Impaired Mitochondrial Respiration Research Articles

Mitochondrial-dependent oxidative phosphorylation is key for postnatal metabolic adaptation of alveolar macrophages in the lung

Alveolar macrophages (AMs) seed in lung during embryogenesis and become mature in perinatal period. Establishment of acclimatization to environmental challenges is important, whereas the detailed mechanisms that drive metabolic adaptation of AMs remains to be elucidated. Here, we showed that energy metabolism of AMs was transformed from glycolysis prenatally to oxidative phosphorylation (OXPHOS) postnatally accompanied by up-regulated expression of mitochondrial transcription factor A (TFAM). TFAM deficiency disturbed mitochondrial stability and decreased OXPHOS, which finally impaired AM maintenance and function, but not AM embryonic development. Mechanistically, Tfam-deletion resulted in impaired mitochondrial respiration and decreased ATP production, which triggered endoplasmic reticulum (ER) stress to cause B cell lymphoma 2 ovarian killer (BOK) accumulation and abnormal distribution of intracellular Ca2+, eventually led to induce AM apoptotic death. Thus, our data illustrated mitochondrial-dependent OXPHOS played a key role in orchestrating AM postnatal metabolic adaptation.

Sit4 Genetically Interacts with Vps27 to Regulate Mitochondrial Function and Lifespan in Saccharomyces cerevisiae.

The Sit4 protein phosphatase plays a key role in orchestrating various cellular processes essential for maintaining cell viability during aging. We have previously shown that SIT4 deletion promotes vacuolar acidification, mitochondrial derepression, and oxidative stress resistance, increasing yeast chronological lifespan. In this study, we performed a proteomic analysis of isolated vacuoles and yeast genetic interaction analysis to unravel how Sit4 influences vacuolar and mitochondrial function. By employing high-resolution mass spectrometry, we show that sit4Δ vacuolar membranes were enriched in Vps27 and Hse1, two proteins that are part of the endosomal sorting complex required for transport-0. In addition, SIT4 exhibited a negative genetic interaction with VPS27, as sit4∆vps27∆ double mutants had a shortened lifespan compared to sit4∆ and vps27∆ single mutants. Our results also show that Vps27 did not increase sit4∆ lifespan by improving protein trafficking or vacuolar sorting pathways. However, Vps27 was critical for iron homeostasis and mitochondrial function in sit4∆ cells, as sit4∆vps27∆ double mutants exhibited high iron levels and impaired mitochondrial respiration. These findings show, for the first time, cross-talk between Sit4 and Vps27, providing new insights into the mechanisms governing chronological lifespan.

Targeting Mitochondrial Dynamics during Lower-Limb Ischemia Reperfusion in Young and Old Mice: Effect of Mitochondrial Fission Inhibitor-1 (mDivi-1).

Peripheral arterial disease (PAD) strikes more than 200 million people worldwide and has a severe prognosis by potentially leading to limb amputation and/or death, particularly in older patients. Skeletal muscle mitochondrial dysfunctions and oxidative stress play major roles in this disease in relation with ischemia-reperfusion (IR) cycles. Mitochondrial dynamics through impairment of fission-fusion balance may contribute to skeletal muscle pathophysiology, but no data were reported in the setting of lower-limb IR despite the need for new therapeutic options. We, therefore, investigated the potential protective effect of mitochondrial division inhibitor-1 (mDivi-1; 50 mg/kg) in young (23 weeks) and old (83 weeks) mice submitted to two-hour ischemia followed by two-hour reperfusion on systemic lactate, muscle mitochondrial respiration and calcium retention capacity, and on transcripts specific for oxidative stress and mitochondrial dynamics. At the systemic levels, an IR-related increase in circulating lactate was still major despite mDivi-1 use (+305.9% p < 0.0001, and +269.4% p < 0.0001 in young and old mice, respectively). Further, IR-induced skeletal muscle mitochondrial dysfunctions (more severely impaired mitochondrial respiration in old mice (OXPHOS CI state, -68.2% p < 0.0001 and -84.9% p < 0.0001 in 23- and 83-week mice) and reduced calcium retention capacity (-46.1% p < 0.001 and -48.2% p = 0.09, respectively) were not corrected by mDivi-1 preconditioning, whatever the age. Further, mDivi-1 treatment did not oppose superoxide anion production (+71.4% p < 0.0001 and +37.5% p < 0.05, respectively). At the transcript level, markers of antioxidant enzymes (SOD 1, SOD 2, catalase, and GPx) and fission markers (Drp1, Fis) remained unchanged or tended to be decreased in the ischemic leg. Fusion markers such as mitofusin 1 or 2 decreased significantly after IR in both groups. In conclusion, aging enhanced the deleterious effects or IR on muscle mitochondrial respiration, and in this setting of lower-limb IR, mDivi-1 failed to protect the skeletal muscle both in young and old mice.

