Early epileptiform EEG activity in infants with tuberous sclerosis complex predicts epilepsy and neurodevelopmental outcomes.

OBJECTIVE: To study the association between timing and characteristics of the first electroencephalography (EEG) with epileptiform discharges (ED-EEG) and epilepsy and neurodevelopment at 24 months in infants with tuberous sclerosis complex (TSC). METHODS: Patients enrolled in the prospective Epileptogenesis in a genetic model of epilepsy - Tuberous sclerosis complex (EPISTOP) trial, had serial EEG monitoring until the age of 24 months. The timing and characteristics of the first ED-EEG were studied in relation to clinical outcome. Epilepsy-related outcomes were analyzed separately in a conventionally followed group (initiation of vigabatrin after seizure onset) and a preventive group (initiation of vigabatrin before seizures, but after appearance of interictal epileptiform discharges [IEDs]). RESULTS: Eighty-three infants with TSC were enrolled at a median age of 28 days (interquartile range [IQR] 14-54). Seventy-nine of 83 patients (95%) developed epileptiform discharges at a median age of 77 days (IQR 23-111). Patients with a pathogenic TSC2 variant were significantly younger (P-value .009) at first ED-EEG and more frequently had multifocal IED (P-value .042) than patients with a pathogenic TSC1 variant. A younger age at first ED-EEG was significantly associated with lower cognitive (P-value .010), language (P-value .001), and motor (P-value .013) developmental quotients at 24 months. In the conventional group, 48 of 60 developed seizures. In this group, the presence of focal slowing on the first ED-EEG was predictive of earlier seizure onset (P-value .030). Earlier recording of epileptiform discharges (P-value .019), especially when multifocal (P-value .026) was associated with higher risk of drug-resistant epilepsy. In the preventive group, timing, distribution of IED, or focal slowing, was not associated with the epilepsy outcomes. However, when multifocal IEDs were present on the first ED-EEG, preventive treatment delayed the onset of seizures significantly (P-value <.001). SIGNIFICANCE: Early EEG findings help to identify TSC infants at risk of severe epilepsy and neurodevelopmental delay and those who may benefit from preventive treatment with vigabatrin.

Cellular senescence links aging and diabetes in cardiovascular disease.

Aging is a powerful independent risk factor for cardiovascular diseases such as atherosclerosis and heart failure. Concomitant diabetes mellitus strongly reinforces this effect of aging on cardiovascular disease. Cellular senescence is a fundamental mechanism of aging and appears to play a crucial role in the onset and prognosis of cardiovascular disease in the context of both aging and diabetes. Senescent cells are in a state of cell cycle arrest but remain metabolically active by secreting inflammatory factors. This senescence-associated secretory phenotype is a trigger of chronic inflammation, oxidative stress, and decreased nitric oxide bioavailability. A complex interplay between these three mechanisms results in age- and diabetes-associated cardiovascular damage. In this review, we summarize current knowledge on cellular senescence and its secretory phenotype, which might be the missing link between aging and diabetes contributing to cardiovascular disease.

Childhood obesity-related endothelial dysfunction: an update on pathophysiological mechanisms and diagnostic advancements.

Childhood obesity jeopardizes a healthy future for our society's children as it is associated with increased cardiovascular morbidity and mortality later on in life. Endothelial dysfunction, the first step in the development of atherosclerosis, is already present in obese children and may well represent a targetable risk factor. Technological advancements in recent years have facilitated noninvasive measurements of endothelial homeostasis in children. Thereby this topic ultimately starts to get the attention it deserves. In this paper, we aim to summarize the latest insights on endothelial dysfunction in childhood obesity. We discuss methodological advancements in peripheral endothelial function measurement and newly identified diagnostic markers of vascular homeostasis. Finally, future challenges and perspectives are set forth on how to efficiently tackle the catastrophic rise in cardiovascular morbidity and mortality that will be inflicted on obese children if they are not treated optimally.

A multi-task and multi-channel convolutional neural network for semi-supervised neonatal artefact detection.

