Cardiac conduction system malformations in heterotaxy result from dysregulated Pitx2 expression
Kunihiko Joo, Ryohei Matsuoka, Keiko Kitajima, Kenta Yashiro, Akira Shiose, Ryuji Tominaga, Michael M. Shen, Shinya Oki, Chikara Meno
Kunihiko Joo, Ryohei Matsuoka, Keiko Kitajima, Kenta Yashiro, Akira Shiose, Ryuji Tominaga, Michael M. Shen, Shinya Oki, Chikara Meno
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Research Article Cardiology Development

Cardiac conduction system malformations in heterotaxy result from dysregulated Pitx2 expression

Abstract

The cardiac conduction system (CCS) develops asymmetrically along the body axes. In heterotaxy syndrome — resulting from aberrant left-right axis formation — atrial and atrioventricular conduction defects can cause life-threatening arrhythmias. However, the developmental mechanisms regulating the atrioventricular conduction system (AVCS) disposition and integrity remain unclear. To investigate the etiology of AVCS malformations in laterality defects, we analyzed CCS development and function in mouse mutants for Cryptic and Lefty1, which are key regulators of Pitx2 in the left-right axis formation. Cryptic–/– embryos exhibited bilateral sinoatrial nodes and an ectopic anterior AV node and bundle accompanied by reduced Pitx2 expression. In contrast, Lefty1–/– embryos showed a hypoplastic sinoatrial node and AV node–bundle dissociation with ectopic Pitx2 expression. Single-cell transcriptomic analysis of Pitx2–/– hearts revealed expansion of AV node and bundle populations, consistent with a repressive role of Pitx2 in AVCS specification. Genetic lineage tracing indicated that Pitx2-expressing cells from the left lateral plate mesoderm populate cranioventral cardiac regions, where AVCS development is suppressed. Together, these findings clarify how global left-right axis information is locally integrated to shape AVCS disposition and integrity, providing a mechanistic model for AVCS abnormalities in laterality-associated congenital heart disease.

Authors

Kunihiko Joo, Ryohei Matsuoka, Keiko Kitajima, Kenta Yashiro, Akira Shiose, Ryuji Tominaga, Michael M. Shen, Shinya Oki, Chikara Meno

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Figure 2

Abnormal cardiac conduction system development in the hearts of Cryptic–/– embryos at E12.5–E14.5.

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Abnormal cardiac conduction system development in the hearts of Cryptic–...
(A–J) Representative images of 3D-reconstructed hearts (A and F) and their original images of in situ hybridization with Hcn4 probes (B–E and G–J) in WT (A–E) (n = 8) and Cryptic–/– (F–J) embryos (n = 8) at E14.5, as presented in Figure 1, A–E. The asterisks in F and H show Hcn4 expression in the superior cushion. Scale bars: 200 μm. (K) SA node head volumes of WT and Cryptic–/– embryos at E12.5 (n = 6–7; biological replicates) and E14.5 (n = 4–7; biological replicates). The left and right values of the same embryo are connected. The magenta bars represent mean ± SD. *P < 0.01 (1-way ANOVA followed by Dunnett’s multiple-comparison test). (L) Representative images of voltage mapping with Di-4-ANEPPS in WT (n = 8) and Cryptic–/– hearts (n = 9) at E12.5 (dorsal views). The dotted circles and rectangles indicate the sites where action potential first appeared, around the head and tail of the SA node, respectively. A single heartbeat is shown. (M and N) First breakthrough site of the action potential around the SA node head (M) and atrial side where the action potential first propagated (N). Quantification is based on the final heartbeat from each heart (n = 8–9; biological replicates). Abbreviations are the same as in Figure 1, except for L, left; M, middle; R, right; SB, septal branch.