Preliminary Study Of Endogenous Peptides Related To Heart Regeneration

Part 1 Peptidomics Analysis of Transient Regeneration in the Neonatal Mouse Heart.Objective: To provide possible therapeutic targets for cardiac regeneration,we compare the differential endogenous peptides profiling of heart at the postnatal 1 and 7 days in the mouse.Method: 1)The cardiac regeneration model of mouse was established and verified.2)We construct a differential peptidomic analysis of the neonatal mouse heart between P1 and P7 group.Analysis of six-plex tandem mass tag(TMT)labelled peptides was performed using nanoflow liquid chromatography coupled online with an LTQ-Orbitrap Velos mass spectrometer.After removing the redundancy data we conducted a statistical analysis.A statistical p value <0.05 and a fold change of ?1.5 was applied.In this way,differentially expressed peptides were screened out.3)We explored the physico-chemical properties in all differentially expressed peptides between P1 and P7 group.The functions of the peptides were predicted by GO and pathway analysis of their protein precursors.4)IPA and the Uni Prot database were used to explored the location of different peptides in their precursor protein and functions of precursors protein.Endogenous peptides that may be closely related to cardiac regeneration were screened out.Result: 1)The heart of P1 neonatal mouses can regenerate after apex excision,while the heart of P7 neonatal mouses cannot regenerate.2)A total of 2692 peptide fragments originating from 640 proteins were identified in the six samples,with 236 differentially expressed peptides.A total of 169 pep- tides were upregulated in the P1 group compared with the P7 group,and 67 peptides were downregulated.3)The MW of total differential peptides were between 500~3000Da.The MW of most downregulated peptides was between 700~900Da and 1500~1900Da range,whereas most upregulated peptides fell into the 700~1300Da.For most peptides,the p I ranged between 4-6 and 8-10.The PI of most downregulated peptides was concentrated between 5 and 6.The distribution trend of PI-MW in the up-regulated and down-regulated polypeptide groups was consistent with the distribution trend of all polypeptides.4)The possible biological events of the 236 differentially peptides were predicted by GO and Pathway enrichment analysis of their precursor proteins.GO analysis results show that,the main biological process categories were ATP metabolic process,cardiac muscle tissue,development and contraction,cardiac cell development and differentiation;the main cellular components involved are extracellular exosome and vesicle,cardiac cell specific cytoskeleton,and contractile complex and so on;molecular function level was mainly related to actin binding,poly(A)RNA binding,calcium ion binding,and so on.Canonical pathway enrichment analysis indicated us to focus on the following pathways: cardiac hypertrophy,mitochondrial dysfunction,cardiac fibrosis,cardiac necrosis/cell death,and G2/M DNA damage checkpoint regulation and so on 5)Based on the above two groups of candidate peptides and precursors,36 important peptides were selected,which are located on the functional domain of the precursor and/or contain functional sites(mainly post transcriptional modification).The results showed that the predicted peptides were mainly derived from EF-hand domain(the most common calcium-binding motif found in proteins;the functional sites included in these peptides were mainly phosphorylation and acetylation modification sites,which suggested that these peptides might be involved inimportant signaling pathways.Conclusion:1)Peptides profiling in P1 group were significant changed compared to P7 group,suggesting that the peptides may be involved in the repair and regeneration of heart jnjury.2)dozens of differential peptides were derived from functional domains of closely associated with proteins of cardiac repair and regeneration.They may participate in the repair and regeneration process of cardiac injury.Additionally,our results provide insight that will be useful for future functional studies of heart regeneration.Part 2 A preliminary study of endogenous peptide related to cardiac regenerationObjective: The characteristics of endogenous peptides were systematically investigated by using bioinformatics methods,and the peptides associated with cardiac regeneration were analyzed.a preliminary study of the selected endogenous peptides was carried out,that preliminarily reveal the role of endogenous peptides in cardiac regeneration,according to the result of bioinformatics analysis.Method: 1)The characteristics of the p-Hspb1 peptide was further analyzed using bioinformatics methods 2)The m RNA expression of p-Hspb1's precursor protein HSPB1 in P1 and P7 mouse heart was verified by q RT PCR method.3)peptide p-Hspb1(ARAQIGGPEAGKSEQSGAK)was synthesized: mice were injected with the peptide every day until seventh days starting within12 hours after a mouse birth.Apical excision of P7 mice was implemented at seventh days after birth,and then the mice injected with the peptide every other day.After 21 days of birth,cardiac tissues were removed,and the cardiac regeneration was observed by immunostaining.4)P19 cells were induced to differentiate into cardiomyocytes,and the effects of different concentrations of p-Hspb1 peptide on the differentiation of P19 cells were observed.Result: 1)The m RNA expression of p-Hspb1 precursor protein HSPB1 in the P1 mice heart was higher than that in P7 mice.2)The mice were injected using different doses of p-Hspb1.Scars on the apex of mice(1X dose)can be obviously found at 21 dpr by HE&Masson staining,No fibrous scar was detected in mice(3X and 9X dose),which revealed the apical defect was replaced by normal myocardial tissue.3)Peptide p-Hspb1 can upregulate the expression of cardiac markers in the process of differentiation of P19 cells into cardiomyocytes(with the increase of peptide concentration,the effect is more significant.).Conclusion: 1)Peptide p-Hspb1 can prolong the time window of myocardium tissue regeneration in mice,suggesting that peptide p-Hspb1 may be involved in the repair and regeneration of heart injury.2)P19 cells were embryonic stem cells,which isolated from male C3H/He mice in teratomas and can be cultured in vitro.p-Hspb1 can promote the differentiation of P19 cells into cardiomyocytes,which suggesting that p-Hspb1 extended the time window in the Neonatal Mouse Heart.The mechanism is that p-Hspb1 may promote the differentiation of neonatal mouse cardiac stem cells into adult myocardial cells.our results provide insight that will be useful for future functional studies of heart regeneration.