increases the inotropy of mouse hearts without detrimental remodeling. To investigate how the chronically increased energy demand of RKIP mouse hearts is covered, we analyzed the impact of RKIP on mitochondria, the main energy producers of cardiomyocytes, using mice with cardiac overexpression of RKIP (RKIP-tg) and RKIP knockout mice (RKIP-KO).

To analyze mitochondrial function and morphology, we used electron microscopy images of myocardial sections, respiration measurements of isolated cardiac mitochondria and confocal analysis of the mitochondrial membrane potential (mtMP) of isolated adult cardiomyocytes stained with the mtMP-sensitive dye tetramethylrhodamine (TMRM). Compared to wildtype (WT) mitochondria, the mitochondria of RKIP-KO mice revealed severe alterations such as smaller size and abnormal shape, reduced ADP-stimulated respiration (77.58% ± 7.02% of WT) and hyperpolarization of the mtMP (136.6% ± 11,9% of WT). Moreover, the expression level of several respiratory proteins was reduced. In contrast, mitochondrial content in RKIP-tg mouse hearts was enhanced and mitochondrial function maintained (ADP-stimulated respiration: 101.1% ± 5.76% of WT; mtMP of RKIP-tg cardiomyocytes: 92.3% ± 5.93% of WT). In addition, the expression of proteins involved in respiratory complexes and mitochondria-related transcription (e.g. Tfam, NRF-1) was enhanced in RKIP-tg heart lysates. To test, if the changes in RKIP-tg mice on the cardiac mitochondrial level led to an increased stress resistance, 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 to 67.15% ± 3.17% compared to basal condition and impaired ADP-stimulated respiration of mitochondria isolated from WT hearts after I/R to 69.90% ± 6.61% compared to baseline condition. Mitochondria of RKIP-tg, in contrast, were protected under these conditions, i.e. mtMP remained stable after I/R (111,7% ±7.57% compared to baseline condition) as well as the ADP-stimulated respiration (101,3% ± 11,35% compared to baseline conditions). To understand this protective RKIP-mediated effect and whether βAR signaling was involved, we used neonatal cardiomyocytes of β2AR knockout mice, different βAR ligands, pertussis toxin and inhibitors of the PI3Kinase and AKT, and the mtMP as functional readout. These experiments suggest that RKIP mediates its mitochondrial protection via the β2AR/Gi/PI3Kα/pAKT signaling pathway.

Our study suggests that βAR can protect mitochondria from I/R stress in the presence of RKIP. Further insights in the modulation of βAR signaling may help to find a therapeutic strategy to protect mitochondria and thus the myocardium in ischemia.