Eve Rigal

Postnatal Overfeeding Causes Early Shifts in Gene Expression in the Heart and Long-Term Alterations in Cardiometabolic and Oxidative Parameters

Background Postnatal overfeeding (OF) in rodents induces a permanent moderate increase in body weight in adulthood. However, the repercussions of postnatal OF on cardiac gene expression, cardiac metabolism and nitro-oxidative stress are less well known. Methodology/Principal Findings Immediately after birth, litters of C57BL/6 mice were either maintained at 10 (normal-fed group, NF), or reduced to 3 in order to induce OF. At weaning, mice of both groups received a standard diet. The cardiac gene expression profile was determined at weaning and cardiac metabolism and oxidative stress were assessed at 7 months. The cardiac expression of several genes, including members of the extracellular matrix and apelin pathway, was modified in juvenile OF mice. In adult mice, OF led to an increase in body weight (+30%) and to significant increases in plasma cholesterol, insulin and leptin levels. Myocardial oxidative stress, SOD and catalase activity and mRNA expression were increased in OF mice. In vivo, diastolic and systolic blood pressures were significantly higher and LV shortening and ejection fraction were decreased in OF mice. Ex vivo, after 30 min of ischemia, hearts isolated from OF mice showed lower functional recovery and larger infarct size (31% vs. 54%, p<0.05). Increases in collagen deposition and expression/activity of matrix-metalloproteinase-2 were observed in adult OF mouse hearts. Moreover, an increase in the expression of SOCS-3 and a decrease in STAT-3 phosphorylation were observed in ventricular tissues from OF mice. Conclusions/Significance Our study emphasizes that over-nutrition during the immediate postnatal period in mice leads to early changes in cardiac gene expression, which may permanently modify the heart’s structural organization and metabolism and could contribute to a greater susceptibility to myocardial ischemia-reperfusion injury.

Paradoxically, iron overload does not potentiate doxorubicin-induced cardiotoxicity in vitro in cardiomyocytes and in vivo in mice

Doxorubicin (DOX) is known to induce serious cardiotoxicity, which is believed to be mediated by oxidative stress and complex interactions with iron. However, the relationship between iron and DOX-induced cardiotoxicity remains controversial and the role of iron chelation therapy to prevent cardiotoxicity is called into question. Firstly, we evaluated in vitro the effects of DOX in combination with dextran-iron on cell viability in cultured H9c2 cardiomyocytes and EMT-6 cancer cells. Secondly, we used an in vivo murine model of iron overloading (IO) in which male C57BL/6 mice received a daily intra-peritoneal injection of dextran-iron (15mg/kg) for 3weeks (D0-D20) and then (D21) a single sub-lethal intra-peritoneal injection of 6mg/kg of DOX. While DOX significantly decreased cell viability in EMT-6 and H9c2, pretreatment with dextran-iron (125-1000μg/mL) in combination with DOX, paradoxically limited cytotoxicity in H9c2 and increased it in EMT-6. In mice, IO alone resulted in cardiac hypertrophy (+22%) and up-regulation of brain natriuretic peptide and β-myosin heavy-chain (β-MHC) expression, as well as an increase in cardiac nitro-oxidative stress revealed by electron spin resonance spectroscopy. In DOX-treated mice, there was a significant decrease in left-ventricular ejection fraction (LVEF) and an up-regulation of cardiac β-MHC and atrial natriuretic peptide (ANP) expression. However, prior IO did not exacerbate the DOX-induced fall in LVEF and there was no increase in ANP expression. IO did not impair the capacity of DOX to decrease cancer cell viability and could even prevent some aspects of DOX cardiotoxicity in cardiomyocytes and in mice.