The down-regulation of GADD45B leads to a conversion of cellular oxidative phosphorylation to glycolysis and promotes the progression of bladder cancer

BackgroundThe predominant feature of cancer cells during the process of carcinogenesis is the inclination towards glycolytic metabolism rather than mitochondrial oxidative phosphorylation. Nevertheless, there is a scarcity of research investigating the correlation between bladder cancer and mitochondrial energy metabolism. MethodsA qPCR array comprising 90 genes associated with mitochondrial oxidative phosphorylation was employed to discern metabolic disparities between three sets of bladder cancer tissue and adjacent normal tissue. Wound healing and transwell assays were utilized to assess cell migration and invasion capabilities, respectively. Colony formation assays were conducted to ascertain the tumorigenic potential of the cells. The proliferative capacity of the cells was examined through in vitro CCK-8 assays. Additionally, nude mouse models were established to evaluate the impact of bladder tumor cells on in vivo proliferation. The Seahorse XFe96 Analyzer was utilized to quantify mitochondrial oxidative phosphorylation, while the levels of glucose-6-phosphate and pyruvate were assessed to evaluate glycolysis. ResultsExamination of qPCR array data demonstrated a noteworthy inhibition of mitochondrial oxidative phosphorylation in bladder cancer tissue, as evidenced by the down-regulation of a majority of genes associated with mitochondrial energy metabolism. Notably, GADD45B may potentially exert a significant influence on bladder cancer development, warranting further investigation. The down-regulation of GADD45B in bladder cancer cells resulted in impaired mitochondrial respiration and elevated levels of glycolysis, thereby enhancing cell migration and invasion. Conversely, up-regulation of GADD45B had the opposite effect. Furthermore, over-expression of GADD45B inhibited tumor proliferation and tumorigenesis in both in vitro and in vivo settings. ConclusionThese findings from our study indicate that the down-regulation of GADD45B promotes the shift of cell mitochondrial oxidative phosphorylation towards glycolysis, thereby facilitating the progression of bladder cancer.

CD8+ T-cell metabolism is related to cerebrovascular reactivity in middle-aged adults.

Cerebrovascular reactivity (CVR) decreases with advancing age, contributing to increased risk of cognitive impairment; however, the mechanisms underlying the age-related decrease in CVR are incompletely understood. Age-related changes to T cells, such as impaired mitochondrial respiration, increased inflammation, likely contribute to peripheral and cerebrovascular dysfunction in animals. However, whether T-cell mitochondrial respiration is related to cerebrovascular function in humans is not known. Therefore, we hypothesized that peripheral T-cell mitochondrial respiration would be positively associated with CVR and that T-cell glycolytic metabolism would be negatively associated with CVR. Twenty middle-aged adults (58 ± 5 yr) were recruited for this study. T cells were separated from peripheral blood mononuclear cells. Cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR, a marker of glycolytic activity) were measured using extracellular flux analysis. CVR was quantified using the breath-hold index (BHI), which reflects the change in blood velocity in the middle-cerebral artery (MCAv) during a 30-s breath-hold. In contrast to our hypothesis, we found that basal OCR in CD8+ T cells (β = -0.59, R2 = 0.27, P = 0.019) was negatively associated with BHI. However, in accordance with our hypothesis, we found that basal ECAR (β = -2.20, R2 = 0.29, P = 0.015) and maximum ECAR (β = -50, R2 = 0.24, P = 0.029) were negatively associated with BHI in CD8+ T cells. There were no associations observed in CD4+ T cells. These associations appeared to be primarily mediated by an association with the pressor response to the breath-hold test. Overall, our findings suggest that CD8+ T-cell respiration and glycolytic activity may influence CVR in humans.NEW & NOTEWORTHY Peripheral T-cell metabolism is related to in vivo cerebrovascular reactivity in humans. Higher glycolytic metabolism in CD8+ T cells was associated with lower cerebrovascular reactivity to a breath-hold in middle-aged adults, which is possibly reflective of a more proinflammatory state in midlife.

Role of AMP deaminase in diabetic cardiomyopathy.

