Biology subjects

West, A. R.

Publications and source records attributed to West, A. R..

11 recordsLinked to original sources

bioRxiv2024

The pro-apoptotic effect of chronic contractile activity-induced extracellular vesicles on Lewis Lung Carcinoma cells

Regular exercise reduces tumor growth in vivo and in vitro, but the exact mechanisms have yet to be fully elucidated. We have previously shown that chronic contractile activity (CCA) increases the concentration of skeletal muscle-derived EVs, and these in turn increased mitochondrial biogenesis in myoblasts. Here, we hypothesized that skeletal muscle-EVs derived post-CCA will mediate the anti-tumorigenic effects associated with chronic exercise. C2C12 myoblasts were differentiated into myotubes, electrically paced, and EVs isolated from conditioned media from control and CCA myotubes using differential ultracentrifugation. Lewis lung carcinoma (LLC) cells were treated with the total number of control-EVs or CCA-EVs isolated after each day of contractile activity for 4 days. Permeabilized CCA-EVs with or without proteinase K before co-culture with LLC cells were used as controls. Effect of EV treatment on cell count, viability, apoptosis, senescence, migration, and mitochondrial content was measured. CCA-EV treatment reduced cell count by 18% and cell viability by 6% vs. control-EVs. CCA-EVs increased the incidence of apoptotic hallmarks: DNA fragmentation by 13%, Annexin V+/PI+ cells by 21%, and the expression of pro-apoptotic Bax (by 25%) and Bax/Bcl-2 ratio (by 60%) vs. control-EVs. CCA-EVs increased number of senescent cells by 29%, and senescence markers, HMGB1 (by 49%) and p16 (by 92%) vs. control-EVs. When CCA-EVs were pretreated with Triton X-100 with or without proteinase-K, the increase in apoptosis and senescence was abrogated, confirming the effect is due to intact EVs and likely through EV membrane-proteins. CCA-EVs did not have any effect on cell migration and mitochondrial content vs. control-EVs. This study illustrates for the first time the potential of CCA-induced skeletal muscle-EVs in mediating anti-tumorigenic effects traditionally linked with chronic exercise.

cancer biology↗

bioRxiv2024

Chronic contractile activity induced skeletal muscle-derived extracellular vesicles increase mitochondrial biogenesis in recipient myocytes via transmembrane or peripheral membrane proteins

The effect of chronic contractile activity (CCA) on the biophysical properties and functional activity of skeletal muscle extracellular vesicles (Skm-EVs) is poorly understood due to challenges in distinguishing Skm-EVs originating from exercising muscle in vivo. To address this, myoblasts were differentiated into myotubes, and electrically paced (3 h/day, 4 days @ 14 V). CCA evoked an increase in mitochondrial biogenesis in stimulated vs. non-stimulated (CON) myotubes as expected. EVs were isolated from conditioned media from control and stimulated myotubes using differential ultracentrifugation and characterized biophysically using tunable resistive pulse sensing (TRPS, Exoid), TEM and western blotting. TEM images confirmed isolated round-shaped vesicles of about 30 - 150 nm with an intact lipid bilayer. The mean size of EVs ranged from 98 -138 nm, and was not altered by CCA. Zeta potential and total EV protein yield remained unchanged between groups, and total EV secretion increased after 4 days of CCA. Concomitant analysis of EVs after each day of CCA also demonstrated a progressive increase in CCA-EV concentration, while size and zeta potential remained unaltered, and EV protein yield increased in both CON-EVs and CCA groups. CCA-EVs were enriched with small-EVs vs. CON-EVs, concomitant with higher expression of small-EV markers CD81, Tsg101 and HSP70. In whole cell lysates, CD63 and ApoA1 were reduced with CCA in myotubes, whereas CD81, Tsg101, Flotillin-1 and HSP70 levels remained unchanged. To evaluate the functional effect of EVs secreted post-CCA, we treated C2C12 myoblasts with all EVs isolated from CON or CCA myotubes after each day of stimulation, and measured cell count, cell viability, protein yield and mitochondrial biogenesis in recipient cells. There was no effect on cell count, viability and protein yield. Myoblasts treated with CCA-EVs exhibited increased mitochondrial biogenesis as indicated by enhanced MitoTracker Red staining, cytochrome c oxidase activity, and protein expression of electron transport chain subunit, CIV-MTCO1. Further, CCA-EV treatment enhanced maximal oxygen consumption rates (OCR), and ATP production in treated myoblasts. This increase in maximal OCR was abrogated when CCA-EVs pre-treated with proteinase K were co-cultured with myoblasts, indicating the pro-metabolic effect was likely mediated by transmembrane or peripheral membrane proteins in CCA-EVs. Our data highlight the novel effect of Skm-EVs isolated post-CCA in mediating pro-metabolic effects in recipient cells and thereby transmitting the effects associated with traditional exercise. Further investigation to interrogate the underlying mechanisms involved in downstream cellular metabolic adaptations is warranted.