Objective. Automated artefact detection in the neonatal electroencephalogram (EEG) is crucial for reliable automated EEG analysis, but limited availability of expert artefact annotations challenges the development of deep learning models for artefact detection. This paper proposes a semi-supervised deep learning approach for artefact detection in neonatal EEG that requires few labelled data by training a multi-task convolutional neural network (CNN).Approach. An unsupervised and a supervised objective were jointly optimised by combining an autoencoder and an artefact classifier in one multi-output model that processes multi-channel EEG inputs. The proposed semi-supervised multi-task training strategy was compared to a classical supervised strategy and other existing state-of-the-art models. The models were trained and tested separately on two different datasets, which contained partially annotated multi-channel neonatal EEG. Models were evaluated using the F1-statistic and the relevance of the method was investigated in the context of a functional brain age (FBA) prediction model.Main results. The proposed multi-task and multi-channel CNN methods outperformed state-of-the-art methods, reaching F1 scores of 86.2% and 95.7% on two separate datasets. The proposed semi-supervised multi-task training strategy was shown to be superior to a classical supervised training strategy when the amount of labels in the dataset was artificially reduced. Finally, we found that the error of a brain age prediction model correlated with the amount of automatically detected artefacts in the EEG segment.Significance. Our results show that the proposed semi-supervised multi-task training strategy can train CNNs successfully even when the amount of labels in the dataset is limited. Therefore, this method is a promising semi-supervised technique for developing deep learning models with scarcely labelled data. Moreover, a correlation between the error of FBA estimates and the amount of detected artefacts in the corresponding EEG segments indicates the relevance of artefact detection for robust automated EEG analysis.

The vulnerability of the heart as a pluricellular paracrine organ: lessons from unexpected triggers of heart failure in targeted ErbB2 anticancer therapy.

In this review, we address clinical aspects and mechanisms of ventricular dysfunction induced by anticancer drugs targeted to the ErbB2 receptor. ErbB2 antagonists prolong survival in cancer, but also interfere with homeostatic processes in the heart. ErbB2 is a coreceptor for ErbB4, which is activated by neuregulin-1. This epidermal growth factor-like growth factor is released from endothelial cells in the endocardium and in the myocardial microcirculation, hence contributing to intercellular crosstalk in the ventricle. We look at the physiological aspects of neuregulin-1/ErbB signaling in the ventricle, and review its (mal)adaptive responses in chronic heart failure. We also compare structural aspects of ErbB receptor activation in cancer and cardiac cells, and analyze the mode of action of current ErbB2 antagonists. This allows us to predict how these drugs interfere with paracrine processes in the ventricle. Differences in the mode of action of individual ErbB2 antagonists affect their impact on the function of the ventricle, considered to be "on-target" or "off-target." Establishing the relation between the cardiac side effects of ErbB2 antagonists and their impact on paracrine ventricular control mechanisms may direct the design of a next generation of ErbB2 inhibitors. For cardiologists, there are lessons to be learned from the unexpected side effects of ErbB2-targeted cancer therapy. The vulnerability of the heart as a pluricellular paracrine system appears greater than anticipated and intercellular crosstalk an essential component of its functional and structural integrity.

Automated detection and removal of flat line segments and large amplitude fluctuations in neonatal electroencephalography.

Background: Artefact removal in neonatal electroencephalography (EEG) by visual inspection generally depends on the expertise of the operator, is time consuming and is not a consistent pre-processing step to the pipeline for the automated EEG analysis. Therefore, there is the need for the automated detection and removal of artefacts in neonatal EEG, especially of distinct and predominant artefacts such as flat line segments (mainly caused by instrumental error where contact between electrodes and head box is lost) and large amplitude fluctuations (related to neonatal movements). Method: A threshold-based algorithm for the automated detection and removal of flat line segments and large amplitude fluctuations in neonatal EEG of infants at term-equivalent age is developed. The algorithm applies thresholds to the absolute second difference, absolute amplitude, absolute first difference and the ratio between the frequency content above 50 Hz and the frequency content across all frequencies. Results: The algorithm reaches a median accuracy of 0.91, a median hit rate of 0.91 and a median false discovery rate of 0.37. Also, a significant improvement (≈10%) in the performance of a four-stage sleep classifier is observed after artefact removal with the proposed algorithm as compared to before its application. Significance: An automated artefact removal method contributes to the pipeline of automated EEG analysis. The proposed algorithm has shown to have good performance and to be effective in neonatal EEG applications.

Activation of the neuregulin/ErbB system during physiological ventricular remodeling in pregnancy.

