Foxm1 regulates cardiomyocyte proliferation in adult zebrafish after cardiac injury.

The regenerative capacity of the mammalian heart is poor, with one potential reason being that adult cardiomyocytes cannot proliferate at sufficient levels to replace lost tissue. During development and neonatal stages, cardiomyocytes can successfully divide under injury conditions; however, as these cells mature their ability to proliferate is lost. Therefore, understanding the regulatory programs that can induce post-mitotic cardiomyocytes into a proliferative state is essential to enhance cardiac regeneration. Here, we report that the forkhead transcription factor Foxm1 is required for cardiomyocyte proliferation after injury through transcriptional regulation of cell cycle genes. Transcriptomic analysis of injured zebrafish hearts revealed that foxm1 expression is increased in border zone cardiomyocytes. Decreased cardiomyocyte proliferation and expression of cell cycle genes in foxm1 mutant hearts was observed, suggesting it is required for cell cycle checkpoints. Subsequent analysis of a candidate Foxm1 target gene, cenpf, revealed that this microtubule and kinetochore binding protein is also required for cardiac regeneration. Moreover, cenpf mutants show increased cardiomyocyte binucleation. Thus, foxm1 and cenpf are required for cardiomyocytes to complete mitosis during zebrafish cardiac regeneration.

Cytoophidia safeguard binucleation of Drosophila male accessory gland cells.

Although most cells are mononuclear, the nucleus can exist in the form of binucleate or even multinucleate to respond to different physiological processes. The male accessory gland of Drosophila is the organ that produces semen, and its main cells are binucleate. Here we observe that CTP synthase (CTPS) forms filamentous cytoophidia in binuclear main cells, primarily located at the cell boundary. In CTPSH355A, a point mutation that destroys the formation of cytoophidia, we find that the nucleation mode of the main cells changes, including mononucleates and vertical distribution of binucleates. Although the overexpression of CTPSH355A can restore the level of CTPS protein, it will neither form cytoophidia nor eliminate the abnormal nucleation pattern. Therefore, our data indicate that there is an unexpected functional link between the formation of cytoophidia and the maintenance of binucleation in Drosophila main cells.

Regulation of extracellular matrix composition by fibroblasts during perinatal cardiac maturation.

BACKGROUND: Cardiac fibroblasts are the main non-myocyte population responsible for extracellular matrix (ECM) production. During perinatal development, fibroblast expansion coincides with the transition from hyperplastic to hypertrophic myocardial growth. Therefore, we investigated the consequences of fibroblast loss at the time of cardiomyocyte maturation by depleting fibroblasts in the perinatal mouse. METHODS AND RESULTS: We evaluated the microenvironment of the perinatal heart in the absence of fibroblasts and the potential functional impact of fibroblast loss in regulation of cardiomyocyte cell cycle arrest and binucleation. Cre-mediated expression of diphtheria toxin A in PDGFRα expressing cells immediately after birth eliminated 70-80% of the cardiac fibroblasts. At postnatal day 5, hearts lacking fibroblasts appeared similar to controls with normal morphology and comparable numbers of endothelial and smooth muscle cells, despite a pronounced reduction in fibrillar collagen. Immunoblotting and proteomic analysis of control and fibroblast-deficient hearts identified differential abundance of several ECM proteins. In addition, fibroblast loss decreased tissue stiffness and resulted in increased cardiomyocyte mitotic index, DNA synthesis, and cytokinesis. Moreover, decellularized matrix from fibroblast-deficient hearts promoted cardiomyocyte DNA replication. While cardiac architecture was not overtly affected by fibroblast reduction, few pups survived past postnatal day 11, suggesting an overall requirement for PDGFRα expressing fibroblasts. CONCLUSIONS: These studies demonstrate the key role of fibroblasts in matrix production and cardiomyocyte cross-talk during mouse perinatal heart maturation and revealed that fibroblast-derived ECM may modulate cardiomyocyte maturation in vivo. Neonatal depletion of fibroblasts demonstrated that although hearts can tolerate reduced ECM composition, fibroblast loss eventually leads to perinatal death as the approach simultaneously reduced fibroblast populations in other organs.

