The snake heart pacemaker is localized near the sinoatrial valve

Each contraction of the vertebrate heart is initiated by the spontaneous generation of electrical activity in specialised pacemaker tissue on the venous pole of the heart (reviewed by Burkhard et al., 2017). While both structure and function of the mammalian sinoatrial node are quite well understood, even the general principles of cardiac pacemaker location and function remain uncertain in other vertebrate classes. Such information is obviously essential to understand the evolution of cardiac rhythm generation and its regulation. Invertebrate hearts contain pacemaker cells that are scattered across the heart in a diffuse manner. For example, slow diastolic depolarization (SDD), the main electrophysiological marker of pacemaker activity, can be observed in any part of a lugworm heart (Abramochkin et al., 2010). In fishes, the cardiac pacemaker becomes a circular field located around the sinoatrial valve (SAV; Haverinen and Vornanen, 2007). Finally, the mammalian heart has a compact pacemaker structure: the sinoatrial node. Thus, the concentration of pacemaker cells in an anatomically distinct compact structure appears to be the general trend of pacemaker evolution. However, the particular steps of this evolution are still not well described.

The exact position of the pacemaker driving the cyclostome heart remains to be localised. In hagfishes, there is SDD in the atrial myocardium (Arlock, 1975) and expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in the atrial tissue (Wilson et al., 2013). However, in lamprey, both the atrium and ventricle lack any features of pacemaker electrical activity (Haverinen et al., 2014). In elasmobranch fishes, nodal myocardial tissue appears across the entire sinus venosus (SV) (Ramos et al., 1996; Ramos, 2004), but electrophysiological evidence for pacemaker function in the SV has only been provided in sting-rays (Saito, 1969).

The SV and its myocardial layer are reduced in teleosts compared with those in elasmobranchs. In plaice (Santer and Cobb, 1972) and sculpin (Santer, 1985), the SV consists of myocardial bundles partly separated by connective tissue, and Saito (1969) provided electrophysiological evidence of pacemaker activity in SV of sea eel. However, in various cyprinids, salmonids and Atlantic cod, the SV lacks myocardium and consists mainly of connective tissue (Yamauchi et al., 1973; Yamauchi and Burnstock, 1968; Haverinen and Vornanen, 2007; Lukyanov et al., 1983). In these teleosts, nodal tissue is concentrated in a ring-like structure surrounding the SAV (Haverinen and Vornanen, 2007; Yamauchi, 1980; Yousaf et al., 2012). Consistent with these histological observations, electrical activity typical for primary pacemaker of the heart, with smooth transition from SDD to action potential (AP) upstroke was found in a circle region at the base of the leaflets of the SAV (Lukyanov et al., 1983, 1986; Haverinen and Vornanen, 2007).

Trautwein and Zink (1952) provided classic recordings of pacemaker electrical activity in the SV of frogs using sharp microelectrodes, and a similar location of pacemaker was later confirmed in the toad Bufo marinus (Bramich et al., 1990). Hutter and Trautwein (1956), however, realised that only a small region within the SV works as the primary pacemaker (the place where excitation originates in normal conditions), while the rest is excited by conduction and acts as subsidiary pacemaker, which also has automaticity, but less pronounced than that of the primary pacemaker, at least in normal conditions. Moreover, the exact position of the primary pacemaker within the SV can move (pacemaker shift) under autonomic influence (Hutter and Trautwein, 1956). In mammals, the dominant pacemaker site is located in an anatomically distinct compact sinoatrial node at the border of the right atrium and the right sinus horn or superior caval vein (Monfredi et al., 2010; Boyett et al., 2003).

The location and function of the cardiac pacemaker is not well known in reptiles, and there is conspicuous lack of electrophysiological information. Early pioneering studies by Walter H. Gaskell pointed to the cardiac pacemaker being placed in the SV of turtles (e.g. Gaskell, 1883), which was supported by later electrophysiological studies (Hutter and Trautwein, 1956), but immunohistochemical studies indicate pacemaker tissue at the border between SV and right atrium in Burmese pythons and Anolis lizard (Jensen et al., 2017). This location was supported by recordings with surface electrodes and points to a retrograde electrical activation of the SV (Jensen et al., 2017, 2014a,b). The present study aims to determine the exact location of the leading pacemaker site in the heart of Burmese python using the conventional sharp microelectrode technique and optical mapping.

