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 ET_A-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.     

Normal lactational environment restores cardiomyocyte number after uteroplacental insufficiency: implications for the preterm neonate

A reduced complement of cardiomyocytes in early life can adversely affect life-long cardiac functional reserve. In the present study, using a cross-fostering approach in rats, we examined the contributions of the prenatal and postnatal environments in the programming of cardiomyocyte growth. Rat dams underwent either bilateral uterine vessel ligation (Restricted) or sham surgery (Control) on day 18 of gestation. One day after birth, Control and Restricted pups were cross-fostered onto Control (normal lactation) or Restricted (impaired lactation due to impaired mammary gland formation) mothers. In male offspring, genes involved in cardiomyocyte differentiation, proliferation, hypertrophy and apoptosis were examined at gestational day 20 and postnatal days 1 and 7 to assess effects on cardiomyocyte growth. At postnatal day 7 cardiomyocyte number was determined stereologically. Offspring were examined at age 6 mo for evidence of hypertension and pathological cardiac gene expression. There was an increase in Igf1 and Igf2 mRNA expression in hearts of Restricted pups at gestational day 20. At postnatal day 7, Agtr1a and Agtr1b mRNA expression as well as Bcl2 and Cmyc were elevated in all hearts from offspring that were prenatally or postnatally growth restricted. There was a significant reduction (-29%) in cardiomyocyte number in the Restricted-on-Restricted group. Importantly, this deficit was prevented by optimization of postnatal nutrition (in the Restricted-on-Control group). At 6 mo, blood pressure was significantly elevated in the Restricted-on-Restricted group, but there was no difference in expression of the cardiac hypertrophy, remodeling or angiogenic genes across groups. In conclusion, the findings reveal a critical developmental window, when cardiomyocytes are still proliferating, whereby improved neonatal nutrition has the capacity to restore cardiomyocyte number to normal levels. These findings are of particular relevance to the preterm infant who is born at a time when cardiomyocytes are immature and still dividing.     

RU486 inhibits induction of aromatase by dexamethasone via glucocorticoid receptor in cultured human skin fibroblasts

Effects of RU486 on the induction of aromatase by dexamethasone via glucocorticoid receptor were determined using cultured human skin fibroblasts. Competition of [3H]dexamethasone binding to the cytosol receptor was 7 times stronger with RU486 than with dexamethasone. The order of the strength of competition was RU486 greater than dexamethasone greater than betamethasone greater than prednisolone greater than hydrocortisone. RU486 abolished a specific 8.6 S [3H]dexamethasone binding peak in the cytosol, determined using a sucrose density gradient analysis. Dexamethasone markedly induced aromatase and this event was strongly suppressed by RU486, in a dose-dependent manner, in the cultured skin fibroblasts. A linear correlation between the strength of competition and the induction of aromatase of various glucocorticoids was observed. RU486 non-competitively inhibited aromatase induction by dexamethasone determined from a double reciprocal plot of aromatase activity, with respect to [3H]androstenedione concentration in the presence of RU486. These results show that RU486 is a peripheral noncompetitive antiglucocorticoid on aromatase induction by glucocorticoid in human skin fibroblasts and that aromatase induction is a good marker for the biological function of glucocorticoid receptor in human skin fibroblasts.     