Stereological estimation of cardiomyocyte number and proliferation.

Cardiovascular diseases remain the leading cause of death, largely due to the limited regenerative capacity of the adult mammalian heart. Yet, neonatal mammals were shown to regenerate the myocardium after injury by increasing the proliferation of pre-existing cardiomyocytes. Re-activation of cardiomyocyte proliferation in adulthood has been considered a promising strategy to improve cardiac response to injury. Notwithstanding, quantification of cardiomyocyte proliferation, which occurs at a very low rate, is hampered by inefficient or unreliable techniques. Herein, we propose an optimized protocol to unequivocally assess cardiomyocyte proliferation and/or cardiomyocyte number in the myocardium. Resorting to a stereological approach we estimate the number of cardiomyocytes using representative thick sections of left ventricle fragments. This protocol overcomes the need for spatial-temporal capture of cardiomyocyte proliferation events by focusing instead on the quantification of the outcome of this process. In addition, assessment of cardiomyocyte nucleation avoids overestimation of cardiomyocyte proliferation due to increased binucleation. By applying this protocol, we were able to previously show that apical resection triggers proliferation of pre-existing cardiomyocytes generating hearts with more cardiomyocytes. Likewise, the protocol will be useful for any study aiming at evaluating the impact of neomyogenic therapies.

Deletion of Gas2l3 in mice leads to specific defects in cardiomyocyte cytokinesis during development.

GAS2L3 is a recently identified cytoskeleton-associated protein that interacts with actin filaments and tubulin. The in vivo function of GAS2L3 in mammals remains unknown. Here, we show that mice deficient in GAS2L3 die shortly after birth because of heart failure. Mammalian cardiomyocytes lose the ability to proliferate shortly after birth, and further increase in cardiac mass is achieved by hypertrophy. The proliferation arrest of cardiomyocytes is accompanied by binucleation through incomplete cytokinesis. We observed that GAS2L3 deficiency leads to inhibition of cardiomyocyte proliferation and to cardiomyocyte hypertrophy during embryonic development. Cardiomyocyte-specific deletion of GAS2L3 confirmed that the phenotype results from the loss of GAS2L3 in cardiomyocytes. Cardiomyocytes from Gas2l3-deficient mice exhibit increased expression of a p53-transcriptional program including the cell cycle inhibitor p21. Furthermore, loss of GAS2L3 results in premature binucleation of cardiomyocytes accompanied by unresolved midbody structures. Together these results suggest that GAS2L3 plays a specific role in cardiomyocyte cytokinesis and proliferation during heart development.

Morphological and Developmental Traits of the Binucleation of Male Accessory Gland Cells in the Benthic Water Bug, Aphelocheirus vittatus (Hemiptera: Aphelochiridae).

The male accessory glands (MAGs) in insects are pair(s) of internal reproductive organs that produce and secrete the plasma component of seminal fluid. In various insects, MAG size is important for male reproductive success because the fluid provides physiologically active substances and/or nutrients to females to control sperm as well as female reproductive behaviors. Although the MAG epithelial cells in most insect species are standard mononucleate cells, those in some insect taxa are binucleate due to incomplete cytokinesis (e.g., Drosophila [Fallén] [Diptera: Drosophilidae]) or cell fusion (e.g., Cimex [Linnaeus] [Hemiptera: Cimicidae]). In the case of Drosophila, the apicobasal position of the two nuclei relative to the epithelial plane changes from vertical to horizontal after nutrient intake, which allows the volume of the MAG cavity to expand effectively. On the other hand, in the case of Cimex, the positions of the two nuclei do not change apicobasally in response to feeding, but their position relative to the proximodistal axis varies depending on the tubular/spherical organ morphology. Here, we report that the MAG of the benthic water bug Aphelocheirus vittatus (Matsumura) (Hemiptera: Aphelochiridae) shows binucleation in all epithelial cells. Despite the phylogenetically close relationship between Aphelocheirus and Cimex, the MAG cells in Aphelocheirus showed a Drosophila-like apicobasal change in the position of the two nuclei in response to feeding. Furthermore, the cytological processes during binucleation are more similar to those in Drosophila (incomplete cytokinesis) than to those in Cimex (cell fusion). These results indicate that the physiological role and mechanism of binucleation in MAG cells changed during the evolution of Hemiptera.

