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Intracellular calcium leak as a therapeutic target for RYR1-related myopathies

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Abstract

RYR1 encodes the type 1 ryanodine receptor, an intracellular calcium release channel (RyR1) on the skeletal muscle sarcoplasmic reticulum (SR). Pathogenic RYR1 variations can destabilize RyR1 leading to calcium leak causing oxidative overload and myopathy. However, the effect of RyR1 leak has not been established in individuals with RYR1-related myopathies (RYR1-RM), a broad spectrum of rare neuromuscular disorders. We sought to determine whether RYR1-RM affected individuals exhibit pathologic, leaky RyR1 and whether variant location in the channel structure can predict pathogenicity. Skeletal muscle biopsies were obtained from 17 individuals with RYR1-RM. Mutant RyR1 from these individuals exhibited pathologic SR calcium leak and increased activity of calcium-activated proteases. The increased calcium leak and protease activity were normalized by ex-vivo treatment with S107, a RyR stabilizing Rycal molecule. Using the cryo-EM structure of RyR1 and a new dataset of > 2200 suspected RYR1-RM affected individuals we developed a method for assigning pathogenicity probabilities to RYR1 variants based on 3D co-localization of known pathogenic variants. This study provides the rationale for a clinical trial testing Rycals in RYR1-RM affected individuals and introduces a predictive tool for investigating the pathogenicity of RYR1 variants of uncertain significance.

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Data and material availability

Antibodies and Rycals used in this study can be purchased from companies (as indicated in the methods section) not affiliated with any of the authors (none of the authors receive any income from selling any of the reagents used in this study), some of the reagents can be obtained from the Marks laboratory using an MTA with Columbia University.

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Funding

This work was supported by an RYR1 Foundation Research Grant to AK and by the National Institutes of Health, National Institute of Nursing Research, and Division of Intramural Research. The authors acknowledge the National Disease Research Interchange (NDRI) for supplying control skeletal muscle tissue. This work was also supported by grants from the NIH to ARM (T32HL120826, R01HL145473, R01DK118240, R01HL142903, R01HL061503, R01HL140934, R01AR070194, R25NS076445). This was also supported by a grant from the NIH UL1TR001873 to OC and AK.

Author information

Author notes

  1. Alexander Kushnir, Joshua J. Todd, Jessica W. Witherspoon and Qi Yuan are co-first authors.

Authors and Affiliations

  1. Department of Physiology and Cellular Biophysics, Clyde and Helen Wu Center for Molecular Cardiology, Columbia University Irving Medical Center, New York, NY, USA

    Alexander Kushnir, Qi Yuan, Steven Reiken, Harvey Lin, Ross H. Munce, Benjamin Wajsberg, Zephan Melville, Kaylee Wedderburn-Pugh, Anetta Wronska & Andrew R. Marks

  2. Department of Medicine, Division of Cardiology, Columbia University Irving Medical Center, New York, NY, USA

    Alexander Kushnir & Andrew R. Marks

  3. Neuromuscular Symptoms Unit, Tissue Injury Branch, National Institute of Nursing Research, National Institutes of Health, Bethesda, MD, USA

    Joshua J. Todd, Jessica W. Witherspoon, Muslima S. Razaqyar, Irene C. Chrismer, Monique O. Shelton, Carolyn Allen & Katherine G. Meilleur

  4. Department of Anesthesiology, Columbia University Irving Medical Center, New York, NY, USA

    Oliver B. Clarke

  5. Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA

    Ami Mankodi & Christopher Grunseich

  6. Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada

    Mark A. Tarnopolsky

  7. Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY, USA

    Kurenai Tanji

  8. Department of Neurology, H. Houston Merritt Neuromuscular Research Center, Columbia University Irving Medical Center, New York, NY, USA

    Michio Hirano

  9. Department of Anesthesia, University of Toronto and Malignant Hyperthermia Investigation Unit, Toronto General Hospital, Toronto, Ontario, Canada

    Sheila Riazi & Natalia Kraeva

  10. Department of Neurology, Donders Centre for Neuroscience, Radboud University Medical Center, Nijmegen, The Netherlands

    Nicol C. Voermans

  11. PreventionGenetics, Marshfield, WI, USA

    Angela Gruber

Authors
  1. Alexander Kushnir
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  2. Joshua J. Todd
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  4. Qi Yuan
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  5. Steven Reiken
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  26. Katherine G. Meilleur
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  27. Andrew R. Marks
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Contributions

AK, JJT, JWW, QY: conducted experiments, conceptualization, data analyses, writing. SR, HL, BW, ZM, KW, AW, MSR, ICC, MOS, AM, CG, MT, KT, MH, SR, NK, NCV, AG: conducted experiments, OBC Methodology. ARM, KGM: conceptualization, data analyses, writing the paper, project administration, supervision.

Corresponding authors

Correspondence to Katherine G. Meilleur or Andrew R. Marks.

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Conflict of interest

Columbia University and ARM own stock in ARMGO, Inc. a company developing compounds targeting RyR and have patents on Rycals.

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Kushnir, A., Todd, J.J., Witherspoon, J.W. et al. Intracellular calcium leak as a therapeutic target for RYR1-related myopathies. Acta Neuropathol 139, 1089–1104 (2020). https://doi.org/10.1007/s00401-020-02150-w

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