Regulation of Ca2+ leak and susceptibility to malignant hyperthermia and heat stroke
Data files
Sep 28, 2025 version files 252.81 KB
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Data_for_all_panels_final_annotated2.xlsx
249.34 KB
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README.md
3.46 KB
Abstract
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.
Dataset DOI: 10.5061/dryad.r2280gbpr
Description of the data and file structure
The data were generated to define the role of SR Ca2+ 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 Ca2+ release channel (RYR1), which serves as a model of enhanced sensitivity to heat and heat-induced Ca2+ 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 Ca2+ 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/cm2) 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/cm2) 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 oC
- 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 Ca2+ spark data, has been deposited in Zenodo (https://doi.org/10.5281/zenodo.17023652)
Access information
Other publicly accessible locations of the data:
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.