HNK‐1 in Morphological Study of Development of the Cardiac Conduction System in Selected Groups of Sauropsida

Abstract

Human natural killer (HNK)‐1 antibody is an established marker of developing cardiac conduction system (CCS) in birds and mammals. In our search for the evolutionary origin of the CCS, we tested this antibody in a variety of sauropsid species (Crocodylus niloticus, Varanus indicus, Pogona vitticeps, Pantherophis guttatus, Eublepharis macularius, Gallus gallus, and Coturnix japonica). Hearts of different species were collected at various stages of embryonic development and studied to map immunoreactivity in cardiac tissues. We performed detection on alternating serial paraffin sections using immunohistochemistry for smooth muscle actin or sarcomeric actin as myocardial markers, and HNK‐1 to visualize overall staining pattern and then positivity in specific myocyte populations. We observed HNK‐1 expression of various intensity distributed in the extracellular matrix and mesenchymal cell surface of cardiac cushions in most of the examined hearts. Strong staining was found in the cardiac nerve fibers and ganglia in all species. The myocardium of the sinus venosus and the atrioventricular canal exhibited transitory patterns of expression. In the Pogona and Crocodylus hearts, as well as in the Gallus and Coturnix ones, additional expression was detected in a subset of myocytes of the (inter)ventricular septum. These results support the use of HNK‐1 as a conserved marker of the CCS and suggest that there is a rudimentary CCS present in developing reptilian hearts. Anat Rec, 302:69–82, 2019. © 2018 Wiley Periodicals, Inc.

Myocardial development in crocodylians

“…The spontaneous beating frequency of isolated hearts at constant temperature showed very little changes with development, oscillating within the range of 50 and 90 bpm (Table 1), with the exception of a trend toward heart rate slowing (especially in comparison to in ovo values, Kockova and Sedmera, unpublished observations) in hearts of C. mindorensis at later stages. The pectinate muscles first appeared between 10 and 12 dpo in C. siamensis (Figure 2) and 9 and 16 dpo in C. mindorensis , similar to situation observed previously in C. niloticus (Ferguson stages 6 and 7, 11‐13 dpo) 29 . At later stages, they filled up the majority of the atrial chamber, similar to ventricular trabeculae, giving them a spongy appearance typical of poikilotherm hearts 30 .…”

“…After the mapping, the isolated hearts were fixed in 4% paraformaldehyde in PBS overnight and processed for paraffin histology. Alternating serial sections were cut at 7 to 10 μm and stained with H&E, mouse monoclonal IgG2a isotype to Alpha Smooth Muscle Action (SMA, 1:800; Sigma #A2547), mouse monoclonal IgM isotype to Alpha Sarcomeric Actin (SA, 1:500; Sigma #A2172), mouse monoclonal IgM heavy chains and kappa light chains to Human Natural Killer‐1 (HNK‐1, 1:100; Becton Dickinson #347390), mouse monoclonal IgG2b isotype to myosin heavy chain (MF20,1:5; DSHB #2147781), mouse monoclonal IgG1 isotype to kappa light chain (QH1, 1:600; DSHB #531829) and their selected combinations 29 . On the next day, the sections for visible light staining, all primary antibodies were detected by Horseradish Peroxidase (HRP)‐conjugated Goat Anti‐Mouse secondary antibody (1:200 or 1:500; Jackson ImmunoResearch #115‐035‐068) and the nuclei were counterstained with hematoxylin.…”

Low incidence of atrial septal defects in nonmammalian vertebrates

“…The bound probe was detected immunologically using antidigoxigenin Fab fragment covalently coupled to alkaline phosphatase and NBT/BCIP was used as the chromogenic substrate (Roche). The immunohistochemistry on the Siamese crocodile was performed as in (Kvasilova, Gregorovicova, Kundrat, & Sedmera, 2019 ). Briefly, sections were incubated overnight at +4°C with mouse monoclonal IgM isotype to α sarcomeric actin (SA; 1:500; #A2172; Sigma).…”

Anguimorpha as a model group for studying the comparative heart morphology among Lepidosauria: Evolutionary window on the ventricular septation

“…After more than one century of studying the cardiovascular system in tetrapods, the sauropsid hearts still fascinate the scientists because of the cardiac chambers variable arrangement. Great variation in the heart development and morphology in sauropsids are well described (Hanemaaijer et al, 2019;Jensen et al, 2014;Kvasilova et al, 2018). However, the reasons underlying these variations are poorly understood.…”