| Background:Nesprins (nuclear envelop spectrin repeat) is a novel family of conserved proteins. It is the major component of cytoskeleton and play a role that maintain cell architecture and coordinate cell function. It has been found that there are three subtypes of nesprins: nesprin-1(syne-1/ENAPTIN),nesprin-2(syne-2/NUANCE),nesprin-3. Although nesprins are widely expressed in a variety of tissues and cell types,both nesprin-1 and nesprin-2 are highly expressed in the sarcomeres of both cardiac and skeletal muscle. Several lines of evidence suggest that nesprins play a crucial role in the structure and function of muscle cells. During C2C12 myoblasts differentiation in vitro into myotubes, nesprins localizations change from being predominantly nuclear/NE to cytoplasmic/sarcomeric, suggesting specific roles during muscle differentiation. Mutations in the nesprin-1and nesprin-2 cause EMDM, Patients with EMDM develop cardiomyopathy with conduction defects. So far, it has not been reported that nesprin-1 and nesprin-2 are expressed and play the roles during cardiogenesis. We observe the expressions of both nesprin-1 and nesprin-2 gene during cardiogenesis, to better understand the function of nesprins during cardiogenesis. Objectives:To observe expressions of both nesprin-1 and nesprin-2 gene during mouse heart development, then to discuss the roles of nesprin-1 and nesprin-2 gene during cardiogenesis.Methods1.The hearts were gained from the embryos at embryonic 12.5day(E12.5), E14.5, E16.5, E18.5 as well as postnatal and adult mouse, then to detect the cardiac changes of morphology of every stage by stereopsis anatomical microscope and HE staining.2.The levels of nesprin-1 gene and nesprin-2 gene mRNA expression were determined by RT-PCR.3.The levels of nesprin-1 protein expression were detected by western-blot.4.The distribution of nesprin-1 protein was analysed by immunofluorescence.Results1.At E12.5, atrial septum(AS), interventricular septum(IVS), atrio-rentriculor valve have alreadly started to develop .At E14.5 AS and IVS have been developed, at E16.5 atrio-rentriculor valve have been formed. At E16.5 and E18.5, myocardial cell has been proliferating and largely become bigger in volume, then the walls of atrial and ventricular become thicker. Compared with that of embryonic stage, myocardial cell become bigger, the volume of heart become larger in neonatal mouse. The shape of myocardial cell changed gradually from polygonal embryo myocyte to thin-shape myocyte at the stage between neonatal mouse to adult mouse.2.There are dynamic expression pattern of nesprin-1 and nesprin-2during mouse heart development by RT-PCR. Nesprin-1 mRNA can be detected at E12.5, and the expression gradually enhanced in embryonic developing stages, reach peak at E16.5 and E18.5 then subsequently decrease at postnatal stage. It is express very lowly in adult mouse heart. The expression of nesprin-2 mRNA also can be observed at E12.5, and this is the highest levels of nesprin-2 gene expression in the observation, and the expression decreased gradually in later embryonic stage and postnatal and adult mouse stage.3.There is dynamic expression pattern in nesprin-1 during mouse heart development by western blot, it is identical with result of RT-PCR. An enhanced expression of nesprin-1 protein is found before E16.5, peaking at stage E16.5 and E18.5 and decrease subsequently at postnatal stage, and the protein is expressed lowly in adult mouse heart.4.Nesprin-1 is detected at nuclear envelop(NE) and perinuclear space during mouse heart development by immunoflourescence.Conclusion:The data show that nesprin-1 and nesprin-2 express dynamicly during cardiogenesis. The time of both genes expression reach peak associated with cardiac conduction system and cardiac muscle developing period. So it can be postulated that nesprin-1 and nesprin-2 could play an important role in the acquisition of a development pattern of conduction and cardiac muscle cell development during cardiogenesis.
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