R-CEPIA1er as a new tool to directly measure sarcoplasmic reticulum [Ca] in ventricular myocytes Journal Article


Authors: Bovo, E; Martin, J. L.; Tyryfter, J.; de Tombe, P. P.; Zima, A. V.
Article Title: R-CEPIA1er as a new tool to directly measure sarcoplasmic reticulum [Ca] in ventricular myocytes
Abstract: In cardiomyocytes, [Ca] within the sarcoplasmic reticulum (SR; [Ca]SR) partially determines the amplitude of cytosolic Ca transient that, in turn, governs myocardial contraction. Therefore, it is critical to understand the molecular mechanisms that regulate [Ca]SR handling. Until recently, the best approach available to directly measure [Ca]SR was to use low-affinity Ca indicators (e.g., Fluo-5N). However, this approach presents several limitations, including nonspecific cellular localization, dye extrusion, and species limitation. Recently a new genetically encoded family of Ca indicators has been generated, named Ca-measuring organelle-entrapped protein indicators (CEPIA). Here, we tested the red fluorescence SR-targeted Ca sensor (R-CEPIA1er) as a tool to directly measure [Ca]SR dynamics in ventricular myocytes. Infection of rabbit and rat ventricular myocytes with an adenovirus expressing the R-CEPIA1er gene displayed prominent localization in the SR and nuclear envelope. Calibration of R-CEPIA1er in myocytes resulted in a Kd of 609 muM, suggesting that this sensor is sensitive in the whole physiological range of [Ca]SR [Ca]SR dynamics measured with R-CEPIA1er were compared with [Ca]SR measured with Fluo5-N. We found that both the time course of the [Ca]SR depletion and fractional SR Ca release induced by an action potential were similar between these two Ca sensors. R-CEPIA1er fluorescence did not decline during experiments, indicating lack of dye extrusion or photobleaching. Furthermore, measurement of [Ca]SR with R-CEPIA1er can be combined with cytosolic [Ca] measurements (with Fluo-4) to obtain more detailed information regarding Ca handling in cardiac myocytes. In conclusion, R-CEPIA1er is a promising tool that can be used to measure [Ca]SR dynamics in myocytes from different animal species.
Journal Title: American journal of physiology.Heart and circulatory physiology
Volume: 311
Issue: 1
ISSN: 1522-1539; 0363-6135
Publisher: the American Physiological Society  
Journal Place: United States
Date Published: 2016
Start Page: H268
End Page: 75
Language: eng
DOI/URL:
Notes: LR: 20160713; CI: Copyright (c) 2016; GR: R01 HL130231/HL/NHLBI NIH HHS/United States; JID: 100901228; OTO: NOTNLM; 2016/02/23 [received]; 2016/05/24 [accepted]; 2016/05/27 [aheadofprint]; ppublish