Cellular Context Dictates the Suppression or Augmentation of Triple-Negative Mammary Tumor Metastasis by NLRX1.

Prior studies have defined multiple, but inconsistent, roles for the enigmatic pattern recognition receptor NLRX1 in regulating several cancer-associated biological functions. In this study, we explore the role of NLRX1 in the highly metastatic murine 4T1 mammary tumor model. We describe a functional dichotomy of NLRX1 between two different cellular contexts: expression in healthy host cells versus expression in the 4T1 tumor cells. Using Nlrx1-/- mice engrafted with 4T1 tumors, we demonstrate that NLRX1 functions as a tumor suppressor when expressed in the host cells. Specifically, NLRX1 in healthy host cells attenuates tumor growth and lung metastasis through suppressing characteristics of epithelial-mesenchymal transition and the lung metastatic niche. Conversely, we demonstrate that NLRX1 functions as a tumor promoter when expressed in 4T1 tumor cells using gain- and loss-of-function studies both in vitro and in vivo. Mechanistically, NLRX1 in the tumor cells augments 4T1 aggressiveness and metastasis through regulating epithelial-mesenchymal transition hallmarks, cell death, proliferation, migration, reactive oxygen species levels, and mitochondrial respiration. Collectively, we provide critical insight into NLRX1 function and establish a dichotomous role of NLRX1 in the 4T1 murine mammary carcinoma model that is dictated by cellular context.

The GDF15-GFRAL Pathway in Health and Metabolic Disease: Friend or Foe?

GDF15 is a cell activation and stress response cytokine of the glial cell line-derived neurotrophic factor family within the TGF-ß superfamily. It acts through a recently identified orphan member of the GFRα family called GFRAL and signals through the Ret coreceptor. Cell stress and disease lead to elevated GDF15 serum levels, causing anorexia, weight loss, and alterations to metabolism, largely by actions on regions of the hindbrain. These changes restore homeostasis and, in the case of obesity, cause a reduction in adiposity. In some diseases, such as advanced cancer, serum GDF15 levels can rise by as much as 10-100-fold, leading to an anorexia-cachexia syndrome, which is often fatal. This review discusses how GDF15 regulates appetite and metabolism, the role it plays in resistance to obesity, and how this impacts diseases such as diabetes, nonalcoholic fatty liver disease, and anorexia-cachexia syndrome. It also discusses potential therapeutic applications of targeting the GDF15-GFRAL pathway and lastly suggests some potential unifying hypotheses for its biological role.

Clinical validation of circulating GDF15/MIC-1 as a marker of response to docetaxel and survival in men with metastatic castration-resistant prostate cancer.

BACKGROUND: Elevated circulating growth differentiation factor (GDF15/MIC-1), interleukin 4 (IL4), and IL6 levels were associated with resistance to docetaxel in an exploratory cohort of men with metastatic castration-resistant prostate cancer (mCRPC). This study aimed to establish level 2 evidence of cytokine biomarker utility in mCRPC. METHODS: IntVal: Plasma samples at baseline (BL) and Day 21 docetaxel (n = 120). ExtVal: Serum samples at BL and Day 42 of docetaxel (n = 430). IL4, IL6, and GDF15 levels were measured by ELISA. Monocytes and dendritic cells were treated with 10% plasma from men with high or low GDF15 or recombinant GDF15. RESULTS: IntVal: Higher GDF15 levels at BL and Day 21 were associated with shorter overall survival (OS) (BL; p = 0.03 and Day 21; p = 0.004). IL4 and IL6 were not associated with outcomes. ExtVal: Higher GDF15 levels at BL and Day 42 predicted shorter OS (BL; p < 0.0001 and Day 42; p < 0.0001). Plasma from men with high GDF15 caused an increase in CD86 expression on monocytes (p = 0.03), but was not replicated by recombinant GDF15. CONCLUSIONS: Elevated circulating GDF15 is associated with poor prognosis in men with mCRPC receiving docetaxel and may be a marker of changes in the innate immune system in response to docetaxel resistance. These findings provide a strong rationale to consider GDF15 as a biomarker to guide a therapeutic trial of drugs targeting the innate immune system in combination with docetaxel in mCRPC.

Single-cell RNA sequencing reveals peripheral blood leukocyte responses to spinal cord injury in mice with humanised immune systems.

