Active β-catenin Promotes Hypoxia-induced Polyploidy Cardiomyocyte Cytokinesis By Upregulating ECT2 Directly

The heart is highly dependent on oxygen,and chronic hypoxia leads to long-term damage to the myocardium.Chronic hypoxia appears to be a common pathophysiological process in a number of cardiovascular diseases,including cyanotic congenital heart disease and coronary atherosclerotic heart disease.In these diseases,long-term chronic hypoxia causes cardiomyocyte（CM）damage,CM loss,reduced systolic function,and finally heart failure.Hence,there is an urgent need to find a way to increase the number of CMs and supplement the loss of CM,which should provide a fundamental cure for hypoxia-related cardiovascular diseases.CM loss is common to several conditions,including ageing and ischemic heart disease.Although mammalian CMs actively proliferate during embryonic development,this proliferation decreases to a very low level shortly after birth,following a shift from a hyperplastic to a hypertrophic phenotype.Less than 50%of CMs were found to be exchanged during a normal lifespan.CM renewal gradually reduces from 1%annual turnover at the age of 20 to 0.3%turnover at the age of 75.Accordingly,the adult mammalian heart has a limited ability for CM regeneration,and the subsequent replacement of lost tissue with functionally and electrically inert scar tissue leads to cardiac dysfunction and death.These characteristics have led to considerable research interest in the potential therapeutic simulation of CM proliferation.Despite decades of cardiac regeneration studies,however,the mechanisms that underlie these regenerative processes remain largely unknown.During hypertrophic growth,CMs express cycle proteins and re-enter the cell cycle and synthesize DNA.CMs are forced by multiple pro-hypertrophic factors to re-enter the cell cycle and undergo DNA synthesis in the absence of cytokinesis,leading to polyploidization and multinucleation.Nonetheless,few studies have addressed the limitations of the cytokinesis of polyploid and multinucleated CMs.Most studies have focused on mononucleated diploid CMs,leading to the logical conclusion that new CMs in the adult heart are derived from pre-existing mononucleated diploid CMs.Unfortunately,only a few studies have addressed the reduced proliferative ability of binucleated CMs in vivo or the equivalent ability in vitro,compared with their mononucleated counterparts.In contrast to mononucleated CMs,multinucleated polyploid CMs can regenerate substantial amounts of new CMs via karyokinesis and/or cytokinesis,rather than by cell cycle re-entry,which is theoretically more challenging.Hence,an in-depth understanding of the molecular processes that control polyploid and multinucleated CM cytokinesis is of paramount importance.Cytokinesis is the process whereby one mitotic cell divides into two after chromosome separation.When cytokinesis fails in rat binucleated CMs,the chromosomes are arranged and separated correctly,but the actomyosin constriction ring is incompletely and asymmetrically assembled with delayed cleavage furrow ingression and no abscission.As noted previously,defective anillin localization is the key determinant of the incorrect assembly of the actomyosin constriction ring in CMs.Anillin is an evolutionally conserved multi-domain protein,which binds to multiple cytoskeleton proteins and acts as a scaffold in the assembly of the actomyosin constriction ring.The regulatory mechanism of the location of anillin remains unclear.As far as we know,anillin binds to ECT2,and co-regulates the downstream assembly of the actomyosin constriction ring.Meanwhile,the ECT2–Anillin interaction appears to be enhanced by ECT2’s lipid binding.With the onset of cytokinesis,Anillin localizes to the cleavage furrow,close to the membrane.However,the regulatory relationship between Anillin and ECT2 remains obscure.The Wnt/β-catenin signaling pathway is an essential determinant of cardiac development,including precardiac mesoderm induction and subsequent formation of the first heart field and expansion of the second.The differentiation of cardiac precursor cells requires the inhibition ofβ-catenin.In the adult heart,β-catenin promotes cardiac fibroblast proliferation to accelerate fibrosis 58 and is required for stem cell proliferation.Although several studies have reported the beneficial effect ofβ-catenin on CM cell cycle entry,we found no published study on its role in CM（particularly polyploid CM）cytokinesis.Therefore,this study aimed to determine whetherβ-catenin is involved in the cytokinesis of multinucleated polyploid CMs and,if so,to clarify its specific regulatory mechanism.Objectives:1.Investigate the multinucleation,polyploidization,and cell cycle activity of CMs in clinical samples.2.Investigate