Cardiomyocyte Terminal Differentiation Research Articles

Intrauterine growth restriction elevates circulating acylcarnitines and suppresses fatty acid metabolism genes in the fetal sheep heart.

At birth, the mammalian myocardium switches from using carbohydrates as the primary energy substrate to free fatty acids as the primary fuel. Thus, a compromised switch could jeopardize normal heart function in the neonate. Placental embolization in sheep is a reliable model of intrauterine growth restriction (IUGR). It leads to suppression of both proliferation and terminal differentiation of cardiomyocytes. We hypothesized that the expression of genes regulating cardiac fatty acid metabolism would be similarly suppressed in IUGR, leading to compromised processing of lipids. Following 10 days of umbilicoplacental embolization in fetal sheep, IUGR fetuses had elevated circulating long-chain fatty acylcarnitines compared with controls (C14: CTRL 0.012 ± 0.005nmol/ml vs. IUGR 0.018 ± 0.005nmol/ml, P < 0.05; C18: CTRL 0.027 ± 0.009nmol/mol vs. IUGR 0.043 ± 0.024nmol/mol, P < 0.05, n = 12 control, n = 12 IUGR) indicative of impaired fatty acid metabolism. Uptake studies using fluorescently tagged BODIPY-C12-saturated free fatty acid in live, isolated cardiomyocytes showed lipid droplet area and number were not different between control and IUGR cells. mRNA levels of sarcolemmal fatty acid transporters (CD36, FATP6), acylation enzymes (ACSL1, ACSL3), mitochondrial transporter (CPT1), β-oxidation enzymes (LCAD, HADH, ACAT1), tricarboxylic acid cycle enzyme (IDH), esterification enzymes (PAP, DGAT) and regulator of the lipid droplet formation (BSCL2) gene were all suppressed in IUGR myocardium (P < 0.05). However, protein levels for these regulatory genes were not different between groups. This discordance between mRNA and protein levels in the stressed myocardium suggests an adaptive protection of key myocardial enzymes under conditions of placental insufficiency. KEY POINTS: The fetal heart relies on carbohydrates in utero and must be prepared to metabolize fatty acids after birth but the effects of compromised fetal growth on the maturation of this metabolic system are unknown. Plasma fatty acylcarnitines are elevated in intrauterine growth-restricted (IUGR) fetuses compared with control fetuses, indicative of impaired fatty acid metabolism in fetal organs. Fatty acid uptake and storage are not different in IUGR cardiomyocytes compared with controls. mRNA levels of genes regulating fatty acid transporter and metabolic enzymes are suppressed in the IUGR myocardium compared with controls, while protein levels remain unchanged. Mismatches in gene and protein expression, and increased circulating fatty acylcarnitines may have long-term implications for offspring heart metabolism and adult health in IUGR individuals. This requires further investigation.

Dynamics of nuclear-cytoplasmic relations in cardiomyocytes in the rat`s heart in the early postnatal period in normal conditions and experiment

Dexamethasone is administered to pregnant women with high risk of preterm delivery, but his impact during intrauterine development can program progression of undifferentiated connective tissue dysplasia and a tendency to cardiovascular diseases in adulthood. The aim of the study was to determine the changes of the nuclear-cytoplasmic relations (NCR) of cardiomyocytes (CMC) in the myocardium in the postnatal period after intrauterine administration of dexamethasone. 144 hearts of laboratory rats were studied from 1 to 45 days of life, which were divided into 3 groups: first — intact group, animals of second group were injected by 0.05 ml dexamethasone solution on the 18th day of intrauterine development, and III — control group. Tissue sections were stained with hematoxylin and eosin. Statistical evaluation was carried out with Mann-Whitney U-test. p&lt;0,05 was accepted as statistically significant. It has been found that, the NCR increases 1.56-fold from birth till 14-th day and decreases 2.55-fold till 45-th day in the myocardium of the left ventricle. There changes correspond for the transition from a proliferative to a hypertrophic growth, which occurs on 14-th day after birth in the heart of rodents. In animals of the experimental group, the NCR was significantly increased at 3, 5, 21, 30, and 45, but decreased on the 14-th day (p&lt;0,01) in comparison with control group. The maximum value of the index was observed on the 5-th day, which indicates on early terminal differentiation of CMC in experimental animals. Thus, intrauterine administration of dexamethasone leads to early maturation of CMC, which reduces their number and may increase the risk of developing cardiovascular diseases in adulthood. In the future we plan to study the proliferative activity of CMC of rat`s heart in the postnatal period in normal conditions and after intrauterine administration of dexamethasone.