Next-generation humanised mouse models and single-cell RNA sequencing (scRNAseq) approaches enable in-depth studies into human immune cell biology. Here we used NSG-SGM3 mice engrafted with human umbilical cord haematopoietic stem cells to investigate how human immune cells respond to and/or are changed by traumatic spinal cord injury (SCI). We hypothesised that the use of such mice could help advance our understanding of spinal cord injury-induced immune depression syndrome (SCI-IDS), and also how human leukocytes change as they migrate from the circulation into the lesion site. Our scRNAseq experiments, supplemented by flow cytometry, demonstrate the existence of up to 11 human immune cell (sub-) types and/or states across the blood and injured spinal cord (7 days post-SCI) of humanised NSG-SGM3 mice. Further comparisons of human immune cell transcriptomes between naïve, sham-operated and SCI mice identified a total of 579 differentially expressed genes, 190 of which were 'SCI-specific' (that is, genes regulated only in response to SCI but not sham surgery). Gene ontology analysis showed a prominent downregulation of immune cell function under SCI conditions, including for T cell receptor signalling and antigen presentation, confirming the presence of SCI-IDS and the transcriptional signature of human leukocytes in association with this phenomenon. We also highlight the activating influence of the local spinal cord lesion microenvironment by comparing the transcriptomes of circulating versus infiltrated human immune cells; those isolated from the lesion site were enriched for genes relating to both immune cell activity and function (e.g., oxidative phosphorylation, T cell proliferation and antigen presentation). We lastly applied an integrated bioinformatics approach to determine where immune responses in humanised NSG-SGM3 mice appear congruent to the native responses of human SCI patients, and where they diverge. Collectively, our study provides a valuable resource and methodological framework for the use of these mice in translational research.

Neurons, Receptors, and Channels.

Here, I recount some adventures that I and my colleagues have had over some 60 years since 1957 studying the effects of drugs and neurotransmitters on neuronal excitability and ion channel function, largely, but not exclusively, using sympathetic neurons as test objects. Studies include effects of centrally active drugs on sympathetic transmission; neuronal action and neuroglial uptake of GABA in the ganglia and brain; the action of muscarinic agonists on sympathetic neurons; the action of bradykinin on neuroblastoma-derived cells; and the identification of M-current as a target for muscarinic action, including experiments to determine its distribution, molecular composition, neurotransmitter sensitivity, and intracellular regulation by phospholipids and their hydrolysis products. Techniques used include electrophysiological recording (extracellular, intracellular microelectrode, whole-cell, and single-channel patch-clamp), autoradiography, messenger RNA and complementary DNA expression, antibody injection, antisense knockdown, and membrane-targeted lipidated peptides. I finish with some recollections about my scientific career, funding, and changes in laboratory life and pharmacology research over the past 60 years.

Targeting the Endothelin-1 pathway to reduce invasion and chemoresistance in gallbladder cancer cells.

BACKGROUND: Gallbladder cancer (GBC) is a prevalent and deadly biliary tract carcinoma, often diagnosed at advanced stages with limited treatment options. The 5-year survival rate varies widely from 4 to 60%, mainly due to differences in disease stage detection. With only a small fraction of patients having resectable tumors and a high incidence of metastasis, advanced GBC stages are characterized by significant chemoresistance. Identification of new therapeutic targets is crucial, and recent studies have shown that the Endothelin-1 (ET-1) signaling pathway, involving ETAR and/or ETBR receptors (ETRs), plays a crucial role in promoting tumor aggressiveness in various cancer models. Blocking one or both receptors has been reported to reduce invasiveness and chemoresistance in cancers like ovarian, prostate, and colon. Furthermore, transcriptomic studies have associated ET-1 levels with late stages of GBC; however, it remains unclear whether its signaling or its inhibition has implications for its aggressiveness. Although the role of ET-1 signaling in gallbladder physiology is minimally understood, its significance in other tumor models leads us to hypothesize its involvement in GBC malignancy. RESULTS: In this study, we investigated the expression of ET-1 pathway proteins in three GBC cell lines and a primary GBC culture. Our findings demonstrated that both ETAR and ETBR receptors are expressed in GBC cells and tumor samples. Moreover, we successfully down-regulated ET-1 signaling using a non-selective ETR antagonist, Macitentan, which resulted in reduced migratory and invasive capacities of GBC cells. Additionally, Macitentan treatment chemosensitized the cells to Gemcitabine, a commonly used therapy for GBC. CONCLUSION: For the first time, we reveal the role of the ET-1 pathway in GBC cells, providing insight into the potential therapeutic targeting of its receptors to mitigate invasion and chemoresistance in this cancer with limited treatment options. These findings pave the way for further exploration of Macitentan or other ETR antagonists as potential therapeutic strategies for GBC management. In summary, our study represents a groundbreaking contribution to the field by providing the first evidence of the ET 1 pathway's pivotal role in modulating the behavior and aggressiveness of GBC cells, shedding new light on potential therapeutic targets.