调节性T细胞参与调控新生小鼠心肌再生

为探究调节性T（regulatory T,Treg）细胞在新生小鼠心肌损伤后再生中的作用,首先建立新生小鼠心肌再生模型。C57BL/6J（C57）新生1 d小鼠20只随机分成2组。实验组进行心尖切除（apex resection,AR）,假手术（Sham,SH）组只进行开胸。术后7 d取心脏组织,利用在细胞核表达的增殖标志物磷酸化组蛋白H3（phospho-histone H3,pH3）和Ki67分别与在心肌细胞胞质特异表达的α-辅肌动蛋白（alpha-actinin cytoskeletal isoform,α-actinin）,进行免疫共染检测心肌细胞增殖。结果显示,与SH组相比,AR组pH3+及Ki67+的心肌细胞明显增多。而且Masson三色染色结果显示,术后21 d被切除的心肌组织完全再生。为研究Treg细胞是否参与调控新生小鼠心肌损伤后的再生,Western印迹检测Treg细胞特异转录因子叉头/翼状螺旋转录因子3（forkhead box P3,Foxp3）蛋白表达水平。结果显示,术后7 d、14 d,AR组心和脾中Foxp3与SH组相比显著升高（P<0.05）。同时,免疫组化染Foxp3结果显示,术后7 d、14 d, AR组与SH组相比,心尖处有大量的Treg细胞富集。为更直观地检测AR后Treg细胞的数目变化,利用流式细胞仪检测术后7 d Treg细胞数目。结果显示,AR组心和脾中Treg细胞数目与SH组相比显著增多（P<0.01）。为研究Treg细胞对AR后心肌再生的影响,引入注射白喉毒素（diphtheria toxin,DT）的Foxp3DTR小鼠,可特异性敲除Treg细胞。实时定量PCR结果显示,AR+DT组与AR+PBS组相比,抑炎因子白介素IL（interleukin,IL）-10、IL-13与转化生长因子TGF（transforming growth factor,TGF）-β表达均降低（P<0.05,P<0.01,P<0.01）。而促炎因子IL-6、IL-1β和肿瘤坏死因子-α（tumor necrosis factor,TNF-α）表达均升高（P<0.01,P<0.001,P<0.01）。免疫荧光染色检测结果显示,AR+DT组与AR+PBS组相比,术后7 d pH3+及Ki67+的心肌细胞明显减少;并且Masson三色染色结果显示,术后21 d AR+DT组被切除的心肌组织不能再生。综上所述,敲除Treg细胞会加剧AR后的炎症反应,抑制心肌细胞增殖,最终导致新生小鼠心肌再生能力丢失。     

LRP6 downregulation promotes cardiomyocyte proliferation and heart regeneration

The adult mammalian heart is thought to be a terminally differentiated organ given the postmitotic nature of cardiomyocytes. Consequently, the potential for cardiac repair through cardiomyocyte proliferation is extremely limited. Low-density lipoprotein receptor-related protein 6 (LRP6) is a Wnt co-receptor that is required for embryonic heart development. In this study we investigated the role of LRP6 in heart repair through regulation of cardiomyocyte proliferation. Lrp6 deficiency increased cardiomyocyte cell cycle activity in neonatal, juvenile and adult mice. Cardiomyocyte-specific deletion of Lrp6 in the mouse heart induced a robust regenerative response after myocardial infarction (MI), led to reduced MI area and improvement in left ventricular systolic function. In vivo genetic lineage tracing revealed that the newly formed cardiomyocytes in Lrp6-deficient mouse hearts after MI were mainly derived from resident cardiomyocytes. Furthermore, we found that the pro-proliferative effect of Lrp6 deficiency was mediated by the ING5/P21 signaling pathway. Gene therapy using the adeno-associated virus (AAV)9 miRNAi-Lrp6 construct promoted the repair of heart injury in mice. Lrp6 deficiency also induced the proliferation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Our study identifies LRP6 as a critical regulator of cardiomyocyte proliferation, which may lead to the development of a novel molecular strategy to promote myocardial regeneration and repair.Subject terms: Cell-cycle exit, Cytokinesis     

Alcohol exposure during late gestation adversely affects myocardial development with implications for postnatal cardiac function

