Search

Aims Degenerative mitral valve dystrophy (MVD) leading to mitral valve prolapse is the most frequent form of MV disease, and there is currently no pharmacological treatment available. The limited understanding of the pathophysiological mechanisms leading to MVD limits our ability to identify therapeutic targets. This study aimed to reveal the main pathophysiological pathways involved in MVD via the multimodality imaging and transcriptomic analysis of the new and unique knock-in (KI) rat model for the FilaminA-P637Q (FlnA-P637Q) mutation associated-MVD. Methods and results Wild-type (WT) and KI rats were evaluated morphologically, functionally, and histologically between 3-week-old and 3-to-6-month-old based on Doppler echocardiography, 3D micro-computed tomography (microCT), and standard histology. RNA-sequencing and Assay for Transposase-Accessible Chromatin (ATAC-seq) were performed on 3-week-old WT and KI mitral valves and valvular cells, respectively, to highlight the main signalling pathways associated with MVD. Echocardiographic exploration confirmed MV elongation (2.0 ± 0.1 mm vs. 1.8 ± 0.1, P = 0.001), as well as MV thickening and prolapse in KI animals compared to WT at 3 weeks. 3D MV volume quantified by microCT was significantly increased in KI animals (+58% vs. WT, P = 0.02). Histological analyses revealed a myxomatous remodelling in KI MV characterized by proteoglycans accumulation. A persistent phenotype was observed in adult KI rats. Signalling pathways related to extracellular matrix homeostasis, response to molecular stress, epithelial cell migration, endothelial to mesenchymal transition, chemotaxis and immune cell migration, were identified based on RNA-seq analysis. ATAC-seq analysis points to the critical role of transforming growth factor-β and inflammation in the disease. Conclusion The KI FlnA-P637Q rat model mimics human myxomatous MVD, offering a unique opportunity to decipher pathophysiological mechanisms related to this disease. Extracellular matrix organization, epithelial cell migration, response to mechanical stress, and a central contribution of immune cells are highlighted as the main signalling pathways leading to myxomatous MVD. Our findings pave the road to decipher underlying molecular mechanisms and the specific role of distinct cell populations in this context.

RATIONALECardiac rhythm, conduction and synchronization of electrical activity require the coordinated action of different types of ion channels that differ according to transmural and regional specificities. Classical pharmacology affects these ion channels in a non-regionalized way which explains why treating arrhythmias, that often occur in specific foci, has often limited efficacy in addition to negative side-effects on non-targeted organs. Photopharmacology is an emergent technology that has the potential to counteract all the negative aspects of classical pharmacology by restricting drug activity in a spatio-temporal manner.OBJECTIVEWe tested the potential of photopharmacology in specifically regulating heart activity by using a caged derivative of a natural peptide inhibitor of the ERG channel, BeKm1. The peptide was uncaged and activity monitoredin vitroon a cell line expressing the hERG channel, on human cardiomyocytes derived from iPS cells, andex vivoandin vivoon zebrafish larvae and rat hearts.METHODS AND RESULTSCaged BeKm-1 is inactive and fully active upon uncaging. Uncaging of the peptide on human iPS-derived cardiomyocytes enlarges the action potential duration and triggers arrhythmias. Uncaging also triggers bradycardia and disturbs cardiac conduction within the atria in perfused rat hearts upon illumination. The potency of photopharmacology for cardiac electrical modulation was further validated in zebrafish larvae where illumination of the caged compound induces bradycardia and atrio-ventricular desynchrony. Finally, in anesthetized rats, illumination of the caged peptide in the right atria, containing the sino-atrial node, leads to bradycardia without arrhythmia.CONCLUSIONSThis report demonstrates that photopharmacology, using the caged peptide strategy, can be used for dynamically regulating cardiac electrical activityin vivoand that spatial illumination restriction can dissociate the bradycardic effect from the arrhythmic one. The technology is applicable to all kinds of cardiac ion channels and regions of interest to create arrhythmogenic models or investigate new clinical applications.

Introduction. Septic shock is a systemic inflammatory response syndrome associated with organ failures. Earlier clinical diagnosis would be of benefit to a decrease in the mortality rate. However, there is currently a lack of predictive biomarkers. The secretome is the set of proteins secreted by a cell, tissue, or organism at a given time and under certain conditions. The plasma secretome is easily accessible from biological fluids and represents a good opportunity to discover new biomarkers that can be studied with nontargeted “omic” strategies. Aims. To identify relevant deregulated proteins (DEP) in the secretome of a rat endotoxemic shock model. Methods. Endotoxemic shock was induced in rats by intravenous injection of lipopolysaccharides (LPS, S. enterica typhi, 0.5 mg/kg) and compared to controls (Ringer Lactate, iv). Under isoflurane anesthesia, carotid cannulation allowed mean arterial blood pressure (MAP) and heart rate (HR) monitoring and blood sampling at different time points (T0 and T50 or T0 and T90, with EDTA and protease inhibitor). Samples were prepared for large-scale tandem mass spectrometry (MS-MS) based on a label-free quantification to allow identification of the proteins deregulated upon endotoxemic conditions. A Gene Ontology (GO) analysis defined several clusters of biological processes (BP) in which the DEP are involved. Results. Ninety minutes after shock induction, the LPS group presents a reduction in MAP (-45%, p < 0.05 ) and increased lactate levels (+27.5%, p < 0.05 ) compared to the control group. Proteomic analyses revealed 10 and 33 DEP in the LPS group, respectively, at 50 and 90 minutes after LPS injection. At these time points, GO-BP showed alterations in pathways involved in oxidative stress response and coagulation. Conclusion. This study proposes an approach to identify relevant DEP in septic shock and brings new insights into the understanding of the secretome adaptations upon sepsis.

