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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY

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

School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom

Submitted 28 July 2004 ; accepted in final form 11 January 2005

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.

calcium sparks; calcium inhomogeneities; calcium transients; ryanodine receptor isoform; sarcoplasmic reticulum

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