Total Number Of Cardiomyocytes Research Articles

Abstract 17359: UCP2 Induced Glycolysis Promotes Histone Acetylation to Regulate Cardiomyocyte Cell Cycle After Injury

Developmental cardiac tissue is proliferative capable of robust regenerative response after myocardial injury, but this ability is lost by P7 when cardiomyocytes (CMs) exit the cell cycle and become terminally differentiated. Postnatal CMs exhibit a specialized metabolic state that supports rapid proliferation and is altered with loss of regeneration in the heart. Studies have shown that reliance on glycolysis for energy generation is preferred by proliferating CMs and activation of glycolysis promotes cardiac repair. Nevertheless, the role of glycolysis in regulating CM gene program linked to cell cycle and repair in the heart remains poorly studied. Here, we identify a novel role for mitochondrial uncoupling protein 2 (UCP2) in the heart regulating CM cell cycle dynamics. UCP2 was found to be primarily active during cardiac development, rapidly decreases after birth and is expressed at low levels in the adult heart. UCP2 overexpression in human IPS derived CMs increases cell cycle progression as assessed by immunostaining and flow cytometry-based DNA content analysis. In parallel, neonatal rat ventricular myocytes (NRVMs) overexpressing UCP2 showed increased glycolysis and glycolytic metabolites, reduced mitochondrial membrane potential and mitochondrial activity compared to controls. Next, we generated αMHC-UCPKO mice that showed reduction in total number of CMs and pHH3 levels, increased CM size and ploidy during postnatal development. Adult αMHC-UCPKO subjected to myocardial infarction injury showed significant cardiac dysfunction and fibrosis at 4 weeks compared to controls. Mechanistically, UCP2 induced glycolysis increases ATP-citrate lyase (ACLY) mediated acetyl-CoA generation in the CMs. Additionally, CMs isolated from P1 αMHC-UCPKO showed several histone modifications including decrease in histone acetylation marks. Similarly, NRVMs overexpressing UCP2 showed increased histone H3 acetylation and expression of histone acetyltransferases (HATs) and corresponding decrease in histone deacetylases (HDACs). In conclusion, UCP2 is a novel developmental metabolic regulator of CM cell cycle in the neonatal and adult heart. Moreover, UCP2 induced glycolysis increases cell cycle via altering CM histone acetylation.

Swimming exercise modifies oxidative stress in skeletal and cardiac muscles of diabetic rats

Introduction: Oxidative stress is a key factor leading to the deterioration of diabetes. Oxidative stress exacerbates diabetes and induction of the activity of the antioxidant system may be required to prevent this effect. Objetive: The aim of the present study was to evaluate the redox state in the skeletal and cardiac muscles in a diabetes rat model subjected to swimming exercise for 4 weeks. Methods: Wistar rats were divided into four groups: untrained control (C), trained control (T), untrained alloxan-induced diabetes (D), and trained alloxan-induced diabetes (TD). The redox state of the skeletal and cardiac muscles was assessed by analyzing TBARS, -SH groups, H2O2 production, and SOD and catalase activity. The total number of cardiomyocytes and the total area of collagen fibers in the cardiac muscle were measured by histomorphometry. Results: In the Soleus muscles, the TD group showed increased H2O2 levels and catalase activity compared to the T group, and SOD activity compared to the D group. Regarding the red gastrocnemius, the TD group presented higher SOD and lower catalase activities than the D group. Regarding the cardiac muscle, the TD group presented lower TBARS and higher levels of -SH groups and catalase activity than the D group. Swimming exercise decreased hyperglycemia and reduced pathology, as evidenced by the reduced number of cardiomyocytes and the area of collagen fibers. Conclusion: Swimming exercise in diabetic rats controlled hyperglycemia and oxidative damage, and the reduced fibrosis in the cardiac muscle of diabetic rats.

Targeting calcineurin induces cardiomyocyte proliferation in adult mice.

