The Role Of POP1 In Cardiomyocyte Hypertrophy

Heart development and its disease development are extremely complicated procedures including a series of morphogenesis, which are regulated by cardiac transcription and regulated and control network. Abnormal expression of these genes will result in mortality in pregnancies and forms of cardiac disease in which cardiac hypertrophy is so prevalent. So, it is a hot field to explore the mechanism of cardiovascular diseases including cardiac hypertrophy.The human POP1 gene has been cloned and polyclonal antibody of POP1 gene has been prepared previously by our lab. Moreover, our lab has certified initially that POP1 has effect in cardiomyocyte hypertrophy to a certain degree.Our researches continue studying the role of POP1 in cardiomyocyte hypertrophy deeply. First, the expression pattern of POP1 protein in adult mouse is analyzed by western blot. The results show that POP1 protein abundantly expresses in heart tissue. Second, it is confirmed that cardiac hypertrophic mice were obtained by intraperitoneal injecting L-tetraiodothyronine. We extracted RNA and protein. POP1 is up-regulated, like cardiac hypertrophic markers, including ANF,β-MHC, SK-α-actin, both by RT-PCR and Western blot in cardiac hypertrophic mice. We obtained the result that POP1 is up-regulated in primary cardiomyocytes of neonatal rat stimulated by FBS from RNA level.All these results support the idea that POP1 may play an important role in regulation pathways of cardiac hypertrophy.In addition, we detected the ability of POP1 to stimulate expression of a luciferase reporter controlled by the ANF promoter or NFAT promoter when HEK293 cells were overexpressed POP1 protein. The POP1 gene which is cloned into pCMV-tag2B vector, normally activate the ANF /NFAT promoter reporter> 10-fold, suggesting that the enhanced ANF/NFAT luciferase reporter gene expression is attributable to an increase in POP1 transactivity. The transcriptional activity assays show that POP1 significantly activates transcriptional activity of ANF-luciferase and NFAT-luciferase, both are hypertrophy transcription factor. It is indicated that POP1 might be an activator in the signal pathways of cardiac hypertrophy.What’s more, H9C2 cells derived from rat cadiocyte were transiently transfected with the POP1 constructs to obtain POP1-overexpressing cells line. Immunocytochemistry was utilized to determine whether the gross cellular distribution of POP1. Immunoreactive protein was detected with anti-POP1. Cytoplasmic localization of POP1 is a common property of this protein in single cells. After staining, the cells transfected with POP1 was observed at a higher level than control ones which transfected with pCMV-tag2B vector. To assess a possible role for POP1 in H9C2 cells line, western blots were performed on proteins extracted using the standard extraction conditions from POP1-overexpressing H9C2 cells line to detect the expression of hypertrophic marker genes, like ANF,β -MHC, SK-α-actin,. Apparently, protein expression of hypertrophic cardiac genes was much higher in POP1-overexpressing H9C2 cells line than that in the control. The reveals that POP1 indeed has relation with cardiomyocyte hypertrophy.To test whether POP1 can induce hypertrophy in cardiomyocytes, we overexpressed POP1 in neonatal rat cardiomyocytes by adenoviral delivery. We overexpressed GFP in neonatal rat cardiomyocytes by adenoviral delivery as the control. When cardiomyocytes were infected with Ad-POP1 at a moi of 100, virtually every cell was found to express POP1 protein. Within 24 hours of infection, cardiomyocytes showed a morphological phenotype of hypertrophic growth, which was enhanced by 48 hours. In contrast, cardiomyocytes infected with a control adenovirus encoding GFP (Ad-GFP) showed no signs of hypertrophy. Cell size, measured by surface area, was increased comparably by Ad-POP1, which is statistically significant. The observations suggest that POP1 may directly promote cardiomyocyte hypertrophy.Moreover, up-regulated expression of POP1 is detected in the heart tissues from a patient with hypertrophic cardiomyopathy, which suggests that POP1 can induce cardiomyocyte hypertrophy in different species. All this providing fundamental theoretical basis for curing human hypertrophic cardiomyopathy.