Binucleated Cardiomyocytes Research Articles

Quantitative label-free digital holographic imaging of cardiomyocyte optical volume, nucleation, and cell division

Cardiac regeneration in newborn rodents depends on the ability of pre-existing cardiomyocytes to proliferate and divide. This capacity is lost within the first week of postnatal development when these cells rapidly switch from hyperplasia to hypertrophy, withdraw from the cell cycle, become binucleated, and increase in size. How these dynamic changes in cell size and nucleation impact cardiomyocyte proliferative potential is not well understood. In this study, we innovate the application of a commercially available digital holographic imaging microscope, the Holomonitor M4, to evaluate the proliferative responses of mononucleated and binucleated cardiomyocytes after CHIR99021 treatment, a model proliferative stimulus. This system enables long-term label-free quantitative tracking of primary cardiomyocyte dynamics in real-time with single-cell resolution. Our results confirm that chemical inhibition of glycogen synthase kinase 3 with CHIR99021 promotes complete cell division of both mononucleated and binucleated cardiomyocytes with high frequency. Quantitative tracking of cardiomyocyte volume dynamics during these proliferative events revealed that both mononucleated and binucleated cardiomyocytes reach a similar size-increase threshold prior to attempted cell division. Binucleated cardiomyocytes attempt to divide with lower frequency than mononucleated cardiomyocytes, which may be associated with inadequate increases in cell size. By defining the interrelationship between cardiomyocyte size, nucleation, and cell cycle control, we may better understand the cellular mechanisms that drive the loss of mammalian cardiac regenerative capacity after birth.

Mitochondrial respiration is lower in the intrauterine growth-restricted fetal sheep heart.

Fetuses affected by intrauterine growth restriction have an increased risk of developing heart disease and failure in adulthood. Compared with controls, late gestation intrauterine growth-restricted (IUGR) fetal sheep have fewer binucleated cardiomyocytes, reflecting a more immature heart, which may reduce mitochondrial capacity to oxidize substrates. We hypothesized that the late gestation IUGR fetal heart has a lower capacity for mitochondrial oxidative phosphorylation. Left (LV) and right (RV) ventricles from IUGR and control (CON) fetal sheep at 90% gestation were harvested. Mitochondrial respiration (states 1-3, LeakOmy, and maximal respiration) in response to carbohydrates and lipids, citrate synthase (CS) activity, protein expression levels of mitochondrial oxidative phosphorylation complexes (CI-CV), and mRNA expression levels of mitochondrial biosynthesis regulators were measured. The carbohydrate and lipid state 3 respiration rates were lower in IUGR than CON, and CS activity was lower in IUGR LV than CON LV. However, relative CII and CV protein levels were higher in IUGR than CON; CV expression level was higher in IUGR than CON. Genes involved in lipid metabolism had lower expression in IUGR than CON. In addition, the LV and RV demonstrated distinct differences in oxygen flux and gene expression levels, which were independent from CON and IUGR status. Low mitochondrial respiration and CS activity in the IUGR heart compared with CON are consistent with delayed cardiomyocyte maturation, and CII and CV protein expression levels may be upregulated to support ATP production. These insights will provide a better understanding of fetal heart development in an adverse in utero environment. KEY POINTS: Growth-restricted fetuses have a higher risk of developing and dying from cardiovascular diseases in adulthood. Mitochondria are the main supplier of energy for the heart. As the heart matures, the substrate preference of the mitochondria switches from carbohydrates to lipids. We used a sheep model of intrauterine growth restriction to study the capacity of the mitochondria in the heart to produce energy using either carbohydrate or lipid substrates by measuring how much oxygen was consumed. Our data show that the mitochondria respiration levels in the growth-restricted fetal heart were lower than in the normally growing fetuses, and the expression levels of genes involved in lipid metabolism were also lower. Differences between the right and left ventricles that are independent of the fetal growth restriction condition were identified. These results indicate an impaired metabolic maturation of the growth-restricted fetal heart associated with a decreased capacity to oxidize lipids postnatally.

