Sun Shiqun, Yu Wenjun, Xu Haixia, Li Congye, Zou Rongjun, Wu Ne N, Wang Li, Ge Junbo, Ren Jun, Zhang Yingmei
Department of Cardiology and Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
Department of Cardiology and Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai 200032, China; Department of Cardiovascular Surgery, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, China.
Metabolism. 2022 Sep;134:155239. doi: 10.1016/j.metabol.2022.155239. Epub 2022 Jun 6.
Mitochondria are essential for myocardial ischemia/reperfusion (I/R) injury. TBC domain family member 15 (TBC1D15) participates in the regulation of mitochondrial homeostasis although its role remains elusive in I/R injury.
This study examined the role of TBC1D15 in mitochondrial homeostasis under myocardial I/R injury using inducible cardiac-specific TBC1D15 knockin (TBC1D15) and knockout (TBC1D15) mice.
TBC1D15 mRNA/protein levels were downregulated in human ischemic cardiomyopathy samples, mouse I/R hearts and neonatal mouse cardiomyocytes with H/R injury, consistent with scRNA sequencing finding from patients with coronary heart disease. Cardiac-specific knockin of TBC1D15 attenuated whereas cardiac-specific knockout of TBC1D15 overtly aggravated I/R-induced cardiomyocyte apoptosis and cardiac dysfunction. TBC1D15 mice exhibited reduced mitochondrial damage and mitochondrial fragmentation following myocardial I/R injury, while TBC1D15 mice displayed opposite results. TBC1D15 preserved mitochondrial function evidenced by safeguarding MMP and oxygen consumption capacity, antagonizing ROS accumulation and cytochrome C release, which were nullified by TBC1D15 knockdown. Time-lapse confocal microscopy revealed that TBC1D15 activated asymmetrical mitochondrial fission through promoting mitochondria-lysosome contacts untethering in NMCMs under H/R injury, whereas overexpression of TBC1D15 mutants (R400K and ∆231-240) failed to regulate asymmetrical fission and knockdown of TBC1D15 slowed down asymmetrical fission. Moreover, TBC1D15-offered benefits were mitigated by knockdown of Fis1 and Drp1. Mechanistically, TBC1D15 recruited Drp1 to mitochondria-lysosome contact sites via direct interaction with Drp1 through its C terminus (574-624) domain. Interfering with interaction between TBC1D15 and Drp1 abrogated asymmetrical mitochondrial fission and mitochondrial function. Cardiac phenotypes of TBC1D15 mice upon I/R injury were rescued by adenovirus-mediated overexpression of wild-type but not mutants (R400K, ∆231-240 and ∆574-624) TBC1D15.
TBC1D15 ameliorated I/R injury through a novel modality to preserve mitochondrial homeostasis where mitochondria-lysosome contacts (through the TBC1D15/Fis1/RAB7 cascade) regulate asymmetrical mitochondrial fission (TBC1D15/Drp1 interaction), suggesting promises of targeting TBC1D15 in the management of myocardial I/R injury.
线粒体对心肌缺血/再灌注（I/R）损伤至关重要。TBC结构域家族成员15（TBC1D15）参与线粒体稳态的调节，但其在I/R损伤中的作用仍不明确。
本研究使用诱导型心脏特异性TBC1D15基因敲入（TBC1D15）和基因敲除（TBC1D15）小鼠，研究TBC1D15在心肌I/R损伤下线粒体稳态中的作用。
在人类缺血性心肌病样本、小鼠I/R心脏以及遭受缺氧/复氧（H/R）损伤的新生小鼠心肌细胞中，TBC1D15 mRNA/蛋白水平下调，这与冠心病患者的单细胞RNA测序结果一致。心脏特异性敲入TBC1D15可减轻损伤，而心脏特异性敲除TBC1D15则明显加重I/R诱导的心肌细胞凋亡和心脏功能障碍。TBC1D15小鼠在心肌I/R损伤后线粒体损伤和线粒体碎片化减少，而TBC1D15小鼠则呈现相反结果。TBC1D15通过维持线粒体膜电位（MMP）和氧消耗能力、对抗活性氧（ROS）积累和细胞色素C释放来保留线粒体功能，而TBC1D15基因敲低则消除了这些作用。延时共聚焦显微镜显示，在H/R损伤下，TBC1D15通过促进新生小鼠心肌细胞（NMCMs）中线粒体-溶酶体接触解聚来激活不对称线粒体分裂，而TBC1D15突变体（R400K和∆231-240）的过表达未能调节不对称分裂，TBC1D15基因敲低则减缓了不对称分裂。此外，Fis1和Drp1基因敲低减轻了TBC1D15带来的益处。机制上，TBC1D15通过其C末端（574-624）结构域与Drp1直接相互作用，将Drp1招募到线粒体-溶酶体接触位点。干扰TBC1D15与Drp1之间的相互作用可消除不对称线粒体分裂和线粒体功能。腺病毒介导的野生型而非突变体（R400K、∆231-240和∆574-624）TBC1D15过表达挽救了TBC1D15小鼠在I/R损伤后的心脏表型。
TBC1D15通过一种新的方式改善I/R损伤，以维持线粒体稳态，即线粒体-溶酶体接触（通过TBC1D15/Fis1/RAB7级联反应）调节不对称线粒体分裂（TBC1D15/Drp1相互作用），这表明靶向TBC1D15在心肌I/R损伤管理中具有前景。
