CALCIUM GRADIENTS DURING EXCITATION-CONTRACTION COUPLING IN CAT ATRIAL MYOCYTES

1. Confocal microscopy in combination with the calcium-sensitive fluor escent probe fluo-3 was used to study spatial aspects of intracellular Ca2+ signals during excitation-contraction coupling in isolated atria l myocytes from cat heart. 2. Imaging of [Ca2+](i) transients evoked b y electrical stimulation revealed that Ca2+ release started at the per iphery and subsequently spread towards the centre of the myocyte. 3. B locking sarcoplasmic reticulum (SR) Ca2+ release with 50 mu M ryanodin e unmasked spatial inhomogeneities in the [Ca2+](i) signal caused sole ly by voltage-dependent Ca2+ influx. During the first 70-100 ms after stimulation [Ca2+](i) was higher in the periphery than in central regi ons of the myocyte. 4. Positive (or negative) staircase or postrest po tentiation of the 'whole-cell' [Ca2+](i) transients were paralleled by characteristic changes in the spatial profile of the [Ca2+](i) signal . With low SR Ca2+ load [Ca2+](i) transients in the subsarcolemmal spa ce were small and no Ca2+ release in the centre of the cell was observ ed. Loading of the SR increased subsarcolemmal [Ca2+](i) transient amp litude and subsequently triggered further release in more central regi ons of the cell. 5. Spontaneous Ca2+ release from functional XR units, i.e. Ca2+ sparks, occurred at higher frequency in the subsarcolemmal space than in more central regions of the myocyte. 6. Visualization of the surface membrane using the membrane-selective dye Di-8-ANEPPS dem onstrated that transverse tubules (t-tubules) were absent in atrial ce lls. 7. It is concluded that in atrial myocytes voltage-dependent Ca2 entry triggers Ca2+-release from peripheral coupling SR that subseque ntly induces further Ca2+ release from stores in more central. regions of the myocyte. Spreading of Ca2+ release from the cell periphery to the centre accounts for [Ca2+](i) gradients underlying the whole-cell [Ca2+](i) transient. The finding that cat atrial myocytes lack t-tubul es demonstrates the functional importance of Ca2+ release from extende d junctional (corbular) SR in these cells.