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Temperature dependence of cardiac sarcoplasmic reticulum function in rainbow trout myocytes

Holly A. Shiels^1,*, Matti Vornanen² and Anthony P. Farrell¹

¹ Simon Fraser University, Biological Sciences, Burnaby, British Columbia, V5A 1S6, Canada
² University of Joensuu, Department of Biology, PO Box 111, 80101 Joensuu, Finland

View larger version (15K): [in a new window]   Fig. 1. The atrial action potential (AP) from isolated rainbow trout myocytes. AP waveforms were elicited by current-clamp at a rate that corresponded to the heart rate for rainbow trout in vivo at each temperature (0.6 Hz at 7°C, 1.0 Hz at 14°C and 1.4 Hz at 21°C). The steady-state APs from 6-10 cells at each temperature were averaged, and the resultant waveforms were used to stimulate L-type Ca2+ channel current (ICa) and sarcoplasmic reticulum (SR) Ca2+ accumulation in subsequent experiments where the inter-pulse holding level was -80 mV.

View larger version (13K): [in a new window]   Fig. 2. A representative recording of the inward Na+/Ca2+ exchanger (NCX) current (INCX) resulting from caffeine application in a trout atrial myocyte at 14°C. This current would be accompanied by a large contraction (not shown). The inset shows the time integral of the current that was used to calculate the Ca2+ content of the sarcoplasmic reticulum (SR) at the time that caffeine was applied.

View larger version (20K): [in a new window]   Fig. 3. The effect of pulse number, temperature, frequency and stimulus waveform on sarcoplasmic reticulum (SR) Ca2+ accumulation in rainbow trout atrial myocytes. At each temperature, myocytes were exposed to caffeine to release the SR Ca2+ stores (not shown) and then stimulated with an increasing number of stimulation pulses between 1 and 100. Stimulation pulse waveform and frequency are given in each panel. SR Ca2+ content is expressed in pC pF-1, and the corresponding values for Ca2+ concentration (µmol l-1) are given in the text. (A) The effect of temperature and pulse number on SR Ca2+ content when cells were stimulated with square (SQ) pulses applied at 1.0 Hz. The results at 21°C are given by a broken line, as only four out of eight cells achieved any SR Ca2+ content under these loading conditions (see text for details). (B) The effect of applying SQ pulses at a frequency that corresponds to the heart rate of rainbow trout in vivo at each test temperature on SR Ca2+ accumulation. # indicates an increase in SR Ca2+ content between 25 and 100 pulses at 7°C, and also a difference between 100 pulses at 0.6 Hz and 100 pulses at 1.0 Hz (part A) at 7°C. (C) The effect of temperature and frequency on SR Ca2+ content when action potentials (APs) were applied at a frequency that corresponds to the heart rate of rainbow trout in vivo at each test temperature. * indicates that the SR content after AP pulses is significantly greater than after SQ pulses. All values are means ± S.E.M. of 4-16 cells at each temperature. In (A) and (B), the lines were drawn by fitting the data to an asymptotic exponential function. In (C), the line is plotted through the data without a curve fit.

View larger version (11K): [in a new window]   Fig. 4. Consecutive measurement of L-type Ca2+ channel current (ICa), 1-25 pulses after caffeine application in a representative rainbow trout atrial myocyte at 21°C receiving square (SQ) stimulation pulses (-80 mV to + 10 mV, 200 ms). The green traces indicate the original curve, and the solid black lines are the double exponential fit (Clampfit, Axon Instruments). The slow inactivation kinetics (s) for this cell were unchanged by progressive SR Ca2+ loading (s = approximately 25 ms at each pulse). However, the fast inactivation kinetics (f) became faster with an increasing number of pulses going from 7.1 ms, 6.7 ms, 5.2 ms, 4.5 ms, 3.9 ms to 4.1 ms after 1 pulse, 5 pulses, 10 pulses, 15 pulses, 20 pulses and 25 pulses, respectively.

View larger version (15K): [in a new window]   Fig. 5. Sarcoplasmic reticulum (SR) Ca2+-dependent inactivation of L-type Ca2+ channel current (ICa) in representative rainbow trout atrial myocytes with square (SQ) and action potential (AP) pulses at 14°C. The green traces indicate the original curve, and the solid black lines are the double exponential fits (Clampfit 6.0, Axon Instruments). (A) ICa elicited by a SQ-voltage clamp pulse immediately after caffeine application [SR empty; fast inactivation kinetics (f)=24.7 ms, and slow inactivation kinetics (s)=113 ms] and after the SR Ca2+ content had reached a steady-state (SR steady-state; f=10.7 ms and s=80 ms). (B) The effect of steady-state SR Ca2+ content on ICa elicited by an AP pulse (green traces). The kinetics of inactivation were best fit with a single exponential (solid black line) over the first 200 ms after peak current. For this cell, decreased from 57 ms after caffeine application to 50 ms with a steady-state SR Ca2+ content. Means and statistics for SR-Ca2+-dependent inactivation of ICa at 7°C, 14°C and 21°C are given in Tables 1, 2.