Identification of drug modifiers for RYR1 related myopathy using a multi-species discovery pipeline

  1. Jonathan R Volpatti
  2. Yukari Endo
  3. Jessica Knox
  4. Linda Groom
  5. Stephanie Brennan
  6. Ramil Noche
  7. William J Zuercher
  8. Peter Roy
  9. Robert T Dirksen
  10. James J Dowling  Is a corresponding author
  1. The Hospital for Sick Children, Canada
  2. University of Toronto, Canada
  3. University of Rochester, United States
  4. University of North Carolina at Chapel Hill, United States
  5. University of Rochester School of Medicine and Dentistry, United States

Abstract

Ryanodine receptor type I-related myopathies (RYR1-RMs) are a common group of childhood muscle diseases associated with severe disabilities and early mortality for which there are no available treatments. The goal of this study is to identify new therapeutic targets for RYR1-RMs. To accomplish this, we developed a discovery pipeline using nematode, zebrafish, and mammalian cell models. We first performed large-scale drug screens in C. elegans which uncovered 74 hits. Targeted testing in zebrafish yielded positive results for two p38 inhibitors. Using mouse myotubes, we found that either pharmacological inhibition or siRNA silencing of p38 impaired caffeine-induced Ca2+ release from wild type cells while promoting intracellular Ca2+ release in Ryr1 knockout cells. Lastly, we demonstrated that p38 inhibition blunts the aberrant temperature-dependent increase in resting Ca2+ in myotubes from an RYR1-RM mouse model. This unique platform for RYR1-RM therapy development is potentially applicable to a broad range of neuromuscular disorders.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source files are available for all figures.

Article and author information

Author details

  1. Jonathan R Volpatti

    Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
  2. Yukari Endo

    Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
  3. Jessica Knox

    Department of Molecular Genetics, University of Toronto, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
  4. Linda Groom

    Department of Pharmacology, University of Rochester, Rochester, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Stephanie Brennan

    Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
  6. Ramil Noche

    Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
  7. William J Zuercher

    UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Peter Roy

    Molecular Genetics, University of Toronto, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
  9. Robert T Dirksen

    Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3182-1755
  10. James J Dowling

    Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Canada
    For correspondence
    james.dowling@sickkids.ca
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3984-4169

Funding

Muscular Dystrophy Association

  • Robert T Dirksen
  • James J Dowling

RYR1 Foundation

  • Robert T Dirksen
  • James J Dowling

Canadian Institutes of Health Research (363863)

  • Robert T Dirksen
  • James J Dowling

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All zebrafish experiments were performed in accordance with all relevant ethical regulations, specifically following the policies and guidelines of the Canadian Council on Animal Care and an institutionally reviewed and approved animal use protocol (#41617). No additional ethical approval was required for our experiments with the invertebrate nematode worm C. elegans.

Reviewing Editor

  1. Jeff S Mumm, Johns Hopkins University, United States

Publication history

  1. Received: October 22, 2019
  2. Accepted: March 29, 2020
  3. Accepted Manuscript published: March 30, 2020 (version 1)
  4. Version of Record published: May 6, 2020 (version 2)

Copyright

© 2020, Volpatti et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Jonathan R Volpatti
  2. Yukari Endo
  3. Jessica Knox
  4. Linda Groom
  5. Stephanie Brennan
  6. Ramil Noche
  7. William J Zuercher
  8. Peter Roy
  9. Robert T Dirksen
  10. James J Dowling
(2020)
Identification of drug modifiers for RYR1 related myopathy using a multi-species discovery pipeline
eLife 9:e52946.
https://doi.org/10.7554/eLife.52946
  1. Further reading

Further reading

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    Background:

    Abiraterone acetate is an effective treatment for metastatic castrate-resistant prostate cancer (mCRPC), but evolution of resistance inevitably leads to progression. We present a pilot study in which abiraterone dosing is guided by evolution-informed mathematical models to delay onset of resistance.

    Methods:

    In the study cohort, abiraterone was stopped when PSA was <50% of pretreatment value and resumed when PSA returned to baseline. Results are compared to a contemporaneous cohort who had >50% PSA decline after initial abiraterone administration and met trial eligibility requirements but chose standard of care (SOC) dosing.