Three-dimensional co-culturing of stem cell-derived cardiomyocytes and cardiac fibroblasts reveals a role for both cell types in Marfan-related cardiomyopathy

Pathogenic variants in the FBN1 gene, which encodes the extracellular matrix protein fibrillin-1, cause Marfan syndrome (MFS), which affects multiple organ systems, including the cardiovascular system. Myocardial dysfunction has been observed in a subset of patients with MFS and in several MFS mouse models. However, there is limited understanding of the intrinsic consequences of FBN1 variants on cardiomyocytes (CMs). To elucidate the CM-specific contribution in Marfan's cardiomyopathy, cardiosphere cultures of CMs and cardiac fibroblasts (CFs) are used. CMs and CFs were derived by human induced pluripotent stem cell (iPSC) differentiation from MFS iPSCs with a pathogenic variant in FBN1 (c.3725G>A; p.Cys1242Tyr) and the corresponding CRISPR-corrected iPSC line (Cor).Cardiospheres containing MFS CMs show decreased FBN1, COL1A2 and GJA1 expression. MFS CMs cultured in cardiospheres have fewer binucleated CMs in comparison with Cor CMs. 13% of MFS CMs in cardiospheres are binucleated and 15% and 16% in cardiospheres that contain co-cultures with respectively MFS CFs and Cor CFs, compared to Cor CMs, that revealed up to 23% binucleation when co-cultured with CFs. The sarcomere length of CMs, as a marker of development, is significantly increased in MFS CMs interacting with Cor CF or MFS CF, as compared to monocultured MFS CMs. Nuclear blebbing was significantly more frequent in MFS CFs, which correlated with increased stiffness of the nuclear area compared to Cor CFs.Our cardiosphere model for Marfan-related cardiomyopathy identified a contribution of CFs in Marfan-related cardiomyopathy and suggests that abnormal early development of CMs may play a role in the disease mechanism.

The orchestration of cell-cycle reentry and ribosome biogenesis network is critical for cardiac repair.

Rationale: Myocardial infarction (MI) is a severe global clinical condition with widespread prevalence. The adult mammalian heart's limited capacity to generate new cardiomyocytes (CMs) in response to injury remains a primary obstacle in developing effective therapies. Current approaches focus on inducing the proliferation of existing CMs through cell-cycle reentry. However, this method primarily elevates cyclin dependent kinase 6 (CDK6) and DNA content, lacking proper cytokinesis and resulting in the formation of dysfunctional binucleated CMs. Cytokinesis is dependent on ribosome biogenesis (Ribo-bio), a crucial process modulated by nucleolin (Ncl). Our objective was to identify a novel approach that promotes both DNA synthesis and cytokinesis. Methods: Various techniques, including RNA/protein-sequencing analysis, Ribo-Halo, Ribo-disome, flow cytometry, and cardiac-specific tumor-suppressor retinoblastoma-1 (Rb1) knockout mice, were employed to assess the series signaling of proliferation/cell-cycle reentry and Ribo-bio/cytokinesis. Echocardiography, confocal imaging, and histology were utilized to evaluate cardiac function. Results: Analysis revealed significantly elevated levels of Rb1, bur decreased levels of circASXL1 in the hearts of MI mice compared to control mice. Deletion of Rb1 induces solely cell-cycle reentry, while augmenting the Ribo-bio modulator Ncl leads to cytokinesis. Mechanically, bioinformatics and the loss/gain studies uncovered that circASXL1/CDK6/Rb1 regulates cell-cycle reentry. Moreover, Ribo-Halo, Ribo-disome and circRNA pull-down assays demonstrated that circASXL1 promotes cytokinesis through Ncl/Ribo-bio. Importantly, exosomes derived from umbilical cord mesenchymal stem cells (UMSC-Exo) had the ability to enhance cardiac function by facilitating the coordinated signaling of cell-cycle reentry and Ribo-bio/cytokinesis. These effects were attenuated by silencing circASXL1 in UMSC-Exo. Conclusion: The series signaling of circASXL1/CDK6/Rb1/cell-cycle reentry and circASXL1/Ncl/Ribo-bio/cytokinesis plays a crucial role in cardiac repair. UMSC-Exo effectively repairs infarcted myocardium by stimulating CM cell-cycle reentry and cytokinesis in a circASXL1-dependent manner. This study provides innovative therapeutic strategies targeting the circASXL1 signaling network for MI and offering potential avenues for enhanced cardiac repair.

