Abstract
With ultra-fast 1D-(x-t) and 2D-(x-y-t) confocal microscopy (Zeiss LSM 5 Live) we studied the spatio-temporal properties of Ca sparks and Ca transients. Ca sparks which originated from subsarcolemmal sarcoplasmic reticulum (SR) release sites in atrial myocytes were elongated in the longitudinal direction of the cell. Ca sparks corresponding to Ca release from non-junctional SR in atrial myocytes and junctional SR in ventricular myocytes were variable spatially with some events being symmetrical and others asymmetrical. Anisotropic sparks occurred in transverse as well as longitudinal direction. Ca sparks originating from non-junctional SR and recorded in line-scan (x-t) mode at 40,000 lines/s revealed a step-like appearance in space (time-dependent step-like increase of width from the point of origin) and amplitude during the activation phase of the spark. These steps in space and amplitude may represent the sequential opening of individual ryanodine receptor (RyR) channels in a release cluster and support the notion that sparks represent Ca release from a group of RyRs. Mathematical analysis of global Ca transients recorded from field-stimulated ventricular myocytes at high temporal resolution allowed separation of Ca entry from Ca release flux. After the electrical stimulus a latency period of 2.5 ms was required to activate sarcolemmal Ca channels. SR Ca release was initiated with an additional delay of 3.0 ms. Maximal Ca release flux was observed 3.9 ms after initiation of Ca release when all release sites became active. Ca entry flux was ten times smaller than Ca release flux. When SR Ca release was eliminated after treatment with thapsigargin and caffeine, identical Ca entry flux was observed as in control conditions. In summary, ultra-fast confocal imaging allows investigation of Ca signals with time resolution similar to patch clamp technique, however in a much less invasive fashion.
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