cell biology↗

bioRxiv2023

The mitophagy receptor Nix coordinates nuclear calcium signaling to modulate the muscle phenotype.

Mitochondrial quality control is critical in muscle to ensure both contractile and metabolic function. Nix is a BCL-2 family member, mitophagy receptor, and has been implicated in muscle atrophy. Human GWAS suggests altered Nix expression could predispose to manifestations of mitochondrial disease. To interrogate Nix function, we generated a muscle-specific knockout model. Nix knockout mice displayed a ragged-red fibre phenotype, along with accumulation of mitochondria and endo/sarcoplasmic reticulum with altered morphology. Intriguingly, Nix knockout mice were more insulin sensitive with a corresponding increase in glycogen-rich muscle fibres. Kinome- and gene expression analyses revealed that Nix knockout impairs NFAT and canonical myostatin signaling, with alterations in muscle fibre-type composition and evidence of regeneration. Mechanistic experiments demonstrated that Nix modulates mitophagy, along with ER-phagy leading to altered nuclear calcium signaling. Collectively, these observations identify novel roles for Nix coordinating selective autophagy, oxidative gene expression, and signaling pathways that maintain the muscle phenotype.

cell biology↗

bioRxiv2022

Development of a 3D bioprinted airway smooth muscle model for manipulating structure and measuring contraction

The contractile function of airway smooth muscle (ASM) is inextricably linked to its mechanical properties and interaction with the surrounding mechanical environment. As tissue engineering approaches become more commonplace for studying lung biology, the inability to replicate realistic mechanical contexts for ASM will increasingly become a barrier to a fulsome understanding of lung health and disease. To address this knowledge gap, we describe the use of 3D bioprinting technology to generate a novel experimental model of ASM with a wide scope for modulating tissue mechanics. Using a stiffness modifiable alginate-collagen-fibrinogen bioink, we demonstrate that modulating the stiffness of free-floating ASM bare rings is unfeasible; bioink conditions favorable for muscle formation produce structures that rapidly collapse. However, the creation of novel sandwich and spiderweb designs that encapsulate the ASM bundle within stiff acellular load bearing frames successfully created variable elastic loads opposing tissue collapse and contraction. Sandwich and spiderweb constructs demonstrated realistic actin filament organisation, generated significant baseline tone, and responded appropriately to acetylcholine, potassium chloride and cytochalasin D. Importantly, the two designs feasibly simulate different mechanical contexts within the lung. Specifically, the sandwich was relatively compliant and subject to plastic deformation under high contractile loads, whereas the stiffer spiderweb was more robust and only deformed minimally after repeated maximal contractions. Thus, our model represents a new paradigm for studying ASM contractile function in a realistic mechanical context. Moreover, it holds significant capacity to study the effects of ECM composition, multiple cell types and fibrosis on lung health and disease. GRANTSO_LINatural Sciences and Engineering Research Council, Discovery Grant (Adrian West) C_LIO_LIResearch Manitoba, New Investigator Operating Grant (Adrian West) C_LIO_LIChildrens Hospital Research Institute of Manitoba, Operating Grant (Adrian West) C_LIO_LICanadian Foundation for Innovation, John R. Evans Leaders Fund (Adrian West) C_LIO_LIUniversity of Manitoba, Manitoba Graduate Scholarship (Jeffery Osagie) C_LIO_LIResearch Manitoba, Masters Studentship Award (Jeffery Osagie) C_LIO_LIResearch Manitoba, Masters Studentship Award (Sanjana Syeda) C_LIO_LIChildrens Hospital Research Institute of Manitoba, Summer Studentship (Michelle Guimond) C_LIO_LIUniversity of Manitoba, Jack Prior Memorial Undergraduate Student Research Award (Lumiere Parrenas) C_LIO_LIUniversity of Manitoba, Undergraduate Research Award (Ahsen Haroon) C_LIO_LIUniversity of Manitoba, UMSU Undergraduate Research Award (Philip Imasuen) C_LI The grant bodies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

physiology↗

bioRxiv2022

Skeletal muscle-derived extracellular vesicles are altered with chronic contractile activity