An obstetrician-gynecologist's review of hernias: risk factors, diagnosis, prevention, and repair.

Management of obstetrical and gynecologic patients with hernias poses challenges to providers. Risks for hernia development include well-described factors that impair surgical wound healing and increase abdominal pressure. Among the diverse populations cared for by obstetricians and gynecologists, pregnant patients and those with gynecologic malignancies are at the highest risk for hernia formation. This article provides an overview of the existing literature, with a focus on patients cared for by obstetrician-gynecologists and commonly encountered preoperative and intraoperative scenarios. We highlight scenarios when a hernia repair is not commonly performed, including those of patients undergoing nonelective surgeries with known or suspected gynecologic cancers. Finally, we offer multidisciplinary recommendations on the timing of elective hernia repair with obstetrical and gynecologic procedures, with attention to the primary surgical procedure, the type of preexisting hernia, and patient characteristics.

Blood-Based Transcriptomic Biomarkers Are Predictive of Neurodegeneration Rather Than Alzheimer's Disease.

Alzheimer's disease (AD) is a growing global health crisis affecting millions and incurring substantial economic costs. However, clinical diagnosis remains challenging, with misdiagnoses and underdiagnoses being prevalent. There is an increased focus on putative, blood-based biomarkers that may be useful for the diagnosis as well as early detection of AD. In the present study, we used an unbiased combination of machine learning and functional network analyses to identify blood gene biomarker candidates in AD. Using supervised machine learning, we also determined whether these candidates were indeed unique to AD or whether they were indicative of other neurodegenerative diseases, such as Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). Our analyses showed that genes involved in spliceosome assembly, RNA binding, transcription, protein synthesis, mitoribosomes, and NADH dehydrogenase were the best-performing genes for identifying AD patients relative to cognitively healthy controls. This transcriptomic signature, however, was not unique to AD, and subsequent machine learning showed that this signature could also predict PD and ALS relative to controls without neurodegenerative disease. Combined, our results suggest that mRNA from whole blood can indeed be used to screen for patients with neurodegeneration but may be less effective in diagnosing the specific neurodegenerative disease.

Effects of OP2113 on Myocardial Infarct Size and No Reflow in a Rat Myocardial Ischemia/Reperfusion Model.

PURPOSE: The present study was to determine whether OP2113 could limit myocardial infarction size and the no-reflow phenomenon in a rat myocardial ischemia/reperfusion model. METHODS: Rat heart-isolated mitochondria (RHM) were used to investigate mitochondrial respiration and mitochondrial reactive oxygen species (mtROS) generation both in normal conditions and in ischemia/reperfusion-mimicking conditions (using high concentrations of succinate). Human skeletal muscle myoblasts (HSMM) in culture were used to investigate the cellular intermittent deprivation in energy substrates and oxygen as reported in ischemia/reperfusion conditions. In vivo, rats were anesthetized and subjected to 30 min of left coronary artery occlusion followed by 3 h of reperfusion. Rats were randomized to receive OP2113 as an intravenous infusion starting either 5 min prior to coronary artery occlusion (preventive), or 5 min prior to reperfusion (curative), or to receive vehicle starting 5 min prior to coronary artery occlusion. Infusions continued until the end of the study (3 h of reperfusion). RESULTS: RHM treated with OP2113 showed a concentration-dependent reduction of succinate-induced mtROS generation. In HSMM cells, OP2113 treatment (5-10 µM) during 48H prevented the reduction in the steady-state level of ATP measured just after reperfusion injuries and decreased the mitochondrial affinity to oxygen. In vivo, myocardial infarct size, expressed as the percentage of the ischemic risk zone, was significantly lower in the OP2113-treated preventive group (44.5 ± 2.9%) versus that in the vehicle group (57.0 ± 3.6%; p < 0.05), with a non-significant trend toward a smaller infarct size in the curative group (50.8 ± 3.9%). The area of no reflow as a percentage of the risk zone was significantly smaller in both the OP2113-treated preventive (28.8 ± 2.4%; p = 0.026 vs vehicle) and curative groups (30.1 ± 2.3%; p = 0.04 vs vehicle) compared with the vehicle group (38.9 ± 3.1%). OP2113 was not associated with any hemodynamic changes. CONCLUSIONS: These results suggest that OP2113 is a promising mitochondrial ROS-modulating agent to reduce no-reflow as well as to reduce myocardial infarct size, especially if it is on board early in the course of the infarction. It appears to have benefit on no-reflow even when administered relatively late in the course of ischemia.