In clinical practice, extracorporeal circulation (ECC) is associated with coagulopathy and inflammation, eventually leading to organ injuries without preventive systemic pharmacological treatment. Relevant models are needed to reproduce the pathophysiology observed in humans and preclinical tests. Rodent models are less expensive than large models but require adaptations and validated comparisons to clinics. This study aimed to develop a rat ECC model and to establish its clinical relevance. One hour of veno-arterial ECC or a sham procedure were achieved on mechanically ventilated rats after cannulations with a mean arterial pressure objective > 60 mmHg. Five hours post-surgery, the rats’ behavior, plasmatic/blood biomarkers, and hemodynamics were measured. Blood biomarkers and transcriptomic changes were compared in 41 patients undergoing on-pump cardiac surgery. Five hours post-ECC, the rats presented hypotension, hyperlactatemia, and behavioral alterations. The same patterns of marker measurements (Lactate dehydrogenase, Creatinine kinase, ASAT, ALAT, and Troponin T) were observed in both rats and human patients. Transcriptome analyses showed similarity in both humans and rats in the biological processes involved in the ECC response. This new ECC rat model seems to resemble both ECC clinical procedures and the associated pathophysiology, but with early organ injury corresponding to a severe phenotype. Although the mechanisms at stake in the post-ECC pathophysiology of rats or humans need to be described, this new rat model appears to be a relevant and costless preclinical model of human ECC.

O-GlcNAcylation is a post-translational modification which affects approximately 5000 human proteins. Its involvement has been shown in many if not all biological processes. Variations in O-GlcNAcylation levels can be associated with the development of diseases. Deciphering the role of O-GlcNAcylation is an important issue to (i) understand its involvement in pathophysiological development and (ii) develop new therapeutic strategies to modulate O-GlcNAc levels. Over the past 30 years, despite the development of several approaches, knowledge of its role and regulation have remained limited. This review proposes an overview of the currently available tools to study O-GlcNAcylation and identify O-GlcNAcylated proteins. Briefly, we discuss pharmacological modulators, methods to study O-GlcNAcylation levels and approaches for O-GlcNAcylomic profiling. This review aims to contribute to a better understanding of the methods used to study O-GlcNAcylation and to promote efforts in the development of new strategies to explore this promising modification.

International audience; All five muscarinic receptors have important physiological roles. The endothelial M2 and M3 subtypes regulate arterial tone through direct coupling to Gq or Gi/o proteins. Yet, we lack selective pharmacological drugs to assess the respective contribution of muscarinic receptors to a given function. We used mamba snake venoms to identify a selective M2R ligand to investigate its contribution to arterial contractions. Using a bio-guided screening binding assay, we isolated MT9 from the black mamba venom, a three-finger toxin active on the M2R subtype. After sequencing and chemical synthesis of MT9, we characterized its structure by X-ray diffraction and determined its pharmacological characteristics by binding assays, functional tests, and ex vivo experiments on rat and human arteries. Although MT9 belongs to the three-finger fold toxins family, it is phylogenetically apart from the previously discovered muscarinic toxins, suggesting that two groups of peptides evolved independently and in a convergent way to target muscarinic receptors. The affinity of MT9 for the M2R is 100 times stronger than that for the four other muscarinic receptors. It also antagonizes the M2R/G i pathways in cell-based assays. MT9 acts as a non-competitive antagonist against acetylcholine or arecaine, with low nM potency, for the activation of isolated rat mesenteric arteries. These results were confirmed on human internal mammary arteries. In conclusion, MT9 is the first fully characterized M2R-specific natural toxin. It should provide a tool for further understanding of the effect of M2R in various arteries and may position itself as a new drug candidate in cardiovascular diseases

Best strategies for managing severe pediatric traumatic brain injury (TBI) are not established, with wide variations among professional practices. The main objective of this study was to assess compliance with updated pediatric TBI management guidelines (2019). A survey was distributed among French-speaking pediatric intensive care physicians from April 1 to June 30, 2019. The survey was based on a clinical case with a total of 70 questions that cover the 15 items of the 2019 TBI guidelines. The questions evaluated the assessment and management of TBI during the acute and intensive care phases. Of 487 e-mails sent, 78 surveys were included. Guidelines were adhered to (> 60%) for 10 of 15 items in the guidelines. Strong adherence to recent guideline changes was achieved for seizure prophylaxis with levetiracetam (n = 21/33, 64%) and partial pressure of carbon dioxide threshold (n = 52, 67%). However, management of the sodium and glucose thresholds and the role of transcranial Doppler were not consistent with the guidelines. Assessment of brain tissue oxygenation (n = 12, 16%) and autoregulation (n = 35, 45%) was not a common practice. There was strong agreement among clinicians on the intracranial pressure (> 80%) and cerebral perfusion pressure (> 70%) thresholds used according to age. Overall, stated practices for the management of TBI appear to be relatively standardized among responders. Variations persist in areas with a lack of evidence and pediatric-specific recommendations.

50% of patients with heart failure have a preserved ejection fraction (HFpEF). Numerous studies have investigated the pathophysiological mechanisms of HFpEF and have shown that endothelial dysfunction plays an important role in HFpEF. Yet no studies answered whether endothelial dysfunction could be the cause or is the consequence of HFpEF. Recently, we have shown that the endothelial overexpression of human β3-adrenoreceptor (Tgβ3) in rats leads to the slow development of diastolic dysfunction over ageing. The aim of the study is to decipher the involvement of endothelial dysfunction in the HFpEF development. For that, we investigated endothelial and cardiac function in 15-, 30-, and 45-week-old wild-type (WT) and Tgβ3 rats. The aortic expression of •NO synthase (NOS) isoforms was evaluated by Western blot. Finally, electron paramagnetic resonance measurements were performed on aortas to evaluate •NO and O2•- production. Vascular reactivity was altered as early as 15 weeks of age in response to isoproterenol in Tgβ3 aortas and mesenteric arteries. NOS1 (neuronal NOS) expression was higher in the Tgβ3 aorta at 30 and 45 weeks of age (30 weeks: WT: 1.00 ± 0.21 ; Tgβ3: 6.08 ± 2.30 ; 45 weeks: WT: 1.00 ± 0.12 ; Tgβ3: 1.55 ± 0.17 ; p < 0.05 ). Interestingly, the endothelial NOS (NOS3) monomer form is increased in Tgβ3 rats at 45 weeks of age (ratio NOS3 dimer/NOS3 monomer; WT: 1.00 ± 0.37 ; Tgβ3: 0.13 ± 0.05 ; p < 0.05 ). Aortic •NO production was increased by NOS2 (inducible NOS) at 15 weeks of age in Tgβ3 rats (+52% vs. WT). Aortic O2•- production was increased in Tgβ3 rats at 30 and 45 weeks of age (+75% and+76%, respectively, vs. WT, p < 0.05 ). We have shown that endothelial dysfunction and oxidative stress are present as early as 15 weeks of age and therefore conclude that endothelial dysfunction could be a cause of HFpEF development.