Systemic arterial hypertension but not IGF-I treatment stimulates cardiomyocyte enlargement in neonatal lambs.

Although cardiomyocyte terminal differentiation is nearly complete at birth in sheep, as in humans, very limited postnatal expansion of myocyte number may occur. The capacity of newborn cardiomyocytes to respond to growth stimulation by proliferation is poorly understood. Our objective was to test this growth response in newborn lambs with two stimuli shown to be potent inducers of cardiomyocyte growth in fetuses and adults: increased systolic load (Load) and insulin-like growth factor I (IGF-I). Vascular catheters and an inflatable aortic occluder were implanted in lambs. Hearts were collected for analysis at 18 days of age after a 7-day experiment and compared with control hearts. Load hearts, but not IGF-I hearts, were heavier ( P = 0.001) because of increased mass of the left ventricle (LV), septum, and left atrium (40-50%, P = 0.004). Terminal differentiation and cell cycle activity were not different between groups. Myocyte length was 7% greater in Load lamb hearts ( P < 0.05), and binucleated myocytes, which comprise ~90% of LV cells, were 25% larger in volume ( P = 0.03). Myocyte number per gram of myocardium was decreased in all ventricles of Load lambs ( P = 0.01). Cells from the IGF-I group were not different by any comparison. These results suggest that the newborn sheep LV responds to systolic stress with cardiomyocyte hypertrophy, not proliferation. Furthermore, IGF-I is ineffective at stimulating cardiomyocyte proliferation at this age (despite effectiveness when administered before birth). Thus, to expand cardiomyocyte number in the newborn heart, therapies other than systolic pressure load and IGF-I treatment need to be developed.

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.

SIRT1 plays a novel role in the regulation of cardiomyocyte terminal differentiation in the developing heart

SIRT1 plays a novel role in the regulation of cardiomyocyte terminal differentiation in the developing heart

Cardiac injury of the newborn mammalian heart accelerates cardiomyocyte terminal differentiation

After birth cardiomyocytes undergo terminal differentiation, characterized by binucleation and centrosome disassembly, rendering the heart unable to regenerate. Yet, it has been suggested that newborn mammals regenerate their hearts after apical resection by cardiomyocyte proliferation. Thus, we tested the hypothesis that apical resection either inhibits, delays, or reverses cardiomyocyte centrosome disassembly and binucleation. Our data show that apical resection rather transiently accelerates centrosome disassembly as well as the rate of binucleation. Consistent with the nearly 2-fold increased rate of binucleation there was a nearly 2-fold increase in the number of cardiomyocytes in mitosis indicating that the majority of injury-induced cardiomyocyte cell cycle activity results in binucleation, not proliferation. Concurrently, cardiomyocytes undergoing cytokinesis from embryonic hearts exhibited midbody formation consistent with successful abscission, whereas those from 3 day-old cardiomyocytes after apical resection exhibited midbody formation consistent with abscission failure. Lastly, injured hearts failed to fully regenerate as evidenced by persistent scarring and reduced wall motion. Collectively, these data suggest that should a regenerative program exist in the newborn mammalian heart, it is quickly curtailed by developmental mechanisms that render cardiomyocytes post-mitotic.

Proteomic Analysis of Endothelin-1 Targets in the Regulation of Cardiomyocyte Proliferation.

Hypoxia is a fetal stressor that leads to the production of endothelin-1 (ET-1). Previous work has shown that ET-1 treatment leads to the premature terminal differentiation of fetal cardiomyocytes. However, the precise mechanism is unknown. We tested the hypothesis that the fetal cardiomyocyte proteome will be greatly altered due to ET-1-treatment, which reveals a potential molecular mechanism of ET-1-induced terminal differentiation. Over a thousand proteins were detected in the fetal cardiomyocytes and among them 75 proteins were significantly altered due to ET-1 treatment. Using IPA pathway analysis, the merged network depicted several key proteins that appeared to be involved in regulating proliferation, including: EED, UBC, ERK1/2, MAPK, Akt, and EGFR. EED protein, which is associated with regulating proliferation via epigenetic mechanisms, is of particular interest. Herein we propose a model of the molecular mechanism by which ET-1 induced cardiomyocyte terminal differentiation occurs.

