Store-operated Calcium Entry in Skeletal Muscle

Skeletal muscle contraction is caused by the intracellular release of calcium (Ca2+) upon nervous excitation, a process termed excitation-contraction (EC) coupling. In addition to the large release of Ca2+ from internal stores, small amounts of Ca2+ are known to enter the muscle cell via store-operated Ca2+ entry (SOCE). Dysfunction of SOCE was shown to increase the susceptibility of skeletal muscle to fatigue and loss as well as gain of function induces different forms of myopathy. Despite the importance of SOCE, the mechanisms of activation and physiological role have remained enigmatic. Evidence from non-excitable cells suggests that SOCE is activated by the drop of Ca2+ in the lumen of the internal stores upon intracellular Ca2+ release. However, it is currently unclear if and how this activation can occur in skeletal muscle, in which the internal stores are never thoroughly depleted of Ca2+ under physiological conditions. This raises three key issues that the proposed project intends to address: (i) Is SOCE activated under physiological conditions in skeletal muscle? (ii) How is SOCE activated? (iii) What is the physiological role of SOCE in skeletal muscle? The currently available techniques are insufficient to record the brief and minute SOCE fluxes across the muscle cell membrane. To overcome previous limitations, I herein present a novel microscopy technique to measure SOCE during single muscle twitches for the very first time. Preliminary data shown within this proposal suggest that SOCE was indeed activated under such conditions. This phasic SOCE activation was directly linked to the process of EC coupling. The existence of such a phasic activation of SOCE sheds a completely new light on the role of this important pathway in skeletal muscle. Thus, with the novel technique at hand, we plan to characterise SOCE in mature skeletal muscle fibres, determine the mechanistic basis of SOCE activation, and define its physiological role. Experiments in mice suffering from myopathy will identify a potential role of SOCE in muscle disease. Finally, we will translate our findings to human muscle, measuring SOCE in freshly biopsied fibres.

The release of calcium (Ca2+) from the sarcoplasmic reticulum (SR) is essential for initiating skeletal muscle contraction. In addition to this major Ca2+ release, smaller amounts of Ca2+ enter the muscle cell through store-operated Ca2+ entry (SOCE), a pathway activated when SR Ca2+ levels are low. Dysfunction in SOCE is associated with increased muscle fatigue and can result in various forms of myopathy through both loss and gain of function. However, it remains unclear how SOCE is activated in skeletal muscle, where the SR is never completely depleted of Ca2+ under normal physiological conditions. Our recent findings have demonstrated a phasic form of SOCE (pSOCE) that occurs during single muscle twitches in rat skeletal muscle, suggesting this pathway operates under normal physiological conditions. In the current study, by advancing of the available technique, we further demonstrate that pSOCE is present in mouse muscle, including both fast and slow twitch fibers. The discovery of pSOCE across different mammalian species and muscle fiber types highlights its fundamental role in muscle physiology. Using genetically modified mice and pharmacological tools, we show that pSOCE is likely mediated by the proteins Stim1 and Orai1, which are key players in SOCE across various tissues. Interestingly, a long splice variant of Stim1 does not appear to be involved in pSOCE activation but plays a significant role in regulating SR Ca2+ levels by influencing the function of the SR Ca2+-ATPase, the main protein that mediates the reuptake of Ca2+ into the SR. Overall, this project has provided important insights into the role of SOCE in skeletal muscle function. Throughout the project, we developed new techniques that made this research possible and established several valuable collaborations that contributed to the successful achievement of our goals. The key results from this work emphasize the critical role of SOCE in maintaining skeletal muscle function and open new avenues for understanding its broader physiological significance.