title: Real-time intra-store confocal Ca2+imaging in isolated mouse cardiomyocytes.
abstract: To initiate the contraction of cardiomyocytes, Ca2+is released from the SR to the cytosol via ryanodine receptors (RyRs), which are activated by the Ca2+-induced Ca2+release mechanism (CICR). The activity of RyRs is regulated by both, cytosolic and SR luminal Ca2+. Deregulation of the CICR, by dysfunctional SR Ca2+release or uptake, is frequently associated with cardiac pathologies (e.g. arrhythmias, CPVT, heart failure). Recently, the interest to directly measure changes of the free Ca2+concentration within the SR ([Ca2+]SR) has led to the application of low affinity Ca2+indicators (mag-fluo-4, Fluo-5N) to follow changes of [Ca2+]SRin cardiomyocytes from some species. However, direct measurement of Ca2+signals from the SR have not been possible in freshly isolated mouse cardiomyocytes. Here, we show a new protocol optimized to measure changes of [Ca2+]SRin mouse cardiomyocytes using fluorescent Ca2+indicators and confocal microscopy. The application of this protocol permits the design of experimental studies with direct evaluation of SR Ca2+in real time in various mouse models of cardiac disease, including transgenic animals harboring mutants of RyRs or other Ca2+signaling proteins. The technique, in combination with these models, will help to understand how these diseases and mutations affect Ca2+signals within the SR and the Ca2+sensitivity of the RyRs for cytosolic and SR luminal Ca2+, thereby contributing to arrhythmias or weak heart beat.
authors.affiliation: Department of Physiology, University of Bern, 3012 Bern, Switzerland.
authors.forename: Miguel
authors.initials: M
authors.lastname: Fernandez-Tenorio
authors.affiliation: Department of Physiology, University of Bern, 3012 Bern, Switzerland. Electronic address: niggli@pyl.unibe.ch.
authors.forename: Ernst
authors.initials: E
authors.lastname: Niggli
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D020013:Calcium Signaling; D051379:Mice; D018613:Microscopy, Confocal; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013997:Time Factors
chemical_list: D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium
keywords: Calcium signaling; Cardiac myocytes; Confocal calcium imaging; Excitation-contraction coupling; Sarcoplasmic reticulum