Abstract P2056: A Micropeptide Encoded By An Endogenous Retroviral-derived Lncrna Regulates Cardiomyocyte Multinucleation

Endogenous retroelements (REs) are pervasive in mammalian genomes, and consist mostly of silenced mobile genetic sequences long considered to be ‘junk DNA.’ In a few known instances, mammalian genomes have captured and repurposed endogenous retroviral sequences to play important roles in regulating mammalian cell functions, notably cell fusion. Our previous work has shown that some mammalian transcripts misannotated as lncRNAs can be translated into small functional peptides, called micropeptides. Here we report the discovery of a cardiac-specific micropeptide encoded by a misannotated lncRNA transcribed from a murine endogenous retroviral element. We named this transcript CARDI, and the encoded micropeptide CARDI-B, since translation of the micropeptide occurs from the second ATG start codon. We show that CARDI-B shares homology with a singular domain of a retroviral protein and localizes to the nucleus. Genetic deletion of CARDI-B in mice from an isogenic background significantly increased the frequency of binucleated cardiomyocytes, at the expense of mononucleated cardiomyocytes. In line with previous reports that the frequency of mononucleated cardiomyocytes is correlated with cardiac regeneration, we show that CARDI-B-deficient hearts perform worse following myocardial infarction injury. These data reveal that functional micropeptides can be concealed within mammalian retroelements, and suggests that RE-derived micropeptides may contribute to the natural variation in heart development and regeneration observed across species.

Use of stable isotope-tagged thymidine and multi-isotope imaging mass spectrometry (MIMS) for quantification of human cardiomyocyte division.

Quantification of cellular proliferation in humans is important for understanding biology and responses to injury and disease. However, existing methods require administration of tracers that cannot be ethically administered in humans. We present a protocol for the direct quantification of cellular proliferation in human hearts. The protocol involves administration of non-radioactive, non-toxic stable isotope 15Nitrogen-enriched thymidine (15N-thymidine), which is incorporated into DNA during S-phase, in infants with tetralogy of Fallot, a common form of congenital heart disease. Infants with tetralogy of Fallot undergo surgical repair, which requires the removal of pieces of myocardium that would otherwise be discarded. This protocol allows for the quantification of cardiomyocyte proliferation in this discarded tissue. We quantitatively analyzed the incorporation of 15N-thymidine with multi-isotope imaging spectrometry (MIMS) at a sub-nuclear resolution, which we combined with correlative confocal microscopy to quantify formation of binucleated cardiomyocytes and cardiomyocytes with polyploid nuclei. The entire protocol spans 3-8 months, which is dependent on the timing of surgical repair, and 3-4.5 researcher days. This protocol could be adapted to study cellular proliferation in a variety of human tissues.