Burmese pythons (Python bivittatus Kuhl 1920) of either sex with a body mass of 220–350 g were kept at Aarhus University for several months in individual containers with free access to water, a temperature range of 26–32°C, and a humidity of 80%. They were fed weekly and gained mass in captivity, but were fasted for 2–3 weeks before the hearts were studied. The experiments were performed in accordance with the Danish Law for Animal Experimentation.

The animals were killed by a sharp blow to the head, and the heart was rapidly excised and rinsed in physiological solution containing (in mmol l⁻¹) 115 NaCl, 2.5 KCl, 1 MgSO₄, 1.2 NaH₂PO₄, 5 glucose, 5 pyruvate, 2.5 CaCl₂ and 25 NaHCO₃ and had a pH of 7.7–7.8 when equilibrated with 2 kPa CO₂ in O₂ at 25–30°C. The preparation containing the SV, the sinoatrial junction and the proximal piece of right atrium was cut off and pinned on the bottom of the Sylgard-coated chamber (3 ml in volume), where it was superfused with a constant flow of physiological saline solution (6 ml min⁻¹) at 30°C. The SV with right horn and posterior horn was opened and the left horn was cut off. The microelectrodes were always impaled from the endocardial side of SV. All preparations exhibited spontaneous contractions with stable rhythm throughout the experiments.

The recording of electrical activity commenced when the preparation had stabilized in the Ringer solution for 40 min. Conventional sharp glass microelectrodes were used for intracellular recording of electrical activity in the myocardium of SV. The microelectrodes were filled with 3 mol l⁻¹ KCl (40–80 MΩ) and connected to a high input impedance amplifier (Model 3100, A-M Systems, Carlsborg, WA, USA); microelectrodes needed a resistance above 40 MΩ for stable impalements. The signal was digitized and recorded using the Powergraph 3.3 (DI-Soft, Moscow, Russia), and analysed using MiniAnalysis 3.0 software (Synaptosoft, Fort Lee, NJ, USA). If acetylcholine or noradrenaline were applied, the stable impalements were maintained during whole period of drug action. AP duration at 50% of repolarization (APD₅₀), resting membrane potential, AP amplitude, AP upstroke velocity (dV/dt_max) and beating rate of the preparations were determined during off-line analysis.

Electrophysiological experiments were conducted on enzymatically isolated myocytes from the region surrounding the sinoatrial valve. The isolation procedure resembled our earlier methods for fish hearts (Abramochkin and Vornanen, 2015), but high enzyme concentrations (1.5 mg ml⁻¹ collagenase Type IA and 1 mg ml⁻¹ Trypsin IV) were needed to digest the considerable amount of connective tissue. A stainless steel cannula was inserted via the right aorta into the ventricle and secured with a fine thread. The left aorta and pulmonary artery were ligated to avoid leak and ensure completely retrograde flow from the ventricle to the atria. Initially, the heart was perfused at room temperature with Ca²⁺-free solution for 20 min, whereupon hydrolytic enzymes and fatty acid-free serum albumin (Sigma) were added for next 40 min. Then, the SAV was separated from the rest of the SV, minced and incubated in a similar enzyme solution on a shaker at room temperature. After another 40 min, the solution was replaced with enzyme-free solution of the same composition, the tissue was triturated with Pasteur pipette to obtain isolated cells. Myocytes were stored at 5°C and studied on the same day. The Ca²⁺-free solution contained (in mmol l⁻¹) 100 NaCl, 10 KCl, 1.2 KH₂PO₄, 4 MgSO₄, 50 taurine, 20 glucose and 10 HEPES at pH 6.9 (adjusted with NaOH at 20°C).

