Ca2+ Release Events in Cardiac Myocytes Up Close: Insights from Fast Confocal Imaging

Abstract

The spatio-temporal properties of Ca2+ transients during excitation-contraction coupling and elementary Ca2+ release events (Ca2+ sparks) were studied in atrial and ventricular myocytes with ultra-fast confocal microscopy using a Zeiss LSM 5 LIVE system that allows sampling rates of up to 60 kHz. Ca2+ sparks which originated from subsarcolemmal junctional sarcoplasmic reticulum (j-SR) release sites in atrial myocytes were anisotropic and elongated in the longitudinal direction of the cell. Ca2+ sparks in atrial cells originating from non-junctional SR and in ventricular myocytes were symmetrical. Ca2+ spark recording in line scan mode at 40,000 lines/s uncovered step-like increases of [Ca2+]i. 2-D imaging of Ca2+ transients revealed an asynchronous activation of release sites and allowed the sequential recording of Ca2+ entry through surface membrane Ca2+ channels and subsequent activation of Ca2+-induced Ca2+ release. With a latency of 2.5 ms after application of an electrical stimulus, Ca2+ entry could be detected that was followed by SR Ca2+ release after an additional 3 ms delay. Maximum Ca2+ release was observed 4 ms after the beginning of release. The timing of Ca2+ entry and release was confirmed by simultaneous [Ca2+]i and membrane current measurements using the whole cell voltage-clamp technique. In atrial cells activation of discrete individual release sites of the j-SR led to spatially restricted Ca2+ release events that fused into a peripheral ring of elevated [Ca2+]i that subsequently propagated in a wave-like fashion towards the center of the cell. In ventricular myocytes asynchronous Ca2+ release signals from discrete sites with no preferential subcellular location preceded the whole-cell Ca2+ transient. In summary, ultra-fast confocal imaging allows investigation of Ca2+ signals with a time resolution similar to patch clamp technique, however in a less invasive fashion. © 2013 Shkryl, Blatter.