Prenatal exposure to high levels of ethanol is associated with cardiac malformations, but the effects of lower levels of exposure on the heart are unclear. Our aim was to investigate the effects of daily exposure to ethanol during late gestation, when cardiomyocytes are undergoing maturation, on the developing myocardium. Pregnant ewes were infused with either ethanol (0.75 g/kg) or saline for 1 h each day from gestational days 95 to 133 (term ～145 days); tissues were collected at 134 days. In sheep, cardiomyocytes mature during late gestation as in humans. Within the left ventricle (LV), cardiomyocyte number was determined using unbiased stereology and cardiomyocyte size and nuclearity determined using confocal microscopy. Collagen deposition was quantified using image analysis. Genes relating to cardiomyocyte proliferation and apoptosis were examined using quantitative real-time PCR. Fetal plasma ethanol concentration reached 0.11 g/dL after EtOH infusions. Ethanol exposure induced significant increases in relative heart weight, relative LV wall volume, and cardiomyocyte cross-sectional area. Ethanol exposure advanced LV maturation in that the proportion of binucleated cardiomyocytes increased by 12%, and the number of mononucleated cardiomyocytes was decreased by a similar amount. Apoptotic gene expression increased in the ethanol-exposed hearts, although there were no significant differences between groups in total cardiomyocyte number or interstitial collagen. Daily exposure to a moderate dose of ethanol in late gestation accelerates the maturation of cardiomyocytes and increases cardiomyocyte and LV tissue volume in the fetal heart. These effects on cardiomyocyte growth may program for long-term cardiac vulnerability.     

Glucocorticoid Modulates Angiotensin II Receptor Expression Patterns and Protects the Heart from Ischemia and Reperfusion Injury

Glucocorticoid regulates angiotensin II receptor (ATR) expression via activating glucocorticoid receptors and binding to glucocorticoid response elements. The regulation of ATR by glucocorticoids in the context of myocardial injury from ischemia/reperfusion (I/R) is yet to be elucidated. The present study determined the role of ATR in glucocorticoid-induced cardiac protection. Adult male rats were administered once a day i.p. 1 mg/kg/day dexamethasone or dexamethasone plus 10 mg/kg/day RU486 for 5 days. Hearts were then isolated and subjected to I/R injury in a Langendorff preparation. Dexamethasone treatment significantly decreased I/R injury and improved post-ischemic recovery of cardiac function. Dexamethasone increased glucocorticoid receptor binding to glucocorticoid response elements at AT_1aR and AT₂R promoters, resulting in a significant increase in expression of AT₁R protein but a decrease in AT₂R expression in the heart. In addition, dexamethasone treatment significantly increased PKCε expression and p-PKCε protein abundance. These dexamethasone-mediated effects were blocked by RU486. More importantly, blockade of AT₁R and AT₂R with losartan and PD123319 abrogated dexamethasone-induced protection of the heart from I/R injury. The results indicate that glucocorticoid promotes a cardioprotective phenotype associated with the upregulation of AT₁R and PKCε and downregulation of AT₂R in the heart.     

Heart-type fatty acid binding proteins are upregulated during terminal differentiation of mouse cardiomyocytes,as revealed by proteomic analysis

At birth, the cardiomyocytes in the mouse neonatal heart still retain their ability to proliferate. However, this lasts only a few days and then the cardiomyocytes irreversibly lose their potential to divide. It is still not fully understood what factors are involved in the cessation of cardiomyocyte proliferation. Using proliferating cell nuclear antigen (PCNA) antibodies, we established that cardiomyocytes could divide extensively in 2-day-old mouse neonatal hearts and to a lesser extent in 6-day-old hearts. By 13 days, the cardiomyocytes have mostly stopped dividing. Comparative two-dimensional gel electrophoresis (2-DE) was performed on total proteins extracted from the 2-day- and 13-day-old hearts, in order to identify peptides that might be involved in the inhibition of cardiomyocyte proliferation. Using matrix-assisted laser desorption ionization mass spectroscopy (MALDI-TOF), we identified two protein spots that have the same molecular weight (approximately 14 kDa) but different pIs (5.9 and 6.1). Mass spectra analysis determined the proteins to be isoforms of the heart-type fatty acid binding protein (H-FABP). The pI 6.1 H-FABP is also known as mammary-derived growth inhibitor (MDGI; Specht et al. 1996). MGDI is a breast tumour growth suppressor gene capable of inhibiting tumour cell proliferation (Huynh et al. 1995). Both H-FABP isoforms were expressed in 2-day-old hearts but became strongly upregulated in 13-day-old hearts. We examined whether H-FABPs and PCNA were coexpressed in 2-, 6- and 13-day-old heart histological sections, using MDGI antibodies. The antibody could detect both forms of H-FABPs. It was established that there was a correlation between an increase in H-FABP expression and a decrease in PCNA expression. Hence, we tentatively propose that H-FABP isoforms are involved in regulating cardiomyocyte growth and differentiation in mouse neonatal hearts.This project was supported by a grant from the National Natural Science Foundation of China (Project No. 30340038).     

