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Optical mapping of conduction patterns of normal and tachycardia-like excitations in the rat atrium.

Multiple-site optical recording of transmembrane voltage activity (MSORTV), using a voltage-sensitive dye and a 12 x 12-element photodiode array, was employed to monitor action potentials in the rat atrium. Atrial preparations including the sinus node area, caval area, and atrial septum were dissected from adult rat hearts and stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761). For suppression of optical artefacts due to contractile movements, a bathing medium containing 2,3-butanedione monoxime (BDM: 10-20 mM) was used. The spread of spontaneous excitation from the pacemaker was assessed optically by timing the initiation of the action potential-related extrinsic absorption changes. The optical signals were recorded from more than 300 contiguous loci in the atrium by sliding the photodiode array over the image of the preparation; in this way, the intra-atrial conduction pattern of spontaneous excitation was mapped. The obtained maps revealed a non-radial spread of excitatory waves originating in the pacemaking area over the atrium. Furthermore, we also mapped the conduction pattern of long-lasting tachycardia-like excitation evoked by a short train of electrical stimulation (5-10 Hz, 1-2s) applied with a bipolar electrode. These maps suggest that excitatory waves are propagated in a circular pathway which often surrounds the ostium of the superior or inferior vena cava. Various patterns for the pathway were also demonstrated optically.

Action Potentials↗

Optical mapping of the early development of the response pattern to vagal stimulation in embryonic chick brain stem.

1. In both intact and slice preparations of vagus-brain stem isolated from 3- to 8-day-old chick embryos, the spatial pattern of neural responses to vagal stimulation and its development were assessed by means of multiple-site optical recording of electrical activity, using a voltage-sensitive merocyanine-rhodanine dye (NK2761) and a 12 x 12-element photodiode array. 2. The first neural responses, viz. fast optical signals (related to the action potential), were recorded in the 4-day-old brain stem preparation, and slow optical signals (related to excitatory postsynaptic potentials) were detected from late 7- and 8-day-old brain stem preparations. 3. The evoked optical signals appeared to be concentrated longitudinally in the central region of the stimulated side of the intact brain stem preparation and in a limited dorsal area in the slice preparation. The signal size gradually increased and the response area expanded as development proceeded. 4. Based on the above results, we have constructed developmental maps of the spatial patterns of the fast and slow optical responses. In the maps, the positions of the peak-size regions of the fast and slow signals were assessed and we have found that there were differences in the location of these areas for the fast vs. the slow signals in the late 7- and 8-day-old embryonic brain stem preparations. 5. In the maps for the late 7- and 8-day-old embryonic brain stems, the fast signal response area seems to correspond to the dorsal motor nucleus of the vagus nerve and the slow response area to the nucleus tractus solitarii.

Action Potentials↗

Optical mapping of neural responses in the embryonic rat brainstem with reference to the early functional organization of vagal nuclei.

We examined the functional organization of the vagal nuclei of the rat embryo during morphogenesis, using multiple-site optical recording with a voltage-sensitive dye. Slice preparations with vagus nerve fibers were dissected from 13- to 16-d-old embryonic (E13-E16) rat brainstems, and they were stained with the dye. Electrical activity in response to vagal stimulation was recorded optically from many sites. In the E13-E14 preparations, two types of spike-like optical signals were recorded: one was a narrow signal (type I), and the other was a broader signal (type II). Comparison with the morphology revealed by DiI labeling suggests that the type I signal response area corresponds to the nucleus of the tractus solitarius, and the type II signal response area corresponds to the dorsal motor nucleus of the vagus nerve. In the E15-E16 preparations, type I signals were followed by a slow signal related to glutamate-mediated excitatory postsynaptic potentials, suggesting that synaptic function is organized in the nucleus of the tractus solitarius by the 15-d-old embryonic stage. In the E14 preparation, a small, slow signal was evoked only in Mg2+-free solution, implying that postsynaptic function related to NMDA receptors emerges, in latent form, at the 14-d-old embryonic stage. In the E15 and E16 preparations, although the nucleus ambiguus is identified morphologically, no neural response-related optical signal was observed there, indicating that the embryonic organization of morphology and physiological function is not necessarily temporally coincident. We have mapped the dynamic spatiotemporal patterns of the evoked optical signals and have outlined the early phase of the functional organization of the cranial nuclei related to the vagus.

