Biology subjects

Smyth, J. W.

Publications and source records attributed to Smyth, J. W..

8 recordsLinked to original sources

bioRxiv2025

An inducible genetic model of chronic hypoxic signaling in cardiomyocytes precipitates severe cardiomyopathy and remodeling.

Cardiovascular disease remains the leading cause of death globally, underscoring the need for physiologically relevant models to investigate mechanisms of heart failure and arrhythmia. Chronic activation of hypoxic signaling pathways, particularly via the hypoxia-inducible factor (HIF) axis, is a key contributor to cardiac remodeling under stress. A major regulator of HIF signaling is the von Hippel-Lindau tumor suppressor (VHL) which, under normoxic conditions, targets HIF for proteasomal degradation. Loss of VHL results in HIF accumulation and persistent hypoxic signaling, but constitutive cardiomyocyte-specific Vhl knockout models are confounded by developmental effects and early mortality. Here, we develop and characterize an inducible, cardiomyocyte-specific Vhl knockout mouse model as a non-invasive and temporally controlled system to study chronic hypoxic stress and its contribution to cardiac remodeling and disease. VhlLoxP/LoxP;MHC-MerCreMer+/- mice were administered tamoxifen to induce Vhl deletion in adult cardiomyocytes. Within 5-7 days post-induction, mice displayed reduced ejection fraction, increased cardiac diameter, and elevated expression of cardiac stress markers. Transcriptomic and protein analyses revealed downregulation of key genes involved in cardiac structure and electrophysiology, including Gja1 (Cx43), Cdh2 (N-cadherin), Cacna1c (CaV1.2), and Kcnq1. Importantly, these changes preceded overt cardiac remodeling, as confirmed in an abbreviated tamoxifen protocol. This inducible Vhl knockout model recapitulates hallmark features of dilated cardiomyopathy and highlights a subset of cardiac structural and ion channel genes as sensitive early responders to chronic hypoxic stress. This platform enables mechanistic dissection of disease onset and progression in ischemic heart disease and serves as a well-controlled and reproducible model for evaluating novel therapeutic strategies.

pathology↗

bioRxiv2024

Astrocytic connexin43 phosphorylation contributes to seizure susceptibility after mild Traumatic Brain Injury

Astrocytes play a crucial role in maintaining brain homeostasis through functional gap junctions (GJs) primarily formed by connexin43 (Cx43) in the cortical gray matter. These GJs facilitate electrical and metabolic coupling between astrocytes, allowing the passage of ions, glucose, and metabolites. Dysregulation of Cx43 has been implicated in various pathologies, including traumatic brain injury (TBI) and acquired epilepsy. After mild TBI/concussion, we previously identified a subset of atypical astrocytes, which are correlated with the development of spontaneous seizures. These astrocytes exhibit reduced Cx43 expression and coupling. However, atypical astrocytes represent a relatively small subset of astrocytes within the cortical gray matter and previous studies suggest an overall increase of Cx43 protein after TBI. Additionally, Cx43 also has non-junctional and channel-independent functions, which include hemichannel communication with the extracellular milieu, cell adhesion, protein trafficking, protein-protein interactions, and intracellular signaling. In the present study, we set out to determine how mild TBI initiates alterations to Cx43 protein expression and localization, how they may be regulated, and whether they contribute to seizure susceptibility. We demonstrate remarkable heterogeneity of Cx43 protein levels from astrocyte to astrocyte. In accordance with our previous findings, a subset of astrocytes lost Cx43 expression, yet total cortical Cx43 protein increased. At the subcellular level, junctional Cx43 protein levels remained stable, while hemichannels and/or cytoplasmic Cx43 were increased. Phosphorylation of Cx43 at serine 368, a key regulatory site influencing GJ assembly and function, increased after mild TBI. Critically, Cx43S368A mutant mice, lacking this phosphorylation, exhibited reduced susceptibility to pentylenetetrazol-induced seizures. These findings suggest that TBI-induced Cx43 phosphorylation enhances seizure susceptibility, while inhibiting this modification presents a potential therapeutic avenue for mitigating neuronal hyperexcitability and seizure development. Significance statementConnexin43 (Cx43) is the main protein comprising astrocyte gap junctions which mediate astrocyte coupling into cellular networks, but it also has other non-junctional functions. Many pathologies present with altered Cx43 regulation. In this study, we assessed Cx43 alterations after mild traumatic brain injury (TBI) in a mouse model. We found that while some astrocytes lost Cx43 expression, other astrocytes had increased cytoplasmic and hemichannel Cx43. This increase correlated with an increase in phosphorylated Cx43 at serine 368. Cx43S368A mutant mice, lacking this phosphorylation, exhibited reduced susceptibility to seizures induced by pentylenetetrazol (PTZ). These findings suggest that TBI-induced Cx43 phosphorylation enhances seizure susceptibility.