The CapZ interacting protein Rcsd1 is required for cardiogenesis downstream of Wnt11a in Xenopus laevis

Wnt proteins are critical for embryonic cardiogenesis and cardiomyogenesis by regulating different intracellular signalling pathways. Whereas canonical Wnt/β-catenin signalling is required for mesoderm induction and proliferation of cardiac progenitor cells, β-catenin independent, non-canonical Wnt signalling regulates cardiac specification and terminal differentiation. Although the diverse cardiac malformations associated with the loss of non-canonical Wnt11 in mice such as outflow tract (OFT) defects, reduced ventricular trabeculation, myofibrillar disorganization and reduced cardiac marker gene expression are well described, the underlying molecular mechanisms are still not completely understood. Here we aimed to further characterize Wnt11 mediated signal transduction during vertebrate cardiogenesis. Using Xenopus as a model system, we show by loss of function and corresponding rescue experiments that the non-canonical Wnt signalling mediator Rcsd1 is required downstream of Wnt11 for ventricular trabeculation, terminal differentiation of cardiomyocytes and cardiac morphogenesis. We here place Rcsd1 downstream of Wnt11 during cardiac development thereby providing a novel mechanism for how non-canonical Wnt signalling regulates vertebrate cardiogenesis.

Dexamethasone Induces Cardiomyocyte Terminal Differentiation via Epigenetic Repression of Cyclin D2 Gene.

Dexamethasone treatment of newborn rats inhibited cardiomyocyte proliferation and stimulated premature terminal differentiation of cardiomyocytes in the developing heart. Yet mechanisms remain undetermined. The present study tested the hypothesis that the direct effect of glucocorticoid receptor-mediated epigenetic repression of cyclin D2 gene in the cardiomyocyte plays a key role in the dexamethasone-mediated effects in the developing heart. Cardiomyocytes were isolated from 2-day-old rats. Cells were stained with a cardiomyocyte marker α-actinin and a proliferation marker Ki67. Cyclin D2 expression was evaluated by Western blot and quantitative real-time polymerase chain reaction. Promoter methylation of CcnD2 was determined by methylated DNA immunoprecipitation (MeDIP). Overexpression of Cyclin D2 was conducted by transfection of FlexiCcnD2 (+CcnD2) construct. Treatment of cardiomyocytes isolated from newborn rats with dexamethasone for 48 hours significantly inhibited cardiomyocyte proliferation with increased binucleation and decreased cyclin D2 protein abundance. These effects were blocked with Ru486 (mifepristone). In addition, the dexamethasone treatment significantly increased cyclin D2 gene promoter methylation in newborn rat cardiomyocytes. 5-Aza-2'-deoxycytidine inhibited dexamethasone-mediated promoter methylation, recovered dexamethasone-induced cyclin D2 gene repression, and blocked the dexamethasone-elicited effects on cardiomyocyte proliferation and binucleation. In addition, the overexpression of cyclin D2 restored the dexamethasone-mediated inhibition of proliferation and increase in binucleation in newborn rat cardiomyocytes. The results demonstrate that dexamethasone acting on glucocorticoid receptors has a direct effect and inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via epigenetic repression of cyclin D2 gene.

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.

Newborn hypoxia/anoxia inhibits cardiomyocyte proliferation and decreases cardiomyocyte endowment in the developing heart: role of endothelin-1.

In the developing heart, cardiomyocytes undergo terminal differentiation during a critical window around birth. Hypoxia is a major stress to preterm infants, yet its effect on the development and maturation of the heart remains unknown. We tested the hypothesis in a rat model that newborn anoxia accelerates cardiomyocyte terminal differentiation and results in reduced cardiomyocyte endowment in the developing heart via an endothelin-1-dependent mechanism. Newborn rats were exposed to anoxia twice daily from postnatal day 1 to 3, and hearts were isolated and studied at postnatal day 4 (P4), 7 (P7), and 14 (P14). Anoxia significantly increased HIF-1α protein expression and pre-proET-1 mRNA abundance in P4 neonatal hearts. Cardiomyocyte proliferation was significantly decreased by anoxia in P4 and P7, resulting in a significant reduction of cardiomyocyte number per heart weight in the P14 neonates. Furthermore, the expression of cyclin D2 was significantly decreased due to anoxia, while p27 expression was increased. Anoxia has no significant effect on cardiomyocyte binucleation or myocyte size. Consistently, prenatal hypoxia significantly decreased cardiomyocyte proliferation but had no effect on binucleation in the fetal heart. Newborn administration of PD156707, an ETA-receptor antagonist, significantly increased cardiomyocyte proliferation at P4 and cell size at P7, resulting in an increase in the heart to body weight ratio in P7 neonates. In addition, PD156707 abrogated the anoxia-mediated effects. The results suggest that hypoxia and anoxia via activation of endothelin-1 at the critical window of heart development inhibits cardiomyocyte proliferation and decreases myocyte endowment in the developing heart, which may negatively impact cardiac function later in life.

