The effective use of blebbistatin to study the action potential of cardiac pacemaker cells of zebrafish (Danio rerio) during incremental warming.

Blebbistatin potently inhibits actin-myosin interaction, preventing contractile activity of excitable cells including cardiac myocytes, despite electrical excitation of an action potential (AP). We collected intracellular microelectrode recordings of pacemaker cells located in the sinoatrial region (SAR) of the zebrafish heart at room temperature and during acute warming to investigate whether or not blebbistatin inhibition of contraction significantly alters pacemaker cell electrophysiology. Changes were evaluated based on 16 variables that characterized the AP waveform. None of these AP variables nor the spontaneous heart rate were significantly modified with the application of 10 µM blebbistatin when recordings were made at room temperature. Compared with the control group, the blebbistatin-treated group showed minor changes in the rate of spontaneous diastolic depolarization (P = 0.027) and the 50% and 80% repolarization (P = 0.008 and 0.010, respectively) in the 26°C-29°C temperature bin, but not at higher temperatures. These findings suggest that blebbistatin is an effective excitation-contraction uncoupler that does not appreciably affect APs generated in pacemaking cells of the SAR and can, therefore, be used in zebrafish cardiac studies.

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 (fHmax ) to acute warming from 18°C until signs of cardiac failure appeared (up to c. 40°C). Because fHmax 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 fHmax 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.