Advances in heart regeneration based on cardiomyocyte proliferation and regenerative potential of binucleated cardiomyocytes and polyploidization.

One great achievement in medical practice is the reduction in acute mortality of myocardial infarction due to identifying risk factors, antiplatelet therapy, optimized hospitalization and acute percutaneous coronary intervention. Yet, the prevalence of heart failure is increasing presenting a major socio-economic burden. Thus, there is a great need for novel therapies that can reverse damage inflicted to the heart. In recent years, data have accumulated suggesting that induction of cardiomyocyte proliferation might be a future option for cardiac regeneration. Here, we review the relevant literature since September 2015 concluding that it remains a challenge to verify that a therapy induces indeed cardiomyocyte proliferation. Most importantly, it is unclear that the detected increase in cardiomyocyte cell cycle activity is required for an associated improved function. In addition, we review the literature regarding the evidence that binucleated and polyploid mononucleated cardiomyocytes can divide, and put this in context to other cell types. Our analysis shows that there is significant evidence that binucleated cardiomyocytes can divide. Yet, it remains elusive whether also polyploid mononucleated cardiomyocytes can divide, how efficient proliferation of binucleated cardiomyocytes can be induced, what mechanism regulates cell cycle progression in these cells, and what fate and physiological properties the daughter cells have. In summary, we propose to standardize and independently validate cardiac regeneration studies, encourage the field to study the proliferative potential of binucleated and polyploid mononucleated cardiomyocytes, and to determine whether induction of polyploidization can enhance cardiac function post-injury.

Pregnancy and perinatal outcomes after transfer of binucleated or multinucleated frozen-thawed embryos: a case-control study.

Blastomere multinucleation in human embryos is a common phenomenon, but data on its effect on pregnancy outcome and the health of newborns are scarce. In this case-control study, we assessed pregnancy and perinatal outcomes from 136 binucleated and multinucleated frozen-thawed embryo transfer cycles against a control group of 136 non-binucleated and multinucleated frozen embryo transfer cycles. Clinical pregnancy and live birth rates were lower among the case group (29.4% versus 44.1%, P = 0.012; 22.1% versus 36.0%, P = 0.011, respectively), but perinatal outcomes (gestational week at delivery, birth weight, placental weight and occurrence of congenital anomalies) were similar. Live birth rates among patients receiving embryos with multinucleation compared with binucleation was not significantly different (24.7% versus 13.2%). Consequently, frozen-thawed cleavage-stage embryos with bi- or multinucleation have lower than normal but still acceptable implantation potential and ability to produce healthy pregnancies and newborns. The study is limited by its retrospective nature. Time-lapse monitoring would be a more sensitive method of detecting multinucleation. Controls and cases were matched only by age at the time of oocyte retrieval, and other characteristics were only interpreted statistically. Although larger than previously reported, the number of cases is limited.

Dynamic changes in the cardiac methylome during postnatal development.

