Mechanisms Of PDGFR? Signaling Pathway Regulating Epicardial Function And Cardiac Regeneration

Background Ischemic heart disease greatly threatens human life and health in the past few decades.Cardiomyocytes lose progressively and fibrotic replacement occurs after heart injury,eventually leading to heart failure.Clinically,interventional therapy or bypass surgery are conventional treatments.Even so,no better rescue strategy for the lost cardiomyocytes has been introduced.Numerous studies in regenerative medicine have confirmed that the mammal heart possesses regenerative capacity at an early age.Studies in zebrafish and mice models have shown that fast revascularization in the acute phase of injury contributes to cardiac repair,and the epicardium participates in the regulation of this process by releasing paracrine signals,thus myocardial regeneration becomes a potential therapeutic way.PDGFR? signaling pathway promotes proliferation,survival,and migration of various cell types,as well as regulates the progression of developmental and damage repair.However,the role of PDGFR? in mammal heart regeneration is still unclear.Objective To clarify whether PDGFR? signaling pathway is involved in the regulation of cardiac regeneration in neonatal mice and how it affects epicardial function and the potential molecular mechanisms.Methods and Results 1.Models of cardiac regeneration in neonatal mice.Postnatal day 1(P1),postnatal day 7(P7),and postnatal day 56(P56)C57 mice were treated with left anterior descending coronary artery(LAD)ligation(MI group)or sham operation(Sham group).Specimens obtained on day 21 post infarction were subjected to paraffin-embedded sections,and then Picrosirius red staining was used for end event assessment.Picrosirius red staining showed that P1 mice heart were completely regenerated with hardly any collagen deposition on day 21 after MI;P7 and P56 mice failed to regenerate with infarct area weakened and fibrotic scar formation.2.The levels of PDGFR? and its phosphorylation were significantly increased in the injured area after myocardial infarction/apical resection in neonatal mice.Inhibition of PDGFR? signaling interfered with the process of neonatal regenerative repair,resulting in impaired cardiac function,scar formation,and cardiomyocyte hypertrophy.We constructed Sham/MI group,apical resection group(AR group)and sham operation group(Sham group)using P1 mice.Specimens were collected on day 7 after operation,and total protein from the damaged region was extracted for Western Blot.The results showed that PDGFR? and its phosphorylated form dramatically increased after neonatal MI/AR.Then MI/AR models of P1 mice were established,and each of the two models were stochastically divided into two groups to receive PDGFR? inhibitors or DMSO injection intraperitoneally.Cardiac function was evaluated by echocardiography on day 21 after MI/AR,and then specimens were taken.The injured tissue were paraffin-embedded for Picrosirius red staining and immunofluorescence staining(IF).Ultrasound showed that PDGFR? inhibitors impaired the physiological function of the neonatal heart suffered from MI/AR;Moreover,Picrosirius red staining confirmed that PDGFR? inhibitors eventually caused fibrous scar formation and thinning of injured ventricle wall;IF demonstrated that the myocardial cells became significantly larger in the unregenerated heart.3.The epicardium was activated and expressed large amounts of PDGFR? after neonatal AR.We constructed Sham/AR group using P1 mice.Specimens were collected at 3 time points on day 1,4,and 7 post injury.After paraffin-embedded sections,the expression of WT1 and PDGFR? in the incision region were detected by IF.It showed that compared with the Sham group,the levels of WT1 and PDGFR? in the incision area were significantly increased in AR group,and colocalization occurred.4.The epicardial activation,epithelial-mesenchymal transition(EMT)and neovascularization dynamic changed after neonatal MI,while the epicardium secreted a variety of paracrine factors to promote regeneration.Sham/MI group of P1 mice was constructed,and specimens were collected at 5 time points on day 1,4,7,14,and 21 after operation,then total RNA was extracted from the injured tissue for quantitative real-time PCR(q RT-PCR)to detect epicardial