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

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 tri...

Source: Autophagy - July 5, 2023 Category: Cytology Authors: Gustav Godtliebsen Kenneth Bowitz Larsen Zambarlal Bhujabal Ida S Opstad Mireia Nager Abhinanda R Punnakkal Trine B Kalstad Randi Olsen Trine Lund Dilip K Prasad Krishna Agarwal Truls Myrmel Asa Birna Birgisdottir Source Type: research