SARS-CoV-2 neutralizing antibodies: Longevity, breadth, and evasion by emerging viral variants.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antibody neutralization response and its evasion by emerging viral variants and variant of concern (VOC) are unknown, but critical to understand reinfection risk and breakthrough infection following vaccination. Antibody immunoreactivity against SARS-CoV-2 antigens and Spike variants, inhibition of Spike-driven virus-cell fusion, and infectious SARS-CoV-2 neutralization were characterized in 807 serial samples from 233 reverse transcription polymerase chain reaction (RT-PCR)-confirmed Coronavirus Disease 2019 (COVID-19) individuals with detailed demographics and followed up to 7 months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization, was associated with COVID-19 severity. A subgroup of "high responders" maintained high neutralizing responses over time, representing ideal convalescent plasma donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants and VOC. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal responders and vaccine monitoring and design.

The latitude-dependent autoimmune disease risk genes ZMIZ1 and IRF8 regulate mononuclear phagocytic cell differentiation in response to vitamin D.

Epidemiological, molecular and genetic studies have indicated that high serum vitamin D levels are associated with lower risk of several autoimmune diseases. The vitamin D receptor (VDR) binding sites in monocytes and dendritic cells (DCs) are more common in risk genes for diseases with latitude dependence than in risk genes for other diseases. The transcription factor genes Zinc finger MIZ domain-containing protein 1 (ZMIZ1) and interferon regulatory factor 8 (IRF8)-risk genes for many of these diseases-have VDR binding peaks co-incident with the risk single nucleotide polymorphisms (SNPs). We show these genes are responsive to vitamin D: ZMIZ1 expression increased and IRF8 expression decreased, and this response was affected by genotype in different cell subsets. The IL10/IL12 ratio in tolerogenic DCs increased with vitamin D. These data indicate that vitamin D regulation of ZMIZ1 and IRF8 in DCs and monocytes contribute to latitude-dependent autoimmune disease risk.

Bioenergetics underlying single-cell migration on aligned nanofiber scaffolds.

Cell migration is centrally involved in a myriad of physiological processes, including morphogenesis, wound healing, tissue repair, and metastatic growth. The bioenergetics that underlie migratory behavior are not fully understood, in part because of variations in cell culture media and utilization of experimental cell culture systems that do not model physiological connective extracellular fibrous networks. In this study, we evaluated the bioenergetics of C2C12 myoblast migration and force production on fibronectin-coated nanofiber scaffolds of controlled diameter and alignment, fabricated using a nonelectrospinning spinneret-based tunable engineered parameters (STEP) platform. The contribution of various metabolic pathways to cellular migration was determined using inhibitors of cellular respiration, ATP synthesis, glycolysis, or glucose uptake. Despite immediate effects on oxygen consumption, mitochondrial inhibition only modestly reduced cell migration velocity, whereas inhibitors of glycolysis and cellular glucose uptake led to striking decreases in migration. The migratory metabolic sensitivity was modifiable based on the substrates present in cell culture media. Cells cultured in galactose (instead of glucose) showed substantial migratory sensitivity to mitochondrial inhibition. We used nanonet force microscopy to determine the bioenergetic factors responsible for single-cell force production and observed that neither mitochondrial nor glycolytic inhibition altered single-cell force production. These data suggest that myoblast migration is heavily reliant on glycolysis in cells grown in conventional media. These studies have wide-ranging implications for the causes, consequences, and putative therapeutic treatments aimed at cellular migration.

