Study On The Role Of PARP1 In Heart Regeneration And Cardiomyocyte Proliferation

Part Ⅰ PARP1 participates in neonatal mice heart regeneration and cardiomyocyte proliferation Background: The adult mammalian heart cannot regenerate in response to damage,resulting in scar formation and heart remodeling.The hearts of lower vertebrates,such as zebrafish,maintain regenerative capacity for all lifetime,which is often used as the animal model of cardiac regeneration.However,the evolutionary gap has prevented its transformation to clinical usage.It has recently been found that the heart of newborn mice can also regenerate,which provides a more efficient way for the study of cardiac regeneration.PARP1,a protein-modifying enzyme,mostly exists in the nucleus and plays an important role in cardiac development and cardiomyocyte hypertrophy.However,until now it has not been studied in cardiac regeneration.Methods: Mouse heart tissues at different developmental time points were used to detect cardiomyocyte proliferation,meanwhile,the expression and activity of PARP1 were detected by Western blot,q PCR,tissue immunofluorescence and co-immunoprecipitation.At one day after birth,the left anterior descending coronary artery(LAD)of the mouse heart was ligated to induce the model of cardiac regeneration.To validate the success of this model,cardiomyocytes proliferation was evaluated by tissue immunofluorescence co-staining of proliferation markers Brd U or p H3 with cardiomyocyte marker c TNT.Cardiac ultrasound,tissue Masson staining were used to evaluate cardiac repair.The differences in cardiac repair and cardiomyocyte proliferation between PARP1 knockout(KO)mice and wild type(WT)mice were compared.The number of cardiomyocytes in the adult mouse heart under physiological conditions was evaluated by WGA staining and heart-to-body weight ratio,and the difference in cardiomyocytes number between PARP1 knockout mice and WT mice was compared.The effect of PARP1 inhibitor on cardiac regeneration and cardiomyocyte proliferation in mice was observed in the same manner.The neonatal rat cardiomyocytes were isolated and the effects of PARP1 knockdown and PARP1 activity inhibition on cardiomyocyte proliferation were examined by immunofluorescence co-staining proliferation markers and cardiomyocyte markers.Results: The activity and expression levels of PARP1 were positively associated with cardiomyocyte proliferation.This model was validated by increased cardiomyocyte proliferation and fully cardiac repair.Compared with WT mice,PARP1 knockout mice showed the decreased capacity of cardiomyocyte proliferation after P1 mice LAD ligation,evidenced by a decreased proportion of Brd U-positive cardiomyocytes and p H3-positive cardiomyocytes.However,there was no obvious fibrosis and decreased cardiac function at P28 in PARP1 knockout mice compared with WT mice.In addition,under physiological conditions,PARP1 knockout mice had a fewer number of cardiomyocytes compared with WT mice.Inhibition of PARP1 activity also inhibited cardiac regeneration and cardiomyocyte proliferation in neonatal mice subjected to LAD ligation.In in vitro experiments,both PARP1 knockdown and inhibition of PARP1 activity could inhibit cardiomyocyte proliferation.Conclusion: PARP1 plays an important role in cardiac regeneration and cardiomyocyte proliferation in neonatal mice,and this may due to the role of PARP1 in the regulation of the cell cycle.Part Ⅱ PAPR1 overexpression promotes cardiomyocyte proliferation and heart regeneration Background: Heart failure caused by acute myocardial infarction is a common disease that threatening the health of people.Promoting heart cardiac regeneration to fully repair the heat is a promising treatment.In the past decades,the studies to promote cardiac regeneration have focused on stem cell transplantation,but the results have not been satisfactory.Many recent studies have begun to try the method of promoting cardiomyocyte proliferation.We have demonstrated that PARP1 is involved in cardiac regeneration and cardiomyocyte proliferation,then we will examine the role of PARP1 in promoting cardiomyocyte proliferation and cardiac regeneration.Methods: 1-day-old neonatal