Diabetes mellitus is one of the major causes of ischemic and nonischemic heart failure. While hypertension and coronary artery disease are frequent comorbidities in patients with diabetes, cardiac contractile dysfunction and remodeling occur in diabetic patients even without comorbidities, which is referred to as diabetic cardiomyopathy. Investigations in recent decades have demonstrated that the production of reactive oxygen species (ROS), impaired handling of intracellular Ca2+, and alterations in energy metabolism are involved in the development of diabetic cardiomyopathy. AMP deaminase (AMPD) directly regulates adenine nucleotide metabolism and energy transfer by adenylate kinase and indirectly modulates xanthine oxidoreductase-mediated pathways and AMP-activated protein kinase-mediated signaling. Upregulation of AMPD in diabetic hearts was first reported more than 30years ago, and subsequent studies showed similar upregulation in the liver and skeletal muscle. Evidence for the roles of AMPD in diabetes-induced fatty liver, sarcopenia, and heart failure has been accumulating. A series of our recent studies showed that AMPD localizes in the mitochondria-associated endoplasmic reticulum membrane as well as the sarcoplasmic reticulum and cytosol and participates in the regulation of mitochondrial Ca2+ and suggested that upregulated AMPD contributes to contractile dysfunction in diabetic cardiomyopathy via increased generation of ROS, adenine nucleotide depletion, and impaired mitochondrial respiration. The detrimental effects of AMPD were manifested at times of increased cardiac workload by pressure loading. In this review, we briefly summarize the expression and functions of AMPD in the heart and discuss the roles of AMPD in diabetic cardiomyopathy, mainly focusing on contractile dysfunction caused by this disorder.

Impaired mitochondrial respiration in upper compared to lower body differentiated human adipocytes and adipose tissue.

Abdominal obesity is associated with increased cardiometabolic disease risk, while lower body fat seems to confer protection against obesity-related complications. The functional differences between upper and lower body adipose tissue (AT) remain poorly understood. We aimed to examine whether mitochondrial respiration is impaired in abdominal as compared to femoral differentiated human multipotent adipose-derived stem cells (hMADS; primary outcome) and AT in postmenopausal women. In this cross-sectional study, 23 postmenopausal women with normal weight or obesity were recruited at the University of Birmingham/Queen Elizabeth Hospital Birmingham (Birmingham, UK). We collected abdominal and femoral subcutaneous AT biopsies to determine mitochondrial oxygen consumption rates in differentiated abdominal and femoral hMADS. Furthermore, we assessed OXPHOS protein expression and mtDNA content in abdominal and femoral AT as well as hMADS. Finally, we explored in vivo fractional oxygen extraction and carbon dioxide release across abdominal and femoral subcutaneous AT in a subgroup of the same individuals with normal weight or obesity. We found lower basal and maximal uncoupled mitochondrial oxygen consumption rates in abdominal compared to femoral hMADS. In line, in vivo fractional oxygen extraction and carbon dioxide release were lower across abdominal than femoral AT. OXPHOS protein expression and mtDNA content did not significantly differ between abdominal and femoral differentiated hMADS and AT. The present findings demonstrate that in vitro mitochondrial respiration and in vivo oxygen fractional extraction are lower in upper compared to lower body differentiated hMADS and AT, respectively, in postmenopausal women.

Cumulative Deleterious Effects of Tetrahydrocannabinoid (THC) and Ethanol on Mitochondrial Respiration and Reactive Oxygen Species Production Are Enhanced in Old Isolated Cardiac Mitochondria.

Delta 9 tetrahydrocannabinol (THC), the main component of cannabis, has adverse effects on the cardiovascular system, but whether concomitant ethanol (EtOH) and aging modulate its toxicity is unknown. We investigated dose responses of THC and its vehicle, EtOH, on mitochondrial respiration and reactive oxygen production in both young and old rat cardiac mitochondria (12 and 90 weeks). THC dose-dependently impaired mitochondrial respiration in both groups, and such impairment was enhanced in aged rats (-97.5 ± 1.4% vs. -75.6 ± 4.0% at 2 × 10-5 M, and IC50: 0.7 ± 0.05 vs. 1.3 ± 0.1 × 10-5 M, p < 0.01, for old and young rats, respectively). The EtOH-induced decrease in mitochondrial respiration was greater in old rats (-50.1 ± 2.4% vs. -19.8 ± 4.4% at 0.9 × 10-5 M, p < 0.0001). Further, mitochondrial hydrogen peroxide (H2O2) production was enhanced in old rats after THC injection (+46.6 ± 5.3 vs. + 17.9 ± 7.8%, p < 0.01, at 2 × 10-5 M). In conclusion, the deleterious cardiac effects of THC were enhanced with concomitant EtOH, particularly in old cardiac mitochondria, showing greater mitochondrial respiration impairment and ROS production. These data improve our knowledge of the mechanisms potentially involved in cannabis toxicity, and likely support additional caution when THC is used by elderly people who consume alcohol.

