Introduction Myocardial development is dependent on the concomitant growth of cardiomyocytes and a supporting vascular network. The coupling of myocardial and coronary vascular development is mediated in part by VEGFA signalling. Cited2 is a transcriptional co-factor that can inhibit hypoxia-activated transcription and also acts as a co-activator for transcription factors such as TFAP2. Genetic evidence indicates that Cited2 is essential for cardiac left-right patterning via regulation of the Nodal-Pitx2c left-right patterning pathway. Zygotic and epiblastic deletion of Cited2 results in atrioventricular septation, outflow tract and aortic arch abnormalities, as well as left-right patterning defects such as right-isomerism. Cited2 is also essential for adrenal, neural crest, liver, lung, lens and placental development. However, the early requirement of Cited2 in left-right patterning and placental development makes it difficult to identify a later specific role for Cited2 in myocardial development. To overcome this problem we therefore investigated the role of Cited2 in the myocardium by conditional deletion in cardiomyocyte precursors.
Methods Cited2 was selectively deleted from cardiomyocytes by intercrossing mice transgenic for Cited2 and Nkx2-5Cre. Embryos were collected and processed for analysis by histology, MRI, X-Gal staining, quantitative reverse transcriptase PCR (Q-RTPCR), chromatin immunoprecipitation and transient transfection assays.
Results The cardiomyocyte specific knockout of Cited2 results in abnormal myocardial compact zone growth and ventricular septal defects. This is associated with a decreased ratio in the number of small vessels to large vessels, and a reduction in Vegfa expression. We also show that CITED2 is present at the Vegfa promoter in mouse embryonic hearts, and that it stimulates human VEGFA promoter activity in cooperation with TFAP2 transcription factors in transient transfection assays. However, we observed no change in the myocardial expression of the left-right patterning gene Pitx2c, a known target of Cited2.
Conclusions The myocardial and capillary defects observed in myocardial loss of Cited2 are not associated with Pitx2c deficiency and suggests that Cited2 can cause myocardial and vascular defects via a mechanism that is distinct from its effect on the left-right patterning pathway. Our results delineate a novel mechanism of Vegfa regulation by CITED2 and TFAP2 transcription factors, and indicate that coupling of myocardial and coronary vascular growth in the developing mouse heart occurs, at least in part, through a Cited2->Vegfa pathway. This pathway may be targeted for the treatment of heart failure resulting from ischaemic heart disease.
- Myocardial development
- vascular growth