Kelsey H. Fisher-Wellman

Trametinib (Trm) is a highly selective mitogen-activated protein kinase kinase (MEK) inhibitor that potently and persistently abrogates extracellular signal–regulated kinase 1/2 activation. Trm initially was used to treat BRAF Val ⁶⁰⁰ →Glu (V600E)-mutated melanoma, but its Food and Drug Administration–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 signal transducer and activator of transcription 3–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 Trm’s anticancer efficacy.

Dextran sulfate sodium (DSS) is widely used to chemically-induce both colitis and colorectal cancer when administered alongside azoxymethane (AOM). DSS functions by disrupting the colonic epithelial barrier, triggering widespread inflammation within the colon. While DSS is a valuable tool for studying colitis-related diseases, its impact on mitochondrial bioenergetics and the proteomic landscape of colonic tissue remains poorly understood. To assess the chronic effects of DSS-induced colitis, we administered three rounds of 3% DSS in drinking water (5-day treatment periods) to C57BL/6 J mice and analyzed resected colonic tissue from DSS-treated and control (non-DSS treated) mice. Longitudinally opened colon segments were cleaned and subjected to high-resolution respirometry and mass spectrometry-based proteomic profiling. DSS treatment led to a global lowering of mitochondrial respiration, with the most pronounced impairments observed in complex I-supported respiration. Proteomic analysis revealed that these functional deficits occurred largely independently of changes in the mitochondrial proteome, except for an apparent upregulation of NIPSNAP1, a mitophagy-related protein. However, lentiviral knockdown of NIPSNAP1 in HCT116 cells did not rescue the observed bioenergetic defects, suggesting it is not the primary driver. Collectively, our findings show that DSS impairs mitochondrial respiration in the colon, most notably at complex I, without major alterations to the mitochondrial proteome. Given the role of mitochondrial dysfunction in various diseases, these effects should be carefully considered when using DSS-based models to study colitis pathophysiology.

Targeting mitochondrial oxidative phosphorylation (OxPhos) has become a recurring strategy in the treatment of cancer, particularly in acute myeloid leukemia (AML). Early reports suggested that leukemic blasts, and especially leukemia stem cells, depend disproportionately on mitochondrial respiration, implying a therapeutic window for systemic inhibition of the electron transport system (ETS) and OxPhos. Yet, the clinical record of broad OxPhos inhibition has been disappointing. In the present review, we argue that the pivotal question is not whether mitochondria matter for cancer, but whether specific mitochondrial processes are disproportionately essential to malignant cells compared with the organism’s most OxPhos-dependent organs. We clarify what OxPhos is (and is not), emphasizing why oxygen consumption rate (OCR) is an incomplete surrogate for ATP-producing OxPhos flux and why transcriptomic ‘OxPhos signatures’ often confound energetic demand with compensatory responses to mitochondrial damage. We then benchmark OxPhos capacity and flux across normal tissues versus tumors, highlighting that highly oxidative organs typically operate at far higher respiratory flux than most cancers. Using the Complex I inhibitor IACS-010759 as a case study, we discuss why systemic ETS inhibition predictably collided with dose-limiting toxicity. Finally, focusing on AML, we dissect how OxPhos ‘dependency’ was inferred from indirect assays, how the failure to normalize for mitochondrial content may invert conclusions, and how ATP synthase reversal can masquerade as ‘ATP-linked respiration.’ We conclude with practical criteria for identifying mitochondrial liabilities that are targetable rather than merely essential, and we outline alternative strategies, which may better align mitochondrial biology with a realistic therapeutic index.