The whole-cell voltage clamp recording of I_K1 and I_f was performed using an Axopatch 200B amplifier (Molecular Devices, San Jose, CA, USA). Myocytes were superfused at room temperature (23±0.5°C) with an external saline solution containing (in mmol l⁻¹): 150 NaCl, 5.4 KCl, 1.8 CaCl₂, 1.2 MgCl₂, 10 glucose, 10 HEPES; pH adjusted to 7.6 at 20°C with NaOH. E-4031 (5×10⁻⁶ mol l⁻¹) was continuously present in external solution to block I_Kr. Patch pipettes of 1.5–2.5 MΩ resistance were pulled from borosilicate glass (Sutter Instruments, Novato, CA, USA) and filled with K⁺-based electrode solution containing (in mmol l⁻¹): 140 KCl, 1 MgCl₂, 5 EGTA, 4 MgATP, 0.3 Na₂GTP and 10 HEPES with pH adjusted to 7.2 with KOH. Series resistance and capacitances of pipette and cell were routinely compensated. Current amplitudes were normalized to the capacitive cell size (pA/pF).

The spread of excitation in the SAV region was visualised by optical mapping, di-4-ANEPPS-based technique in multicellular preparations of SV, which were dissected as described previously (Abramochkin et al., 2020). The optical mapping rig included a photodiode array (PDA, WuTech H-469V, Gaithersburg, MD, USA) designed for high-speed data acquisition (1.63 kfps). Macroscopic projections of the cardiac tissue preparations were transferred to the PDA with aid of the optical system including adapters and Computar V5013 (CBC Group, Japan) camera lens (focal length 50 mm, aperture ratio 1:1.3) mounted in distance of 24 mm from tissue surface. The optical system allowed to project an area of 5 mm in diameter to a hexagonal array of 464 PDA photodiodes; each photodiode covered the surface of 0.23 mm in diameter approximately. The view-field was projected also to the monitoring CCD camera (NexImage, Celestron, USA) used to match the mapping area and sites in the tissue preparations. An excitation light was emitted by LED (520±40 nm) arrays surrounding the experimental chamber with mapped preparation. A long-pass emission filter (λ>650 nm) was positioned in front of the camera lens.

After dissection, SV preparations were placed into experimental chamber and superfused with a constant flow of 30°C physiological saline solution (6 ml min⁻¹) of the same composition as used for microelectrode experiments. Voltage-sensitive dye di-4-ANEPPS (5 mg ml⁻¹, dissolved in DMSO) was added to physiological solution (final concentration 5×10⁻⁵ mol l⁻¹) for 30 min staining of preparation. Then preparations were equilibrated for 20 min prior to mapping procedure. To suppress mechanical artefacts, the electromechanical uncoupler blebbistatin (5×10⁻⁵ mol l⁻¹) was added to the perfusion solution.

In all experiments fluorescent signals (optical APs) were recorded continuously for 5 s with 0.614 ms frame intervals, digitized using a data acquisition system (CardioPDA-III; RedShirtImaging, Decatur, GA, USA) and analyzed using Cardioplex (v.8.2.1, RedShirtImaging) software. Resting fluorescence was determined before each recording. Signals were processed via Savitsky-Golay filter using custom algorithm to remove noise and were normalized to the resting fluorescence level. Also, minimal high-pass filter was applied to remove slow photodiode-derived basal drift. The maximum upstroke derivative (dF/dt_max) for each optical AP was calculated to determine the activation times in the mapped areas. Isochronic activation maps were constructed from activation times using an in-house developed software. The area of an initial activation was defined as area covered by depolarization in first 2 ms after the start of excitation. The localization of the primary activation sites was defined as the center of the 2 ms activated area.

E-4031 was purchased from Tocris (Bristol, UK). Collagenase type IA, trypsin, noradrenaline (NA), blebbistatin, BaCl₂ and acetylcholine chloride (ACh) were purchased from Sigma (St Louis, MO, USA), and Di-4-ANNEPS from Thermo Fisher Scientific.

All data are expressed as means±s.e.m. Effects of ACh and NA on AP waveform and optical mapping parameters were evaluated using paired t-test. A P-value of 0.05 was regarded as the limit of statistical significance.

