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Journal of Experimental Biology, Vol 203, Issue 3 493-504, Copyright © 2000 by Company of Biologists
JOURNAL ARTICLES |
CL Harwood, FC Howarth, JD Altringham and E White
School of Biomedical Sciences and School of Biology, University of Leeds, Leeds LS2 9JT, UK. charwood@sas.upenn.edu
The effects of increasing stimulation frequency (from 0.2 to 1.4 Hz) on the contractility, intracellular Ca(2+) concentration ([Ca(2+)](i)) and membrane potential of single ventricular myocytes isolated from the heart of rainbow trout (Oncorhynchus mykiss) were measured. Cell shortening, expressed as a percentage of resting cell length, was our index of contractility. The fluorescent Ca(2+) indicator Fura-2 was used to monitor changes in [Ca(2+)](i). Action potentials and L-type Ca(2+) currents (I(Ca)) were recorded using the whole-cell patch-clamp technique. Experiments were performed at 15 degrees C. Increasing the stimulation frequency caused a significant increase in diastolic [Ca(2+)](i) and a significant decrease in diastolic cell length and membrane potential. During systole, there was a significant fall in the amplitude of the [Ca(2+)](i) transient, cell shortening and action potential with a decrease in the duration of the action potential at both 20 % and 90 % repolarisation. Caffeine was used to assess the Ca(2+) content of the sarcoplasmic reticulum. We observed that sarcoplasmic reticulum Ca(2+) load was greater at 1.0 Hz than at 0.6 Hz, despite a smaller electrically evoked [Ca(2+)](i) transient. The amplitude of I(Ca) was found to decrease with increased stimulation frequency. At 0.6 Hz, electrically evoked [Ca(2+)](i) transients in the presence of 10 mmol l(-)(1) caffeine or 10 micromol l(-)(1) ryanodine and 2 micromol l(-)(1) thapsigargin were reduced by approximately 15 %. We have described the changes in contractility, [Ca(2+)](i) and action potential configuration in a fish cardiac muscle system. Under the conditions tested (0.6 Hz, 15 degrees C), we conclude that the sarcoplasmic reticulum contributes at least 15 % of the Ca(2+) associated with the [Ca(2+)](i) transient. The rate-dependent decrease in contraction amplitude appears to be associated with the fall in the amplitude of the [Ca(2+)](i) transient. This, in turn, may be influenced by changes in the action potential configuration via mechanisms such as altered Ca(2+) efflux and Ca(2+) influx. In support of our conclusions, we present evidence that there is a rate-dependent decrease in Ca(2+) influx via I(Ca) but that the Ca(2+) load of the sarcoplasmic reticulum is not reduced at increased contraction frequencies.
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