The neuregulin-1 (NRG1)/ErbB system has emerged as a paracrine endothelium-controlled system in the heart, which preserves left ventricular (LV) performance in pathophysiological conditions. Here, we analyze the activity and function of this system in pregnancy, which imparts a physiological condition of LV hemodynamic overload. NRG1 expression and ErbB receptor activation were studied by Western blot analyses in rats and mice at different stages of pregnancy. LV performance was evaluated by transthoracic echocardiography, and myocardial performance was assessed from twitches of isolated papillary muscles. NRG1/ErbB signaling was inhibited by oral treatment of animals with the dual ErbB1/ErbB2 tyrosine kinase inhibitor lapatinib. Analyses of LV tissue revealed that protein expression of different NRG1 isoforms and levels of phosphorylated ErbB2 and ErbB4 significantly increased after 1-2 wk of pregnancy. Lapatinib prevented phosphorylation of ErbB2 and ERK1/2, but not of ErbB4 and protein kinase B (Akt), revealing that lapatinib only partially inhibited NRG1/ErbB signaling in the LV. Lapatinib did not prevent pregnancy-induced changes in LV mass and did not cause apoptotic cell death or fibrosis in the LV. Nevertheless, lapatinib led to premature maternal death of ∼25% during pregnancy and it accentuated pregnancy-induced LV dilatation, significantly reduced LV fractional shortening, and induced abnormalities of twitch relaxation (but not twitch amplitude) of isolated papillary muscles. This is the first study showing that the NRG1/ErbB system is activated, and plays a modulatory role, during physiological hemodynamic overload associated with pregnancy. Inhibiting this system during physiological overload may cause LV dysfunction in the absence of myocardial cell death.

Effects of nebivolol on vascular endothelial and myocardial function in diabetes mellitus.

BACKGROUND: Nebivolol is a ß1-adrenergic receptor (ß1-AR) antagonist, inducing endothelial nitric oxide (NO) release, most likely due to ß3-AR agonism. Nebivolol is vasculoprotective and cardioprotective in the setting of hypertension. In this study, we investigated the effects of nebivolol, compared with those of bisoprolol, on vascular and myocardial function in diabetic mice. METHODS: Diabetic (Lepr) and nondiabetic mice (Lepr) were treated with vehicle, nebivolol, or bisoprolol for 16 weeks. Endothelium-dependent and endothelial-independent relaxations were studied in isolated aortic segments. Myocardial twitch performance was studied in isolated right ventricular papillary muscles. RESULTS: In aortic segments of diabetic mice, endothelium-dependent relaxations were significantly shifted to the right. This shift was not prevented by chronic nebivolol or bisoprolol treatment. Papillary muscle twitches of diabetic mice displayed a significant delay in the onset of relaxation, and an increased time from peak active force to 50% relaxation, leading to prolonged twitch activity, without changes in twitch amplitude. These changes were not prevented in nebivolol- and bisoprolol-treated diabetic mice. Surprisingly, in nebivolol-treated mice, regardless of diabetic status, twitch duration was further increased. Applying nebivolol to papillary muscles in the organ bath reproduced the changes induced by chronic treatment in vivo; this was not the case for bisoprolol. Nebivolol-induced changes were blunted by a ß3-AR antagonist and by NO synthase (NOS) inhibition. CONCLUSIONS: Diabetes-induced changes in vascular and myocardial function were not prevented by treatment with nebivolol or bisoprolol. Strikingly, myocardial effects of nebivolol differed from those of bisoprolol, by inducing a ß3-AR and NOS-dependent prolongation of twitch activity.

Prediction of Neurodevelopment in Infants With Tuberous Sclerosis Complex Using Early EEG Characteristics.