Protein O-GlcNAcylation is increasingly recognized as an important cellular regulatory mechanism, in multiple organs including the heart. However, the mechanisms leading to O-GlcNAcylation in mitochondria and the consequences on their function remain poorly understood. In this study, we used an in vitro reconstitution assay to characterize the intra-mitochondrial O- GlcNAc system without potential cytoplasmic confounding effects. We compared the O-GlcNAcylome of isolated cardiac mitochondria with that of mitochondria acutely exposed to NButGT, a specific O-GlcNAcylation inducer. Amongst the 409 O-GlcNAcylated mitochondrial proteins identified, 191 displayed increased O-GlcNAcylation in response to NButGT. This was associated with enhanced Complex I (CI) activity, increased maximal respiration in presence of CI substrates, and a striking reduction of mitochondrial ROS release, which could be related to O- GlcNAcylation of subunits within the NADH dehydrogenase module of CI. In conclusion, our work underlines the existence of a dynamic mitochondrial O-GlcNAcylation system capable of rapidly modifying mitochondrial function.

International audience; Implantable pressure sensors represent an important part of the research activity in laboratories. Unfortunately, their use is limited by cost, autonomy and temperature-related drifts. The cost of use depends on several parameters, particularly their low battery life and the need for miniaturization to be able to implant the animals and monitor them over a time that is long enough to be physiologically relevant. This paper studied the possibility of reducing the thermal drift of implantable sensors. To quantify and compensate for the thermal drift, we developed the equivalent model of the piezoresistive probe by using the Cadence software. Our model takes into account the temperature (34–39 °C) as well as the pressure (0–300 mmHg). We were thus able to identify the source of the drift and thanks to our model, we were able to compensate for it thanks to the compensation circuits added to the conditioning circuits of the sensor. The maximum relative drift of the sensor is (0.1 mV/°C)/3.6 mV (2.7%), a drift of the conditioning circuit is (0.98 mV/°C)/916 mV (0.1%) and the whole is (13.4 mV/°C)/420 mV (32%). The compensated sensor shows a relative maximum drift of (0.371 mV/°C)/405 mV (0.09%). The output voltage remains stable over the measurement temperature range.

Background Mitogen‐activated protein kinase–activated protein kinase‐2 (MK2) is a protein serine/threonine kinase activated by p38α/β. Herein, we examine the cardiac phenotype of pan MK2‐null (MK2 −/− ) mice. Methods and Results Survival curves for male MK2 +/+ and MK2 −/− mice did not differ (Mantel‐Cox test, P =0.580). At 12 weeks of age, MK2 −/− mice exhibited normal systolic function along with signs of possible early diastolic dysfunction; however, aging was not associated with an abnormal reduction in diastolic function. Both R‐R interval and P‐R segment durations were prolonged in MK2‐deficient mice. However, heart rates normalized when isolated hearts were perfused ex vivo in working mode. Ca 2+ transients evoked by field stimulation or caffeine were similar in ventricular myocytes from MK2 +/+ and MK2 −/− mice. MK2 −/− mice had lower body temperature and an age‐dependent reduction in body weight. mRNA levels of key metabolic genes, including Ppargc1a , Acadm , Lipe , and Ucp3, were increased in hearts from MK2 −/− mice. For equivalent respiration rates, mitochondria from MK2 −/− hearts showed a significant decrease in Ca 2+ sensitivity to mitochondrial permeability transition pore opening. Eight weeks of pressure overload increased left ventricular mass in MK2 +/+ and MK2 −/− mice; however, after 2 weeks the increase was significant in MK2 +/+ but not MK2 −/− mice. Finally, the pressure overload–induced decrease in systolic function was attenuated in MK2 −/− mice 2 weeks, but not 8 weeks, after constriction of the transverse aorta. Conclusions Collectively, these results implicate MK2 in (1) autonomic regulation of heart rate, (2) cardiac mitochondrial function, and (3) the early stages of myocardial remodeling in response to chronic pressure overload.

The use of animal models in fundamental or pre-clinical research remains an absolute requirement for understanding human pathologies and developing new drugs. In order to transpose these results into clinical practice, many parameters must be taken into account to limit bias. Attention has recently been focused on the sex, age or even strain of each animal, but the impact of diet has been largely neglected. Soy, which is commonly used in the diet in varying quantities can affect their physiology. In order to assess whether the presence of soy can impact the obtained results, we studied the impact of a soy-based diet versus a soy-free diet, on diastolic function in a rat model based on transgenic overexpression of the β3-adrenergic receptors in the endothelium and characterized by the appearance of diastolic dysfunction with age. Our results show that the onset of diastolic dysfunction is only observed in transgenic male rats fed with a soy-free diet in the long term. Our study highlights the importance of the diet’s choice in the study design process, especially regarding the proportion of soy, to correctly interpret the outcome as low-cost diets are more likely to be highly concentrated in soy.

