Activation and propagation of Ca2+release from inside the sarcoplasmic reticulum network of mammalian skeletal muscle

Key points Prolonged Ca2+ transients and Ca2+ waves have been observed in mammalian skeletal muscle upon reducing the normal resting inhibition of the ryanodine receptor (RyR)/Ca2+ release channels of the sarcoplasmic reticulum (SR) by lowering cytoplasmic [Mg2+] ([Mg2+]cyto). The mechanism driving Ca2+ release under these conditions may have pathophysiological implications but is poorly understood. The lowering of [Mg2+]cyto induced a leak of Ca2+ from the SR that triggered a local reduction of the SR Ca2+ buffering power to induce the rise in [Ca2+]SR, an intra-SR Ca2+ transient. Prolonged Ca2+ transients always developed following this event and evolved as propagating Ca2+ waves. No classical hallmarks of cytoplasmic Ca2+ propagation mechanisms were observed in conjunction with these waves. In conjunction with the spread of Ca2+ release, Ca2+ was observed to diffuse through the SR away from the site of the intra-SR Ca2+ transient. Ca2+ release was sustained by the action of SR Ca2+ pumps, presumably to maintain [Ca2+]SR above an inactivation threshold for closure of the RyRs. Inactivation of prolonged Ca2+ release allowed [Ca2+]SR to recover to threshold for activation of further Ca2+ waves, which were always briefer in duration than the initial Ca2+ release transients, consistent with a reduced SR Ca2+ buffering capacity. These observations reveal activation of Ca2+ release in mammalian skeletal muscle by [Ca2+]SR in the absence of the normal resting inhibition of the RyR and changes in voltage. Diffusion of Ca2+ within SR promotes propagation of Ca2+ release. Abstract Skeletal muscle fibres are large and highly elongated cells specialized for producing the force required for posture and movement. The process of controlling the production of force within the muscle, known as excitation–contraction coupling, requires virtually simultaneous release of large amounts of Ca2+ from the sarcoplasmic reticulum (SR) at the level of every sarcomere within the muscle fibre. Here we imaged Ca2+ movements within the SR, tubular (t-) system and in the cytoplasm to observe that the SR of skeletal muscle is a connected network capable of allowing diffusion of Ca2+ within its lumen to promote the propagation of Ca2+ release throughout the fibre under conditions where inhibition of SR ryanodine receptors (RyRs) was reduced. Reduction of cytoplasmic [Mg2+] ([Mg2+]cyto) induced a leak of Ca2+ through RyRs, causing a reduction in SR Ca2+ buffering power argued to be due to a breakdown of SR calsequestrin polymers, leading to a local elevation of [Ca2+]SR. The local rise in [Ca2+]SR, an intra-SR Ca2+ transient, induced a local diffusely rising [Ca2+]cyto. A prolonged Ca2+ wave lasting tens of seconds or more was generated from these events. Ca2+ waves were dependent on the diffusion of Ca2+ within the lumen of the SR and ended as [Ca2+]SR dropped to low levels to inactivate RyRs. Inactivation of RyRs allowed re-accumulation of [Ca2+]SR and the activation of secondary Ca2+ waves in the persistent presence of low [Mg2+]cyto if the threshold [Ca2+]SR for RyR opening could be reached. Secondary Ca2+ waves occurred without an abrupt reduction in SR Ca2+ buffering power. Ca2+ release and wave propagation occurred in the absence of Ca2+-induced Ca2+ release. These observations are consistent with the activation of Ca2+ release through RyRs of lowered cytoplasmic inhibition by [Ca2+]SR or store overload-induced Ca2+ release. Restitution of SR Ca2+ buffering power to its initially high value required imposing normal resting ionic conditions in the cytoplasm, which re-imposed the normal resting inhibition on the RyRs, allowing [Ca2+]SR to return to endogenous levels without activation of store overload-induced Ca2+ release. These results are discussed in the context of how pathophysiological Ca2+ release such as that occurring in malignant hyperthermia can be generated.