Tuberous Sclerosis Complex (TSC) is a multisystem genetic disorder with a high risk of early-onset epilepsy and a high prevalence of neurodevelopmental comorbidities, including intellectual disability and autism spectrum disorder (ASD). Therefore, TSC is an interesting disease model to investigate early biomarkers of neurodevelopmental comorbidities when interventions are favourable. We investigated whether early EEG characteristics can be used to predict neurodevelopment in infants with TSC. The first recorded EEG of 64 infants with TSC, enrolled in the international prospective EPISTOP trial (recorded at a median gestational age 42 4/7 weeks) was first visually assessed. EEG characteristics were correlated with ASD risk based on the ADOS-2 score, and cognitive, language, and motor developmental quotients (Bayley Scales of Infant and Toddler Development III) at the age of 24 months. Quantitative EEG analysis was used to validate the relationship between EEG background abnormalities and ASD risk. An abnormal first EEG (OR = 4.1, p-value = 0.027) and more specifically a dysmature EEG background (OR = 4.6, p-value = 0.017) was associated with a higher probability of ASD traits at the age of 24 months. This association between an early abnormal EEG and ASD risk remained significant in a multivariable model, adjusting for mutation and treatment (adjusted OR = 4.2, p-value = 0.029). A dysmature EEG background was also associated with lower cognitive (p-value = 0.029), language (p-value = 0.001), and motor (p-value = 0.017) developmental quotients at the age of 24 months. Our findings suggest that early EEG characteristics in newborns and infants with TSC can be used to predict neurodevelopmental comorbidities.

Ventricular ErbB2/ErbB4 activation and downstream signaling in pacing-induced heart failure.

The neuregulin-1 (NRG-1)/ErbB system has emerged as a cardioprotective system that becomes activated during myocardial stress, most convincingly shown in response to cardiotoxic chemotherapy. Direct evidence of increased ventricular ErbB receptor activity in heart failure unrelated to cardiotoxic drugs is, however, limited. We investigated changes in NRG-1 expression, ErbB receptor phosphorylation and downstream activation of intracellular ErbB targets during rapid pacing and progressive ventricular dysfunction in the dog. Heart failure was induced in dogs by 7 weeks of rapid pacing. Ventricular function was assessed by echocardiography. Messenger RNA expression was investigated in ventricular biopsies using quantitative PCR. Activation of NRG-1/ErbB signaling and of downstream targets was investigated using immunoprecipitation and/or Western blotting. Over the course of 7 weeks of pacing and ventricular dilatation, ventricular levels of NRG-1, but not of other ErbB4 ligands, and of ADAM19, a protease promoting NRG-1 release, progressively increased. In parallel, levels of activated ErbB2 and ErbB4, phosphorylated at tyrosine residues 877/1248 and 1284 respectively, became progressively higher. Similarly, levels of total and phosphorylated PI-3 kinase increased. Surprisingly, however, and in contrast with activation of downstream targets of ErbB receptors in normal hearts, Akt and ERK1/2, remained inactivated. This study shows that ventricular ErbB2 and ErbB4 receptors become activated during the development of pacing-induced heart failure, but that downstream signaling is, at least partly, abrogated. Abrogation of cardioprotective signaling after ErbB activation is an unanticipated phenomenon in the progression of heart failure with possibly major pathophysiological significance. The underlying mechanisms should be further elucidated.

Role of neuregulin-1/ErbB signaling in cardiovascular physiology and disease: implications for therapy of heart failure.

Since the discovery that neuregulin-1 (NRG-1)/ErbB signaling is indispensable in cardiac development, evidence has shown that this system also plays a crucial role in the adult heart. In patients, an inhibitory ErbB2 antibody, trastuzumab, used in the treatment of mammary carcinomas, increases the risk for the development of cardiotoxic cardiomyopathy. Postnatal disruption of NRG-1/ErbB signaling by gene targeting in mice leads to dilated cardiomyopathy. Initially, the search for the mechanisms behind these observations focused mainly on the effects of NRG-1 on cardiomyocyte growth and survival and revealed that NRG-1 has Akt-dependent antiapoptotic effects in cultured cardiomyocytes. In vivo studies, however, did not uniformly reinforce a role for apoptosis in the development of cardiomyopathy induced by impaired NRG-1/ErbB signaling. More recent studies have revealed that NRG-1 is involved in the regulation of cardiac sympathovagal balances by counterbalancing adrenergic stimulation of the adult myocardium and through an obligatory interaction with the muscarinic cholinergic system. NRG-1 is synthesized and released by the endocardial and cardiac microvascular endothelium, dynamically controlled by neurohormonal and biomechanical stimuli. The physiology of the cardiac NRG-1/ErbB system has implications for the treatment of both cancer and heart failure. Clinical studies in breast cancer with novel ErbB inhibitors are currently underway. Novel oncological indications for ErbB inhibition are emerging; cardiovascular side effects need to be carefully monitored. On the other hand, pharmacological activation of ErbB signaling is likely an unrecognized and beneficial effect of currently used drugs in heart failure and a promising therapeutic approach to prevent or reverse myocardial dysfunction.

