Newborn Rat Heart Research Articles

Age-Specific Features of the Levels of Reference Proteins Actin, Tubulin, and GAPDH in Different Organs of Male Wistar Rats.

Western blot analysis is used for evaluation of the level of proteins production in organs and tissues, and housekeeping proteins GAPDH, actin, and tubulin are usually used as the reference proteins. The signal of the target protein is normalized to the corresponding signal of the reference protein. The data on the intensity of actin, tubulin, and GAPDH synthesis are fragmentary: their expression differs in different organs and can vary depending on age, which is often not taken into account in experimental studies. We studied the features of the production of reference proteins in the liver, heart, brain, and lungs of newborn, prepubertal, and adult male Wistar rats. Age-related differences in the expression of β-actin, β-tubulin, and GAPDH in the myocardium and dorsal prefrontal cortex were revealed. GAPDH expression in the dorsal prefrontal cortex in adult rats was significantly higher than in prepubertal rats; GAPDH expression in the myocardium of adult rats was significantly higher than in newborns. The level of actin in the dorsal prefrontal cortex in newborn rats was significantly higher than in prepubertal and adult rats. In the liver and lungs, the expression of actin, tubulin, and GAPDH did not differ in newborn, prepubertal, and adult rats. When choosing the reference protein for Western blotting, animals age and the studied organ should be taken into account.

Targeting Myadm to Intervene Pulmonary Hypertension on Rats Before Pregnancy Alleviates the Effect on Their Offspring's Cardiac-Cerebral Systems.

Pregnancy with pulmonary hypertension (PH) seriously threatens the life and safety of mothers and infants. Here, the long-term effect of maternal PH on the postpartum growth of rat offspring was focused for the first time, as well as explored the role of Myadm in PH rats before pregnancy based upon the previous findings. Patients with PH are prone to hypoxemia, leading to insufficient placental structure and function, which affects the organ function of fetuses, followed by evidence that differently expressed genes (DEGs) existed in the heart of maternal PH newborn rats and enriched in pathways related to cardiac and nerve development on human infants with similar birth outcome: low birth weight (LBW). LBW was one of the possible birth outcomes of pregnancy with PH, especially severe PH, accompanied by evidence that offspring derived from mothers with PH presented lower birth weights and slower growth rates than those derived from normal control mothers in a rat model. Besides, maternal PH rat offspring showed cardiac remodeling and a significant elevation of the expression levels of hypoxia- and inflammation-related markers in the cerebral cortex at both 10 and 14 weeks of age, respectively. What is more, the previous studies found that the overexpression of Myadm could result in the remodeling of the pulmonary artery. And targeting Myadm to intervene PH before pregnancy could alleviate sustained low weight growth in maternal PH rat offspring, and the pathological changes of the cardiac–cerebral system caused by maternal PH, including enlarged right heart cavity, loss of cardiomyocytes, abnormal heart index, as well as cerebral cortex hypoxia and the inflammatory state as they grew up to a certain extent. The findings show the pathological significance of maternal PH on offspring growth and the cardiac–cerebral development in a rat model, as well as point out the potential treatment target, which may provide a further reference for pregnancy outcomes in women with PH and healthy development of offspring to some extent.

Effect of Clonidine Hydrochloride on Isolated Newborn Rat Heart.

The concentration dependenies of the chronotropic response and changes in blood supply to the isolated heart of 7-day-old newborn rats induced by application of α2-adrenergic receptor agonist clonidine hydrochloride in concentrations of 10-9-10-6 M were revealed. The minimum concentration of α2-adrenergic receptor agonist caused tachycardia, while higher concentrations led to bradycardia. The maximum effect manifesting in a decrease in coronary flow was recorded at the minimum concentration of the agonist, while the highest concentration had no effect on the coronary flow. When comparing these results with those obtained in control adult rats, we found that the most pronounced differences in the chronotropic effects were observed after addition of the minimum concentration of the α2-adrenergic receptor agonist: bradycardia in adult rats and tachycardia in newborns. The maximum differences in coronary flow parameters were observed after addition of α2-adrenergic receptor agonist in the maximum concentration that induced a two-phase response in adult rats and had no effect on the blood supply in newborns.

