The Hypertrophic Response Of Neonatal Cardiomyocytes By The Treatment Of Gq-coupled Receptor Agonist In Fukutin Knockout Mice

Backgroud:Fukuyama congenital dystrophy (FCMD) is one common congenital mucular dystrophies. It is one of the most common autosomal recessive disorders in the Japanese population, following the Duchenne muscular dystrophy (DMD). FCMD is characterized by congenital muscular dystrophy in combination with cortical dysgenesis, and most of the patients get illness within 6 months after birth. Patients show hypotonia, motor development delay, facial muscle is obvious, especially the cheek. Respiratory distress is not so often, and pseudohypertrophy becomes evident in the cheeks and calves in early infancy, as well as joint contracture. The blood CK of patients is also 10-60 times higher than normal children, and most of the patients die between 2-23 years old.Now the FCMD gene was identified in the 9q31, and the product of this gene is fukutin, after Fukuyama-type. Fukutin is a protein of 461 amino acids with molecular weight of 53.7KDa. This protein has an N-terminal hydrophobic signal sequence and one putative N-glycosilation site, and it is thought to be a glycosyltransferase but the function of fukutin and the molecular mechanism of FCMD is now unclear. Transfection experiments indicated that the the fukutin protein is transferred to an extracellular region, co-locolizing with a Golgi protein.Objective:Use the myocardium as the object in this experiment, to check the changes of related protein of the membrane of myofibers, to identify the molecular mechanism of cardiac muscle degeneration and necrosis in FCMD.Methods:Take the FCMD gene in the cardiomyofibers as the target gene, make conditional FCMD gene knockout mice with a Cre-Lox recombinase, then hybrid them to get F1. Choose the homozygous FCMD gene+/+ and FCMD gene-/-groups in the F1. The F2 is fukutin+/+(WT) and fukutin-/-(KO) contrast groups in the heart. Induce the hypertrophic response of neonatal cardiomyocytes (1-3 days) of WO and KO groups with a Gq-receptor agonist to observe the difference of the KO and WT types, and check the related proteins with immunofluorescence. Make a contrast of adult WT and KO groups of cardiac muscle to affirm the conclusion.Results:1. After 48 hours induction of different concentration of Gq-receptor agonist, fukutin KO group did not show an obvious change of cytomorphology (P>0.05), However, in WT group, we saw the compensatory response:the cell area enlarged obviously (P<0.05). After 72 hours induction, all the cardiomyocytes of KO group became cell death and WT groups still show the compensatory response.2. Use 10um/L Gq-receptor agonist phenylephrine to induce the hypertrophy of neonatal cardiomyocytes of both groups, after 48hours, clear sarcomere is detected in the WT group while in KO group, we did not see the regular sarcomere.3. The immunofluorescence indicates that NCX1 is not located in the membrane of cardiomyocytes of fukutin KO mice.Conclusions:1. The neonatal cardiomyocytes of fukutin KO mice cannot response to the Gq-receptor agonist.2. Immnofluorescence shows that NCX1 is not localized in the membrane of the cardiomyofiber which indicates that the reason of cardiac muscle necrosis is Ca+ overload.