The mammalian neonatal heart can regenerate for 1 week after birth, after which, the majority of cardiomyocytes exit the cell cycle. Recent studies demonstrated that calcineurin mediates cell-cycle arrest of postnatal cardiomyocytes, partly through induction of nuclear translocation of the transcription factor Hoxb13 (a cofactor of Meis1). Here we show that inducible cardiomyocyte-specific deletion of calcineurin B1 in adult cardiomyocytes markedly decreases cardiomyocyte size and promotes mitotic entry, resulting in increased total cardiomyocyte number and improved left ventricular (LV) systolic function after myocardial infarction (MI). Similarly, pharmacological inhibition of calcineurin activity using FK506 promotes cardiomyocyte proliferation in vivo and increases cardiomyocyte number; however, FK506 administration after MI in mice failed to improve LV systolic function, possibly due to inhibition of vasculogenesis and blunting of the post-MI inflammatory response. Collectively, our results demonstrate that loss of calcineurin activity in adult cardiomyocytes promotes cell cycle entry; however, the effects of the calcineurin inhibitor FK506 on other cell types preclude a significant improvement of LV systolic function after MI.

Glucocorticoids rapidly promote YAP phosphorylation via the cAMP-PKA pathway to repress mouse cardiomyocyte proliferative potential

Adult mammalian cardiomyocytes (CMs) lose their proliferative potential due to cell-cycle withdrawal and polyploidization and fail to mount a proliferative response to regenerate new CMs after cardiac injury. The decline in the proliferative potential of mammalian CMs occurs in the neonatal period when the endocrine system undergoes drastic changes for adaptation to extra-uterine life. There is an increase in circulating glucocorticoid (GC) levels shortly after birth in mammals, and thus, we sought to determine the roles and mechanisms of GCs in regulating CM proliferation. Here, we showed that GCs suppressed CM proliferation in vitro and in vivo, decreased the total number of CMs, and increased the cross-sectional area of CMs. However, the glucocorticoid receptor antagonist had no effect on CM proliferation. Agonists of adenylate cyclase and protein kinase A (PKA) inhibited CM proliferation, while PKA antagonists or knockdown of PKA alleviated the inhibitory effect of GCs on CM proliferation. GCs and the activation of the cyclic adenosine monophosphate (cAMP)/PKA signaling pathway facilitated yes-associated protein (YAP) phosphorylation in mouse CMs and promoted YAP protein translocation from the nucleus to the cytoplasm. Meanwhile, blocking the cAMP/PKA signaling pathway partially blocked the effect of GCs on YAP protein phosphorylation and YAP protein translocation. Thus, our findings suggest that GCs suppress mouse CM proliferation in vitro and in vivo, through a mechanism that involves targeting the cAMP-PKA-YAP signaling pathway.

ЭКСПЕРИМЕНТАЛЬНОЕ ИССЛЕДОВАНИЕ ВЛИЯНИЯ ПОЛИФЕНОЛОВ ВИНОГРАДА НА МИОКАРД НА ФОНЕ ХОЛОДОВОЙ ГИПОБАРИИ

The aim of this research was to reveal the cardioprotective properties of food concentrate polyphenols grapes during experimental hypobaric hypoxia. The study was conducted on mature male Wistar rats (n = 25) aged 1-1.5 months divided into three experimental groups. In the I (control) group 0,9% NaCl solution was administered intragastrically to intact animals. Hypobaric cold hypoxia was modeled for rats of groups II and III once a day for 30 days. Animals III series 30 minutes before hypoxia intragastrically through a tube were injected an aqueous solution of «Fenokor» containing grape polyphenols. In rats of II group without correction there was an unreliable decrease in heart mass by 8.71%, a decrease in the diameter of cardiomyocytes by 20.3% was accompanied by a decrease in their concentration in 1 mg of myocardium. Accelerated compensatory physiological growth of cardiomyocytes in animals of group III caused a slight decrease in the diameter of a number of cardiomyocytes by 3.75% with almost no decrease in their concentration, that mostly preserved of the total number of cardiomyocytes in the heart by the end of the experiment. Hypertrophic growth of the number of cardiomyocytes is caused by the end of the experiment statistically nonsignificant compensatory increase in the mass of the heart. Thus, the main morphological signs of hypoxic myocardial damage in young rats were presented by the phenomena of mixed dystrophy, edema and destruction of contractile cardiomyocytes, lysis of myofibrils and mitochondrial cristae, contracture changes. The administration of «Fenokor» demonstarated its cytoprotective properties, contributed to the preservation of myocardial structure of rats exposed to hypobaric hypoxia.