Sepsis in the young population, which is particularly at risk, is rarely studied. O-GlcNAcylation is a post-translational modification involved in cell survival, stress response and metabolic regulation. O-GlcNAc stimulation is beneficial in adult septic rats. This modification is physiologically higher in the young rat, potentially limiting the therapeutic potential of O-GlcNAc stimulation in young septic rats. The aim is to evaluate whether O-GlcNAc stimulation can improve sepsis outcome in young rats. Endotoxemic challenge was induced in 28-day-old rats by lipopolysaccharide injection (E. Coli O111:B4, 20 mg·kg−1) and compared to control rats (NaCl 0.9%). One hour after lipopolysaccharide injection, rats were randomly assigned to no therapy, fluidotherapy (NaCl 0.9%, 10 mL·kg−1) ± NButGT (10 mg·kg−1) to increase O-GlcNAcylation levels. Physiological parameters and plasmatic markers were evaluated 2h later. Finally, untargeted mass spectrometry was performed to map cardiac O-GlcNAcylated proteins. Lipopolysaccharide injection induced shock with a decrease in mean arterial pressure and alteration of biological parameters (p &lt, 0.05). NButGT, contrary to fluidotherapy, was associated with an improvement of arterial pressure (p &lt, 0.05). ATP citrate lyase was identified among the O-GlcNAcylated proteins. In conclusion, O-GlcNAc stimulation improves outcomes in young septic rats. Interestingly, identified O-GlcNAcylated proteins are mainly involved in cellular metabolism.

This article presents an analog method of compensation for thermal drifts of implantable piezoresistive blood pressure sensors used in wireless medical monitoring. A PNP transistor-based circuit in parallel with a variable resistor is used. This circuit is in series with the pressure probe constituted of four piezoresistors in Wheatstone bridge. This analog method is effective in reducing the thermal drift of the sensor and also has a low cost and consumption. The thermal drift of the sensor before compensation is 9.55 mmHg over the temperature range [34°C - 39°C] with the pressure of 300 mmHg and after compensation, the drift is reduced to 2.5 mmHg. This method has been validated with 3 pressure levels (0, 100, 200 and 300 mmHg).

Background: O -GlcNAcylation ( O -GlcNAc) is a post-translational modification involved in the stress response. A single pair of enzymes, O -GlcNAc transferase (OGT) and O -GlcNAcase (OGA) controls the O -GlcNAcylation. Due to the contribution of energy-linked metabolites (e.g. glucose, glutamine, etc.) to produce UDP-GlcNAc, the substrate used by OGT, O -GlcNAc is considered as a nutrient sensor. Metabolic sources vary from carbohydrates in utero , to fatty acids after birth then a mix once adult. Yet the link between diet composition and O -GlcNAc has never been explored. Purpose: Evaluate changes in O -GlcNAc levels and regulatory mechanisms throughout development. Methods: Organs were harvested from Wistar rats (n=6-8 for each group) before and after birth (D-1 and D0), in suckling animals (D12), after weaning with a standard (D28) or a carbohydrate free diet (D28F), and adult (D84). O -GlcNAc levels and regulatory enzymes were evaluated by Western blots. Mass spectrometry (MS) was performed to quantify known regulators of O -GlcNAc (n=6-8) and identify cardiac O -GlcNAcylated proteins at D0 and D28 (n=2). Results: O -GlcNAc levels fell after birth in the heart, brain and liver (2.5, 1.5 and 1.3-fold, respectively). Interestingly, while O -GlcNAc levels decrease progressively from D0 to D84 (5.75-fold pO -GlcNAc changes are opposite and of much smaller magnitude in liver and brain. Regulatory mechanisms appear to be in accordance in heart with a decrease in OGT (4.5-fold; pO -GlcNAc levels in brain and liver. O -GlcNAc levels are unaffected by weaning diet in any tissues. MS analyses identified changes in cardiac levels of specific O -GlcNAcylated proteins, namely those involved in the energy metabolism, such as ACAT1, which is only O -GlcNAcylated at D0. Conclusion: We demonstrate that O -GlcNAc levels are not linked to dietary intake and are regulated in a time and tissue-specific manner during postnatal development. We have identified by MS proteins with a particular O -GlcNAc signature across the development process suggesting specific role for those proteins.

Background: We have shown that increase in O -GlcNAc levels, a post-translational modification involved in the stress response, at the early phase of septic shock in adult (84 days old rats) is a potential new therapeutic strategy. Most studies focus in adults while the population most affected by septic shock, young children, is rarely studied. Considering that O- GlcNAc levels are higher in the young, the impact of O- GlcNAc on septic shock in the young should be tested. Purpose: Evaluate if O- GlcNAc stimulation could improve sepsis outcomes in young. Methods: Endotoxemic shock was induced in 28 days old rats with an i.v. injection of saline (CTRL, n=10) or LPS (O111:B4, 20mg.kg -1 - LPS, n=9). 1 hour after LPS rats were randomly assigned to no therapy (LPS), fluidotherapy (saline, 10ml.kg -1 - LPS+R, n=10) ± NButGT (10 mg.kg -1 - NButGT, n=11) to increase O- GlcNAc levels. 2 hours later, physiological functions and markers of severity were measured and used in adapted Pediatric RISk of Mortality score (PRISM score). The impact of treatment on survival was evaluated on n=16 per group. Mass spectrometry (MS) study was performed to identify O -GlcNAcylated proteins. Results: LPS induce a shock (mean arterial pressure (MAP): CTRL: 67.2 ± 1.9; LPS: 50.7 ± 2.1; mmHg; p-1 ; pH: CTRL: 7.27 ± 0.02; LPS: 7.15 ± 0.02; p-1 ; creatinine: CTRL: 15.9 ± 1.5; LPS: 25.3 ± 2.6; μmol.l -1 ; pO- GlcNAcylation particularly that of mitochondrial proteins. Conclusions: Despite higher O- GlcNAc levels, we demonstrate that O- GlcNAc stimulation is also a potential new therapeutic strategy for septic shock in young. Our results show that it is the difference between the basal levels and the post-stimulation levels which induces a protection against sepsis. Proteins identify by MS will need to be specifically studied to decipher their impact in septic shock.

