The formation of the atrioventricular conduction axis is linked in development to ventricular septation

The vertebrate heart shows great variability in the number of chambers according to animal physiology, life history and phylogeny. Generally in reptiles, ventricular septation varies among clades from a partially septated ventricle in some taxa of non-crocodylian lineages, e.g. in turtles or squamate reptiles, to a fully septated ventricle in extant Archosauria (Koshiba-Takeuchi et al., 2009), comprising crocodylians and birds (Brusatte et al., 2010). Evolution of the four-chambered heart has occurred at least 3 times during vertebrate phylogeny (Moorman and Christoffels, 2003; Summers, 2005): in mammals, and in birds and crocodylians. The atrioventricular conduction system (AVCS) has previously been described in crocodylians (Jensen et al., 2018) and also in some squamates (varanids; Hanemaaijer et al., 2019). Turtles have also been suggested to possess a specialized conducting tissue in the funnel-shaped atrioventricular (AV) canal (Robb, 1952). AVCS function is essential for impulse transmission to the ventricle apex and, therefore, it is responsible for the contraction of the working myocardium (Sedmera et al., 2003). Moreover, a damaged AV junction leads to arrhythmias (Sedmera et al., 2015). However, a completely developed AVCS is traditionally considered to be present only in endotherms (Challice and Virágh, 1974; Szabóa et al., 1986).

Unlike that in other reptiles, the crocodylian AV junction is unique in having an insulating plane that disrupts the ventral part of the myocardial AV continuity (Davies et al., 1952). The pattern of impulse propagation in the developing crocodylian heart could be either primitive, from base to apex via the primary ring, as is also observed in embryonic mammals and birds (Moorman et al., 1998; Olejnickova et al., 2018; Gourdie et al., 1999; Rentschler et al., 2001), or mature, from apex to base. A distinct preferential conduction pathway was demonstrated along the crux of the heart in the juvenile alligator heart (Crick et al., 1998; Jensen et al., 2018). The presence of this pathway results in an apex-to-base activation of these hearts, similar to the situation in mature birds (Chuck et al., 1997; Reckova et al., 2003) and mammals (Morley and Vaidya, 2001). Therefore, we hypothesized that the formation of the AVCS and the full septum is interlinked in crocodylians not only in phylogeny as previously shown (Jensen et al., 2018) but also in development as in mammals (Rothenberg et al., 2005) and birds (Chuck et al., 1997). To test this hypothesis, we studied embryonic and fetal stages of Siamese crocodile (Crocodylus siamensis Schneider 1801) to reveal whether a switch from a primitive (base-to-apex) to a specialized (apex-to-base) pattern occurs, and if so, whether it coincides with ventricular septation. For comparison, embryonic hearts of Amazon toadhead turtle [Mesoclemmys heliostemma (McCord, Joseph-Ouni and Lamar 2001)], with partial ventricular septation, and white leghorn chicken [Gallus gallus domesticus (Linnaeus 1758)] with complete ventricular septum were studied using the same approach. All selected species belong to one evolutionary clade, the Archelosauria (Crawford et al., 2015).

White leghorn chicken eggs (G. gallus domesticus; N=33; Institute of Molecular Genetics, Kolec, Czech Republic; incubation period: 21 days) were incubated at 37.5±1°C in 70% humidity with rotation every 6 h. Embryos were isolated from the eggs in ice-cold Tyrode's buffer (Sankova et al., 2010) and quickly decapitated. Only the isolated chicken hearts were optically mapped (see below).

