title: Sarcomere length nanometry in rat neonatal cardiomyocytes expressed with α-actinin-AcGFP in Z discs.
abstract: Nanometry is widely used in 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 substantial change in contractility, indicating the need for the simultaneous measurement of SL and intracellular Ca(2+) concentration ([Ca(2+)]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 applied nanometry techniques to primary-cultured rat neonatal cardiomyocytes. First, we developed an experimental system for simultaneous nanoscale analysis of single sarcomere dynamics and [Ca(2+)]i changes via the expression of AcGFP in Z discs. We found that the averaging of the lengths of sarcomeres along the myocyte, a method generally used in today's myocardial research, caused marked underestimation of sarcomere lengthening speed because of the superpositioning of different timings for lengthening between sequentially connected sarcomeres. Then, we found that after treatment with ionomycin, neonatal myocytes exhibited spontaneous sarcomeric oscillations (cell-SPOCs) 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 SPOCs. 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.
authors.affiliation: Department of Pure and Applied Physics, Faculty of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 169-8555, Japan.
authors.forename: Seine A
authors.initials: SA
authors.lastname: Shintani
authors.forename: Kotaro
authors.initials: K
authors.lastname: Oyama
authors.forename: Fuyu
authors.initials: F
authors.lastname: Kobirumaki-Shimozawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Ohki
authors.forename: Shin'ichi
authors.initials: S
authors.lastname: Ishiwata
authors.forename: Norio
authors.initials: N
authors.lastname: Fukuda
mesh_terms: D000185:Actinin / Q000096:biosynthesis*; D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000096:biosynthesis*; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000648:ultrastructure; D036103:Nanotechnology / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar; D012518:Sarcomeres / Q000378:metabolism* / Q000648:ultrastructure*
chemical_list: D000185:Actinin; D049452:Green Fluorescent Proteins