Thyroid hormone drives fetal cardiomyocyte maturation

Tri-iodo-l-thyronine (T(3)) suppresses the proliferation of near-term serum-stimulated fetal ovine cardiomyocytes in vitro. Thus, we hypothesized that T(3) is a major stimulant of cardiomyocyte maturation in vivo. We studied 3 groups of sheep fetuses on gestational days 125-130 (term ～145 d): a T(3)-infusion group, to mimic fetal term levels (plasma T(3) levels increased from ～0.1 to ～1.0 ng/ml; t(1/2)～24 h); a thyroidectomized group, to produce low thyroid hormone levels; and a vehicle-infusion group, to serve as intact controls. At 130 d of gestation, sections of left ventricular freewall were harvested, and the remaining myocardium was enzymatically dissociated. Proteins involved in cell cycle regulation (p21, cyclin D1), proliferation (ERK), and hypertrophy (mTOR) were measured in left ventricular tissue. Evidence that elevated T(3) augmented the maturation rate of cardiomyocytes included 14% increased width, 31% increase in binucleation, 39% reduction in proliferation, 150% reduction in cyclin D1 protein, and 500% increase in p21 protein. Increased expression of phospho-mTOR, ANP, and SERCA2a also suggests that T(3) promotes maturation and hypertrophy of fetal cardiomyocytes. Thyroidectomized fetuses had reduced cell cycle activity and binucleation. These findings support the hypothesis that T(3) is a prime driver of prenatal cardiomyocyte maturation.     

Regulation of cardiomyocyte proliferation during development and regeneration

The regulation of cardiomyocyte proliferation is important for heart development and regeneration. The proliferation patterns of cardiomyocytes are closely related to heart morphogenesis, size, and functions. The proliferation levels are high during early embryogenesis; however, mammalian cardiomyocytes exit the cell cycle irreversibly soon after birth. The cell cycle exit inhibits cardiac regeneration in mammals. On the other hand, cardiomyocytes of adult zebrafish and probably newts can proliferate after cardiac injury, and the hearts can be regenerated. Therefore, the ability to reproliferate determines regenerative ability. As in other cells, the relationship between proliferation and differentiation is very interesting, and is closely related to cardiac development, regeneration and homeostasis. In this review, these topics are discussed.     

BMP and Notch Signaling Pathways differentially regulate Cardiomyocyte Proliferation during Ventricle Regeneration

Adult mammalian hearts show limited capacity to proliferate after injury, while zebrafish are capable to completely regenerate injured hearts through the proliferation of spared cardiomyocytes. BMP and Notch signaling pathways have been implicated in cardiomyocyte proliferation during zebrafish heart regeneration. However, the molecular mechanism underneath this process as well as the interaction between these two pathways remains to be further explored. In this study we showed BMP signaling was activated after ventricle ablation and acted epistatic downstream of Notch signaling. Inhibition of both signaling pathways differentially influenced ventricle regeneration and cardiomyocyte proliferation, as revealed by time-lapse analysis using a cardiomyocyte-specific FUCCI (fluorescent ubiquitylation-based cell cycle indicator) system. Further experiments revealed that inhibition of BMP and Notch signaling led to cell-cycle arrest at different phases. Overall, our results shed light on the interaction between BMP and Notch signaling pathways and their functions in cardiomyocyte proliferation during cardiac regeneration.     

