Temporal and spatial properties of cellular Ca2+ flux in trout ventricular myocytes

Confocal microscopy was used to investigate the temporal and spatial properties of Ca2+ transients and Ca2+ sparks in ventricular myocytes of the rainbow trout (Oncorhynchus mykiss). Confocal imaging confirmed the absence of T tubules and the long (similar to 160 mu m), thin (similar to 8 mu m) morphology of trout myocytes. Line scan imaging of Ca2+ transients evoked by electrical stimulation in cells loaded with fluo 4 revealed spatial inhomogeneities in the temporal properties of Ca2+ transients across the width of the myocytes. The Ca2+ wavefront initiated faster, rose faster, and reached larger peak amplitudes in the periphery of the myocyte compared with the center. These differences were exacerbated by stimulation with the L-type Ca2+ channel agonist (-) BAY K 8644 or by sarcoplasmic reticulum (SR) inhibition with ryanodine and thapsigargin. Results reveal that the shape of the trout myocyte allows for rapid diffusion of Ca2+ from the cell periphery to the cell center, with SR Ca2+ release contributing to the cytosolic Ca2+ rise in a time-dependent manner. Spontaneous Ca2+ sparks were exceedingly rare in trout myocytes under control conditions (1 sparking cell from 238 cells examined). This is in marked contrast to the rat where a total of 56 spontaneous Ca2+ sparks were observed in 9 of 11 myocytes examined. Ca2+ sparklike events were observed in a very small number of trout myocytes (15 sparks from 9 of 378 cells examined) after stimulation with either (-) BAY K 8644 or high Ca2+ ( 6 mM). Reducing temperature to 15 degrees C in intact myocytes or perme-abilizing myocytes to adjust intracellular conditions to favor Ca2+ spark detection was without significant effects. Possible reasons for the rarity of Ca2+ sparks in a cardiac myocyte with an active SR are discussed.