Homocysteine and mitochondrial quality control in diabetic retinopathy

BackgroundDiabetic retinopathy is a progressive disease, and one of the key metabolic abnormalities in the pathogenesis of diabetic retinopathy, mitochondrial damage, is also influenced by the duration of hyperglycemia. Mitochondrial quality control involves a coordination of mitochondrial dynamics, biogenesis and removal of the damaged mitochondria. In diabetes, these processes are impaired, and the damaged mitochondria continue to produce free radicals. Diabetic patients also have high homocysteine and reduced levels of hydrogen sulfide, and hyperhomocysteinemia is shown to exacerbate diabetes-induced mitochondrial damage and worsen their dynamics. This study aims to investigate the temporal relationship between hyperhomocysteinemia and retinal mitochondrial quality control in diabetic retinopathy.MethodsHuman retinal endothelial cells incubated in 20 mM d-glucose for 24 to 96 h, in the absence or presence of 100 µM homocysteine, with/without a hydrogen sulfide donor GYY4137, were analyzed for mitochondrial ROS (MitoSox fluorescence), DNA damage (transcripts of mtDNA-encoded ND6 and CytB), copy numbers, oxygen consumption rate (Seahorse XF analyzer) and mitophagy (mitophagosomes immunofluorescence labeling and flow cytometry). Results were confirmed in the retina from mice genetically manipulated for hyperhomocysteinemia (cystathionine β-synthase deficient mice, Cbs+/−), streptozotocin-induced diabetic for 8 to 24 weeks. At 24 weeks of diabetes, vascular health was evaluated by counting acellular capillaries in the trypsin digested retinal vasculature and by fluorescein angiography.ResultsHomocysteine, in high glucose medium, exacerbated mitochondrial ROS production, mtDNA damage and impaired mitochondrial respiration within 24 h, and slowed down/worsened mitochondrial biogenesis and mitophagy, as compared to 48 to 96 h in high glucose alone. GYY4137 supplementation ameliorated homocysteine + high glucose-induced mitochondrial damage and impairment in biogenesis and mitophagy. Similar results were obtained from Cbs+/− mice-mitochondrial ROS, mtDNA damage and decline in biogenesis and mitophagy were observed within eight weeks of diabetes vs. 16 to 24 weeks of diabetes in Cbs+/+ mice, and at 24 weeks of diabetes, Cbs+/− mice had significantly higher acellular capillaries and vascular leakage.ConclusionsHyperhomocysteinemia, in a hyperglycemic environment, overwhelms the mitochondria, accelerating and exacerbating their dysfunction, and also delays/worsens their removal, augmenting the development of diabetic retinopathy. Thus, our results strengthen the importance of maintaining homocysteine-hydrogen sulfide balance during the early stages of diabetes for a patient to prevent/retard vision loss.

Importance of Per2 in Cardiac Mitochondrial Function during Stress

Background and Hypothesis: Ischemic heart disease is the worldwide leading cause of death. Cardiac cellular damage from ischemia is mainly inflicted in the form of mitochondrial dysfunction by inflammatory cytokines and reactive oxidative species (ROS). Mitochondria are critical for metabolic function to maintain cardiac activity. Interventions against inflammatory cytokines and ROS are therefore cardioprotective during ischemic damage. Period Circadian Regulator 2 (Per2) is a circadian rhythm protein involved in metabolic regulation as a time-responsive gene in cardiomyocytes during ischemic damage. Overexpression of Per2 has been shown to decrease infarct size following myocardial infarction. In this study, we hypothesize that Per2 protein plays a regulatory role in the mitochondrial response to inflammatory cytokine TNFα and oxidative stressor H2O2 in human cardiomyocytes. &#x0D; Project Methods: AC16 Human Cardiomyocytes (HCM) transfected with Per2 or control siRNA were subjected to stress treatment of 100ng/mL TNFa or 100μM H2O2. RT-PCR and Western blot were used to detect Per2 expression. After two hours of treatment, mitochondrial membrane potential ([Equation]M) was detected using JC1 fluorescence probe and mitochondrial respiration capacity was evaluated via Seahorse Mito Stress Test. After four hours of treatment, cell death was measured using Annexin V and propidium iodide (PI) apoptosis kit via flow cytometry. &#x0D; Results: Per2 siRNA significantly reduced Per2 mRNA and protein levels in HCM. Increased cell death and decreased [Equation]M were observed in HCM treated with TNFa or H2O2. Knockdown of Per2 potentiated TNFa-induced cell death, TNFa- or H2O2 -disrupted [Equation]M, and TNFa- or H2O2- impaired mitochondrial maximal respiration. &#x0D; Conclusion and Potential Impact: Per2 knockdown increases apoptotic susceptibility and mitochondrial dysfunction in human cardiomyocytes exposed to TNFa or H2O2. Delivery of Per2 may serve as a promising therapeutic strategy to protect cardiomyocyte mitochondrial function during periods of stress, such as myocardial infarction, organ transplantation, and cardiac surgery.