Neuregulin-1 and its potential role in the control of cardiac function.

The rapidly evolving insights into the protective and modulatory function of neuregulin-1 (NRG-1) in the adult heart are discussed in this review. The actions of NRG-1 in the adult heart have begun to be elucidated following the unexpected clinical observation that trastuzumab can cause ventricular dysfunction and increases the risk of cardiomyopathy induced by anthracyclines. Trastuzumab is an inhibitory antibody against the NRG receptor erythroblastic leukemia viral oncogene homolog 2 (ErbB2) and is used in the treatment of breast cancer. In vitro studies have demonstrated that NRG-1 promotes growth and survival of isolated cardiomyocytes. Ventricular dysfunction following anti-ErbB2 treatment was initially explained by a loss of ErbB2-dependent cell survival pathways in the heart. However, in vivo studies in genetically modified mice did not uniformly confirm this finding. More recent studies have revealed that NRG-1 counterbalances the adrenergic inotropic response of the adult myocardium through an obligatory interaction with the muscarinic cholinergic system. In addition, it was demonstrated that cardiac NRG-1 synthesis and release from the cardiac endothelium, the principal source of NRG-1 in the heart, is dynamically controlled by neurohormonal and biomechanical stimuli, allowing adaptive tuning of ErbB signaling during cardiovascular stress. Cardiac NRG-1 is beginning to emerge as a cardioprotective factor implicated in the physiological regulation of myocardial performance and sympathovagal balances. Cardiac NRG-1/ErbB signaling has implications for the treatment of both cancer and heart failure. As novel ErbB inhibitors are currently being tested in broader oncological indications, there is a need to better understand their cardiovascular side effects. It is possible that pharmacological activation of ErbB signaling is an indirect, beneficial effect of the drugs currently used in heart failure, and this could be a promising therapeutic approach for prevention or reversal of myocardial dysfunction. Heart Fail Monit 2008;5(4):119-24.

Neuregulin-1 attenuates stress-induced vascular senescence.

Aims: Cardiovascular ageing is a key determinant of life expectancy. Cellular senescence, a state of irreversible cell cycle arrest, is an important contributor to ageing due to the accumulation of damaged cells. Targeting cellular senescence could prevent age-related cardiovascular diseases. In this study, we investigated the effects of neuregulin-1 (NRG-1), an epidermal growth factor with cardioprotective and anti-atherosclerotic effects, on cellular senescence. Methods and results: Senescence was induced in cultured rat aortic endothelial cells (ECs) and aortic smooth muscle cells (SMCs) by 2 h exposure to 30 µM hydrogen peroxide (H2O2). Cellular senescence was confirmed after 72 h using senescence-associated-ß-galactosidase staining (SA-ß-gal), cell surface area, and western blot analyses of SA pathways (acetyl-p53, p21). Recombinant human NRG-1 (rhNRG-1, 20 ng/mL) significantly reduced H2O2-induced senescence, as shown by a lower number of SA-ß-gal positive cells, smaller surface area and lower expression of acetyl-p53. In C57BL/6 male mice rendered diabetic with streptozotocin (STZ), rhNRG-1 attenuated cellular senescence in aortic ECs and SMCs. Next, we created mice with SMC-specific knockdown of the NRG-1 receptor ErbB4. Aortic SMCs isolated from SMC-specific ErbB4 deficient mice (ErbB4f/+ SM22α-Cre+) showed earlier cellular senescence in vitro compared with wild-type (ErbB4+/+ SM22α-Cre+) SMCs. Furthermore, when rendered diabetic with STZ, ErbB4f/+ SM22α-Cre+ male mice showed significantly more vascular senescence than their diabetic wild-type littermates and had increased mortality. Conclusions: This study is the first to explore the role of NRG-1 in vascular senescence. Our data demonstrate that NRG-1 markedly inhibits stress-induced premature senescence in vascular cells in vitro and in the aorta of diabetic mice in vivo. Consistently, deficiency in the NRG-1 receptor ErbB4 provokes cellular senescence in vitro as well as in vivo.

Endogenous inhibitors of hypertrophy in concentric versus eccentric hypertrophy.

