Mechanical stress-induced pathomechanisms in BAG3-associated myopathies

辅伴蛋白BAG3(Bcl2-Association athanogene 3)在横纹肌中有较强的表达，在机械应激下维持肌肉的结构和功能方面起着关键作用，因为它能够平衡转录、翻译和自噬。BAG3基因第209位的脯氨酸到亮氨酸的替代导致患者出现严重的进行性限制性心肌病和骨骼肌营养不良，随后死于心力衰竭或呼吸功能不全。这种与BAG3相关的肌原纤维肌病的特征是Z-Disc崩解和蛋白质聚集。在第一个资助期，我们完成了我们的人源化BAG3P209L过表达小鼠的彻底分析，这些小鼠患有限制性心肌病和骨骼肌肌原纤维肌病，从而概括了人类疾病的表型。我们能够证明该病的机制是由于BAG3的积累和蛋白质质量控制系统和自噬机制的组件的隔离导致Z-Disc组件的稳定性和功能组织的降低。目前报道的BAG3P209L-肌纤维性肌病患者中存在很大程度的异质性。因此，目前尚不清楚哪些因素影响危及生命的心肌和骨骼肌表型的发展，以及心脏表型是否影响骨骼肌表型以及如何影响骨骼肌表型，反之亦然。因此，在第二个资助期，我们将使用心脏和骨骼肌特异性诱导hBAG3P209L-表达的小鼠系来分析骨骼肌表型是否会影响心脏表型，反之亦然，以及hBAG3P209L表达的开始如何影响心肌/骨骼肌表型的发育。此外，我们将使表达心脏和骨骼肌特异性hBAG3P209L的小鼠在跑步机上受到运动诱导的机械应激，并研究心肌和骨骼肌细胞中聚集体的形成和肌节结构的扭曲。我们将进一步在我们的hBAG3P209L小鼠模型中测试BAG3P209L-肌纤维肌病的治疗方法，使用腺相关病毒(AAVs)通过CRISPR/Cas9敲除hBAG3P209L或通过RNA干扰敲除hBAG3P209L。为了了解与BAG3相关的肌肉疾病的全谱，我们将分析带有BAG3P209L突变的患者来源或基因组编辑的iPS细胞系，将它们分化为心肌细胞和骨骼肌管，并对其表型、特性进行表征并测试治疗这种疾病的方法。这项拟议的工作旨在阐明机械应力是否以及在多大程度上导致BAG3P209L-肌纤维肌病的病理，以及基因治疗方法是否适合于这种致命疾病的治疗。

The cochaperone BAG3 (Bcl-2 associated athanogene 3) is strongly expressed in cross striated muscles and plays a key role in maintaining muscle architecture and function under mechanical stress owing to its ability to balance transcription, translation, and autophagy. The proline to leucine substitution at position 209 within BAG3 leads to severe and progressively restrictive cardiomyopathy and skeletal muscle dystrophy in patients, followed by death due to heart failure or respiratory insufficiency. Hallmarks of this BAG3-associated myofibrillar myopathy are Z-disc disintegration and protein aggregation. In the first funding period, we finished the thorough analysis of our humanized BAG3P209L overexpressing mice, which suffer from restrictive cardiomyopathy and skeletal muscle myofibrillar myopathy, thereby recapitulating the human disease phenotype. We were able to demonstrate that the disease mechanism is due to the accumulation of BAG3 and sequestering of components of the protein quality control system and autophagy machinery leading to a reduction of the stability and functional organization of Z-disc components. In patients suffering from BAG3P209L-myofibrillar myopathy reported so far there is a large degree of heterogeneity. Accordingly, it is not clear which factors influence the development of the life-threatening cardiac and skeletal muscle phenotype and whether and how the cardiac phenotype influences the skeletal muscle phenotype and vice versa. Therefore, in the second funding period, we will use inducible mouse lines for heart and skeletal muscle-specific induction of hBAG3P209L-expression to analyze if and how the skeletal muscle phenotype influences the cardiac phenotype and vice versa and how the onset of hBAG3P209L expression influences the development of the cardiac/skeletal muscle phenotype. Furthermore, we will subject heart and skeletal muscle-specific hBAG3P209L-expressing mice to mechanical stress induced by physical exercise on a treadmill and investigate the formation of aggregates and the distortion of sarcomeric structures in cardiac and skeletal muscle cells.We will further test therapeutic approaches for BAG3P209L-myofibrillar myopathy in our hBAG3P209L mouse model by using adeno-associated viruses (AAVs) to either knockout hBAG3P209L by CRISPR/Cas9 or knockdown hBAG3P209L by RNA-interference.To understand the full spectrum of BAG3-related muscular diseases we will analyze patient-derived or genome-edited human iPS cell lines with the BAG3P209L mutation, differentiate them to cardiomyocytes and skeletal myotubes, characterize their phenotype, and test therapeutic approaches to treat the disease. The proposed work aims to clarify whether and to what extent mechanical stress contributes to the pathology of BAG3P209L-myofibrillar myopathy and whether gene-therapy approaches are suitable for the treatment of this lethal disease.