High-resolution visualization and assessment of basal and OXPHOS-induced mitophagy in H9c2 cardiomyoblasts.

ABSTRACT

Mitochondria are susceptible to damage resulting from their activity as energy providers. Damaged mitochondria can cause harm to the cell and thus mitochondria are subjected to elaborate quality-control mechanisms including elimination via lysosomal degradation in a process termed mitophagy. Basal mitophagy is a house-keeping mechanism fine-tuning the number of mitochondria according to the metabolic state of the cell. However, the molecular mechanisms underlying basal mitophagy remain largely elusive. In this study, we visualized and assessed the level of mitophagy in H9c2 cardiomyoblasts at basal conditions and after OXPHOS induction by galactose adaptation. We used cells with a stable expression of a pH-sensitive fluorescent mitochondrial reporter and applied state-of-the-art imaging techniques and image analysis. Our data showed a significant increase in acidic mitochondria after galactose adaptation. Using a machine-learning approach we also demonstrated increased mitochondrial fragmentation by OXPHOS induction. Furthermore, super-resolution microscopy of live cells enabled capturing of mitochondrial fragments within lysosomes as well as dynamic transfer of mitochondrial contents to lysosomes. Applying correlative light and electron microscopy we revealed the ultrastructure of the acidic mitochondria confirming their proximity to the mitochondrial network, ER and lysosomes. Finally, exploiting siRNA knockdown strategy combined with flux perturbation with lysosomal inhibitors, we demonstrated the importance of both canonical as well as non-canonical autophagy mediators in lysosomal degradation of mitochondria after OXPHOS induction. Taken together, our high-resolution imaging approaches applied on H9c2 cells provide novel insights on mitophagy during physiologically relevant conditions. The implication of redundant underlying mechanisms highlights the fundamental importance of mitophagy.Abbreviations ATG autophagy related; ATG7 autophagy related 7; ATP adenosine triphosphate; BafA1 bafilomycin A1; CLEM correlative light and electron microscopy; EGFP enhanced green fluorescent protein; MAP1LC3B microtubule associated protein 1 light chain 3 beta; OXPHOS oxidative phosphorylation; PepA pepstatin A; PLA proximity ligation assay; PRKN parkin RBR E3 ubiquitin protein ligase; RAB5A RAB5A, member RAS oncogene family; RAB7A RAB7A, member RAS oncogene family; RAB9A RAB9A, member RAS oncogene family; ROS reactive oxygen species; SIM structured illumination microscopy; siRNA short interfering RNA; SYNJ2BP synaptojanin 2 binding protein; TEM transmission electron microscopy; TOMM20 translocase of outer mitochondrial membrane 20; ULK1 unc-51 like kinase 1.