Disruption of MAM integrity in mutant FUS oligodendroglial progenitors from hiPSCs

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder, characterized by selective loss of motor neurons (MNs). A number of causative genetic mutations underlie the disease, including mutations in the fused in sarcoma (FUS) gene, which can lead to both juvenile and late-onset ALS. Although ALS results from MN death, there is evidence that dysfunctional glial cells, including oligodendroglia, contribute to neurodegeneration. Here, we used human induced pluripotent stem cells (hiPSCs) with a R521H or a P525L mutation in FUS and their isogenic controls to generate oligodendrocyte progenitor cells (OPCs) by inducing SOX10 expression from a TET-On SOX10 cassette. Mutant and control iPSCs differentiated efficiently into OPCs. RNA sequencing identified a myelin sheath-related phenotype in mutant OPCs. Lipidomic studies demonstrated defects in myelin-related lipids, with a reduction of glycerophospholipids in mutant OPCs. Interestingly, FUSR521H OPCs displayed a decrease in the phosphatidylcholine/phosphatidylethanolamine ratio, known to be associated with maintaining membrane integrity. A proximity ligation assay further indicated that mitochondria-associated endoplasmic reticulum membranes (MAM) were diminished in both mutant FUS OPCs. Moreover, both mutant FUS OPCs displayed increased susceptibility to ER stress when exposed to thapsigargin, and exhibited impaired mitochondrial respiration and reduced Ca2+ signaling from ER Ca2+ stores. Taken together, these results demonstrate a pathological role of mutant FUS in OPCs, causing defects in lipid metabolism associated with MAM disruption manifested by impaired mitochondrial metabolism with increased susceptibility to ER stress and with suppressed physiological Ca2+ signaling. As such, further exploration of the role of oligodendrocyte dysfunction in the demise of MNs is crucial and will provide new insights into the complex cellular mechanisms underlying ALS.

VISTA promotes the metabolism and differentiation of myeloid-derived suppressor cells by STAT3 and polyamine-dependent mechanisms

Myeloid-derived suppressor cells (MDSCs) impair antitumor immune responses. Identifying regulatory circuits during MDSC development may bring new opportunities for therapeutic interventions. We report that the V-domain suppressor of Tcell activation (VISTA) functions as a key enabler of MDSC differentiation. VISTA deficiency reduced STAT3 activation and STAT3-dependent production of polyamines, which causally impaired mitochondrial respiration and MDSC expansion. In both mixed bone marrow (BM) chimera mice and myeloid-specific VISTA conditional knockout mice, VISTA deficiency significantly reduced tumor-associated MDSCs but expanded monocyte-derived dendritic cells (DCs) and enhanced Tcell-mediated tumor control. Correlated expression of VISTA and arginase-1 (ARG1), a key enzyme supporting polyamine biosynthesis, was observed in multiple human cancer types. In human endometrial cancer, co-expression of VISTA and ARG1 on tumor-associated myeloid cells is associated with poor survival. Taken together, these findings unveil the VISTA/polyamine axis as a central regulator of MDSC differentiation and warrant therapeutically targeting this axis for cancer immunotherapy.

FT895 Impairs Mitochondrial Function in Malignant Peripheral Nerve Sheath Tumor Cells.

Neurofibromatosis type 1 (NF1) stands as a prevalent neurocutaneous disorder. Approximately a quarter of NF1 patients experience the development of plexiform neurofibromas, potentially progressing into malignant peripheral nerve sheath tumors (MPNST). FT895, an HDAC11 inhibitor, exhibits potent anti-tumor effects on MPNST cells and enhances the cytotoxicity of cordycepin against MPNST. The study aims to investigate the molecular mechanism underlying FT895's efficacy against MPNST cells. Initially, our study unveiled that FT895 disrupts mitochondrial biogenesis and function. Post-FT895 treatment, reactive oxygen species (ROS) in MPNST notably increased, while mitochondrial DNA copy numbers decreased significantly. Seahorse analysis indicated a considerable decrease in basal, maximal, and ATP-production-coupled respiration following FT895 treatment. Immunostaining highlighted FT895's role in promoting mitochondrial aggregation without triggering mitophagy, possibly due to reduced levels of XBP1, Parkin, and PINK1 proteins. Moreover, the study using CHIP-qPCR analysis revealed a significant reduction in the copy numbers of promoters of the MPV17L2, POLG, TFAM, PINK1, and Parkin genes. The RNA-seq analysis underscored the prominent role of the HIF-1α signaling pathway post-FT895 treatment, aligning with the observed impairment in mitochondrial respiration. In summary, the study pioneers the revelation that FT895 induces mitochondrial respiratory damage in MPNST cells.

Pentagamavunone-1 inhibits aggressive breast cancer cell proliferation through mitotic catastrophe and ROS-mediated activities: in vitro and in vivo studies