Animals↗

Optical mapping of pontine chemosensitive regions of neonatal rat.

We analyzed the neuronal response to hypercapnic acidosis, using an optical recording technique with a fluorescent voltage-sensitive dye (di-4-ANEPPS), in pontine slice preparations of neonatal rats, containing the locus coeruleus (LC), which has been electrophysiologically demonstrated to be chemosensitive. The dye-stained preparation was continuously superfused with artificial cerebrospinal fluid. Epifluorescence of the slice was detected using a high-sensitivity optical recording system. Changes in the intensity of fluorescence were serially analyzed while switching artificial cerebrospinal fluid from control to hypercapnic acidosis, or vice versa. The optical recording method revealed that the LC, as reported in previous studies, reversibly showed a depolarizing response to hypercapnic acidosis in 56% of the examined preparations. The A5 area (56%) also exhibited a reversible, depolarizing response to hypercapnic acidosis. The response was preserved under conditions in which chemical synaptic transmission was blocked by low Ca(2+)-high Mg(2+) solution. These results suggest that the optical recording method is applicable to identification of potentially chemosensitive areas, which deserve further electrophysiological analysis, and that the A5 area could be chemosensitive.

Acidosis↗

Optical mapping of early embryonic expressions of Mg(2+)-/APV-sensitive components of vagal glutaminergic EPSPs in the chick brainstem.

Multiple-site optical recording of transmembrane voltage activity, using a voltage-sensitive dye (NK2761) and a 12 x 12 element photodiode array, was employed to monitor neural responses in the slice preparations that were dissected from 6-9-d-old embryonic brainstems. Transmembrane voltage-related optical responses evoked by a brief square current pulse applied with a microsuction electrode to the vagus nerve were recorded simultaneously from many sites in the preparation. In preparations from 6-d-old embryos, only action potential-related spike-like optical signals were often detected in the normal bathing solution containing 0.5 mM Mg2+. However, in several loci of some preparations, excitatory postsynaptic potential-related slow signals were elicited in a Mg(2+)-free bathing medium, and these elicited slow signals were blocked by DL-2-amino-5-phosphonovaleric acid (2-APV). These results show a possible embryonic expression of NMDA receptor-mediated excitatory postsynaptic potentials. The slow signals were usually detected from tested preparations from 7-9-d-old embryos in normal bathing solution. The later phase of the slow signals was enhanced in a Mg(2+)-free bathing medium, and the enhanced component was also sensitive to 2-APV and insensitive to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). There were regional differences in the degree of the enhancement of the slow signals in the Mg(2+)-free bathing medium, and of the 2-APV-induced reduction of the slow signals. We have constructed early developmental maps of the spatial patterns of the slow signals related to Mg(2+)- and 2-APV-sensitive postsynaptic potentials. These maps reflect the spatial distribution of functional expression of the NMDA receptors during early development of the chick brainstem. The present findings are new data that have not been reported previously, and they have been obtained for the first time using the multiple-site optical recording technique with a voltage-sensitive dye.

2-Amino-5-phosphonovalerate↗

Optical mapping of neural network activity in chick spinal cord at an intermediate stage of embryonic development.

