Partial Inhibition of Ca 2+ Current by Methoxyverapamil (D600) Reveals Spatial Nonuniformities in [Ca 2+ ] i During Excitation-Contraction Coupling in Cardiac Myocytes

Abstract

The laser scanning confocal microscope was used in conjunction with the Ca²⁺ indicator fluo 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.