developing-a-clinically-relevant-hemorrhagic-shock-model-in-rats

Developing a Clinically Relevant Hemorrhagic Shock Model in Rats

Over the recent decades, the development of animal models allowed us to better understand various pathologies and identify new treatments. Hemorrhagic ...

Nantes Université

α-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of  snake. Like most large toxins, chemical synthesis of α-bungarotoxin is challenging, explaining why all previous reports use purified or recombinant α-bungarotoxin. However, only chemical synthesis allows easy insertion of non-natural amino acids or new chemical functionalities. Herein, we describe a procedure for the chemical synthesis of a fluorescent-tagged α-bungarotoxin. The full-length peptide was designed to include an alkyne function at the amino-terminus through the addition of a pentynoic acid linker. Chemical synthesis of α-bungarotoxin requires hydrazide-based coupling of three peptide fragments in successive steps. After completion of the oxidative folding, an azide-modified Cy5 fluorophore was coupled by click chemistry onto the toxin. Next, we determined the efficacy of the fluorescent-tagged α-bungarotoxin to block acetylcholine (ACh)-mediated currents in response to muscle nicotinic receptor activation in TE671 cells. Using automated patch-clamp recordings, we demonstrate that fluorescent synthetic α-bungarotoxin has the expected nanomolar affinity for the nicotinic receptor. The blocking effect of fluorescent α-bungarotoxin could be displaced by incubation with a 20-mer peptide mimicking the α-bungarotoxin binding site. In addition, TE671 cells could be labelled with fluorescent toxin, as witnessed by confocal microscopy, and this labelling was partially displaced by the 20-mer competitive peptide. We thus demonstrate that synthetic fluorescent-tagged α-bungarotoxin preserves excellent properties for binding onto muscle nicotinic receptors.

Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin.

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  -adrenoreceptor (Tg ) 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 Tg  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 O  production. Vascular reactivity was altered as early as 15 weeks of age in response to isoproterenol in Tg  aortas and mesenteric arteries. NOS1 (neuronal NOS) expression was higher in the Tg  aorta at 30 and 45 weeks of age (30 weeks: WT: 1.00 ± 0.21; Tg : 6.08 ± 2.30; 45 weeks: WT: 1.00 ± 0.12; Tg : 1.55 ± 0.17;  < 0.05). Interestingly, the endothelial NOS (NOS3) monomer form is increased in Tg  rats at 45 weeks of age (ratio NOS3 dimer/NOS3 monomer; WT: 1.00 ± 0.37; Tg : 0.13 ± 0.05;  < 0.05). Aortic NO production was increased by NOS2 (inducible NOS) at 15 weeks of age in Tg  rats (+52% vs. WT). Aortic O  production was increased in Tg  rats at 30 and 45 weeks of age (+75% and+76%, respectively, vs. WT,  < 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.

The Endothelial Dysfunction Could Be a Cause of Heart Failure with Preserved Ejection Fraction Development in a Rat Model.

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 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 cardio-vascular diseases.

MT9, a natural peptide from black mamba venom antagonizes the muscarinic type 2 receptor and reverses the M2R-agonist-induced relaxation in rat and human arteries.

The young population, which is particularly at risk of sepsis, is, paradoxically, rarely studied. Acute stimulation of O-GlcNAcylation, a post-translational modification involved in metabolic regulation, cell survival and stress response, is beneficial in young rats with sepsis. Considering that sepsis impacts the gene expression profile and that O-GlcNAcylation is a regulator of transcription, the aims of this study are to (i) unveil beneficial mechanisms of O-GlcNAcylation and (ii) decipher the relationship between O-GlcNAcylation and transcription during sepsis. Endotoxemic challenge was induced in 28-day-old male rats using a lipopolysaccharide injection (E. coli O111:B4, 20 mg·kg−1) and compared to control rats (NaCl 0.9%). One hour after, rats were assigned to no therapy or fluidotherapy (NaCl 0.9%, 10 mL.kg−1) ± NButGT (10 mg·kg−1) to stimulate O-GlcNAc levels. Cardiac O-GlcNAcylation levels were evaluated via Western blot and gene transcription using 3′ SRP analysis. Lipopolysaccharide injection favorizes inflammatory state with the overexpression of genes involved in the NF-κB, JAK/STAT and MAPK pathways. NButGT treatment increased cardiac O-GlcNAcylation levels (p < 0.05). Yet, the mRNA expression was not impacted two hours after fluidotherapy or NButGT treatment. In conclusion, O-GlcNAc stimulation-induced beneficial effects are not dependent on the gene expression profile at the early phase of sepsis.