We have applied multiple-site optical recording of transmembrane potential changes to recording of neuronal pathway/network activity from embryonic chick spinal cord slice preparations. Spinal cord preparations were dissected from 8-day-old chick embryos at Hamburger-Hamilton stage 33, and transverse slice preparations were prepared with the 13th cervical spinal nerve or with the 2nd or 5th lumbosacral spinal nerve intact. The slice preparations were stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761). Transmembrane voltage-related optical (dye-absorbance) changes evoked by spinal nerve stimulation with positive square-current pulses using a suction electrode were recorded simultaneously from many loci in the preparation, using a 128- or 1,020-element photodiode array. Optical responses were detected from dorsal and ventral regions corresponding to the posterior (dorsal) and anterior (ventral) gray horns. The optical signals were composed of two components, fast spike-like and slow signals. In the dorsal region, the fast spike-like signal was identified as the presynaptic action potential in the sensory nerve and the slow signal as the postsynaptic potential. In the ventral region, the fast spike-like signal reflects the antidromic action potential in motoneurons, and the slow signal is related to the postsynaptic potential evoked in the motoneuron. In preparations in which the ventral root was cut microsurgically, the antidromic action potential-related optical signals were eliminated. The areas of the maximal amplitude of the evoked signals in the dorsal and ventral regions were located near the dorsal root entry zone and the ventral root outlet zone, respectively. Quasiconcentric contour-line maps were obtained in the dorsal and ventral regions, suggesting the functional arrangement of the dorsal and ventral synaptic connections. Synaptic fatigue induced by repetitive stimuli in the ventral synapses was more rapid than in the dorsal synapses. The distribution patterns of the signals were essentially similar among C13, LS2, and LS5 preparations, suggesting that there is no difference in the spatiotemporal pattern of the neural responses along the rostrocaudal axis of the spinal cord at this developmental stage. In the ventral root-cut preparations, comparing the delay times between the ventral slow optical signals, we have been able to demonstrate that neural network-related synaptic connections are generated functionally in the embryonic spinal cord at Hamburger-Hamilton stage 33.

Animals↗

Optical mapping of DNA polymerase I action and products.

Single molecule approaches to the characterization of biochemical systems offer an intrinsically simple and direct approach to address difficult, previously unyielding problems. Optically based approaches have recently been used to construct high resolution, ordered restriction maps from a variety of clone types. Advancements in surface technologies have enabled the reliable elongation and fixation of large DNA molecules onto specially derivatized substrates with retention of biochemical accessibility. In this study, the addition of fluorescently labeled nucleotides to surface-mounted DNA molecules by the action of DNA polymerase I is investigated using fluorescence microscopy to image individual template molecules. Molecules undergoing nick translation and containing only a few fluorochromes are readily imaged. These novel results suggest that surface-bound molecules may serve as a substrate for a broad range of enzymatic actions, and may offer new routes to analysis when coupled to advanced imaging techniques.

Bacteriophage lambda↗

Optical mapping reveals the functional organization of the trigeminal nuclei in the chick embryo.

The functional organization of the trigeminal nuclei during embryogenesis was investigated using multiple-site optical recording with a fast voltage-sensitive dye. Brainstem preparations with three classified trigeminal nerve afferents, the ophthalmic, maxillary and mandibular nerves, together with motor nerve fibers, were dissected from five- to eight-day-old chick embryos. Electrical responses evoked by trigeminal nerve stimulations were optically recorded simultaneously from many loci of the stained preparations. We identified three response areas related to the trigeminal nerve: area I, located cephalic to the level of the trigeminal ganglion; area II, located caudal to the level of the trigeminal ganglion; and area III, located at the level of the trigeminal root. The neural responses in areas I and II were evoked by ophthalmic, maxillary or mandibular nerve stimulation, while the responses in area III were detected when the stimulation was applied to the trigeminal motor nerve. In comparison with the morphology indicated by DiI labeling, the results suggest that areas I, II and III correspond to the principal sensory nucleus of the trigeminal nerve, the spinal sensory nucleus of the trigeminal nerve and the trigeminal motor nucleus, respectively. We identified two components of the optical response: a fast and a slow signal. In five-day-old preparations, fast spike-like signals related to action potentials were recorded from the three response areas. In six-day-old preparations, slow optical signals which reflect glutamate-mediated excitatory postsynaptic potentials were detected from area II only when the ophthalmic nerve was stimulated: no slow signal was evoked by maxillary or mandibular nerve stimulation. In seven- and eight-day-old preparations, slow signals were detected from both areas I and II with every nerve stimulation. These results suggest that synaptic function is first generated in the spinal trigeminal nucleus by the six-day embryonic stage, and the developmental organization of synaptic function is not the same in the three trigeminal nerves or in the two sensory nuclei. Contour line maps of the signal amplitude revealed that the size and the area of the neural responses within the trigeminal nuclei changed dramatically with development. We compared the spatial distribution and temporal dynamics of the optical signals between the ophthalmic, maxillary and mandibular nerve stimulations, and we found that somatotopic organization is less clear in a rostrocaudal/mediolateral X-Y plane, although the areas of the maxillary and mandibular nerves appeared to separate in the lateral direction.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

Optical mapping of repolarization and refractoriness from intact hearts.

