Biology subjects

McClung, J. M.

Publications and source records attributed to McClung, J. M..

5 recordsLinked to original sources

bioRxiv2025

Generation and Validation of a Lower Limb Muscle Single-Cell RNA Sequencing Data Set Identifies Pathogenic Endothelial Metabolism in Peripheral Arterial Disease

BackgroundPeripheral arterial disease (PAD) results from atherosclerotic occlusion(s) in leg arteries. Chronic limb-threatening ischemia (CLTI) is the most severe form of PAD. Patients with CLTI suffer from rest pain, ulcers, or gangrene. Clinical outcomes remain poor in patients with CLTI and many investigational approaches, such as promoting angiogenesis, have failed. Understanding cell-specific vs. bulk-RNA changes within muscle offers an opportunity to better understand this disease. ObjectiveTo assess cell-specific alterations in gene and metabolism pathways in endothelial and muscle cells from leg muscle between patients with PAD vs. non-PAD controls. MethodsWe analyzed publicly available bulk RNA-seq leg muscle data from non-PAD controls, patients with intermittent claudication (IC) and CLTI. An emphasis was on glycolysis and the hexosamine biosynthetic pathway (HBP). Two single-cell RNA-seq datasets were integrated, cell types identified, and metabolism pathway scores calculated across identified cell types. Validations included quantitative mRNA in flow-sorted ECs, and immunofluorescence from clinically-phenotyped human samples. ResultsBulk RNA-seq revealed downregulation of both glycolysis and HBP in CLTI vs. IC or control tissues. Single-cell analysis uncovered cell-type specific changes. In patients with CLTI, gene expression in ECs showed increased glycolysis and decreased HBP. In myonuclei gene expression of both pathways were reduced. Flow-sorted ECs confirmed higher glycolysis and reduced HBP in CLTI vs. controls. Immunofluorescence analysis of muscle revealed significantly lower HBPECs (9.5% vs. 31.3%, n=5/group, p=0.004) and increased glycolytic ECs (42.0% vs. 14.4%, n=7/group, p<0.001) in CLTI vs. control. The findings were independent of capillary density. In IC patients, supervised exercise vs. optimal medical care reduced ischemic muscle PFKFB3+ ECs (2.67% vs. 14.61%, n=11 vs. 9, p<0.001). ConclusionsWe generated and validated a single-cell RNA-seq data set that confirms maladaptive metabolic reprogramming in CLTI ECs. Strategies designed to alter metabolism need to account for differences between bulk vs. single-cell analyses.

bioinformatics↗

bioRxiv2025

The MEK inhibitor trametinib incurs mitochondrial injury and induces innate immune responses in the mouse heart

Trametinib (Trm) is a highly selective MEK inhibitor that potently and persistently abrogates ERK1/2 activation. Trm initially was used to treat BRAF V600E-mutated melanoma but its FDA-approved indications are expanding rapidly. Trm generally is well tolerated but it can cause dose-limiting cardiomyopathy and heart failure. Here we characterize a mouse model of Trm cardiotoxicity using complementary in vitro approaches to show that Trm induces mitochondrial dysfunction in cardiomyocytes and some cancer cell types. In vivo, Trm caused contractile dysfunction within 3 days and heart failure within 2 weeks. High resolution respirometry using isolated cardiac mitochondria revealed that Trm compromises oxidative metabolism, in part through blunted activity of Electron Transport System Complexes. Trm-mediated mitochondrial injury led to the release of mitochondrial Damage-Associated Molecular Patterns including mitochondrial DNA in both mice and humans, triggering activation of canonical innate immune pathways including cGAS-STING. In multiple rodent and human cardiomyocyte platforms, Trm diminished mitochondrial respiratory capacity at nanomolar concentrations but this lesion was reversed by expression of a phosphomimetic STAT3-S727 construct. We also found that Trm induced mitochondrial dysfunction in some but not all cancer cell lines, identifying a previously unrecognized effect that could contribute to Trms anti-cancer efficacy.

physiology↗

bioRxiv2024

Cell state transition analysis reveals a causal network that controls critical limb ischemia

