Role Of PDGF Signaling In Neonatal Mice Heart Regeneration And Its Mechanism

Acute myocaridial infarction causes loss of cardiomyocytes in a short time, and induces impaired heart function, even heart failure. Cardiac regenerative medicine targeting the restoration of cardiomyocyte is always the focus of medicine. A series of studies showed that zebrafish and salamanders can perform a wonderful cardiac regeneration after15-20%removal of heart ventricle. Recently a study showed that the neonatal mice can also regenerate the heart after apex resection, which is similar to the phenomenon of zebrafish heart. But the regenerative capacity of the neonatal mouse heart is lost within the first week of postnatal life. It will be important to determine the mechanisms of heart regeneration for enhancing the limited regenerative ability of adult mammal response to the injuries.In the process of zebrafish heart regeneration after injury, PDGF signaling plays an important role in replicating the cardiomyocyte. Also, PDGF signaling regulates the pancreatic beta cell proliferation by Ezh2in mice pancreas regeneration. Due to the role of PDGF signaling in mice pancreas regeneration and zebrafish heart regeneration, we hypothesis PDGF signaling may be involved in neonatal mice heart regeneration after apex resection. To test the hypothesis, we determined whether the PDGF signaling is involved in neonatal mice heart regeneration in vivo, and investigate the detailed mechanisms, key molecules and pathways which is involved in promoting cardiomyocyte proliferation by PDGF signaling in vitro.The main findings are as follows:The first part:The expression changes of PDGF signaling related genes in the postnatal mice heart development Objective:To observe the expression of PDGF related genes in the mice heart development after birth.Methods:real time-PCR was used to detect the expression of PDGF related genes and Ezh2related genes in the mice heart development after birth. The protein levels of PDGF receptors and Ezh2were detected by Western blot. The mice heart proliferative ability after birth was investigated by high content screening.Results:The mice heart maintained the proliferative ability within a week after birth, and the peak appeared at postnatal4days, but the proliferative ability lost after7days postnatally. The expression of PDGFA and PDGFB peaked at4-7days during the postnatal development, and then gradually decreased. The expression of PDGFRB decreased after birth. The expression of Ezh2reached a peak at postnatal4days, and gradually declined to the undetected level in the adulthood. The expression of Suz12and Eed were similar to that of Ezh2. The expressions of cell cycle inhibitor p16and p19were increased after birth, whereas the expression of CDKN2B decreased gradually in the postnatal period.Conclusions:During the mice heart postnatal development, the proliferative ability reached a peak at4days after birth, and sharply declined after7days. The trends of PDGF related genes and Ezh2related genes were consistent with that of the proliferative ability, which is associated with the regulation of proliferative ability, but the exact relationship needs further confirmation.The second part:The expression changes of PDGF signaling related genes in the neonatal mice heart regenerationObjective:To detect whether PDGF signaling is involved in the process of newborn mice myocardial regeneration.Methods:The heart regeneration model in newborn mice was constructed. The real time-PCR was used to detect the levels of gene associated with PDGF. The immunohistochemistry and immunofluorescence staining was used to detect the PDGFRB and Ezh2distribution in the process of newborn mice myocardial regeneration.Results:The neonatal heart after apex resection was completely regenerated, with the similar results of cardiac function test between apex resection group and sham group. The levels of PDGF associated genes, such as PDGFB, PDGFC, PDGFD and PDGFRB were changed significantly, and the time were1day and4day post surgery. The immunohistochemistry showed the PDGFRB abundantly expressed at the incision in the seven days after surgery. The levels of PRC2core members, such as Ezh2, Suzl2and Eed were changed significantly, and the time is1day post surgery. The immunohistochemistry showed the Ezh2abundantly expressed in the regenerating area.Conclusion:The newborn mice have the ability to completely regenerate the heart. The expression of PDGF receptor and Ezh2changed regularly in the regenerative response after apex resection in the newborn.The third part:The effect of PDGF related genes on cardiomyocyteObjective:To investigate the specific role of PDGF signaling and Ezh2in cardiomyocytes.Methods:H9C2cell line was cultured in standard. Cell was stimulated by PDGF-BB following high content cell analysis. Flow cytometry was used to detect the cell cycle phases. Real time-PCR was used to detect the level of Ezh2and relative genes. Western blot was used to detect the change of Ezh2protein. After Ezh2knockdown by siRNA, flow cytometry was used to detect the cell phases and apoptosis.Real time-PCR was used to detect the level of Ezh2and relative genes.Results:PDGF-BB had a growth-promoting effect on H9C2cardiomyocytes. Flow cytometry showed H9C2cardiomyocytes reentered the cell cycle under the PDGF-BB stimulation. In the process of H9C2myocardial cell proliferation, the protein level of Ezh2was increased with the declining level of CDKN2A. When Ezh2was knockdown by siRNA, the level of CDKN2A elevated. The cell cycle was not affected by the knockdown of Ezh2, but the apoptosis occurred. Ezh2knockdown eliminated the proliferation effect of PDGF-BB on H9C2myocardial cells. Under the stimulation of PDGF-BB, PLCy pathway, PI3K/Akt pathway and Erkl/2pathway were activated. The level of Ezh2decreased under the blocking of Erkl/2pathway, but the blocking of Akt pathway did not affect the level of Ezh2.Conclusion:PDGF-BB had a growth-promoting effect on H9C2cardiomyocytes, and increased the level of Ezh2. After knockdown of Ezh2, H9C2cells showed apoptosis, and affected the proliferation effect of PDGF-BB. Under the stimulation of PDGF-BB, many pathways were activated, but the Ezh2was under the control of Erkl/2pathway. Ezh2performed its function by regulating the level of CDKN2A, which could be involved in heart regeneration.