Pentagamavunone-1 (PGV-1), an analog of curcumin, has been studied for its cytotoxic effects in 4T1, MCF7, MCF7/HER2, and T47D breast cancer cells. Its antiproliferative effect is partly mediated through G2/M arrest; however, its molecular mechanism during cell cycle progression remains unknown. In this study, we aimed to determine whether PGV-1 has any anticancer effects on highly aggressive breast cancer cells, with a focus on cell cycle regulatory activity, reactive oxygen species (ROS) generation, and their mediated effects on cancer cells. MDA-MB-231 (triple-negative) and HCC1954 (overexpressed HER2) immortalized human breast cancer cells were used in the study. PGV-1 exhibited cytotoxic activity with an irreversible antiproliferative impact on treated cells and had good selectivity when tested in fibroblast cells. Oral PGV-1 administration suppressed tumor growth in a cell-derived xenograft mouse model. PGV-1 induced the phosphorylation of Aurora A kinase and PLK1 in MDA-MB-231 cells, while PLK1 and cyclin B1 phosphorylation were enhanced in the PGV-1-treated HCC1954 cells during prometaphase arrest. Intracellular ROS production was substantially higher upon PGV-1 treatment following mitotic arrest, and this activity caused impairment of mitochondrial respiration, induced senescence, and subsequently triggered early-to-late apoptosis. Collectively, these results suggest that the molecular mechanism of PGV-1 involves the regulation of mitotic kinases to cause cell cycle arrest and the enhancement of ROS production to impair mitochondrial activity and induce cellular senescence. The therapeutic activities demonstrated by PGV-1 in this study show its potential as an appealing candidate for chemotherapy in breast cancer treatment.

The Atpase Domain of LONP1 Is Necessary for Mitochondrial Protein Solubility and the Viability of Acute Myeloid Leukemia (AML) Cells

Compared to normal hematopoietic cells, we and others have shown that AML cells have a heightened reliance on oxidative phosphorylation and mitochondrial metabolism for survival and proliferation. To support this unique metabolic phenotype, we previously demonstrated that AML cells have increased import of nuclear-encoded mitochondrial proteins. These newly imported proteins must be properly folded by mitochondrial chaperones, proteases, and heat shock proteins, and failure to properly process and fold these precursors leads to protein aggregation, mitochondrial dysfunction, and cell death. To evaluate the reliance of AML cells on this family of mitochondrial chaperones, proteases, and heat shock proteins, we assessed their dependencies using the gene dependency datasets (eg: depmap.org). From this analysis, we identified the mitochondrial protease, LONP1, as the top hit, and among the top 10% of all essential genes for AML. Localized to the mitochondrial matrix, LONP1 is a nuclear encoded AAA+ serine protease. Proteins are unfolded by its ATPase domain and degraded by its serine-catalyzed proteolytic domain. Compared to normal hematopoietic cells, LONP1 mRNA was overexpressed in AML across three publicly available datasets. Compared to normal hematopoietic cells (n=8) and CD34+ cells (n=3), LONP1 protein was increased in 16/30 primary AML samples by immunoblotting. Using shRNA and CRISPR, LONP1 knockdown and knockout reduced the growth and viability of OCI-AML2, OCI-M2, NB4, and TEX cells. Genetic knockdown or knockout of LONP1 increased levels of insoluble, aggregated mitochondrial proteins as measured by mass spectrometry, proteostat fluorescence and confocal microscopy, and immunoblotting of soluble and insoluble protein fractions. LONP1 knockdown/knockout also reduced mitochondrial respiration, depolarized the mitochondria, and induced AML cell death. We next determined the domain of LONP1 that was necessary for mitochondrial protein solubility, mitochondrial function, and AML survival. We over-expressed wild type, ATPase dead (E591A), or proteolytically dead (S855A) LONP1 cDNA in OCI-AML2 cells and knocked down endogenous LONP1 with shRNA targeting the 3'UTR of the endogenous gene. We then measured mitochondrial protein solubility/aggregation, mitochondrial respiration and cell viability. Wild type LONP1 and the proteolytically dead (S855A) mutant, but not the ATPase mutant (E591A), rescued mitochondrial protein solubility, mitochondrial respiration and cell viability, thus demonstrating that the ATPase domain of LONP1 is necessary for these functions. Bardoxolone methyl (CDDO-Me) is a synthetic triterpenoid that inhibits the ATPase activity of LONP1 by binding to an allosteric site near the ATPase domain of the enzyme. CDDO-Me killed OCI-AML2 and NB4 cells with IC 50 values of 178.5±29.7 and 156.5±39.7 nM, respectively. CDDO-Me (200nM) also killed &amp;gt;50% of cells in 3 out of 4 high-LONP1 expressing primary AML patient samples, but 10 of 10 tested primary AML samples with low levels of LONP1 were insensitive to the drug. Likewise, CDDO-Me induced mitochondrial protein aggregation in OCI-AML2 cells and in a primary AML patient sample with high LONP1 expression, while a primary AML patient sample with undetectable LONP1 expression showed no protein aggregation. In summary, the mitochondrial serine AAA+ protease LONP1 is over-expressed in a subset of AML cells and primary samples. The ATPase domain is necessary for LONP1's function in maintaining mitochondrial protein solubility. Selective inhibition of this domain leads to mitochondrial protein aggregation, impaired mitochondrial respiration and AML cell death. Thus, inhibiting the ATPase domain of LONP1 may be a novel therapeutic strategy for AML.

