Measurement of Free Ca2+ in Sarcoplasmic Reticulum in Perfused Rabbit Heart Loaded with 1,2-Bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N′,N′-tetraacetic Acid by 19F NMR

Abstract

Measurements of free calcium ion concentration in the sarcoplasmic reticulum ([Ca2+]SR) and an evaluation of its relationship to changes in cytosolic free calcium and energy state of the cell, as well as heterogeneity of the SR calcium pool, were performed using 19F NMR in Langendorff perfused rabbit hearts loaded with acetoxymethyl ester of 1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid. We report a base-line time-average [Ca2+]SR value of 1.5 mM (n = 13) in the beating heart, similar to the value measured at diastole. We further report that [Ca2+]SR decreases by approximately 30% at the start of systole and that there is no evidence of spacial heterogeneity in [Ca2+]SR during the contraction cycle. However, there appears to be a heterogeneous response to SR calcium channel release activator (caffeine) and SR calcium-ATPase inhibitor (cyclopiazonic acid), consistent with studies suggesting that there are subpopulations of SR. Raising cytosolic free calcium by depolarizing the cell with 30 mM extracellular KCl, resulted in an increase in [Ca2+]SR; however, the calcium gradient was unchanged. Lowering cell phosphorylation potential, which would reduce the free energy available for the SR Ca2+-ATPase, leads to a decrease in the calcium gradient across the SR, but this reduced gradient was primarily due to an increase in cytosolic free calcium and not a net release of SR calcium.