Background Implantable pressure sensors are important tools of preclinical research activity in laboratories. Unfortunately, their use is limited by cost, autonomy and temperature-related drifts. Cost and ease of use depend on several parameters, in particular battery life and miniaturization required to be able to implant animals and monitor them over time that is long enough to be physiologically relevant. While thermal drifts inherent in implantable sensors due to animals’ body temperature variation vary depending on their operating principe. Aim Reduction of thermal drift and energy consumption and overall miniaturization. Methods and results The blood pressure sensor studied is based on a Honeywell NPB030 piezoresistive probe chosen due to its low cost and compactness. We look for reducing its thermal drifts over temperature range [34 °C–39 °C] and pressure range [0 - 300 mmHg]. With a classical conditioning circuit, the sensor's overall relative drift is 3,2%/°C (13.4 mV/°C/420 mV), resulting from two contributions, one of 2.7%/°C (0.1 mV/°C/3.6 mV) due to the piezoresistive probe alone, another of 0.1% (0.98 mV/°C/916 mV) due to the conditioning circuit alone. An NPN transistor is added in series with the Wheaston Bridge formed by the piezoresistors of the pressure probe to correct the thermal drift. Thanks to optimization of bias resistors of the transistor using Cadence/Pspice, the measured overall thermal drift of the compensated sensor is reduced to 0.09%/°C (0.371 mV/°C/405 mV), in very good agreement with simulation. It's worth noting that the optimization is made possible due to our original method to determine the value of each piezoresistor in the Wheaston Bridge. Conclusion Our approach proves effective to reduce thermal drift of the studied pressure sensor. Additional space required, cost and consumption will be very low. This method should be assessed and validated with CMS components.

Septic shock is a systemic inflammatory response syndrome associated with circulatory failure leading to organ failure with a 40% mortality rate. Early diagnosis and prognosis of septic shock are necessary for specific and timely treatment. However, no predictive biomarker is available. In recent years, improvements in proteomics-based mass spectrometry have improved the detection of such biomarkers. This approach can be performed on different samples such as tissue or biological fluids. Working directly from human samples is complicated owing to interindividual variability. Indeed, patients are admitted at different stages of disease development and with signs of varying severity from one patient to another. All of these elements interfere with the identification of early, sensitive, and specific septic shock biomarkers. For these reasons, animal models of sepsis, although imperfect, are used to control the kinetics of the development of the pathology and to standardise experimentation, facilitating the identification of potential biomarkers. These elements underline the importance of the choice of animal model used and the sample to be studied during preclinical studies. The aim of this review is to discuss the relevance of different approaches to enable the identification of biomarkers that could indirectly be relevant to the clinical setting.

It remains unknown whether β-blockers are useful and safe in acute myocardial infarction (MI). Owing to its pharmacological profile and vasodilating action, nebivolol (N) is useful in MI. The aim of the present study was to assess in rat whether early nebivolol treatment could be beneficial in MI. It remains unknown whether β-blockers are useful and safe in acute MI. On day (D) 0, male Sprague-Dawley rats underwent left coronary artery ligation (MI) or simple thoracotomy (SHAM). On D1 and D2, the rats were treated with either nebivolol (5 mg.kg-1 .day-1 , MI-N and Sham-N) or vehicle (V, MI-V and Sham-V). On D3, heart rate, left ventricle (LV) intrinsic contractility (PESmid) and arterial elastance were measured. Cardiac and aortic β-Adrenoceptor (AR) subtype mRNA were quantified using real time quantitative RT-qPCR. Catecholamine response was assessed on isolated heart and aortic rings with isoproterenol. PESmid was decreased in MI without worsening the decrease nebivolol. In LV, β1 - and β3 -AR mRNA were respectively decreased and increased in all MI. β3 -AR mRNA increase was partly limited by nebivolol. Ex vivo, basal contractility was less decreased in MI-N than in MI-V. Isoproterenol response was only altered in MI-V. In MI aorta, Nebi prevented β2 - and β3 -AR mRNA increases. In addition, Acetylcholine-induced relaxation was lowered in MI-V but preserved with nebivolol. We demonstrated an early modulation of cardiovascular β3 -AR transcription early MI. Despite its putative negative inotropic properties, nebivolol did not worsen cardiac function in basal conditions and preserved LV catecholamine response.

MAP kinase-activated protein kinase-2 (MK2) is protein serine/threonine kinase activated by p38α/β. Herein we examined the cardiac phenotype of pan MK2-null (MK2-/-) mice. Survival curves for male MK2+/+ and MK2-/- mice did not differ (Mantel-Cox test, P = 0.580). At 12-weeks of age, MK2-/- mice exhibited normal systolic function along with signs of possible early diastolic dysfunction; however, ageing was not associated with an abnormal reduction in diastolic function. Both R-R interval and P-R segment durations were prolonged in MK2-deficient mice. However, heart rates normalized when isolated hearts were perfused ex vivo in working mode. Ca2+ transients evoked by field stimulation or caffeine were similar in ventricular myocytes from MK2+/+ and MK2-/- mice. MK2-/- mice had lower body temperature and an age-dependent reduction in body weight. mRNA levels of key metabolic genes, including Ppargc1a, Acadm, Lipe, and Ucp3 were increased in hearts from MK2-/- mice. For equivalent respiration rates, mitochondria from MK2-/- hearts showed a significant decrease in Ca2+-sensitivity to mitochondrial permeability transition pore (mPTP) opening. Finally, the pressure overload-induced increase in heart weight/tibia length and decrease in systolic function were attenuated in MK2-/- mice two weeks, but not eight weeks, after constriction of the transverse aorta. Collectively, these results implicate MK2 in (i) autonomic regulation of heart rate, (ii) cardiac mitochondrial function, and (iii) the early stages of myocardial remodeling in response to chronic pressure overload.

