In situ visualization of spontaneous calcium waves within perfused whole rat heart by confocal imaging

We describe the first direct visualization of Ca2+ oscillations in the perfused whole rat heart. Dye loading at a low temperature and enhanced optical-section techniques of confocal microscopy by elimination of the refractive index mismatch with use of saline-immersible objective lens enabled us to image multiple Ca2+ waves in the subepicardial myocardium of the fluo 3-loaded heart. These Ca2+ waves were sporadically seen even with a physiological extracellular Ca2+ perfusion in either a paced or an arrested heart and propagated beyond cellular boundaries within the three-dimensional structures of cardiac muscle. Under these conditions, the velocity of wave propagation was 60-100 microns/s and the frequency of initiation was relatively low (< 2 Hz). With an increase in extracellular Ca2+ concentration, however, the waves became more prevalent and tended to be multifocal, and an increasing fraction of the waves exhibited faster propagation velocities and higher frequencies. These results suggest that perfused rat hearts exhibit spontaneous Ca2+ waves in an apparently resting state and that under Ca(2+)-overload conditions the multifocal and high-frequency waves become more widespread in the heart syncytium, which may provide an understanding of the ionic basis for the summation of afterdepolarizations and triggering of arrhythmias seen under pathological conditions.