Kimberly J. Dunham-Snary

How cells sense disruption of mitochondrial architecture and couple it to adaptive metabolic stress responses remains poorly understood. Here, we investigated the consequences of perturbing mitochondrial fusion and fission in breast cancer cells and found that loss of the inner membrane fusion protein Opa1, but not the fission regulator Drp1, elicited profound metabolic and transcriptional remodeling. Opa1 silencing disrupted cristae architecture, impaired mitochondrial respiration and induced an ATF4-dependent reprogramming of amino acid metabolism pathways. Mechanistically, ATF4 induction was heightened upon removal of extracellular pyruvate, phenocopied by pharmacological inhibition of electron transport chain (ETC) complex I and required the amino acid-dependent stress kinase GCN2. In contrast, inhibition of ATP synthase (complex V) activated distinct signaling mechanisms that were independent of pyruvate availability or aspartate supplementation, indicating that individual respiratory defects engage mechanistically distinct stress responsive pathways. Functionally, complex I-dependent mitochondrial stress attenuated DNA damage caused by chemotherapeutic agents that target one-carbon metabolism. Together, our findings identify mitochondrial cristae integrity as a critical regulator linking ETC dysfunction to a GCN2-dependent ISR, thereby reshaping metabolic adaptation and therapeutic responsiveness in cancer cells.

RNA-seq quantifies the abundance of transcripts within a biological sample and performs differential analysis between different conditions to reveal regulated gene signatures. Three challenges exist: (1) different analytical packages can often report different expression patterns and false-discovery-rates and P-values; (2) the effective use of these analytical packages requires substantial knowledge of programming and bioinformatics; and (3) there are a lack of intuitive methods to prioritize target genes for further investigation. To address these challenges, we developed Confidence, a web-based application to perform simultaneous statistical analysis of RNA-seq count data. Confidence incorporates the Confidence Score (CS), ranging from 1 to 4 to aid in gene prioritization, where 1 represents low confidence and 4 represents high confidence. The Confidence web-based application was designed for rapid and intuitive analysis of standard experimental metadata and gene count inputs providing a web-based, ‘wide-net’ approach to RNA-seq analysis. Gene scoring allows for unbiased gene selection and identification of novel genes strongly associated with disease/treatment models across multiple species. Pathway analysis has been integrated so that highly confident genes can be placed into biological context. Confidence provides a new strategy for target prioritization in RNA-seq analysis and the generation of publication-quality figures, which we demonstrate here using a published database.

Introduction: Metabolic Dysfunction‐Associated Steatotic Liver Disease (MASLD) has emerged as the most prevalent chronic liver disease worldwide, affecting ~25%–30% of the adult population, with higher prevalence observed in individuals with obesity and type 2 diabetes. Among reported MASLD cases, prevalence is consistently higher in men than in women, and global incidence has risen by ~50% over the past two decades, mirroring the global rise in obesity and metabolic syndrome. MASLD encompasses a spectrum of hepatic pathologies ranging from simple steatosis to steatohepatitis, fibrosis and cirrhosis. Despite its high prevalence, the heterogeneity in disease progression and relative absence of approved pharmacological therapies pose challenges for effective clinical management. Methods and Results: This review synthesises current literature on MASLD across epidemiology, pathophysiology, clinical presentation and treatment. Key molecular mechanisms, including lipid metabolism dysregulation, insulin resistance and mitochondrial dysfunction, are examined with a focus on understanding the basis for progression to metabolic dysfunction‐associated steatohepatitis (MASH). Clinical manifestations, diagnostic tools and risk stratification systems for MASLD are summarised. Current and emerging therapies such as lifestyle interventions, pharmacological agents and microbiome‐targeted strategies are reviewed. The review also highlights ongoing challenges, including diagnostic limitations, disease heterogeneity and disparities in care. Conclusion: MASLD is a complex, multifactorial liver disease with a growing public health impact, driven by the rising prevalence of metabolic syndrome. Mitochondrial dysfunction is a critical nexus linking genetic susceptibility to metabolic stress and inflammatory responses. Preclinical models that capture these mitochondrial contributions are vital for therapeutic discovery and for advancing personalised medicine approaches in MASLD care.