Background We have shown that increase in O-GlcNAc levels, a post-translational modification, at the early phase of septic shock in adult (84 days old rats) resulted in a 3-fold increase in survival time. Most studies focus only in adults, and the population most affected by septic shock, young children, is rarely studied. Sepsis, with a mortality rate of 29%, is an important but preventable cause of pediatric death worldwide. Considering that basal O-GlcNAc levels are higher in the young, the potential impact of O-GlcNAc stimulation at the early phase of septic shock in the young should be evaluated. Purpose Evaluate if O-GlcNAc stimulation could improve sepsis outcomes in young. Methods Endotoxemic shock was induced in 28 days old rats with an i.v. injection of saline (CTRL) or LPS (O111:B4, 20 mg.kg−1 – LPS). 1 hour after LPS rats were randomly assigned to no therapy (LPS), fluidotherapy (saline, 10 mL.kg−1 – LPS + R) ± NButGT (10 mg.kg−1 – NButGT) to increase O-GlcNAc levels. 2 hours later, physiological functions and plasmatic markers were measured and used in adapted Pediatric RISk of Mortality score (PRISM score). The impact of treatment on survival was evaluated on n = 64. Results LPS induced a shock (mean arterial pressure (MAP): CTRL: 67.2 ± 1.9; LPS: 50.7 ± 2.1; mmHg; P Conclusion Despite higher O-GlcNAc levels in young population, we demonstrate that O-GlcNAc stimulation is able to improve pups’ response to endotoxemic shock. Our results show that it is the difference in O-GlcNAcylation between the basal levels and the post-stimulation levels that induces protection against septic shock.

Background Mitochondria is a key cellular component. During pathophysiological stress mitochondrial function is regulated dynamically to provide cell with sufficient energy. O-GlcNAcylation, a post-translational modification associated with stress response, has been linked to beneficial effect when stimulated in shock situation, yet the link between O-GlcNAc and mitochondrial function remains poorly understood. Aim To evaluate mitochondrial impact of O-GlcNAc modulation. Methods Hearts were harvested from Wistar rat from Ctrl, shocked (LPS) and treated by NButGT, a pharmacological O-GlcNAcylation inducer. Respiration rate was assessed polarographically in isolated mitochondria. O-GlcNAcylomic was performed by mass spectrometry on cardiac tissue from rats at D0 (after birth) and D28 (suckling weaning) as well as from LPS and NButGT treated animals. Gene Ontology (GO) for cellular components (CC) of identified cardiac O-GlcNAcylated proteins from both groups were obtained by running the clusterProfileR package. Results Within D0/28 groups, 647 O-GlcNAcylated proteins were found and 1012 in Ctrl/LPS/NButGT groups. Protein for subsequent analysis were selected by the log(FoldChange) > 2 or Conclusion Mitochondrial O-GlcNAcylation appears to play a major role in pathophysiology. Targeting mitochondrial O-GlcNAcylation could represent a new therapeutic challenge and should be further explored.

Introduction Obesity has become a prevalent pathology, especially in the developed world, being associated with the activation of numerous pathophysiologic mechanisms which may lead to cardiovascular diseases. Objective Our study aimed at assessing cardiac function in a rat model of heart failure and obesity. Methods Sprague-Dawley male rats (wild type, WT) and transgenic rats overexpressing β3-adrenergic receptors in endothelial cells (TGβ3) were either fed with standard diet (WT + SD, n = 12; TGβ3 + SD, n = 12) or with hypercaloric diet (SD + HD, n = 12; TGβ3 + HD, n = 12). For 35 weeks body-weight (BW), systolic blood pressure (SBP), echocardiographic structural and functional parameters were monitored. Oral glucose tolerance test (OGTT) was also performed. Results HD induced an increase in BW in HD groups (∼10.08% for WT and ∼10.45% for TGβ3) since week 10 (W10), trend maintained until the end of the study. Regardless of WT or TGβ3 status, SBP (mmHg) was significantly increased by HD (∼12%), as measured at W35. Also, in the HD groups, hyperglycemia during OGTT was observed, suggesting insulinoresistance. Starting with W25, a decrease in early-to-late filing ratio (E/A ratio) in TGβ3 + HD vs. TGβ3 + SD was observed (0.97 ± 0.04 vs. 1.11 ± 0.07), while for WT + HD vs. WT + SD, reduction occurred at W35 (0.99 ± 0.06 vs. 1.25 ± 0.05). E’/A’ ratio followed the same trend, at W25: TGβ3 + HD vs. TGβ3 + SD (0.93 ± 0.05 vs. 1.07 ± 0.05), at W35: WT + HD vs. WT + SD (0.99 ± 0.06 vs. 1.19 ± 0.04). At W35, no significant change was registered in ejection fraction (%) between all groups (∼63%), suggesting no systolic dysfunction at this timepoint. Conclusions HD increased BW, SBP and glycemia. Alteration of diastolic function occurs at around W35 in obese WT rats, whereas in obese TGβ3 rats diastolic function alteration occurred earlier (W25).

Introduction Understanding endothelial dysfunction is at the heart of current concerns due to its involvement in many cardiovascular diseases such as heart failure (HF). The β3-adrenergic receptor (β3-AR) could be at the origin of this endothelial dysfunction. Indeed, it is expressed at the level of the endothelial cell and induces vasorelaxation by the release of nitric oxide (NO), a central actor in endothelial dysfunction. Objective Our objective was therefore to study the overexpression of the receptor in endothelial cells to understand its involvement in endothelial dysfunction. Method We used the Tgβ3 rat at 45 weeks of age, which overexpresses the human β3-AR gene in the endothelium. Endothelium-dependent aortic relaxation versus isoproterenol was measured with or without L-VNIO an inhibitor of nNOS at 10 μM. The protein expression of NOS was evaluated by Western-Blot and the production of NO and O2- was evaluated by EPR on aorta. Results Vascular reactivity was significantly reduced in response to isoproterenol in Tgβ3 aorta (−10% P Conclusion The increase in β3-AR expression induces endothelial dysfunction with a key role of eNOS and nNOS. Further studies will be conducted to understand the causal links between endothelial dysfunction and the alteration of the cardiac function.

