Dose-Dependent Effect of Caffeine on Intracellular Calcium Concentration of Cultured Skeletal Myotubes Derived From Equine Skin
Muscle from Thoroughbreds with the genetic disorder Recurrent Exertional Rhabdomyolysis (RER) displays altered sensitivity to caffeine in comparison with muscle from normal horses, suggesting that affected animals may have an inherited defect in the regulation of skeletal muscle intracellular calcium. At present, definitive diagnosis of the disorder requires an in vitro muscle contraction test that is not widely available to practitioners. With a view to investigating alternative and less invasive diagnostic methods, we hypothesised that fibroblasts derived from equine skin biopsy samples, when converted to muscle cells through forced expression of an equine muscle-specific transcription factor (MyoD), would display a dose-dependent increase in intracellular calcium concentration in response to caffeine.
Fibroblasts cultured from 4mm punch skin biopsy samples, aseptically obtained from ponies, were subsequently transduced with a lentivirus (pCMV-eqMyoD-IRES-EGFP) that would force the co-expression of equine MyoD and green fluorescent protein. Unlike untransduced cells, virus treated cells were transformed to myotubes as verified by the expression of muscle-specific proteins over 7-15 days by immunocytochemistry and western blot. Myotubes were subsequently loaded with Indo-1 (10ug/ml) for 30 minutes and intracellular calcium responses to increasing doses of caffeine (0-10mM) in the bathing solution (37°C) were measured by fluorescence. Caffeine was washed out between each test dose.
A dose-dependent and reproducible effect of caffeine on intracellular calcium concentration was detected. Specifically, a rise in intracellular calcium concentration was first detected with 1mM caffeine and a maximum response was detected with 10mM caffeine. On washout, intracellular calcium concentration returned to baseline.
These preliminary findings suggest that observation of responses of cultured skin cells that have been converted to muscle cells through forced expression of MyoD, may provide a less invasive method than muscle biopsy for the study of muscle intracellular calcium regulation and the pathophysiology of RER. Further study is required to determine whether skin-derived myotubes can be used in a practical diagnostic assay for RER in Thoroughbreds.