BACKGROUND: Heterogeneities of repolarization (R) across the myocardium have been invoked to explain most reentrant arrhythmias. The measurement of refractory periods (RPs) has been widely used to assess R, but conventional electrode and extrastimulus mapping techniques have not provided reliable maps of RPs. METHODS AND RESULTS: Guinea pig hearts were stained with a voltage-sensitive dye to measure fluorescence (F) action potentials (APs) from 124 sites with a photodiode array. AP duration (APD) was defined as the time between depolarization (dF/dt)max and R time points (ie, the time when AP returns to baseline or some percent thereof). However, R time points are difficult to determine because AP downstrokes are often encumbered by drifting baselines and motion artifacts, which make this definition ambiguous. In optical and microelectrode recordings, the second derivative of AP downstrokes is shown to contain an easily detected, unique local maximum. The correlation between the position of this maximum (d2F/dt2)max and R has been tested during altered AP characteristics induced by changes in cycle length, ischemia, and hypoxia. Under these various modifications of the AP, the time points of (d2F/dt2)max fell at 97.0 +/- 2.1% of recovery to baseline. Extrastimulus techniques applied to (1) isolated myocytes, (2) intact hearts, and (3) mathematical simulations indicated that (d2V/dt2)max coincided with the effective RPs of APs. The coincidence of RPs and (d2V/dt2)max was valid within 5 milliseconds, for resting potentials of -75 to -90 mV and extrastimuli three times threshold voltage. CONCLUSIONS: Thus, optical APs and (d2F/dt2)max can be used to map activation, R, and RPs with AP recordings from a single heartbeat.

Animals↗

Simultaneous optical mapping of transmembrane potential and intracellular calcium in myocyte cultures.

INTRODUCTION: Fast spatially resolved measurements of transmembrane potential (Vm) and intracellular calcium (Ca(i)2+) are important for studying mechanisms of arrhythmias and defibrillation. The goals of this work were (1) to develop an optical technique for simultaneous multisite optical recordings of Vm and Ca(i)2+, and (2) to determine the relationship between Vm and Ca(i)2+ during normal impulse propagation in myocyte cultures. METHODS AND RESULTS: Monolayers of neonatal rat myocytes were stained with fluorescent dye RH-237 (Vm) and Fluo-3AM (Ca(i)2+). Both dyes were excited at the same wavelength range. The emitted fluorescence was optically separated into components corresponding to changes in Vm and Ca(i)2+ and measured using two 16 x 16 photodiode arrays at a spatial resolution of up to 27.5 microm per diode and sampling rate of 2.5 kHz. The optical setup was adjusted so that there was no optical cross-talk between the two types of measurements, which was validated in experiments involving staining with either RH-237 or Fluo-3. The amplitude of Fluo-3 signals rapidly decreased during experiments due to dye leakage. Dye leakage was substantially reduced by application of 1 mM probenecid, a blocker of organic anion transport, which had no effect on action potential duration and only minor effect on conduction velocity. In double-stained preparations, during regular pacing Ca(i)2+ transients had a rise time of 14.2 +/- 2 msec, and they followed Vm upstrokes with a delay of 5.3 +/- 1 msec (n = 9). Durations of Vm and Ca(i)2+ transients determined at 50% level of signal recovery were 54.6 +/- 10 msec and 136 +/- 8 msec, respectively. Application of 2 microM nifedipine reduced the amplitude and duration of Ca(i)2+ transients without significantly affecting conduction velocity. CONCLUSION: The results demonstrate feasibility of simultaneous optical recordings of Vm and Ca(i)2+ transients with high spatial and temporal resolution.

Aniline Compounds↗

Anisotropic activation spread in heart cell monolayers assessed by high-resolution optical mapping. Role of tissue discontinuities.