Proximity to injury, but neither number of nuclei nor ploidy define pathological adaptation and plasticity in cardiomyocytes

The adult mammalian heart consists of mononuclear and binuclear cardiomyocytes (CMs) with various ploidies. However, it remains unclear whether a variation in ploidy or number of nuclei is associated with distinct functions and injury responses in CMs, including regeneration. Therefore, we investigated transcriptomes and cellular as well as nuclear features of mononucleated and binucleated CMs in adult mouse hearts with and without injury. To be able to identify the role of ploidy we analyzed control and failing human ventricular CMs because human CMs show a larger and disease-sensitive degree of polyploidization.Using transgenic Myh6-H2BmCh to identify mononucleated and binucleated mouse CMs, we found that cellular volume and RNA content were similar in both. On average nuclei of mononuclear CMs showed a 2-fold higher ploidy, as compared to binuclear CMs indicating that most mononuclear CMs are tetraploid. After myocardial infarction mononucleated and binucleated CMs in the border zone of the lesion responded with hypertrophy and corresponding changes in gene expression, as well as a low level of induction of cell cycle gene expression. Human CMs allowed us to study a wide range of polyploidy spanning from 2n to 16n. Notably, basal as well as pathological gene expression signatures and programs in failing CMs proved to be independent of ploidy.In summary, gene expression profiles were induced in proximity to injury, but independent of number of nuclei or ploidy levels in CMs.

Placenta-targeted treatment in hypoxic dams improves maturation and growth of fetal cardiomyocytes in vitro via the release of placental factors.

What is the central question of this study? Does treatment of hypoxic dams with a placenta-targeted antioxidant prevent the release of placenta-derived factors that impair maturation or growth of fetal cardiomyocytes in vitro? What is the main finding and its importance? Factors released from hypoxic placentae impaired fetal cardiomyocyte maturation (induced terminal differentiation) and growth (increased cell size) in vitro, which was prevented by maternal treatment with a placenta-targeted antioxidant (nMitoQ). Moreover, there were no sex differences in the effects of placental factors on fetal cardiomyocyte maturation and growth. Overall, our data suggest that treatment targeted against placental oxidative stress could prevent fetal programming of cardiac diseases via the release of placental factors. Pregnancy complications associated with placental oxidative stress may impair fetal organ development through the release of placenta-derived factors into the fetal circulation. We assessed the effect of factors secreted from placentae previously exposed to prenatal hypoxia on fetal cardiomyocyte development and developed a treatment strategy that targets placental oxidative stress by encapsulating the antioxidant MitoQ into nanoparticles (nMitoQ). We used a rat model of prenatal hypoxia (gestational day (GD) 15-21), which was treated with saline or nMitoQ on GD15. On GD21, placentae were harvested, placed in culture, and conditioned medium (containing placenta-derived factors) was collected after 24h. This conditioned medium was then added to cultured cardiomyocytes from control dam fetuses. Conditioned medium from prenatally hypoxic placentae increased the percentage of binucleated cardiomyocytes (marker of terminal differentiation) and the size of mononucleated and binucleated cardiomyocytes (sign of hypertrophy), effects that were prevented by nMitoQ treatment. Our data suggest that factors derived from placentae previously exposed to prenatal hypoxia lead to abnormal fetal cardiomyocyte development, and show that treatment against placental oxidative stress may prevent fetal programming of cardiac disease.

Direct Comparison of Mononucleated and Binucleated Cardiomyocytes Reveals Molecular Mechanisms Underlying Distinct Proliferative Competencies

SUMMARYThe mammalian heart is incapable of regenerating a sufficient number of cardiomyocytes to ameliorate the loss of contractile muscle after acute myocardial injury. Several reports have demonstrated that mononucleated cardiomyocytes are more responsive than are binucleated cardiomyocytes to proproliferative stimuli. We have developed a strategy to isolate and characterize highly enriched populations of mononucleated and binucleated cardiomyocytes at various times of development. Our results suggest that an E2f/Rb transcriptional network is central to the divergence of these two populations and that remnants of the differences acquired during the neonatal period remain in adult cardiomyocytes. Moreover, inducing binucleation by genetically blocking the ability of cardiomyocytes to complete cytokinesis leads to a reduction in E2f target gene expression, directly linking the E2f pathway with nucleation. These data identify key molecular differences between mononucleated and binucleated mammalian cardiomyocytes that can be used to leverage cardiomyocyte proliferation for promoting injury repair in the heart.

