Pan-tissue mitochondrial phenotyping reveals lower OXPHOS expression and function across cancer types.

Targeting mitochondrial oxidative phosphorylation (OXPHOS) to treat cancer has been hampered due to serious side-effects potentially arising from the inability to discriminate between non-cancerous and cancerous mitochondria. Herein, comprehensive mitochondrial phenotyping was leveraged to define both the composition and function of OXPHOS across various murine cancers and compared to both matched normal tissues and other organs. When compared to both matched normal tissues, as well as high OXPHOS reliant organs like heart, intrinsic expression of the OXPHOS complexes, as well as OXPHOS flux were discovered to be consistently lower across distinct cancer types. Assuming intrinsic OXPHOS expression/function predicts OXPHOS reliance in vivo, these data suggest that pharmacologic blockade of mitochondrial OXPHOS likely compromises bioenergetic homeostasis in healthy oxidative organs prior to impacting tumor mitochondrial flux in a clinically meaningful way. Although these data caution against the use of indiscriminate mitochondrial inhibitors for cancer treatment, considerable heterogeneity was observed across cancer types with respect to both mitochondrial proteome composition and substrate-specific flux, highlighting the possibility for targeting discrete mitochondrial proteins or pathways unique to a given cancer type.

Bioenergetic Phenotyping of DEN-Induced Hepatocellular Carcinoma Reveals a Link Between Adenylate Kinase Isoform Expression and Reduced Complex I-Supported Respiration.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer worldwide. Increasing evidence suggests that mitochondria play a central role in malignant metabolic reprogramming in HCC, which may promote disease progression. To comprehensively evaluate the mitochondrial phenotype present in HCC, we applied a recently developed diagnostic workflow that combines high-resolution respirometry, fluorometry, and mitochondrial-targeted nLC-MS/MS proteomics to cell culture (AML12 and Hepa 1-6 cells) and diethylnitrosamine (DEN)-induced mouse models of HCC. Across both model systems, CI-linked respiration was significantly decreased in HCC compared to nontumor, though this did not alter ATP production rates. Interestingly, CI-linked respiration was found to be restored in DEN-induced tumor mitochondria through acute in vitro treatment with P1, P5-di(adenosine-5') pentaphosphate (Ap5A), a broad inhibitor of adenylate kinases. Mass spectrometry-based proteomics revealed that DEN-induced tumor mitochondria had increased expression of adenylate kinase isoform 4 (AK4), which may account for this response to Ap5A. Tumor mitochondria also displayed a reduced ability to retain calcium and generate membrane potential across a physiological span of ATP demand states compared to DEN-treated nontumor or saline-treated liver mitochondria. We validated these findings in flash-frozen human primary HCC samples, which similarly displayed a decrease in mitochondrial respiratory capacity that disproportionately affected CI. Our findings support the utility of mitochondrial phenotyping in identifying novel regulatory mechanisms governing cancer bioenergetics.

Alterations in sphingolipid composition and mitochondrial bioenergetics represent synergistic therapeutic vulnerabilities linked to multidrug resistance in leukemia.

Modifications in sphingolipid (SL) metabolism and mitochondrial bioenergetics are key factors implicated in cancer cell response to chemotherapy, including chemotherapy resistance. In the present work, we utilized acute myeloid leukemia (AML) cell lines, selected to be refractory to various chemotherapeutics, to explore the interplay between SL metabolism and mitochondrial biology supportive of multidrug resistance (MDR). In agreement with previous findings in cytarabine or daunorubicin resistant AML cells, relative to chemosensitive wildtype controls, HL-60 cells refractory to vincristine (HL60/VCR) presented with alterations in SL enzyme expression and lipidome composition. Such changes were typified by upregulated expression of various ceramide detoxifying enzymes, as well as corresponding shifts in ceramide, glucosylceramide, and sphingomyelin (SM) molecular species. With respect to mitochondria, despite consistent increases in both basal respiration and maximal respiratory capacity, direct interrogation of the oxidative phosphorylation (OXPHOS) system revealed intrinsic deficiencies in HL60/VCR, as well as across multiple MDR model systems. Based on the apparent requirement for augmented SL and mitochondrial flux to support the MDR phenotype, we explored a combinatorial therapeutic paradigm designed to target each pathway. Remarkably, despite minimal cytotoxicity in peripheral blood mononuclear cells (PBMC), co-targeting SL metabolism, and respiratory complex I (CI) induced synergistic cytotoxicity consistently across multiple MDR leukemia models. Together, these data underscore the intimate connection between cellular sphingolipids and mitochondrial metabolism and suggest that pharmacological intervention across both pathways may represent a novel treatment strategy against MDR.

Mitochondrial Diagnostics: A Discovery-Based Biochemical Platform for Phenotyping Human Peripheral Blood Cell Mitochondria.

In vitro experiments using permeabilized cells and/or isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across multiple scientific fields to shed valuable insight on mitochondrial-linked pathologies. The present chapter is intended to serve as a methodological blueprint for comprehensively phenotyping peripheral blood cell mitochondria. While primarily adapted for peripheral blood cells, the protocols outlined herein could easily be made amenable to most all cell types with minimal modifications.

Novel approach to quantify mitochondrial content and intrinsic bioenergetic efficiency across organs.

