Latent Effect Of Congenital Heart Disease Gene On Cardiac Development And Its Role In Adult Cardiovascular Disease

Congenital heart disease（CHD）is a major cause for pediatric morbidity and mortality,ranking the top in birth defect incidence and mortality.Genetic mutation is considered as the major cause.But for CHD individuals,the underlying genetic causes are often unidentifiable.Thanks to the improvement in CHD therapy,pediatric CHD patients often survie to adulthood.The therapeutic surgery can correct the anatomic abnormalities,but the underlying genetic lesions still exist.It is unclear whether these genetic lesions would affect the occurrence and prognosis of adult disease.Therefore,there are numerous unknowns to be studied about the roles of CHD genes in CHD pathogenesis and their effects on events of the whole life cycle.MEGF8（Multiple epidermal growth factor-like domains protein 8）encodes a highly conserved protein with 2789 amino acids and a molecular weight of 300 k D.MEGF8 point mutation causes Carpenter syndrome,which is characterized by a number of anomalies in craniofacial structures,limb and heart.Knockout of mouse ortholog Megf8 recapitulates the defects seen in Carpenter syndrome.These work reveals the role of Megf8 in embryogenesis,However,the underlying mechanism is unknown.Chromosome 22q11.2 microdeletion syndrome（22q11.2DS,also called velocardiofacial syndrome,conotruncal anomaly face syndrome or Di George syndrome）is the most common human chromosomal microdeletion with an incidence of 1 in 2,000 live births.TBX1 is the major disease gene within this interval.Knockout of Tbx1 in mice recapitulates the majority of clinical manifestation including cardiovascular abnormalities（persistent truncus arteriosus,interrupted aortic arch,Tetrology of Fallot etc.）,craniofacial anomalies,thymic/parathyroid hypoplasia etc..Currently,there are quite a few 22q11.2 DS patients in adult population.Previous studies have shown that adult CHD patients are more prone for myocardial infarct（MI）and they have worse outcome than general population.These observations indicate that genetic lesions underlying CHD may have a role in the occurrence and prognosis of acquired adult cardiovascular disease.Objectives:（1）To dissect the mechanism how Megf8 regulates cardiovascular development.（2）To study whether Tbx1 has a role in post-MI repair.（3）To dissect the molecular mechanisms how Tbx1 regulates post-MI repair.（4）To invent measures for counteract the damage caused by Tbx1 deficiency.Methods:Construct Megf8^{lac Z} allele and collect embryos and hearts at different stages for X-gal staing to examine the spatiotemporal expression dynamics of Megf8;Temporal and spacial knockout of Megf8 using various tissue-specific and inducible Cre lines to identify the critical tissue and time window essential for the regulation of Megf8 on cardiac development.Using Tbx1^{Lac Z} allele to examine the expression pattern of Tbx1 after MI;Use conditional Tbx1 knockout mouse as genetic model of 22q11.2DS and permanent coronary ligation as MI model to dissect the role of Tbx1 in post-MI repair.Hearts were collected at the corresponding days after MI.Histopathological examination,bulk RNA-seq,and single-cell RNA-seq were performed to explore the molecular mechanism how Tbx1 regulates post-MI repair and to identify the potential therapeutic measures.Results:（1）X-gal staining of Megf8^{lac Z/+}embryos revealed the Megf8 expression dynamics during embryogenesis.（2）Megf8^{lac Z/lac Z} mice recapitulated the phenotype of Megf8 knockout mice and presented previously unreported anomalies including persistent truncus arteriosus（PTA）,double outlet right ventricle（DORV）and exencephaly.（3）Five different tissue-specific deletion of Megf8 did not recapitulate the cardiac defects of Megf8 knockout mice.（4）Timed deletion of Megf8 with Tg CAGG-Cre ^ER from E6.5 on recapitulate knockout phenotype except the left-right patterning and cardiac defects.（5）Tbx1 was mainly activated in cardiac lymphatic endothelial cells（LECs）after MI.（6）Endothelium-specific knockout of Tbx1 led to worse cardiac function and increased myocardial infarct size after MI.（7）Tbx1deficiency disrupted MI-induced lymphangiogenesis,which is a consequence of disturbed Dtx1/Notch1/Vegfr3 axis.（8）Immunosuppressive tolerogenic dendritic cells（t DC）and immunosuppressive regulatory T cells（Treg）were significantly reduced,thereby leading to increased cytotoxic T cell and reduced reparative M2 macrophages in the Tbx1Cko heart.（9）Targeting the aforemention cellular defects,the application of exogenous VEGFC（Cys156Ser）,BMP4 or CD8⁺T cell blocking antibody rescued the impaired cardiac function of Tbx1Cko mice.Conclusions:The critical time interval for Megf8 to regulate cardiac development is before E6.5,which indicates that the regulation of Megf8 on cardiac development is indirect and might be mediated through a latent effect of left-right patterning.Current model indicates that the left-right asymmetry is established at E7.75.However,our results suggest that the temporal requirement of Megf8 on left-right patterning is much earlier than the known symmetry break event in mouse.Our data,for the first time,show that Tbx1 is upregulated in cardiac LECs at post-MI day 4 and promote post-MI repair.It promotes lymphangiogenesis through Dtx1/Notch1/Vegfr3 axis and regulates immunomodulatory gene expressin to enhance immunosuppressive environment characterized by increased immunosuppressive cells,thereby promoting reparative macrophage transition and maintaining tolerance to facilitate cardiac repair.Overall,our studies indicate that CHD genes can regulate cardiac development and affect the occurrence and prognosis of the related cardiovascular deseases at different stages of life cycle.