Ryanodine Receptor Ca ²⁺ Leak‐Induced Redistribution of Ca ²⁺ in Dystrophic mdx Mouse Muscle

Figures

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  FIGURE 1. T‐system Ca 2+ handling and RyR1 Ca 2+ leak in mdx muscle fibers. (A) schematic diagram of rhod‐5 N trapped in the sealed t‐system as used to assess RyR1 Ca2+ leak. The functional RyR1 leaks Ca2+ into the junctional space (JS), increasing the local [Ca2+]JS above that of the bulk cytoplasm, which is set by the internal bathing solution. The [Ca2+]JS directly influences PMCA activity to pump Ca2+ into the sealed t‐system until an equilibrium is reached with t‐system Ca2+ leak to set the steady [Ca2+]t‐sys. The [Ca2+]t‐sys is reported by the rhod‐5 N fluorescence signal. Introducing a RyR1 leak blocker, tetracaine (A'), causes a decrease in the [Ca2+]JS, and subsequently a decrease in the [Ca2+]t‐sys to a new steady state as t‐system Ca2+ leak equilibrates with the adjusted Ca2+ pumping activity of the PMCA. (B) and (C) representative traces of [Ca2+]t‐sys from WT and mdx respectively. The t‐system is fully depleted following the application of 30 mM caffeine. Substituting caffeine for a solution containing 85 nM Ca2+ in the presence of tetracaine (first transient) and then absence of tetracaine (second transient) allows the assessment of RyR1 Ca2+ leak to the [Ca2+]t‐sys…

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  FIGURE 2. Intracellular Ca 2+ redistribution in EDL fibers from WT and mdx mice. (A) force recording from mdx muscle mechanically skinned fiber to measure fiber endogenous Ca2+ content. The fiber was first bathed in a 0.1 mM EGTA resting solution, then moved to 0.3 mM BAPTA, followed by membrane lysing in TX‐oil, which released all compartmentalized Ca2+ to generate a force response. TX‐oil was then washed out to relax the fiber. Finally, the fiber is equilibrated in a 50 mM EGTA relaxing solution prior to activation of maximal Ca2+‐activated force. (B) summary of endogenous (left), non‐caffeine releasable (middle) and non‐caffeine and on‐FCCP releasable Ca2+ content (right) in WT and mdx fibers. (C) total SR Ca2+ content and (D) total mitochondrial Ca2+ content in WT and mdx muscle fibers, as calculated from the data in B (see Methods). (E) Relationship between free [Ca2+]cyto vs. total [Ca2+]mito used to determine resting [Ca2+]cyto in WT and mdx resting fibers. (F) Relationship between mitochondrial Ca2+ content vs. free [Ca2+]cyto used to determine cytoplasmic Ca2+ content in WT and mdx fibers, see Lamboley et al. [9]. (G) SR, cytosol, and mitochondria total Ca2+ represented as a…

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  FIGURE 3. Redistribution of Ca2+ in dystrophic mdx mouse muscle. RyR1 Ca2+ leak is responsible for depleting SR Ca2+ content. This triggers a net influx of Ca2+ across the t‐system membrane to persistently raise cytoplasmic Ca2+ levels. Raised cytoplasmic Ca2+ levels drive Ca2+ into the mitochondria to raise the basal level of Ca2+. These alterations from the healthy (wild‐type) state may drive the generation of reactive oxygen species inside the mitochondria.