Fatty Acid Oxidation Promotes Cardiomyocyte Proliferation Rate but Does Not Change Cardiomyocyte Number in Infant Mice.

Cardiomyocyte proliferation accounts for the increase of cardiac muscle during fetal mammalian heart development. Shortly after birth, cardiomyocyte transits from hyperplasia to hypertrophic growth. Here, we have investigated the role of fatty acid β-oxidation in cardiomyocyte proliferation and hypertrophic growth during early postnatal life in mice. A transient wave of increased cell cycle activity of cardiomyocyte was observed between postnatal day 3 and 5, that proceeded as cardiomyocyte hypertrophic growth and maturation. Assessment of cardiomyocyte metabolism in neonatal mouse heart revealed a myocardial metabolic shift from glycolysis to fatty acid β-oxidation that coincided with the burst of cardiomyocyte cell cycle reactivation and hypertrophic growth. Inhibition of fatty acid β-oxidation metabolism in infant mouse heart delayed cardiomyocyte cell cycle exit, hypertrophic growth and maturation. By contrast, pharmacologic and genetic activation of PPARα, a major regulator of cardiac fatty acid metabolism, induced fatty acid β-oxidation and initially promoted cardiomyocyte proliferation rate in infant mice. As the cell cycle proceeded, activation of PPARα-mediated fatty acid β-oxidation promoted cardiomyocytes hypertrophic growth and maturation, which led to cell cycle exit. As a consequence, activation of PPARα-mediated fatty acid β-oxidation did not alter the total number of cardiomyocytes in infant mice. These findings indicate a unique role of fatty acid β-oxidation in regulating cardiomyocyte proliferation and hypertrophic growth in infant mice.

Is the cardioprotective effect of LCZ696 linked to increased cardiac regeneration?

LCZ696 treatment reduces the mortality and the risk of hospitalization of patients with hypertension or heart failure with a preserved ejection fraction by 20%. The LCZ696 molecule associates both an angiotensin receptor blocker (valsartan) and an inhibitor of neprilysin (NEP, sacubitril). NEP is an endopeptidase able to degrade several factors such as the natriuretic peptides, angiotensin II, bradykinin, endothelin-1. Despite the cardioprotective effect of LCZ696, its exact mechanism in the heart is not elucidated yet. The aim of this project is to determine whether a part of the cardioprotective effect of this treatment is due to the stimulation of heart regeneration. For this purpose, myocardial infarction was induced in 8–10 weeks-old C57BL/6 mice by ligature of the left anterior descending coronary artery. Mice were treated or not with LCZ696 (at 6 or 60 mg/kg) given by oral gavage once per day and sacrificed 10 days after surgery. For all mice, BrdU (1 mg/ml) was added to drinking water 1 day after surgery and during all the treatment period. The effect of the treatment on heart function, cardiomyocyte proliferation and neovascularization was evaluated. High dose of LCZ696 increases the ejection fraction (+23%) and the fractional shortening (+14%) of infarcted hearts. Heart remodeling (the percentage of increase of the left ventricular diameter) was decreased in systole (−49%). At the cellular level, the number of BrdU+ cardiomyocytes was increased with both doses of LCZ696 (+110% in the infarcted and border zone and + 216% in the remote zone for 6 mg/kg LCZ696). The total number of cardiomyocytes was also increased in the infarcted and border zone (+105%) and in the remote zone (+156%). In contrast, neovascularization measured after CD31 staining didn’t differ between groups. Understanding the cellular mechanisms underlying the beneficial effects of LCZ696 in patients suffering from chronic heart failure is crucial for its further clinical use.

Structural Analysis of the Myocardium in Experimental Anthracycline-Induced Cardiomyopathy Combined with Adrenergic Stimulation.

