Different Capabilities for CICR of Skeletal Muscle of Amphibians and Mammals, Demonstrated through the Response to Artificial Ca Sparks

Abstract

To probe CICR (Endo, Phys. Rev. 2009), single skeletal muscle cells were stimulated with artificial Ca2+ sparks generated by spot 2-photon breakdown of NDBF-EGTA, while their Ca2+ response was monitored by confocal imaging of fluorescence of fluo 4FF. The cells had their plasmalemma permeabilized. Sparks were applied either inside cells or slightly outside, a method that avoids photodamage and allows measurement of the triggering [Ca2+] at precisely the time when the cellular response is elicited. Frog skeletal muscle fibers usually responded to stimuli > 1 μM with a propagating, all-or-none Ca2+ wave. The moving wavefront peaked at 3-5 μM; the calculated Ca2+ release flux peaked at 50-100 mM/s and the wave velocity was ∼100 μm/s (and slightly faster in the axial direction). In other cases a “frustrated” response was generated, which stopped after propagating for 20-30 μm, usually following progressive reduction in Ca2+ release flux. In striking contrast, the same stimuli failed to elicit a response when applied to cells of the mouse FDB muscle. Variants of the stimulus included “whorls” (2-P radiation in scribbles that covered regions of ∼3 μm diameter), involving much greater amounts of Ca2+ than an artificial spark. Whether placed outside or inside the cells, even at intensities that caused persistent cell damage, whorls elicited neither the fully propagated nor the frustrated response. These results directly demonstrate that frog muscle is capable of CICR but do not prove a physiologic role, as they were obtained on permeabilized cells. In conclusion, mammalian muscle does not have the CICR capability, which other works have associated with the presence of RyR isoform 3 (Pouvreau et al., PNAS 2007; Legrand et al., J. Physiol. 2008). Supported by NIAMS and NHLBI (NIH).