Abstract 132: Imaging-Based Assay for Screening of Cell Cycle Modifying Substances in Postnatal Cardiomyocytes

Cardiovascular diseases are the main cause of death in the industrial world. Especially large myocardial infarctions and the potentially resulting left ventricular dysfunction are chronical diseases with a poor prognosis. Due to limited endogenous cardiac repair capacity, we are investigating the regulation of the cell cycle in cardiomyocytes (CMs) as a potential treatment strategy. We hypothesize that controlled induction of cardiomyocyte proliferation will lead to an increase in muscle mass and improvement of cardiac function. We are using CMs from double transgenic αMHC-H2B-mCherry/CAG-eGFP-anillin mice for the analysis of postnatal CM development. This double transgenic system enables us to unequivocally identify CM nuclei as well as cell cycle variations like acytokinetic mitosis (mitosis without cytokinesis), resulting in binucleated CMs, and endoreplication (mitosis without cytokinesis and karyokinesis), leading to polyploid CMs. With the goal of inducing authentic cell division, we are screening for pro-proliferative substances in P1 and P6 postnatal CMs of αMHC-H2B-mCherry/CAG-eGFP-anillin mice. Here we will present the detailed analysis of several screening hits demonstrating an increase in cell cycle activity leading mainly to binucleation but only to limited cell division in CMs.

Pseudo-bipolar spindle formation and cell division in postnatal binucleated cardiomyocytes

BackgroundThe majority of adult human, mouse and rat cardiomyocytes is not diploid mononucleated. Nevertheless, the current literature on heart regeneration based on cardiomyocyte proliferation focuses mainly on the proliferation capacity of diploid mononucleated cardiomyocytes, instead of the more abundant mononucleated polyploid or binucleated cardiomyocytes. Here, we aimed at a better understanding of the process of mitosis and cell division in postnatal binucleated cardiomyocytes. Methods and resultsPostnatal rat binucleated cardiomyocytes were stimulated to re-enter the cell cycle either by fetal bovine serum or a combination of fibroblast growth factor 1 and p38 MAP kinase inhibitor. Phase-contrast videos revealed that binucleated cardiomyocytes form one metaphase plate and preferentially undergo afterwards cytokinesis failure. The maximum rate of cell division of video-recorded binucleated cardiomyocytes was around 6%. Immunofluorescence analyses of centriole number in mitotic binucleated cardiomyocytes revealed that these cells contain more than four centrioles, which can be paired as well as unpaired. In agreement with multiple and/or unpaired centrioles, multipolar spindle formation was observed in mitotic binucleated cardiomyocytes using fluorescence live imaging of tubulin-GFP. Multipoles were transient and resolved into pseudo-bipolar spindles both in case of cell division and cytokinesis failure. Notably, centrioles were in most cases unevenly distributed among daughter cells. ConclusionsOur results indicate that postnatal binucleated cardiomyocytes upon stimulation can enter mitosis, cope with their multiple and/or unpaired centrioles by forming pseudo-bipolar spindles, and divide.

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.

Does BNP modulate cardiomyocyte proliferation?