Conophylline Suppresses Angiotensin II-Induced Myocardial Fibrosis In Vitro via the BMP4/JNK Pathway.

We studied the effects and mechanisms of action of conophylline in different concentrations in the original in vitro model of myocardial fibrosis (treatment of cardiac fibroblasts isolated form the hearts of newborn rats with angiotensin II). Viability, collagen content, and expression of related protein in cardiac fibroblasts were assessed using the MTT-test, Sircol assay, and Western blotting, respectively. Conophylline markedly protected the cultured cells against the development of angiotensin II-induced fibrosis, which was seen from reduced viability of fibroblasts, decreased collagen content, and down-regulation of the expression of α-smooth muscle actin (α-SMA). Conophylline did not affect the TGF-β pathway altered by angiotensin II, but markedly decreased the level of bone morphogenetic protein-4 (BMP4) enhanced by angiotensin II and BMP4 itself. Conophylline produced no effect on phosphorylation of α-SMA and Smad homologue-1/5/8, the classic BMP4 downstream pathway elements, but reduced the level of c-Jun N-terminal kinase (JNK) elevated by BMP4. Conophylline did not inhibit the development of myocardial fibrosis in the presence of JNK activator anisomycin. Thus, conophylline inhibited angiotensin II-provoked myocardial fibrosis via the BMP4/JNK pathway.

Optical Metabolic Imaging of Mitochondrial Dysfunction on HADH Mutant Newborn Rat Hearts.

Background: Mitochondrial n}{}beta n-oxidation of fatty acids is the primary energy source for the heart and carried out by Hydroxy Acyl-CoA Dehydrogenase (HADH) encoded trifunctional protein. Mutations in the genes encoding mitochondrial proteins result in functionally defective protein complexes that contribute to energy deficiencies, excessive reactive oxygen species (ROS) production, and accumulation of damaged mitochondria. We hypothesize that a dramatic alternation in redox state and associated mitochondrial dysfunction is the underlying cause of Fatty Acid Oxidation (FAO) deficiency mutant, resulting in heart failure. Mitochondrial co-enzymes, NADH and FAD, are autofluorescent metabolic indices of cells when imaged, yield a quantitative assessment of the cells’ redox status and, in turn, that of the tissue and organ. Method: We utilized an optical cryo-imager to quantitively evaluate the three-dimensional distribution of mitochondrial redox state in newborn rats’ hearts and kidneys. Redox ratio (RR) assessment shows that mitochondrial dysfunction is extreme and could contribute to severe heart problems and eventual heart failure in the mutants. Results: Three-dimensional redox ratio (NADH/FAD) rendering, and the volumetric mean value calculations confirmed significantly decreased cardiac RR in mutants by 31.90% and 12.32%, in renal mitochondrial RR compared to wild-type control. Further, histological assessment of newborn heart myocardial tissue indicated no significant difference in myocardial tissue architecture in both control and severe (HADHAe4−/−) conditions. Conclusion: These results demonstrate that optical imaging can accurately estimate the redox state changes in newborn rat organs. It is also apparent that the FAO mutant’s heart tissue with a low redox ratio is probably more vulnerable to cumulative damages than kidneys and fails prematurely, contributing to sudden death.

Maternal High Fat Diet and Diabetes Disrupts Transcriptomic Pathways That Regulate Cardiac Metabolism and Cell Fate in Newborn Rat Hearts.

