Simultaneous imaging of local calcium and single sarcomere length in rat neonatal cardiomyocytes using yellow Cameleon-Nano140

In cardiac muscle, contraction is triggered by sarcolemmal depolarization, resulting in an intracellular Ca2+ transient, binding of Ca2+ to troponin, and subsequent cross-bridge formation (excitation-contraction [EC] coupling). Here, we develop a novel experimental system for simultaneous nano-imaging of intracellular Ca2+ dynamics and single sarcomere length (SL) in rat neonatal cardiomyocytes. We achieve this by expressing a fluorescence resonance energy transfer (FRET)-based Ca2+ sensor yellow Cameleon-Nano (YC-Nano) fused to a-actinin in order to localize to the Z disks. We find that, among four different YC-Nanos, alpha-actinin-YC-Nano140 is best suited for high-precision analysis of EC coupling and a-actinin-YC-Nano140 enables quantitative analyses of intracellular calcium transients and sarcomere dynamics at low and high temperatures, during spontaneous beating and with electrical stimulation. We use this tool to show that calcium transients are synchronized along the length of a myofibril. However, the averaging of SL along myofibrils causes a marked underestimate (similar to 50%) of the magnitude of displacement because of the different timing of individual SL changes, regardless of the absence or presence of positive inotropy (via beta-adrenergic stimulation or enhanced actomyosin interaction). Finally, we find that beta-adrenergic stimulation with 50 nM isoproterenol accelerated Ca2+ dynamics, in association with an approximately twofold increase in sarcomere lengthening velocity. We conclude that our experimental system has a broad range of potential applications for the unveiling molecular mechanisms of EC coupling in cardiomyocytes at the single sarcomere level.