H. Tanaka, M. Oyamada, E. Tsujii, T. Nakajo and T. Takamatsu. Excitation-Dependent Intracellular Ca²⁺ Waves at the Border Zone of the Cryo-injured Rat Heart Revealed by Real-time Confocal Microscopy. Journal of Molecular and Cellular Cardiology (2002) 34, 1501–1512. Intracellular Ca²⁺ waves, which develop under Ca²⁺-overloaded conditions of the injured myocardium, are regarded as an important substrate for triggered arrhythmias. However, little is known about whether Ca²⁺ waves arise or become proarrhythmic in the injured heart in situ. On the hypothesis that injured myocardium manifests frequent Ca²⁺ waves and produce an oscillatory [Ca²⁺]_i rise leading to triggered activity, we applied cryo-injury to the epicardial surface of fluo 3-AM-loaded perfused rat hearts and analyzed spatiotemporal [Ca²⁺]_i changes at border zones of the injured myocardium using real-time confocal microscopy. In intact regions Ca²⁺ waves barely emerged, whereas the border zone myocardium exhibited frequent Ca²⁺ waves, propagating randomly within the individual cells. Two different types of Ca²⁺ waves were identified: highly frequent waves (159.6±86.5waves/min/cell, n=266) adjacent to the cryo-ablated regions, and less frequent waves (79.0±50.1waves/min/cell, n=160) slightly farther (>2 cells) away from the ablated regions (vicinities). The former Ca²⁺ waves emerged asynchronously to Ca²⁺ transients. Contrariwise, the latter depended on ventricular excitation: they vanished instantaneously on Ca²⁺ transients, but emerged more frequently and propagated more swiftly after cessation of higher-frequency pacing. Immediately after 3-Hz pacing, some cryo-injured hearts exhibited oscillatory [Ca²⁺]_i rises; an instantaneous and synchronous elevation of [Ca²⁺]_i followed by burst occurrence of Ca²⁺ waves with a gradual decrease in incidence and propagation velocity in a considerable number of cells. These observations indicate that myocardial injury induces Ca²⁺ waves in the heart, and that their synchronous occurrence could become a substrate for triggered arrhythmias.