the role of hypoxia in multinucleation,polyploidization,and cell cycle activity of CMs in vivo and in vitro.3.Screen for and identify the potential key regulator in hypoxia-induced CM multinucleation and polyploidization and investigate the mechanism for the inhibition ofβ-catenin by hypoxia.4.Investigate the role of activeβ-catenin in the multinucleation,polyploidization,and cell cycle activity of CMs in vivo and in vitro.5.Investigate the role of activeβ-catenin-ECT2 on sorted tetraploid CM cytokinesis.Methods:1.Investigate the differences in CM multinucleation,polyploidization and cell cycle activity between patients with cyanotic congenital heart disease and patients with acyanotic congenital heart disease.From October 2014 to October 2016,patients with either cyanotic or acyanotic congenital heart disease who underwent cardiac surgery in the Department of Cardiovascular Surgery at Xinqiao Hospital were included in this study.All procedures involving human tissue samples were performed according to the principles outlined in the Declaration of Helsinki and approved by the Human Ethics Committee of Xinqiao Hospital（Chongqing,China）.Informed consent was obtained from all patients whose samples were included in this study.A total of 20 patients agreed to participate:10 patients with cyanotic and 10 with acyanotic congenital heart disease.The diagnosis of disease was according to preoperative echocardiography or CT and was confirmed during the operation.Inclusion criteria:patients with either cyanotic or acyanotic congenital heart disease undergoing right ventricular outflow tract dredging;the difference of blood oxygen saturation（SpO₂）no more than 2%between the limbs;and aged below 18 years.Patients undergoing multiple cardiac surgery or other diseases were excluded.Patients were divided into the cyanotic and acyanotic group according to their SpO₂ levels:the acyanotic group included patients with an SpO₂ level of 95%or higher,while the cyanotic group included patients with an SpO₂level no more than 85%.Immunofluorescence was performed to measure the nuclear number and cell surface area of CMs,and the percentage of pH3+CMs,Aurora B+CMs,and kif20a+CMs.Flow cytometry was applied to detect the ploidy of CMs in both acyanotic group and cyanotic group.2.Examine the effect of hypoxia on the CM cell cycle leading to polyploidization and multinucleation.Twelve 8-week C57BL/6J male mice were randomly divided into a hypoxic（n=6）and a normoxic（n=6）group.Mice from the hypoxic group were housed in a hypoxic chamber（oxygen content 9.9-10.1%）for 4 weeks,while those from the normoxic group were placed in normoxic conditions for the same length of time.Neonatal rat CMs（NRCMs）were obtained and divided into a hypoxic（n=6）and a normoxic（n=6）group,and the NRCMs were cultured in a hypoxic cell incubator（94%N2,5%CO2,1%O2,37℃）or normoxic cell incubator（74%N2,5%CO2,21%O2,37℃）for 48 h,respectively.CM smears were isolated from mice and NRCMs to perform immunofluorescence for cellular ploidy,nuclear number,and nuclear ploidy of CMs.Immunofluorescence was carried out to determine the cell surface area of the CMs and the percentage of Ki67+CMs,pH3+CMs,Aurora B+CMs,and kif20a+CMs in the NRCMs and mice heart slides.Flow cytometry was applied to evaluate the ploidy of CM nuclei in both NRCMs and mice heart.3.Determine the mechanism for hypoxia-inhibited regulatory activity ofβ-catenin on cell cycle genes during CM ploidization and multinucleation.Twelve 8-week C57BL/6J male mice and 12 NRCM cell culture plates were randomly divided into a hypoxic（n=6 mice or 6 plates of NRCMs）and a normoxic（n=6 mice or 6plates of NRCMs）group,as in Part 2.Bioinformatic analyses were conducted to identify the differentially expressed gene for GO analysis and KEGG pathway analysis,which could help to identify the potential key gene.The HIF-1αandβ-catenin contents of the nucleus and cytoplasm were evaluated by Western blot and confirmed by immunofluorescence.The mRNA levels of Cyclin D1 and c-Myc,the downstream target gene ofβ-catenin,were detected by RT-qPCR.Co-immunoprecipitation was used to assess the binding amongβ-catenin,HIF-1α,and TCF4 under hypoxia.4.Detect the regulatory action ofβ-catenin on the CM cell cycle leading to CM diploidization and mononucleation.Eighteen 8-week C57BL/6J male mice were randomly divided into activator（n=6）,inhibitor（n=6）and vehicle（n=6）groups.After 3 weeks of hypoxia,activator CHIR99021（2 mg/kg）,inhibitor IWR-1（1.75 mg/kg）,and dimethyl sulfoxide（DMSO）at the same volume as the inhibitor and activator were injected intraperitoneally into the three groups of mice,respectively,according to their body weight in the hypoxic chamber.Given that the multiplicity of infection（MOI）is 10,adenovirus-β-catenin（Adβ-catenin,overexpression