COX4I1 Controls Mitochondrial Electron Transport Chain Complex IV Assembly and Leukemia Progression in Acute Myeloid Leukemia

Aberrant cellular signaling plays a pivotal role in the pathogenesis of acute myeloid leukemia (AML). Identifying key signaling components and their dependencies is crucial for advancing AML therapy. Here, we conducted a focused CRISPR library screen targeting 427 cell signaling-related genes with 3,416 sgRNAs (8 sgRNAs per gene) in the Cas9-expressing Molm13 AML cells. In addition to the known leukemia essential genes (MYC, RPS6, CCND3, GAB2, and AURKB), we discovered COX4I1 (Cytochrome c oxidase subunit 4 isoform 1) as a novel essential gene for AML maintenance (Fig. 1). CRISPR-knockout of COX4I1 in AML cells led to inhibited cell proliferation, reduced colony formation, and attenuated leukemia progression in vivo. Transcriptomic profiling revealed elevated mitochondrial stress upon COX4I1 knockout, accompanied by dysregulated mitochondrial cristae structure observed through transmission electron microscopy. Functional assays demonstrated impaired mitochondrial respiration (oxygen consumption rate, i.e., OCR, measured by Seahorse XF) and depleted cellular ATP (measured by metabolomic mass spectrometry) upon COX4I1 knockout in AML cells, highlighting the pivotal role of COX4I1 in mitochondrial homeostasis and energy metabolism. COX4I1 is a nuclear-encoded mitochondrial protein located on the mitochondrial inner membrane. Using a high-density CRISPR gene tiling scan (Fig. 2; targeting density ~2.1 aa per sgRNA), we identified two critical COX4I1 peptide regions (P1: K67 - L73 and P2: E136 - K169) required for leukemia cell proliferation and mitochondrial respiration. Mechanistically, we found that expression of COX4I1 is essential for the assembly of mitochondrial complex IV, a crucial component of the electron transport chain involved in establishing the proton electrochemical potential across the mitochondrial inner membrane. Structural analysis revealed that facing the mitochondrial matrix, COX4I1's P1 region establishes the interface between COX4I1 and COX5A, which is necessary for the formation of the COX1 module. Conversely, COX4I1's P2 region (C-terminal 34 residues) is dispensable to the COX1 module formation; nonetheless, the P2 region co-localizes with the intermembrane space domains of the COX2/COX3 modules and is required for the assembling of an intact complex IV. Finally, we found that knockout of COX4I1 induced mitochondrial stress further triggers cellular ferroptosis in AML cells, which is accompanied by an imbalanced cellular redox index (reduced GSH/GSSG ratio) and elevated lipid peroxidation (increased malondialdehyde). These notions led us to discover the critical role of COX4I1 as an opportunity for targeted AML therapy, especially when combined with mitochondria-targeting therapeutics such as Venetoclax (i.e., ABT-199). In conclusion, our study highlights COX4I1 as a novel vulnerability in AML, controlling mitochondrial complex IV assembly and ATP biosynthesis. This finding holds promise for improving AML therapy and may be extended to identify therapeutically relevant signaling components in other hematopoietic malignancies.

Histidine Triad Nucleotide-Binding Protein 1 Improves Critical Limb Ischemia by Regulating Mitochondrial Homeostasis

Critical limb ischemia (CLI) is a common complication of diabetes mellitus that typically occurs in the later stages of the disease. Vascularization is indeed an important physiological process involving the formation of new blood vessels from existing ones. It occurs in response to various normal and pathophysiological conditions, and one of its critical roles is to compensate for inadequate oxygen supply, which is often seen in situations like chronic limb ischemia (CLI). Histidine triad nucleotide-binding protein 1 (Hint1) is a member of the Hint family that has been shown to attenuate cardiac hypertrophy, but its role in vascularization still needs to be clarified. In this study, we investigated the role of Hint1 in CLI. We found that Hint1 is significantly reduced in the muscle tissue of STZ-induced diabetic mice and high-glucose (HG)-treated endothelial cells (ECs). Hint1 deletion impaired blood flow recovery and vascularization, whereas Hint1 overexpression promoted these processes. In addition, our in vitro study showed that Hint1 deficiency aggravated mitochondrial dysfunction in ECs, as evidenced by impaired mitochondrial respiration, decreased mitochondrial membrane potential, and increased reactive oxygen species. Our findings suggest that Hint1 deficiency impairs blood perfusion by damaging mitochondrial function and that Hint1 may represent a potential therapeutic target for treating CLI.

