Mechanical stress-induced pathomechanisms in BAG3-associated myopathies

Cochaperone BAG 3（Bcl-2 associated athanogene 3）在横纹肌中强烈表达，并且由于其平衡转录、翻译和自噬的能力而在机械应力下维持肌肉结构和功能中起关键作用。在BAG 3内的位置209处的脯氨酸至亮氨酸的取代导致患者的严重和进行性限制性心肌病和骨骼肌营养不良，随后由于心力衰竭或呼吸功能不全而死亡。这种BAG 3相关肌原纤维肌病的特征是Z盘崩解和蛋白质聚集。在第一个资助期内，我们完成了对人源化BAG 3 P209 L过表达小鼠的全面分析，这些小鼠患有限制性心肌病和骨骼肌肌原纤维肌病，从而重现了人类疾病表型。我们能够证明疾病机制是由于BAG 3的积累和蛋白质质量控制系统和自噬机制的组分的隔离导致Z盘组分的稳定性和功能组织的降低。在迄今为止报道的患有BAG 3 P209 L-肌原纤维肌病的患者中，存在很大程度的异质性。因此，尚不清楚哪些因素影响危及生命的心脏和骨骼肌表型的发展，以及心脏表型是否和如何影响骨骼肌表型，反之亦然。因此，在第二个资助期，我们将使用诱导型小鼠系进行hBAG 3 P209 L表达的心脏和骨骼肌特异性诱导，以分析骨骼肌表型是否以及如何影响心脏表型，反之亦然，以及hBAG 3 P209 L表达的开始如何影响心脏/骨骼肌表型的发育。此外，委员会认为，我们将对心脏和骨骼肌特异性表达hBAG 3 P209 L的小鼠进行跑步机运动诱导的机械应激，并研究心脏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hBAG 3 P209 L小鼠模型中使用腺相关病毒（AAV）测试BAG 3 P209 L-肌原纤维肌病的治疗方法。为了了解BAG 3相关肌肉疾病的全谱，我们将分析具有BAG 3 P209 L突变的患者来源的或基因组编辑的人iPS细胞系，将它们分化为心肌细胞和骨骼肌管，表征它们的表型，并测试治疗疾病的治疗方法。这项工作旨在阐明机械应力是否以及在多大程度上有助于BAG 3 P209 L-肌原纤维肌病的病理学，以及基因治疗方法是否适合治疗这种致命疾病。

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.