Abstract
In human muscle with ryanodine receptor (RyR) variants, there is expected to be a change in the basal RyRs Ca
2+
leak that may alter local Ca
2+
dynamics. It is not currently possible to assess these basal Ca
2+
dynamics in human muscle. By trapping a Ca
2+
-sensitive fluorescent dye in the transverse tubular (t)-system of freshly biopsied human skeletal muscle fibers we could detect tiny local RyR-dependent Ca
2+
movements. We show that malignant hyperthermia (MH) causative RyR variant human muscle displays a chronically different RyR Ca
2+
leak which reconfigures the Ca
2+
-handling properties by the t-system. This approach can be used to assess human muscle for function and pathogenicity of other RyR variants of uncertain significance, MH susceptibility, and assessment of drugs targeted to the Ca
2+
-handling proteins.
We used the nanometer-wide tubules of the transverse tubular (t)-system of human skeletal muscle fibers as sensitive sensors for the quantitative monitoring of the Ca
2+
-handling properties in the narrow junctional cytoplasmic space sandwiched between the tubular membrane and the sarcoplasmic reticulum cisternae in single muscle fibers. The t-system sealed with a Ca
2+
-sensitive dye trapped in it is sensitive to changes in ryanodine receptor (RyR) Ca
2+
leak, the store operated calcium entry flux, plasma membrane Ca pump, and sodium–calcium exchanger activities, thus making the sealed t-system a nanodomain Ca
2+
sensor of Ca
2+
dynamics in the junctional space. The sensor was used to assess the basal Ca
2+
-handling properties of human muscle fibers obtained by needle biopsy from control subjects and from people with a malignant hyperthermia (MH) causative RyR variant. Using this approach we show that the muscle fibers from MH-susceptible individuals display leakier RyRs and a greater capacity to extrude Ca
2+
across the t-system membrane compared with fibers from controls. This study provides a quantitative way to assess the effect of RyR variants on junctional membrane Ca
2+
handling under defined ionic conditions.