Background: Children born to diabetic or obese mothers have a higher risk of heart disease at birth and later in life. Using chromatin immunoprecipitation sequencing, we previously demonstrated that late-gestation diabetes, maternal high fat (HF) diet, and the combination causes distinct fuel-mediated epigenetic reprogramming of rat cardiac tissue during fetal cardiogenesis. The objective of the present study was to investigate the overall transcriptional signature of newborn offspring exposed to maternal diabetes and maternal H diet.Methods: Microarray gene expression profiling of hearts from diabetes exposed, HF diet exposed, and combination exposed newborn rats was compared to controls. Functional annotation, pathway and network analysis of differentially expressed genes were performed in combination exposed and control newborn rat hearts. Further downstream metabolic assessments included measurement of total and phosphorylated AKT2 and GSK3β, as well as quantification of glycolytic capacity by extracellular flux analysis and glycogen staining.Results: Transcriptional analysis identified significant fuel-mediated changes in offspring cardiac gene expression. Specifically, functional pathways analysis identified two key signaling cascades that were functionally prioritized in combination exposed offspring hearts: (1) downregulation of fibroblast growth factor (FGF) activated PI3K/AKT pathway and (2) upregulation of peroxisome proliferator-activated receptor gamma coactivator alpha (PGC1α) mitochondrial biogenesis signaling. Functional metabolic and histochemical assays supported these transcriptome changes, corroborating diabetes- and diet-induced cardiac transcriptome remodeling and cardiac metabolism in offspring.Conclusion: This study provides the first data accounting for the compounding effects of maternal hyperglycemia and hyperlipidemia on the developmental cardiac transcriptome, and elucidates nuanced and novel features of maternal diabetes and diet on regulation of heart health.

Spermine attenuates high glucose-induced myocardial fibrosis by regulating the cell cycle

Objective: To investigate the protective effect of spermine (Sp) on diabetic cardiomyopathy (DCM) and high glucose-induced cardiac fibroblasts (CFs), and to explore its mechanism.Methods: ①Animal experiments: 24 male Wistar rats were randomly divided into control group, type 1 diabetes group (TID) and spermine group (TID+Sp, each group n=8). TID rats were induced by streptozocin (STZ, 60 mg/kg), and TID+Sp rat were pretreated with spermine (Sp, 5 mg/(kg·d)) for 2 weeks before STZ injection. After 12 weeks of modeling, blood glucose, insulin levels, ejection fraction (EF) and shortening fraction (FS) were measured, and Masson staining and Sirius red staining were performed in the rat cardiac tissues. ②Cell experiments: primary CFs were extracted from newborn (1-3 d) Wistar rat hearts, and were randomly divided into control group, high-glucose group (HG) and HG+Sp group (n=6 per group). HG group was treated with 40 mmol/L glucose, and the HG+Sp group was pretreated with 5 μmol/L Sp for 30 min before HG treatment. The cell viability of CFs was detected by CCK8, the content of collagen in culture medium was analyzed by ELISA, and protein expressions of cell cycle related proteins (PCNA, CyclinD1 and P27) were detected by Western blot. Results: Compared with control group, the blood glucose and collagen content were increased, and the insulin level and heart function were decreased in the T1D group. Meanwhile, HG induced an increasing of the cell viability, the collagen content in the medium and the expressions of PCNA and CyclinD1, while the expression of P27 was down-regulated. Spermine could reduce the above changes, manifested as improving the cardiac function, regulating the expression of cyclin and reducing the level of myocardial fibrosis. Conclusion: Spermine can alleviate myocardial fibrosis in diabetic cardiomyopathy, which mechanism is related to the regulation of cell cycle.

The role of nitric oxide in the antioxidant effect of the non-opiate analogue of leu-enkephalin in the heart of newborn albino rats

The eff ect of the non-opiate analog of leu-enkephalin (peptide NALE: Phe – D – Ala – Gly – Phe – Leu – Arg) on the reactive oxygen species generation in the heart of albino rats in the early postnatal period was studied. Peptide NALE was administered intraperitoneally in the dose of 100 μ/kg daily from 2 to 6 days of life. Reactive oxygen species generation was assessed by chemiluminescence in the heart homogenates of 7-day-old animals. Decreasing of reactive oxygen species generation nearly by 30 % and an increasing in antioxidant system activity by the 20-27 %, compared with the control parameters, were found. The antioxidant eff ect of peptide NALE is associated with the presence of the amino acid Arg in the structure of the peptide. An analogue of NALE peptide, devoid of Arg (peptide Phe – D – Ala – Gly – Phe – Leu – Gly), had a signifi cant lower antioxidant eff ect. The NO-synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) in the dose 50 mg/kg, administered with NALE peptide, reduced the severity of the NALE antioxidant eff ect. The results of the study suggest that the pronounced antioxidant eff ect of NALE peptide in the heart of albino rats, at least in part, is due to the interaction with the nitric oxide system.

