ABSTRACT

Dominant de novo mutations in the co-chaperone BAG3 cause a severe form of myofibrillar myopathy, exhibiting progressive muscle weakness, muscle structural failure, and protein aggregation. To elucidate the mechanism of disease in, and identify therapies for, BAG3 myofibrillar myopathy, we generated two zebrafish models, one conditionally expressing BAG3^P209L and one with a nonsense mutation in bag3. While transgenic BAG3^P209L-expressing fish display protein aggregation, modeling the early phase of the disease, bag3^-/- fish exhibit exercise dependent fiber disintegration, and reduced swimming activity, consistent with later stages of the disease. Detailed characterization of the bag3^-/- fish, revealed an impairment in macroautophagic/autophagic activity, a defect we confirmed in BAG3 patient samples. Taken together, our data highlights that while BAG3^P209L expression is sufficient to promote protein aggregation, it is the loss of BAG3 due to its sequestration within aggregates, which results in impaired autophagic activity, and subsequent muscle weakness. We therefore screened autophagy-promoting compounds for their effectiveness at removing protein aggregates, identifying nine including metformin. Further evaluation demonstrated metformin is not only able to bring about the removal of protein aggregates in zebrafish and human myoblasts but is also able to rescue the fiber disintegration and swimming deficit observed in the bag3^−/- fish. Therefore, repurposing metformin provides a promising therapy for BAG3 myopathy.

Abbreviations:ACTN: actinin, alpha; BAG3: BAG cochaperone 3; CRYAB: crystallin alpha B; DES: desmin; DMSO: dimethyl sulfoxide; DNAJB6: DnaJ heat shock protein family (Hsp40) member B6; dpf: days post fertilization; eGFP: enhanced green fluorescent protein; FDA: Food and Drug Administration; FHL1: four and a half LIM domains 1; FLNC: filamin C; hpf: hours post-fertilization; HSPB8: heat shock protein family B [small] member 8; LDB3/ZASP: LIM domain binding 3; MYOT: myotilin; TTN: titin; WT: wild-type.

摘要

辅助伴侣BAG3中的主要从头突变导致严重的肌原纤维肌病，表现出进行性肌肉无力、肌肉结构衰竭和蛋白质聚集。为了阐明 BAG3 肌原纤维肌病的疾病机制并确定治疗方法，我们生成了两种斑马鱼模型，一种有条件地表达 BAG3 ^{P209L ，另一种在}bag3中具有无义突变。虽然表达转基因 BAG3 ^P209L的鱼表现出蛋白质聚集，模拟疾病的早期阶段，但bag3 ^-/-鱼表现出运动依赖的纤维分解和游泳活动减少，这与疾病的后期阶段一致。的详细表征bag3 ^-/-鱼，显示巨自噬/自噬活性受损，这是我们在BAG3患者样本中证实的缺陷。总之，我们的数据强调，虽然 BAG3 ^{P209L 的}表达足以促进蛋白质聚集，但由于 BAG3 被隔离在聚集体中而导致 BAG3 的损失，这导致自噬活性受损，以及随后的肌肉无力。因此，我们筛选了促进自噬的化合物在去除蛋白质聚集体方面的有效性，确定了九种包括二甲双胍在内的化合物。进一步的评估表明，二甲双胍不仅能够去除斑马鱼和人类成肌细胞中的蛋白质聚集体，而且还能够挽救在bag3 ^-/-鱼。因此，重新利用二甲双胍为 BAG3 肌病提供了一种有希望的治疗方法。

缩写： ACTN：肌动蛋白，α；BAG3：BAG cochaperone 3；CRYAB：晶状体蛋白αB；DES：结蛋白；DMSO：二甲亚砜；DNAJB6：DnaJ 热休克蛋白家族 (Hsp40) 成员 B6；dpf：受精后天数；eGFP：增强型绿色荧光蛋白；FDA：食品药品监督管理局；FHL1：四个半LIM域1；FLNC：细丝蛋白 C；hpf：受精后的小时数；HSPB8：热休克蛋白家族B[小]成员8；LDB3/ZASP：LIM 域绑定 3；MYOT：肌动蛋白；TTN: 提丁; WT：野生型。