group）,adenovirus-shRNAβ-catenin（Adshβ-catenin,silenced group）,and adenovirus-GFP（AdGFP,empty vector group）were transfected into the NRCMs.After transfection,the NRCMs were cultured in a hypoxic cell incubator for 48 h.Increasedβ-catenin at the nucleus was confirmed by Western blot and the transcriptional activity ofβ-catenin was detected by RT-qPCR.CM smears were isolated from mice and NRCMs to perform immunofluorescence for assessing the cellular ploidy,nuclear number,and nuclear ploidy of CMs.Immunofluorescence was carried out to measure the cell surface area of CMs,and the percentage of Ki67+CMs,pH3+CMs,Aurora B+CMs,and kif20a+CMs in the NRCMs and mice heart slides.Flow cytometry was applied to evaluate the ploidy of the CM nuclei in both NRCMs and mice heart.A cell counting kit–8（CCK-8）and CM counting were used to detect the number of CMs.Hemodynamics and cardiac function were evaluated by cardiac catheterization.5.Explore the effect of ECT2 upregulated by activeβ-catenin on tetraploid CM cytokinesis.Eighteen 8-week C57BL/6J male mice were randomly divided into activator（n=6）,inhibitor（n=6）,and vehicle（n=6）groups,as in Part 4.As the MOI is 10,adenovirus-β-catenin（Adβ-catenin,overexpression group）,adenovirus-shRNAβ-catenin（Adshβ-catenin,silenced group）,and adenovirus-GFP（AdGFP,empty vector group）were transfected into the NRCMs,as in Part 4.The transcriptional and translational levels of ECT2 were detected by RT-qPCR and Western blot,respectively.The binding site of TCF4on the non-coding region of Ect2 was predicted via bioinformatics and confirmed by chromatin immunoprecipitation–quantitative PCR（ChIP-qPCR）.Immunofluorescence was conducted to assess the co-location of ECT2 and Anillin or ECT2 and MgcRacGAP.The cellular ploidy of sorted tetraploid CMs was evaluated via flow cytometry.Results:1.Differences in the multinucleation,polyploidization,and cell cycle of CMs between patients with cyanotic and acyanotic congenital heart disease.Ten patients with cyanotic CHD（tetralogy of Fallot）and 10 age-matched patients with acyanotic CHD（ventricular septal defects and right ventricular outflow tract stenosis）were included.The two groups exhibited a significant difference in arterial blood oxygen saturation（P<0.05）.Immunofluorescence analysis revealed significantly higher numbers of binucleated CMs and a higher mean ploidy in CMs from cyanotic CHD samples than from acyanotic CHD samples.Moreover,the cyanotic group showed higher mean nuclear ploidy in CMs（P<0.05）.In addition,we observed larger CMs in samples from cyanotic patients（P<0.05）.We investigated the CM cell cycle in both groups and observed significant increases in pH3+CMs（P<0.05）in cyanotic tissues compared with acyanotic tissues,but the numbers of aurora B+CMs（P>0.05）and kif20a+CMs（P>0.05）were not significantly different.2.Effect of hypoxia on the CM cell cycle leading to polyploidization and multinucleation.Fluorescence microscope cytometry analysis revealed a dramatic increase in the mean cellular ploidy of CMs in mice exposed to hypoxia（P<0.05）,consistent with the hypoxic cell model（P<0.05）.The mean nuclear numbers of CMs were significantly higher in the hypoxic mice（P<0.05）and the cell model（P<0.05）.The mean nuclear ploidy of CMs was also higher in the hypoxic mice（P<0.05）and the cell model（P<0.05）.Notably,we observed a significant increase in the percentage of Ki67+CMs under hypoxia in the hypoxic mice（P<0.05）and the cell model（P<0.05）.Hypoxia induced more pH3+CMs in vivo（P<0.05）and in vitro（P<0.05）.However,hypoxia did not increase the frequency of Aurora B+CMs and kif20a+CMs in the mice（P>0.05）or cell cultures（P>0.05）.3.Mechanism for hypoxia-inhibited regulatory activity ofβ-catenin on cell cycle genes during CM ploidization and multinucleation.We found 420 differentially expressed genes（DEGs）between the diploid and polyploid CMs.The main function of the DEGs was regulation of the cell cycle and cell division.Moreover,comparison ofβ-catenin ChIP-seq data revealed that this transcription factor regulated 20.08%of the cell cycle genes and 22.03%of cell division genes.Accordingly,the nuclear protein content ofβ-catenin increased in cyanotic CHD samples（P<0.05）.A similar phenomenon was observed both in vivo（P<0.05）and in vitro（P<0.05）.Immunofluorescence confirmed the nuclear location ofβ-catenin.The downstream genes Cyclin D1 showed non-significant changes between the hypoxic and normoxic models in vivo（P>0.05）and in vitro（P>0.05）.There was moreβ-catenin/HIF-1αbinding thanβ-catenin/TCF4 binding under hypoxia（P<0.05）,and reverse co-IPs support these data（P<0.05）.4.The regulation of activeβ-catenin on the CM cell cycle leading to CM diploidization and mononucleation.