Endocardial-Myocardial Interactions During Early Cardiac Differentiation and Trabeculation.

Throughout the continuum of heart formation, myocardial growth and differentiation occurs in concert with the development of a specialized population of endothelial cells lining the cardiac lumen, the endocardium. Once the endocardial cells are specified, they are in close juxtaposition to the cardiomyocytes, which facilitates communication between the two cell types that has been proven to be critical for both early cardiac development and later myocardial function. Endocardial cues orchestrate cardiomyocyte proliferation, survival, and organization. Additionally, the endocardium enables oxygenated blood to reach the cardiomyocytes. Cardiomyocytes, in turn, secrete factors that promote endocardial growth and function. As misregulation of this delicate and complex endocardial-myocardial interplay can result in congenital heart defects, further delineation of underlying genetic and molecular factors involved in cardiac paracrine signaling will be vital in the development of therapies to promote cardiac homeostasis and regeneration. Herein, we highlight the latest research that has advanced the elucidation of endocardial-myocardial interactions in early cardiac morphogenesis, including endocardial and myocardial crosstalk necessary for cellular differentiation and tissue remodeling during trabeculation, as well as signaling critical for endocardial growth during trabeculation.

Tie2 regulates endocardial sprouting and myocardial trabeculation.

The ang1-Tie2 pathway is required for normal vascular development, but its molecular effectors are not well-defined during cardiac ontogeny. Here we show that endocardial specific attenuation of Tie2 results in mid-gestation lethality due to heart defects associated with a hyperplastic but simplified trabecular meshwork (fewer but thicker trabeculae). Reduced proliferation and production of endocardial cells (ECs) following endocardial loss of Tie2 results in decreased endocardial sprouting required for trabecular assembly and extension. The hyperplastic trabeculae result from enhanced proliferation of trabecular cardiomyocyte (CMs), which is associated with upregulation of Bmp10, increased retinoic acid (RA) signaling, and Erk1/2 hyperphosphorylation in the myocardium. Intriguingly, myocardial phenotypes in Tie2-cko hearts could be partially rescued by inhibiting in utero RA signaling with pan-retinoic acid receptor antagonist BMS493. These findings reveal two complimentary functions of endocardial Tie2 during ventricular chamber formation: ensuring normal trabeculation by supporting EC proliferation and sprouting, and preventing hypertrabeculation via suppression of RA signaling in trabecular CMs.

Myocardial differentiation is dependent upon endocardial signaling during early cardiogenesis in vitro.

The endocardium interacts with the myocardium to promote proliferation and morphogenesis during the later stages of heart development. However, the role of the endocardium in early cardiac ontogeny remains under-explored. Given the shared origin, subsequent juxtaposition, and essential cell-cell interactions of endocardial and myocardial cells throughout heart development, we hypothesized that paracrine signaling from the endocardium to the myocardium is crucial for initiating early differentiation of myocardial cells. To test this, we generated an in vitro, endocardial-specific ablation model using the diphtheria toxin receptor under the regulatory elements of the Nfatc1 genomic locus (NFATc1-DTR). Early treatment of NFATc1-DTR mouse embryoid bodies with diphtheria toxin efficiently ablated endocardial cells, which significantly attenuated the percentage of beating EBs in culture and expression of early and late myocardial differentiation markers. The addition of Bmp2 during endocardial ablation partially rescued myocyte differentiation, maturation and function. Therefore, we conclude that early stages of myocardial differentiation rely on endocardial paracrine signaling mediated in part by Bmp2. Our findings provide novel insight into early endocardial-myocardial interactions that can be explored to promote early myocardial development and growth.

Critical roles of miRNA-mediated regulation of TGFß signalling during mouse cardiogenesis.

AIMS: MicroRNAs (miRNAs) play critical roles during the development of the cardiovascular system. Blocking miRNA biosynthesis in embryonic hearts through a conditional gene inactivation approach led to differential cardiac defects depending on the Cre drivers used in different studies. The goal of this study is to reveal the cardiogenic pathway that is regulated by the miRNA mechanism at midgestation, a stage that has not been evaluated in previous publications. METHODS AND RESULTS: We specifically inactivated Dicer1, which is essential for generation of functional mature miRNAs, in the myocardium by crossing cTnt-Cre mice with Dicer1(loxP) mice. cTnt-Cre efficiently inactivates target genes in cardiomyocytes at midgestation. All mutants died between E14.5 and E16.5 with severe myocardial wall defects, including reduced cell proliferation, increased cell death, and spongy myocardial wall. Expression of TGFß type I receptor (Tgfbr1), which encodes the Type I receptor of TGFß ligands, was up-regulated in mutant hearts. As expected, TGFß activity was increased in Dicer1-inactivated hearts. Our further molecular analysis suggested that Tgfbr1 is a direct target of three miRNAs. Reducing TGFß activities using a pharmacological inhibitor on in vitro cultured hearts, or through an in vivo genetic approach, partially rescued the cardiac defects caused by Dicer1 inactivation. CONCLUSIONS: We show for the first time that TGFß signalling is directly regulated by the miRNA mechanism during myocardial wall morphogenesis. Increased TGFß activity plays a major role in the cardiac defects caused by myocardial deletion of Dicer1. Thus, miRNA-mediated regulation of TGFß signalling is indispensable for normal cardiogenesis.