Relatively little is known about the epigenetic control mechanisms that guide postnatal organ maturation. The goal of this study was to determine whether DNA methylation plays an important role in guiding transcriptional changes during the first 2 wk of mouse heart development, which is an important period for cardiomyocyte maturation, loss of proliferative capacity and loss of regenerative potential. Gene expression profiling (RNA-seq) and genome-wide sequencing of methylated DNA (MBD-seq) identified dynamic changes in the cardiac methylome during postnatal development [2545 differentially methylated regions (DMRs) from P1 to P14 in the mouse]. The vast majority (~80%) of DMRs were hypermethylated between P1 and P14, and these hypermethylated regions were associated with transcriptional shut down of important developmental signaling pathways, including Hedgehog, bone morphogenetic protein, TGF-ß, fibroblast growth factor, and Wnt/ß-catenin signaling. Postnatal inhibition of DNA methylation with 5-aza-2'-deoxycytidine induced a marked increase (~3-fold) in cardiomyocyte proliferation and ~50% reduction in the percentage of binucleated cardiomyocytes compared with saline-treated controls. This study provides novel evidence for widespread alterations in DNA methylation during postnatal heart maturation and suggests that cardiomyocyte cell cycle arrest during the neonatal period is subject to regulation by DNA methylation.

Regulation of cytokinesis and its clinical significance.

Dysregulation of the cell cycle leads to polyploid cells, which are classified into mononuclear or binuclear polyploid cells depending on the number of nuclei. Polyploidy is common in plants and in animals. Physiologically, polyploidy and binucleation are differentiation markers and also features of the aging process. In fact, although they provide multiple copies of genes required for survival, a negative correlation between growth capacity and polyploidy has been reported, and thus, suppression or reversal of this phenomenon may be a growth advantage. On the other hand, unscheduled polyploidization may cause genomic instability that might lead to neoplastic aneuploidy. The aim of this review is to analyze the mechanisms that lead to polyploidy, and particularly binucleation, and highlight the potential of ploidy as a marker of illness severity or the success of the adaptive response for an injury, with special emphasis in the liver under physiological and pathological conditions. Hepatocyte binucleation occurs in late fetal development and postnatal maturation, especially after weaning via phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt). It also increases upon aging of the liver as well as in liver cirrhosis and cancer. Liver binucleation mainly indicates the severity of the damage. Furthermore, the eventual increase in hepatocyte binucleation points out compensatory proliferation associated with liver injury. Ploidy conveyor would also permit hepatocyte adaptation to xenobiotic or nutritional injury. In contrast, polyploidy is a feature of many human cancers, and it may predispose to genomic instability and generation of aneuploidization that play a major role in carcinogenesis. Finally, a better understanding of the polyploidization process is needed in order to approach clinical research but also, to get deeper knowledge of cell cycle control. The fascinating regulation of cell cycle in liver and the generation and reversal of ploidies will provide more clues for the mystery of liver regeneration.

Cardiomyocyte cytokinesis score: a potential method for cardiomyocyte proliferation.

One of the most important indicators of myocardial regeneration is cardiomyocyte proliferation. However, it is difficult to distinguish cardiomyocytes in the regenerating stage from binucleated or multinucleated myocytes by conventional morphometric techniques. As cell cycle progression (CCP) scores have been successfully applied to the evaluation of the proliferation of cancer cells, we sought to establish a multi-gene score to evaluate cardiomyocyte proliferation in this study. Given the disturbances of nuclear division without cell division that occurs in cardiomyocytes, ten cytokinesis-correlated genes (Anln, Aurkb, Cenpa, Kif4, Kif23, Prc1, RhoA, Spin1, TACC2, and CDC42) were chosen to establish the cardiomyocyte cytokinesis score (CC-Score). The expression levels of these genes in H9C2 rat cardiomyoblast cells, the proliferation of which were stimulated or inhibited, were detected using qRT-PCR. To confirm the feasibility of the CC-Score system, four conventional methods for evaluating cardiomyocyte proliferation, including the MTT assay, BrdU assay, immunofluorescence, and flow cytometry analysis, were used in each group. The results of the CC-Score in the assessment of the proliferation of H9C2 cells were consistent with those of four commonly used proliferative assay methods. We conclude that the CC-Score can be used to assess the proliferation status of H9C2 cells, and suggest that the CC-Score may be a potential method for the assessment of cardiomyocyte proliferation in myocardial regeneration. However, validation studies utilizing primary cultured rat cardiomyocytes and heart tissue are warranted.

