The Role Of Epicardium During Heart Regeneration In Neonatal Mice

Rationale:Mammalian epicardium generated epicardium-derived cells (EPDCs) through epithelial to mesenchymal transition (EMT) and made essential cellular contributions to the development of the fetal myocardium. However, it was controversial whether EPDCs differentiate into cardiomyocytes in postnatal mammalian heart under the condition of faint regeneration after injury. Recent study exhibited that the hearts of1-day-old mice could regenerate after partial surgical resection, this capacity was lost by7days of age, which provided a ideal model to study the fate of EPDCs during the procedure of heart regeneration. Even previous study supported a role for myocyte proliferation in the neonatal cardiac regenerative potential, in the procedure of1-day-old mice heart regeneration Wt1marked EPDCs were observed to migrate into myocardial area. These studies suggested that EPDCs of neonatal mice remained some capability of fetal epicardium. So that it was value to explore the contribution of EPDCs in heart regeneration of neonatal mice.Objective:To explore whether Wtl marked EPDCs can adopt cardiomyocyte fate during the heart regeneration of neonatal mice and study the possibility of extending the cardiac regeneration potential of1-day-old mouse to the7days age with maintaining Wtl expression primed by Tβ4.Methods and Results:Part one:De novo cardiomyocytes derive from epicardium after partial surgical resection of neonatal heart.1) We repeated the model of neonatal heart resection and the entire apical defect was replaced by cardiomyocytes without fibrillation, as detected by Masson and HE staining.2) Western-blot and immunofluorescence stain showed the expression level of Wilm’s tumour1(Wtl), a embryonic epicardial marker, increased after apical resection compared from the sham groups, peaking between7and14days post-resection (dpr) and declining to near-baseline levels by21dpr. Most of Wtl+cells localized in thickened epicardium nearby injury area, covered the resection site and migrated into subepicardium.3) Snail, an EMT marker, was found to be in both the Wtl+epicardial cells and EPDCs at2-4dpr (day post-resection). E-cadherin, the marker of epithelium, was suppressed in Wtl+EPDCs.4) The P-Smad mediated BMP signaling, which directs the differentiation of these epicardiac progenitors into myocardial lineage during development. P-Smad was found to be expressed in nucleus of mesenchymal cells at both epicardium and myocardiac area.5) Wtl+/Isletl+cells were localized in both subepicardiac and myocardiac area during7dpr to14dpr and Wtl+/cTnT+or Wtl+/SαA+cells were beginning appearance from14dpr and still existed in21dpr, which indicated Wtl+EPDCs differentiated into de novo cardiomyocytes. Flow cytometry revealed that7.9%of all cardiac cells were Wt+/cTOT+at14dpr.Part two:Extending the time window of mammalian heart regeneration by activating epicardium.1) Tβ4administration:The1-day-old neonates were given intraperitoneal injection of Tβ4or vehicle (PBS) on daily basis for7days. Apical resection was performed on these7-day-old Tβ4-and PBS-treated mice. Two days after the resection, the9-day-old mice continued to receive intraperitoneal injection of Tβ4or PBS on alternate days until the19th day after birth.2) At21dpr, Masson and HE staining revealed in Tβ4-primed mice the lost ventricular apex regenerated with minimal fibrosis, but in PBS-treated group the7-day-old mice failed to regenerate their myocardium after apical resection and developed significant fibfosis. Echocardiography was used to assess left ventricular ejection fraction (EF) and fractional shortening (FS) in Tβ4-and PBS-primed mice at7,14and60dpr, Tβ4-primed mice had significantly better heart function than mice in PBS group (p<0.05).3) According to immunohistochemically staining, after partial ventricular amputation, in both groups, Wtl+epicardial cells were increased from one layer to several layers across the epicardium. In Tβ4group, Wtl+EPDCs migrated into sub-epicardial and myocardial areas. At7dpr, some Wtl+EPDCs were found to be positive for Isll, and these Wtl+/Isll+EPDCs were significantly increased in the regions of regenerated epicardium and sub-epicardium covering the resection site and across the entire injury area. But in PBS-treated mice no Isll was expressed. At7and14dpr, some Wtl+EPDCs in the injury area expressed the markers of cardiomyocytes, cTnT and SaA. On the other hand, in PBS group the Wtl+EPDCs stayed on the surface of the heart and did not migrate to myocardial areas. We failed to detect the expression of cTnT or SaA in Wtl+EPDCs from7to14dpr.4) Cardiomyocyte self-proliferation were assessed by colocalization of phospho-histone H3(pH3) with cTnT in PBS and Tβ4hearts at7and14dpr. Only a small number of pH3were expressed in the nucleus of cardiomyocytes in both groups. Most of pH3were expressed in interstitial cells and part of pH3+cells stayed at epicardial region. Tβ4-may have no effect in improving cardiomyocyte self-proliferation.5) We stained for smooth muscle a-actin (SMA), Wtl, and nuclei in PBS and Tβ4-treated hearts at7dpr. Quantification of SMA+cells showed that at7dpr the percentage of SMA-cells in two third of the ventricle containing the regeneration plane was6.13±1.41in Tβ4-treated hearts and for PBS-treated hearts the percentage was2.39±0.49(p=0.0038). The results showed Wtl marked EPDCs adopted smooth muscle cell fate and T(34improved angiogenesis after heart injury.Conclusions:1) Our results indicate that EPDCs can adopt the cardiomyocyte fate in the injury neonatal heart.2) Heart regeneration potential of neonatal mice could be maintained to the7th postnatal day by mobilizing epicardium-derived cells with thymosin β4.