We studied dynamic changes in the total number of cardiomyocytes and the character of structural lesions in the myocardium in rats with modeled anthracycline-induced cardiomyopathy provoked by a single injection of doxorubicin in a dose of 10 mg/kg alone or in combination with subsequent adrenergic stimulation. The injections of epinephrine during the development of anthracycline-induced cardiomyopathy resulted in more pronounced loss of body weight, stronger decrement of the heart weight, and more severe decrease of the cardiomyocyte count in comparison with the corresponding changes induced by doxorubicin alone. The basic lesions of cardiomyocytes in anthracycline-induced cardiomyopathy are the lytic alterations and subsegmental contractures; in contrast, combined use of doxorubicin and epinephrine provoked degree II and III contractures. The revealed necrobiotic changes of cardiomyocytes resulted in their death and pronounced decrease of their number at the initial terms of the study. Hypertrophy observed at later terms of the experiments in parallel with partial recovery of cardiomyocyte number reflected the development of regenerative and adaptivecompensatory processes induced by massive death and elimination of the parenchymatous cells (up to 36-37% of population).

The lifelong impact of fetal growth restriction on cardiac development.

BackgroundMaternal nutrient restriction (MNR) is a widespread cause of fetal growth restriction (FGR), an independent predictor of heart disease and cardiovascular mortality. Our objective was to examine the developmental and long-term impact of MNR-induced FGR on cardiac structure in a model that closely mimics human development.MethodsA reduction in total caloric intake spanning pre-gestation through to lactation in guinea pig sows was used to induce FGR. Proliferation, differentiation, and apoptosis of cardiomyocytes were assessed in late-gestation fetal, neonatal, and adult guinea pig hearts. Proteomic analysis and pathway enrichment were performed on fetal hearts.ResultsCardiomyocyte proliferation and number of mononucleated cells was enhanced in the MNR-FGR fetal and neonatal heart, suggesting a delay in cardiomyocyte differentiation. In fetal hearts of MNR-FGR animals, apoptosis was markedly elevated and the total number of cardiomyocytes reduced, the latter remaining so throughout neonatal and into adult life. A reduction in total cardiomyocyte number in adult MNR-FGR hearts was accompanied by exaggerated hypertrophy and a disorganized architecture. Pathway analysis identified genes related to cell proliferation, differentiation, and survival.ConclusionsFGR influences cardiomyocyte development during critical windows of development, leading to a permanent deficiency in cardiomyocyte number and compensatory hypertrophy in a rodent model that recapitulates human development.

Does BNP modulate cardiomyocyte proliferation?

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

The acceleration of cardiomyogenesis in embryonic stem cells in vitro by serum depletion does not increase the number of developed cardiomyocytes.

The differentiation of pluripotent embryonic stem (ES) cells into various lineages in vitro represents an important tool for studying the mechanisms underlying mammalian embryogenesis. It is a key technique in studies evaluating the molecular mechanisms of cardiomyogenesis and heart development and also in embryotoxicology. Herein, modest modifications of the basic protocol for ES cell differentiation into cardiomyocytes were evaluated in order to increase the yield and differentiation status of developed cardiomyocytes. Primarily, the data show that ES cell cultivation in the form of non-adherent embryoid bodies (EBs) for 5 days compared to 8 days significantly improved cardiomyogenic differentiation. This is illustrated by the appearance of beating foci in the adherent EBs layer at earlier phases of differentiation from day 10 up to day 16 and by the significantly higher expression of genes characteristic of cardiomyogenic differentiation (sarcomeric alpha actinin, myosin heavy chain alpha and beta, myosin light chain 2 and 7, and transcriptional factor Nkx2.5) in EBs cultivated under non-adherent conditions for 5 days. The ratio of cardiomyocytes per other cells was also potentiated in EBs cultivated in non-adherent conditions for only 5 days followed by cultivation in adherent serum-free culture conditions. Nevertheless, the alteration in the percentage of beating foci among these two tested cultivation conditions vanished at later phases and also did not affect the total number of cardiomyocytes determined as myosin heavy chain positive cells at the end of the differentiation process on day 20. Thus, although these modifications of the conditions of ES cells differentiation may intensify cardiomyocyte differentiation, the final count of cardiomyocytes might not change. Thus, serum depletion was identified as a key factor that intensified cardiomyogenesis. Further, the treatment of EBs with N-acetylcysteine, a reactive oxygen species scavenger, did not affect the observed increase in cardiomyogenesis under serum depleted conditions. Interestingly, a mild induction of the ventricular-like phenotype of cardiomyocytes was observed in 5-day-old EBs compared to 8-day-old EBs. Overall, these findings bring crucial information on the mechanisms of ES cells differentiation into cardiomyocytes and on the establishment of efficient protocols for the cardiomyogenic differentiation of ES cells. Further, the importance of determining the absolute number of formed cardiomyocyte-like cells per seeded pluripotent cells in contrast to the simple quantification of the ratios of cells is highlighted.