Effect of If Current Blockade on Newborn Rat Heart Isolated According to Langendorff.

The study examined the effects of hyperpolarization-activated funny current (If) on HR and coronary flow in Langendorff-isolated hearts from newborn rats. Blockade of If current with ZD7288 changed the examined cardiac parameters. The blocker in a concentration of 10-9 M decreased HR by 26.8% (p≤0.05). In concentrations 10-8, 10-7, 10-6, and 10-5 M ZD7288 produced minor differently directed effects. In a concentration of 10-5 M, ZD7288 reduced coronary flow in the isolated heart (p≤0.01). In other concentrations, the blocker produced no significant effects on coronary flow.

Structural and functional characteristics of endothelial cells of vessels of the heart of newborn rats (immunohistochemical study)

The study is aimed to investigate the pattern of cytodifferentiation of developing endothelial cells (EC) of microcirculatory bed of the heart of newborn rats using light microscopy and immunohistochemical labeling of von Willebrand factor (vWF). Immunohistochemical study was carried out on Wistar rats on the postnatal day 1 (n=6). For the first time, EC of the developing vessels of the heart of newborn rats were found to be on different stages of cytodifferentiation. Most pronounced structural and functional maturity at this developmental stage was typical for EC of intima of aorta, coronary vessels, middle and small arteries and arterioles of the myocardium in the upper third of the ventricles. Endothelium of these vessels demonstrates strong vWF-immunoreactivity, while in the developing capillaries of the sinusoidal type of the subepicardial region and ventricular endocardium, the vWF-immunoreaction at this time period is still weak or absent. Morphological pattern and variability of sizes of vWF-immunoreactive granules of endothelium has been established. Signs of secretion in the EC were revealed. Synthesis, accumulation, and exocytosis of vWF are suggested to be associated with the degree of cytodifferentiation of EC.

Extracellular NAD+ Suppresses Adrenergic Effects in the Atrial Myocardium of Rats during the Early Postnatal Ontogeny.

The effects of sympathetic cotransmitter NAD+ (10 μM) on bioelectric activity of the heart under conditions of adrenergic stimulation were studied on isolated spontaneously contracting preparations (without stimulation) of the right atrium from 2-7-day-old rats. Action potentials were recorded in the working myocardium using standard microelectrode technique. Perfusion of the right atrium with norepinephrine solution (1 μM) altered the configuration and significantly lengthened the action potentials. NAD + against the background of norepinephrine stimulation significantly decreased the duration of action potentials, in particular, at 25% repolarization. The effect of purine compounds NAD + , ATP, and adenosine on bioelectrical activity of the heart of newborn rats was studied under basal conditions (without norepinephrine stimulation). The effect of NAD + against the background of adrenergic stimulation was more pronounced than under basal conditions and was probably determined by suppression of ICaL, which can be the main mechanism of NAD + action on rat heart.

Intrauterine hyperglycemia-induced inflammatory signalling via the receptor for advanced glycation end products in the cardiac muscle of the infants of diabetic mother rats.

Gestational diabetes is associated with increased risk to the health of the mother and her offspring. In particular, the infants of diabetic mothers (IDMs) exhibit elevated levels of preterm birth, macrosomia, hypoglycemia, hypocalcemia, and cardiomyopathy. We have previously reported that IDMs showed abnormalities in cardiac Akt-related insulin signalling, and that these deficiencies in Akt-related signalling were attenuated by supplementing the maternal diet with fish-oil. Herein, we investigated whether the eicosapentaenoic acid (EPA) found in fish oil can be used to attenuate diabetes associated impairments in cardiomyocyte signalling. Pregnant diabetic rats were administered streptozotocin before receiving EPA or water, and their infants were designated IDM/EPA, IDM/W. We assessed the potential molecular pathway for this effect in the primary cardiac cell from newborn rat hearts. Insulin resistance as determined by diminished GLUT4 translocation following insulin stimulation, the levels of advanced glycation end products (AGEs) and reactive oxygen species were elevated in the neonatal hearts of IDM/W compared with that seen in the offspring born from non-diabetic control animals. Similarly, the receptor of AGEs (RAGE) mRNA levels, reactive oxygen species and the amount of nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6) mRNA were higher in the hearts from the IDM/W when compared to that observed in the hearts of offspring born to non-diabetic animals. These deleterious effects of gestational diabetes were significantly decreased in the offspring of diabetic mothers receiving EPA supplementation. Taken together, our data suggest that the EPA in fish oil may improve the impaired signalling and the excessive protein glycation in the cardiac muscles of infants exposed to intrauterine hyperglycemia.

