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 4

Altered cardiac conduction system population in Pitx2–/– embryos.

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Altered cardiac conduction system population in Pitx2–/– embryos. (A) UM...
(A) UMAP visualization of Tbx3-expressing cardiomyocytes at E13.5 (control, 9739 cells; Pitx2–/–, 8247 cells). The graph shows the proportions of CCS-related cells among the cardiomyocytes: cluster 4 (control, 55 cells; Pitx2–/–, 54 cells); clusters 0, 1, and 5 (control, 155 cells; Pitx2–/–, 327 cells); and the remaining clusters (control, 9,529 cells; Pitx2–/–, 7,866 cells). (B) Expression patterns of Cacna2d2, Smoc2, Shox2, Kcne1, and Gja1 visualized by feature and violin plots derived from the UMAP in A. (C) Subclustering analysis of clusters 0, 1, and 5 from A. The graph shows the ratios of subclusters among the cardiomyocytes: cluster A (control, 89 cells; Pitx2–/–, 49 cells); cluster D (control, 10 cells; Pitx2–/–, 60 cells); and cluster E (control, 11 cells; Pitx2–/–, 49 cells). P values were calculated using the Fisher’s exact test. (D) Feature and violin plots showing the expression of Myh6, Myh7, Kcne1, Gnao1, Irx3, and Etv1 based on the UMAP analysis in C. LNC, lower nodal cells.