Left ventricular (LV) hypertrophy (LVH) is an adaptive response to hemodynamic overload, but also contributes to the pathogenesis of heart failure. LVH can be concentric (cLVH) but subsequent dilatation and progression to eccentric hypertrophy (eLVH) may lead to global pump failure. Recently, several endogenous molecular inhibitors of hypertrophy have been identified. Using real-time PCR, we compared the myocardial mRNA expression of these inhibitors in pressure-overload induced cLVH (severe aortic stenosis) and in volume overload-induced eLVH (severe mitral regurgitation) in patients, and during the progression from cLVH to eLVH in pressure overload in rat. Each of these genes showed a unique temporal expression profile. Strikingly, except for SOCS-3, changes in gene expression of these negative regulators in rat cLVH and eLVH vs sham were recapitulated in human cLVH and eLVH. In particular, VDUP-1 and MCIP-1 were high in cLVH but expression levels were normal in eLVH, both in rat and human. These data indicate that during the progression of LVH, both in pressure and volume overload, expression levels of endogenous inhibitors of hypertrophy are modified and that these changes may have pathophysiological significance. In particular, MCIP-1 (the endogenous calcineurin inhibitor) and VDUP-1 (the endogenous inhibitor of thioredoxin) are potential molecular switches in the progression of LV hypertrophy.

Targeting Endothelial Function to Treat Heart Failure with Preserved Ejection Fraction: The Promise of Exercise Training.

Although the burden of heart failure with preserved ejection fraction (HFpEF) is increasing, there is no therapy available that improves prognosis. Clinical trials using beta blockers and angiotensin converting enzyme inhibitors, cardiac-targeting drugs that reduce mortality in heart failure with reduced ejection fraction (HFrEF), have had disappointing results in HFpEF patients. A new "whole-systems" approach has been proposed for designing future HFpEF therapies, moving focus from the cardiomyocyte to the endothelium. Indeed, dysfunction of endothelial cells throughout the entire cardiovascular system is suggested as a central mechanism in HFpEF pathophysiology. The objective of this review is to provide an overview of current knowledge regarding endothelial dysfunction in HFpEF. We discuss the molecular and cellular mechanisms leading to endothelial dysfunction and the extent, presence, and prognostic importance of clinical endothelial dysfunction in different vascular beds. We also consider implications towards exercise training, a promising therapy targeting system-wide endothelial dysfunction in HFpEF.

Endothelial Senescence Contributes to Heart Failure With Preserved Ejection Fraction in an Aging Mouse Model.

BACKGROUND: Because of global aging, the prevalence of heart failure with preserved ejection fraction (HFpEF) continues to rise. Although HFpEF pathophysiology remains incompletely understood, endothelial inflammation is stated to play a central role. Cellular senescence is a process of cellular growth arrest linked with aging and inflammation. We used mice with accelerated aging to investigate the role of cellular senescence in HFpEF development. METHODS AND RESULTS: Senescence-accelerated mice (SAM, n=18) and control mice with normal senescence (n=15) were fed normal chow or a high-fat, high-salt diet (WD). Vascular and cardiac function was assessed at 8, 16, and 24 weeks of age. At 24 weeks, both SAM on WD (SAM-WD) and SAM on regular diet displayed endothelial dysfunction, as evidenced by impaired acetylcholine-induced relaxation of aortic segments and reduced basal nitric oxide. At week 24, SAM-WD had developed HFpEF, characterized by diastolic dysfunction, left ventricular hypertrophy, left atrial dilatation, and interstitial fibrosis. Also, exercise capacity was reduced and lung weight increased. Cardiovascular inflammation and senescence were assessed by immunohistochemical and immunofluorescence staining of hearts and aortas. SAM-WD showed increased endothelial inflammation (intercellular adhesion molecule 1 expression) and increased endothelial senescence (acetyl-p53/CD31 costaining). The latter correlated with diastolic function and intercellular adhesion molecule 1 expression. CONCLUSIONS: SAM develop endothelial dysfunction. Adding a high-salt, high-fat diet accelerates endothelial senescence and instigates endothelial inflammation. This coincides with hemodynamic and structural changes typical of HFpEF. Targeting endothelial senescence could be a new therapeutic avenue in HFpEF.

Continuous administration of the mTORC1 inhibitor everolimus induces tolerance and decreases autophagy in mice.