Extracellular signal-regulated kinase and phosphoinositol-3 kinase mediate IGF-1 induced proliferation of fetal sheep cardiomyocytes

Growth of the fetal heart involves cardiomyocyte enlargement, division, and maturation. Insulin-like growth factor-1 (IGF-1) is implicated in many aspects of growth and is likely to be important in developmental heart growth. IGF-1 stimulates the IGF-1 receptor (IGF1R) and downstream signaling pathways, including extracellular signal-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3K). We hypothesized that IGF-1 stimulates cardiomyocyte proliferation and enlargement through stimulation of the ERK cascade and stimulates cardiomyocyte differentiation through the PI3K cascade. In vivo administration of Long R3 IGF-1 (LR3 IGF-1) did not stimulate cardiomyocyte hypertrophy but led to a decreased percentage of cells that were binucleated in vivo. In culture, LR3 IGF-1 increased myocyte bromodeoxyuridine (BrdU) uptake by three- to five-fold. The blockade of either ERK or PI3K signaling (by UO-126 or LY-294002, respectively) completely abolished BrdU uptake stimulated by LR3 IGF-1. LR3 IGF-1 did not increase footprint area, but as expected, phenylephrine stimulated an increase in binucleated cardiomyocyte size. We conclude that 1) IGF-1 through IGF1R stimulates cardiomyocyte division in vivo; hyperplastic growth is the most likely explanation of IGF-1 stimulated heart growth in vivo; 2) IGF-1 through IGF1R does not stimulate binucleation in vitro or in vivo; 3) IGF-1 through IGF1R does not stimulate hypertrophy either in vivo or in vitro; and 4) IGF-1 through IGF1R requires both ERK and PI3K signaling for proliferation of near-term fetal sheep cardiomyocytes in vitro.     

Evidence from pulse-chase labeling studies that the antiglucocorticoid hormone RU486 stabilizes the nonactivated form of the glucocorticoid receptor in mouse lymphoma cells

A pulse-chase labeling technique was used to determine the properties of glucocorticoid receptors occupied by the antiglucocorticoid hormone RU486 in S49.1 mouse lymphoma cells. Cells were pulse-labeled with [35S]methionine and then at the beginning of the chase, either no hormone (control), dexamethasone, or RU486 was added to cells. At 4 h into the chase, cytosol was prepared and receptors were immunoadsorbed to protein A-Sepharose using the BuGR2 antireceptor antibody. Immunoadsorbed proteins were resolved by gel electrophoresis and analyzed by autoradiography. The 90 kDa heat shock protein (hsp90) coimmunoadsorbed with receptors from control cells when protein A-Sepharose pellets were washed with 250 mM NaCl but not when protein A-Sepharose pellets were washed with 500 mM NaCl, indicating that hsp90-receptor complexes are disrupted by a high concentration of salt in the absence of molybdate. hsp90 coimmunoadsorbed with receptors from RU486-treated cells even when protein A-Sepharose pellets were washed with 500 mM NaCl, indicating that RU486 stabilizes the association of hsp90 with the glucocorticoid receptor. In contrast, hsp90 did not coimmunoadsorb with receptors from dexamethasone-treated cells, consistent with earlier evidence that hsp90 dissociates from the receptor when the receptor binds glucocorticoid hormone. Dexamethasone induced a rapid quantum decrease in the amount of normal receptor recovered from cytosol but did not induce a decrease in the amount of nuclear transfer deficient receptor recovered from cytosol, consistent with tight nuclear binding of normal receptors occupied by dexamethasone. In contrast, RU486 did not induce a quantum decrease in the recovery of normal receptors from cytosol, indicating that receptors occupied by RU486 are not tightly bound in the nuclear fraction. We conclude that the antiglucocorticoid hormone RU486, in contrast to the glucocorticoid hormone dexamethasone, stabilizes the association between the glucocorticoid receptor and hsp90. The decreased affinity of receptors occupied by RU486 for the nuclear fraction may be due to their association with hsp90 and may account for the failure of RU486 to exert agonist activity.     