URSODEOXYCHOLIC ACID REGULATES CASPASE-9 AND ROS PRODUCTION IN PROTECTING CARDIOMYOCYTES AGAINST HYPOXIA

Ursodeoxycholic acid (UDCA) is known as a therapeutic agent in treating cholestasis and liver diseases. Recently, UDCA has been suggested as a therapeutic drug for heart related diseases. Cardioprotective effect of UDCA against the development of ischemia has been studied. Yet, the mechanism of UDCA-cardioprotection is not clearly understood. Therefore, this study aimed to elucidate the mechanisms of UDCA cardioprotection against hypoxia by investigating the expression of caspase -3/-9 and ROS generation using an in vitro hypoxic heart model. A newborn (0-2 days old) rat heart was isolated for primary cell culture of cardiomyocytes. Hypoxia was chemically induced by using CoCl2. Cardiomyocytes were then incubated with UDCA. The treated cardiomyocytes were subjected for ROS generation detection assay, QuantiGene Plex assay for caspase-3/-9 gene expression and ELISA for caspase-3/-9 protein expression. The data were analyzed by using sample paired t-test and One-way ANOVA. Our results showed that UDCA abolishes the effects on CoCl2 in ROS production and UDCA downregulates caspase-9 protein expression in CoCl2 treated cardiomyocytes. This study provides an insight of UDCA in protecting cardiomyocytes against hypoxia mediated by anti-apoptosis mechanism.

N-3 long chain polyunsaturated fatty acids increase the expression of PPARγ-target genes and resistance of isolated heart and cultured cardiomyocytes to ischemic injury.

The goal of present investigation is to check the hypothesis that cardioprotective effect of polyunsaturated fatty acids (PUFAs) is mediated by influence on mRNA expression level of the FATP, IL-1ra and GJP43 through stimulation of PPARγ. Animals obtained n-3 PUFAs orally during 4 weeks (0.1ml/100g b.w. per day) or during prenatal period. In experiments, isolated perfused hearts were subjected to 20-min regional ischemia and 40min reperfusion. The hearts of newborn rats (2-3days old) were used for isolated cardiomyocytes culture preparing. Culture cells underwent 30min of anoxia followed by 60min of reoxygenation. Using rtPCR the level of FATP, IL-1ra, GJP43 and BCL2 mRNA in isolated cardiomyocyte and hearts was evaluated. The data obtained indicate that in heart tissues from pups with prenatal n-3 PUFAs application the level of LA and DHA acids were increased in 3.6-fold and 2-fold correspondingly comparing to control. In adult hearts the level of DHA was increased in 1.4-fold, and the level of EPA-in 6.9-fold. We observed the increase in mRNA level of PPARγ-target genes: FATP in 2.25 times, and IL-1ra in 8.4 times. At the same time the level of mRNA of antiapoptotic gene BCL2 was increased in 2.13 times and Connexin-43 gene in 2.2 times after n-3 PUFAs application. These effects were accompanied by significantly improved cardiac function, and increase of living cardiomyocytes number at modeling of ischemia-reperfusion. n-3 PUFAs application has cardioprotective effects and increases mRNA level of FATP, IL-1ra, GJP43, and BCL2 genes in culture of neonatal cardiomyocytes and in adult hearts.

Morphological changes of rat atrial cardiomyocytes contractile apparatus in normal conditions and under the influence of acute and chronic prenatal hypoxia. Role of α-smooth muscle actin in myofibrilogenesis.

