Purpose: The heart is one of the most energy consuming organs. This energy is used to maintain proper contractile function and is produced mainly in the mitochondria by oxidative phosphorylation (OXPHOS). The production of reactive oxygen species (ROS) is an unavoidable byproduct of OXPHOS. Increased ROS production has detrimental effects on the cells, inducing DNA damage and apoptosis. EndonucleaseG-like-1 (EXOG) and EndonucleaseG are mitochondrial endo/exonucleases with poorly investigated functions in the heart. Whereas EXOG has been implicated in mitochondrial DNA repair, EndonucleaseG appears to play a role in apoptosis, but a recent study in cardiomyocytes showed its importance in mitochondrial function and cardiac hypertrophy. Whether EXOG has additional functions as well, is not clear, but it is interesting to note that EXOG is present in a human genomic locus linked to cardiovascular disease. The goal of this study was to elucidate the role of EXOG in mitochondrial function in cardiomyocytes.
Methods: EXOG adenoviral mediated knock-down was performed in neonatal rat cardiomyocytes. The Seahorse XF24 Extracellular Flux Analyzer was used to measure bio-energetic functions, including OCR (oxygen consumption rate). Mitochondrial parameters including citrate synthesis, mtDNA amount, mtDNA damage and reactive oxygen species (ROS) production were determined. Hypertrophy was assessed by 3H-leucine incorporation and cell surface measurements.
Results: Knock-down of EXOG did not induce mtDNA damage in neonatal cardiomyocytes. Mitochondrial respiration was improved and revealed a 2.4 fold increase in basal mitochondrial OCR (n=6, P< 0.05). Moreover, the ATP-linked OCR was 5.2 fold higher. Western blotting did not showed changes in components of the OXPHOS complex and no changes in mitochondrial biogenesis were observed, suggesting that specific activities in the mitochondria are modulated. Also the membrane potential was not altered, but mitochondrial specific ROS production was strongly increased (5.4 fold) indicating that proper electron transport chain (ETC) flux was impaired. These changes were accompanied by an increase in cellular growth. Interestingly, this hypertrophic response could be attenuated by specific mitochondrial ROS scavengers.
Conclusion: In cardiomyocytes EXOG silencing did not have a direct effect on mitochondrial DNA integrity. However, absence of EXOG increased mitochondrial activity and ROS production, which could be linked to cardiomyocyte hypertrophy.