Ratio-metric measurement of intracellular calcium in visceral muscles via selective expression of a yellow cameleon calcium sensor

Visceral muscles exhibit various patterns of contractions depending on their locations and functions. Most visceral muscles are comprised of smooth muscle, and an increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) induces contraction, as in striated muscle. In order to critically assess the mechanisms underlying a variety of visceral movements, it is important to measure [Ca²⁺]_i. However, due to limitations of sensing methods, it is difficult to compare [Ca²⁺]_i between different organs and individuals. In this study, we thus measured [Ca²⁺]_i in visceral muscles using transgenic mice that selective expressed a genetically encoded ratiometric Ca²⁺ indicator (YC-Nano50) in both skeletal and smooth muscles. We acquired CFP and YFP fluorescence images of YC-Nano50 separately, to evaluate the fluorescence resonance energy transfer (FRET), which reflects [Ca²⁺]_i. In the resting condition, the YFP/CFP ratio measured in isolated muscularis was lower in smooth muscle than in skeletal muscle, corresponding to [Ca²⁺]_i in the range of approximately 20–40 nM and 50–80 nM, respectively. Among visceral smooth muscles, the YFP/CFP ratio was highest in the urinary bladder, and lowest in the stomach (antrum). In the esophagus, comprised of skeletal muscle, the YFP/CFP ratio was higher than in other visceral smooth muscles, and was comparable to that in abdominal wall and diaphragm skeletal muscles. In addition, we were able to measure Ca²⁺ transients and oscillations in muscle sheets and intact segments isolated from the intestine even during movement. This method is able to elucidate the biological significance of Ca²⁺ signaling in diverse functions of visceral muscles.