Mama and papa of the early spontaneous electrical activity of the embryo sinoatrial node
Authors: Gonotkov Mikhail , Lebedeva Elena , Ryvkin Alexander 
Published: May 2022

In adult mammalian hearts the spontaneous diastolic depolarization of the sinoatrial node (SAN) cells is generated by two interacting systems, a so‐called “membrane‐clock” and “calcium‐clock”. Hyperpolarization‐activated pacemaker current (If) plays a key role in the “membrane‐clock”, while the “calcium‐clock” is based on rhythmic spontaneous Ca2+ release from sarcoplasmic reticulum via ryanodine receptors (RyRs). These complementing systems provide regular pacemaking even in case of damage of one of them. The aim of this study was to evaluate a role of If current and ryanodine receptors in the electrical activity of the developing mouse SAN.

Methods

We used embryos of mice, aged of E9.5, E12.5 and newborn to perform our experiments. After release from the amniotic membrane, the embryos were transferred with a pipette to a clean Petri dish with oxygenated physiological solution containing (in mM/L) 140 NaCl, 5.4 KCl, 0.1 CaCl2, 0.33 NaH2PO4, 1 MgCl2, 10 HEPES, 10 Glucose, (pH 7.38) at room temperature. To record spontaneous action potential (AP) we used conventional microelectodes technique at 37◦C. Differences were considered significant at p < 0.05

Results

Microelectrode mapping at E9.5 revealed that the SAN cells are located in the inflow tract of the venous pole. At E12 the SAN cells are located in the cavo‐auricular junction. In neonatal mice, SAN cells were detected at intercaval area near to the crista terminalis. At E9.5, in 6 experiments, on 25‐ 30 minute exposure of 3 mkM ivabradine (Iva) an irregularity in AP generation by SAN cells was occurred. The same effects were obtained in 4 experiments on 20‐30 minutes exposure of 3 mkM ryanodine. At E12.5 exposure of Iva 3 mkM for 30 minutes reduced the pacemaker firing rate by 34% (p < 0.05) but did not break the rhythmic generation. In contrast, the exposure of 3 mkM ryanodine for 30 minutes, in 4 experiments, still caused an irregularity in AP generation by SAN cells. In neonatal isolated SAN preparations, exposure of Iva 3 mkM and 3 mkM ryanodine for 30 minutes reduced the pacemaker firing rate equally (on 32%; p < 0.05). However, co‐application of Iva and ryanodine induced an irregularity in AP generation by SAN cells (Figure).

Numerical experiments within the framework of mouse SANC model (Kharche, AJP, 11) adopted for embryonic cells (Markov, IEEE, 21) have shown the decrease in the frequency of the AP generation with decreasing of the “funny” currents permeability caused by Iva action.

The supposed complete RyRs opening, caused by a relatively low ryanodine concentration, led to a decrease of the AP generation frequency, but not to the rhythm disturbances. However a decrease in RyRs permeability by an order of magnitude led to the appearance of “bursts‐like” irregularity in AP generation.

Conclusion

Thus, the membrane‐clock and the Ca2+‐clock mechanisms is critical for spontaneous electrical activity E9.5 mouse embryos, whereas the Ca2+‐clock mechanisms is critical for increase rate AP generation in development SAN cells.