BAG3 (Bcl-2 associated athanogene 3) is a multifunctional protein with pleiotropic effects in multiple cell types. Despite our knowledge of its role in cardiovascular disease, its specific role in endothelial cells (ECs) is unknown. The purpose of this study was to identify differences in the EC proteome of multiple tissues before and after cell specific deletion of Bag3 . We hypothesized that BAG3 loss would uniquely alter the baseline proteome landscape of ECs in each tissue. Cdh5(PAC)-CreERT2; Bag3 ^f/f mice received tamoxifen (KO; n=18) or vehicle (WT; n=18) and tissues (brain, heart, lung, and peripheral skeletal muscle-skm) were collected for FACS sorting of ECs from equal numbers of male and female mice at >22 weeks of age, followed by LC-MS/MS label-free proteomics. Initial comparisons of the WT proteomes between tissues revealed differential abundance of EC proteins (p<0.05), including: 379 brain v heart; 325 brain v lung; 501 brain v skm; 354 heart v lung; 107 heart v skm; and 442 lung v skm. Additionally, the EC mitochondrial proteome was unique to each tissue of origin, with significantly (p<0.05) higher proportions dedicated to complexes I, III, IV and V, in heart compared to the other tissues. KO demonstrated the largest effect on skm ECs, increasing 30 proteins (Mybpc2 L2FC=4.81; PFKM L2FC=3.02) and decreasing 65 (CDC42 L2FC=-2.44; Prpf8 L2FC=-2.24). Overall, these results demonstrate that the EC proteome of different tissues is unique and that the loss of BAG3 in these cells differentially alters a small proportion of the EC specific proteome.

Mitochondrial function varies widely across kidney nephron segments, yet conventional approaches lack the resolution and control needed to assess cell-type-specific bioenergetics in situ. We present a methodological platform that enables segment-resolved profiling of mitochondrial respiration, conductance, and membrane potential in freshly isolated mouse and human nephron segments. Combining mechanical sieving and adhesion-based enrichment with permeabilized high-resolution respirometry, we adapted the creatine kinase clamp to quantify oxygen flux and mitochondrial membrane potential across defined free energies. Using this approach, we found that proximal tubules exhibit high respiratory conductance and dynamic mitochondrial polarization, while distal tubules and glomeruli maintain static membrane potential and low conductance. In a model of adenine-induced nephropathy, only proximal tubule mitochondria showed marked reductions in respiration and ATP production. This segment-specific dysfunction was not detectable in bulk mitochondrial isolates. We then demonstrate that these methods can be applied in human kidney samples. Our approach provides thermodynamically anchored, segment-resolved insight into mitochondrial adaptation under physiological and pathological conditions. It is broadly applicable to other tissues with metabolic heterogeneity and compatible with disease models, genetic tools, and pharmacological interventions. This platform bridges a critical gap between conventional respirometry and functional mitochondrial phenotyping in native tissue structures.

OxPhos inhibitors have struggled to show a clinical benefit because of their inability to distinguish healthy from cancerous mitochondria. Herein, we describe an actionable bioenergetic mechanism unique to acute myeloid leukemia (AML) mitochondria. Unlike healthy cells that couple respiration to ATP synthesis, AML mitochondria support inner-membrane polarization by consuming ATP. Matrix ATP consumption allows cells to survive bioenergetic stress. Thus, we hypothesized AML cells may resist chemotherapy-induced cell death by reversing the ATP synthase reaction. In support, BCL-2 inhibition with venetoclax abolished OxPhos flux without affecting mitochondrial polarization. In surviving AML cells, sustained mitochondrial polarization depended on matrix ATP consumption. Mitochondrial ATP consumption was further enhanced in AML cells made refractory to venetoclax, consequential to down-regulations in the endogenous F ₁ -ATPase inhibitor ATP5IF1. Knockdown of ATP5IF1 conferred venetoclax resistance, while ATP5IF1 overexpression impaired F ₁ -ATPase activity and heightened sensitivity to venetoclax. These data identify matrix ATP consumption as a cancer cell–intrinsic bioenergetic vulnerability actionable in the context of BCL-2 targeted chemotherapy.

Mesenchymal stem cells (MSCs) of infants born to women with gestational diabetes mellitus (GDM) treated by metformin display lower rates of oleate oxidation despite no limitations in lipid availability compared with GDM treated by diet. Mitochondrial capacity was also lower among infant MSCs from metformin-treated GDM.