Development of the excitation-contraction coupling machinery and its relation to myofibrillogenesis in human iPSC-derived skeletal myocytes.

Background: Human induced pluripotent stem cells-derived myogenic progenitors develop functional and ultrastructural features typical of skeletal muscle when differentiated in culture. Besides disease-modeling, such a system can be used to clarify basic aspects of human skeletal muscle development. In the present study, we focus on the development of the excitation-contraction (E-C) coupling, a process that is essential both in muscle physiology and as a tool to differentiate between the skeletal and cardiac muscle. The occurrence and maturation of E-C coupling structures (Sarcoplasmic Reticulum-Transverse Tubule (SR-TT) junctions), key molecular components, and Ca signaling were examined, along with myofibrillogenesis.

Methods: Pax7-myogenic progenitors were differentiated in culture, and developmental changes were examined from a few days up to several weeks. Ion channels directly involved in the skeletal muscle E-C coupling (RyR1 and Cav1.1 voltage-gated Ca channels) were labeled using indirect immunofluorescence. Ultrastructural changes of differentiating cells were visualized by transmission electron microscopy. On the functional side, depolarization-induced intracellular Ca transients mediating E-C coupling were recorded using Fura-2 ratiometric Ca imaging, and myocyte contraction was captured by digital photomicrography.

Results: We show that the E-C coupling machinery occurs and operates within a few days post-differentiation, as soon as the myofilaments align. However, Ca transients become effective in triggering myocyte contraction after 1 week of differentiation, when nascent myofibrils show alternate A-I bands. At later stages, myofibrils become fully organized into adult-like sarcomeres but SR-TT junctions do not reach their triadic structure and typical A-I location. This is mirrored by the absence of cross-striated distribution pattern of both RyR1 and Cav1.1 channels.

Conclusions: The E-C coupling machinery occurs and operates within the first week of muscle cells differentiation. However, while early development of SR-TT junctions is coordinated with that of nascent myofibrils, their respective maturation is not. Formation of typical triads requires other factors/conditions, and this should be taken into account when using in-vitro models to explore skeletal muscle diseases, especially those affecting E-C coupling.