Sarcomere Length Nanometry in Cardiomyocytes Expressed with α-Actinin-AcGFP in Z-Discs

Abstract

Nanometry is widely used in today's biological sciences to analyze the movement of molecules or molecular assemblies in cells and in vivo. In cardiac muscle, a change in sarcomere length (SL) by a mere ∼100 nm causes a dramatic change in contractility (i.e., the Frank-Starling mechanism), indicating the need for the simultaneous measurement of SL and intracellular Ca2+ concentration ([Ca2+]i) in cardiomyocytes at high spatial and temporal resolution. To accurately analyze the motion of individual sarcomeres with nanometer precision during excitation-contraction coupling, we in the present study applied nanometry techniques to primary-cultured rat neonatal cardiomyocytes. First, we developed an experimental system for simultaneous nano-scale analysis of single sarcomere dynamics and [Ca2+]i changes via the expression of AcGFP in Z-discs. We found that the averaging of the lengths of sarcomeres along the myocyte caused marked underestimation of sarcomere lengthening speed due to superpositioning of different timings for lengthening between sequentially connected sarcomeres. Then, we found that following the treatment with ionomycin, neonatal myocytes exhibited spontaneous sarcomeric oscillations (Cell-SPOC) at partial activation with blockage of sarcoplasmic reticulum functions, and the waveform properties were indistinguishable from those obtained in electric field stimulation. The myosin activator, omecamtiv mecarbil, markedly enhanced Z-disc displacement during Cell-SPOC. Finally, we interpreted the present experimental findings in the framework of our mathematical model of spontaneous sarcomeric oscillations (Sato, K. et al., Prog Biophys Mol Biol. 2011; Sato, K. et al., Phys. Rev. Lett. 2013). The present experimental system has a broad range of application possibilities for unveiling single sarcomere dynamics during excitation-contraction coupling in cardiomyocytes under various settings.