The role of osteoprotegerin in the crosstalk between vessels and bone: Its potential utility as a marker of cardiometabolic diseases

Abstract Among the numerous molecules that are being studied for their potential utility as biomarkers of cardiovascular diseases, much interest has been shown in the superfamily of tumor necrosis factor (TNF) receptors. Members of this family include osteoprotegerin (OPG) and its ligands, which are receptor activators of nuclear factor &kgr;B ligand (RANKL) and TNF‐related apoptosis‐inducing ligand (TRAIL). These signals may be expressed and regulated, and their functions could be involved in several physiological and pathological processes. The relationship between bone regulatory proteins and vascular biology has attracted attention, and it has been suggested that OPG may mediate vascular calcification and cardiometabolic diseases. OPG is steadily released from vascular endothelial cells in response to inflammatory stimuli, suggesting that it plays a modulatory role in vascular injury, inflammation, and atherosclerosis. Vascular calcification, a hallmark of atherosclerosis, is similar to bone remodeling. It is an actively regulated mechanism that includes both inductive and inhibitory processes. There is a temporal link between the development of osteoporosis and vascular calcification, which is particularly marked in post‐menopausal women and the elderly. The precise nature of the link between bone metabolism, vascular calcification and cardiovascular disease is largely unknown but increasing evidence suggests that the triad of RANK/RANKL/OPG may be important in the initiation of various diseases. An increased release of OPG is associated with increased cardiovascular risk and it is suggested that increased OPG levels resulting from vascular damage correspond to a protective mechanism. Circulating OPG levels could be used as independent biomarkers of cardiovascular disease in patients with acute or chronic cardiometabolic disease and thus an improved prognosis.

Short-term moderate diet restriction in adulthood can reverse oxidative, cardiovascular and metabolic alterations induced by postnatal overfeeding in mice

We aimed to determine whether moderate diet restriction could restore cardiac, oxidative and metabolic alterations induced by postnatal overfeeding (PNOF). Litters of C57BL/6 male mice were either maintained at 9 (normal litter, NL), or reduced to 3 (small litter, SL) in order to induce PNOF. At 6 months, half of the NL and SL mice were subjected to 20% calorie-restriction (CR: NLCR, SLCR) for one month, while the other half continued to eat ad libitum (AL: NLAL, SLAL). Six-month old SL mice presented overweight, fat accumulation, hyperleptinemia, glucose intolerance, insulin resistance, increased cardiac ROS production and decreased left ventricular ejection fraction (LVEF). After CR, SL mice body weight was normalized; however, their fat mass and leptinemia were not decreased, glucose metabolism was improved and LVEF was increased. In SL mice, CR increased the cardiac mitochondrial respiratory rate and decreased cardiac ROS production. Hearts from SLCR mice showed better recovery and smaller postischemic infarct size. Intriguingly, no difference was observed between NLAL and NLCR mice for most of the parameters investigated. Short-term moderate CR not only normalized body weight in SL mice but also improved metabolic programming and reversed oxidative and cardiac dysfunction induced by PNOF.

0132 : Oxidative stress and cardio-metabolic alterations induced by postnatal programming can be reversed in adulthood by a short-term moderate caloric restriction

Postnatal overfeeding (PNOF) in rodents induces early programming of cardio-metabolic risk. Our aim was to determine if a moderate diet restriction could restore cardio-metabolic alterations induced by PNOF. Immediately after birth, litters of C57BL/6 mice were either maintained at 9 (normal litter, NL), or reduced to 3 (small litter, SL) to induce PNOF. At weaning, all mice received a standard diet ad libitum (AL). At 6 month of age, half of the NL and SL mice were assigned to a moderate 20% calorie restriction (CR: NLCR, SLCR) for one month, while the other mice continued to eat AL (AL: NLAL, SLAL). Glucose and insulin tolerance tests, cardiac function (echocardiography), body composition (Echo-MRI), cardiac sensitivity to ischemia-reperfusion injury, mitochondrial function, reactive oxygen species (ROS) generation (EPR spectroscopy) and insulin signaling were assessed before and/or after one month of CR. Adult SL mice presented overweight, fat accumulation, hyperleptinemia, glucose intolerance, insulin resistance and decreased left ventricular ejection fraction (LVEF). After one month of moderate CR, body weight of SLCR was normalized to this of NLAL however their fat mass and leptinemia were not decreased. Glucose metabolism was improved and LVEF was increased In SLCR. After 30 min of global ischemia, hearts isolated from SLCR mice showed better recovery and smaller infarct size than this of others groups. CR increased the cardiac mitochondrial respiratory rate in SLCR mice whereas cardiac ROS production was significantly decreased in SLCR mice. Insulin signaling in heart was affected neither by PNOF nor by CR. Intriguingly, no difference was observed in NLCR mice for most of the parameters investigated. Our results confirmed the programming of early overfeeding on metabolic and cardiac function. A short-term moderate CR in not only normalized body weight in SL mice but also ameliorate the metabolic programming and reverse the cardiac dysfunction induced by PNOF. The author hereby declares no conflict of interest