BACKGROUND AND PURPOSE: Everolimus is an allosteric inhibitor of the mechanistic target of rapamycin complex 1 (mTORC1) widely known for its potent autophagy stimulating properties. Because everolimus shows poor solubility and stability in aqueous solutions, long-term in vivo administration in preclinical models is challenging. The aim of the present study was to evaluate the effects of short-term and long-term everolimus administration on mTORC1 inhibition and autophagy induction in mice. EXPERIMENTAL APPROACH: We developed a vehicle in which everolimus was solubilized and stable at 37°C for at least 1 month. Using osmotic minipumps, GFP microtubule-associated protein light chain 3 transgenic mice were treated continuously either with vehicle or everolimus (1.5 mg·kg-1 per day) for 3 or 28 days. Alternatively, a regimen consisting of intermittent everolimus administration (every other day) for 56 days by oral gavage was used. Autophagy markers and mTORC1 activation status were investigated in the liver. KEY RESULTS: As expected, everolimus inhibited mTORC1 and stimulated autophagy in the liver after 3 days of treatment. However, continuous administration for 28 days resulted in hyperactivation of the Akt1-mTORC1 pathway accompanied by a remarkable decrease in autophagy markers. Everolimus given intermittently for 56 days partially rescued mTORC1 sensitivity to the drug but without inducing autophagy. The failure to induce autophagy following long-term everolimus administration was due to uncoupling of the mTORC1 substrate unc-51 like autophagy activating kinase 1. CONCLUSIONS AND IMPLICATIONS: Our data encourage the use of intermittent everolimus regimens to prevent tolerance and to extend its activity.

Neuregulin-1 induces a negative inotropic effect in cardiac muscle: role of nitric oxide synthase.

BACKGROUND: Deficient cardiac neuregulin/ErbB signaling increases susceptibility to heart failure. In this study, we examined the effects of neuregulin-1 (NRG-1) on myocardial contractility. METHODS AND RESULTS: NRG-1 (alpha and beta isoforms) induced a negative inotropic effect in isolated rabbit papillary muscles and a rightward shift of the dose-response curve to isoproterenol. Both effects were attenuated by L-NMMA, which suggests a role for NO synthase. In cultured rat cardiomyocytes, NRG-1beta enhanced nitrite production and resulted in phosphorylation of endothelial NO synthase and the serine/threonine kinase Akt. CONCLUSIONS: NRG-1 has negative inotropic effects that are preserved during beta-adrenergic stimulation and activates endothelial NO synthase in cardiomyocytes.

Chronic intermittent mental stress promotes atherosclerotic plaque vulnerability, myocardial infarction and sudden death in mice.

Vulnerable atherosclerotic plaques are prone to plaque rupture leading to acute cardiovascular syndromes and death. Elucidating the risk of plaque rupture is important to define better therapeutic or preventive strategies. In the present study, we investigated the effect of chronic intermittent mental stress on atherosclerotic plaque stability and cardiovascular mortality in apolipoprotein E-deficient (ApoE(-/-)) mice with a heterozygous mutation in the fibrillin-1 gene (Fbn1(C1039G+/)(-)). This mouse model displays exacerbated atherosclerosis with spontaneous plaque ruptures, myocardial infarction and sudden death, when fed a Western-type diet (WD). Female ApoE(-/-)Fbn1(C1039G+/-) mice were fed a WD for up to 25 weeks. After 10 weeks WD, mice were divided in a control (n = 27) and mental stress (n = 29) group. The chronic intermittent mental stress protocol consisted of 3 triggers: water avoidance, damp bedding and restraint stress, in a randomly assigned order lasting 6 h every weekday for 15 weeks. Chronic intermittent mental stress resulted in a significant increase in the amount of macrophages in atherosclerotic plaques of the proximal ascending aorta, whereas type I collagen and fibrous cap thickness were decreased. The coronary arteries of mental stress-treated mice showed larger plaques, more stenosis, and an increased degree of perivascular fibrosis. Moreover, myocardial infarctions occurred more frequently in the mental stress group. As compared to the control group, the survival of stressed ApoE(-/-)Fbn1(C1039G+/-) mice decreased from 67% to 52% at 25 weeks WD, presumably due to myocardial infarctions. In conclusion, chronic intermittent mental stress promotes plaque instability, myocardial infarctions, and mortality of ApoE(-/-)Fbn1(C1039G+/-) mice.