Ablation Of Periostim Inhibits Post-infarction Myocardial Regeneration In Neonatal Mice Mediated By The Phosphatidylinositol 3 Kinase/glycogen Synthase Kinase 3?/cyclin D1 Signalling Pathway

Background:The death of heart failure caused by coronary heart disease has become the leading cause of death in the world.The non-regeneration of myocardial cells aftermyocardial infarction in adult mammals is the main reason for limiting the treatment of heart failure.Since the first time porrello reported that neonatal mice showed a capacity for myocardial regeneration after injuries such as myocardial infarction(MI)or resection of the left ventricular apex,provides a new idea for the treatment of necrotic cardiomyocyte regeneration,recent studies have revealed that certain relevant cytokines,proteins,physical and chemical factors,and genes may be involved in myocardial regeneration.Periostin plays important roles during cardiac development and in the epithelial-mesenchymal transition.It was also linked to cardiovascular diseases such as dilated cardiomyopathy and MI.Several studies demonstrated function for periostin in regeneration of tissues including the myocardium.However,it remains controversial whether periostin can promote myocardial regeneration.A previous study using an adult MI model reported no significant differences in myocardial regeneration in periostin defecient or periostin transgenic mice,as compared with their corresponding wildtype strains.Intriguingly,using a neonatal heart resection model,a recent study demonstrated that signal transducers and activators of transcription 3/periostin signalling is a critical mediator of interleukin 13 signalling in the regenerating mouse heart.Therefore,it may be a good way to resolve this controversy using an MI model in neonatal periostin knockout mice.We therefore used this approach to test our hypothesis that periostin is necessary for postinfarction myocardial regeneration in the neonatal heart.Objective:We compared the myocardial regenerative capacity of neonatal periostin knockout(KO)and wildtype(WT)mice and further clarified the influence of periostin on the phosphatidylinositol 3-kinase(PI3K)/glycogen synthase kinase 3?(GSK 3?)/cyclin D1 signalling pathway.Methods:(1)To measure expression of periostin in mice subjected to MI.Two groups were included(sham and MI groups),we measured mRNA and protein expressions at 1,3,7,14 and 21 days after MI.(2)To clarify whether neonatal mice have the capacity to completely regenerate their hearts after MI at 21 days.We observed fibrosis areas and regenerate markers in 5 time points.(3)To verify the effects of periostin knockout on heart regenerate and investigate the underlies mechanism.Four groups were consisted,(WT + sham group,WT + MI group,KO + sham group,KO +MI group).Cardiac function,fibrosis areas,regenerate markers(pH3 and aurora B),and vessel were measured at 7 days after MI,and protein expressions of PI3K/Akt/GSK3(3/cyclin D1 were measured in both neonatal heart and isolated neonatal mouse ventricular myocytes.(4)Rescue experiments were performed todemonstrate whether treatment with GSK3? inhibitor SB216763(short of SB)improves periostin knockout induced regenerate impairation.Four groups were included(KO + sham + vehicle,KO + MI + vehicle,KO + sham + SB,KO + MI +SB).We intraperitoneal injected SB to the mice from the 2nd day and last for one week.Cardiac function,fibrosis areas,regenerate markers of heart tissure and vessels were measured thereafter.Results:1.Periostin expression was upregulated in neonatal and adult mice response to MI.Periostin was upreglulated at the 1St day after MI,and reached to peak at 7 days in border zone in adult mice.In neonatal mice,periostin mRNA and protein were increased 7 days after MI.2.The neonatal mice heart have the capacity to regenerate themselves at 21 days after MI.7 days after MI,phospho-histone 3(pH3)and aurora B,both are regenerate markers,were significant increased in infarcted,border and remote zones.Fibrosis area was increased from the 1st day after MI,and reached to a top level at 7 days,dissolved in 10th,14th,and 17th day,and complety disappeared at 21 days,which means a completely regenerate occurs.3.Periostin deficiency impared regenerate ability,increased post-infarction fibrosis and lower cardiac function in neonatal miceCompared to sham group,MI mice have decreased cardiac function,increased fibrosis area and heart weight/body weight ratio.Wildtype mice present completely disappeared fibrosis area and can regenerate themselves,while periostin knockout showed impaired regenerate capacity,indicated by regenerate marker,and increased fibrosis,and lowered cardiac function.4.Ablation of periostin decreased expression of PI3K/Akt/cyclin D1 protein and increased expression of GSK3P proteinAblation of periostin decreased expression of PI3K/Akt/cyclin D1 protein,increased expression of GSK3? protein in neonatal mice hearts subjected to MI at 7 days,while,same results were also obtained in neonatal mouse ventricular myocytes response to hypixia and reoxygenation injury.5.Periostin knockout inhibited post-infarction angiogenesisImmunohistochemical results showed periostin deficiency reduced endothelial cell marker CD31 and vessel smooth muscle cell marker ?-SMA expression at 7 days after MI,that suggest periostin knockout inhibits angiogenesis.6.GSK3? inhibitor blocked effect of periostin knockout induced impaired regenerate capacity,decreaded fibrosis areaWe performed sham or MI surgery in periostin knockout mice,GSK3? inhibitor SB216763(SB,10 mg/kg/day/time)was intraperitoneal injection at the 2nd day after MI,and last for 7 days.Treatment with SB significant inhibited periostin deficiency induced decrease of pH3 and aurora B,lowered enhanced fibrosis area,improved impaired angiogenesis and deteriorated cardiac function.Conclusion:Our findings indicate that a lack of periostin impairs post-MI regeneration of cardiomyocytes and angiogenesis,those effects mediated by the PI3K/GSK3?/cyclin D1 signalling pathway.