Chronic hypoxemia in late gestation decreases cardiomyocyte number but does not change expression of hypoxia-responsive genes.

BackgroundPlacental insufficiency is the leading cause of intrauterine growth restriction in the developed world and results in chronic hypoxemia in the fetus. Oxygen is essential for fetal heart development, but a hypoxemic environment in utero can permanently alter development of cardiomyocytes. The present study aimed to investigate the effect of placental restriction and chronic hypoxemia on total number of cardiomyocytes, cardiomyocyte apoptosis, total length of coronary capillaries, and expression of genes regulated by hypoxia.Methods and ResultsWe induced experimental placental restriction from conception, which resulted in fetal growth restriction and chronic hypoxemia. Fetal hearts in the placental restriction group had fewer cardiomyocytes, but interestingly, there was no difference in the percentage of apoptotic cardiomyocytes; the abundance of the transcription factor that mediates hypoxia‐induced apoptosis, p53; or expression of apoptotic genes Bax and Bcl2. Likewise, there was no difference in the abundance of autophagy regulator beclin 1 or expression of autophagic genes BECN1, BNIP3, LAMP1, and MAP1LC3B. Furthermore, fetuses exposed to normoxemia (control) or chronic hypoxemia (placental restriction) had similar mRNA expression of a suite of hypoxia‐inducible factor target genes, which are essential for angiogenesis (VEGF, Flt1, Ang1, Ang2, and Tie2), vasodilation (iNOS and Adm), and glycolysis (GLUT1 and GLUT3). In addition, there was no change in the expression of PKC‐ε, a cardioprotective gene with transcription regulated by hypoxia in a manner independent of hypoxia‐inducible factors. There was an increased capillary length density but no difference in the total length of capillaries in the hearts of the chronically hypoxemic fetuses.ConclusionThe lack of upregulation of hypoxia target genes in response to chronic hypoxemia in the fetal heart in late gestation may be due to a decrease in the number of cardiomyocytes (decreased oxygen demand) and the maintenance of the total length of capillaries. Consequently, these adaptive responses in the fetal heart may maintain a normal oxygen tension within the cardiomyocyte of the chronically hypoxemic fetus in late gestation.

Proliferative activity of cadriomyocytes in chronic hypercholesterolemia.

We studied proliferative activity of cardiomyocytes (using proliferation marker Ki-67) and compared it with their total number in the heart under conditions of experimental chronic hypercholesterolemia and its combination with hypothyroidism. It was found that Ki-67-positive cells are primarily located in the subepicardial layer near the heart base in both intact and experimental animals. Replicative cardiomyocyte pool in intact rats constituted 1.67 ± 0.33‰ of the total cardiomyocyte population; after 68-day atherogenic diet with exogenous cholesterol alone, the replicative cardiomyocyte pool decreased by 16 % (to 1.40 ± 0.24‰). Treatment with mercazolil against the background of exogenous cholesterol increased this parameter by 40 % (to 2.33 ± 0.88‰). Changes in replicative activity of cardiomyocytes correlated with their total number in the heart and organ weight. We conclude that replicative cardiomyocyte pool primarily includes non-terminally differentiated cardiomyocytes (small mononuclear cardiomyocytes) and their proliferation maintains the total number of cardiomyocytes in the heart under conditions of cytopathic influences and provides the basis for physiological and reparative regeneration of the myocardium.

