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

Abstract

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.

Keywords: Action potential; Blebbistatin; Electrophysiology; Pacemaker cell; Zebrafish.

Fig. 1

A schematic protocol of action potential (AP) variable extraction from raw AP traces recorded from pacemaker cells. Numbers correspond to the following AP variables: 1, spontaneous depolarization duration (ms); 2, spontaneous depolarization potential (mV); 3, overshoot potential (mV); 4, beat to beat period (ms; heart rate in beats min^-1 was calculated as the inverse of the period); 5, AP amplitude (mV); 6, depolarization potential (mV); depolarization duration was taken as time from threshold to peak depolarization (ms); 7, threshold potential (mV); 8, 9, 10: total repolarization potential (10) was the measured difference between peak AP potential and the maximum hyperpolarization potential (mV). 50% AP repolarization potential (8) was calculated as half the total repolarization potential (mV) and 50% AP duration was taken as the time between this potential and the equivalent point on the depolarizing phase of the AP. 90% AP repolarization potential (9, mV) was that at 90% of the total repolarization potential; 90% repolarization duration (ms) was the time (ms) between this potential and the equivalent point on the depolarizing phase of the AP.

Fig. 2

The effect of blebbistatin and temperature on the rate of diastolic depolarization (A, B), the rate of depolarization (C, D), and the rate of repolarization (E, F). Pairwise comparison of AP variables within different temperature bins is illustrated in panels A, C, and E. Linear regression analysis over the range of warming is shown in panels B, D, and F. Data points represent individuals. Mean data values are presented with their respective standard deviations. Statistical significance of pairwise comparisons was determined using a t-test (P ≤ 0.05).

Fig. 3

Linear regression analysis of the effect of blebbistatin and temperature on the rate of diastolic depolarization (A), the rate of depolarization (B), and the rate of repolarization (C). Data points represent individuals where solid-colored points represent recorded measures and open data points represent predicted values.