In vivo cytosolic H2O2 changes and Ca2+ homeostasis in mouse skeletal muscle

Hydrogen peroxide (H2O2) and calcium ions (Ca2+) are functional regulators of skeletal muscle contraction and metabolism. Although H2O2 is one of the activators of the type-1 ryanodine receptor (RyR1) in the Ca2+ release channel, the interdependence between H2O2 and Ca2+ dynamics remains unclear. This study tested the following hypotheses using an in vivo model of mouse tibialis anterior (TA) skeletal muscle. 1) Under resting conditions, elevated cytosolic H2O2 concentration ([H2O2](cyto)) leads to a concentration-dependent increase in cytosolic Ca2+ concentration ([Ca2+](cyto)) through its effect on RyR1; and 2) in hypoxia (cardiac arrest) and muscle contractions (electrical stimulation), increased [H2O2](cyto) induces Ca2+ accumulation. Cytosolic H2O2 (HyPer7) and Ca2+ (Fura-2) dynamics were resolved by TA bioimaging in young C57BL/6J male mice under four conditions: 1) elevated exogenous H2O2; 2) cardiac arrest; 3) twitch (1 Hz, 60 s) contractions; and 4) tetanic (30 s) contractions. Exogenous H2O2 (0.1-100 mM) induced a concentration-dependent increase in [H2O2](cyto) (+55% at 0.1 mM; +280% at 100 mM) and an increase in [Ca2+](cyto) (+3% at 1.0 mM; +8% at 10 mM). This increase in [Ca2+](cyto) was inhibited by pharmacological inhibition of RyR1 by dantrolene. Cardiac arrest-induced hypoxia increased [H2O2](cyto) (+33%) and [Ca2+](cyto) (+20%) 50 min postcardiac arrest. Compared with the exogenous 1.0 mM H2O2 condition, [H2O2](cyto) after tetanic muscle contractions rose less than one-tenth as much, whereas [Ca2+](cyto) was 4.7-fold higher. In conclusion, substantial increases in [H2O2](cyto) levels evoke only modest Ca2+ accumulation via their effect on the sarcoplasmic reticulum RyR1. On the other hand, contrary to hypoxia secondary to cardiac arrest, increases in [H2O2](cyto) from muscle contractions are small, indicating that H2O2 generation is unlikely to be a primary factor driving the significant Ca2+ accumulation after, especially tetanic, muscle contractions.<bold>NEW & NOTEWORTHY</bold> We developed an in vivo mouse myocyte H2O2 imaging model during exogenous H2O2 loading, ischemic hypoxia induced by cardiac arrest, and muscle contractions. In this study, the interrelationship between cytosolic H2O2 levels and Ca2+ homeostasis during muscle contraction and hypoxic conditions was revealed. These results contribute to the elucidation of the mechanisms of muscle fatigue and exercise adaptation.