Embryonated eggs of the Amazon toadhead turtle (M. heliostemma; N=2; National Museum, Prague, Czech Republic; incubation period: ∼230 days at 28±1°C with short irregular fluctuations from 18 to 31°C, and 90% humidity; for details, see Moravec, 2017) and the Siamese crocodile (C. siamensis; N=16; The Crocodile Zoo, Protivin, Czech Republic; incubation period: ∼68 days at 32±1°C and 70% humidity; M. Prochazka, personal communication; Bezuijen et al., 2013; Platt et al., 2012) were used as a tissue source. Reptilian eggs were incubated without rotation on vermiculite substrate (a hydrous phyllosilicate mineral, which holds humidity and is widely used for incubation of reptilian eggs; Deeming, 2004). The embryos isolated from the eggs were handled according to the Czech law (Gregorovicova et al., 2012) and, in addition, sedated in MS 222 according to Conroy et al. (2009). Under the Czech Animal Protection Law, usage of embryos in eggs is exempt from regulation; furthermore, only samplings followed by in vitro measurements on isolated hearts were performed. After quick sedation in 0.2% solution of MS 222 (higher permeability of the solution for the reptilian embryos, similar to amphibians; V.O., unpublished observations) in ice-cold reptilian buffer (in mmol l⁻¹: NaCl 95, Tris 5, NaH₂PO₄ 1, KCl 2.5, MgSO₄ 1, CaCl₂ 1.5, glucose 5, pH adjusted to 7.5 with HCl; Jensen et al., 2012), the embryos were quickly decapitated. The hearts were then extracted from the decapitated embryos with the posterior thoracic wall attached and stained at room temperature for 15 min in di-4-ANEPPS (2.5 mmol l⁻¹ in species-appropriate buffer; Invitrogen, Carlsbad, CA, USA). After brief rinsing, the samples were pinned at various orientations (anterior, posterior, left and right lateral view) on the silicone-covered bottom of a custom-made dish filled with oxygenated Tyrode's or reptilian buffer with the addition of 0.15 μmol l⁻¹ blebbistatin (Vostarek et al., 2016) for chicken or of 40–60 μmol l⁻¹ cytochalasin D (Sedmera et al., 2003) for reptiles to reduce motion artefacts. Embryonic day 14 (ED14) chicken hearts were perfused in Langendorff mode adjusted as described elsewhere (Hlaváčová et al., 2017; Olejnickova and Sedmera, 2020). Temperature during optical mapping differed among the tested species according to their physiological optimum: M. heliostemma 24°C, C. siamensis 32°C, and G. gallus domesticus 37°C. Optical mapping was performed at 0.25–1 kHz using an ULTIMA L setup (SciMedia, Tokyo, Japan; Sankova et al., 2010; Gregorovicova et al., 2018; Hanemaaijer et al., 2019) for all tested species. BV_Ana software (SciMedia Brain Vision, Tokyo, Japan) was used to generate epicardial activation maps and analyse ventricular activation patterns.

Double staining was carried out according to Kvasilova et al. (2018). Mouse monoclonal human natural killer-1 (HNK-1) primary antibody IgM (1:50; Becton Dickinson #347390) detected by cyanine 5 (Cy5)-conjugated goat anti-mouse IgM secondary antibody (1:200; Jackson ImmunoResearch #115-175-075) was used together with mouse monoclonal IgG2b isotype to myosin heavy chain (MF20) primary antibody (1:5; DSHB #2147781) detected by Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (1:200; Invitrogen # A11001). The nuclei were counterstained with Hoechst (1:80,000, Sigma #33342).

Two complementary approaches, optical mapping and immunohistochemistry, were chosen to reveal the signal propagation pattern through the crocodylian heart and its potential morphological substrate. Optical mapping revealed a primitive pattern of electrical impulse spreading (base to apex) on the ventral and dorsal side of the ventricle at 124 days post-oviposition (dpo) and near the completion of development (214 dpo) in the Amazon toadhead turtle (Fig. 1A). A similar activation pattern was observed in the Siamese crocodile at 21 dpo, before ventricular septation was completed: the signal spread from base to apex via the primary ring (Fig. 1B). Around septation (30 dpo), the first site of ventricular activation was localized in the future right ventricle and activation proceeded in the mature specialized pattern in the apex-to-base direction (Fig. 1C). Finally, in the post-septation stages (after 42 dpo), two centres of epicardial activation appeared in the right and left ventricle, and there were almost no changes in spreading of the electrical impulse until hatching (65 dpo, not shown). In chicken, the signal ran via the immature primary ring pattern before septation on ED04 in the left part of the ventricle (Fig. 1D). At the septation stage (ED07), both patterns were observed: from base to apex as well as from apex to base. After ED07, exclusively the mature apex-to-base activation was present (Fig. 1E). Cardiac parameters showed similar embryonic development in the crocodile and chicken. The difference between the crocodile and chicken was a longer (approximately threefold) incubation period in the crocodile. From this perspective, all important cardiovascular changes occur in the first half of development in both species. Baseline physiological parameters obtained from optical mapping during embryonic development of the chosen species are summarized in Table 1.