Abstract 133: Novel Transgenic Mouse Lines for Identification of Cardiomyocyte Nuclei and Visualization of Their Cell Cycle Status In-vitro and In-vivo

The typical remodelling process after cardiac injury is scarring and compensatory hypertrophy. The limited regeneration potential of the adult heart is due to the post-mitotic status of cardiomyocytes (CMs), which are mostly binucleated. Nevertheless, there is evidence for CM turnover in the adult heart, but its extent is still under debate. One technical limitation of quantitations is the unequivocal identification of CMs and of CM cell division. In order to enable a clear identification of CM nuclei in-vivo, we have developed a transgenic mouse line in which a fusion protein of the human histone 2B and the red fluorescence protein mCherry is specifically and persistently expressed in CM nuclei (αMHC-H2B-mCh). The fluorescence label allowed the investigation of CM percentages in native tissue slices, which properly reflect the cellular composition of the heart. We focused on regional (atrium versus left and right ventricle, apical versus basal slices) and developmental-stage dependent changes in the percentage of CM nuclei and binuclearity. We therefore analyzed time-points before/during and after terminal differentiation of CMs (postnatal day 3 (P3), P7 and 9 weeks). In addition, we also investigated endoreduplication and acytokinetic mitosis using time lapse microscopy in postnatal CMs to better understand cell biological mechanisms leading to terminal differentiation. As currently huge efforts are invested for the search of substances that increase the regeneration potential of the heart, we established a novel screening assay for cell-cycle modifying substances in isolated, postnatal CMs. We crossed the αMHC-H2B-mCh with the CAG-eGFPanillin mouse line, which marks cell-cycle activity with a high resolution of M-phase. Analysis of binuclearity and of different eGFPanillin subcellular localizations will be helpful to understand, whether CMs complete cytokinesis. As a proof of principle we investigated the effects of cell cycle activating micro RNAs199 and 590. Thus, our double transgenic mouse line will be useful to examine the plasticity of mono- and binuclear CMs and to unravel cell biological mechanisms leading to terminal differentiation of CMs.

A Proliferative Burst during Preadolescence Establishes the Final Cardiomyocyte Number

It is widely believed that perinatal cardiomyocyte terminal differentiation blocks cytokinesis, thereby causing binucleation and limiting regenerative repair after injury. This suggests that heart growth should occur entirely by cardiomyocyte hypertrophy during preadolescence when, in mice, cardiac mass increases many-fold over a few weeks. Here, we show that a thyroid hormone surge activates the IGF-1/IGF-1-R/Akt pathway on postnatal day 15 and initiates a brief but intense proliferative burst of predominantly binuclear cardiomyocytes. This proliferation increases cardiomyocyte numbers by ~40%, causing a major disparity between heart and cardiomyocyte growth. Also, the response to cardiac injury at postnatal day 15 is intermediate between that observed at postnatal days 2 and 21, further suggesting persistence of cardiomyocyte proliferative capacity beyond the perinatal period. If replicated in humans, this may allow novel regenerative therapies for heart diseases.

