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 Ca(2+) transient, binding of Ca(2+) to troponin, and subsequent cross-bridge formation (excitation-contraction [EC] coupling). Here, we develop a novel experimental system for simultaneous nano-imaging of intracellular Ca(2+) dynamics and single sarcomere length (SL) in rat neonatal cardiomyocytes. We achieve this by expressing a fluorescence resonance energy transfer (FRET)-based Ca(2+) sensor yellow Cameleon-Nano (YC-Nano) fused to α-actinin in order to localize to the Z disks. We find that, among four different YC-Nanos, α-actinin-YC-Nano140 is best suited for high-precision analysis of EC coupling and α-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 (∼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 β-adrenergic stimulation or enhanced actomyosin interaction). Finally, we find that β-adrenergic stimulation with 50 nM isoproterenol accelerated Ca(2+) 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.
Department of Cell Physiology, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.
Seiichi
S
Tsukamoto
Department of Cell Physiology, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.
Teruyuki
T
Fujii
Department of Cell Physiology, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.
Kotaro
K
Oyama
Department of Physics, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
Seine A
SA
Shintani
Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University, Sinjuku-ku, Tokyo 162-8480, Japan.
Togo
0000-0003-2292-8785
T
Shimozawa
Department of Cell Physiology, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.
Fuyu
0000-0001-7054-1935
F
Kobirumaki-Shimozawa
Department of Physics, Faculty of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 169-8555, Japan.
Shin'ichi
S
Ishiwata
Department of Cell Physiology, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan noriof@jikei.ac.jp.
Norio
N
Fukuda
D000185:Actinin / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism*; D008164:Luminescent Proteins / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000166:cytology*; D036103:Nanotechnology; D051381:Rats; D012518:Sarcomeres / Q000378:metabolism*
D008164:Luminescent Proteins; D000185:Actinin; D002118:Calcium
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