Background. There are only a few researches considering the influence of hypoxia on myofibrils formation and cardiomyocytes differentiation. However the role of α-smooth muscle actin on sarcomyogenesis is still unclear, that leads to the further study of this question. Objective. The purpose of the work was to determine ontogenetic changes of myofibrils and cardiomyocytes differentiation in rat atrial myocardium in normal conditions and under the influence of acute and chronic prenatal hypoxia. Methods. Embryo hearts were investigated on 14th, 16th days of prenatal ontogenesis, newborn rat hearts and the hearts of rats – on 30th day of postnatal ontogenesis. Animals were subdivided into three groups: 1stexperimental group of animals was exposed to acute prenatal hypoxia, 2nd experimental group animals was exposed to chronic prenatal hypoxia and control group animals. Hypoxia modeling was conducted on pregnant females by injection of 1% sodium nitrite intraperitonealy in doses that lead to moderate hypoxia. During the work complex of histological, immunohistochemical and electron microscopy methods were used. Antibodies to α-smooth muscle actin (α-SMA) were used to study cardiomyocytes differentiation. The digital images were analyzed using a computer with NIH ImageJ program. Results and conclusion. The results show that myofibrillar differentiation is associated with reduction of α-SMA expression. Ultrastructural changes of rat atrial cardiomyocytes in 3 days after the influence of acute prenatal hypoxia manifested in disruption of myofibrillar orientation, accompanied with decreasing α-SMA expression. Chronic prenatal hypoxia from 16thday of prenatal ontogenesis to the 1stday of postnatal ontogenesis manifestated in myofibrillar defragmentation and disorientation, associated with decreased α-SMA expression compared with the control group. Conclusion. α-SMA can be used as a marker of cardiomyocytes differentiation.

Ultrastructural and tissue restructuring of the rat atrial myocardium under the influence of acute and chronic prenatal hypoxia.

Background. There are relatively few researches that focused on the study of the influence of hypoxia on atrial myocardium. However the results of previous researches are contradictory and fragmentary, that leades to the further study of this item. Objective. Purpose of the work was to determine the tissue, cellular and ultrastructural changes of the rat atrial myocardium during the stages of cardiogenesis under the conditions of acute and chronic prenatal hypoxia. Methods. Embryo hearts were investigated on 14th, 16th and 18th day of prenatal ontogenesis, newborn rat hearts and the hearts of rats on the 3rd, 14th and 30th day of postnatal ontogenesis. Animals were subdivided into three groups: first experimental group animals were exposed to acute prenatal hypoxia, second experimental group animals were exposed to chronic prenatal hypoxia and control group animals. Hypoxia modeling was conducted on pregnant females by injection of 1% sodium nitrite intraperitonealy in doses that lead to moderate hypoxia. During the work complex of histological, immunohistochemical and morphometric methods was used. Results and conclusion. Effect of acute hypoxia leads to stimulation of proliferative activity of fibroblasts and endothelial cells on the right atrium, which causes to increasing of the relative volume of connective tissue and blood vessels of microvasculature of myocardium. Relative volume of connective tissue of the atrial myocardium exceeds significantly the control level of newborn rats. Ultrastructural changes of the atrial cardiomyocytes in 3 days after the influence of acute prenatal hypoxia reveals on violation of mitochondrial structure, significant inhibition of myofibrils accumulation. Until the birth of most of morphometric parameters renewal and reach control levels, however changes of the degree of myofibrils orientation on the left atrium proves stability of hypoxic damages. Chronic prenatal hypoxia causes a significant increase of relative volume of connective tissue and microvessels content of the myocardium compared with control values. Proliferative activity of fibroblasts and endothelial cells stays increased on the atrial myocardium of newborn rats. Relative volume of connective tissue observed higher significantly in 30 days after the birth. Persistent intracellular hypoxic changes of cardiomyocytes, which observed in 30 days after the rats birth, are increase numerical density of mitochondria, decrease of relative volume of myofibrils and degree of myofibrils orientation on the left atrium.

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.

Developmental determinants of cardiac sensitivity to hypoxia.

