Biology subjects

Foster, D. B.

Publications and source records attributed to Foster, D. B..

5 recordsLinked to original sources

bioRxiv2026

Optimized Mn2+-Phos-tag Gels Reveal Sarcomeric Protein Dephosphorylation upon Myofibril Preparation

Precise quantification of myofilament protein phosphorylation is essential for understanding the regulation of cardiac contractility in health and disease. Although Phos-tag SDS-PAGE is widely used to resolve phosphorylated protein isoforms, its reproducibility and quantitative reliability are often limited by variability in the key experimental factors, including gel composition, electrophoretic conditions, protein loading, and sample preparation. Here, we present a standardized manganese (Mn2+)-Phos-tag SDS-PAGE workflow optimized for cardiac myofilament proteins, using myosin regulatory light chain 2 (MLC2) and cardiac troponin I (cTnI) as model targets. We systematically evaluated critical parameters - including Mn2+ and Phos-tag concentrations, acrylamide composition, electrophoretic regime, buffer chemistry, protein loading, and EDTA-mediated transfer - to define conditions that maximize phospho-species resolution while preserving quantitative fidelity. We further demonstrate that electrophoresis rate, sample loading, and extraction strategy significantly influence band morphology, signal intensity, and the apparent distribution of phospho-species. As a use case scenario, we compared Trichloroacetic acid (TCA) extracted mouse left ventricular homogenates with myofibrils prepared using a widely adopted Triton-X-100 tissue-demembranization protocol. Myofibril preparation was associated with profound MLC2 dephosphorylation at the earliest stages of preparation, whereas cTnI exhibited a marked reduction in higher-order, low-stoichiometry phosphoforms. Further evaluation of Myosin-binding protein C (MyBP-C) showed progressive loss of phosphorylation over the course of 24 hours. We submit that TCA-extracted heart standards in combination with Phos-tag gels can provide valuable quality control for the phosphorylation status of myofibril preparations, and that inclusion of a high-affinity PP2A and PP1 phosphatase inhibitor like okadaic acid may benefit future myofibril mechanics studies.

biochemistry↗

bioRxiv2026

SureQuant IS-PRM Enables Cross-Species Targeted Quantification of Retinoid Metabolism and Signaling Proteins in the Heart

Retinoic acid signaling is critical for cardiac development and homeostasis. Dysregulation of all-trans retinoic acid metabolism contributes to vascular atherogenesis, restenosis, calcification, and heart failure. Therefore, assessment of proteins involved in retinoid metabolism and signaling has gained interest for identifying potential biomarkers and therapeutic targets in cardiovascular disease. However, quantifying these proteins remains challenging due to limitations of antibody-based methods. We developed a targeted proteomics approach using SureQuant internal standard-triggered parallel reaction monitoring mass spectrometry to profile these proteins. We designed a panel of 80 stable isotope-labeled (heavy) peptides representing proteins involved in retinoid signaling and metabolism, with sequences applicable to human samples and conserved across multiple species. Survey experiments using directed data-dependent acquisition on the Orbitrap Fusion Lumos mass spectrometer determined precursor and product ion masses for each heavy peptide, which were programmed into the SureQuant method for continuous monitoring. Upon detection of these heavy internal standards, the instrument transitions to a targeted PRM acquisition mode in which repeated high-resolution MS/MS spectra of both endogenous (light) and heavy peptides are acquired. Using this method, retinoid pathway-associated proteins were quantified to as low as 10 attomoles for selected targets across multiple tissues and developmental stages. Distinct tissue-specific retinoid metabolic networks were identified across lung, liver, retinal cell lines and cardiac tissues. Developmental profiling of mouse and rat hearts revealed remodeling of retinoid pathway proteins from embryonic to postnatal and adult stages, suggesting a functional transition from retinoid-driven cardiac development toward maintenance of retinoid homeostasis in the mature heart.

biochemistry↗

bioRxiv2025

Tuberculous meningitis alters the proteomic landscape of brain-derived extracellular vesicles

Tuberculous meningitis (TB meningitis), the deadliest form of Mycobacterium tuberculosis infection, leads to mortality and severe neurological disability despite standard therapy. Brain injury and microglial activation are major determinants of outcome, yet the mechanisms linking infection, inflammation and neuronal injury remain poorly understood. Extracellular vesicles (EVs), key mediators of cell-to-cell communication, have been investigated in pulmonary TB but their role in TB meningitis remains unexplored. We used our young rabbit model of TB meningitis to isolate pure, intact EVs from brain tissue (i.e., brain-derived EVs) from infected and uninfected rabbits and used nanoflow cytometry, transmission electron microscopy and protein quantification to characterize the EVs. Comparative proteomic profiling was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by in silico pathway, cell-type and protein-protein interaction analyses using DAVID, Enrichr, and STRING databases. We found that EV isolation from fresh and frozen tissue was equivalent and demonstrated that M. tuberculosis infection activated EV biogenesis. Despite preserved vesicle morphology, EVs from infected brain showed a significant proteomic shift characterized by enrichment of TB host defense, microglial and immune activation, metabolic excitotoxicity, and neuronal injury. These proteome dysregulations suggest that infection reprograms brain EV cargo toward proinflammatory and metabolic stress responses while depleting neuronal and mitochondrial components. Collectively, these data demonstrate that M. tuberculosis infection alters the cargo and abundance of brain-derived EV, highlighting their potential as biomarkers and mediators of host-pathogen interactions in TB meningitis.

