Tbx20 regulation of cardiac cell proliferation and lineage specialization during embryonic and fetal development in vivo

Abstract

TBX20 gain-of-function mutations in humans are associated with congenital heart malformations and myocardial defects. However the effects of increased Tbx20 function during cardiac chamber development and maturation have not been reported previously. CAG-CAT-Tbx20 transgenic mice were generated for Cre-dependent induction of Tbx20 in myocardial lineages in the developing heart. βMHCCre-mediated overexpression of Tbx20 in fetal ventricular cardiomyocytes results in increased thickness of compact myocardium, induction of cardiomyocyte proliferation, and increased expression of Bmp10 and pSmad1/5/8 at embryonic day (E) 14.5. βMHCCre-mediated Tbx20 overexpression also leads to increased expression of cardiac conduction system (CCS) genes Tbx5, Cx40, and Cx43 throughout the ventricular myocardium. In contrast, Nkx2.5Cre mediated overexpression of Tbx20 in the embryonic heart results in reduced cardiomyocyte proliferation, increased expression of a cell cycle inhibitor, p21CIP1, and decreased expression of Tbx2, Tbx5, and N-myc1 at E9.5, concomitant with decreased phospho-ERK1/2 expression. Together, these analyses demonstrate that Tbx20 differentially regulates cell proliferation and cardiac lineage specification in embryonic versus fetal cardiomyocytes. Induction of pSmad1/5/8 at E14.5 and inhibition of dpERK expression at E9.5 are consistent with selective Tbx20 regulation of these pathways in association with stage-specific effects on cardiomyocyte proliferation. Together, these in vivo data support distinct functions for Tbx20 in regulation of cardiomyocyte lineage maturation and cell proliferation at embryonic and fetal stages of heart development.

Tbx20 promotes cardiomyocyte proliferation and persistence of fetal characteristics in adult mouse hearts

“…Female CAG-CAT-Tbx20 (CC-Tbx20) transgenic mice [20] were bred with β-Myosin Heavy Chain(βMHC)Cre males [22] (both on a FVBN background) to generate double transgenic neonates and adult offspring. All studies were performed on cohorts of CCTbx20;βMHCCre double transgenic (DTG) animals compared to CC-Tbx20 and/or βMHCCre single transgenic (STG) littermate controls.…”

“…CC-Tbx20;βMHCCre DTG animals were obtained at expected Mendelian ratios and no significant morbidity or mortality was observed. Genotyping for the Cre and Tbx20 transgenes was performed as described previously [20]. All experiments involving animals were carried out with experimental protocols and procedures reviewed and approved by the Cincinnati Children's Medical Center Biohazard Safety Committee and Institutional Animal Care and Use Committee.…”

“…During mouse development, Tbx20 is required for cardiomyocyte cell proliferation, and loss of Tbx20 leads to cardiac hypoplasia and embryonic death at mid-gestation [16][17][18][19]. Likewise, Tbx20 gain-of-function in developing cardiomyocytes leads to increased cell proliferation and thickening of the myocardium in fetal hearts [20]. In adult mice, induced ablation of Tbx20 in cardiomyocytes leads to severe cardiomyopathy with associated arrhythmias and death soon thereafter [21].…”

Recent Advances in Cardiovascular Development

“…[13][14][15][16] Thus far, >2 dozen TFs that regulate the differentiation, proliferation, and lineage specification of cardiac progenitor cells have been identified and characterized. [17][18][19][20] Notable features of the TFs are cardiac regional expression and functionality, which enable migration and differentiation of the embryonic cells in a manner that commits them to different cardiac regions and cell types. [21][22][23] Furthermore, TFs function cooperatively to modify the chromatin, assemble to the promoter regions, and regulate the expression of their target genes.…”

Circulation Research Thematic Synopsis

“…As for the heart, transcription factors that determine lineage specification for many cell types, including cardiac muscle, Purkinje cells, smooth muscle cells, and endothelial cells are partially defined and characterized, mostly through the reductionist approach of candidate gene knockout experiments. 1,[4][5][6] Similarly, there have been considerable advances in our understanding of cooperative and coordinated interactions among the regulatory networks, including genomics, through holistic approaches 1,3,7 Building on knowledge garnered through reductionist and holistic approaches, defined sets of transcription factors and noncoding RNAs have been used to repair damaged hearts, to induce cellular transdifferentiation to a myocyte-like phenotype, or to commit stem cells to specific lineages, such as myocytes or endothelial cells. 8,9 Perturbations of the exquisite process of cardiovascular development, whether due to genetic mutations or the environmental factors, if not embryonically lethal, often lead to congenital heart defect with serious medical consequences.…”