Brain natriuretic peptide (BNP) treatment protects the heart after myocardial infarction (MI). As cardiomyocytes (CMs) express BNP receptors, the aim of this study was to determine the role of BNP on CM proliferation. We studied the BNP effect on the hearts of newborn (NB) and adult infarcted mice. NB mice were injected with BNP and with BrdU two days after birth. Eleven days post-birth, the mice were sacrificed. MI was induced by ligation of the left anterior descending coronary artery. BNP was injected directly into the myocardium after surgery, then intraperitoneally every 2 days up to 10 days. BrdU was added to drinking water. Mice were sacrificed 3 and 10 days after surgery. The infarction zone (ZI) was separated from the rest of the heart (remote zone, RZ). The number of CMs expressing BrdU + (+22%, P = 0.04) or Aurora B + (+47%, P = 0.001) was increased in NB BNP-treated mice compared to the control. In addition, the total number of CMs was also increased (+21%) in BNP-treated mice. After MI, the number of BrdU + CMs and the number of Aurora B + CMs were increased in ZI, 3 days after MI in BNP-treated hearts compared to the infarcted untreated hearts (+172%, P < 0.001; +69%, P = 0.01, respectively). This seems to be also the case 10 days after MI in ZI for the BrdU + CMs (preliminary results: +194%, n = 2 mice/group). In parallel, we isolated CMs from the hearts of NB. These cells were cultured with or without BNP. After two weeks of culture with BNP, the number of Aurora B + CMs increased (+87%, P = 0.003) but not the number of binucleated CMs. In addition, among Aurora B + CMs, BNP increased the number of cells undergoing cytokinesis (+6%). Interestingly, the total number of CMs was increased in BNP-treated cells compared to control (+26%, P = 0.01). BNP treatment leads to increased number of Aurora B + CMs in vivo in NB hearts, and in adult hearts after MI. This was confirmed by in vitro studies. Whether this reflects an increase of cytokinesis remains to be established in vivo.

SIRT1 increases cardiomyocyte binucleation in the heart development.

SIRT1 regulates cell senescence. We investigated a novel role of SIRT1 in the regulation of cardiomyocyte terminal differentiation in the developing heart. Retinoic acid (RA)-induced binucleation of H9c2 cells was associated with increased SIRT1 expression. Inhibition of SIRT1 activity or expression significantly decreased RA-induced binucleation. SIRT1 expression was minimal in the fetal heart and significantly upregulated in the hearts of postnatal day 7 (P7) rat pups. In contrast, heart-specific miR-133a expression was high in the fetal heart but significantly reduced in P7 pup hearts. The miR-133a promoter contains a canonical HRE element and hypoxia upregulated miR-133a gene expression in the heart. SIRT1 mRNA 3′UTR has miR-133a binding sequences and miR-133a and hypoxia suppressed SIRT1 expression in cardiomyocytes. Of importance, inhibition of SIRT1 significantly reduced binucleated cardiomyocytes in the hearts of P7 pups. Taken together, the present study reveals a novel role of SIRT1 and its regulation by miR-133a in cardiomyocyte terminal differentiation of the developing heart, and suggests a potential therapeutic strategy that may impact cardiac function later in life.

Sustained cardiac programming by short-term juvenile exercise training in male rats.

Cardiac hypertrophy following endurance-training is thought to be due to hypertrophy of existing cardiomyocytes. The benefits of endurance exercise on cardiac hypertrophy are generally thought to be short-lived and regress to sedentary levels within a few weeks of stopping endurance training. We have now established that cardiomyocyte hyperplasia also plays a considerable role in cardiac growth in response to just 4weeks of endurance exercise in juvenile (5-9weeks of age) rats. The effect of endurance exercise on cardiomyocyte hyperplasia diminishes with age and is lost by adulthood. We have also established that the effect of juvenile exercise on heart mass is sustained into adulthood. The aim of this study was to investigate if endurance training during juvenile life 'reprogrammes' the heart and leads to sustained improvements in the structure, function, and morphology of the adult heart. Male Wistar Kyoto rats were exercise trained 5daysweek-1 for 4weeks in either juvenile (5-9weeks of age), adolescent (11-15weeks of age) or adult life (20-24weeks of age). Juvenile exercise training, when compared to 24-week-old sedentary rats, led to sustained increases in left ventricle (LV) mass (+18%; P<0.05), wall thickness (+11%; P<0.05), the longitudinal area of binucleated cardiomyocytes (P<0.05), cardiomyocyte number (+36%; P<0.05), and doubled the proportion of mononucleated cardiomyocytes (P<0.05), with a less pronounced effect of exercise during adolescent life. Adult exercise training also increased LV mass (+11%; P<0.05), wall thickness (+6%; P<0.05) and the longitudinal area of binucleated cardiomyocytes (P<0.05), despite no change in cardiomyocyte number or the proportion of mono- and binucleated cardiomyocytes. Resting cardiac function, LV chamber dimensions and fibrosis levels were not altered by juvenile or adult exercise training. At 9weeks of age, juvenile exercise significantly reduced the expression of microRNA-208b, which is a known regulator of cardiac growth, but this was not sustained to 24weeks of age. In conclusion, juvenile exercise leads to physiological cardiac hypertrophy that is sustained into adulthood long after exercise training has ceased. Furthermore, this cardiac reprogramming is largely due to a 36% increase in cardiomyocyte number, which results in an additional 20 million cardiomyocytes in adulthood.

