The Effect And Mechanism Of LncRNA-DRR And Wnt Pathway Inhibitor CGX1321 On Cardiomyocyte Proliferation And Heart Regeneration

Background:Heart failure after myocardial infarction is one of the main cause of the global disease mortality,although early reperfusion therapy can save most of the injuried myocardium,but the subsequent ventricular remodeling and heart failure is still irreversible,which is attributed to the loss of functional cardiomyocytes,therefore the realization of ultimate restoration of cardiac function and heart failure must rely on cardiomyocte proliferation and heart tissue regeneration.Traditional view demonstrated that mammalian heart was terminal differentiated without the function of repair and regeneration after injury,but recent studies have found that adult cardiomyocytes possess self-renewal potential in physiological and pathological progresses,but their regeneration ability is very limited,which cannot reach significantly repaired effect under the condition of non intervention.In recent years,fate-mapping technology has been used to find that the main source of myocardial regeneration is the proliferation of pre-existed cardiomyocytes,while the differentiation of"in-situ cardiac stem cells"represented by c-kit~+cells into cardiomyocytes has been proved to play a negligible role.At the same time,the fate-mapping technique of the double fluorescent reporter gene proved that other non-cardiomyocytes in heart tissue could not transform into cardiomyocytes.The use of fate-mapping combined with live cell real-time tracking technique has demonstrated the phenomenon of dedifferentiation,proliferation and redifferentiation of adult cardiomyocytes in mammals,further confirming that endogenous cardiomyocyte proliferation is the main source of myocardial regeneration.However,the proliferation potential of adult cardiomyocytes is very limited,far from achieving the regeneration after myocardial injury.Therefore,it is a key scientific problem in the field of cardiac regeneration to explore the molecular mechanism of cardiomyocyte proliferation and find targets to stimulate the proliferation of adult mammalian cardiomyocytes.Mammalian hearts at the early embryo and birth stage have complete regeneration ability,which drops rapidly to extremely low levels after birth,the main reason is due to rapid reduction of cardiomycyte proliferation.Apical resection on P1 mouse heart can further activate the ability of cardiomyocyte proliferation.Recent studies have found that epigenetic regulation of myocardium is an important factor affecting the shutdown and restart of cardiomyocyte proliferation.Starting from epigenetic regulation to identify the molecular"switch"of cardiomyocyte proliferation is a key link to find out how to activate the proliferation of adult cardiomyocytes and promote cardiac repair after myocardial infarction.As a novel discovered non-transcriptome gene and epigenetic molecule,long non-coding RNA is crucial in the heart development and the progression of myocardial diseases.In this study,the effect and mechanism of lncRNA-DRR on cardiomyocyte proliferation both in physiological and pathological conditions were revealed by high-throughput screening of apical resection model.Wnt signaling pathway is a key molecule in cardiac embryonic development.Through systematic analysis of human embryonic stem cells,it was found that Wnt/β-catenin could promote the differentiation of h ESCs into mesodermal cells and inhibit the differentiation of mesodermal cells into cardiomyocytes.Wnt signaling also plays an important regulatory role in the myocardium of adult mammals.Under physiological conditions,Wnt signal in cardiomyocytes is inhibited,while under pathological conditions such as ischemia and hypoxia,Wnt signal can be reactivated.Recent studies have shown that a variety of small molecule compounds have significant therapeutic effects in mouse models of myocardial infarction by inhibiting the Wnt signaling pathway.Activation of the Wnt signaling pathway mainly depends on the binding of secreted Wnt proteins to membrane receptors.Intracellular Wnt proteins can only be secreted to the extracellular membrane through palmitoylation,so palmitoylation is an effective target for