Beneficial Effects of O-GlcNAc Stimulation in a Young Rat Model of Sepsis: Beyond Modulation of Gene Expression.

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.

Multimodality imaging and transcriptomics to phenotype mitral valve dystrophy in a unique knock-in Filamin-A rat model.

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.

An overview of tools to decipher O-GlcNAcylation from historical approaches to new insights.

Sepsis is a life-threatening disease defined as an organ dysfunction caused by a dysregulated host response to an infection. Early diagnosis and prognosis of sepsis are necessary for specific and timely treatment. However, no predictive biomarkers or therapeutic targets are available yet, mainly due to the lack of a pertinent model. A better understanding of the pathophysiological mechanisms associated with sepsis will allow for earlier and more appropriate management. For this purpose, experimental models of sepsis have been set up to decipher the progression and pathophysiology of human sepsis but also to identify new biomarkers or therapeutic targets. These experimental models, although imperfect, have mostly been performed on a murine model. However, due to the different pathophysiology of the species, the results obtained in these studies are difficult to transpose to humans. This underlines the importance of identifying pertinent situations to improve patient care. As humans, horses have the predisposition to develop sepsis spontaneously and may be a promising model for spontaneous sepsis. This review proposes to give first an overview of the different animal species used to model human sepsis, and, secondly, to focus on adult equine sepsis as a spontaneous model of sepsis and its potential implications for human and veterinary medicine.

Spontaneous Sepsis in Adult Horses: From Veterinary to Human Medicine Perspectives.

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.

A Rat Model of Clinically Relevant Extracorporeal Circulation Develops Early Organ Dysfunctions.

Optical Control of Cardiac Rhythm by In Vivo Photoactivation of an ERG Channel Peptide Inhibitor.

Histones display a wide variety of post-translational modifications, including acetylation, methylation, and phosphorylation. These epigenetic modifications can influence chromatin structure and function without altering the DNA sequence. Histones can also undergo post-translational O-GlcNAcylation, a rather understudied modification that plays critical roles in almost all biological processes and is added and removed by O-linked N-acetylglucosamine transferase and O-GlcNAcase, respectively. This review provides a current overview of our knowledge of how O-GlcNAcylation impacts the histone code both directly and by regulating other chromatin modifying enzymes. This highlights the pivotal emerging role of O-GlcNAcylation as an essential epigenetic marker.

Article	Year
Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin. Toxins\| PubMed ID: 35202107	2022
The Endothelial Dysfunction Could Be a Cause of Heart Failure with Preserved Ejection Fraction Development in a Rat Model. Oxidative medicine and cellular longevity\| PubMed ID: 35633883	2022
MT9, a natural peptide from black mamba venom antagonizes the muscarinic type 2 receptor and reverses the M2R-agonist-induced relaxation in rat and human arteries. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie\| PubMed ID: 35658242	2022
Beneficial Effects of O-GlcNAc Stimulation in a Young Rat Model of Sepsis: Beyond Modulation of Gene Expression. International journal of molecular sciences\| PubMed ID: 35742875	2022
Multimodality imaging and transcriptomics to phenotype mitral valve dystrophy in a unique knock-in Filamin-A rat model. Cardiovascular research\| PubMed ID: 36001550	2023
An overview of tools to decipher O-GlcNAcylation from historical approaches to new insights. The international journal of biochemistry & cell biology\| PubMed ID: 36031106	2022
Spontaneous Sepsis in Adult Horses: From Veterinary to Human Medicine Perspectives. Cells\| PubMed ID: 37048125	2023
A Rat Model of Clinically Relevant Extracorporeal Circulation Develops Early Organ Dysfunctions. International journal of molecular sciences\| PubMed ID: 37108501	2023
Optical Control of Cardiac Rhythm by In Vivo Photoactivation of an ERG Channel Peptide Inhibitor. Circulation research\| PubMed ID: 37593901	2023
O-GlcNAcylation: the sweet side of epigenetics. Epigenetics & chromatin\| PubMed ID: 38093337	2023

Benjamin Lauzier

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