Investigation of Arrhythmogenic Calcium Events by Initiating Local Calcium Release in Cardiomyocytes

In cardiac muscle the sarcoplasmic reticulum (SR) contains the Ca2+ that is released during excitation-contraction coupling to initiate cross-bridge cycling, sarcomere shortening and force generation. Under physiological conditions SR Ca2+ uptake by the SR/ER Ca2+ ATPase is balanced by Ca2+ “leak” out of the SR through the SR Ca2+ release channels (ryanodine receptors, RyR2). Under diverse conditions SR Ca2+ overload can develop (i.e. elevation of [Ca2+]SR) and this is associated with an increase in the open probability of the RyR2s which leads to SR Ca2+ instability. SR Ca2+ overload is thus associated with an increased Ca2+ spark rate, increased “invisible” SR Ca2+ leak and the development of a propagating chain-reaction of Ca2+-induced Ca2+ release (Ca2+ wave) within the ventricular myocyte. This abnormal Ca2+ signaling activates the sarcolemmal Na+/Ca2+ exchanger to produce an arrhythmic inward current. In order to investigate SR Ca2+ release under these conditions, local Ca2+ release was produced by photolysis of a caged paraxanthine (BiNiX, 3-(4,5-bis(carboxymethyl)-2-nitrobenzyl)-paraxanthine), a caffeine-like “activator” of RyR2. Using a newly developed photolysis system, laser flashes (354 nm) of different sizes and shapes were rapidly (millisecond) positioned on the focused image plane of a confocal microscope. A spectrum of responses in murine cardiac myocytes was observed ranging from instigating Ca2+ sparks to triggering Ca2+ waves. Eliciting a fully propagating Ca2+ wave required SR Ca2+ overload and/or an increased sensitivity of RyR2s to cytosolic Ca2+. This study was uniquely successful in triggering multiple Ca2+ waves simultaneously. Creating multiple Ca2+ wave fronts in a myocyte enabled the generation of an extrasystole triggered by a delayed after-depolarization. This new experimental technology allows systematic investigation of rare extrasystoles triggered by Ca2+ waves.