Ibuprofen plus isosorbide dinitrate treatment in the mdx mice ameliorates dystrophic heart structure

Background: Co-administration of ibuprofen (IBU) and isosorbide dinitrate (ISDN) provides synergistic beneficial effects on dystrophic skeletal muscle. Whether this treatment has also cardioprotective effects in this disease was still unknown. Aims: To evaluate the effects of co-administration of IBU and ISDN (a) on left ventricular (LV) structure and function, and (b) on cardiac inflammatory response and fibrosis in mdx mice. Methods: Three groups of mice were studied: mdx mice treated with IBU (50mgkg−1)+ISDN (30mgkg−1) administered daily in the diet, mdx mice that received standard diet without drugs and wild type aged-matched mice. Animals were analysed after 10–11 months of treatment. Structural and functional parameters were evaluated by echocardiography while histological analyses were performed to evaluate inflammatory response, collagen deposition, cardiomyocyte number and area. Results: Treatment for 10–11 months with IBU+ISDN preserved LV wall thickness and LV mass. Drug treatment also preserved the total number of cardiomyocytes in the LV and attenuated the increase in cardiomyocyte size, when compared to untreated mdx mice. Moreover, a trend towards a decreased number of inflammatory cells, a reduced LV myocardial interstitial fibrosis and an enhanced global LV function response to stress was observed in treated mdx mice. Conclusions: Treatment for 10–11 months with IBU+ISDN is effective in preventing the alterations in LV morphology of mdx mice while not reaching statistical significance on LV function and cardiac inflammation.

How Cardiomyocytes Make the Heart Old

Naturally occurring decline in cardiovascular reserve with age associates with a combination of the reduction in cardiomyocyte number and altered cardiomyocyte function. Recent investigations suggested that about half of the cardiomyocytes is the same as at birth, while the other half of the cardiomyocytes is the result of cardiomyocyte renewal in the senescent heart. In addition, the total number of cardiomyocytes is estimated to be less by one third in the old heart than the number of cardiomyocytes at birth. Thus, the reduction in cardiomyocyte number of the aging heart cannot be fully compensated by cardiomyocyte renewal. Aging of long-lived differentiated myocardial cells, as well as of cardiac progenitor stem cells may contribute to an increased rate of apoptosis, and decreased capacity of cell duplication and/or differentiation. In addition, differentiated cardiomyocytes are prone for accumulating biological by-products of cellular metabolism and of incompletely processed oxidative insults. In this context, interactions between lysosomes and mitochondria may provide a mechanistic background for the age-dependent alterations in cardiac macromolecules. This reasoning postulates a direct relationship between the number of pro-oxidative, ill-functioning mitochondria and the amount of ballast- overloaded lysosomes in long-lived cardiomyocytes. Accumulation of biological garbage and telomere shortening might be considered as hallmarks of cardiomyocyte aging with implications for depressed cardiac function and cardiomyocyte renewal. Changes in protein expression together with posttranslational modifications of myocardial proteins affect excitation-contraction coupling and explain the declining mechanical function of the cardiomyocytes. Altogether, these changes represent a significant part of the reduced cardiovascular reserve in aged individuals. Keywords: Aging, cardiomyocyte cell death, cardiomyocyte renewal, telomere

How Cardiomyocytes Make the Heart Old

Naturally occurring decline in cardiovascular reserve with age associates with a combination of the reduction in cardiomyocyte number and altered cardiomyocyte function. Recent investigations suggested that about half of the cardiomyocytes is the same as at birth, while the other half of the cardiomyocytes is the result of cardiomyocyte renewal in the senescent heart. In addition, the total number of cardiomyocytes is estimated to be less by one third in the old heart than the number of cardiomyocytes at birth. Thus, the reduction in cardiomyocyte number of the aging heart cannot be fully compensated by cardiomyocyte renewal. Aging of long-lived differentiated myocardial cells, as well as of cardiac progenitor stem cells may contribute to an increased rate of apoptosis, and decreased capacity of cell duplication and/or differentiation. In addition, differentiated cardiomyocytes are prone for accumulating biological by-products of cellular metabolism and of incompletely processed oxidative insults. In this context, interactions between lysosomes and mitochondria may provide a mechanistic background for the age-dependent alterations in cardiac macromolecules. This reasoning postulates a direct relationship between the number of pro-oxidative, ill-functioning mitochondria and the amount of ballast- overloaded lysosomes in long-lived cardiomyocytes. Accumulation of biological garbage and telomere shortening might be considered as hallmarks of cardiomyocyte aging with implications for depressed cardiac function and cardiomyocyte renewal. Changes in protein expression together with posttranslational modifications of myocardial proteins affect excitation-contraction coupling and explain the declining mechanical function of the cardiomyocytes. Altogether, these changes represent a significant part of the reduced cardiovascular reserve in aged individuals.