Neural marker HNK-1, previously described as a potential marker of the conduction system in mammals (Gorza et al., 1988) and birds (Luider et al., 1993), was chosen for characterization of the specialized AVCS tissue through co-localization with an established cardiomyocyte marker (González-Sánchez and Bader, 1985), MF20 (Fig. 2). In turtles, we did not find any AVCS in the ventricle during development and the only co-localization was present in the AV canal myocardium. In contrast, AVCS was clearly observed in the middle part of the forming ventricular septum in Siamese crocodiles and in chickens.

Here, we show that the formation of the AVCS is linked to full ventricular septation in development. In the clade Archelosauria (Crawford et al., 2015), both birds and crocodylians have a four-chambered heart with complete septation of the ventricle (Eme et al., 2010). Turtles, as an outgroup taxon to Archosauria (Chiari et al., 2012), show only partial ventricular septation (Koshiba-Takeuchi et al., 2009). However, some similarity during embryonic development could be shown as an ancestral state, such as the presence of morphologically very active structures (e.g. in AV canal), an early ventricular activation pattern, or positivity for different markers in Archelosauria overall (e.g. conserved HNK-1 immunostaining in the AV canal myocardium).

Activation pattern of the ventricle is determined primarily by three factors: the nature of the AV connections (Sedmera et al., 2003), the presence of a specialized (differentiated and possibly morphologically distinct) conduction pathway within the ventricle (Reckova et al., 2003; Rentschler et al., 2001), and the specific shape of the heart (Gregorovicova et al., 2018). Propagation of signal in crocodylians was described in detail recently as a dorsal preferential AV conduction pathway (Jensen et al., 2018). In turtles, trabeculae transfer electrical impulses until hatching and the propagation of signal itself is strictly in the base-to-apex direction. In contrast, the impulse is spread in the first third of embryonic crocodylian development via trabeculae and in about the middle of the incubation period (30 dpo) it changes direction of spreading from a base-to-apex to an apex-to-base pattern, similar to that in chickens. Therefore, mature signal propagation occurs in the apex-to-base direction (van Weerd and Christoffels, 2016) in crocodiles after septation, similar to that in birds and mammals (Gourdie et al., 1999; Chen et al., 2010; Anderson et al., 2018).

Completely developed AVCS was classically considered only in endothermic vertebrates – birds and mammals (Davies, 1942). During embryonic development, the propagation of the electrical impulse through AVCS shows similar patterns. Around septation stage in the crocodylian AVCS, structures are linked with the developing ventricular septum.

HNK-1 is an established marker of developing AVCS in birds (Luider et al., 1993) and mammals (Aoyama et al., 1995; Wenink et al., 2000). While the interpretation of HNK-1 localization has limitations because of the specificity of expression of its epitopes (Kvasilova et al., 2018), its co-localization with an established myocardial marker MF20 (González-Sánchez and Bader, 1985; Chuck and Watanabe, 1997) clearly shows similarity in positive areas of the heart between the chosen ectothermic and endothermic model species. In these areas, such as in the AV canal, we observed a similar positive pattern in all tested species. However, co-localization of neural marker HNK-1 and cardiomyocyte marker MF20 identified the AVCS on the top of the ventricular septum in crocodile and chicken. We thus confirmed a lack of specialized conducting tissue in developing turtle heart, in contrast to earlier speculations about its presence in adult turtle Trachemys scripta (Robb, 1952). This result could be due to a postnatal transition between hatching and adulthood periods, which may explain differences among presence/observations of these fine structures.

It thus appears that during embryonic development the AVCS is tied to completion of ventricular septation in crocodylians, similar to endotherms – birds and mammals – in both functional and morphological aspects.

We would like to thank Jiri Moravec, National Museum, Czech Republic, for providing Mesoclemmys heliostemma eggs; Miroslav Prochazka, The Crocodile Zoo Protivin, for providing Crocodylus siamensis eggs; and Jarmila Svatunkova, Blanka Topinkova, Eva Zabrodska and Patrik Stych for excellent technical assistance.