regulating Wnt signaling.CGX1321 is a novel palmitoylation inhibitor of Wnt protein,which has been approved for clinical trials.This study elucidates the effect and molecular mechanism of CGX1321 on cardiac regeneration after myocardial infarction.Methods:Part I:1.High-throughput transcriptome and non-transcriptome sequencing was performed on the mouse apical resection model to screen the candidate lncRNAs regulating the proliferation of cardiomyocytes.2.Primary cardiomyocytes from 1-day old rats were isolated and cultured for intervention using siRNA or overexpressed plasmids of candidate lnc RNAs.LncRNAs that regulate proliferation of cardiomyocytes were further screened by immunofluorescence staining of nuclear division(Ki67 and pH3)and cytokinesis(Aurkb)markers.3.Fluorescence in situ hybridization(FISH),RNA coding potential analysis website and qRT-PCR were used to study the conservation,coding potential,tissue/cell distribution and subcellular localization of target lnc RNA.4.The effect of target lncRNA on mature or immature genes of cardiomyocytes was detected by qRT-PCR.Part II:1.LncRNA-DRR knockout transgenic mice were established,and qRT-PCR was used to identify whether the mouse systerm was successfully established.2.Through immunofluorescence staining of cell proliferation and other indicators,the effects of lnc RNA-DRR on cardiac development and proliferation of cardiomyocytes in mice were studied,and apical resection model was established to study the effects of lnc RNA-DRR on cardiomyocytes proliferation and cardiac repair after injury.3.A model of myocardial infarction was established,and the effects of lnc RNA-DRR on cardiomyocytes proliferation and post-MI repair were determined by immunofluorescence staining of cell proliferation and other indicators,cardiac ultrasound,masson staining.etc.Part III:1.High-throughput transcriptometric analysis of wild-type and lnc RNA-DRR knockout mice after myocardial infarction was performed to identify the downstream signaling pathway regulated by lncRNA-DRR,and the changes of DNA damage in cardiomyocytes were quantified by immunofluorescence staining in frozen sections of heart tissue.2.Bioinformatics predictive analysis was used to screen protein molecules that directly interact with lncRNA-DRR.3.The signaling molecules interacting with lnc RNA-DRR were further determined by fluorescence energy resonance transfer,and the molecular mechanism of lnc RNA-DRR acting on the proliferation of cardiomyocytes was clarified.Part IV:1.The inhibitory effect of CGX1321 on wnt-5a/b protein secretion in primary cardiomyocytes was verified.2.A model of myocardial infarction was established,and the effects of CGX1321 on cardiomyocytes proliferation and post-MI repair in mice were determined through cardiac ultrasound,TTC staining,masson staining and immunofluorescence staining of cell proliferation indicators.3.Primary cardiomyocytes were isolated and treated in hypoxia environment,total and nuclear proteins of cardiomyocytes were collected 48 hours after CGX1321 treatment.The canonical Wnt signaling pathway was marked by the nuclear translocation ofβ-catenin,while the non-canonical Wnt signaling pathway was marked by the nuclear translocation of NFATc3.Results:Part I:1.Cardiomyocytes showed significant differences in proliferation capacity 7 days after sham surgery or apical resection.The selected target lnc RNA-DRR was located on mouse chromosome 4 and belonged to antisense long non-coding RNA,with three exons and two introns,mainly distributed in the nucleus and a small amount in cytoplasm of cardiomyocyte.2.Down-regulation of lnc RNA-DRR expression significantly increased the ratio of nuclear division and cytokinesis in primary cardiomyocytes,while up-regulation of lnc RNA-DRR significantly decreased the ratio of nuclear division and cytokinesis in primary cardiomyocytes.3.When lnc RNA-DRR was down-regulated,MYOM2,COX6A2 and GJA1,which are genes related to cardiomyocyte maturation,were significantly decreased,indicating cardiomyocyges tended to be immature state.Part II:1.After lnc RNA-DRR knockout under physiological conditions,P7 mice showed no significant changes in cardiac anatomical structure,proliferation index of cardiomyocytes and WGA staining.2.After