Measurements of calcium transients

For Ca²⁺ transient measurements, myobundles were incubated with 50 μM of calcium-sensitive dye Fluo-8 AM (Abcam, ab142773) in DM in an incubator for 1 hour while rocking, followed by washing in dye-free media for 30 min. Electrically induced Ca²⁺ transients were recorded as previously described (24, 40). Myobundles were transferred into a glass-bottom live-imaging chamber with Tyrode’s solution warmed at 37°C in a heated live-imaging chamber. Fluorescence images were acquired at ×4 magnification on a Nikon microscope using a high speed EMCCD (electron multiplying charge-coupled device) camera (Andor iXon 860) and Andor Solis software. Ca²⁺ transient amplitudes were calculated as the maximum relative change in fluorescence signal, ΔF/F = (Peak − Trough)/(Trough − Background).