neuroscience↗

bioRxiv2024

Optimized Enrichment of Murine Blood-Brain Barrier Vessels with a Critical Focus on Network Hierarchy in Post-Collection Analysis

Cerebrovascular networks contain a unique region of interconnected capillaries known as the blood-brain barrier (BBB). Positioned between upstream arteries and downstream veins, these microvessels have unique structural features, such as the absence of vascular smooth muscle cells (vSMCs) and a relatively thin basement membrane, to facilitate highly efficient yet selective exchange between the circulation and the brain interstitium. This vital role in neurological health and function has garnered significant attention from the scientific community and inspired methodology for enriching BBB capillaries. Extensive characterization of the isolates from such protocols is essential for framing the results of follow-on experiments and analyses, providing the most accurate interpretation and assignment of BBB properties. Seeking to aid in these efforts, here we visually screened output samples using fluorescent labels and found considerable reduction of non-vascular cells following density gradient centrifugation (DGC) and subsequent filtration. Comparatively, this protocol enriched brain capillaries, though larger diameter vessels associated with vSMCs could not be fully excluded. Protein analysis further underscored the enrichment of vascular markers following DGC, with filtration preserving BBB-associated markers and reducing - though not fully removing - arterial/venous contributions. Transcriptional profiling followed similar trends of DGC plus filtration generating isolates with less non-vascular and non- capillary material included. Considering vascular network hierarchy inspired a more comprehensive assessment of the material yielded from brain microvasculature isolation protocols. This approach is important for providing an accurate representation of the cerebrovascular segments being used for data collection and assigning BBB properties specifically to capillaries relative to other regions of the brain vasculature. HIGHLIGHTS- We optimized a protocol for the enrichment of murine capillaries using density gradient centrifugation and follow-on filtration. - We offer an approach to analyzing post-collection cerebrovascular fragments and cells with respect to vascular network hierarchy. - Assessing arterial and venous markers alongside those associated with the BBB provides a more comprehensive view of material collected. - Enhanced insight into isolate composition is critical for a more accurate view of BBB biology relative to larger diameter cerebrovasculature. MOTIVATIONThe recent surge in studies investigating the cerebrovasculature, and the blood-brain barrier (BBB) in particular, has inspired a broad range of approaches to target and observe these specialized blood vessels within murine models. To capture transcriptional and molecular changes during a specific intervention or disease model, techniques have been developed to isolate brain capillary networks and collect their cellular constituents for downstream analysis. Here, we sought to highlight the benefits and cautions of isolating and enriching microvessels from murine brain tissue. Specifically, through rigorous assessment of the output material following application of specific protocols, we presented the benefits of specific approaches to reducing the inclusion of non-vascular cells and non-capillary vessel segments, verified by analysis of vascular-related proteins and transcripts. We also emphasized the levels of larger- caliber vessels (i.e. arteries/arterioles and veins/venules) that are collected alongside cerebral capillaries with each method. Distinguishing these vascular regions with greater precision is critical for attributing specific characteristics exclusively to the BBB where metabolic, ion, and waste exchange occurs. While the addition of larger vessels to molecular / transcriptional analyses or follow-on experiments may not be substantial for a given protocol, it is essential to gauge and report their level of inclusion, as their contributions may be inadvertently assigned to the BBB. Therefore, we present this optimized brain microvessel isolation protocol and associated evaluation methods to underscore the need for increased rigor in characterizing vascular regions that are collected and analyzed within a given study. Graphical AbstractConstructed using resources from BioRender.com. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/613898v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@7b4448org.highwire.dtl.DTLVardef@bff92borg.highwire.dtl.DTLVardef@17fa8b3org.highwire.dtl.DTLVardef@1c950d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Novel Pannexin 1 isoform is increased in cancer