the establishment of apical resection model on lncRNA-DRR knockout mice,the proliferation rate of cardiomyocytes was increased significantly,and the cardiomyocyte size was significantly decreased.The scar of myocardial tissue was significantly decreased both 7 days and 28 days after surgery,and the complete repair of myocardial tissue was basically achieved at 28 days after surgery.3.After the establishment of myocardial infarction model on lnc RNA-DRR knockout mice,there was no significant change in c TnT 24 hours after surgery.The left ventricular ejection fraction and short-axis shortening rate were significantly increased at 28 days after surgery,while the left ventricular diastolic and systolic diameters were significantly decreased.The level of fibrosis after myocardial infarction in lncRNA-DRR knockout group was significantly decreased.The indicators of nuclear division(Edu,Ki67 and p H3)and cytoplasmic division(Aurkb)were significantly increased,while the size of cardiomyocyte did not change significantly..Part III:1.High-throughput transcriptome analysis of cardiac tissue samples from adult wild-type and lncRNA-DRR knockout transgenic mice two weeks after myocardial infarction revealed significant changes in cell cycle and DNA damage response signaling pathways.2.The catRAPID database was used to predict the binding of lnc RNA-DRR to SFPQ protein,which is a protein associated with DNA repair after damage.RNA immunoprecipitation confirmed the direct binding of lncRNA-DRR and SFPQ protein in cardiomyocytes.3.Fluorescence energy resonance transfer was used to detect a significant increase of SFPQ/NONO protein complex in lnc RNA-DRR knockout transgenic mouse cardiomyocytes,and a significant decrease in the enrichment of DNA damage indicators(γH2A and p-ATM)in cardiomyocytes.Part IV:1.After treatment of cardiomyocytes for 48 hours,Wnt-5a/b in CGX1321 group was significantly decreased bothe in cells and supernatant medium.2.In the myocardial infarction model,CGX1321 can significantly improve the survival rate,increase the left ventricular ejection fraction,left ventricular short axis shortening rate and left ventricular posterior wall diastolic thickness.3.In the model of myocardial infarction,TTC staining showed that CGX1321 could significantly reduce the infarct area and the level of fibrosis both in infarct area and infarct border zone,while significantly increase the number of EdU positive cardiomyocytes.4.After CGX1321 treatment of cardiomyocytes for 48h,western blot showed that CGX1321 had no significant effect on the expression ofβ-catenin and NFATc3 under non-hypoxia conditions,while under hypoxia conditions,CGX1321 could significantly inhibit the expression ofβ-catenin and NFATc3 in nuclear proteins,while it had no significant effect onβ-catenin and NFATc3 in total proteins.Conclusion:1.The mouse model of apical resection can be used as an animal model for screening the proliferation-related lncRNAs of cardiomyocytes.Lnc RNA-DRR screened by high-throughput sequencing in the model of apical resection has a negative regulatory effect on the proliferation and immaturation of cardiomyocytes.2.Under physiological conditions,lncRNA-DRR had no significant effect on the cardiac function,cardiac anatomical structure,proliferation and size of cardiomyocytes in the early development stage of mice.After apical resection,lncRNA-DRR knockout can significantly promote the cardiomyocytes proliferation and improve the ability of repair after resection.After myocardial infarction,lncRNA-DRR knockout can significantly promote the proliferation of adult mouse cardiomyocytes,improve cardiac function,and inhibit myocardial fibrosis.3.After myocardial infarction,lnc RNA-DRR knockout transgenic mice had significantly increased SFPQ/NONO protein complexes in cardiomyocytes,promoting post-DNA damage repair and increasing the proliferation capacity of cardiomyocytes.4.CGX1321 can inhibit Wnt-5a/b secretion in cardiomyocytes;CGX1321 can significantly improve the survival rate of adult mouse myocardial infarction,improve cardiac function,reduce the area of infarction through increasing nuclear translocation ofβ-catenin and NFATc3 protein and activating the canonical and non-canonical Wnt signaling pathways.