Increased endothelial cell (EC) permeability significantly contributes to peripheral arterial disease and its severe manifestation, critical limb ischemia (CLI). CLI remains refractory to current pharmacological interventions, often requiring limb amputation and showing how limited our knowledge is about signaling pathways that control EC states. Here, we utilize a two-pronged approach, employing cell State Transition Assessment and Regulation (cSTAR) analyses to both the published knowledge (e.g., LINCS data portal) and our own transcriptomics data collected from CLI patients and healthy adult (HA) control. We identified CLI and HA transcriptomics patterns and reconstructed causal network that controls transitions between EC states associated with CLI and HA. Contrary to general belief, our study demonstrates that VEGF administration slightly promotes the CLI EC states, indicating VEGF ineffectiveness in enhancing perfusion of CLI tissue. Based on the unveiled control network, we suggest targets whose inhibition would reverse CLI EC states closer to HA states.

bioinformatics↗

bioRxiv2020

Muscle progenitor cells are required for the regenerative response and prevention of adipogenesis after limb ischemia

Peripheral artery disease (PAD) is nearly as common as coronary artery disease, but few effective treatments exist, and it is associated with significant morbidity and mortality. Although PAD studies have focused on the vascular response to ischemia, skeletal muscle cells play a critically important role in determining the phenotypic manifestation of PAD. Here, we demonstrate that genetic ablation of Pax7+ muscle progenitor cells (MPCs, or satellite cells) in a murine model of hind limb ischemia (HLI) resulted in a complete absence of normal muscle regeneration following ischemic injury, despite a lack of morphological or physiological changes in resting muscle. Compared to ischemic muscle of control mice (Pax7WT), the ischemic limb of Pax7-deficient mice (Pax7{Delta}) was unable to generate significant force 7- or 28-days after HLI in ex vivo force measurement studies. A dramatic increase in adipose infiltration was observed 28 days after HLI in Pax7{Delta} mice, which replaced functional muscle. To investigate the mechanism of this adipogenic change, mice with inhibition of fibro/adipogenic precursors (FAPs), another pool of MPCs, were subjected to HLI. Inhibition of FAPs decreased muscle adipose fat but increased fibrosis. MPCs cultured from mouse muscle tissue failed to form myotubes in vitro following depletion of satellite cells in vivo, and they displayed an increased propensity to differentiate into fat in adipogenic medium. Importantly, this phenotype was recapitulated in patients with critical limb ischemia (CLI), the most severe form of PAD. Skeletal muscle samples from CLI patients demonstrated an increase in adipose deposition in more ischemic regions of muscle, which corresponded with a decrease in the number of satellite cells in those regions. Collectively, these data demonstrate that Pax7+ MPCs are required for normal muscle regeneration after ischemic injury, and they suggest that targeting muscle regeneration may be an important therapeutic approach to prevent muscle degeneration in PAD.

pathology↗

bioRxiv2019

Effects of fasting induced carbohydrate depletion on murine ischemic skeletal muscle function.

Stored muscle carbohydrate supply and energetic efficiency constrain muscle functional capacity during exercise and are influenced by common physiological variables (e.g. age, diet, and physical activity level). Whether these constraints affect overall functional capacity or the timing of muscle energetic failure during acute ischemia is not known. We interrogated skeletal muscle contractile properties in two anatomically distinct hindlimb muscles that have well characterized differences in energetic efficiency (locomotory-extensor digitorum longus (EDL) and postural-soleus muscles) under conditions of reduced carbohydrate supply. 180 mins of acute ischemia resulted in complete energetic failure in all muscles tested, indicated by: loss of force production, substantial reductions in total adenosine nucleotide pool intermediates, and increased adenosine nucleotide degradation product - inosine monophosphate (IMP). These changes occurred in the absence of apparent myofiber structural damage assessed histologically by both transverse section and whole mount. Restriction of the available intracellular carbohydrate pool by fasting (~50% decrease in skeletal muscle) did not significantly alter the timing to muscle functional impairment or affect the overall force/work capacities of either muscle type. Fasting did cause rapid development of passive tension in both muscle types, which may have implications for optimal timing of reperfusion or administration of precision therapeutics.

physiology↗