237 CARDIOMYOCYTE NUMBER AND EXPRESSION OF FATTY ACID β-OXIDATIVE GENES ARE DIFFERENTIALLY AFFECTED BY THE CAUSE OF FETAL GROWTH RESTRICTION AND SEX

Background: Fetal growth restriction (FGR) is associated with an increased risk of cardiovascular disease (CVD) in adulthood and this may be due to reduced number and/or altered metabolism of cardiomyocytes. We hypothesise that FGR due to reduced maternal oxygen (MH) and/or nutrition (MNR) will decrease cardiomyocyte number and increase expression of FAO genes in offspring. Method: Date-mated guinea pigs at 35d gestation (term, 69d) were assigned to Control (21% O2; food ad libitum, n=16), MH (12% O2; food ad libitum, but ate less, n=11) or MNR (21% O2; food weight matched to MH, n=9). The heart of offspring was collected at 120d of age and total number of cardiomyocytes and mRNA expression of genes involved in transport, activation and oxidation of FAs was determined by stereological techniques and real time RT-PCR in the left ventricle (LV), respectively. Data was analysed by two-way ANOVA for Treatment and Sex and by Duncan post-hoc tests. Results: MH and MNR reduced birth weight, but did not affect adult body or LV weight. In females, MH reduced the number of cardiomyocytes and MNR increased expression of long chain acyl-CoA dehydrogenase, however, in males MH and MNR decreased expression of AMPK. Conclusion: These data suggest FGR may lead to increased vulnerability to CVD in females due to decreased cardiomyocyte number if the FGR was due to MH, but greater cardiac FAO if the FGR was due to MNR. In contrast, male offspring exposed to MH and MNR may have decreased cardiac FAO in adulthood.

Effects of high-fat chow on heart tissue in acute and chronic experimental murine schistosomiasis mansoni

This study aimed to investigate myocardial injuries in mice infected with Schistosoma mansoni and fed a high-fat chow. Sections of myocardial tissue from S. mansoni-infected mice, and controls that had been killed 9 and 17 weeks post-infection, were stained with H&E and Picrosirius red. Histopathological examination, stereological design-based method (optical disector) and morphometry (vessels, cardiomyocytes and an amount of collagen) were used. Data were analysed using two-way ANOVA. Regardless of time of infection, myocardial tissue from the infected mice fed high-fat chow showed myocarditis characterized by a higher number of inflammatory foci, several areas displaying coagulation of cardiac fibres, a greater loss of cardiomyocytes and fibroblast proliferation than in the standard chow control. Comparing infected mice from acute and chronic infections, a higher cardiomyocyte hyperplasia (P < 0·0001) and higher amounts of collagen (P < 0·05) were observed than in standard chow control. In addition, all animals fed high-fat chow showed lower numerical density and total number of cardiomyocytes (P < 0·05), thicker vessel walls and narrowed luminal intramyocardial vessels (P > 0·05) than in the standard chow control. Altogether the data supported the view that a double burden has a synergistic deleterious effect on the myocardial tissue.

Abstract P136: E2F2/4 Regulated by Serine 21 Phosphorylation of GSK-3α Play Compensatory Roles During Pressure Overload

