Rawnsley David R, Islam Moydul, Zhao Chen, Kargar Gaz Kooh Yasaman, Mendoza Adelita, Navid Honora, Kumari Minu, Guan Xumin, Murphy John T, Nigro Jess, Kovacs Attila, Mani Kartik, Huebsch Nathaniel, Ma Xiucui, Diwan Abhinav
bioRxiv. 2024 Nov 26:2024.11.24.624947. doi: 10.1101/2024.11.24.624947.
Protein quality control (PQC) is critical for maintaining sarcomere structure and function in cardiac myocytes, and mutations in PQC pathway proteins, such as CRYAB (arginine to glycine at position 120, R120G) and BAG3 (proline to lysine at position 209, P209L) induce protein aggregate pathology with cardiomyopathy in humans. Novel observations in yeast and mammalian cells demonstrate mitochondrial uptake of cytosolic protein aggregates. We hypothesized that mitochondrial uptake of cytosolic protein aggregates and their removal by mitophagy, a lysosomal degradative pathway essential for myocardial homeostasis, facilitates cytosolic protein quality control in cardiac myocytes.
Mice with inducible cardiac myocyte specific ablation of TRAF2 (TRAF2icKO), which impairs mitophagy, were assessed for protein aggregates with biochemical fractionation and super-resolution imaging in comparison to floxed controls. Induced pluripotent stem cell (iPSC)-derived cardiac myocytes with R120G knock-in to the locus were assessed for localization of the CRYAB protein. Transgenic mice expressing R120G CRYAB protein (R120G-TG) were subjected to both TRAF2 gain-of-function (with AAV9-cardiac Troponin T promoter-driven TRAF2 transduction) and TRAF2 loss-of-function (with tamoxifen-inducible ablation of one allele) in cardiac myocytes to determine the effect of mitophagy modulation on cardiac structure, function, and protein aggregate pathology.
Cardiomyocyte-specific ablation of TRAF2 results accumulation of mitochondrial and cytosolic protein aggregates and DESMIN mis-localization to protein aggregates. Isolated mitochondria take up cardiomyopathy-associated aggregate-prone cytosolic chaperone proteins, namely arginine to glycine (R120G) CRYAB mutant and proline to lysine (P209L) BAG3 mutant. R120G-CRYAB mutant protein increasingly localizes to mitochondria in human and mouse cardiomyocytes. R120G-TG mice demonstrate upregulation of TRAF2 in the mitochondrial fraction with increased mitophagy as compared with wild type. Adult-onset inducible haplo-insufficiency of TRAF2 resulted in accelerated mortality, impaired left ventricular systolic function and increased protein aggregates in R120G-TG mice as compared with controls. Conversely, AAV9-mediated TRAF2 transduction in R120G-TG mice reduced mortality and attenuated left ventricular systolic dysfunction, with reduced protein aggregates and restoration of normal localization of DESMIN, a cytosolic scaffolding protein chaperoned by CRYAB, as compared with control AAV9-GFP group.
TRAF2-mediated mitophagy in cardiac myocytes facilitates removal of cytosolic protein aggregates and can be stimulated to ameliorate proteotoxic cardiomyopathy.
蛋白质质量控制（PQC）对于维持心肌细胞中的肌节结构和功能至关重要，PQC途径蛋白的突变，如CRYAB（第120位精氨酸突变为甘氨酸，R120G）和BAG3（第209位脯氨酸突变为赖氨酸，P209L），会在人类中诱发蛋白质聚集病理并导致心肌病。酵母和哺乳动物细胞中的新观察结果表明，线粒体可摄取胞质蛋白聚集体。我们推测，线粒体对胞质蛋白聚集体的摄取及其通过线粒体自噬（一种对心肌稳态至关重要的溶酶体降解途径）的清除，有助于心肌细胞中的胞质蛋白质质量控制。
与对照小鼠相比，通过生物化学分级分离和超分辨率成像评估了可诱导心肌细胞特异性敲除TRAF2（TRAF2icKO）的小鼠的蛋白质聚集体，TRAF2的敲除会损害线粒体自噬。对在 位点敲入R120G的诱导多能干细胞（iPSC）衍生的心肌细胞进行CRYAB蛋白定位评估。对表达R120G CRYAB蛋白的转基因小鼠（R120G-TG）的心肌细胞进行TRAF2功能增强（通过AAV9-心肌肌钙蛋白T启动子驱动的TRAF2转导）和TRAF2功能丧失（通过他莫昔芬诱导敲除一个 等位基因）处理，以确定线粒体自噬调节对心脏结构、功能和蛋白质聚集病理的影响。
TRAF2的心肌细胞特异性敲除导致线粒体和胞质蛋白聚集体的积累以及结蛋白错误定位于蛋白聚集体。分离的线粒体摄取与心肌病相关的易聚集胞质伴侣蛋白，即精氨酸突变为甘氨酸（R120G）的CRYAB突变体和脯氨酸突变为赖氨酸（P209L）的BAG3突变体。R120G-CRYAB突变蛋白在人和小鼠心肌细胞中越来越多地定位于线粒体。与野生型相比，R120G-TG小鼠线粒体部分的TRAF2上调，线粒体自噬增加。与对照组相比，成年期诱导的TRAF2单倍体不足导致R120G-TG小鼠死亡率加速、左心室收缩功能受损和蛋白质聚集体增加。相反，与对照AAV9-GFP组相比，R120G-TG小鼠中AAV9介导的TRAF2转导降低了死亡率，减轻了左心室收缩功能障碍，减少了蛋白质聚集体，并恢复了由CRYAB伴侣蛋白介导的胞质支架蛋白结蛋白的正常定位。
心肌细胞中TRAF2介导的线粒体自噬有助于清除胞质蛋白聚集体，并可被刺激以改善蛋白毒性心肌病。