Mitochondrial bioenergetic research in skeletal muscle is limited by the need for biopsies. We executed a proof-of-concept study to evaluate whether blood platelets could serve as a minimally invasive surrogate for skeletal muscle mitochondrial respiration in mice. Using Seahorse extracellular flux analysis, platelet respiration was measured in healthy C57BL/6J and leptin receptor-null db/db mice, while high-resolution respirometry (Oroboros O2k) assessed mitochondrial function in white gastrocnemius muscle of the same animals. A critical component of this study was extensive methodological optimization for platelet bioenergetics analysis in mice. We provide comprehensive methodological details and guiding principles for performing Seahorse bioenergetic assays on mouse platelets. Our foundational findings also suggest platelet mitochondria can reflect tissue-level mitochondrial health, pointing to a potential “liquid biopsy” approach for assessing metabolic status. Multiple key metrics of respiration showed significant correlations between platelets and muscle in the same animals, indicating that platelet bioenergetic profiles mirror the metabolic status of skeletal muscle in healthy and genetically diabetic mice. This work lays the conceptual and methodological foundation for future studies in human metabolic diseases where muscle bioenergetic dysfunction is implicated but current methods are not implementable for clinical surveillance. This study provides foundational proof-of-concept in healthy and diabetic mice, motivating validation in human studies as the next step toward biomarker development and precision medicine strategies.

Metabolic syndrome and type II diabetes pose a significant international health burden, with the latter characterized by insulin resistance. Patients must rely on therapies that maintain glucose homeostasis when endogenous systems become dysfunctional. Skeletal muscle, as the largest insulin-sensitive tissue in the body, plays a critical role in maintaining glucose homeostasis. During disease progression, chronic nutrient overload shifts redox balance to a pro-oxidant state, further exacerbating metabolic dysfunction. First-line treatments, such as metformin and insulin, along with newly adopted incretin-based therapies, modulate the redox state of skeletal muscle. This review explores how the redox state of healthy skeletal muscle is altered throughout metabolic disease progression and how these changes contribute to a worsening phenotype. We also highlight how each class of regularly prescribed medications targets redox-sensitive systems in skeletal muscle, identifying literature gaps and areas for future investigation.

BACKGROUND: The BMPR2 gene encodes the BMPR-II (bone morphogenetic protein receptor type-II) and is a known regulator of endothelial proliferation, apoptosis, and translational stress responses. While these effects are generally attributed to the actions of BMPR-II protein, we used circular RNA profiling to identify circ3218 and circ5078 as new BMPR2 -derived functional RNAs. METHODS: Circular RNAs were profiled by ultradeep RNA sequencing of human pulmonary artery endothelial cells. Novel BMPR2 -derived circular RNAs were assessed for their effects on endothelial proliferation, apoptosis, and translational stress responses in human pulmonary artery endothelial cells and endothelial cells from patients with pulmonary arterial hypertension with heterozygous loss-of-function BMPR2 mutations. circ5078 to linear BMPR2 mRNA ratios were quantified in cultured lymphocytes from patients with pulmonary arterial hypertension with BMPR2 mutations versus unaffected mutation carriers. RESULTS: Depletion of circ3218 enhanced human pulmonary artery endothelial cell apoptosis, whereas circ5078 silencing increased proliferation. Enhanced proliferation with circ5078 depletion was eliminated by cosilencing of circ5078 with either linear BMPR2 mRNA or the stress granule protein, Caprin-1, indicating a potential interdependence of BMPR2 transcripts in the regulation of endothelial function. Patients with pulmonary arterial hypertension with BMPR2 mutations exhibited increased circ5078 to linear BMPR2 mRNA ratios, alongside impaired stress responses in patient endothelial cells that were deficient in linear BMPR2 transcripts but not circ5078 . circ5078 depletion enhanced stress responses in human pulmonary artery endothelial cells and rescued stress granule formation in patient endothelial cells, independent of BMPR-II protein levels. Assessment of translational regulation by polysome profiling did not identify any impact of linear or circular BMPR2 transcript loss on global protein synthesis or stress-induced eIF2α (eukaryotic initiation factor 2α) phosphorylation but did identify the enhanced translational efficiency of select nuclear-encoded mitochondrial ribosome proteins with circ5078 depletion, offering a link between mitochondrial function and the circ5078 -deficient endothelial phenotype. CONCLUSIONS: The identification of circ3218 and circ5078 as novel BMPR2 -derived gene products reveals interdependent roles for coding and noncoding BMPR2 transcripts as regulators of endothelial function.