Cardiac sensitivity to oxygen deprivation changes significantly during ontogenetic development. However, the mechanisms for the higher tolerance of the immature heart, possibilities of protection, and the potential impact of perinatal hypoxia on cardiac tolerance to oxygen deprivation in adults have not yet been satisfactorily clarified. The hypoxic tolerance of an isolated rat heart showed a triphasic pattern: significant decrease from postnatal day 1 to 7, followed by increase to the weaning period, and final decline to adulthood. We have observed significant ontogenetic changes in mitochondrial oxidative phosphorylation and mitochondrial membrane potential, as well as in the role of the mitochondrial permeability transition pores in myocardial injury. These results support the hypothesis that cardiac mitochondria are deeply involved in the regulation of cardiac tolerance to oxygen deprivation during ontogenetic development. Ischemic preconditioning failed to increase tolerance to oxygen deprivation in the highly tolerant hearts of newborn rats. Chronic hypoxic exposure during early development may cause in-utero or neonatal programming of several genes that can change the susceptibility of the adult heart to ischemia-reperfusion injury; this effect is sex dependent. These results would have important clinical implications, since cardiac sensitivity in adult patients may be significantly affected by perinatal hypoxia in a sex-dependent manner.

NF-κB involvement in hyperoxia-induced myocardial damage in newborn rat hearts

Premature newborns are frequently exposed to hyperoxia ventilation and some literature data indicate the possibility of hyperoxia-induced myocardial damage. Since nuclear factor κB (NF-κB) is a crucial signaling molecule involved in physiological response to hyperoxia in different cell types as well as in various tissues, our attention has been focused on the role played by NF-κB pathway in response to moderate and severe hyperoxia exposure in rat neonatal heart tissue. Akt and IκBα levels, involved in NF-κB activation, along with the balance between apoptotic and survival pathways have also been investigated. Experimental design of the study has involved exposure of newborn rats to room air (controls), 60 % O2 (moderate hyperoxia), or 95 % O2 (severe hyperoxia) for the first two postnatal weeks. Morphological analysis shows a less compact tissue in rat heart exposed to moderate hyperoxia and a decreased number of nuclei in samples exposed to severe hyperoxia. A significant increase of NF-κB positive nuclei percentage and p-IκBα expression in samples exposed to 95 % hyperoxia compared to control and to 60 % hyperoxia is evidenced; in parallel, an increase of pAkt/Akt ratio in both samples exposed to 95 and 60 % hyperoxia is shown. Furthermore, a more evident cytochrome c/Apaf-1 immunocomplex and a decreased Bcl2 expression in 95 % hyperoxia-exposed sample compared to 60 % exposed one is evidenced. In conclusion, our findings suggest the involvement of the NF-κB pathway and Akt signaling in the mechanisms of myocardial hyperoxic damage in the newborns, with particular reference to the induction of oxidative stress-related apoptosis.

A maternal diet rich in fish oil may improve cardiac Akt-related signaling in the offspring of diabetic mother rats

ObjectiveNewborns of diabetic mothers have abnormal circulatory organs, so in this study, we explore insulin signaling in the newborn rat heart. MethodsPregnant rats were divided into streptozotocin-induced diabetic groups (DM) and control groups (CM). Rats were fed lard (21% fat), fish oil (21% fat), or a control diet (7% fat). To examine changes in insulin signaling in the hearts of infants of diabetic mothers (IDM) in relation to diet, we isolated the hearts from the IDM and control infants and determined the phosphorylation levels of Akt308, Akt473, p38, c-jun-NH2-terminal protein kinase (JNK), and extracellular signal-regulated protein kinase (ERK), and the expression levels of phosphoinositide-dependent protein kainase1 (PDK1) and mammalian target of rapamycin (mTOR). ResultsThe mean blood glucose levels in the DM group and their infants were significantly higher than those in the CM group (P < 0.05) and their infants (P < 0.05), but the mean blood glucose levels of all infants was normal on postnatal d 4. Phosphorylation levels of Akt Thr 308 (P < 0.05) and Akt Ser 473 and the expression levels of PDK1 and mTOR were lower in infants of diabetic mothers (IDM) than in control infants. The phosphorylation level of Akt Ser 473 and the expression level of mTOR increased in IDM fed the fish oil diet compared with those fed the lard diet (P < 0.05). ConclusionA maternal diet rich in fish oil improves cardiac Akt-related signaling in the offspring of diabetic rats.