Endothelin‐1 promotes fetal cardiomyocyte terminal differentiation via heightened DNA methylation (862.2)

Hypoxia is a major stress on fetal development and leads to induction of endothelin‐1 (ET‐1) expression. We tested the hypothesis that ET‐1 stimulates the terminal differentiation of cardiomyocytes from mononucleate to binucleate in the developing heart. In fetal hearts, prepro‐ET‐1 mRNA expression was significantly increased by hypoxia (10.5% O2) treatment of pregnant rats from day 15 to day 21. Under both control and hypoxic conditions, the ET‐1 ex vivo treatment of fetal rat cardiomyocytes increased percent binucleate cells and decreased Ki‐67 expression, a proliferation marker. Hypoxia alone was able to decrease Ki‐67 expression and cardiomyocyte size. A non‐selective ET‐receptor antagonist, PD145065, blocked the ET‐1 induced changes in binucleation and proliferation. ET‐1 treatment significantly increased DNA methylation in fetal cardiomyocytes, which was further blocked by 5‐aza‐2’‐deoxycytidine, a DNA methylation inhibitor. In addition, 5‐aza‐2’‐deoxycytidine treatment abrogated the increase in binucleation and decrease in proliferation induced by ET‐1. Hypoxic stress and synthesis of ET‐1 increases DNA methylation and promotes terminal differentiation of cardiomyocytes in the developing heart. This premature exit of the cell cycle may lead to a reduced cardiomyocyte endowment in the heart and have a negative impact on cardiac function.

Mutations in Alström protein impair terminal differentiation of cardiomyocytes.

Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognise homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at two weeks postnatal compared to wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest.

Endothelin-1 Promotes Cardiomyocyte Terminal Differentiation in the Developing Heart via Heightened DNA Methylation

Aims: Hypoxia is a major stress on fetal development and leads to induction of endothelin-1 (ET-1) expression. We tested the hypothesis that ET-1 stimulates the terminal differentiation of cardiomyocytes from mononucleate to binucleate in the developing heart.Methods and results: Hypoxia (10.5% O2) treatment of pregnant rats from day 15 to day 21 resulted in a significant increase in prepro-ET-1 mRNA expression in fetal hearts. ET-1 ex vivo treatment of fetal rat cardiomyocytes increased percent binucleate cells and decreased Ki-67 expression, a marker for proliferation, under both control and hypoxic conditions. Hypoxia alone decreased Ki-67 expression and in conjunction with ET-1 treatment decreased cardiomyocyte size. PD145065, a non-selective ET-receptor antagonist, blocked the changes in binucleation and proliferation caused by ET-1. DNA methylation in fetal cardiomyocytes was significantly increased with ET-1 treatment, which was blocked by 5-aza-2'-deoxycytidine, a DNA methylation inhibitor. In addition, 5-aza-2'-deoxycytidine treatment abrogated the increase in binucleation and decrease in proliferation induced by ET-1.Conclusions: Hypoxic stress and synthesis of ET-1 increases DNA methylation and promotes terminal differentiation of cardiomyocytes in the developing heart. This premature exit of the cell cycle may lead to a reduced cardiomyocyte endowment in the heart and have a negative impact on cardiac function.

Degradation of p21Cip1 through Anaphase-promoting Complex/Cyclosome and Its Activator Cdc20 (APC/CCdc20) Ubiquitin Ligase Complex-mediated Ubiquitylation Is Inhibited by Cyclin-dependent Kinase 2 in Cardiomyocytes

Cyclin-dependent kinase inhibitor p21Cip1 plays a crucial role in regulating cell cycle arrest and differentiation. It is known that p21Cip1 increases during terminal differentiation of cardiomyocytes, but its expression control and biological roles are not fully understood. Here, we show that the p21Cip1 protein is stabilized in cardiomyocytes after mitogenic stimulation, due to its increased CDK2 binding and inhibition of ubiquitylation. The APC/CCdc20 complex is shown to be an E3 ligase mediating ubiquitylation of p21Cip1 at the N terminus. CDK2, but not CDC2, suppressed the interaction of p21Cip1 with Cdc20, thereby leading to inhibition of anaphase-promoting complex/cyclosome and its activator Cdc20 (APC/CCdc20)-mediated p21Cip1 ubiquitylation. It was further demonstrated that p21Cip1 accumulation caused G2 arrest of cardiomyocytes that were forced to re-enter the cell cycle. Taken together, these data show that the stability of the p21Cip1 protein is actively regulated in terminally differentiated cardiomyocytes and plays a role in inhibiting their uncontrolled cell cycle progression. Our study provides a novel insight on the control of p21Cip1 by ubiquitin-mediated degradation and its implication in cell cycle arrest in terminal differentiation.

