Electrophysiological Mapping of Embryonic Mouse Hearts: Mechanisms for Developmental Pacemaker Switch and Internodal Conduction Pathway

Abstract

Introduction Understanding sinoatrial node (SAN) development could help in developing therapies for SAN dysfunction. However, electrophysiological investigation of SAN development remains difficult because mutant mice with SAN dysfunctions are frequently embryonically lethal. Most research on SAN development is therefore limited to immunocytochemical observations without comparable functional studies. Methods and Results We applied a multi-electrode array (MEA) recording system to study SAN development in mouse hearts acutely isolated at embryonic ages (E) 8.5 to 12.5 days. Physiological heart rates were routinely restored, enabling accurate functional assessment of SAN development. We found that dominant pacemaking activity originated from the left inflow tract (LIFT) region at E8.5, but switched to the right SAN by E12.5. Combining MEA recordings and pharmacological agents, we show that intracellular calcium (Ca2+)-mediated automaticity develops early and is the major mechanism of pulse generation in the LIFT of E8.5 hearts. Later in development at E12.5, sarcolemmal ion channels develop in the SAN at a time when pacemaker channels are down regulated in the LIFT, leading to a switch in the dominant pacemaker location. Additionally, low micromolar concentrations of tetrodotoxin (TTX), a sodium channel blocker, minimally affect pacemaker rhythm at E8.5–12.5; but suppress atrial activation and reveal a tetrodotoxin-resistant SAN-atrioventricular node (internodal) pathway that mediates internodal conduction in E12.5 hearts. Conclusions Using a physiological mapping method, we demonstrate that differential mechanistic development of automaticity between the left and right inflow tract regions confers the pacemaker location switch. Moreover, a tetrodotoxin-resistant pathway mediates preferential internodal conduction in E12.5 mouse hearts.

Pitx2 -microRNA pathway that delimits sinoatrial node development and inhibits predisposition to atrial fibrillation

“…In chick, discrete cells within lateral plate also contribute to the pacemaker (29). Recent work indicates that a dominant pacemaker activity is found in the E8.5 left inflow tract, where Pitx2c is expressed, but switches to the right SAN by E12.5 (15,30). Our data are consistent with the notion that Pitx2 directly regulates this developmental switch in part by regulating miR-17-92 and miR-106b-25.…”

Genetic Inhibition of Na + -Ca 2+ Exchanger Current Disables Fight or Flight Sinoatrial Node Activity Without Affecting Resting Heart Rate

“…26 Second, the existence of a pacemaker/conduction system within embryonic hearts is a contentious issue, as conduction system components are not morphologically recognizable until embryonic age of E8.5 in mice. 27, 28 Third, NCX1 is likely to be necessary for normal cardiomyocyte development as hearts with constitutive Ncx1 deletion lead to myocyte apoptosis and lack an organized contractile apparatus. 13, 22 It is possible that loss of pacemaking activity in the recently described constitutive Ncx1 −/− mice 18 might be secondary to the structural disease.…”

Abnormal sinoatrial node development resulting from disturbed vascular endothelial growth factor signaling

“…The poor electrical coupling to the surrounding myocardium is essential for the spontaneous depolarization, because the trigger is then only due to the funny current (channel related) and not due to ionic fluxes from the myocardium (gap junction related) [40]. Cx43 is a gap junction protein associated with fast conduction, mainly expressed in the working myocardium [41], which has a lower intrinsic automaticity as compared with the SAN. We propose that the increase of Cx43, observed in this study, improves electrical coupling in the SAN, causing a lower resistance for ions to move from one cell to another.…”

“…Expansion of Cx43 into the SAN, as observed in the Vegf mutants, can disturb this conduction barrier. Further electrophysiological investigation of the SAN using high resolution optical mapping or micro-electrode arrays [41] is required to investigate a shift of pacemaker initiation within the SAN.…”