Aims Endothelial dysfunction is one of the earliest symptoms in septic patients and plays an important role in the cardiovascular alterations. However, the endothelial mechanisms involved in the impaired sympathetic regulation of the cardiovascular system are not clear. This study aimed to determine the role of the endocardial endothelium (EE) in the cardiac β-adrenergic (β-AR) remodeling at the early phase of endotoxemic shock. Main methods Rats received either lipopolysaccharide (LPS) or saline (control) intravenously. Three hours later, β-AR cardiac contractility was evaluated on papillary muscles with or without a functional EE. Key findings Isoproterenol-induced contractility was strongly increased in papillary muscles from LPS rats. A similar increase was observed with a β1-AR stimulation, whereas β2-AR and β3-AR produced similar contractility in control and LPS treatments. The removal of the EE did not modify β1-AR-induced contractility in controls, whereas it abolished the increased β1-AR response in LPS-treated muscles. In LPS-treated papillary muscle, the increased β1-AR-induced contractility was not modified by pretreatment with a NOS inhibitor or an endothelin receptor antagonist. Conversely, the increased β1-AR-induced contractility was abolished by indomethacin, a non-selective cyclooxygenase (COX) inhibitor, as well as by selective inhibitors of COX1 and COX2. An early treatment with indomethacin improved the survival of LPS rat. Significance Our results suggest that the EE is involved in the increased cardiac β1-AR contractility in the early phase of endotoxemic shock. This effect is mediated through the activation of COX1 and COX2 and suggests these may be novel putative therapeutic targets during endotoxemic shock.

Endothelial dysfunction is one of the earliest symptoms in septic patients and plays an important role in the cardiovascular alterations. However, the endothelial mechanisms involved in the impaired sympathetic regulation of the cardiovascular system are not clear. This study aimed to determine the role of the endocardial endothelium (EE) in the cardiac β-adrenergic (β-AR) remodeling at the early phase of endotoxemic shock.Rats received either lipopolysaccharide (LPS) or saline (control) intravenously. Three hours later, β-AR cardiac contractility was evaluated on papillary muscles with or without a functional EE.Isoproterenol-induced contractility was strongly increased in papillary muscles from LPS rats. A similar increase was observed with a βOur results suggest that the EE is involved in the increased cardiac β

Introduction The secretome is a set of proteins secreted by a cell at a given time and under certain conditions. The secretome, easily accessible from plasma and analyzable by a proteomic approach, represents a good opportunity to identify biomarkers in septic shock. This pathology corresponds to a systemic inflammatory response syndrome related to an infection and associated with cardiocirculatory failure leading to organ dysfunction with a mortality rate of 40%. The identification of diagnostic/prognostic biomarkers is crucial to improve patient outcome. Objective Study the secretome of a rat model of endotoxemic shock to identify relevant deregulated proteins (DEP). Methods Endotoxemic shock was induced by lipopolysaccharides injection (LPS, S. enterica typhi, 0.5 mg/kg, iv) and compared to controls (Ringer lactate, iv). Under isoflurane anesthesia, carotid cannulation allowed evaluation of mean arterial blood pressure (MAP), heart rate and blood sampling (T0, and T50 or T90 min on EDTA and protease inhibitor). Plasma was analyzed with tandem MS based on a label free quantification. Gene ontology (GO) analyses defined several groups of biological process (BP) in which the DEP were involved. Results Ninety minutes after shock induction, the LPS group present a reduction in MAP (−45%, P Conclusion This study proposes an approach to identify DEP in septic shock. DEP need to be tested in cellular models in order to understand their role in the physiopathology of shock and to improve understanding of adaptations of the secretome during sepsis. The link between our results and patient prognosis will need to be studied for clinical use.

Introduction Cardiopulmonary bypass (CPB) is a common procedure to maintain body perfusion during cardiac surgery. It is associated with a systemic inflammatory response, hemostasis and circulatory dysfunction that can lead to organ dysfunction and seldomly to death. O-GlcNAcylation is a post-translational modification modulated in response to stress in different acute and chronic situations. Objective Decipher if blood O-GlcNAc levels are modified during CPB induced stress. Method Vingt adult patients with CPB time > 60 minutes were included. Blood samples were collected on EDTA at (i) anesthesia induction (Ind), (ii) aortic declamping (AD) and (iii) 5 hours post-declamping (AD + 5). Whole blood and plasma were recovered from samples. O-GlcNAc levels in blood were evaluated by western blot while plasmatic biological markers were measured on automatic laboratory analysis system to correlate O-GlcNAc level with systemic response to CPB. Demographics and One-Way ANOVA analysis were performed with RStudio. Results CPB induced an early increase of plasmatic inflammatory marker IL-6 (Ind: 5.7 ± 3.2, AD: 120.0 ± 26.5 pg/mL, P Conclusion In this preliminary study, we highlighted that the inflammation and tissue stress consecutive to CPB are associated with an increase in blood O-GlcNAc levels 5 hours after CPB. Inclusion of more patients and correlations between O-GlcNAc level variation and demographics and/or patients’ outcome might reveal a new biomarker of risk after CPB.