Alternative translation initiation (ATI) is a process of increasing protein diversity from one transcript, allowing cells to rapidly respond to signals, which is particularly important in cancer cells. Here, we report potential internal translation start sites exist in PANX1 which have implications in trafficking and channel function. Using mouse (mPANX1) constructs for each internal methionine, we saw that these PANX1 isoforms were N-glycosylated, could traffic to the cell surface and mPANX1-M37 formed functional channels activated by C-terminus cleavage or 1-adrenoceptor stimulation. We also identified a ~25 kDa isoform of mPANX1 (mPANX1-25K) endogenously expressed in mouse melanoma cell lines that could be confirmed with a cognate peptide. mPANX1-25K lacks the mPANX1 N-terminus and most likely corresponds to the M210 internal translation start site since we could not identify any alternative transcripts that would produce this ATI product. When we expressed the human equivalent of M210 in Hs578T PANX1 KO cells with and without wildtype human PANX1, we determined M211 exhibits a predominantly intracellular localization, is N-glycosylated and can interact with full-length human PANX1. Collectively, these findings indicate species specific differences in the abundance of PANX1 ATI isoforms which could act independently or in conjunction with the canonical full-length protein in melanoma.

cell biology↗

bioRxiv2024

Connexin 43 Regulates Intercellular Mitochondrial Transfer from Human Mesenchymal Stromal Cells to Chondrocytes

BackgroundThe phenomenon of intercellular mitochondrial transfer from mesenchymal stromal cells (MSCs) has shown promise for improving tissue healing after injury and has potential for treating degenerative diseases like osteoarthritis (OA). Recently MSC to chondrocyte mitochondrial transfer has been documented, but the mechanism of transfer is unknown. Full-length connexin43 (Cx43, encoded by GJA1) and the truncated internally translated isoform GJA1-20k have been implicated in mitochondrial transfer between highly oxidative cells, but have not been explored in orthopaedic tissues. Here, our goal was to investigate the role of Cx43 in MSC to chondrocyte mitochondrial transfer. In this study, we tested the hypotheses that (a) mitochondrial transfer from MSCs to chondrocytes is increased when chondrocytes are under oxidative stress and (b) MSC Cx43 expression mediates mitochondrial transfer to chondrocytes. MethodsOxidative stress was induced in immortalized human chondrocytes using tert-Butyl hydroperoxide (t-BHP) and cells were evaluated for mitochondrial membrane depolarization and reactive oxygen species (ROS) production. Human bone-marrow derived MSCs were transduced for mitochondrial fluorescence using lentiviral vectors. MSC Cx43 expression was knocked down using siRNA or overexpressed (GJA1+ and GJA1-20k+) using lentiviral transduction. Chondrocytes and MSCs were co-cultured for 24 hrs in direct contact or separated using transwells. Mitochondrial transfer was quantified using flow cytometry. Co-cultures were fixed and stained for actin and Cx43 to visualize cell-cell interactions during transfer. ResultsMitochondrial transfer was significantly higher in t-BHP-stressed chondrocytes. Contact co-cultures had significantly higher mitochondrial transfer compared to transwell co-cultures. Confocal images showed direct cell contacts between MSCs and chondrocytes where Cx43 staining was enriched at the terminal ends of actin cellular extensions containing mitochondria in MSCs. MSC Cx43 expression was associated with the magnitude of mitochondrial transfer to chondrocytes; knocking down Cx43 significantly decreased transfer while Cx43 overexpression significantly increased transfer. Interestingly, GJA1-20k expression was highly correlated with incidence of mitochondrial transfer from MSCs to chondrocytes. ConclusionsOverexpression of GJA1-20k in MSCs increases mitochondrial transfer to chondrocytes, highlighting GJA1-20k as a potential target for promoting mitochondrial transfer from MSCs as a regenerative therapy for cartilage tissue repair in OA.

bioengineering↗

bioRxiv2024

Increased interaction between connexin43 and microtubules is critical for glioblastoma stem-like cell maintenance and tumorigenicity.

