Membrane and calcium clock mechanisms contribute variably as a function of temperature to setting cardiac pacemaker rate in zebrafish Danio rerio

Here, we show that heart rate in zebrafish Danio rerio is dependent upon two pacemaking mechanisms and it possesses a limited ability to reset the cardiac pacemaker with temperature acclimation. Electrocardiogram recordings, taken from individual, anaesthetised zebrafish that had been acclimated to 18, 23 or 28°C were used to follow the response of maximum heart rate (f_Hmax) to acute warming from 18°C until signs of cardiac failure appeared (up to c. 40°C). Because f_Hmax was similar across the acclimation groups at almost all equivalent test temperatures, warm acclimation was limited to one significant effect, the 23°C acclimated zebrafish had a significantly higher (21%) peak f_Hmax and reached a higher (3°C) test temperature than the 18°C acclimated zebrafish. Using zatebradine to block the membrane hyperpolarisation-activated cyclic nucleotide–gated channels (HCN) and examine the contribution of the membrane clock mechanisms to cardiac pacemaking, f _Hmax was significantly reduced (by at least 40%) at all acute test temperatures and significantly more so at most test temperatures for zebrafish acclimated to 28°C vs. 23°C. Thus, HCN channels and the membrane clock were not only important, but could be modified by thermal acclimation. Using a combination of ryanodine (to block sarcoplasmic calcium release) and thapsigargin (to block sarcoplasmic calcium reuptake) to examine the contribution of sarcoplasmic reticular handling of calcium and the calcium clock, f _Hmax was again consistently reduced independent of the test temperature and acclimation temperature, but to a significantly lesser degree than zatebradine for zebrafish acclimated to both 28 and 18°C. Thus, the calcium clock mechanism plays an additional role in setting pacemaker activity that was independent of temperature. In conclusion, the zebrafish cardiac pacemaker has a limited temperature acclimation ability compared with known effects for other fishes and involves two pacemaking mechanisms, one of which was independent of temperature.