Direct measurement of Ca2+ concentration in the SR of living cardiac myocytes |
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| Authors: | Kasai Hiroki Yao Atsushi Oyama Tomomi Hasegawa Hiroshi Akazawa Hiroshi Toko Haruhiro Nagai Toshio Kinugawa Koichiro Kohmoto Osami Maruyama Kei Takahashi Toshiyuki Nagai Ryozo Miyawaki Atsushi Komuro Issei |
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| Affiliation: | Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan. |
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| Abstract: | Although abnormal sarcoplasmic reticulum (SR) Ca(2+) handling may cause heart failure, there has been no method to directly measure Ca(2+) concentration in SR ([Ca(2+)](SR)) of living cardiomyocytes. We have measured [Ca(2+)](SR) by expressing novel fluorescent Ca(2+) indicators yellow cameleon (YC) 2.1, YC3er, and YC4er in cultured neonatal rat cardiomyocytes. The distribution of YC2.1 was uniform in the cytoplasm, while that of YC3er/YC4er, containing the signal sequence which recruits them to SR, showed reticular pattern and was co-localized with SERCA2a. The treatment with caffeine reversibly decreased the emission ratio (R) in YC3er/YC4er-expressing myocytes, and the treatment with ryanodine and thapsigargin decreased R irreversibly. During the contraction-relaxation cycle, R was changed periodically in the YC2.1- and YC3er-expressing myocytes, but its direction of the change was opposite. These results suggest that YC3er/YC4er were specifically localized and functioned in SR as a [Ca(2+)](SR) indicator. This technique would be useful to understand the function of SR in failing myocardium. |
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| Keywords: | Sarcoplasmic reticulum Fluorescent Ca2+ indicators Yellow cameleon Real-time monitoring Cardiomyocyte Caffeine Thapsigargin SERCA Heart failure |
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