microbiology↗

bioRxiv2024

Inhibition of Cardiac p38 Highlights the Role of the Phosphoproteome in Heart Failure Progression

Heart failure (HF) is a complex condition characterized by the inability of the heart to pump sufficient oxygen to the organs to meet their metabolic needs. Among the altered signal transduction pathways associated with HF pathogenesis, the p38 mitogen-activated protein kinase (p38 MAPK) pathway--activated in response to stress-- has attracted considerable attention for its potential role in HF progression and cardiac hypertrophy. However, the exact mechanisms by which p38 MAPK influences HF remain unclear. Addressing knowledge gaps may provide insight on why p38 inhibition has yielded inconsistent outcomes in clinical trials. Here we investigate the effects of p38 MAPK inhibition via SB203580 on cardiac remodeling in a guinea pig model of HF and sudden cardiac death. Using a well-established HF model with ascending aortic constriction and daily isoproterenol (ACi) administration, we assessed proteomic changes across three groups: sham-operated controls, untreated ACi, and ACi treated with SB203580 (ACiSB). Cardiac function was evaluated by M-mode echocardiography, while proteome and phosphoproteome profiles were analyzed using multiplexed tandem mass tag labeling and LC-MS/MS. Our findings demonstrate that chronic SB203580 treatment offers protection against progressive decline in cardiac function in HF. The proteomic data indicate that SB203580-treatment exerts broad protection of the cardiac phosphoproteome, beyond inhibiting maladaptive p38-dependent phosphorylation, extending to PKA and AMPK networks among others, ultimately protecting the phosphorylation status of critical myofibrillar and Ca2+-handling proteins. Though SB203580 had a more restricted impact on widespread protein changes in HF, its biosignature was consistent with preserved mitochondrial energetics as well as reduced oxidative and inflammatory stress.

molecular biology↗

bioRxiv2024

Innate Immune Activation and Mitochondrial ROS Invoke Persistent Cardiac Conduction System Dysfunction after COVID-19

BackgroundCardiac risk rises during acute SARS-CoV-2 infection and in long COVID syndrome in humans, but the mechanisms behind COVID-19-linked arrhythmias are unknown. This study explores the acute and long term effects of SARS-CoV-2 on the cardiac conduction system (CCS) in a hamster model of COVID-19. MethodsRadiotelemetry in conscious animals was used to non-invasively record electrocardiograms and subpleural pressures after intranasal SARS-CoV-2 infection. Cardiac cytokines, interferon-stimulated gene expression, and macrophage infiltration of the CCS, were assessed at 4 days and 4 weeks post-infection. A double-stranded RNA mimetic, polyinosinic:polycytidylic acid (PIC), was used in vivo and in vitro to activate viral pattern recognition receptors in the absence of SARS-CoV-2 infection. ResultsCOVID-19 induced pronounced tachypnea and severe cardiac conduction system (CCS) dysfunction, spanning from bradycardia to persistent atrioventricular block, although no viral protein expression was detected in the heart. Arrhythmias developed rapidly, partially reversed, and then redeveloped after the pulmonary infection was resolved, indicating persistent CCS injury. Increased cardiac cytokines, interferon-stimulated gene expression, and macrophage remodeling in the CCS accompanied the electrophysiological abnormalities. Interestingly, the arrhythmia phenotype was reproduced by cardiac injection of PIC in the absence of virus, indicating that innate immune activation was sufficient to drive the response. PIC also strongly induced cytokine secretion and robust interferon signaling in hearts, human iPSC-derived cardiomyocytes (hiPSC-CMs), and engineered heart tissues, accompanied by alterations in electrical and Ca2+ handling properties. Importantly, the pulmonary and cardiac effects of COVID-19 were blunted by in vivo inhibition of JAK/STAT signaling or by a mitochondrially-targeted antioxidant. ConclusionsThe findings indicate that long term dysfunction and immune cell remodeling of the CCS is induced by COVID-19, arising indirectly from oxidative stress and excessive activation of cardiac innate immune responses during infection, with implications for long COVID Syndrome.

pathology↗