The isolated SV preparations from pythons exhibited stable, spontaneous and rhythmic contractions (40.2±2.3 min⁻¹; n=13) with stable AP waveform for several hours when superfused with oxygenated physiological solution (see Abramochkin et al., 2020).

In the major part of the SV (whole posterior horn, right horn, see Fig. 1A), we only observed electrical activities with features of the working myocardium; i.e. a stable negative resting potential of −81.3±1.5 mV, large AP amplitude of 109.2±3.1 mV and fast AP upstroke with dV/dt_max of 56±3.2 V s⁻¹ (n=7). The APD50 was 350.2±5.9 ms. In this type of SV myocardium, ACh (10⁻⁶ mol l⁻¹) greatly shortened APD50 by 91.5±3.1% of the control value and caused a small, but significant decrease in AP amplitude by 17.9±2.1% without significant changes in resting membrane potential (n=6). However, the rate of spontaneous APs only decreased by 6.4±1.8% of control (n=6). Higher ACh concentration (10⁻⁵ mol l⁻¹) induced almost similar effects (Fig. 1B) with exception of slower rhythm by as much as a 21.4±4.2% reduction (n=5). NA (10⁻⁶ M) transiently prolonged the APs (Fig. 1C). Within 1 min of NA application, APD50 was 10.6±2.3% longer than before NA (n=5), but this effect subsided within 7 min, although AP amplitude tended to increase. NA accelerated the spontaneous rhythm 1 min after drug application, and reached 133.2±7.4% after 7 min.

We successfully impaled six preparations with microelectrodes to identify the specific location for pacemaker activity, i.e. SDD and APs with slow upstroke. In all six preparations, we could record electrical activity with SDD in a circular region around the SAV, but not further than 1.5 mm from the SAV (Fig. 1A). However, in SAV leaflets, which consist of myocardial tissue, we found no evidence for SDD. Furthermore, the AP waveform was the same as in posterior or right horn of SV. Electrical activity in the region around SAV was very heterogeneous in terms of SDD velocity, AP amplitude and upstroke velocity, as well as AP duration (Fig. 1A). Therefore, for further investigation we selected the pattern of electrical activity characterized by smooth transition from SDD to upstroke of AP (primary pacemaker at Fig. 1A). This pattern was first hypothesized by Hutter and Trautwein (1956) to be characteristic for the primary (leading) pacemaker site and that assumption was confirmed in numerous mapping studies (Bleeker et al., 1980; Vinogradova et al., 1998; Abramochkin et al., 2009). Electrical activity in primary pacemaker sites of python SV was also characterized by low dV/dt_max of 1.42±0.13 V s⁻¹, low AP amplitude of 74.4±3.8 mV and very long APs with APD₅₀ of 602.6±57.5 ms (n=6 for each parameter). The stable resting membrane potential was absent in these sites owing to SDD, but the value of maximal diastolic potential was −63.7±4.2 mV (n=6). The velocity of SDD was 27.3±2.48 mV s⁻¹ (n=6). In all 6 preparations the primary pacemaker sites were located near the basement of the left leaflet of SAV (Fig. 1A)

At the leading pacemaker site, ACh and NA caused principally different effects to those observed in the major (‘working’) part of the SV. ACh (10⁻⁵ M) produced prominent hyperpolarization, a decrease in AP amplitude and slowed the SDD by 66.7±7.4% (n=5) of control SDD velocity; leading to slower rhythm generation (Fig. 1D). At the same time, no drastic AP shortening, as seen in working SV myocardium, was observed. NA (10⁻⁶ mol l⁻¹) did not affect maximal diastolic potential and AP amplitude, but increased SDD velocity by 82.8±7.5% of the control level and accelerated the rhythm (Fig. 1E).

Thus, electrical activity at the base of the SAV left leaflet differed entirely from the working myocardium in the SV and responded differently to ACh and NA: neurotransmitters greatly affect the SDD slope, but changed AP waveform less than in the working myocardium. Since the microelectrodes only allow recordings of electrical activity at a single point of the preparation, we also used optical mapping to analyse the activation sequence to identify the position of leading pacemaker in the SV from pythons.