Experimentally induced intrauterine growth restriction in rabbits leads to differential remodelling of left versus right ventricular myocardial microstructure.

Intrauterine growth restriction (IUGR) is associated with foetal cardiac remodelling and dysfunction together with increased risk of cardiovascular disease in adulthood. Experimental data concerning effects of IUGR on cardiomyocyte and microvascularization anatomy are inconsistent and it is unknown whether both ventricles are similarly susceptible to in utero undersupply. Foetal IUGR was induced in pregnant rabbits at 25days of gestation by selective ligation of uteroplacental vessels. Foetal echocardiography showed systolic and diastolic dysfunction of both ventricles and body and heart weight were significantly reduced in response to IUGR. Design-based stereology revealed a decrease in cardiomyocyte number in both ventricles which was only in the left ventricle accompanied by a significantly higher cardiomyocyte mean volume. The proportion of mono- and bi-nucleated cardiomyocytes was unaltered between the groups indicating a similar maturation status. The number and length of cardiac capillaries in IUGR offspring was diminished in left but not in right ventricles. Foetal left and right ventricles are differently affected by placental insufficiency. While cardiomyocyte numbers are diminished in both ventricles, hypertrophic remodelling of cardiomyocytes and alterations in microvascularization is rather a left ventricular adaptation to IUGR. These unequal structural changes may be related to loading and developmental differences of the left and right ventricles.

Chronic hypoxia during development does not trigger pathologic remodeling of the chicken embryonic heart but reduces cardiomyocyte number.

Fetal growth restriction programs an increased risk of cardiovascular disease in adulthood, but the actual mechanisms of this developmental programming are not fully understood. Previous studies in mammalian models suggest that hearts of growth-restricted fetuses have reduced cardiomyocyte number due to reduced proliferation and premature cardiomyocyte maturation. Chicken embryos incubated under chronic hypoxia are also growth-restricted, have smaller hearts, and show signs of cardiac insufficiency posthatching. The aim of the present study was to investigate how chronic hypoxia (14% O2) during development affects cardiomyocyte mass and how myocardial structure is altered. Hypoxic incubation reproduced the well-characterized embryonic growth restriction and an increased ventricle-to-body mass ratio (at E11, E15, E17, and E19) with reduced absolute heart mass only at E19. Cell density, apoptosis, and cardiomyocyte size were insensitive to hypoxia at E15 and E19, and no signs of ventricular wall remodeling or myocardial fibrosis were detected. Bayesian modeling provided strong support for hypoxia affecting absolute mass and proliferation rates at E15, indicating that the growth impairment, at least partly, occurs earlier in development. Neither E15 nor E19 hearts contained binucleated cardiomyocytes, indicating that fetal hypoxia does not trigger early maturation of cardiomyocytes in the chicken, which contrasts with previous results from hypoxic rat pups. In conclusion, prenatal hypoxia in the chick embryo results in a reduction in the number of cardiomyocytes without inducing ventricular remodeling, cell hypertrophy, or premature cardiomyocyte maturation.

Dexamethasone Treatment of Newborn Rats Decreases Cardiomyocyte Endowment in the Developing Heart through Epigenetic Modifications.

