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.NEW & NOTEWORTHY 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.

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