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 μM, 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.
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois.
authors.forename: Elisa
authors.initials: E
authors.lastname: Bovo
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois.
authors.forename: Jody L
authors.initials: JL
authors.lastname: Martin
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois.
authors.forename: Jollyn
authors.initials: J
authors.lastname: Tyryfter
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois.
authors.forename: Pieter P
authors.initials: PP
authors.lastname: de Tombe
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois azima@luc.edu.
authors.forename: Aleksey V
authors.initials: AV
authors.lastname: Zima
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D032383:Myocytes, Cardiac / Q000378:metabolism*; D011817:Rabbits; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D014162:Transfection; D014966:Xanthenes / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D000814:Aniline Compounds; D002118:Calcium; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014966:Xanthenes; D000097573:Red Fluorescent Protein; C409648:Fluo 4
keywords: calcium indicators; confocal microscopy; excitation-contraction coupling; sarcoplasmic reticulum; ventricular myocytes