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase with two isoforms, alpha and beta, which have distinct functions in cardiomyocytes (CMs). GSK-3alpha is phosphorylated at S21 during pressure overload (PO), and inhibition of S21 phosphorylation in GSK-3alpha S21A knock-in (alpha−KI) mice promotes hypertrophy and heart failure in response to PO, accompanied by decreases in the total number of CMs in the heart. Since GSK-3alpha downregulates cyclinD1 in the nucleus, GSK-3alpha may negatively regulate E2F-mediated transcription. Reporter gene assays showed that the transcriptional activity of E2F was increased by GSK-3alpha knockdown (1.75 fold, p&lt;0.05). To evaluate the role of E2F isoforms in regulating cardiac hypertrophy and function during PO, E2F1−/−, E2F2−/+, E2F4−/+, and wild type (WT) mice were subjected to transverse aortic constriction (TAC). Left ventricular (LV) weight/ tibial length (LVW/TL) was significantly greater and LV ejection fraction (LVEF) was significantly decreased in both E2F2−/+ and E2F4−/+ after 2 weeks of TAC (LVW/TL: E2F2−/+=7.1±0.3, E2F4−/+=7.0±0.4, WT=5.9±0.3, p&lt;0.05 vs. WT; LVEF: E2F2−/+=53±1%, E2F4−/+=61±2%, WT=75±1%, p&lt;0.05 vs. WT). Thus, downregulation of either E2F2 or E2F4 induced a phenotype similar to that of alpha−KI in response to TAC. To examine the causative role of E2F2/E2F4 downregulation in mediating the cardiac phenotype in alpha-KI mice, adenovirus (Ad) harboring either E2F2 or E2F4 was injected into alpha-KI hearts. Rescue with E2F2 or E2F4 attenuated cardiac hypertrophy (LVW/TL: alpha−KI+E2F2=7.1±0.4, alpha−KI+E2F4=7.3±0.3, alpha−KI+LacZ=8.7±0.4, p&lt;0.05 vs. alpha−KI+LacZ) and improved LV dysfunction (LVEF: alpha−KI+E2F2=66±3%, alpha−KI+E2F4=60±2%, alpha−KI+LacZ=39±2%, p&lt;0.05 vs. alpha−KI+LacZ) in alpha−KI mice under PO conditions. Injection of either Ad-E2F2 or Ad-E2F4, but not of Ad-LacZ, significantly increased the number of Ki67-positive myocytes in the alpha-KI mice (alpha-KI+LacZ =0.7±0.3%, alpha-KI+E2F2=10.4±2.3%, alpha-KI+E2F4=9.2±1.5%, p&lt;0.05 vs. alpha-KI+LacZ). These results suggest that maintaining the activity of E2F2 and E2F4 through S21 phosphorylation of GSK-3alpha plays an essential role in preserving cardiac function during PO.

Myocardial infarction quantification with late gadolinium-enhanced magnetic resonance imaging in rats using a 7-T scanner

The objective of this study was to noninvasively measure the volume of myocardial infarction in rats, using delayed enhancement magnetic resonance imaging (MRI) in a coronary occlusion/reperfusion model on a 7-T scanner. At 24 h after cardiac ischemia, contrast-enhanced MRI was performed. Two distinct experimental groups were compared: one was subjected to permanent ischemia (PL) and the other was subjected to 30 min of ischemia followed by 24 h of reperfusion (IR). The sizes of enhanced regions were compared to triphenyltetrazolium chloride (TTC)-stained sections of the excised rat heart. Cardiomyocyte apoptosis was analyzed by TUNEL methods, and neutrophils and macrophages were quantitated after histology and immunohistochemical staining. Twenty-four hours after ischemia, delayed hyperenhancement imaging was clearly visualized in the anterior left ventricular walls corresponding to the infarcted myocardium. In the PL group, infarct size was 37.2±9.8% (LV %) as measured by MRI and 38.8±9% (LV %) by TTC (P=NS). In the IR group, infarct size was 23.2±8.8% (LV %) as measured by MRI and 24.4±9.2% (LV %) by TTC (P=NS). Infarction volume measured with MRI was strongly correlated to TTC staining (R=0.82 for PL, R=0.973 for IR). Increased inflammatory cell infiltration was detected in the infarct area of the heart after reperfusion compared to permanent ligation (P<.01). The ratio of TUNEL-positive cardiomyocytes to total number of cardiomyocytes in the IR group was significantly reduced as compared to the PL group (P<.01). MRI can accurately assess infarct size in intact rats early after MI. After transient arterial occlusion, the size of the myocardial infarct was found to be significantly smaller as compared to permanent occlusion.