rat cardiomyocytes were isolated and transfected with PARP1-overexpressing adenovirus and negative control adenovirus.Cardiomyocyte proliferation was detected by cellular immunofluorescence co-staining proliferative markers such as Ed U,Ki67,p H3 or Aurora B with cardiomyocyte marker Actn2.The size of cardiomyocytes was calculated by the ratio of the cardiomyocytes area to the number of myocardial nuclei.TUNEL staining was used to detect cardiomyocyte apoptosis.Then7-day-old rat cardiomyocytes were isolated and transfected with PARP1-overexpressing adenovirus and negative control adenovirus.The cell proliferation was also detected by cellular immunofluorescence.Adult mice were injected with PARP1 overexpressing adeno-associated virus or negative control virus via the tail vein.After two weeks the mice were performed myocardial infarction.Cardiac regeneration was assessed by Masson staining of the heart sections and echocardiography,and cardiomyocyte proliferation was detected by tissue immunofluorescence co-stained with Brd U and cardiomyocytes marker c Tn T.Result: 1-day-old neonatal rat cardiomyocytes transfected with PARP1-overexpression adenovirus increased the proportion of proliferating cardiomyocytes compared with negative control by Ed U,Ki67,p H3 and Aurora B staining.Meanwhile,PARP1 overexpression did not lead to cardiomyocyte hypertrophy or apoptotic.In accordance with this,overexpression of PARP1 in 7-day-old neonatal rat cardiomyocytes also increased the proportion of Ed U positive cardiomyocytes and Ki67 positive cardiomyocytes.Overexpression of PARP1 in the heart of adult mice can improve cardiac repair after myocardial infarction,and increase the number of Brd U-positive cardiomyocytes.Conclusion: PARP1 overexpression can not only promote the proliferation of neonatal mice cardiomyocyte but also promote cardiac regeneration and cardiomyocyte proliferation in adult mice after myocardial infarction.Part Ⅲ PARP1 modulates cardiomyocyte proliferation by regulating Hand2 activity Background: Our previous studies have demonstrated that overexpressing PARP1 promotes cardiomyocyte proliferation,while the mechanisms remain unclear.Being a nuclear protein,one of the main functions of PARP1 is transcriptional regulation.Transcription factors involved in heart development mostly promote cardiomyocyte proliferation.Therefore,we speculate that PARP1 may promote cardiomyocyte proliferation through one of the transcriptional factors associated with cardiac development.Methods: The co-immunoprecipitation method was used to determine possible interactional target proteins of PARP1 in the heart tissue of neonatal mice.After overexpressing PARP1 and its target protein in 293 T cells,their interaction was verified by co-immunoprecipitation.PARP1 was segmented into different functional domain fragments using GST fusion protein,and the binding fragment of target protein was detected by GST pull-down.After overexpression of Hand2 in 293 T cells,the co-immunoprecipitation method was used to verify whether the target protein could be poly(ADP-ribosyl)ated(PARylated).The effect of PARP1 on the transcriptional activity of the target protein was detected by the dual luciferase reporter gene.The role of the target protein in cardiomyocyte proliferation-promoting effects of PARP1 was examined in isolated neonatal rat cardiomyocytes.Cell proliferation was evaluated by immunofluorescence staining.Results: PARP1 bound to cardiac development-associated transcription factor Hand2 in neonatal mouse heart tissue.Their interaction was also verified in 239 T cells.Further studies revealed that the DNA binding domain of PARP1 bound to Hand2.Hand2 could be PARylated and its PAR level was reduced by inhibition of PARP1 activity.Inhibition of PARP1 activity reduced the transcriptional activity of Hand2,while overexpression of PARP1 increased its transcriptional activity.Knockdown of Hand2 in neonatal rat cardiomyocytes inhibited the proliferation-promoting effects of PARP1 in cardiomyocyte.Conclusion:PARP1 combines with Hand2 and modifies it by PARlation.PARP1 exhibits its role in promoting cardiomyocyte proliferation partially by regulating Hand2 activity.