PARTIAL INHIBITION OF CA2+ CURRENT BY METHOXYVERAPAMIL (D600) REVEALS SPATIAL NONUNIFORMITIES IN [CA2+](I) DURING EXCITATION-CONTRACTION COUPLING IN CARDIAC MYOCYTES

The laser scanning confocal microscope was used in conjunction with the Ca2+ indicator flue 3 to examine the spatiotemporal properties of free Ca2+ ([Ca2+](i)) transients in isolated rat cardiac myocytes. We show that localized increases in [Ca2+](i) (Ca2+ sparks) can be triggered by membrane depolarization in cardiac myocytes when the sarcolemmal Ca2+ current amplitude is reduced by methoxyverapamil (D600). These depolarization-evoked Ca2+ sparks are similar in amplitude and spatiotemporal properties to spontaneous Ca2+ sparks previously observed at rest. These observations support the idea that Ca2+ sparks are the result of the activation of functional elementary units of sarcoplasmic reticulum (SR) Ca2+ release. The synchronous activation of a large number of Ca2+ sparks can explain the increased amplitude and slower time course of the electrically evoked [Ca2+](i) transient as well as the presence of spatial nonuniformities in [Ca2+](i) during its rise. The data shown here suggest a model for excitation-contraction coupling in which the amplitude of the [Ca2+](i) transient is regulated by variations in the probability of recruitment of elementary SR Ca2+ release units as well as the amount of Ca2+ released by each unit. Since the activation of each release unit will depend on the local amplitude of the Ca2+ current, this model can explain the regulation of the amplitude of the [Ca2+](i) transient by the Ca2+ current. In addition, these data indicate that caution should be applied to the interpretation of signals obtained with nonlinear Ca2+ indicators during the rising phase of the [Ca2+](i) transient, when the nonuniformities in [Ca2+](i) are largest.