The potential adverse effect of synthetic glucocorticoid, dexamethasone therapy on the developing heart remains unknown. The present study investigated the effects of dexamethasone on cardiomyocyte proliferation and binucleation in the developing heart of newborn rats and evaluated DNA methylation as a potential mechanism. Dexamethasone was administered intraperitoneally in a three day tapered dose on postnatal day 1 (P1), 2 and 3 to rat pups in the absence or presence of a glucocorticoid receptor antagonist Ru486, given 30 minutes prior to dexamethasone. Cardiomyocytes from P4, P7 or P14 animals were analyzed for proliferation, binucleation and cell number. Dexamethasone treatment significantly increased the percentage of binucleated cardiomyocytes in the hearts of P4 pups, decreased myocyte proliferation in P4 and P7 pups, reduced cardiomyocyte number and increased the heart to body weight ratio in P14 pups. Ru486 abrogated the effects of dexamethasone. In addition, 5-aza-2'-deoxycytidine (5-AZA) blocked the effects of dexamethasone on binucleation in P4 animals and proliferation at P7, leading to recovered cardiomyocyte number in P14 hearts. 5-AZA alone promoted cardiomyocyte proliferation at P7 and resulted in a higher number of cardiomyocytes in P14 hearts. Dexamethasone significantly decreased cyclin D2, but not p27 expression in P4 hearts. 5-AZA inhibited global DNA methylation and blocked dexamethasone-mediated down-regulation of cyclin D2 in the heart of P4 pups. The findings suggest that dexamethasone acting on glucocorticoid receptors inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via increased DNA methylation in a gene specific manner.

0202 : Adult cardiomyocytes proliferation blockage by nuclear ephrin-B1: involvement of the PI3Kγ pathway

The possibility to reboot the proliferation of adult resident cardiomyocytes (CMs) has emerged as a promising avenue in cardiac regenerative medicine. For that purpose, there is an urgent need for identifying the molecular mechanisms involved in the natural blockage of adult CMs proliferation. Recently, we recently demonstrated that ephrin-B1 expression in the CMs nuclei acts as a specific inhibitor of the adult CMs proliferation (unpublished data). Here, we have explored the specific role of the PI3Kγin the nuclearization of ephrin-B1. Fluorescent immunostaining showed a specific loss of nuclear ephrin-B1 in p110-γKO but not in KI mice, suggesting a specific role of the PKAanchoring function of the p110-γcatalytic subunit of PI3Kγin ephrin-B1 nuclearization. Interestingly, p110-γKO mice demonstrated a significant increase of mononucleated CMs (19,1 ± 1,1% vs 13,3 ± 1,0% in WT) correlated to a decrease in binucleated CMs (70,9 ± 1,9% vs 79,7 ±1,0% in WT) possibly indicative of a proliferative potential. In agreement with a proliferative potential, we measured a higher level of replicative-BrdU± CMs in KO PI3Kγmice (WT: 6,23±2,2% ; KO: 19,3±4,9%; KI: 3,0±0,8%), but also a higher CMs density in cardiac tissue from 8 month old mice (WT: 2633±79,4cm/mm 2 ; KO: 3208±155,6CM/mm 2 ; KI: 2659,7±99,3CM/mm 2 ) mosy likely reflecting CMs hyperplasia. To confirm the involvement of a cAMP/PKA pathway in ephrin-B1 nuclearization, we studied a neonatal cardiomyoblast cell line (H9C2) naturally expressing a nuclear ephrin-B1 pool. We showed that H89 treatment prevented ephrin-B1 nuclearization while forskolin promoted nuclearization of a recombinant ephrin-B1-GFP. These results demonstrated for the first time the specific role of the cAMP/ PKA/PI3Kγ pathway in ephrin-B1 nuclearization in the CM. In the future, the accurate characterization of this pathway could allow identifying new and more specific targets for heart regenerative medicine strategies.

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-β, fibroblast growth factor, and Wnt/β-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.