The relative importance of the SR in contributing Ca2+ to force development in isolated ventricular trabeculae from rainbow trout (Oncorhynchus mykiss) : the effects of temperature, adrenaline and ryanodine

The sarcoplasmic reticulum (SR) is central in regulating intracellular Ca2+ during excitationcontraction (E-C) coupling in mammalian cardiac tissue. The role of the SR in E-C coupling in lower vertebrates is less certain. This uncertainty can be partially attributed to the temperature dependency of the Ca"-release channel and to the varying ryanodine sensitivity of ectotherm cardiac muscle. Additionally, adrenaline (AD) is known to > influence sarcolemmal (SL) (extracellular) Ca2+ influx in lower vertebrates, further obscuring the relative significance of SR (intracellular) Ca2+ contribution. As such, the objective of this thesis was to assesses the relative importance of the SR versus the SL in contributing Ca2+ to force development in isolated ventricular trabeculae from rainbow trout performing both isometric and oscillatory contractions. Ryanodine, a noted blocker of SR Ca2+ release in mammals, was used to assess SR involvement. Additionally, adrenergic stimulation was used to assess the effect of modulating sarcolemmal Ca2+ flux, on the relative importance of SR Ca2+ contribution. The muscles were tested to two temperatures (12•‹C and 22•‹C) with and without acclimation to determine the modulating effect of temperature. Further, unlike previous studies, a tonic level (10 nM) of adrenaline was used as a control to better approximate the in vivo Ca2+ availability to the muscle. The SR Ca2+-release channel in rainbow trout was temperature dependent, being more operative at 22•‹C than 12•‹C and displaying a greater sensitivity to test temperature than to acclimation temperature. At 22"C, ryanodine caused significant reductions in peak tension over a range of pacing frequencies (40-50% at 0.2 Hz and 25-30% at 1.2-2.0 Hz), including physiologically realistic pacing frequencies. This suggests significant, but secondary, involvement of SR Ca2+ in force development under acute exposure to warm temperatures. At a colder test temperatures (12"C), ryanodine sensitivity is observed only at low, sub-physiological (0.2 Hz) pacing frequencies regardless of temperature acclimation status. This finding suggests that at lower temperatures the involvement of the SR is not physiologically important and that the sarcolemma plays the primary role in supplying Ca2+ to the contractile elements of the fish heart. At both warm and cold test and acclimation temperatures the positive inotropy associated with 10 pM AD ameliorated the negative effects of ryanodine, demonstrating that the ca2+ contribution from the SR can be overwhelmed by adrenergic stimulation of SL Ca2+ influx. Additionally, results from the muscles performing oscillatory contractions suggest that at 12OC, the optimum frequency for power production is between 0.8 Hz and 1.2 Hz. This optimum frequency appears to be ryanodine-sensitive, a finding which may provide insight into a possible link between SR Ca" recruitment and cardiac performance at high temperatures and high cardiac frequencies. Thus, unlike mammals, E-C coupling in trout predominantly relies on transsarcolemmal Ca2+ influx with SR-Ca2+ playing a significant secondary role at 22OC but not 12OC. Regardless of temperature, this secondary role can be easily overwhelmed by increased Ca2+ influx upon adrenergic stimulation of the sarcolemma Certainly this relationship between the relative predominance of SL versus SR Ca2+ flux observed in trout, may vary in stenothermic warm-water fish, whose body temperatures are more compatible with those of mammals.