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. 2012 Mar;23(3):309-18.
doi: 10.1111/j.1540-8167.2011.02191.x. Epub 2011 Oct 10.

Electrophysiological mapping of embryonic mouse hearts: mechanisms for developmental pacemaker switch and internodal conduction pathway

Tongyin Yi  1 , Johnson Wong, Eric Feller, Samantha Sink, Ouarda Taghli-Lamallem, Jianyan Wen, Changsung Kim, Martin Fink, Wayne Giles, Walid Soussou, Huei-Sheng V Chen
Affiliations

Electrophysiological mapping of embryonic mouse hearts: mechanisms for developmental pacemaker switch and internodal conduction pathway

Tongyin Yi et al. J Cardiovasc Electrophysiol. 2012 Mar.

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 multielectrode array (MEA) recording system to study SAN development in mouse hearts acutely isolated at embryonic ages (E) 8.5-12.5 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 (Ca(2+))-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-E12.5, but suppress atrial activation and reveal a TTX-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 TTX-resistant pathway mediates preferential internodal conduction in E12.5 mouse hearts.

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Figures

Figure 1
Figure 1. MEA-based electrophysiological mapping of embryonic mouse hearts
A: Image of an E8.5 heart with a dense Hexa array (Supplemental Fig. 1). B: Image of an E12.5 heart with a standard 30 × 200 µm array. The yellow circle indicates SAN. C: Field potentials (FPs) recorded from external cardiac surface of the E8.5 heart in A. The arrows (red or blue) indicate the sequence of activation from the dominant pacemaker site at the LIFT. D: FPs from B. Black arrows indicate the distinct sinal (S), atrial (A) and ventricular (V) signals respectively in the E12.5 heart. E: The method to determine LAT (labeled with a red vertical bar) is explained in Text. F: Activation maps of SAN, bilateral atria (A), and bilateral ventricles (V) demonstrate electrical propagation sequences (red is early and blue is late). G: Summary of the locations of dominant pacemakers, determined by both MEA and video recordings, at E8.5, E10.5 and E12.5 is shown. RIFT and LIFT indicate right and left inflow tract; eA and eV, embryonic atrium and ventricle; AVC, atrioventricular canal; RA and LA, right and left atrium; RV and LV, right and left ventricle; OFT, outflow tract; LVA, LV apex. Vertical bars represent the amplitudes of FP voltage and horizontal bars, time.
Figure 2
Figure 2. Intracellular Ca2+ handling proteins control the dominant automaticity of E8.5 hearts
A: Raw MEA tracings from the LIFT of E8.5 hearts and from the SAN of E12.5 hearts at baseline (Pre; before drug application) and 10 minutes after drug application (Post) are shown. B: Raw tracings from left and right pacemaker sites are shown to illustrate the effect on HRs by co-application of Ryanodine (Rya) and KB after either Rya (E8.5) or KB (E12.5) blockade. At E8.5, combination of Rya and KB completely arrested LIFT pacemaking activity in all hearts, and 3 of 8 hearts displayed residual pacemaker activity in the RIFT (pacemaker location switch, left bottom tracing). C: Summary of effects of 10 Rya, 50 KB, and 10 APB (in µM) on HRs of E8.5 and E12.5 hearts. Values of residual HRs after drugs relative to controls in this histogram for E8.5 and E12.5 hearts are: KB: 95.3±4.9 and 85.8±4.3%; APB: 92.1±8.1 and 95.2±3.0%; Rya: 81.6±6.9 and 72.1±12.1%; Rya+APB: 67.0±13.7 and 61.5±8.8%; and Rya+KB: 15.9±25.0 and 55.0±9.9%, respectively. Asterisks indicate p<0.05. The number in each column represents the number of hearts tested. Vertical bars represent 50 µV at E8.5 and 250 µV at E12.5 hearts in this and following figures unless otherwise indicated. Horizontal bars denote 200 ms.
Figure 3
Figure 3. HCN and T-type Ca2+ channels, but not Na+ channels, play increasing roles in dominant automaticity with cardiac differentiation
A: Raw MEA tracings from the LIFT of E8.5 hearts and from the SAN of E12.5 hearts at baseline (Pre) and after drug application (Post) are shown. ZD7288 (top) and Mibefradil (bottom) reduced HRs of E12.5 hearts much more than E8.5 hearts. B: Summary of effects of ZD7288 and Mibefradil on heart rates. Values of residual HRs after drugs (in µM) in this histogram for E8.5 and E12.5 hearts are 1 ZD: 84.7±6.7 and 69.8±9.4%; 10 ZD, 72.6±10.8 and 34.0±11.0%; 0.5 Mibefradil, 94.8±2.7 and 79.5±7.0%; 1 Mibefradil, 90.6±8.0% and 71.7±7.5% respectively. Asterisks indicate difference between E8.5 and E12.5 with p<0.01 in this figure. C: Raw MEA tracings from the LIFT of E8.5 hearts (left) and from the SAN of E12.5 hearts (middle) at baseline (Pre) and after TTX application (Post) are shown. Red circles indicate sinus signals. D: TTX minimally decreased HRs of E8.5 (n= 7) and E12.5 (n=14) hearts to 95.6±6.1% and 93.5±5.9% of controls respectively. E: Schematic demonstration