Cardiomyocyte Ploidy, Metabolic Reprogramming and Heart Repair.

The adult heart is made up of cardiomyocytes (CMs) that maintain pump function but are unable to divide and form new myocytes in response to myocardial injury. In contrast, the developmental cardiac tissue is made up of proliferative CMs that regenerate injured myocardium. In mammals, CMs during development are diploid and mononucleated. In response to cardiac maturation, CMs undergo polyploidization and binucleation associated with CM functional changes. The transition from mononucleation to binucleation coincides with unique metabolic changes and shift in energy generation. Recent studies provide evidence that metabolic reprogramming promotes CM cell cycle reentry and changes in ploidy and nucleation state in the heart that together enhances cardiac structure and function after injury. This review summarizes current literature regarding changes in CM ploidy and nucleation during development, maturation and in response to cardiac injury. Importantly, how metabolism affects CM fate transition between mononucleation and binucleation and its impact on cell cycle progression, proliferation and ability to regenerate the heart will be discussed.

RBPMS is an RNA-binding protein that mediates cardiomyocyte binucleation and cardiovascular development.

Noncompaction cardiomyopathy is a common congenital cardiac disorder associated with abnormal ventricular cardiomyocyte trabeculation and impaired pump function. The genetic basis and underlying mechanisms of this disorder remain elusive. We show that the genetic deletion of RNA-binding protein with multiple splicing (Rbpms), an uncharacterized RNA-binding factor, causes perinatal lethality in mice due to congenital cardiovascular defects. The loss of Rbpms causes premature onset of cardiomyocyte binucleation and cell cycle arrest during development. Human iPSC-derived cardiomyocytes with RBPMS gene deletion have a similar blockade to cytokinesis. Sequencing analysis revealed that RBPMS plays a role in RNA splicing and influences RNAs involved in cytoskeletal signaling pathways. We found that RBPMS mediates the isoform switching of the heart-enriched LIM domain protein Pdlim5. The loss of Rbpms leads to an abnormal accumulation of Pdlim5-short isoforms, disrupting cardiomyocyte cytokinesis. Our findings connect premature cardiomyocyte binucleation to noncompaction cardiomyopathy and highlight the role of RBPMS in this process.

Binucleated embryos at the two-cell stage show higher blastocyst formation rates and higher pregnancy and live birth rates compared to non-multinucleated embryos.