The role of tissue discontinuities in anisotropic impulse propagation was assessed in two-dimensional anisotropic monolayers of neonatal rat myocytes cultured on a growth-directing substrate of collagen. Activation spread and distribution of maximal upstroke rate of rise (Vmax) of the action potential were measured with an optical system using a voltage-sensitive fluorescent dye (RH-327) and a 10x10 photodiode array with a spatial resolution ranging from 7 to 15 microns. Activation maps were compared with the cellular architecture and the distribution of gap junctions obtained from immunostaining the gap junction protein connexin43 (Cx43). Four types of structures were studied: (1) dense cell cultures, (2) cultures with anisotropic intercellular clefts of variable size, (3) discontinuities created by inclusion of nonmyocyte cells, and (4) discontinuities resulting from nonuniform expression of gap junctions. In dense monolayers, activation spread was continuous with microinhomogeneities in both longitudinal and transverse directions. The average cell dimensions in such monolayers were smaller than in adult canine myocardium. However, the degree of cellular anisotropy (length-to-width ratio of 5.3 +/- 1.4) and connectivity were similar. The presence of small intercellular clefts (less than one cell in length) did not disturb the general pattern of transverse or longitudinal activation spread, but it was associated with wave front microcollisions during transverse propagation and a concomitant increase of Vmax beyond the cleft. Long intercellular clefts caused discontinuous transverse propagation. Conduction velocity and Vmax decreased significantly at narrow isthmuses formed by closely apposed clefts, rendering such sites susceptible for conduction block. In contrast Vmax increased when the wave front faced the borders of the clefts. Nonmyocyte cells were electrically connected to myocytes and served as sinks for electrotonic currents, thereby producing localized conduction slowing and a decrease in Vmax. Localized inhomogeneity in Cx43 distribution correlated accurately with circumscribed conduction block and changes in Vmax. Our results provide direct experimental evidence that the cellular structure and gap junction distribution correlate with action potential propagation and distribution of Vmax. We suggest that in tissue with a nonuniform anisotropy, connective tissue separating fiber bundles or sites of inhomogeneous connexin distribution may represent predilective sites for block in transverse direction.

Animals↗

Optical mapping of the functional organization of the rat trigeminal nucleus: initial expression and spatiotemporal dynamics of sensory information transfer during embryogenesis.

We examined the functional organization of the rat trigeminal nuclear complex and its developmental dynamics using a multiple-site optical recording technique. Brainstem preparations were dissected from embryonic day 12 (E12)-E16 rat embryos, and stimulation was applied individually to the three branches of the trigeminal nerve (V1-V3). The action potential activity of presynaptic fibers was detected from E13, and the glutamate-mediated postsynaptic response was significantly observed from E15 on. At E14, the evoked signals usually consisted of only the action potential-related fast component. However, when extracellular Mg2+ was removed, a significant dl-2-amino-5-phosphonovaleric acid-sensitive slow component appeared. These results suggest that postsynaptic function mediated by NMDA receptors is latently generated as early as E14. The response area of the three branches of the trigeminal nerve showed some functional somatotopic organization, with the ophthalmic (V1) nerve area medially located and the mandibular (V3) nerve area laterally located. The center of the trigeminal nuclear complex in which the activity of neurons and synaptic function was greatest shifted caudally with development, suggesting that the functional architecture of the trigeminal nuclear complex is not fixed but changes dynamically during embryogenesis. By electron microscopy, we could not observe clear correlations between functional data and morphological information; when we surveyed E16 preparations, we could not identify typical synaptic structures between the 1,1'-dioctyldecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled trigeminal nerve terminals and the neurons in the trigeminal nuclear complex. This implies that postsynaptic function in the trigeminal nuclear complex is generated before the appearance of the morphological structure of conventional synapses.

Animals↗

Optical mapping reveals developmental dynamics of Mg2+-/APV-sensitive components of glossopharyngeal glutamatergic EPSPs in the embryonic chick NTS.

