Ca2+ Sparks Are Initiated by Ca2+ Entry in Embryonic Mouse Skeletal Muscle and Decrease in Frequency Postnatally

"Spontaneous" Ca2+ sparks and ryanodine receptor type 3 (RyR3) expression are readily detected in embryonic mammalian skeletal muscle but not in adult mammalian muscle, which rarely exhibits Ca2+ sparks and expresses predominantly RyR1. We have used confocal fluorescence imaging and systematic sampling of enzymatically dissociated single striated muscle fibers containing the Ca2+ indicator dye fluo 4 to show that the frequency of spontaneous Ca2+ sparks decreases dramatically from embryonic day 18 (E18) to postnatal day 14 (P14) in mouse diaphragm and from P1 to P14 in mouse extensor digitorum longus fibers. In contrast, the relative levels of RyR3 to RyR1 protein remained constant in diaphragm muscles from E18 to P14, indicating that changes in relative levels of RyR isoform expression did not cause the decline in Ca2+ spark frequency. E18 diaphragm fibers were used to investigate possible mechanisms underlying spark initiation in embryonic fibers. Spark frequency increased or decreased, respectively, when E18 diaphragm fibers were exposed to 8 or 0 mM Ca2+ in the extracellular Ringer solution, with no change in either the average resting fiber fluo 4 fluorescence or the average properties of the sparks. Either CoCl2 (5 mM) or nifedipine (30 microM) markedly decreased spark frequency in E18 diaphragm fibers. These results indicate that Ca2+ sparks may be triggered by locally elevated [Ca2+] due to Ca2+ influx via dihydropyridine receptor L-type Ca2+ channels in embryonic mammalian skeletal muscle.