Metformin rescues muscle function in BAG3 myofibrillar myopathy models

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.

KEYWORDS:

• Autophagy
• BAG3
• metformin
• muscle
• myofibrillar myopathy
• zebrafish

Acknowledgments

We would like to thank Prof Bernard Brais for the myoblast cell lines utilized, Dr Aidan Sudbury for statistical advice, Coco Bayly-Jones for assistance with the maintenance of mutant and transgenic strains, Oleksandr Chernyavskiy and Monash Micro Imaging for assistance with super resolution imaging, and the patients who contributed samples to this study. The hybridoma product A4.1025 was developed by Blau, H.M., obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242.

Disclosure statement

No potential conflict of interest was reported by the authors.

Compliance with ethical standards

Fish maintenance and handling were carried out as per standard operating procedures approved by the Monash Animal Services Ethics Committee. The generation of transgenic and mutant strains was approved by the School of Biological Sciences Animal Ethics Committee (BSCI/2013/27 and BSCI/2015/06 respectively). This study was performed according to the guidelines of the Committee on the Use of Human Subjects in Research of the Policlinico Hospital of Milan (Milan, Italy) or the Institute of Myology, Pitié-Salpêtriere hospital (Paris, France). Informed consent was obtained from all family members. The authors have no conflicts of interest to declare.

Data availability statement

Raw data and files for statistical analysis are available at Bridges and can be accessed using the following link: https://bridges.monash.edu/projects/Metformin_rescues_muscle_function_in_BAG3_myofibrillar_myopathy_models/60959.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was funded by the Bellini Foundation and Fondazione Roby.