To examine whether there are any differences in functional organization between the glossopharyngeal nerve (N. IX)- and vagus nerve (N. X)-projecting areas in the nucleus of the tractus solitarius (NTS), we performed optical recording of neural responses evoked by N. IX stimulation in 5- to 9-day-old embryonic chick brain stem preparations and compared the results with those in our previous studies concerning the N. X-related NTS. First, we investigated DL-2-amino-5-phosphonovaleric acid (APV)/Mg2+ sensitivity of the glutamatergic excitatory postsynaptic potentials (EPSPs) in the N. IX-related NTS. In 7- to 9-day-old preparations, we found regional differences in the degree of both the APV-induced reduction and Mg2+-free-induced enhancement of the EPSPs. We constructed developmental maps of spatial patterns of the APV- and Mg2+-sensitive components and showed that functional expression of the N-methyl-D-aspartate (NMDA) receptor dynamically changed during development. Second, we studied initial expression of synaptic functions in the N. IX-related NTS. In 6-day-old preparations, although action potentials alone were usually detected in normal Ringer solution, small EPSPs were elicited in a Mg2+-free solution. This result suggests that the NMDA receptor-mediated synaptic function is latently generated in the N. IX-related NTS at the 6-day-old embryonic stage and that external Mg2+ regulates the onset of synaptic functions. Developmental patterns of APV/Mg2+ sensitivity and the stage of initial expression of the glossopharyngeal EPSP were similar to those of the N. X, suggesting that the developmental sequence of the synaptic function in the NTS is the same for the N. IX- and N. X-related NTS.

2-Amino-5-phosphonovalerate↗

Role of microscopic tissue structure in shock-induced activation assessed by optical mapping in myocyte cultures.

INTRODUCTION: Termination of ventricular fibrillation by electric shocks is believed to be due to the direct activation of large tissue mass that may be caused by microscopic virtual electrodes formed at discontinuities in tissue structure. Here, microscopic shock-induced activation was measured optically in myocyte cultures; spatially averaged microscopic Vm measurements were compared with macroscopic measurements from left ventricular (LV) tissue. METHODS AND RESULTS: Experiments were performed in linear cell strands of different width (approximately 0.1 and 0.8 mm) and isolated porcine LV preparations. Uniform field shocks were applied across strands or LV preparations during diastole and action potential (AP) plateau. Depending on shock strength, three different types of activation were observed in cell strands. Weakest shocks produced "delayed make" activation that started on the cathodal strand side after long latency and rapidly spread to the anodal side. Stronger shocks caused "make" activation with short latency and rapid spread across strands. Strongest shocks caused nonuniform "make-break" activation where the cathodal side was activated with a short latency but activation of the anodal side was delayed until after the shock end due to a large negative shock-induced polarization. Spatial averaging of Vm responses across 0.1-mm (but not 0.8-mm) strands resulted in AP upstrokes and plateau polarizations that closely resembled the Vm responses measured in LV myocardium. The shock strength for the transition between fast and delayed activation in 0.1-mm cell strands and LV myocardium was similar as well. CONCLUSION: These data provide evidence that microscopic tissue structures with dimensions of approximately hundred microns are responsible for shock-induced activation of ventricular tissue.

Action Potentials↗

Correction of motion artifact in cardiac optical mapping using image registration.

Cardiac motion is one of the main sources of artifacts in epifluorescence imaging experiments. It can cause significant error in electrophysiological measurements such as action potential duration. We present a novel approach that uses image registration based on maximization of mutual information to correct for in-plane cardiac motion in such experiments. The approach is relatively fast (a few seconds per frame) and is performed entirely post acquisition. The image registration approach is an alternative to traditional approaches such as mechanical restraint of the heart or addition of chemical uncouplers. Our results show that the image registration method significantly reduces motion-related artifacts in experimental data.

Algorithms↗

Optical mapping of activation patterns in an animal model of congenital heart block.

Congenital heart block (CHB) is associated with high mortality and affects children of mothers with autoantibodies (IgG) to ribonucleoproteins SSB/La and SSA/Ro. IgG from mothers of children with CHB (positive IgG) was used to assess activation patterns in both the right atrium (RA) and right ventricle (RV) of Langendorff-perfused young rabbit hearts. Optical action potentials (AP) were obtained by using a 124-site photodiode array with 4-[-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium. Optical APs were recorded to simultaneously image activation patterns from the RA and RV. Perfusion of positive IgG (800--1,200 micro resulted in sinus bradycardia and varying degrees of heart block. Activation maps revealed marked conduction delay at the sinoatrial junction but only minor changes in overall atrial and ventricular activation patterns. No conduction disturbances were seen in the presence of IgG from mothers with healthy children. In conclusion, besides atrioventricular (AV) block, positive IgG induces sinus bradycardia. These results establish that the sequelae of CHB are associated with impaired intrasinus and/or sinoatrial conduction. The findings raise the possibility that sinus bradycardia in the developing heart may indicate the potential for AV conduction disturbances.

Action Potentials↗