THE COMBINATORIAL ROLE OF IROQUOIS HOMEOBOX GENES 3 AND 4 IN THE COMPACTION OF THE VENTRICULAR MYOCARDIUM

Abstract

BACKGROUND Left ventricular non-compaction cardiomyopathy (LVNC) is a congenital cardiomyopathy, characterized by excessive ventricular trabeculation, with a ‘spongy’ appearance of the myocardium, deep intertrabecular recesses, and a thin compacted myocardial layer. LVNC has recently gained more attention because it has been increasingly recognized as a cause of heart failure, thromboembolism, or ventricular arrhythmia, which can all lead to sudden cardiac death. However, due to its heterogeneous anatomical and clinical features, LVNC is still a poorly understood condition. In addition, LVNC is suggested to be due to a premature arrest in the myocardial compaction process, but its genetic basis and pathologic mechanism remain largely unknown. METHODS AND RESULTS Here we introduce novel genetic factors implicated in ventricular compaction. Specifically, combinatorial loss of two Iroquois homeobox (Irx) transcription factors, Irx3, critical for development and function of the ventricular conduction system (e.g., His-bundle, bundle branches and Purkinje fibers) and Irx4, essential for establishing ventricular identity, recapitulates the clinical hallmarks of LVNC in mice. While mice lacking either Irx3 or Irx4 do not exhibit gross congenital cardiac malformations, mice lacking both Irx3 and Irx4 (Irx3;Irx4DKO) show postnatal lethality with abnormal muscular growths that resemble the polypoid pattern of LVNC, characterized by multiple muscular nodes in the left ventricle. Optical projection tomography in postnatal day 14 (P14) mutant hearts unveiled increased trabeculation, abnormal bridging below the papillary muscles, and disorganized muscle structures (Fig. 1). During embryogenesis, Irx3 and Irx4 exhibit overlapping expression in the developing ventricles. Consistent with the hypothesis that LVNC can be caused by a defect in embryonic heart growth, Irx3;Irx4DKO embryonic hearts showed abnormal trabeculation with thinner ventricular walls as early as E14.5. RNA sequencing on E14.5 Irx3;Irx4DKO ventricles revealed altered ventricular identity with atrial gene activation and enriched Bmp2/Tbx2 pathway that is critical for endocardial cushion development by suppressing cardiomyocyte differentiation. Moreover, co-immunoprecipitation discovered that Irx4 physically interacts with Irx3 and Gata4, and promoter assay further showed that Irx3, Irx4, and Gata4 synergistically suppress Tbx2 promoter activity, suggesting the repressive role of this transcriptional regulatory complex in the developing myocardium. CONCLUSION Our study demonstrates a novel mechanistic interaction of Irx3 and Irx4, in cooperation with Gata4, during the ventricular compaction process, thereby ensuring proper ventricular development. This finding can promote improvement of genetic testing for LVNC and prediction of individual outcomes with LVNC. Left ventricular non-compaction cardiomyopathy (LVNC) is a congenital cardiomyopathy, characterized by excessive ventricular trabeculation, with a ‘spongy’ appearance of the myocardium, deep intertrabecular recesses, and a thin compacted myocardial layer. LVNC has recently gained more attention because it has been increasingly recognized as a cause of heart failure, thromboembolism, or ventricular arrhythmia, which can all lead to sudden cardiac death. However, due to its heterogeneous anatomical and clinical features, LVNC is still a poorly understood condition. In addition, LVNC is suggested to be due to a premature arrest in the myocardial compaction process, but its genetic basis and pathologic mechanism remain largely unknown. Here we introduce novel genetic factors implicated in ventricular compaction. Specifically, combinatorial loss of two Iroquois homeobox (Irx) transcription factors, Irx3, critical for development and function of the ventricular conduction system (e.g., His-bundle, bundle branches and Purkinje fibers) and Irx4, essential for establishing ventricular identity, recapitulates the clinical hallmarks of LVNC in mice. While mice lacking either Irx3 or Irx4 do not exhibit gross congenital cardiac malformations, mice lacking both Irx3 and Irx4 (Irx3;Irx4DKO) show postnatal lethality with abnormal muscular growths that resemble the polypoid pattern of LVNC, characterized by multiple muscular nodes in the left ventricle. Optical projection tomography in postnatal day 14 (P14) mutant hearts unveiled increased trabeculation, abnormal bridging below the papillary muscles, and disorganized muscle structures (Fig. 1). During embryogenesis, Irx3 and Irx4 exhibit overlapping expression in the developing ventricles. Consistent with the hypothesis that LVNC can be caused by a defect in embryonic heart growth, Irx3;Irx4DKO embryonic hearts showed abnormal trabeculation with thinner ventricular walls as early as E14.5. RNA sequencing on E14.5 Irx3;Irx4DKO ventricles revealed altered ventricular identity with atrial gene activation and enriched Bmp2/Tbx2 pathway that is critical for endocardial cushion development by suppressing cardiomyocyte differentiation. Moreover, co-immunoprecipitation discovered that Irx4 physically interacts with Irx3 and Gata4, and promoter assay further showed that Irx3, Irx4, and Gata4 synergistically suppress Tbx2 promoter activity, suggesting the repressive role of this transcriptional regulatory complex in the developing myocardium. Our study demonstrates a novel mechanistic interaction of Irx3 and Irx4, in cooperation with Gata4, during the ventricular compaction process, thereby ensuring proper ventricular development. This finding can promote improvement of genetic testing for LVNC and prediction of individual outcomes with LVNC.