: Rationale: Mitochondria in ductus arteriosus (DA) smooth muscle cells (DASMC) are oxygen sensors that trigger O ₂ -induced vasoconstriction at birth; however, the molecular mechanisms of mitochondrial oxygen sensing are not fully understood. Many redox sensor proteins are conserved in the mammalian adult homeostatic oxygen sensing system, including NDUFS2 (NADH:Ubiquinone oxidoreductase core subunit S2), a component of mitochondrial Complex I that contributes to oxygen sensing in adult pulmonary arteries. Here we compared the role of NDUFS2 in DA oxygen sensing, to that of other Complex I subunits and putative O ₂ -sensor subunits, including: NADH:Ubiquinone oxidoreductase core subunit S1 (NDUFS1), NADH:Ubiquionone oxidoreductase core subunit S7 (NDUFS7), Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 (UQCRFS1), and Cytochrome c oxidase subunit 4I2 (COX4I2). Methods: Human DASMC were grown in hypoxia (2.5% O ₂ , pO ₂ =41mmHg). Oxygen responsiveness of DASMC was assessed, measuring O ₂ -induced changes in intracellular calcium, [Ca ²⁺ ] _i , cell length, and mitochondrial reactive oxygen species (mROS) production. DASMC were treated for 48-hours with silencing RNA (siRNA) targeting NDUFS2, NDUFS1, NDUFS7, UQCRFS1, or COX4I2; knockdown was confirmed using qPCR and immunoblot. Mitochondrial metabolic consequences were assessed with micropolarimetry and Complex I, III, and IV activity assays. 3’RNA sequencing was used to explore the impact of gene knockdown on the DASMC molecular signature. Results: The O ₂ -induced increase in [Ca ²⁺ ] _i in siControl-treated cells (+18.6±2.3%) was reduced by siNDUFS2 (+5.5±1.5%, p2 -induced DASMC shortening (from -18.4±1.1% to -8.9±0.77%, p<0.0001), and mROS generation (+24±4.9% untreated versus -6.6±5.4% siNDUFS2, p<0.0001). The mitochondrial antioxidant MitoTEMPO also inhibited mROS (+2.9±4.5%, p=0.001) and attenuated oxygen-induced cell shortening (8.43±0.91%, p=0.0003). Knockdown of NDUFS2 and other ETC subunits did not inhibit mitochondrial respiration or ETC activity. Transcriptomics revealed unique changes in mitochondrial pathways with siNDUFS2. Conclusions: NDUFS2 regulates mROS and acts as a mitochondrial oxygen sensor in human DASMC.

RNA sequencing (RNA-seq) is used to quantify transcript levels through measurement of nucleotide sequences. To evaluate statistically significant changes in gene expression, transcript counts between samples are compared using differential expression analysis methods. However, three of the most pressing challenges in transcriptomics analyses are: 1) analytical packages produce a distinct number of differentially expressed genes with varied P -value and fold-change values; 2) the effective use of these analytical packages requires substantial knowledge of programming and bioinformatics; and 3) there are a lack of intuitive methods to select target genes for further investigation in an unbiased manner. To address these challenges, we developed Confidence, a web-based application to perform simultaneous statistical analysis of RNA-seq count data. Confidence incorporates the Confidence Score (CS), ranging from 1 to 4 to aid in gene prioritization, where 1 represents low confidence and 4 represents high confidence. The Confidence web-based application was designed for rapid and intuitive analysis of standard experimental metadata and gene count inputs. Confidence provided a web-based, ‘wide-net’ approach to differential gene expression analysis. Gene scoring allows for unbiased gene selection and identification of novel genes strongly associated with disease and treatment models across multiple species. Additionally, pathway analysis tools have been integrated so that highly confident genes can be placed into biological context in terms of functions and pathways. Confidence provides a new strategy for target prioritization in RNA-seq analysis and the generation of publication-quality figures.

Mitochondrial-driven diseases encompass a diverse group of single-gene and complex disorders, all linked to mitochondrial dysfunction, with significant impacts on human health. While there are rare mitochondrial diseases in which the primary defect resides in mutations in mitochondrial DNA, it is increasingly clear that acquired mitochondrial dysfunction, both genetically- and epigenetically-mediated, complicates common complex diseases, including diabetes, cardiovascular disease and ischemia reperfusion injury, cancer, pulmonary hypertension, and neurodegenerative diseases. It is also recognized that mitochondrial abnormalities not only act by altering metabolism but, through effects on mitochondrial dynamics, can regulate numerous cellular processes including intracellular calcium handling, cell proliferation, apoptosis and quality control. This review examines the crucial role of preclinical models in advancing our understanding of mitochondrial genetic contributions to these conditions. It follows the evolution of models of mitochondrial-driven diseases, from earlier in vitro and in vivo systems to the use of more innovative approaches, such as CRISPR-based gene editing and mitochondrial replacement therapies. By assessing both the strengths and limitations of these models, we highlight their contributions to uncovering disease mechanisms, identifying therapeutic targets, and facilitating novel discoveries. Challenges in translating preclinical findings into clinical applications are also addressed, along with strategies to enhance the accuracy and relevance of these models. This review outlines the current state of the field, the future trajectory of mitochondrial disease modeling, and its potential impact on patient care.