Nox4 - RyR1 - Nox2: Regulators of micro-domain signaling in skeletal muscle

The ability for skeletal muscle to perform optimally can be affected by the regulation of Ca²⁺ within the triadic junctional space at rest. Reactive oxygen species impact muscle performance due to changes in oxidative stress, damage and redox regulation of signaling cascades. The interplay between ROS and Ca²⁺ signaling at the triad of skeletal muscle is therefore important to understand as it can impact the performance of healthy and diseased muscle. Here, we aimed to examine how changes in Ca²⁺ and redox signaling within the junctional space micro-domain of the mouse skeletal muscle fibre alters the homeostasis of these complexes. The dystrophic mdx mouse model displays increased RyR1 Ca²⁺ leak and increased NAD(P)H Oxidase 2 ROS. These alterations make the mdx mouse an ideal model for understanding how ROS and Ca²⁺ handling impact each other. We hypothesised that elevated t-tubular Nox2 ROS increases RyR1 Ca²⁺ leak contributing to an increase in cytoplasmic Ca²⁺, which could then initiate protein degradation and impaired cellular functions such as autophagy and ER stress. We found that inhibiting Nox2 ROS did not decrease RyR1 Ca²⁺ leak observed in dystrophin-deficient skeletal muscle. Intriguingly, another NAD(P)H isoform, Nox4, is upregulated in mice unable to produce Nox2 ROS and when inhibited reduced RyR1 Ca²⁺ leak. Our findings support a model in which Nox4 ROS induces RyR1 Ca²⁺ leak and the increased junctional space [Ca²⁺] exacerbates Nox2 ROS; with the cumulative effect of disruption of downstream cellular processes that would ultimately contribute to reduced muscle or cellular performance.