STUDY QUESTION: How does nucleus status at the two-cell stage predict blastocysts formation and clinical outcome after single blastocyst transfer? SUMMARY ANSWER: Binucleated embryos at the two-cell stage (2BI) show higher rates of good quality blastocyst formation, pregnancy and live birth compared to those with one nucleus in each blastomere (2MONO), whereas true multinucleated embryos at the two-cell stage (2MULTI) show lower rates of good quality blastocyst formation and pregnancy compared to 2MONO embryos. WHAT IS KNOWN ALREADY: The introduction of time-lapse culture has made it possible to study nucleus status at the two-cell stage more consistently and it shows that multinucleation at the two-cell stage (2MN) is a common event. The effect of 2MN is still unclear. High numbers of 2MN with the potential to develop to blastocysts that become clinical pregnancies and result in birth of healthy babies with no impaired perinatal outcome have been reported. However, some studies have found 2MN to be associated with impaired implantation and live birth. Furthermore, knowledge on how the different subgroups of multinucleation affects the IVF outcome is limited. STUDY DESIGN SIZE DURATION: A non-interventional retrospective study was performed in a public fertility clinic. Blastocyst formation data from 223 women attending their first IVF cycle between May 2016 and December 2018, and clinical outcome data from 1314 single blastocyst transfers between May 2014 and December 2018 were used for the study. Fresh and frozen-thawed embryo transfers were included. PARTICIPANTS/MATERIALS SETTING METHODS: Embryos were cultured until the blastocyst stage in a time-lapse incubator and nucleus status at the two-cell stage, the Gardner score and other morphokinetic parameters were annotated. We compared blastocyst development and clinical outcome, including positive hCG, ongoing pregnancy and live birth, of embryos with 2BI and/or 2MULTI blastomeres to 2MONO embryos. MAIN RESULTS AND THE ROLE OF CHANCE: Embryos with 2BI in one blastomere (2BI1) were twice as likely to develop to good quality blastocysts (odds ratio (OR) 2.54, 95% CI 1.30-4.95, P = 0.006) compared to 2MONO embryos. Embryos with 2MULTI in both blastomeres (2MULTI2) were significantly less able to develop to good quality blastocysts (OR 0.38, 95% CI 0.23-0.63, P < 0.001) compared to 2MONO embryos. Embryos with 2BI in both blastomeres (2BI2) had a significantly better chance of resulting in a positive hCG (OR 2.40, 95% CI 1.11-5.20, P = 0.027), ongoing pregnancy (OR 2.79, 95% CI 1.29-6.04, P = 0.009) and live birth (OR 3.16, 95% CI 1.43-6.95, P = 0.004) compared to 2MONO blastocysts after single blastocyst transfer. In contrast, 2MULTI2 embryos were significantly less likely to result in a positive hCG (OR 0.58, 95% CI 0.35-0.97, P = 0.036) and ongoing pregnancy (OR 0.51, 95% CI 0.28-0.94, P = 0.030) compared to 2MONO blastocysts. LIMITATIONS REASONS FOR CAUTION: Discrepancies among the existing studies regarding the definition of multinucleation may lead to different conclusions. Even though the distinction between binucleation and true multinucleation was a strength in our study design, a further distinction between true multinucleated and micronucleated embryos could be interesting to investigate in future studies. Also, we included any anucleated embryos in the 2MONO group. For the study of clinical outcomes, the patients were allowed to be included with more than one transfer cycle. Both fresh and thawed transfers were included. WIDER IMPLICATIONS OF THE FINDINGS: We find it important to discriminate between binucleation and true multinucleation when evaluating embryo nucleus status at the two-cell stage. Embryos displaying 2BI1 and 2BI2 have significantly better good quality blastocyst formation rates and clinical outcome after single blastocyst transfers, respectively. 2MULTI2 embryos have impaired blastocyst development potential and poorer clinical outcomes. STUDY FUNDING/COMPETING INTERESTS: H.S.N. received an unrestricted grant from Merck for 3 months' normal salary for a medical Doctor (A.L.T.) to write the manuscript. Merck was not involved in the study design, analysis, interpretation of data, writing the paper or the decision to submit the manuscript for publication. H.S.N. has received speaker's fees from Ferring Pharmaceuticals, Merck Denmark A/S, Astra Zeneca, Cook Medical and Ibsa Nordic (outside the submitted work). N.l.C.F. has received a grant from Gedeon Richter (outside the submitted work). The other authors did not report any potential conflicts of interest. All authors declared no conflicts of interest regarding this work. TRIAL REGISTRATION NUMBER: N/A.

Sarcomere Disassembly and Transfection Efficiency in Proliferating Human iPSC-Derived Cardiomyocytes.

