Functional Study Of Protein Geranylgeranylation In The Mouse Heart Development During Mid-gestation

Congenital heart disease(CHD)is one of the major type of birth defects.CHD remains a major unresolved public health problem due to its high incidence in newborns in China.Many congenital heart defects originate from the abnormal embryonic heart development.Thus,understanding the mechanisms that underlie embryonic heart development has important implications for the diagnosis,prevention and treatment of congenital heart disease.The mevalonate pathway is a critical metabolic pathway that provides cells with sterol and nonsterol isoprenoids.Two types of nonsterol isoprenoids,famesyl pyrophosphate(FPP)and geranylgeranylpyrophosphate(GGPP)are required for the protein prenylation which is a posttranslational modification that participates in a multitude of cellular processes.Genetic evidence showed that the inactivation of HMG-CoA reductase(a key upstream enzyme in the mevalonate pathway)and geranylgeranyl pyrophosphate synthase(Ggpps,a branch point enzyme in the mevalonate pathway that converts FPP to GGPP)or the pharmacological inhibition of prenyltransferases disrupted heart formation in drosophila and zebrafish.Additionally,our previous study has shown that protein prenylation is involved in the hypertrophy growth of cardiomyocytes at the postnatal stage.Thus,these studies implicate a relationship between protein prenylation and heart development.However,the exact function of protein prenylation in mammalian embryonic heart development is still unknown.In this study,we found that the geranylgeranyl pyrophosphate synthase(Ggpps),which mediates protein geranylgeranylation,expressed in the developing embryonic mouse heart and its expression level significantly increased from E10.5 when placenta becomes functional.Next,we developed two kinds of cardiac specific Ggpps deficient mouse model using Nkx2.5Cre/+ and α-SMA-Cre line.We found that the early cardiac inactivation of Ggpps by the Nkx2.5Cre/+ line disrupted protein geranylgeranylation from E10.5,which resulted in the embryonic lethality between E12.0 and E13.0.The reduction of myocardial proliferation,but not apoptosis was observed in the Ggpps mutant hearts from E11.5,which accounted for smaller size of mutant hearts at E12.5 and also contributed to the poorly developed interventricular septum and the trabeculae at E11.5 and E12.5.Moreover,ectopic expression of trabecular maker ANF and loss expression of compact myocardium maker HEY2 were observed in the compact layer of Ggpps mutant heart from E11.5,suggesting the loss of Ggpps induced abnormal myocardial regional gene expression also involved in the failure of ventricular chamber maturation.In addition,cardiac deletion of Ggpps even caused abnormal epicardium development,evidenced by the excessive EMT and proliferation of epicardial cells.Interestingly,when α-SMA-Cre line mediated the disruption of protein geranylgeranylation from E13.5,the disruption did not affect embryonic heart development.Thus,our present results indicate that GGPPS-mediated protein genraylgeranylation is stage-dependently required for mammalian heart development during embryonic stage.Given the fact that severe heart defects,including aberrant proliferation,poorly developed interventricular septum and trabeculae,were observed in Nkx2.5Cre/+;Ggppsfl/fl hearts at E12.5 during the death time of the mutant embryos,we next focused on analyzing the potential abnormality in mutant hearts at an earlier stage.Notably,the well-organization cardiac cytoarchitecture is required for the heart function and morphological remodeling in ventricular chamber maturation,Further TEM and immunofluorescence staining analysis of Nkx2.5Cre/+;Ggppsfl/fl mutants showed that the loss of Ggpps caused disorganized cardiac cytoarchitecture as early as E11.5 by disturbing cell-cell junctions,including adherens junction and gap junction.Ggpps inactivation decreased Rho GTPase geranylgeranylation and their activity,which might account for the disruption of cell-cell junctions.Moreover,elevating the protein geranylgeranylation by supplement of geranylgeranyl pyrophosphate(GGPP)could partially recover the Ggpps deficient induced defects of cytoarchitecture and cell-cell junctions in vitro and in vivo.In conclusion,our present study demonstrates that GGPPS-mediated protein geranylgeranylation plays an indispensable role in the ventricular chamber maturation through acting as a stage-specific signal to regulate the establishment of cardiac cytoarchitecture.