Pascal Bernatchez

Caveolin-1 (Cav1) is a membrane protein that plays important structural and signalling roles in several mammalian cell types. While best known for the formation of flask-shaped membrane structures known as caveolae, which have been implicated in endocytic, mechanoprotective, and signalling roles, Cav1 also readily oligomerizes within lipid rafts to form Cav1 domains known as scaffolds. Disrupting Cav1 expression promotes tumour survival and migration, and Cav1 knockout animals suffer from pulmonary and cardiovascular conditions. While Cav1 is well-known to interact with multiple cell signalling partners, notably its inhibition of endothelial nitric oxide synthase (eNOS), it remains unclear if this interaction is caveolae- or scaffold-dependent, of if it involves the caveolin scaffolding domain or the tyrosine-14 residue of Cav1.Here, we used confocal and single molecule localization microscopy (SMLM) network analysis of Cav1 and eNOS localization in a transiently transfected Cav1 knockout MDA-MB-231 cell line, as well as whole-mount labeling of Cav1 and eNOS in mouse pulmonary arteries to study Cav1 interaction with eNOS. We found that phosphomimetic Cav1-Y14D mutant caveolae and scaffolds had similar features to caveolin scaffolding domain (CSD) mutants, while phosphorylation-deficient Cav1-Y14F mutant Cav1 structures closely resembled Cav1-WT blobs. Using binary mask analyses of Cav1 and eNOS, we identified that eNOS interacts with caveolae and scaffolds approximately equally, and that CSD mutant and Y14D mutant Cav1 had a slight but non-significant increase in Cav1-eNOS co-occurrence across all Cav1 domains.Additionally, to investigate changes in Cav1 and eNOS localization and behaviour with age, we used confocal microscopy to image Cav1, eNOS, and the Golgi marker GM130 in the pulmonary artery endothelium young and old mice. We observed that eNOS expression decreases with age (p
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Limb-Girdle Muscular Dystrophy (MD) Type 2B (LGMD2B) is a rare form of MD caused by mutations in the dysferlin (DYSF) gene. As an autosomal recessive disorder, LGMD2B can occur in both males and females and is characterized by progressive muscle wasting, eventually leading to ambulatory dysfunction. Unfortunately, no treatment options can cure patients of LGMD2B. One potential factor in the lack of therapies is the absence of humanized animal models for preclinical studies. Current LGMD2B mouse models lack the disease severity typically observed in the human condition. Additionally, LGMD2B mice remain mobile, whereas most patients are non-ambulatory or exhibit a marked decrease in mobility. Developing more humanized animal models of LGMD2B is vital for improvements in LGMD2B research. Recently, our lab has shown that crossing a DYSF knockout mouse with a mouse lacking apolipoprotein E (ApoE), a surface transport lipoprotein, significantly raises plasma cholesterol and correlates with a severe muscle wasting phenotype and marked ambulatory dysfunction. However, LGMD2B patients have an intact ApoE gene, demonstrating a major limitation of this model. Regardless, the DYSF/ApoE double knockout (DKO) mouse provides proof of principle that dyslipidemia plays a factor in LGMD2B pathology and warrants further investigations. Since thermoneutral (TN) housing of wild type mice has been shown to increase plasma lipids significantly when fed a western diet (WD), we hypothesized that TN housing of BLAJ mice, a mild model of LGMD2B, would lead to an exacerbation of muscle wasting when fed a cholesterol-rich WD. Herein, we report that TN BLAJ mice fed a cholesterol-rich WD exhibited the greatest level of fibrofatty replacement, despite not increasing plasma cholesterol. Additionally, thermoneutrality affected LGMD2B mice in a sex-specific manner, with females having significantly more fibrofatty replacement than males. Finally, TN BLAJ mice fed a cholesterol-rich WD had increases in mTORC1 regulated protein synthesis, in addition to a decrease in LC3 mediated autophagy, suggesting an overall decrease in protein turnover within DYSF-null skeletal muscle. In conclusion, this study reveals BLAJ mice to be sensitive to changes in ambient temperature and suggests that alterations in metabolism could be a contributing factor in LGMD2B pathology.