Contractility of the adult heart relates to the architectural degree of sarcomeres in individual cardiomyocytes (CMs) and appears to be inversely correlated with the ability to regenerate. In this study we utilized multiple imaging techniques to follow the sequence of sarcomere disassembly during mitosis resulting in cellular or nuclear division in a source of proliferating human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We observed that both mono- and binuclear hiPSC-CMs give rise to mononuclear daughter cells or binuclear progeny. Within this source of highly proliferative hiPSC-CMs, treated with the CHIR99021 small molecule, we found that Wnt and Hippo signaling was more present when compared to metabolic matured non-proliferative hiPSC-CMs and adult human heart tissue. Furthermore, we found that CHIR99021 increased the efficiency of non-viral vector incorporation in high-proliferative hiPSC-CMs, in which fluorescent transgene expression became present after the chromosomal segregation (M phase). This study provides a tool for gene manipulation studies in hiPSC-CMs and engineered cardiac tissue. Moreover, our data illustrate that there is a complex biology behind the cellular and nuclear division of mono- and binuclear CMs, with a shared-phenomenon of sarcomere disassembly during mitosis.

Cellular and Biochemical Changes in Different Categories of Periodontitis: A Patient-based Study.

OBJECTIVES: The aim of this study was to study the effects of periodontitis, diabetes mellitus (DM), and tobacco smoking and chewing habits (TBSCH) on the oxidative stress biomarker levels, namely malondialdehyde (MDA), and the mucosal genotoxic nuclear damage in the marginal gingival cells of subjects. Furthermore, the correlation of the biomarkers, MDA, and nuclear changes in the form of micronucleation (Mn) and binucleation (Bn) was investigated. MATERIALS AND METHODS: Forty study participants were divided into five subject categories, which were established based on the presence of periodontitis, DM, and TBSCH. Whole saliva and marginal gingival smears collected from subjects were used to determine MDA levels and nuclear changes, respectively. A full-mouth assessment of periodontal pocket depth, clinical attachment loss, and bleeding on probing was performed for each subject to determine periodontal status. RESULTS: MDA and Mn levels between control group and subjects with only periodontitis (MDA: P < 0.9990; Mn: P < 0.8200) showed no significant difference, whereas levels among subjects with DM, TBSCH, and periodontitis, and all other categories were statistically significant (MDA: P < 0.001). DM and/or TBSCH superimposed on periodontitis cause an exponential increase in biomarker levels. Furthermore, MDA and Mn showed poor correlation (r = 0.162; P = 0.318). Periodontitis alone did not significantly increase oxidative stress levels compared to healthy controls, whereas DM and TBSCH resulted in augmented oxidative stress levels, implying that increased stress produced by DM and TBSCH aggravates or exaggerates periodontal inflammation. CONCLUSION: Poor correlation between MDA and Mn indicated that the mechanisms involved in their production are independent of each other.

Aqueous extract of bulbus Fritillaria cirrhosa induces cytokinesis failure by blocking furrow ingression in human colon epithelial NCM460 cells.

Bulbus Fritillariacirrhosa D. Don (BFC) has been widely used as an herbal medicament for respiratory diseases in China for over 2000 years. The ethnomedicinal effects of BFC have been scientifically verified, nevertheless its toxicity has not been completely studied. Previously, we have reported that the aqueous extract of BFC induces mitotic aberrations and chromosomal instability (CIN) in human colon epithelial NCM460 cells via dysfunctioning the mitotic checkpoint. Here, we extend this study and specifically focus on the influence of BFC on cytokinesis, the final step of cell division. One remarkable change in NCM460 cells following BFC treatment is the high incidence of binucleated cells (BNCs). More detailed investigation of the ana-telophases reveals that furrow ingression, the first stage of cytokinesis, is inhibited by BFC. Asynchronous cultures treatment demonstrates that furrow ingression defects induced by BFCs are highly associated with the formation of BNCs in ensuing interphase, indicating the BNCs phenotype after BFC treatment was resulted from cytokinesis failure. In line with this, the expression of genes involved in the regulation of furrow ingression is significantly de-regulated by BFC (e.g., LATS-1/2 and Aurora-B are upregulated, and YB-1 is downregulated). Furthermore, long-term treatment of BFC elucidates that the BNCs phenotype is transient and the loss of BNCs is associated with increased frequency of micronuclei and nuclear buds, two biomarkers of CIN. In supporting of these findings, the Nin Jiom Pei Pa Koa and Chuanbei Pipa Gao, two commercially available Chinese traditional medicines containing BFC, are able to induce multinucleation and CIN in NCM460 cells. Altogether, these data provide the first in vitro experimental evidence linking BFC to cytokinesis failure and suggest the resultant BNCs may be intermediates to produce CIN progenies.