    Results:

    17 subjects were enrolled in the adaptive therapy group and 16 in the SOC group. All SOC subjects have progressed, but four patients in the study cohort remain stably cycling (range 53–70 months). The study cohort had significantly improved median time to progression (TTP; 33.5 months; p<0.001) and median overall survival (OS; 58.5 months; hazard ratio, 0.41, 95% confidence interval (CI), 0.20–0.83, p<0.001) compared to 14.3 and 31.3 months in the SOC cohort. On average, study subjects received no abiraterone during 46% of time on trial. Longitudinal trial data demonstrated the competition coefficient ratio (αRSSR) of sensitive and resistant populations, a critical factor in intratumoral evolution, was two- to threefold higher than pre-trial estimates. Computer simulations of intratumoral evolutionary dynamics in the four long-term survivors found that, due to the larger value for αRSSR, cycled therapy significantly decreased the resistant population. Simulations in subjects who progressed predicted further increases in OS could be achieved with prompt abiraterone withdrawal after achieving 50% PSA reduction.

    Conclusions:

    Incorporation of evolution-based mathematical models into abiraterone monotherapy for mCRPC significantly increases TTP and OS. Computer simulations with updated parameters from longitudinal trial data can estimate intratumoral evolutionary dynamics in each subject and identify strategies to improve outcomes.

    Funding:

    Moffitt internal grants and NIH/NCI U54CA143970-05 (Physical Science Oncology Network).

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    2. Medicine
    Eric N Jimenez-Vazquez et al.
    Research Article

    Background:

    Patients with cardiomyopathy of Duchenne Muscular Dystrophy (DMD) are at risk of developing life-threatening arrhythmias, but the mechanisms are unknown. We aimed to determine the role of ion channels controlling cardiac excitability in the mechanisms of arrhythmias in DMD patients.

    Methods:

    To test whether dystrophin mutations lead to defective cardiac NaV1.5–Kir2.1 channelosomes and arrhythmias, we generated iPSC-CMs from two hemizygous DMD males, a heterozygous female, and two unrelated control males. We conducted studies including confocal microscopy, protein expression analysis, patch-clamping, non-viral piggy-bac gene expression, optical mapping and contractility assays.

    Results:

    Two patients had abnormal ECGs with frequent runs of ventricular tachycardia. iPSC-CMs from all DMD patients showed abnormal action potential profiles, slowed conduction velocities, and reduced sodium (INa) and inward rectifier potassium (IK1) currents. Membrane NaV1.5 and Kir2.1 protein levels were reduced in hemizygous DMD iPSC-CMs but not in heterozygous iPSC-CMs. Remarkably, transfecting just one component of the dystrophin protein complex (α1-syntrophin) in hemizygous iPSC-CMs from one patient restored channelosome function, INa and IK1 densities, and action potential profile in single cells. In addition, α1-syntrophin expression restored impulse conduction and contractility and prevented reentrant arrhythmias in hiPSC-CM monolayers.

    Conclusions:

    We provide the first demonstration that iPSC-CMs reprogrammed from skin fibroblasts of DMD patients with cardiomyopathy have a dysfunction of the NaV1.5–Kir2.1 channelosome, with consequent reduction of cardiac excitability and conduction. Altogether, iPSC-CMs from patients with DMD cardiomyopathy have a NaV1.5–Kir2.1 channelosome dysfunction, which can be rescued by the scaffolding protein α1-syntrophin to restore excitability and prevent arrhythmias.

    Funding:

    Supported by National Institutes of Health R01 HL122352 grant; ‘la Caixa’ Banking Foundation (HR18-00304); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); Instituto de Salud Carlos III/FEDER/FSE; Horizon 2020 - Research and Innovation Framework Programme GA-965286 to JJ; the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). American Heart Association postdoctoral fellowship 19POST34380706s to JVEN. Israel Science Foundation to OB and MA [824/19]. Rappaport grant [01012020RI]; and Niedersachsen Foundation [ZN3452] to OB; US-Israel Binational Science Foundation (BSF) to OB and TH [2019039]; Dr. Bernard Lublin Donation to OB; and The Duchenne Parent Project Netherlands (DPPNL 2029771) to OB. National Institutes of Health R01 AR068428 to DM and US-Israel Binational Science Foundation Grant [2013032] to DM and OB.