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

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. <b>Abbreviations:</b> 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.

线粒体作为能量供给者，其活性状态下极易受到损伤。受损线粒体会对细胞造成损害，因此细胞演化出精密的质量控制机制，其中包括通过溶酶体降解清除受损线粒体，这一过程被称为线粒体自噬（mitophagy）。基础线粒体自噬是一种持家机制，可根据细胞的代谢状态微调线粒体的数量。然而，目前关于基础线粒体自噬的分子机制仍在很大程度上尚不明确。
本研究可视化并评估了基础状态下，以及经半乳糖适应诱导氧化磷酸化（OXPHOS）后，H9c2心肌成肌细胞中的线粒体自噬水平。我们使用了稳定表达pH敏感型荧光线粒体报告基因的细胞，并应用了前沿成像技术与图像分析方法。我们的数据显示，半乳糖适应后酸性线粒体的数量显著增加。通过机器学习方法，我们还证实氧化磷酸化诱导会加剧线粒体碎片化。
此外，活细胞超分辨率显微镜技术成功捕捉到了溶酶体内的线粒体片段，以及线粒体内容物向溶酶体的动态转运过程。通过关联光镜与电镜（CLEM）技术，我们揭示了酸性线粒体的超微结构，证实其与线粒体网络、内质网（Endoplasmic Reticulum, ER）以及溶酶体紧密邻近。
最后，我们结合小干扰RNA（siRNA）敲低策略与溶酶体抑制剂介导的通量扰动实验，证实了经典与非经典自噬介导因子在氧化磷酸化诱导后的线粒体溶酶体降解过程中均发挥重要作用。
综上，我们应用于H9c2细胞的高分辨率成像方法，为生理相关条件下的线粒体自噬研究提供了全新的见解。其冗余的潜在机制凸显了线粒体自噬的核心重要性。

<b>缩写说明:</b> ATG：自噬相关基因（autophagy related）；ATG7：自噬相关基因7（autophagy related 7）；ATP：三磷酸腺苷（adenosine triphosphate）；BafA1：巴弗洛霉素A₁（bafilomycin A₁）；CLEM：关联光电子显微镜（correlative light and electron microscopy）；EGFP：增强型绿色荧光蛋白（enhanced green fluorescent protein）；MAP1LC3B：微管相关蛋白1轻链3β（microtubule associated protein 1 light chain 3 beta）；OXPHOS：氧化磷酸化（oxidative phosphorylation）；PepA：胃酶抑素A（pepstatin A）；PLA：邻近连接测定法（proximity ligation assay）；PRKN：帕金RBR E3泛素蛋白连接酶（parkin RBR E3 ubiquitin protein ligase）；RAB5A：RAS癌基因家族成员RAB5A（RAB5A, member RAS oncogene family）；RAB7A：RAS癌基因家族成员RAB7A（RAB7A, member RAS oncogene family）；RAB9A：RAS癌基因家族成员RAB9A（RAB9A, member RAS oncogene family）；ROS：活性氧（reactive oxygen species）；SIM：结构照明显微镜（structured illumination microscopy）；siRNA：小干扰RNA（short interfering RNA）；SYNJ2BP：突触多聚磷酸肌醇磷酸酶2结合蛋白（synaptojanin 2 binding protein）；TEM：透射电子显微镜（transmission electron microscopy）；TOMM20：线粒体外膜转位酶20（translocase of outer mitochondrial membrane 20）；ULK1：unc-51样激酶1（unc-51 like kinase 1）。