Safety of drug use in patients with a primary mitochondrial disease: An international Delphi-based consensus.

Clinical guidance is often sought when prescribing drugs for patients with primary mitochondrial disease. Theoretical considerations concerning drug safety in patients with mitochondrial disease may lead to unnecessary withholding of a drug in a situation of clinical need. The aim of this study was to develop consensus on safe medication use in patients with a primary mitochondrial disease. A panel of 16 experts in mitochondrial medicine, pharmacology, and basic science from six different countries was established. A modified Delphi technique was used to allow the panellists to consider draft recommendations anonymously in two Delphi rounds with predetermined levels of agreement. This process was supported by a review of the available literature and a consensus conference that included the panellists and representatives of patient advocacy groups. A high level of consensus was reached regarding the safety of all 46 reviewed drugs, with the knowledge that the risk of adverse events is influenced both by individual patient risk factors and choice of drug or drug class. This paper details the consensus guidelines of an expert panel and provides an important update of previously established guidelines in safe medication use in patients with primary mitochondrial disease. Specific drugs, drug groups, and clinical or genetic conditions are described separately as they require special attention. It is important to emphasise that consensus-based information is useful to provide guidance, but that decisions related to drug prescribing should always be tailored to the specific needs and risks of each individual patient. We aim to present what is current knowledge and plan to update this regularly both to include new drugs and to review those currently included.

A theory of the piezoelectric slotted cylinder projector.

The energy method is used on the radial and circumferential displacement mode shapes of tapered piezoelectric slotted cylinder projectors to determine the electro-mechanical equivalent circuit parameters for the transducer. Results are determined for acoustically unloaded conditions for any degree of shell tapering. The resonance frequency (fr), mechanical quality factor (Qm), and electro-mechanical coupling factor (keff) are calculated and compared to measured data.

Effects of circumferential stress on tangentially polarized piezoelectric cylinders.

This paper investigates the dependence of the electromechanical properties of the tangential polarized (stripe-electroded) cylindrical piezoelements made of the hard (PZT-4 like) and soft (PZT-5A like) ceramics on the compressive circumferential stress produced by hydrostatic pressure. Results are presented for the relative change of the effective dielectric constants, elastic constants, coupling coefficients, and piezoelectric moduli. The sensitivity of dielectric constant and piezoelectric modulus to the compressive stress is shown to be less than radially polarized ceramic.

Increasing piezoelectric effect in radially polarized soft piezoelectric cylinders by pressure treating and its practical applications.

Subjecting soft piezoelectric ceramics such as lead zirconate titanate (PZT)-5 to uniaxial stress in a direction perpendicular to the principal polarization axis is known to increase its transverse piezoelectric modulus [Krueger, J. Acoust. Soc. Am. 43 (3), 583-559 (1968)], however, practical transducer applications of this effect remain undeveloped. This study explores uniaxial pressure-treatment to develop experimental methods to realize practical pressure treatment, to use dynamic measurements of the electromechanical coupling coefficient k32, piezoelectric modulus, and dielectric constants, and to enhance piezoelectric properties and performance of underwater acoustic cylindrical transducer designs. The results of axial pressure treatment on parameters of the cylinders are shown to increase the coupling coefficient by about 20% and remained stable. The applicability of using pressure treated cylinders in transducer designs is investigated. The performance was analyzed under one-dimensional circumferential static compression and under the hydrostatic compression. When the improved piezoelectric cylinders are used in air-backed transducer designs, the benefit is only useful at shallow depths as it is observed that when subjected to increased hydrostatic pressure, the corresponding induced circumferential stress reduces the piezoelectric properties, namely, the coupling coefficient, to approximately its original pre-treated value. However, when the improved cylinders are used in transducers of the pressure-equalized design, such as liquid filled or free-flooded, the increase in coupling coefficient remains stable with environmental hydrostatic pressure.

Comparison of Resistance-Based Walking Cardiorespiratory Test to the Bruce Protocol.

