Activation and propagation of Ca2+ release during excitation-contraction coupling in atrial myocytes

Fast two-dimensional confocal microscopy and the Ca2+ indicator fluo-4 were used to study excitation-contraction (E-C) coupling in cat atrial myocytes which lack transverse tubules and contain both subsarcolemmal junctional (I-SR) and central nonjunctional (nj-SR) sarcoplasmic reticulum. Action potentials elicited by field stimulation induced transient increases of intracellular Ca2+ concentration ([Ca2+](i)) that were highly inhomogeneous. Increases started at distinct subsarcolemmal release sites spaced similar to2 mum apart. The amplitude and the latency of Ca2+ release from these sites varied from beat to beat. Subsarcolemmal release fused to build a peripheral ring of elevated [Ca2+](i), which actively propagated to the center of the cells via Ca2+-induced Ca2+ release. Resting myocytes exhibited spontaneous Ca2+ release events, including Ca2+ sparks and local (microscopic) or global (macroscopic) [Ca2+](i) waves. The microscopic [Ca2+]l waves propagated in a saltatory fashion along the sarcolemma ("coupled" Ca2+ sparks) revealing the sequential activation of Ca2+ release sites of the j-SR. Moreover, during global [Ca2+](i) waves, Ca2+ release was evident from individual nj-SR sites. Ca2+ release sites were arranged in a regular three-dimensional grid as deduced from the functional data and shown by immunostaining of ryanodine receptor Ca2+ release channels. The longitudinal and transverse distances between individual Ca2+ release sites were both similar to2 tm. Furthermore, electron microscopy revealed a continuous sarcotubular network and one peripheral coupling of j-SR with the sarcolemma per sarcomere. The results demonstrate directly that, in cat atrial myocytes, the action potential-induced whole-cell [Ca2+](i) transient is the spatio-temporal summation of Ca2+ release from subsarcolemmal and central sites. First, j-SR sites are activated in a stochastic fashion by the opening of voltage-dependent sarcolemmal Ca2+ channels. Subsequently, nj-SR sites are activated by Ca2+-induced Ca2+ release propagating from the periphery.