The Ontogeny of Cardiac Regeneration

### Transient Regenerative Potential of the Neonatal Mouse Heart Porello et al Science . 2011;331:1078–1080 The regenerative capacity of adult human hearts after infarction seems vestigial at best, perhaps because of the poor proliferative capacity of terminally differentiated cardiomyocytes and desecration of the local environment that would otherwise be conducive of stem cell-mediated repair or loss of stem cells, or both. In a recent edition of the journal Science , Porello et al set a new benchmark in this field by showing that the hearts of neonatal mice have, in contrast, an impressive regenerative capacity after surgical resection, albeit one that is lost almost immediately as cardiomyocytes exit the cell cycle and mature. In a technical tour de force, Porello et al1 report that day 1 neonatal mice subjected to resection of the apex of the left ventricle (amounting to ∼15% of the ventricular myocardium) not only survive but also show complete regeneration of the myocardium and restoration of cardiac function. Furthermore, they provide evidence that the predominant mechanism involved is not differentiation of stem cells, but transient dedifferentiation and proliferation of cardiomyocytes in both the border and remote zones of the heart. In contrast, resection of a lesser amount of apical myocardium in day 7 neonates, at a time after terminal differentiation of cardiomyocytes has already taken place, resulted in markedly increased mortality and failure of myocardial regeneration, with wound repair by scar formation only. It has long been known that in adult mammals, the heart is one of the least regenerative organs in the body. As a result, repair after massive tissue injury, as occurs with myocardial infarction, is limited to replacement of the myocardium by dense collagenous scar tissue. This impairs contractile function, resulting in adverse remodelling and, in severe cases, ventricular dilation, heart failure, and death. To explore if intracardiac …

Oxidative stress-induced formation of a positive-feedback loop for the sustained activation of p38 MAPK leading to the loss of cell division in cardiomyocytes soon after birth

Shortly after birth, mammalian cardiomyocytes irreversibly exit from the cell cycle and become terminally differentiated. The cellular mechanisms responsible for the cessation of cell division and terminal differentiation of cardiomyocytes soon after birth have intrigued developmental biologists as well as cardiovascular physicians, but the genetic cues for the irreversible exit from the cell cycle soon after birth remain largely unknown. We examined whether and if so how oxidative stress to mammalian hearts during fetal-neonatal transition produces changes in the proliferative activity and terminal differentiation of cardiomyocytes. Scavenging of reactive oxygen species (ROS) during fetal-neonatal transition, especially after birth, resulted in an increase in the proliferative activity and a decrease in the ratio of binucleated cardiomyocytes. Exposure to ROS in cultured cardiomyocytes increased the activity of p38 MAPK and the expression of connexin 43 (Cx43). Not only knockdown of Cx43 using siRNA but also the inhibition of p38 MAPK activity resulted in a significant decrease in the production of ROS in cardiomyocytes, suggesting that the signaling pathway ROS-p38 MAPK-Cx43 (especially, Cx43 at mitochondria, mtCx43) constituted a closed regulatory system with positive feedback. In addition, continuous scavenging of ROS or suppression of p38 MAPK activity for 4days after birth resulted in a significant decrease in the expression of mtCx43 and in the number of binucleated cardiomyocytes. This study demonstrated that the ROS-induced formation of a positive-feedback loop ROS-p38 MAPK-mtCx43 for the sustained activation of p38 MAPK soon after birth possibly contributes to the loss of cell division and binucleation in mammalian cardiomyocytes.

Epigenetic regulation of neonatal cardiomyocytes differentiation

The relationship between DNA methylation, histone modifications and terminal differentiation in cardiomyocytes was investigated in this study. The upregulation of methylation-related proteins, including DNA methyltransferase (DNMT) 1, methyl-CpG binding domain proteins 1, 2 and 3, and the increase in global methylation during rat neonatal heart development were observed. Moreover, an increase in DNA synthesis and a delay in differentiation were found in 5-azacytidine (5-azaC)-treated cardiomyocytes. Increase in acetylation of H3-K9, H4-K5, H4-K8 and methylation of H3-K4 suggested a more accessible chromatin structure in 5-azaC-treated cells. Furthermore, methyl-CpG-binding protein 2 was found to be upregulated in differentiated cardiomyocytes. Overexpression of this protein resulted in an increase of global methylation levels. Therefore, we suggest that a hypermethylated genome and a more compact chromatin structure are formed during terminal differentiation of cardiomyocytes.