CHD7 interacts with BMP R-SMADs to epigenetically regulate cardiogenesis in mice.

Haploinsufficiency for CHD7, an ATP-dependent nucleosome remodeling factor, is the leading cause of CHARGE syndrome. While congenital heart defects (CHDs) are major clinical features of CHARGE syndrome, affecting >75% of patients, it remains unclear whether CHD7 can directly regulate cardiogenic genes in embryos. Our complementary yeast two-hybrid and biochemical assays reveal that CHD7 is a novel interaction partner of canonical BMP signaling pathway nuclear mediators, SMAD1/5/8, in the embryonic heart. Moreover, CHD7 associates in a BMP-dependent manner with the enhancers of a critical cardiac transcription factor, Nkx2.5, that contain functional SMAD1-binding elements. Both the active epigenetic signature of Nkx2.5 regulatory elements and its proper expression in cardiomyocytes require CHD7. Finally, inactivation of Chd7 in mice impairs multiple BMP signaling-regulated cardiogenic processes. Our results thus support the model that CHD7 is recruited by SMAD1/5/8 to the enhancers of BMP-targeted cardiogenic genes to epigenetically regulate their expression. Impaired BMP activities in embryonic hearts may thus have a major contribution to CHDs in CHARGE syndrome.

Myocardial Mycn is essential for mouse ventricular wall morphogenesis.

MYCN is a highly conserved transcription factor with multifaceted roles in development and disease. Mutations in MYCN are associated with Feingold syndrome, a developmental disorder characterized in part by congenital heart defects. Mouse models have helped elucidate MYCN functions; however its cardiac-specific roles during development remain unclear. We employed a Cre/loxp strategy to uncover the specific activities of MYCN in the developing mouse myocardium. Myocardial deletion of Mycn resulted in a thin-myocardial wall defect with dramatically reduced trabeculation. The mutant heart defects strongly resemble the phenotype caused by disruption of BMP10 and Neuregulin-1 (NRG1) signaling pathways, two central mediators of myocardial wall development. Our further examination showed that expression of MYCN is regulated by both BMP and NRG1 signaling. The thin-wall defect in mutant hearts is caused by a reduction in both cell proliferation and cell size. MYCN promotes cardiomyocyte proliferation through regulating expression of cell cycle regulators (including CCND1, CCND2, and ID2) and promotes cardiomyocyte growth through regulating expression of p70S6K. In addition, expression of multiple sarcomere proteins is altered in Mycn myocardial-inactivation embryos, indicating its essential role for proper cardiomyocyte differentiation. In summary, Mycn acts downstream of BMP and NRG1 cardiogenic signaling pathways to promote normal myocardial wall morphogenesis.

Characterization of the novel interaction between muskelin and TBX20, a critical cardiogenic transcription factor.

The genetic regulation necessary for the formation of a four-chambered heart is tightly regulated by transcription factors such as TBX20, a member of the T-box (TBX) transcription factor family. TBX20 is critical for proper cardiogenesis and is expressed in the heart throughout development. Missense mutations in TBX20 have been found in patients with congenital heart defects (CHD). Characterization of modifiers of TBX20 activity will help elucidate the genetic mechanisms of heart development and CHD. A yeast two-hybrid assay screening an embryonic mouse heart cDNA library with TBX20b as bait was used to identify potential modifiers of TBX20 activity and identified an interaction with muskelin (MKLN1), a primarily cytoplasmic protein with potential roles in signal transduction machinery scaffolding and nucleocytoplasmic protein shuttling. In cellular studies, MKLN1 directly binds to the T-box DNA-binding domain of only the TBX20b isoform by its kelch repeats domain. Immunostaining of mammalian cells transfected with tagged TBX20b and MKLN1 revealed colocalization primarily in the cytoplasm. Immunohistochemistry analysis of embryonic mouse hearts reveals coexpression in the developing endocardial valvular and myocardial interventricular cells. This novel interaction between TBX20b and MKLN1 may help elucidate new regulatory mechanisms within heart development.