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 5

Hypoplastic sinoatrial node and atrioventricular block in Lefty1–/– embryos.

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Hypoplastic sinoatrial node and atrioventricular block in Lefty1–/– embr...
(A–E) Representative 3D-reconstructed hearts (A) and corresponding Hcn4 in situ hybridization of a Lefty1–/– embryo (n = 7) (B–E) at E14.5, as in Figure 1, A–E. (F) SA node head volumes in control (Lefty1+/–, Ctl) and Lefty1–/– hearts at E12.5 (n = 4–12; biological replicates). The magenta bars denote mean ± SD. *P < 0.001 (unpaired 2-sided t test with Welch’s correction). (G) Atrial side where the action potential first propagated (n = 9–14). (H) 3D-reconstructed Hcn4 expression in control and Lefty1–/– hearts at E14.5 (different heart from A). The AV conduction systems are viewed from the right craniodorsal side. Asterisks and dotted areas: discontinuous Hcn4 expression at the AV node/AV bundle junction and within the AV bundle, respectively. Arrowhead: continuous expression between the AV bundle and ring. (I) Action potentials in the atrium (Atr) and ventricle (Vnt) of control and Lefty1–/– hearts at E12.5, traced using voltage mapping. (J–M) Transverse sections showing Pitx2 expression in control (J and K) and Lefty1–/– (L and M) hearts. Green bars indicate Pitx2 expression boundary. Arrowheads indicate ectopic Pitx2 expression. Asterisks mark Pitx2 expression in the septal branch. Scale bars: 200 μm; bars in E, J, and K apply to B–D, L, and M, respectively. (N) RT–qPCR of Pitx2c in laser-microdissected heart tissues from control and Lefty1–/– embryos at E12.5, quantified in the SA node region, including the adjacent superior caval vein and right atrial tissue, and in the right caudodorsal AV canal (rcAVC) region. The magenta bars indicate mean ± SD. **P < 0.05 (unpaired 2-sided t test with Welch’s correction). Abbreviations as in Figure 1 except for iAVC, inferior atrioventricular cushion; (L/R)SH, (left/right) sinus horn; MLA, morphologically left atrium.