0110 : Experimental cerebral ischemia in rats increases myocardial vulnerability to ischemia-reperfusion injury ex vivo

For years, the relationship between cardiac and neurological ischemic events has been mainly attributed to overlapping pathophysiological mechanisms and common risk factors. However, acute stroke may induce dramatic alterations of cardiovascular function. The aim of this work was to evaluate how prior cerebrovascular lesions affect myocardial function in vivo and ex vivo , as well as myocardial vulnerability to ischemic injury. Cerebral embolization was performed in adult Wistar male rats by the injection of microspheres into the left internal carotid artery. Left ventricular function, investigated in vivo using echocardiography (1 hour, 24 hours and 7 days after the embolization), was not significantly impaired; however, the heart rate was significantly increased in the stroke group (+7.2%). Epinephrine (E) and norepinephrine (NE) plasma levels increased in rats from the stroke group (E: 47.3±2.1 vs. 24.3±8.7 and NE: 22.7±4.2 vs. 10.9±3.7). One hour after stroke or sham embolization, hearts were isolated and perfused ex vivo in the Langendorff mode. In hearts from the stroke group, the baseline left ventricular developed pressure was diminished (-11%); moreover, a greater myocardial vulnerability to ischemic injury was observed, with impaired coronary flow recovery after 40 minutes of total global normothermic ischemia. Our study provides original exciting data indicating that myocardial vulnerability to ischemia can be worsened by prior ischemic stroke, a situation that does not agree with the concept of remote preconditioning. The underlying molecular mechanisms of the stroke-induced myocardial alterations after cerebral embolization remain to be established, insofar as they may involve the sympathetic nervous system. The author hereby declares no conflict of interest

0126: New regulators of iron metabolism, Hepcidin and Erythroferrone, in acute myocardial infarction.

Background Dysfunctional iron storage and transport are common in patients with chronic heart failure and associated with poor prognosis. Body iron could contribute to the pathogenesis of coronary artery disease (CAD) through its ability to induce oxidative stress. However, studies on the relationship between iron metabolism and CAD have yielded conflicting results. Patients and Methods From the obseRvatoire des Infarctus de Cote d’Or (RICO) survey, 31 consecutive patients admitted in Intensive Care Unit for a first AMI were included. Serum concentrations of iron, transferrin, ferritin, the iron-regulatory hormone hepcidin and erythroferrone (a new hepcidin-regulating hormone), transferrin saturation and total iron binding capacity were assessed on admission. Results Mean age was 65±16 yrs, 61% were male, 51% had hypertension, 23% diabetes, 45% dyslipidemia and 32% were smokers. There was a trend toward a higher serum hepcidin concentration in men (99.8 versus 56.3 ng/ml, p=0.181). Heart rate on admission was negatively associated with an erythroferrone concentration (r=–0.428, p=0.023). Haemoglobin level and hematocrit were positively correlated with erythroferrone concentration (p=0.027 and p=0.021). Moreover, a lower serum transferrin concentration was found in patients with heart failure on admission (1.93±0.16g/l, vs 2.32±0.42, p=0.001). Ferritin concentration was positively related with infarct size, as assessed by Creatine Kinase peak (r=0.535, p=0.002) and there was a trend toward a positive correlation with erythroferrone concentration (r=0.314, p=0.085). Conclusion Elucidating the metabolic circuits regulated by peptidic hormones will provide valuable insights into complex networks governing iron availability in acute myocardial infarction.

0131 : Impact of overweight on anthracycline and trastuzumab-induced cardiotoxicity: experimental study in mice