Human disease pathophysiology commonly involves metabolic disruption at both the cellular and subcellular levels. Isolated mitochondria are a powerful model for separating global cellular changes from intrinsic mitochondrial alterations. However, common laboratory practices for isolating mitochondria (e.g., differential centrifugation) routinely results in organelle preparations with variable mitochondrial purity. To overcome this issue, we developed a mass spectrometry-based method that quantitatively evaluates sample-specific percent mitochondrial enrichment. Sample-specific mitochondrial enrichment was then used to correct various biochemical readouts of mitochondrial function to a 'fixed' amount of mitochondrial protein, thus allowing for intrinsic mitochondrial bioenergetics, relative to the underlying proteome, to be assessed across multiple mouse tissues (e.g., heart, brown adipose, kidney, liver). Our results support the use of mitochondrial-targeted nLC-MS/MS as a method to quantitate mitochondrial enrichment on a per-sample basis, allowing for unbiased comparison of functional parameters between populations of mitochondria isolated from metabolically distinct tissues. This method can easily be applied across multiple experimental settings in which intrinsic shifts in the mitochondrial network are suspected of driving a given physiological or pathophysiological outcome.

Microvascular Endothelial Dysfunction in Sedentary, Obese Humans Is Mediated by NADPH Oxidase: Influence of Exercise Training.

OBJECTIVE: The objectives of this study were to determine the impact of in vivo reactive oxygen species (ROS) on microvascular endothelial function in obese human subjects and the efficacy of an aerobic exercise intervention on alleviating obesity-associated dysfunctionality. APPROACH AND RESULTS: Young, sedentary men and women were divided into lean (body mass index 18-25; n=14), intermediate (body mass index 28-32.5; n=13), and obese (body mass index 33-40; n=15) groups. A novel microdialysis technique was utilized to detect elevated interstitial hydrogen peroxide (H2O2) and superoxide levels in the vastus lateralis of obese compared with both lean and intermediate subjects. Nutritive blood flow was monitored in the vastus lateralis via the microdialysis-ethanol technique. A decrement in acetylcholine-stimulated blood flow revealed impaired microvascular endothelial function in the obese subjects. Perfusion of apocynin, an NADPH oxidase inhibitor, lowered (normalized) H2O2 and superoxide levels, and reversed microvascular endothelial dysfunction in obese subjects. After 8 weeks of exercise, H2O2 levels were decreased in the obese subjects and microvascular endothelial function in these subjects was restored to levels similar to lean subjects. Skeletal muscle protein expression of the NADPH oxidase subunits p22phox, p47phox, and p67phox was increased in obese relative to lean subjects, where p22phox and p67phox expression was attenuated by exercise training in obese subjects. CONCLUSIONS: This study implicates NADPH oxidase as a source of excessive ROS production in skeletal muscle of obese individuals and links excessive NADPH oxidase-derived ROS to microvascular endothelial dysfunction in obesity. Furthermore, aerobic exercise training proved to be an effective strategy for alleviating these maladies.

Comparison of Risk of Atrial Fibrillation in Black Versus White Patients After Coronary Artery Bypass Grafting.

Obesity has been identified as a risk factor for postoperative atrial fibrillation (POAF) after coronary artery bypass grafting (CABG). However, no studies have addressed the influence of race on this association. A total of 13,594 patients undergoing first-time, isolated CABG without preoperative AF between 1992 and 2011 were included in our study. The association between body mass index and POAF was compared by race. Relative risk and 95% CIs were computed using maximum likelihood log-binomial regression. Increasing levels of body mass index were associated with higher POAF risk after CABG in black but not white patients (pinteraction = 0.0009).

The "Goldilocks Zone" from a redox perspective-Adaptive vs. deleterious responses to oxidative stress in striated muscle.

Consequences of oxidative stress may be beneficial or detrimental in physiological systems. An organ system's position on the "hormetic curve" is governed by the source and temporality of reactive oxygen species (ROS) production, proximity of ROS to moieties most susceptible to damage, and the capacity of the endogenous cellular ROS scavenging mechanisms. Most importantly, the resilience of the tissue (the capacity to recover from damage) is a decisive factor, and this is reflected in the disparate response to ROS in cardiac and skeletal muscle. In myocytes, a high oxidative capacity invariably results in a significant ROS burden which in homeostasis, is rapidly neutralized by the robust antioxidant network. The up-regulation of key pathways in the antioxidant network is a central component of the hormetic response to ROS. Despite such adaptations, persistent oxidative stress over an extended time-frame (e.g., months to years) inevitably leads to cumulative damages, maladaptation and ultimately the pathogenesis of chronic diseases. Indeed, persistent oxidative stress in heart and skeletal muscle has been repeatedly demonstrated to have causal roles in the etiology of heart disease and insulin resistance, respectively. Deciphering the mechanisms that underlie the divergence between adaptive and maladaptive responses to oxidative stress remains an active area of research for basic scientists and clinicians alike, as this would undoubtedly lead to novel therapeutic approaches. Here, we provide an overview of major types of ROS in striated muscle and the divergent adaptations that occur in response to them. Emphasis is placed on highlighting newly uncovered areas of research on this topic, with particular focus on the mitochondria, and the diverging roles that ROS play in muscle health (e.g., exercise or preconditioning) and disease (e.g., cardiomyopathy, ischemia, metabolic syndrome).