Introduction During post-natal development, the organism is subjected to 2 metabolic transitions. The weaning transition is particularly interesting as it is associated with a change in glucose metabolism. O-GlcNAcylation is an ubiquitous post-translational modification, described as a nutrient sensor. The GlcNAc moiety is added and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Interestingly, cardiac O-GlcNAc levels decrease throughout post-natal development but its regulatory mechanism and impact on proteins have never been studied under this condition. Objective Study the regulatory mechanism involved in O-GlcNAc regulation and its potential impact on proteins implied in cardiac metabolism. Method Hearts from 28 day-olds (D28) Wistar rats fed with a standard or low-carbohydrate (low-CHO) diet (D28F: 50% fat, 30% protein, 3% carbohydrates) during the weaning period (D12 to D28) were studied. O-GlcNAc levels and regulatory enzymes expression were evaluated by western blot. The OGA activity was evaluated with a spectrophotometric assay. The impact of selected protein expression in response to O-GlcNAc levels variations (Thiamet-G, OGA inhibitor) was evaluated on cell culture (HEK and COS). Results No change was reported on O-GlcNAc levels between the diets (D28: 1.00 ± 0.06; D28F: 0.99 ± 0.08). Cardiac OGA expression was 5 times lower in D28F however OGA activity remained stable (D28: 1.00 ± 0.03; D28F: 0.83 ± 0.16 AU). Protein expression, acetyl-CoA transferase 1 (ACAT1) and acetyl-CoA synthetase 1 (ACSS1), was reduced both in HEK and COS cells in responses to O-GlcNAc stimulation with Thiamet-G. Conclusion While OGA expression is lower with the low-CHO diet, OGA activity and O-GlcNAc levels remain stable. This indicates a strong regulatory mechanism to control O-GlcNAc levels throughout post-natal development. This regulatory mechanism could help to modulate the expression of key metabolic enzymes such as ACAT1 or ACSS1.

Introduction The young population, particularly at risk of septic shock, is rarely studied. The O-GlcNAcylation is a post-translational modification involved in cell survival, metabolism and stress response. We have shown that O-GlcNAc stimulation at the early phase of septic shock is beneficial in adult rats. Considering that O-GlcNAc levels are higher in the young, the impact of O-GlcNAc stimulation on sepsis in the young should be tested. Objective Evaluate if O-GlcNAc stimulation could improve sepsis outcomes in young. Method Endotoxemic shock was induced in 28 days old rats by injection of LPS (O111 :B4, 20 mg.kg−1–LPS) and compared to control rats (injection of saline–CTRL). One hour after LPS injection rats were randomly assigned to no therapy (LPS), fluidotherapy (saline, 10 mL.kg−1–LPS + R) ± NButGT (10 mg.kg−1–NButGT) to increase O-GlcNAc levels (n = 11 per group). Physiological functions and plasmatic markers were evaluated 2 hours later. Impact of treatment was evaluated with an adapted Pediatric RISk of Mortality score (PRISM score) and mortality was evaluated over 36 hours (n = 16 per group). Untargeted mass spectrometry was performed to identify cardiac O-GlcNAcylated proteins. Results LPS induced a shock (mean arterial pressure (MAP): CTRL: 67.2 ± 1.9; LPS: 50.7 ± 2.1 mmHg; P Conclusion O-GlcNAc stimulation acutely improves outcome in young septic shock rat. The impact of O-GlcNAcylation on ACLY is a potential issue to decipher the role of this protein during sepsis.

Obesity induces hemodynamic and humoral changes that are associated with functional and structural cardiac remodeling, which ultimately result in the development of heart failure (HF) with preserved ejection fraction (HFpEF). In recent years, pharmacological studies in patients with HFpEF were mostly unsatisfactory. In these conditions, alternative new therapeutic approaches are necessary. The aim of our study was (1) to assess the effects of obesity on heart function in an experimental model and (2) to evaluate the efficacy of an alpha-lipoic acid (ALA) antioxidant treatment. Sprague-Dawley rats (7 weeks old) were either included in the control group (n = 6) or subjected to abdominal aortic banding (AAB) and divided into three subgroups, depending on their diet: standard (AAB + SD, n = 8), hypecaloric (AAB + HD, n = 8) and hypercaloric with discontinuous ALA treatment (AAB + HD + ALA, n = 9). Body weight (BW), glycemia, echocardiography parameters and plasma hydroperoxides were monitored throughout the study. After 36 weeks, plasma adiposity (leptin and adiponectin) and inflammation (IL-6 and TNF-alpha) markers, together with B-type natriuretic peptide and oxidative stress markers (end-products of lipid peroxidation and endogenous antioxidant systems) were assessed. Moreover, cardiac fiber diameters were measured. In our experiment, diet-induced obesity generated cardiometabolic disturbances, and in association with pressure-overload induced by AAB, it precipitated the onset of heart failure, cardiac hypertrophy and diastolic dysfunction, while producing a pro-oxidant and pro-inflammatory plasmatic status. In relationship with its antioxidant effects, the chronic ALA-discontinuous treatment prevented BW gain and decreased metabolic and cardiac perturbations, confirming its protective effects on the cardiovascular system.

Background O-GlcNAcylation is a well-preserved, ubiquitous, post-translational modification involved in response to stress, considered as a nutrient sensor. GlcNAc moiety is added and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Metabolic sources vary from carbohydrate in utero, to fatty acid after birth then a mix once adult. Purpose Study the evolution of O-GlcNAc levels during the first period of life and decipher the impact of O-GlcNAc on cardiac proteins. Methods Heart and brain were harvested from Wistar rats before and after birth (D-1 and D0), in suckling animals (D12), after weaning (D28) with a standard or a carbohydrate free diet, and when adults (D84). O-GlcNAc levels, GFAT, OGT and OGA protein expression were evaluated by western blot. Mass spectrometry approaches were performed to (i) quantify known regulator of O-GlcNAc and (ii) identify cardiac O-GlcNAcylated proteins at D0 and D28. Results Cardiac protein O-GlcNAc levels decrease progressively from D-1 to D84 (13 fold, P Conclusion We demonstrate for the first time that O-GlcNAc levels are not linked to the dietary sources. Our results suggest that glutamine and UDP-GlcNAc are not the main regulatory element of O-GlcNAc levels. Proteins identified by untargeted mass spectrometry will need to be specifically studied to better understand the impact of O-GlcNAcylation during development, growth or stress response.