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

Abstract

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 (∼160 μm), thin (∼8 μm) 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°C in intact myocytes or permeabilizing 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.