The effect of ventricular microvascular dysfunction on cardiomyocyte excitation-contraction coupling and mitochondria

Abstract Introduction Microvascular dysfunction is a hallmark feature of cardiac allograft vasculopathy in HTx patients and associated with heart failure and coronary artery disease. On a cellular level, impaired mitochondrial respiration has been associated with the disease, however the exact mechanisms remain elusive. Objective The aim of this study was to determine the effect of microvascular dysfunction on functional and structural properties of cardiomyocytes isolated from ventricular biopsies of HTx patients. Methods We functionally assessed coronary artery microvasculature using guidewire based coronary flow reserve(CFR)/microvasculatory resistance (IMR) measurements (Abbott) in 13 post-HTx patients with angiographically normal coronary arteries or normal FFR. Ventricular (RV septum) myocardial biopsies were obtained for in-vitro measurements and single ventricular cardiomyocytes were isolated using enzymatic digestion. Cells were electrically stimulated (1 Hz) and subcellular Ca2+ signalling during excitation-contraction coupling was measured using confocal imaging (Fluo-4 AM). In a subset of cardiomyocytes mitochondrial density was quantified upon staining with Mitotracker Red FM (otsu thresholding). Results Microvascular dysfunction (MVD) measured by IMR was present in 6 of 13 patients with a mean IMR of 56±11 and 13±3 in MVD and CTRL, respectively. CFR did not differ between MVD and CTRL. Ca2+ transients during excitation-contraction coupling in isolated ventricular cardiomyocytes from a subset of patients showed unaltered amplitudes (1.2±0.01 vs. 1.3±0.05; a.u. F/F0). In addition, Ca2+ release (i.e. time to peak Ca2+) and Ca2+ removal (i.e. tau) were not significantly different between MVD and CTRL. However, mitochondrial density was significantly increased in MVD vs. CTRL, indicating subcellular changes associated with MVD. Conclusion In-vivo ventricular microvascular dysfunction is associated with preserved excitation-contraction coupling in-vitro, potentially owing to compensatory changes on the mitochondrial level.

Vitronectin promotes TTR deposition and mitochondrial damage in ATTR-CA mice

Abstract Background Transthyretin cardiac amyloidosis (ATTR-CA) is an increasingly recognized disorder that leads to heart failure and cardiovascular death. However, the pathogenesis of ATTR-CA is still poorly understood. Vitronectin (VTN), a common component of the extracellular matrix, has been reported to be associate with amyloidosis. However, the role of VTN in TTR deposition has not been clarified. Purpose Here we aimed to elucidate the TTR deposition mechanism in ATTR-CA and test therapeutic interventions using a gene-edited ATTR-CA mice model. Methods We used CRISPR/Cas9-mediated genome engineering to create humanized wild-type TTR knock-in mice (hTTRWT). Cardiac function was examined by echocardiography and pressure-volume analysis. VTN expression was assessed by immunofluorescence and western blotting. Interactions among VTN and TTR are analyzed with in vitro pull-down and co-immunoprecipitation (Co-IP) assays. Recombinant TTR protein was added in the culture of VTN overexpressed mice primary cardiomyocytes (CMs) to simulate the TTR deposition process in vitro. Mitochondrial function was determined by the Seahorse assay and mitochondrial ultrastructure was evaluated with the transmission electron microscope.Additionally, we assessed the mitochondrial damage with mitoSOX staining. Results In the hTTRWT group, pressure-volume analysis and echocardiography revealed significant diastolic dysfunction and increased left ventricle chamber stiffness at 52 weeks. Compared to WT group, the hTTRWT group showed a dramatic elevation of VTN in hearts. Co-IP and pull-down assays confirmed a direct interaction between VTN and TTR. In cultured CMs exposed to recombinant TTR protein, VTN overexpression promotes the deposition of TTR. Damaged mitochondrial ultrastructure, elevated oxidative stress and decreased mitochondrial numbers were found. In accordance with observed mitochondrial abnormalities, seahorse experiments demonstrated impaired mitochondrial respiration capacity. Conclusion These results suggest that the VTN contributes to ATTR-related pathological and cardiac function changes and mitochondrial dysfunction as a contributor of disease progression from ATTR-CA to heart failure.

Mitochondrial dysfunctions in T cells: focus on inflammatory bowel disease.

Mitochondria has emerged as a critical ruler of metabolic reprogramming in immune responses and inflammation. In the context of colitogenic T cells and IBD, there has been increasing research interest in the metabolic pathways of glycolysis, pyruvate oxidation, and glutaminolysis. These pathways have been shown to play a crucial role in the metabolic reprogramming of colitogenic T cells, leading to increased inflammatory cytokine production and tissue damage. In addition to metabolic reprogramming, mitochondrial dysfunction has also been implicated in the pathogenesis of IBD. Studies have shown that colitogenic T cells exhibit impaired mitochondrial respiration, elevated levels of mROS, alterations in calcium homeostasis, impaired mitochondrial biogenesis, and aberrant mitochondria-associated membrane formation. Here, we discuss our current knowledge of the metabolic reprogramming and mitochondrial dysfunctions in colitogenic T cells, as well as the potential therapeutic applications for treating IBD with evidence from animal experiments.