Simultaneous High-Precision Imaging of Local Calcium and Single Sarcomere Length in Rat Neonatal Cardiomyocytes via Expression of Yellow Cameleon-Nano140 in Z-Discs

Abstract

In the present study, we developed a novel experimental system for simultaneous measurement of changes in the intracellular Ca2+ concentration ([Ca2+]i) at local areas of the T-tubules / Z-discs and single sarcomere length (SL) via expression of FRET-based Ca2+ indicator Yellow Cameleon (YC)-Nano140 in Z-discs (α-actinin) in rat neonatal cardiomyocytes. Under dual-view fluorescence microscopy, local [Ca2+]i was determined by the YFP/CFP fluorescence ratio and SL by the peak-to-peak distance of the YFP fluorescence profile (see Shintani et al., J Gen Physiol 2014 for SL nanometry: S.D., 12 nm in the present study). During spontaneous beating, although [Ca2+]i changed uniformly in a localized area (5 consecutive sarcomeres) of a myocyte, sarcomere shortening / lengthening was not synchronized, but rather occurred in an asynchronous fashion, suggesting that the length of each sarcomere is determined via tug-of-war forces of sarcomeres along the myofibril. Likewise, we successfully quantified amplifications of systolic [Ca2+]i and single sarcomere contraction upon β-adrenergic stimulation. Furthermore, α-actinin-YC-Nano140 enabled high-precision simultaneous measurements of local [Ca2+]i and single sarcomere contraction under electric field stimulation at 5 Hz. The present experimental system has a broad range of application possibilities for analyzing cardiac excitation-contraction (E-C) coupling in detail at the single sarcomere level. Here, we discuss 1) the technical aspects of the application of YC-Nano140 for the analysis of cardiac E-C coupling, and 2) mechanistic implications of single sarcomere dynamics in relation to local [Ca2+]i changes.