Simultaneous Imaging of Local Calcium and Single Sarcomere Length in Rat Neonatal Cardiomyocytes via Expression of Cameleon-Nano in Z-Discs

Abstract

In cardiac muscle, contraction is regulated by micromolar concentrations of Ca2+ released from the sarcoplasmic reticulum (SR) (i.e., Ca2+-induced Ca2+ release), resulting in the binding of Ca2+ to troponin C and subsequent formation of cross-bridges. In order to enhance our understanding of cardiac excitation-contraction coupling, we in the present study developed a novel experimental system for simultaneous measurement of local intracellular Ca2+ ([Ca2+]i) and single sarcomere length via expression of yellow cameleon-Nano (i.e., FRET-based ultrasensitive Ca2+ indicator) fused to the C-terminus of α-actinin in Z-discs in primary-cultured rat neonatal cardiomyocytes. In a given sarcomere, the fluorescence emission ratio (YFP/CFP) of the expressed fusion protein varied in response to a change in local [Ca2+]i. Under dual-view microscopy, we simultaneously measured local [Ca2+]i and sarcomere length (the latter defined as the distance between the YFP fluorescence profiles), and found that sarcomere length varied in response to a change in YFP/CFP in various local regions of the myocyte. These results demonstrate that the present experimental system is useful for elucidating cardiac excitation-contraction coupling at the single sarcomere level.