Malignant hyperthermia‐associated mutation of leaky RyR1 induces mitochondrial Ca2+ overload in the heart
Authors: O‐Uchi Jin , Jhun Bong Sook , Sheu Shey‐Shing 
Published: June 2021

Malignant hyperthermia (MH) is known as a pharmacogenetic disorder of skeletal muscle that shows hypermetabolic responses to anesthetic gases. It is reported that this syndrome is associated with the ryanodine receptor type 1 (RyR1) mutations. Although clinical manifestation of MH is mainly skeletal muscle disorder under anesthesia, sudden cardiac death (SCD) in the MH families in the conscious condition without anesthesia are also reported in the clinical case studies. However, the molecular mechanism underlying SCD in MH is completely unknown. We previously reported that a low level of RyR1 is expressed in the cardiac mitochondria, but not in the sarcoplasmic reticulum (SR) in the hearts, which may serves as an additional mitochondrial Ca2+ influx mechanism in cardiomyocytes distinct from the main pathway by the mitochondrial Ca2+ uniporter. Using knock‐in mice carrying a MH‐related RyR1 mutation Y522S (YS), we showed that YS hearts exhibit disrupted mitochondrial morphology. Moreover, ex‐vivo YS heart developed significantly higher number of multiple ventricular extrasystoles by β‐adrenergic stimulation compared to those observed in the wild‐type (WT) hearts. Therefore, we hypothesize that YS‐RyR1s form “leaky channel” at cardiac mitochondria and induce mitochondrial Ca2+ overload, followed by an increase in the mitochondrial reactive oxygen species (ROS) generation, which alters the cellular Ca2+ handing in cardiomyocytes. In this study, we used isolated cardiac mitochondria and cardiomyocytes from the YS mice to investigate the role of YS‐RyR1 in mitochondrial and cellular Ca2+ handling as well as ROS generation. We loaded the isolated mitochondria with Ca2+ or membrane‐potential sensitive dyes, plated them on the glass‐bottom dishes and assessed mitochondrial Ca2+ concentration ([Ca2+]m) and mitochondrial membrane potential (ψm) under the confocal microscopy. We found that YS mitochondria possess higher basal [Ca2+]m and depolarized ψm. Moreover, pretreatment with RyR1 blocker dantrolene decreased basal [Ca2+]m and hyperpolarized ψm in YS mitochondria. We next loaded cardiomyocytes with Ca2+ sensitive dyes and measured cellular Ca2+ handling. We found that YS myocytes had significantly higher basal cytosolic Ca2+ concentration as well as slower cytosolic Ca2+ clearance compared to WT. Pretreatment of dantrolene normalized the Ca2+ handing profiles in YS cardiomyocytes similar to those in WT. Finally, we found that YS cardiomyocytes exhibited higher mitochondrial superoxide (mSO) level and excessive cellular oxidation assessed by the staining with mSO‐sensitive dye, MitoSox Red and immunoblotting of carbonyl groups by OxyBlot, respectively. In summary, these results suggest that chronic mitochondrial Ca2+ overload via leaky mutant mRyR1 increases mSO production and cellular oxidation, which may alter cytosolic Ca2+ handling and increase the arrhythmogenic events in MH.Support or Funding InformationJ.O.‐U. was supported by NIH/NHLBI R01HL136757, NIH/NIGMS P30GM1114750, American Heart Association (AHA) 4BGIA18830032, AHA 16SDG27260248, Rhode Island Foundation #20164376 Medical Research Grant, American Physiological Society (APS), and 2017 Shih‐Chun Wang Young Investigator Award. B.S.J was supported by NIH/NIGMS U54GM115677 and P30GM1114750. S.S.S was supported by NIH/NHLBI R01HL093671 and R01HL122124.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.