Extracellular vesicles (EVs) are small lipid bilayer-delimited particles that are secreted by all cells, playing a central role in cellular communication. EVs are released from skeletal muscle during exercise, but the effects of contractile activity on skeletal muscle-derived EVs (Skm-EVs) are poorly understood due to the challenges in distinguishing Skm-EVs derived from exercising muscle in vivo. Using tunable resistive pulse sensing (TRPS), we previously demonstrated that chronic contractile activity (CCA) increased the secretion of Skm-EVs from C2C12 myotubes, while their size and zeta potential remained unchanged. Here, we aimed to determine whether similar results could be obtained using an alternative method of EV characterization, dynamic light scattering (DLS). C2C12 myoblasts were differentiated into myotubes, and electrically paced (3h/day x 4days @14V, C-PACE EM, IonOptix) to mimic chronic exercise in vitro. EVs were isolated from conditioned media of control and stimulated myotubes using differential ultracentrifugation, and characterized based on size and zeta potential. The mean size of EVs from chronically stimulated myotubes (CCA-EVs, 132 nm) was 26% smaller than control (CON-EVs, 178 nm). Size distribution analysis revealed that CCA-EVs were enriched in small EVs (100-150 nm), while CON-EVs were largely composed of 200-250 nm sized vesicles. Additionally, zeta potential was 27% lower in CCA-EVs compared to CON-EVs. Our data indicate that the effect of CCA on facilitating the release of smaller, more stable EVs, is a robust finding, reproducible by multiple methods of EV characterization. Future studies investigating the mechanisms by which CCA influences Skm-EV biogenesis and secretion are warranted.

cell biology↗

bioRxiv2020

Misoprostol Treatment Prevents Hypoxia-Induced Cardiac Dysfunction, Aberrant Cardiomyocyte Mitochondrial Dynamics and Permeability Transition Through Bnip3 Phosphorylation

Systemic hypoxia is a common element in most perinatal emergencies and is a known driver of Bnip3 expression in the neonatal heart. Bnip3 plays a prominent role in the evolution of necrotic cell death, disrupting ER calcium homeostasis and initiating mitochondrial permeability transition (MPT). Emerging evidence suggests a cardioprotective role for the prostaglandin E1 analogue misoprostol during periods of hypoxia, but the mechanisms for this protection are not completely understood. Using a combination of mouse and cell models, we tested if misoprostol is cardioprotective during neonatal hypoxic injury by altering Bnip3 function. Here we report that hypoxia elicits mitochondrial-fragmentation, MPT, reduced ejection fraction, and evidence of necroinflammation, which were abrogated with misoprostol treatment or Bnip3 knockout. Through molecular studies we show that misoprostol leads to PKA-dependent Bnip3 phosphorylation at threonine-181, and subsequent redistribution of Bnip3 from mitochondrial Opa1 and the ER through an interaction with 14-3-3 proteins. Taken together, our results demonstrate a role for Bnip3 phosphorylation in the regulation of cardiomyocyte contractile/metabolic dysfunction, and necroinflammation. Furthermore, we identify a potential pharmacological mechanism to prevent neonatal hypoxic injury.

cell biology↗

bioRxiv2019

Nix induced mitochondrial fission, mitophagy, and myocyte insulin resistance are abrogated by PKA phosphorylation.

Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that the mitophagy receptor Nix is responsive to lipotoxicity and accumulates in response to diacylglycerols induced by high-fat (HF) feeding. In addition, previous studies have implicated autophagy and mitophagy in muscle insulin sensitivity. To provide a better understanding of these observations, we undertook gene expression array and shot-gun metabolomics studies in soleus muscle from rodents on an HF diet. Interestingly, we observed a modest reduction in several autophagy-related genes including Beclin-1, ATG3, and -5. Moreover, we observed alterations in the fatty acyl composition of cardiolipins and phosphatidic acids. Given the previously reported roles of these phospholipids and Nix in mitochondrial dynamics, we investigated aberrant mitochondrial fission and turn-over as a mechanism of myocyte insulin resistance. In a series of gain-of-function and loss-of-function experiments in rodent and human myotubes, we demonstrate that Nix accumulation triggers mitochondrial depolarization, fragmentation, calcium-dependent activation of DRP1, and mitophagy. In addition, Nix-induced mitochondrial fission leads to myotube insulin resistance through activation of mTOR-p70S6 kinase inhibition of IRS1, which is contingent on phosphatidic acids and Rheb. Finally, we demonstrate that Nix-induced mitophagy and insulin resistance can be reversed by direct phosphorylation of Nix by PKA, leading to the translocation of Nix from the mitochondria and sarcoplasmic reticulum to the cytosol. These findings provide insight into the role of Nix-induced mitophagy and myocyte insulin resistance during an overfed state when overall autophagy-related gene expression is reduced. Furthermore, our data suggests a mechanism by which exercise or pharmacological activation of PKA may overcome myocyte insulin resistance. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/825828v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@15d0a4org.highwire.dtl.DTLVardef@34f358org.highwire.dtl.DTLVardef@5427f3org.highwire.dtl.DTLVardef@10ae48a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2019

Development of an in vitro Experimental Model for Investigating the Effect of Matrix Stiffness on Epithelial Barrier Permeability

Epithelial cells are well-known to be modulated by extracellular mechanical factors including substrate stiffness. However, the effect of substrate stiffness on an epithelial cells principal function -creating an effective barrier to protect the underlying tissue - cannot be directly measured using existing experimental techniques. We developed a strategy involving ethylenediamine aminolysis and glutaraldehyde crosslinking to chemically graft polyacrylamide hydrogels with tunable stiffness to PET Transwell membranes. Grafting success was evaluated using spectroscopic methods, scrape tests, and extended incubation in culture. By assessing apical to basolateral transfer of fluorescent tracers, we demonstrated that our model is permeable to biologically relevant molecules and usable for direct measurement of barrier function by calculating paracellular permeability.\n\nWe found that BEAS-2B epithelial cells form a more effective barrier on stiff substrates, likely attributable to increased cell spreading. We also observed barrier impairment after treatment with transforming growth factor beta, indicating loss of cell-cell junctions, validating our models ability to detect biologically relevant stimuli. Thus, we have created an experimental model that allows explicit measurement of epithelial barrier function for cells grown on different substrate stiffnesses. This model will be a valuable tool to study mechanical regulation of epithelial and endothelial barrier function in health and disease.

bioengineering↗

bioRxiv2019

Oxidised phosphatidylcholines induce multiple functional defects in airway epithelial cells

Oxidative stress is a hallmark of numerous airway diseases, contributing to extensive cell and tissue damage. Cell membranes and the airway mucosal lining are rich in phospholipids that are particularly susceptible to oxidative attack, producing bioactive molecules including oxidized phosphatidylcholines (OxPC). With the recent discovery of elevated OxPC in asthmatic patients after allergen challenge, we hypothesized that OxPC directly contribute to disease by inducing airway epithelial cell dysfunction. We found that OxPC induced dose-dependent cell stress and loss of viability in BEAS-2B and Calu-3 cell lines and primary human epithelial cells. These responses corresponded with significant epithelial barrier dysfunction, which was further compounded when combining OxPC with an epithelial wound. OxPC inhibited DNA synthesis and migration required to re-establish barrier function, but cells recovered if OxPC were washed off soon after treatment. OxPC induced generation of reactive oxygen species, lipid peroxidation and mitochondrial dysfunction, raising the possibility that OxPC cause pathological lipid metabolism in a self-propagating cycle. The oxidative stress induced by OxPC could not be abrogated by putative OxPC receptor blockers, but partial recovery of barrier function, proliferation and lipid peroxidation could be achieved with the antioxidant n-acetyl cysteine. In summary, we have identified OxPC as a group of bioactive molecules that significantly impair multiple facets of epithelial cell function, consistent with pathological features of asthma. Further characterisation of the mechanisms by which OxPC affect epithelial cells could yield new insights into how oxidative stress contributes to the pathogenesis of airway disease.

