Direct measurement of Ca2+ concentration in the SR of living cardiac myocytes.
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.
Kasai
Hiroki
H
Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
Yao
Atsushi
A
Oyama
Tomomi
T
Hasegawa
Hiroshi
H
Akazawa
Hiroshi
H
Toko
Haruhiro
H
Nagai
Toshio
T
Kinugawa
Koichiro
K
Kohmoto
Osami
O
Maruyama
Kei
K
Takahashi
Toshiyuki
T
Nagai
Ryozo
R
Miyawaki
Atsushi
A
Komuro
Issei
I
D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism*; D006352:Heart Ventricles / Q000378:metabolism*; D007150:Immunohistochemistry; D051381:Rats; D017208:Rats, Wistar; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D002118:Calcium