Histone deacetylase 1 controls cardiomyocyte proliferation during embryonic heart development and cardiac regeneration in zebrafish

In contrast to mammals, the zebrafish maintains its cardiomyocyte proliferation capacity throughout adulthood. However, neither the molecular mechanisms that orchestrate the proliferation of cardiomyocytes during developmental heart growth nor in the context of regeneration in the adult are sufficiently defined yet. We identified in a forward genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screen the recessive, embryonic-lethal zebrafish mutant baldrian (bal), which shows severely impaired developmental heart growth due to diminished cardiomyocyte proliferation. By positional cloning, we identified a missense mutation in the zebrafish histone deacetylase 1 (hdac1) gene leading to severe protein instability and the loss of Hdac1 function in vivo. Hdac1 inhibition significantly reduces cardiomyocyte proliferation, indicating a role of Hdac1 during developmental heart growth in zebrafish. To evaluate whether developmental and regenerative Hdac1-associated mechanisms of cardiomyocyte proliferation are conserved, we analyzed regenerative cardiomyocyte proliferation after Hdac1 inhibition at the wound border zone in cryoinjured adult zebrafish hearts and we found that Hdac1 is also essential to orchestrate regenerative cardiomyocyte proliferation in the adult vertebrate heart. In summary, our findings suggest an important and conserved role of Histone deacetylase 1 (Hdac1) in developmental and adult regenerative cardiomyocyte proliferation in the vertebrate heart.     

Cyclin A2 mediates cardiomyocyte mitosis in the postmitotic myocardium

Cell cycle withdrawal limits proliferation of adult mammalian cardiomyocytes. Therefore, the concept of stimulating myocyte mitotic divisions has dramatic implications for cardiomyocyte regeneration and hence, cardiovascular disease. Previous reports describing manipulation of cell cycle proteins have not shown induction of cardiomyocyte mitosis after birth. We now report that cyclin A2, normally silenced in the postnatal heart, induces cardiac enlargement because of cardiomyocyte hyperplasia when constitutively expressed from embryonic day 8 into adulthood. Cardiomyocyte hyperplasia during adulthood was coupled with an increase in cardiomyocyte mitosis, noted in transgenic hearts at all time points examined, particularly during postnatal development. Several stages of mitosis were observed within cardiomyocytes and correlated with the nuclear localization of cyclin A2. Magnetic resonance analysis confirmed cardiac enlargement. These results reveal a previously unrecognized critical role for cyclin A2 in mediating cardiomyocyte mitosis, a role that may significantly impact upon clinical treatment of damaged myocardium.     

Receptor-mediated effects of glucocorticoids on inflammation: enhancement of the inflammatory response with a glucocorticoid antagonist

Glucocorticoids suppress the inflammatory response by altering leukocyte traffic and function, cytokine secretion and action, and phospholipid metabolism. We employed the glucocorticoid receptor antagonist RU 486, to examine whether glucocorticoids suppress the inflammatory response through a receptor-mediated mechanism and whether basal glucocorticoid secretion exerts antiinflammatory effects in the resting (non-stress) state. To test these hypotheses we evaluated the effects of increasing doses of dexamethasone, RU 486, or dexamethasone plus RU 486 on the exudate volume and concentrations of leukocytes, prostaglandin E2, (PGE2) and leukotriene B4 (LTB4) in intact rats that received subcutaneous carrageenin. Exudate volume, leukocyte concentration and LTB4 and PGE2 levels were all suppressed by dexamethasone in a dose-dependent fashion (P less than 0.005). RU 486 was able to antagonize fully the suppressive effects of dexamethasone on the inflammatory response (P less than 0.001) and to cause increases of exudate volume and leukocyte, PGE2 and LTB4 concentrations when given alone (P less than 0.05). These increases ranged between 30 and 100% above the basal inflammatory response. We conclude that glucocorticoids most likely suppress the inflammatory response by a glucocorticoid receptor-mediated mechanism and under basal conditions exert tonic antiinflammatory effects.     

LRP5 regulates cardiomyocyte proliferation and neonatal heart regeneration by the AKT/P21 pathway