of the measurement of sinus field potential duration (SNd), sinoatrial interval (SAI), and RA to ventricular interval (RA-VI) pre and post TTX application. F: Summary of TTX effects on electrophysiological properties of E8.5 and E12.5 hearts. TTX slightly prolonged field potential durations (FPDs) at E8.5 (left, 444.5±66.5 vs. 485.3±70.5 ms, p=NS). At E12.5, TTX significantly prolonged SNd, atrial field potential duration (AFPD) and SAI (7.9±3.2 vs. 20.4±8.3, 53.2±8.2 vs. 65.7±16.2, and 73.2±18.6 vs. 116.7±26.2 ms, respectively) with minimal effects on ventricular field potential duration (VFPD, 122.2±16.9 vs. 129.8±24.0 ms) and RA-VI (84.0±15.3 vs. 84.7±18.8 ms). Asterisks indicate a difference between pre and post TTX with p-values <0.01 in Fig. 3F. NS indicates no significant difference between comparisons (ANOVA).
Figure 4
Figure 4. TTX revealed a preferential conduction pathway in the RA of E12.5 hearts
A: Raw MEA tracings of an E12.5 heart after TTX application are shown. TTX led to normal sino-atrial-ventricular activation (S-A-V) sequence (1st beat), complete sinoatrial (SA) exit block (2nd beat), and Sino-V conduction without atrial activation (3rd beat). B: Activation maps of these three beats are shown here to demonstrate that the activation sequence of both ventricles is unchanged during Sino-V conduction. Pre-TTX baseline activation maps of this heart are shown in Fig. 1F. C: The morphologies of ventricular field potentials during Sino-V conduction (3rd beat) at LA apex (LVA) and LV base display no pre-excitation and are unchanged from a normally conducted beat (1st beat). The local activation time (LAT) at each location is labeled with a red vertical bar. The LV apex is activated earlier than LV base in both cases.
Figure 5
Figure 5. Sino-ventricular linking during Sino-V conduction and evidence for internodal conducting pathways
A: Raw MEA tracings from an E12.5 heart before and B: after TTX application. Electrical recordings from three consecutive electrodes around the SAN region and at a cranial-to-caudal sequence were named high, mid, or low SAN tracings in this figure (see also Supplements Fig. 3). TTX led to a stable pattern of alternating (2:1) normal sino-atrial-ventricular (S-A-V) (first and third beats) and Sino-V conduction sequences (second and fourth beats). C: During 12 sec recordings of stable 2:1 Sino-V conduction rhythm, the ratio of ventricular intervals (V1V1) to preceding sinus cycle length (S1S1) is close to 1 for all normal (S-A-V) and Sino-V conducted beats, indicating SAN-V linking. D: For another E12.5 heart with sinus arrhythmias, the high-to-low conducting sequence remains the same between normal and Sino-V conducted beats, as well as E, the sino-ventricular interval (SVI) during Sino-V conduction is longer after a short preceding S1S1 interval. F: Using all Sino-V conducted beats from a 30 sec recording period shown in E, a plot of V1V1 intervals and SVIs versus preceding S1S1 intervals showed a progressive increase in SVIs when S1S1 intervals were shorter than 320 ms, which is a classic decremental conduction property of AVN and supports an inter-nodal pathway that mediates Sino-V conduction with AVN as the relay. Red circles indicate sinus signals.
Figure 6
Figure 6. Immunostaining and In situ hybridization of HCN4 channels subunits revealed a potential internodal pathway at E12.5
A: Consecutive sections of E12.5 atria from high to low levels were obtained for immunostaining (only three levels of sections are shown here). Immunostainings of high atria (High A) at SAN level, middle atria (Mid A) and low atrial (Low A) close to AVN level are shown. Magnified views at each level showed a pathway throughout these atrial levels with high HCN4 staining (lower left) and low NaV1.5 expression (lower right) at the RA-right sinus horn (rsh) junction. B: In situ hybridization (dorsal view) is used to show a continuous HCN4 expression pathway in the RA with arrows indicating comparable levels shown in A. C: Schematic summary of location and mechanistic switch of dominant automaticity during cardiac differentiation. Left panel, in E8.5 hearts, intracellular Ca2+-driven mechanisms dominate control of the LIFT pacemaker site (red region) with small contributions of sarcolemmal ion channels to automaticity and an underdeveloped RIFT pacemaker site (yellow circle). Right panel, in E12.5 hearts, ion channels up-regulate (an arrow pointing up) in the right-sided SAN (filled yellow circle) and control the dominant rhythm with concomitant down-regulation (an arrow pointing down) of HCN4 channels in the comparable region at LA/ left sinus horn junction (red circle). An internodal conduction pathway (a yellow tract) could be demonstrated functionally in E12.5 hearts. The normal AVN-bundle branch-Purkinje system is labeled with blue color.
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