β-catenin overexpression enhanced the transcription activity of this protein,as confirmed by Western blot（P<0.05 in vivo and P<0.05 in vitro）and qPCR analysis of downstream genes（P<0.05 in vivo and P<0.05 in vitro）.Notably,both cellular（P<0.05）and nuclear（P<0.05）ploidy and the nuclear number（P<0.05）were very clearly decreased in NRCMs subjected to overexpression,compared with those subjected to silencing or treated with vehicle alone.In vivo,cellular（P<0.05）and nuclear（P<0.05）ploidy and the nuclear number（P<0.05）were consistent with those observed in NRCMs.Regarding cell cycle activity,we observed a significant increase in Ki67+CMs in theβ-catenin overexpression group（P<0.05）,as well as higher percentages of pH3+（P<0.05）,aurora B+（P<0.05）,and mklp2+CMs（P<0.05）.Similarly,in vivo experiments demonstrated obvious increases in Ki67（P<0.05）,pH3（P<0.05）,aurora B（P<0.05）,and mklp2（P<0.05）expression in the CHIR99021 treatment group.The number of CMs increased significantly in theβ-catenin overexpression group（P<0.05 in vivo and P<0.05 in vitro）.In addition,a hemodynamics analysis revealed a significantly higher proportion of left ventricle ejection among mice in the CHIR99021 group than in the IWR-1 and DMSO groups（P<0.05）.5.ECT2 upregulated by activeβ-catenin promotes tetraploid CM cytokinesis,which is involved in heart regeneration.RT-qPCR and Western blot revealed that ECT2 increased in theβ-catenin overexpression group（P<0.05 in vivo and P<0.05 in vitro）.Analyzing the promoter of these genes,we identified two potentialβ-catenin-regulated sites located at the promoter and intron of the Ect2 gene of mice and rat.We then performed a ChIP-qPCR assay to confirm theβ-catenin occupancy of motif 1 in the CHIR99021 group of NRCM and mice.Moreover,β-catenin activation improved the localization of anillin and ECT2（P<0.05）.Immunofluorescence analysis revealed a significantly higher frequency of MgcRacGAP localization in the mid-body（P<0.05）.Flow cytometric analysis revealed a reduction in CM ploidy during hypoxia in the presence of CHIR99021（P<0.05）.Conclusions:1.Differences in the multinucleation,polyploidization,and cell cycle of CMs between patients with cyanotic congenital heart disease and patients with acyanotic congenital heart disease.Compared with the acyanotic group,CMs in the cyanotic group showed obvious polyploidization and multinucleation,which was significantly related to SpO2.The cell surface area of CMs enlarged during CM polyploidization and multinucleation.In the cyanotic group,the S phase activity was enhanced and karykinesis was less obviously increased,with no significant cytokinesis.These findings imply that hypoxia may increase the S phase activity with no effect on cytokinesis,which results in human CM polyploidization and the formation of multinucleated polyploid CMs.2.Effect of hypoxia on the CM cell cycle leading to polyploidization and multinucleation.Hypoxia enhanced the activity of the G1/S phase in CMs,which led to cell-cycle re-entry and completion of DNA synthesis.The activity of the G2/M phase and cytokinesis did not change during hypoxia and the number of multinucleated polyploid CMs increased.These data suggest that hypoxia induces CM polyploidization and multinucleation.Cytokinesis failure is the main determinant of CM proliferation.3.Mechanism for hypoxia-inhibited regulatory activity ofβ-catenin on cell cycle genes during CM ploidization and multinucleation.Regulation of the cell cycle and cell division were the main differences in transcriptome between polyploid and diploid CMs.Meanwhile,β-catenin was the potential key regulator in CM cytokinesis.Under hypoxia,activeβ-catenin entered the nuclei and bound to HIF-1α,which competitively inhibited the binding ofβ-catenin-TCF4 and the transcription activity ofβ-catenin.Therefore,β-catenin may be a promising target for CM division and is inhibited by hypoxia.4.The regulation of activeβ-catenin on the CM cell cycle leads to CM diploidization and mononucleation.Activeβ-catenin induced CMs to re-enter the cell cycle,synthesize DNA,enter mitosis,and complete karykinesis and cytokinesis,which finally resulted in CM diploidization and mononucleation.Furthermore,activeβ-catenin improved cardiac function during hypoxia.β-catenin may induce CM cytokinesis and enhance cardiac function.5.ECT2 upregulated by activeβ-catenin promotes tetraploid CM cytokinesis involved in heart regeneration.Activeβ-catenin directly upregulated the expression of ECT2 and increased the binding of Anillin-ECT2,leading to its correct location in the actomyosin constriction ring,which induced the completion of CM cytokinesis and decreased the percentage of polyploid CM.These findings add to our understanding of the molecular mechanism for the regulation ofβ-catenin in polyploid CM cytokinesis and present a novel phenomenon and mechanism for the failure of cytokinesis in CMs.