DOI: 10.5061/dryad.r2280gbpr
title: Regulation of Ca2+ leak and susceptibility to malignant hyperthermia and heat stroke

description: Sarcoplasmic reticulum (SR) Ca2+ leak is elevated in many myopathies, but
 whether Ca2+ leak is a driver or consequence of the disease process is not
 always clear. The Ryanodine Receptor, RYR1, is the SR Ca2+ release channel
 required for both muscle contraction and Ca2+ leak. SPEG (striated muscle
 preferentially expressed protein kinase) regulates the Ca2+ leak
 properties of RYR1 by phosphorylation of serine 2902. Mutation of Serine
 S2902 to an aspartic acid to mimic SPEG phosphorylation of RYR1 decreases
 muscle temperature-dependent SR Ca2+ leak, restores the levels of
 excitation-contraction coupling proteins, and reduces the heat and
 volatile anesthetic sensitivity of mice with a malignant hyperthermia
 susceptibility mutation in RYR1 (Y524S, Y522S in humans). The S2902D mice
 allow a direct test of the role of SR Ca2+ leak in both normal muscle
 function (aging, exercise) and mice disease pathophysiology.
description: Analysis of phosphoylation sites on RYR1 by immunoprecipitation,
 mass spectrometry, and parallel reaction monitoring of gastrocnemius
 muscle for floxed controls (FL) and SPEG-deficient mice (SPEG KO) data
 were plotted as log2(fold change) versus -log(q), where q is the false
 discovery rate. Other techniques include force with force transducers,
 Western blots, and indirect calorimetry, as described in the
 method.
description: # Regulation of Ca2+ leak and susceptibility to malignant hyperthermia and
 heat stroke Dataset DOI:
 [10.5061/dryad.r2280gbpr](https://doi.org/10.5061/dryad.r2280gbpr) ##
 Description of the data and file structure The data were generated to
 define the role of SR Ca^2+^ leak in muscle heat production and enhanced
 sensitivity to heat stroke. The data were generated with mouse models of
 malignant hyperthermia susceptibility with a mutation in the skeletal
 muscle Ca^2+^ release channel (RYR1), which serves as a model of enhanced
 sensitivity to heat and heat-induced Ca^2+^ leak. A second mouse model had
 a mutation in RYR1 to mimic phosphorylation of RYR1 by the striated muscle
 preferentially expressed protein kinase SPEG. These mice displayed reduced
 SR Ca^2+^ leak. This dataset includes multiple approaches to assessing the
 role of SR Ca2+ leak in the phenotypes of the mice. ### Files and
 variables #### File: Data_for_all_panels_final_annotated2.xlsx The data
 file contains multiple sheets, each corresponding to a specific figure
 presented in the manuscript. Each sheet includes the relevant source data,
 along with a legend explaining the data and a list of any abbreviations
 used. **Description of variables:** * Genotype: WT (wildtype, S2902D HET
 (mice heterozygous for a mutation in RYR1), S2902D HOM (mice homozygous
 for a mutation in RYR1), YS (mice with a heterozygous MHS mutation in
 RYR1), YS/S2902D (mice heterozygous for the YS and S2902D mutation). *
 Muscles: extensor digitorum longus (EDL), soleus, flexor digitorum brevis
 (FDB), tibialis anterior (TA), gastrocnemius. * Gender: male, female *
 phosphorylation sites detected by parallel reaction monitoring
 (iiBAQWesternblots (absorbance) normalized to wildtype controls and
 plotted as % WT * Body composition: weight (g),  Fat (fat weight in g/
 body weight), lean weight/body weight (g/g ). * food intake (g/day),
 activity in wheel turns per day, or cumulative wheel turns over 3 days, *
 heat generated during the day or night  in kcal/hr, * Force (N/cm^2^)
 generated as a function of stimulation frequency (Hz), Fmax is the maximal
 force generated in the force-frequency curve, HZ50 is the half maximal
 stimulation frequency * Force(N/cm^2^) as a function of caffeine
 concentration (mM). * Mag-fluo 4 fluorescence (F-0/F0) * Manganese (Mn)
 quench data with fura2.  Slope of passive influx (quenching of
 fluorescence) * Immunoprecipitation analyzed by volcano plots of log2(fold
 change) vs -log(q) * Bodu and muscle temperature in ^o^C  * Indirect
 calorimetry to measure VO2 (ml/kg/hr), VCO2 (ml/kg/hr), and respiratory
 exchange ratio (RER) ## Code/software Prism, Excel. Code, written in IDL
 to analyze Ca^2+^ spark data, has been deposited in Zenodo
 ([https://doi.org/10.5281/zenodo.17023652](https://doi.org/10.5281/zenodo.17023652)) ## Access information Other publicly accessible locations of the data: * ASSIVE DB [https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=8aff256bffa34d188e9e67a2382b26a6](https://urldefense.proofpoint.com/v2/url?u=https-3A__massive.ucsd.edu_ProteoSAFe_dataset.jsp-3Ftask-3D8aff256bffa34d188e9e67a2382b26a6&d=DwMGaQ&c=ZQs-KZ8oxEw0p81sqgiaRA&r=Nturpmr1P5wHkzgp51urtw&m=DwuXBTxpVrYIjE2jiiF-U9W8YleKeyjb4QigVU04uytFOYaYepY6GqhN3nx3EwuZ&s=ixc9BSacv13t7AHVAb391rPXZ2aPiEkoOHYHusLeazY&e=) * [https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD060589](https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD060589)