The optical mapping was performed on similar preparations as those studied with microelectrodes. The site of the earliest activation, i.e. the leading pacemaker site, was located 1.5–2 mm from the left leaflet of the SAV (Fig. 2B). No specialized conduction pathways or conductive corridors were observed surrounding the primary pacemaker sites under control conditions. However, from there, the excitation spread with low anisotropy directed predominantly circumferentially to the SAV border. A second, minor site of early activation could be found in the mapped region in two of the five preparations (see Fig. 2). The area of the primary activation zone covered 1.7±0.4 mm² (n=5) in control conditions, which is less than 5% of the total SV square.

ACh (10⁻⁵ mol l⁻¹) shifted leading pacemaker site towards the SAV (Fig. 2C). At the same time, the region where the leading pacemaker was located in normal conditions turned into zone of slow conduction (activation with >10 ms delay after leading site). Upon addition of 10⁻⁶ mol l⁻¹ NA, a number of sites located close to the SAV left leaflet started to activate almost synchronously (Fig. 2D), but the region of primary pacemaker activity in control conditions lost its dominance, similarly to results obtained in the presence of ACh. Owing to delocalized primary excitation and lack of zones with a slow conduction in the presence of NA the excitation covered 90% of the mapped area in the SV in a significantly shorter time in comparison to ACh application (13±4 and 22±4 ms, P<0.05).

At the last stage of our experiments, we confirmed the presence of pacemaker cardiac myocytes in the SAV region of pythons. The investigated cells could easily be divided in two groups according to their electrophysiological properties. The first group of cells were usually elongated spindle-shaped (Fig. 3A) with a mean capacitance of 30.1±1.1 pF (n=48) and a clear inward rectifier I_K1 current during the hyperpolarizing ramp protocol (Fig. 3C). Square-pulse hyperpolarization to potentials from −100 to −160 mV induced instantaneous inward current (Fig. 3E), which was attributed to inward component of I_K1. Note also the large peak inward current, I_Na, induced by return of membrane potential to holding level (−35 mV).

Cells of the second group were elongate or ‘short-spindle’ shaped (Fig. 3B) and much smaller (capacitance of 17.1±1.9 pF, n=7, P<0.05). They were very scarce, representing merely 13% of all myocytes studied. They lacked a distinct I_K1 current (Fig. 3D), but hyperpolarization to potentials from −100 to −160 mV induced a time-dependent inward current (Fig. 3F), which was similar in size to I_K1 recorded in the first group of cells. No peak I_Na or just a very small peak I_Na was observed after return to −35 mV (Fig. 3F). The time-dependent hyperpolarization-induced inward current could be abolished by 2 mmol l⁻¹ Cs²⁺ (Fig. 3F, inset). Its mean I–V curve was standard for a pacemaker current (I_f) recorded in mammalian sinoatrial node cells (Verkerk et al., 2007), although shifted left by 10–20 mV (Fig. 3G). Therefore, we assume that recorded current in the second type of cells is I_f.

It is likely that the first group of cells represents working myocytes of the SV, while the second group consists of true pacemaker cells that express I_f current. However, given that the I_f current in the second cell types is negligible at potentials within the physiological limits, the physiological role of these cells remains uncertain.

In summary, both microelectrode and optical mapping data point to the region between left leaflet of SAV and posterior horn of SV as the position of leading pacemaker site in the python heart under normal conditions. However, all of the circular zone around the SAV should be considered to be a subsidiary pacemaker region. Points within that circular zone can work as leading pacemaker sites under the influence of autonomic neurotransmitters, as in mammalian hearts (Bleeker et al., 1980; Shibata et al., 2001; Abramochkin et al., 2009). Cells with specific electrophysiological properties similar to those of mammalian pacemaker cells may be isolated from the SAV region. These cells lack I_K1 and I_Na, but have a pacemaker I_f current.

Authors are grateful to Elin Petersen for excellent technical assistance and Heidi Meldgaard for devoted animal care.