Glioblastoma (GBM) is the most common primary tumor of the central nervous system. One major challenge in GBM treatment is the resistance to chemotherapy and radiotherapy observed in subpopulations of cancer cells, including GBM stem-like cells (GSCs). These cells hold the ability to self-renew or differentiate following treatment, participating in tumor recurrence. The gap junction protein connexin43 (Cx43) has complex roles in oncogenesis and we have previously demonstrated an association between Cx43 and GBM chemotherapy resistance. Here, we report, for the first time, increased direct interaction between non-junctional Cx43 with microtubules in the cytoplasm of GSCs. We hypothesize that non-junctional Cx43/microtubule complexing is critical for GSC maintenance and survival and sought to specifically disrupt this interaction while maintaining other Cx43 functions, such as gap junction formation. Using a Cx43 mimetic peptide of the carboxyl terminal tubulin-binding domain of Cx43 (JM2), we successfully ablated Cx43 interaction with microtubules in GSCs. Importantly, administration of JM2 significantly decreased GSC survival in vitro, and limited GSC-derived tumor growth in vivo. Together, these results identify JM2 as a novel peptide drug to ablate GSCs in GBM treatment.

cancer biology↗

bioRxiv2022

Acute adenoviral cardiac infection elicits an arrhythmogenic substrate prior to inflammatory myocardial remodeling and myocarditis

BackgroundViral cardiac infection represents a significant clinical challenge encompassing several etiological agents, disease stages, complex presentation, and a resulting lack of mechanistic understanding. Myocarditis is a major cause of sudden cardiac death in young adults, where current knowledge in the field is dominated by later disease phases, and pathological immune responses. However, little is known regarding how infection can acutely induce an arrhythmogenic substrate prior to significant immune responses. Adenovirus is a leading cause of myocarditis, but due to species-specificity, models of infection are lacking and it is not understood how adenoviral infection may underlie sudden cardiac arrest. Mouse Adenovirus Type-3 (MAdV-3) was previously reported as cardiotropic, yet has not been utilized to understand mechanisms of cardiac infection and pathology. MethodsWe have developed MAdV-3 infection as a model to investigate acute cardiac infection and molecular alterations to the infected heart prior to an appreciable immune response or gross cardiomyopathy. ResultsOptical mapping of infected hearts exposes decreases in conduction velocity concomitant with increased Cx43Ser368 phosphorylation, a residue known to regulate gap junction function. Hearts from animals harboring a phospho-null mutation at Cx43Ser368 are protected against MAdV-3 induced conduction velocity slowing. Additional to gap junction alterations, patch clamping of MAdV-3-infected adult mouse ventricular cardiomyocytes reveals prolonged action potential duration as a result of decreased IK1 and IKs current density. Turning to human systems, we find human adenovirus type-5 (HAdV-5) increases phosphorylation of Cx43Ser368 and disrupts synchrony in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), indicating common mechanisms with our mouse whole heart and adult cardiomyocyte data. ConclusionsTogether, these findings demonstrate that adenoviral infection creates an arrhythmogenic substrate through direct targeting of gap junction and ion channel function in the heart. Such alterations are known to precipitate arrhythmias and likely contribute to sudden cardiac death in acutely infected patients.

pathology↗

bioRxiv2021

Adenovirus transduction to express human ACE2 causes obesity-specific morbidity in mice, impeding studies on the effect of host nutritional status on SARS-CoV-2 pathogenesis.

The COVID-19 pandemic has paralyzed the global economy and resulted in millions of deaths globally. People with co-morbidities like obesity, diabetes and hypertension are at an increased risk for severe COVID-19 illness. This is of overwhelming concern because 42% of Americans are obese, 30% are pre-diabetic and 9.4% have clinical diabetes. Here, we investigated the effect of obesity on disease severity following SARS-CoV-2 infection using a well-established mouse model of diet-induced obesity. Diet-induced obese and lean control C57BL/6N mice, transduced for ACE2 expression using replication-defective adenovirus, were infected with SARS-CoV-2, and monitored for lung pathology, viral titers, and cytokine expression. No significant differences in tissue pathology, viral replication or cytokine expression were observed between lean and obese groups. Notably, significant weight loss was observed in obese mice treated with the adenovirus vector, independent of SARS-CoV-2 infection, suggesting an obesity-dependent morbidity induced by the vector. These data indicate that the adenovirus-transduced mouse model of SARS-CoV-2 infection is inadequate for performing nutrition studies, and caution should be used when interpreting resulting data.

pathology↗