cell biology↗

bioRxiv2019

Striatal Nurr1 Facilitates the Dyskinetic State and Exacerbates Levodopa-Induced Dyskinesia in a Rat Model of Parkinson’s Disease

BackgroundThe transcription factor Nurr1 has been identified to be ectopically induced in the striatum of dyskinetic rodents expressing L-DOPA-induced dyskinesia (LID). In the present study, we sought to characterize Nurr1 as a causative factor in LID expression.\n\nMethodsWe used rAAV2/5 to overexpress Nurr1 or GFP in the parkinsonian striatum of LID-resistant Lewis or LID-prone Fischer-344 (F344) rats. In a second cohort, rats received the Nurr1 agonist amodiaquine (AQ) together with L-DOPA or ropinirole. All rats received a chronic DA agonist and were evaluated for LID severity. Finally, we performed single unit recordings and dendritic spine analyses in drug-naive rAAV-injected parkinsonian rats.\n\nResultsrAAV-GFP injected LID-resistant Lewis rats displayed mild LID and no induction of striatal Nurr1. However, Lewis rats transduced to overexpress Nurr1 developed severe LID. Nurr11 agonism with AQ exacerbated LID in F344 rats. We additionally determined that in L-DOPA-naive rats striatal rAAV-Nurr1 overexpression 1) increased firing activity in dopamine-depleted striatal direct pathway neurons, and 2) decreased spine density and thin-spine morphology on striatal medium spiny neurons, mimicking changes seen in dyskinetic rats. Finally, we provide post-mortem evidence of Nurr1 expression in the striatum of L-DOPA treated PD patients.\n\nConclusionsOur data demonstrate that ectopic induction of striatal Nurr1 is capable of inducing LID behavior and associated neuropathology, even in resistant subjects. These data support a direct role of Nurr1 in aberrant neuronal plasticity and LID induction, providing a potential novel target for therapeutic development.

neuroscience↗

bioRxiv2019

Misoprostol Attenuates Cardiomyocyte Proliferation in the Neonatal Heart Through Bnip3 and Perinuclear Calcium Signaling

Systemic hypoxia resulting from preterm birth, altered lung development, and cyanotic congenital heart disease is known to impede the regulatory and developmental pathways in the neonatal heart. While the molecular mechanisms are still unknown, hypoxia induces aberrant cardiomyocyte proliferation, which may be initially adaptive, but can ultimately program the heart to fail in early life. Recent evidence suggests that the prostaglandin E1 analogue, misoprostol, is cytoprotective in the hypoxia-exposed neonatal heart by impacting alternative splicing of the Bcl-2 family member Bnip3, resulting in the generation of a variant lacking the third exon (Bnip3{Delta}Exon3 or small Nip; sNip). Using a rodent model of neonatal hypoxia, in combination with rat primary neonatal cardiomyocytes (PVNCs) and H9c2 cells, we sought to determine if misoprostol can prevent cardiomyocyte proliferation and what the key molecular mechanisms might be in this pathway. In PVNCs, exposure to 10% oxygen induced myocyte proliferation concurrent with molecular markers of cell-cycle progression, such as Cyclin-D1, which were prevented by misoprostol treatment. Furthermore, we describe a critical role for sNip in opposing cardiomyocyte proliferation through several mechanisms, including reduced expression of the proliferative MEF2C-myocardin-BMP10 pathway, accumulation of nuclear calcium leading to NFATc3 activation, and increased expression of the cardiac maturation factor BMP2. Intriguingly, misoprostol and sNip inhibited hypoxia-induced glycolytic flux, which directly influenced myocyte proliferation. These observations were further supported by knockdown studies, where hypoxia-induced cardiomyocyte proliferation is restored in misoprostol-treated cells by an siRNA targeting sNip. Finally, in postnatal day (PND)-10 rat pups exposed to hypoxia, we observed histological evidence of increased nuclei number and increased PPH3 staining, which were completely attenuated by misoprostol treatment. Collectively, this data demonstrates how neonatal cardiomyocyte proliferation can be pharmacologically modulated by misoprostol treatment, which may have important implications for both neonatal and regenerative medicine.

cell biology↗