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AICAR prevents heat-induced sudden death in RyR1 mutant mice independent of AMPK activation

Nature Medicine volume 18, pages 244–251 (2012)Cite this article

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Abstract

Mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (Ryr1), a mutation analogous to the Y522S mutation that is associated with malignant hyperthermia in humans, die when exposed to short periods of temperature elevation (≥37 °C). We show here that treatment with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) prevents this heat-induced sudden death in this mouse model. The protection by AICAR is independent of AMP-activated protein kinase (AMPK) activation and results from a newly identified action of the compound on mutant Ryr1 to reduce Ca2+ leak from the sarcoplasmic reticulum to the sarcoplasm. AICAR thus prevents Ca2+-dependent increases in the amount of both reactive oxygen species (ROS) and reactive nitrogen species (RNS) that act to further increase resting Ca2+ concentrations. If unchecked, the temperature-driven increases in resting Ca2+ concentrations and the amounts of ROS and RNS create an amplifying cycle that ultimately triggers sustained muscle contractions, rhabdomyolysis and death. Although antioxidants are effective in reducing this cycle in vitro, only AICAR prevents heat-induced death in vivo. Our findings suggest that AICAR is probably effective in prophylactic treatment of humans with enhanced susceptibility to exercise- and/or heat-induced sudden death associated with RYR1 mutations.

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Figure 1: Effect of AICAR on heat-induced sudden death in YS mice heat challenged at 37 °C.
Figure 2: AICAR rescue of the YS mice is independent of AMPK activation.
Figure 3: The effect of AICAR on Ryr1 in the presence of AMP-PCP.
Figure 4: The effect of AICAR on the concentrations of Ca2+, ROS and RNS in single isolated FDB fibers from WT and YS mice.
Figure 5: The effects of NOX or NOS inhibition in single isolated FDB fibers at 35 °C.
Figure 6: A model for the AICAR prevention of EHR.

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Acknowledgements

This work was supported by grants from US National Institutes of Health (AR053349), the Department of Defense (DAMD W81XWH-10-2-0117) and the Muscular Dystrophy Association of America. J.T.L. was supported by a postdoc fellowship from The Swedish Research Council. A.D.-A. was supported by a postdoctoral fellowship from The Mexican National Council of Science and Technology (150489). The model shown in Figure 6 was created by S.A. Weldon.

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  1. Johanna T Lanner and Dimitra K Georgiou: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Physiology and Biophysics Baylor College of Medicine, Houston, Texas, USA

    Johanna T Lanner, Dimitra K Georgiou, Adan Dagnino-Acosta, Qing Cheng, Aditya D Joshi, Zanwen Chen, Joshua M Oakes, Chang Seok Lee, Tanner O Monroe, Arturo Santillan, Keke Dong, Iskander I Ismailov, George G Rodney & Susan L Hamilton

  2. Department of Pharmacology and Physiology University of Rochester Medical Center, Rochester, New York, USA

    Alina Ainbinder, Viktor Yarotskyy & Robert T Dirksen

  3. Joslin Diabetes Center Research Division, Boston, Massachusetts, USA

    Laurie Goodyear

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Contributions

J.T.L. designed, performed and analyzed the experiments shown in Figures 1a–d and 3a–c, analyzed data, produced Supplementary Figure 1a–f, wrote the initial draft of the paper and edited the final draft. D.K.G. developed the new AMPK assay, designed, performed and analyzed the experiments in Figure 2, wrote an intermediate draft of the paper, prepared the supplementary information, and helped write and edit the final draft of the manuscript. A.D.-A. designed, performed and analyzed the experiments in Figures 4e,f and 5a,b and participated in the writing of the manuscript. A.A. designed, performed and analyzed data from the experiments in Figure 4d. Q.C. made the initial AICAR discovery and performed the experiments in Figure 1e,f and Supplementary Figure 2. A.D.J. generated and analyzed the data in Figure 4g–i and Supplementary Figure 6. Z.C. performed the bilayer experiments. V.Y. designed, performed and analyzed data from the experiments in Supplementary Table 1 and Supplementary Figure 3b,c. J.M.O. performed the experiments shown in Figure 5c,d. C.S.L. designed, performed and analyzed data (using western blots and quantitative RT-PCR) to show that calcium-handling proteins are not changed by the Y524S mutation or by the presence of AICAR. T.O.M. designed the experiments and performed and analyzed many of the pAMPK and AMPK western blots shown in Supplementary Figure 1. A.S. performed all of the mouse dissections, tested the endurance of mice on running wheels, performed indirect calorimetry and contributed to the preparation of the manuscript. K.D. handled all mouse matings and genotyping, performed indirect calorimetry on mice and helped in the manuscript preparation. L.G. provided mice, advised on crucial metabolic experiments and AMPK assays and participated in the writing and critique of the manuscript. I.I.I. designed and performed the experiments in Figure 4a,b and 5e–h, helped in the analysis of the bilayer data and contributed to the manuscript preparation and revision. G.G.R. contributed reagents, supervised, designed and analyzed the experiments to assess the role of NOX and contributed to the manuscript preparation and revision. R.T.D. designed, supervised and analyzed all Ca2+ measurements, critiqued and analyzed all studies and contributed to the manuscript preparation and revision. S.L.H. supervised all experiments, reanalyzed all data for accuracy, plotted all figures and wrote the final draft of the manuscript. All authors reviewed and approved the final version of the manuscript.

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Correspondence to Susan L Hamilton.

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Supplementary Table 1, Supplementary Figures 1–6 and Supplementary Methods (PDF 727 kb)

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Lanner, J., Georgiou, D., Dagnino-Acosta, A. et al. AICAR prevents heat-induced sudden death in RyR1 mutant mice independent of AMPK activation. Nat Med 18, 244–251 (2012). https://doi.org/10.1038/nm.2598

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