Niggli, Ernst , FERNANDEZ TENORIO, MIGUEL
No
Methods Mol. Biol.
Capítulo de un Libro
Científica
0
0
01/01/2019
000893300500006
An increase in the cytosolic Ca2+ concentration triggers the contraction in cardiomyocytes. In these cells sarcoplasmic reticulum (SR) is the major source of Ca2+, and the release from this store is mediated by the ryanodine receptors (RyRs). These receptors are regulated by cytosolic and intra-SR [Ca2+]. The cytosolic Ca2+ regulation is well established, but there are some limitations to determine indirectly the intra-SR Ca2+ concentration and understand its role in the RyRs regulation. Therefore, the interest to directly measure the free intra-SR Ca2+ concentration ([Ca2+](SR)) has led to the application of a low-affinity Ca2+ indicator (Fluo-5N AM) to follow changes of [Ca2+](SR) in cardiomyocytes. However the loading of this AM-ester dye into the SR has remained a challenge in freshly isolated mouse cardiomyocytes. Here, we describe an optimized protocol to measure changes of [Ca2+](SR) in mouse cardiomyocytes using fluorescent Ca2+ indicators and confocal microscopy. The application of this protocol allows to evaluate directly intra-SR Ca2+ in real time in various mouse models of cardiac disease, including transgenic animals harboring mutants of RyRs or other Ca2+ signaling proteins.
Calcium signaling; Cardiomyocytes; Fluo-5N; Confocal calcium imaging; Cell permeabilization; Sarcoplasmic reticulum; Excitation-contraction coupling