activation(Wt1,Aldh1a2,Tbx18,and Tcf21),EMT(Slug,Snail,Smad,and Twist),and neovascularization(Pecam1,Sm22 a,and Acta2)levels,while the epicardial paracrine function was tested on day 7 post MI.The results showed that epicardial activation and EMT enhanced early and then lowered to physiological level,neovascularization-related genes continuously regulated,while considerable angiogenesis factors secreted by the epicarium accelerated the restoration.5.Inhibition of PDGFR? signaling apparently reduced the extent of epicardial reactivation,EMT,angiogenesis,and impaired epicardial paracrine,as well as weakened cardiomyocyte proliferation and inhibited several downstream signaling pathways after neonatal MI/AR.MI/AR models of P1 mice were established,and both of the two models were randomly divided into two groups to give PDGFR? inhibitors or DMSO.Specimens were acquired on day 7 post-surgery,then total RNA,total protein,and paraffin-embedded sections from the injured tissue were utilized for q RT-PCR,Western Blot,and IF detection respectively.q RT-PCR showed that application of PDGFR? inhibitors after neonatal MI/AR led to a decrease of epicardial reactivation and EMT,a reduction of neovascularization-related gene expression,and a decline of the secretion capacity of the epicardium;In addition,Western Blot indicated that PDGFR? inhibitors reduced the levels of PDGFR? and its phosphorylation after injury,and several signaling pathways involved in cell proliferation,survival,and migration were also inhibited;Further,IF confirmed that neovascular network decreased in the injured area and cardiomyocyte proliferation activity went down owing to PDGFR? inhibitors administration.6.Inhibition of PDGFR? signaling directly repressed cardiomyocyte proliferation in vitro.Cardiomyocytes of fetal mice were isolated from pregnant C57 mice on embryonic day(E)14.5,and then treated with PDGFR? inhibitors or DMSO respectively.After 24 hours' incubation,IF was used to assess cardiomyocyte proliferation by co-staining p H3,Ki67,or Aurora B with ?-actinin.It showed that the proportion of p H3+,Ki67+,and Aurora B+ cardiomyocytes in PDGFR? inhibitor group were obviously decreased.7.Epicardium specific knockout Pdgfrb(PdgfrbWt1 KO)impaired the epicardial function,decreased the neovascularization in the injured area,and reduced cardiomyocyte proliferation during heart repair after neonatal MI.Wt1-Cre ERT2 strain was crossed with Pdgfrbfl/fl strain to generate epicardium conditional knockout Pdgfrb(PdgfrbWt1 KO)mice.LAD ligation was performed with 1-day-old PdgfrbWt1 KO mice and wild type littermates,and samples were collected on day 7 after MI.Total RNA and paraffin embedded sections in the injured area were subjected to q RT-PCR and IF detection respectively.q RT-PCR showed that Pdgfrb specific knockout resulted in inhibition of epicardial EMT and paracrine after neonatal MI;IF indicated that the neovascular network in the infarct region grew less and cardiomyocyte proliferation ability decreased.8.PdgfrbWt1 KO neonatal mice cannot successfully regenerate after MI/AR,manifested as cardiac dysfunction,scar formation and myocardial hypertrophy. MI/AR models of 1-day-old PdgfrbWt1 KO mice and wild type littermates were established.Cardiac function was detected by echocardiography on day 21 after MI/AR.Specimens were collected and paraffin-embedded sections of the injured area were used for Picrosirius red staining and IF.Echocardiography showed that Pdgfrb specific knockout resulted in damaged physiological cardiac function after neonatal MI/AR;In addition,Picrosirius red staining confirmed that Pdgfrb specific knockout caused fibrosis repair after injury,and the wall of the injured area became thin;IF demonstrated that cardiomyocytes in the unregenerated heart were significantly enlarged.Conclusions Epicardial PDGFR? signaling pathway is activated in neonatal MI/AR,and inhibition of PDGFR? results in decreased epicardial activation,EMT,and paracrine function,thereby impairing angiogenesis and reducing cardiac myocytes proliferation,leading to the failure of neonatal heart regeneration.PDGFR? signaling pathway plays an important role in maintaining epicardial function in neonatal mice,and this conclusion provides new ideas for translational medicine study of epicardial function.