The neonatal heart can efficiently regenerate within a short period after birth, whereas the adult mammalian heart has extremely limited capacity to regenerate. The molecular mechanisms underlying neonatal heart regeneration remain elusive. Here, we revealed that as a coreceptor of Wnt signalling, low‐density lipoprotein receptor‐related protein 5 (LRP5) is required for neonatal heart regeneration by regulating cardiomyocyte proliferation. The expression of LRP5 in the mouse heart gradually decreased after birth, consistent with the time window during which cardiomyocytes withdrew from the cell cycle. LRP5 downregulation reduced the proliferation of neonatal cardiomyocytes, while LRP5 overexpression promoted cardiomyocyte proliferation. The cardiac‐specific deletion of Lrp5 disrupted myocardial regeneration after injury, exhibiting extensive fibrotic scars and cardiac dysfunction. Mechanistically, the decreased heart regeneration ability induced by LRP5 deficiency was mainly due to reduced cardiomyocyte proliferation. Further study identified AKT/P21 signalling as the key pathway accounting for the regulation of cardiomyocyte proliferation mediated by LRP5. LRP5 downregulation accelerated the degradation of AKT, leading to increased expression of the cyclin‐dependent kinase inhibitor P21. Our study revealed that LRP5 is necessary for cardiomyocyte proliferation and neonatal heart regeneration, providing a potential strategy to repair myocardial injury.     

Effects of adrenal hormones on the expression of adiponectin and adiponectin receptors in adipose tissue, muscle and liver

Background

Adiponectin, an insulin-sensitive hormone that is primarily synthesized in adipose tissue, exerts its effects by binding to two receptors, adipoR1 and adipoR2. Little is known regarding the effects of glucocorticoids on the expression of adiponectin receptors.

Methods

Male Wistar rats were bilaterally adrenalectomized and treated with dexamethasone (0.2 mg/100 g) twice daily for 3 days. To analyze the potential effects of glucocorticoids, rats received two daily injections of the glucocorticoid receptor antagonist (RU-486, 5.0 mg) over the course of 3 days. Additionally, 3T3-L1 adipocytes and C2C12 myotubes were treated with dexamethasone, adrenaline or RU-486. The gene expression of adiponectin, adipoR1 and adipoR2 was determined by real-time PCR, and protein secretion was examined by Western blotting using lysates from retroperitoneal, epididymal and subcutaneous adipose tissue depots, liver and muscle.

Results

In rats, excess glucocorticoids increased the levels of insulin in serum and decreased serum adiponectin concentrations, whereas adrenalectomy decreased the mRNA expression of adiponectin (3-fold) and adipoR2 (7-fold) in epididymal adipose tissue and increased adipoR2 gene expression in muscle (3-fold) compared to control group sham-operated. Dexamethasone treatment did not reverse the effects of adrenalectomy, and glucocorticoid receptor blockade did not reproduce the effects of adrenalectomy. In 3T3-L1 adipocytes, dexamethasone and adrenaline both increased adipoR2 mRNA levels, but RU-486 reduced adipoR2 gene expression in vitro.

Conclusion

Dexamethasone treatment induces a state of insulin resistance but does not affect adiponectin receptor expression in adipose tissue. However, the effects of catecholamines on insulin resistance may be due to their effects on adipoR2.     

Expression analysis of Baf60c during heart regeneration in axolotls and neonatal mice

Some organisms, such as zebrafish, urodele amphibians, and newborn mice, have a capacity for heart regeneration following injury. However, adult mammals fail to regenerate their hearts. To know why newborn mice can regenerate their hearts, we focused on epigenetic factors, which are involved in cell differentiation in many tissues. Baf60c (BRG1/BRM‐associated factor 60c), a component of ATP‐dependent chromatin‐remodeling complexes, has an essential role for cardiomyocyte differentiation at the early heart development. To address the function of Baf60c in postnatal heart homeostasis and regeneration, we examined the detailed expression/localization patterns of Baf60c in both mice and axolotls. In the mouse heart development, Baf60c was highly expressed in the entire heart at the early stages, but gradually downregulated at the postnatal stages. During heart regeneration in neonatal mice and axolotls, Baf60c expression was strongly upregulated after resection. Interestingly, the timing of Baf60c upregulation after resection was consistent with the temporal dynamics of cardiomyocyte proliferation. Moreover, knockdown of Baf60c downregulated proliferation of neonatal mouse cardiomyocytes. These data suggested that Baf60c plays an important role in cardiomyocyte proliferation in heart development and regeneration. This is the first study indicating that Baf60c contributes to the heart regeneration in vertebrates.