Trastuzumab (TRZ), a humanized monoclonal antibody against Human Epidermal Growth Factor Receptor 2 (HER2) oncogene, is believed to potentiate doxorubicin (DOX) cardiotoxicity, resulting in left ventricular dysfunction. Few data indicate that overweight could influence DOX-induced cardiotoxicity, and no study has already evaluated the impact of moderate overweight on the cardiotoxic effect of DOX alone or in combination with TRZ. Immediately after birth, litters of C57BL/6 mice were either maintained at 10 (normal litter, NL), or reduced to 3 (small litter, SL) in order to induce programming of ~15% overweight through postnatal overfeeding. At 4 months, in order to evaluate the potentiation of DOX cardiotoxicity by TRZ, NL and SL mice received a single intraperitoneal injection of either saline, DOX (6mg/kg), TRZ (10mg/kg) or the combination of both (DOX-TRZ). Transthoracic echocardiography was performed 24 hours before, 10 and 20 days after treatments, in order to evaluate the evolution of cardiac function. Twenty days after DOX administration, systolic dysfunction was observed only in overweight-SL group, while NL mice group kept a preserved left ventricular ejection fraction (LVEF). Moreover, in NL group, the function impairment appeared when TRZ was co-administrated. 48 hours after drug administration, gene expression of Erb-B2, the murine analog of HER2, was induced in the myocardium of DOX-treated mice, and its induction was potentiated by co-treatment with TRZ. Expression of natriuretic peptides (ANP, BNP) appeared to be potentiated in DOX-TRZ mice of both NL and SL groups, whereas the expression of b-MHC increased significantly in overweight- SL mice. In an acute model of DOX cardiotoxicity, moderately overweighed adult mice are more sensitive to cardiac systolic impairment. Moreover, our results show an early myocardial induction of TRZ-receptor after DOX and/or TRZ, and confirm the potentiating action of TRZ on DOX-induced cardiotoxicity in mice

0053 : Short-term moderate diet restriction in adulthood can reverse alterations of cardiac function induced by postnatal overfeeding in mice

Postnatal overfeeding (OF) in rodents induces early programming of cardio-metabolic risk: permanent moderate increase in body weight, metabolic disorders and progressive alterations of cardiac function in adulthood. Our aim was to determine whether moderate diet restriction, performed at the time where these disorders are acquired, could restore cardiac function and ameliorate post-ischemic recovery. Immediately after birth, litters of C57BL/6 mice were either maintained at 9 (normal-fed group, NF), or reduced to 3 in order to induce OF. At weaning, mice of both groups received a standard diet ad libitum (AL). At 6 months of age, half of the NF mice and OF mice were assigned to a moderate 20% calorie restriction (CR, NF-CR, OF-CR) for one month, while the other half of mice continued to eat AL (NF-AL, OF-AL). Cardiac function was followed using echocardiography and, at 7 months, the sensitivity to ischemia-reperfusion injury was evaluated in isolated perfused hearts. Six-month-old OF mice weighed 19.3% more than NF mice. Left ventricular fractional shortening (LVFS) and ejection fraction (LVEF, 51.4% vs. 57.6%, p Our study suggests that while short-term moderate CR could diminish body weight in both NF and OF groups of mice, CR was able to improve cardiac function and susceptibility to myocardial ischemia-reperfusion injury only in OF, reversing the deleterious influence of postnatal programming by early overnutrition.

0229: Alterations of cardiac function induced by postnatal overfeeding can be reversed by moderate diet restriction

Postnatal overfeeding (OF) in rodents induces a permanent moderate increase in body weight, metabolic disorders and progressive alterations of cardiac function. Our aim was to determine whether moderate diet restriction could restore cardiac function in mature overfed mice. Immediately after birth, litters of C57BL/6 mice were either maintained at nine (normal-fed group, NF), or reduced to three in order to induce OF. At weaning, mice of both groups received a standard diet ad libitum (AL) . At 6 months of age, half of the OF mice were assigned to a moderate 20% calorie restriction (CR, OF- CR ) for one month, while NF and the other half of the OF mice continued to eat ad libitum (NF-AL , OF- AL) . Cardiac function was followed using echocardiography and, at 7 months, the sensitivity to ischemia-reperfusion injury was evaluated in isolated perfused hearts. Six-month-old OF mice weighed 22.5% more than NF mice. Left ventricular fractional shortening (LVFS) and ejection fraction (LVEF) were decreased in OF mice (25.5% vs. 30.5% for LVFS; 50% vs. 58% for LVEF, P CR than OF-AL (61% vs. 52%, p CR . Ex vivo, after 30 min of global ischemia, hearts isolated from OF CR mice showed better functional recovery than those of the two other groups. Our study suggests that short-term moderate diet restriction could normalize body weight gain induced by postnatal OF and, interestingly, could reverse alterations of cardiac function and susceptibility to myocardial ischemia-reperfusion injury in OF.