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Muscular dystrophy (MD) is a class of diseases marked by progressive muscle wasting and impaired ambulatory function. Dysferlin- and dystrophin-deficiencies lead to MD and both are expressed in myofibers as well as smooth muscle and endothelial cells lining the vasculature, suggesting vascular function may be a contributing factor to muscle pathology seen in MD. However, murine models of MD develop only mild pathology compared to patients, making therapeutic drug development challenging. Since mice are known to have endogenously low lipid levels and superior vascular health, disrupted vascular function may be aggravating MD progression in humans. We seek to investigate the effect of impaired vascular function on MD pathology through the generation and characterization of doubledisease murine models affected by both hyperlipidemia and MD. Firstly, the dysferlin-null (Dysf-KO) model of limb-girdle type 2B MD (LGMD2B) and the dystrophin-null (mdx) model of Duchenne MD (DMD) were crossed with apolipoprotein E-KO (ApoE-KO) hyperlipidemia model to generate Dysf-ApoE and Mdx-ApoE double-knockout mice (DKO). To further confirm the effects vascular disease on MD using a more human-relevant model, the Dysf-KO model was also crossed with the milder low-density-lipoprotein receptor-knockout (LDLRKO) model of hyperlipidemia. Significant worsening of ambulatory function and muscle pathology was observed in the Dysf-ApoE DKO model, displaying reduced stride length, sometimes complete loss of ambulation, and increased muscle wasting, necrosis, fibrosis and fat infiltration. Although the Mdx-ApoE model showed no exacerbation of ambulation decline, significant worsening of muscle necrosis and fibrofatty replacement was observed in MdxApoE DKO mice. However, few Dysf-LDLR DKOs were produced; all were maloccluded runts with abnormal gait, though without observable muscle pathology upon histological analysis. In all, the Dysf-ApoE and Mdx-ApoE mice demonstrate that hyperlipidemia and associated vascular disease can dramatically aggravate muscle pathology in MD. These double-disease models mimic more closely the severity of human MD and may be useful for evaluating therapeutic interventions. In addition, these data suggest that lipid-lowering and vascular-targeted therapies may be beneficial in LGMD2B and DMD.

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Nitric Oxide (NO) produced by the endothelium is a critical mediator of vascular function and plays an important role in the protection against various cardiovascular diseases1. In fact, a central feature of most cardiovascular diseases is reduced bioavailability of NO resulting from impaired endothelial function. Consequently, therapies that improve NO synthesis and availability in disease settings are relevant. Endothelial nitric oxide synthase (eNOS) is a membrane enzyme expressed exclusively in vascular endothelial cells and is responsible for NO production. Improper regulation of the enzyme results in production of eNOS-derived superoxide anion (O₂₋) instead of NO. O₂₋ is an oxidative stress mediator and scavenges NO, thereby contributing to lowered NO bioavailability. Extensive research has demonstrated a number of factors involved in positively regulating eNOS activity. However, one of the few proteins that bind to eNOS under basal conditions and inhibit NO release is Caveolin-1 (Cav-1), the major coat protein of plasma membrane lipid-enriched invaginations known as caveolae². Recently, it was demonstrated that a single amino acid substitution of the Cav-1 protein, mutant known as F92A Cav-1, is unable to inhibit eNOS³. Furthermore, preliminary data indicates that high expression of F92A Cav-1 can increase basal NO release. Due to the significance of NO in vascular function, the current work explores the possible mechanisms by which F92A Cav-1 potentiates eNOS activity and NO release. We report that F92A Cav-1 preserves the unique properties of Cav-1, including targeting to caveolae and forming high molecular weight oligomers, which are essential for caveolae organelle biogenesis. Moreover, F92A Cav-1 still retains the ability to bind to eNOS without altering its subcellular localization, indicating that F92A Cav-1 can prevent eNOS binding to endogenous Cav-1, which could rationalize the increased NO release observed. Lastly, we provide evidence that over-expression of F92A Cav-1 reduces the release of basal O₂₋ in endothelial cells as compared to WT Cav-1, revealing another potential positive effect of the mutant Cav-1. Hence, this report compares the biological properties of WT and F92A Cav-1 and the data collected is aimed at describing a therapeutically relevant pharmacological target to increase NO bioavailability in cardiovascular disease settings.

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Heart disease and cancer are the two leading causes of death worldwide. In heart disease, reperfusion of an ischemic myocardium through increased angiogenesis, or the growth of new blood vessels, is considered the ‘holy grail’ of future therapies. In contrast, inhibiting tumour growth by decreasing angiogenesis through anti-angiogenic therapies is increasingly used in cancer patients, although the therapeutic effect is only partial. Hence, a better comprehension of angiogenesis is clearly warranted. While a plethora of literature suggests that the vascular endothelial growth factor (VEGF) and angiopoietins systems are the most potent endogenous regulators of angiogenesis, an increasing number of recent studies also show that their net angiogenic effects are mostly dictated by membrane expression of their main receptors, VEGFR-2 and tie-2, respectively. Recently, endothelial cells (ECs) were unexpectedly found to express myoferlin, a muscle protein [1] known for its ability to regulate plasma membrane integrity [2]. Moreover, myoferlin was found to be involved in the regulation of VEGFR-2 expression in ECs [3]. In this work, we report that disruption of myoferlin by gene-silencing causes decreased tie-2 expression in cultured ECs. However, myoferlin disruption does not affect the transcriptional levels of tie-2 in cultured ECs. Separation of caveolae/lipid rafts from cytosol in ECs shows presence of myoferlin and tie-2 in caveolae/lipid rafts, suggesting co-localization of the two proteins to form a large signaling complex at caveoli, which are known platforms for clustering of signaling complexes. Moreover, myoferlin is present in a cancer cell model (Lewis Lung Carcinoma) and myoferlin disruption causes decreased cell proliferation, further exploring the involvement of this membrane protein in a completely different cell system. The current work identifies potential pharmacological targets for the regulation of the tie-2 system and since tie-2 expression is almost exclusively found in ECs, this work initiates the characterization of an EC-specific target that could be further exploited to modulate angiogenic responses in an in vivo model.

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