Tugging at the Heart Strings: The Septin Cytoskeleton in Heart Development and Disease.

Septin genes were originally identified in budding yeast in 1971. Since their original discovery, at least 13 mammalian genes have now been found, which give rise to a vast array of alternatively spliced proteins that display unique spatial-temporal function across organs systems. Septin's are now recognized as the 4th major component of the cytoskeleton. Their role in regulating ciliogenesis, actin and microtubule organization and their involvement in mechanotransduction clearly solidify their place as both a responder and driver of cellular activity. Although work on septin's has escalated over the past decades, knowledge of septin function in the heart remains rudimentary. Whereas many cardiovascular diseases have been associated with genetic loci that include septin genes, new and additional concerted efforts will likely uncover previously unrecognized mechanisms by which the septin class of proteins contribute to clinical cardiac phenotypes. In this review, we place known function of septin proteins in the context of heart development and disease and provide perspectives on how increased knowledge of these proteins can mechanistically inform cardiac pathologies.

Precise Tuning of Cortical Contractility Regulates Cell Shape during Cytokinesis.

The mechanical properties of the actin cortex regulate shape changes during cell division, cell migration, and tissue morphogenesis. We show that modulation of myosin II (MII) filament composition allows tuning of surface tension at the cortex to maintain cell shape during cytokinesis. Our results reveal that MIIA generates cortex tension, while MIIB acts as a stabilizing motor and its inclusion in MII hetero-filaments reduces cortex tension. Tension generation by MIIA drives faster cleavage furrow ingression and bleb formation. We also show distinct roles for the motor and tail domains of MIIB in maintaining cytokinetic fidelity. Maintenance of cortical stability by the motor domain of MIIB safeguards against shape instability-induced chromosome missegregation, while its tail domain mediates cortical localization at the terminal stages of cytokinesis to mediate cell abscission. Because most non-muscle contractile systems are cortical, this tuning mechanism will likely be applicable to numerous processes driven by myosin-II contractility.

Septin and Ras regulate cytokinetic abscission in detached cells.

BACKGROUND: Integrin-mediated adhesion is normally required for cytokinetic abscission, and failure in the process can generate potentially oncogenic tetraploid cells. Here, detachment-induced formation of oncogenic tetraploid cells was analyzed in non-transformed human BJ fibroblasts and BJ expressing SV40LT (BJ-LT) ± overactive HRas. RESULTS: In contrast to BJ and BJ-LT cells, non-adherent BJ-LT-Ras cells recruited ALIX and CHMP4B to the midbody and divided. In detached BJ and BJ-LT cells regression of the cytokinetic furrow was suppressed by intercellular bridge-associated septin; after re-adhesion these cells divided by cytofission, however, some cells became bi-nucleated because of septin reorganization and furrow regression. Adherent bi-nucleated BJ cells became senescent in G1 with p21 accumulation in the nucleus, apparently due to p53 activation since adherent bi-nucleated BJ-LT cells passed through next cell cycle and divided into mono-nucleated tetraploids; the two centrosomes present in bi-nucleated BJ cells fused after furrow regression, pointing to the PIDDosome pathway as a possible mechanism for the p53 activation. CONCLUSIONS: Several mechanisms prevent detached normal cells from generating tumor-causing tetraploid cells unless they have a suppressed p53 response by viruses, mutation or inflammation. Importantly, activating Ras mutations promote colony growth of detached transformed cells by inducing anchorage-independent cytokinetic abscission in single cells.