Hurt, CP, Bamman, M, Naidu, A, and Brown, DA. Comparison of resistance-based walking cardiorespiratory test to the Bruce Protocol. J Strength Cond Res 34(12): 3569-3576, 2020-Cardiorespiratory fitness is assessed through graded exercise tests that determine the maximum amount of sustained mechanical work that an individual can perform while also providing health- and fitness-related information. This article describes a novel method to perform graded exercise tests that use posteriorly directed resistive forces. The purpose of this investigation was to validate a novel resistance-based test (RBT) in comparison with a traditional speed- and incline-based test (SIBT) in a cohort of nonimpaired individuals. Twenty nonimpaired individuals, 8 men and 20 women age 28.4 ± 9.6, range 20-54 years old performed 2 maximal exercise tests. The SIBT used the Bruce Protocol and increased treadmill incline and speed every 3 minutes. The RBT used a robotic device interfaced with the treadmill that provided specified horizontal resistive forces at the center of mass calculated to match each Bruce Protocol stage while individuals walked at 1.1 m·s. Subjects obtained ∼3% higher maximum V[Combining Dot Above]O2 measure using the speed- and incline-based method (dependent t-test p = 0.08). V[Combining Dot Above]O2peaks between tests were strongly correlated (r = 0.93, p < 0.001). Peak values of secondary physiologic measures (i.e., max heart rate and respiratory exchange ratio) were within 3% between tests. We found a significant linear relationship between mass-specific work rate and measured V[Combining Dot Above]O2 stage by stage for both tests, but no significant difference between each linear fit (p = 0.84). These data suggest that horizontal resistive forces, while walking on a treadmill, can be used to increase aerobic effort in a way that closely simulates work rates of the Bruce Protocol.

Cardioprotective effects of idebenone do not involve ROS scavenging: Evidence for mitochondrial complex I bypass in ischemia/reperfusion injury.

Novel therapeutic strategies to treat mitochondrial deficiencies in acute coronary syndromes are needed. Complex I of the mitochondrial electron transport system is damaged following ischemia/reperfusion (I/R) injury. This disruption contributes to aberrant electron transport, diminished bioenergetics, an altered redox environment, and mitochondrial damage involved in tissue injury. In this study, we determined the cardiac and mitochondrial effects of idebenone, a benzoquinone currently in several clinical trials with purported 'antioxidant' effects. We employed complimentary models of ischemia/reperfusion injury in perfused hearts, permeabilized cardiac fibers, isolated mitochondria, and in cells to elucidate idebenone's cardioprotective mechanism(s). In ex vivo whole hearts, infarct size was markedly reduced with post-ischemic idebenone treatment (25 ±â€¯5% area at risk, AAR) compared to controls (56 ±â€¯6% AAR, P < .05). Several parameters of hemodynamic function were also significantly improved after idebenone treatment. Parallel studies of anoxia/reoxygenation were conducted using isolated mitochondria and permeabilized ventricular fibers. In isolated mitochondria, we simultaneously monitored respiration and ROS emission. Idebenone treatment modestly elevated succinate-derived H2O2 production when compared to vehicle control (1.34 ±â€¯0.05 vs 1.21 ±â€¯0.05%, H2O2/O2 respectively, P < .05). Isolated mitochondria subjected to anoxia/reoxygenation demonstrated higher rates of respiration with idebenone treatment (2360 ±â€¯69 pmol/s*mg) versus vehicle control (1995 ±â€¯101 pmol/s*mg). Both mitochondria and permeabilized cardiac fibers produced high rates of H2O2 after anoxia/reoxygenation, with idebenone showing no discernable attenuation on H2O2 production. These insights were further investigated with studies in mitochondria isolated from reperfused ventricle. The profound decrease in complex-I dependent respiration after ischemia/reperfusion (701 ±â€¯59 pmolO2/s*mg compared to 1816 ±â€¯105 pmol O2/s*mg in normoxic mitochondria) was attenuated with idebenone treatment (994 ±â€¯76 vs pmol O2/s*mg, P < .05). Finally, the effects of idebenone were determined using permeabilized cell models with chemical inhibition of complex I. ADP-dependent oxidative phosphorylation capacity was significantly higher in complex-I inhibited cells treated acutely with idebenone (89.0 ±â€¯4.2 pmol/s*million cells versus 70.1 ±â€¯8.2 pmol/s*million cells in untreated cells). Taken together, these data indicate that the cardioprotective effects of idebenone treatment do not involve ROS-scavenging but appear to involve augmentation of the quinone pool, thus providing reducing equivalents downstream of complex I. As this compound is already in clinical trials for other indications, it may provide a safe and useful approach to mitigate ischemia/reperfusion injury in patients.

Proteolipid domains form in biomimetic and cardiac mitochondrial vesicles and are regulated by cardiolipin concentration but not monolyso-cardiolipin.

Cardiolipin (CL) is an anionic phospholipid mainly located in the inner mitochondrial membrane, where it helps regulate bioenergetics, membrane structure, and apoptosis. Localized, phase-segregated domains of CL are hypothesized to control mitochondrial inner membrane organization. However, the existence and underlying mechanisms regulating these mitochondrial domains are unclear. Here, we first isolated detergent-resistant cardiac mitochondrial membranes that have been reported to be CL-enriched domains. Experiments with different detergents yielded only nonspecific solubilization of mitochondrial phospholipids, suggesting that CL domains are not recoverable with detergents. Next, domain formation was investigated in biomimetic giant unilamellar vesicles (GUVs) and newly synthesized giant mitochondrial vesicles (GMVs) from mouse hearts. Confocal fluorescent imaging revealed that introduction of cytochrome c into membranes promotes macroscopic proteolipid domain formation associated with membrane morphological changes in both GUVs and GMVs. Domain organization was also investigated after lowering tetralinoleoyl-CL concentration and substitution with monolyso-CL, two common modifications observed in cardiac pathologies. Loss of tetralinoleoyl-CL decreased proteolipid domain formation in GUVs, because of a favorable Gibbs-free energy of lipid mixing, whereas addition of monolyso-CL had no effect on lipid mixing. Moreover, murine GMVs generated from cardiac acyl-CoA synthetase-1 knockouts, which have remodeled CL acyl chains, did not perturb proteolipid domains. Finally, lowering the tetralinoleoyl-CL content had a stronger influence on the oxidation status of cytochrome c than did incorporation of monolyso-CL. These results indicate that proteolipid domain formation in the cardiac mitochondrial inner membrane depends on tetralinoleoyl-CL concentration, driven by underlying lipid-mixing properties, but not the presence of monolyso-CL.

Docosahexaenoic acid lowers cardiac mitochondrial enzyme activity by replacing linoleic acid in the phospholipidome.

Cardiac mitochondrial phospholipid acyl chains regulate respiratory enzymatic activity. In several diseases, the rodent cardiac phospholipidome is extensively rearranged; however, whether specific acyl chains impair respiratory enzyme function is unknown. One unique remodeling event in the myocardium of obese and diabetic rodents is an increase in docosahexaenoic acid (DHA) levels. Here, we first confirmed that cardiac DHA levels are elevated in diabetic humans relative to controls. We then used dietary supplementation of a Western diet with DHA as a tool to promote cardiac acyl chain remodeling and to study its influence on respiratory enzyme function. DHA extensively remodeled the acyl chains of cardiolipin (CL), mono-lyso CL, phosphatidylcholine, and phosphatidylethanolamine. Moreover, DHA lowered enzyme activities of respiratory complexes I, IV, V, and I+III. Mechanistically, the reduction in enzymatic activities were not driven by a dramatic reduction in the abundance of supercomplexes. Instead, replacement of tetralinoleoyl-CL with tetradocosahexaenoyl-CL in biomimetic membranes prevented formation of phospholipid domains that regulate enzyme activity. Tetradocosahexaenoyl-CL inhibited domain organization due to favorable Gibbs free energy of phospholipid mixing. Furthermore, in vitro substitution of tetralinoleoyl-CL with tetradocosahexaenoyl-CL blocked complex-IV binding. Finally, reintroduction of linoleic acid, via fusion of phospholipid vesicles to mitochondria isolated from DHA-fed mice, rescued the major losses in the mitochondrial phospholipidome and complexes I, IV, and V activities. Altogether, our results show that replacing linoleic acid with DHA lowers select cardiac enzyme activities by potentially targeting domain organization and phospholipid-protein binding, which has implications for the ongoing debate about polyunsaturated fatty acids and cardiac health.