Mitochondrial OXPHOS Research Articles

Oxidative stress-mediated mitochondrial fission promotes hepatic stellate cell activation via stimulating oxidative phosphorylation

Previous studies have demonstrated dysregulated mitochondrial dynamics in fibrotic livers and hepatocytes. Little is currently known about how mitochondrial dynamics are involved, nor is it clear how mitochondrial dynamics participate in hepatic stellate cell (HSC) activation. In the present study, we investigated the role of mitochondrial dynamics in HSC activation and the underlying mechanisms. We verified that mitochondrial fission was enhanced in human and mouse fibrotic livers and active HSCs. Moreover, increased mitochondrial fission driven by fis1 overexpression could promote HSC activation. Inhibiting mitochondrial fission using mitochondrial fission inhibitor-1 (Mdivi-1) could inhibit activation and induce apoptosis of active HSCs, indicating that increased mitochondrial fission is essential for HSC activation. Mdivi-1 treatment also induced apoptosis in active HSCs in vivo and thus ameliorated CCl4-induced liver fibrosis. We also found that oxidative phosphorylation (OxPhos) was increased in active HSCs, and OxPhos inhibitors inhibited activation and induced apoptosis in active HSCs. Moreover, increasing mitochondrial fission upregulated OxPhos, while inhibiting mitochondrial fission downregulated OxPhos, suggesting that mitochondrial fission stimulates OxPhos during HSC activation. Next, we found that inhibition of oxidative stress using mitoquinone mesylate (mitoQ) and Tempol inhibited mitochondrial fission and OxPhos and induced apoptosis in active HSCs, suggesting that oxidative stress contributes to excessive mitochondrial fission during HSC activation. In conclusion, our study revealed that oxidative stress contributes to enhanced mitochondrial fission, which triggers OxPhos during HSC activation. Importantly, inhibiting mitochondrial fission has huge prospects for alleviating liver fibrosis by eliminating active HSCs.

Abstract P1036: Role Of Mitochondrial Ribosomal Protein L7/l12 (mrpl12) In Diabetic Ischemic Heart Disease

Myocardial infarction (MI) is a significant cause of death in diabetic patients. In addition, growing evidence suggests that mitochondrial dysfunction contributes to heart failure in diabetes. However, the molecular mechanisms of mitochondrial dysfunction mediating heart failure in diabetes are still poorly understood. The current study aimed to investigate the role of mitochondrial ribosomal protein L7/L12 (MRPL12) in mouse models of type II diabetes (db/db mice)and high-fat diet (HFD) mice with or without induction of MI and human hearts with or without diabetes (n=7) .Data analysis revealed an increase in MRPL12 levels in the LV tissue of HFD fed mice with MI than in LV tissues of low-fat diet-fed mice with MI, whereas MRPL12 levels remained unchanged in the db/db mice with MI. Intriguingly, we found increased MRPL12 levels in atrial appendage tissue of diabetic patients with ischemic heart disease compared to non-diabetic patients. We utilized human cardiomyocyte cell-line (AC-16) as surrogate models to delineate the mechanisms; surprisingly, adenovirus-mediated overexpression of MRPL12 with or without hyperglycemia in AC-16 cardiomyocytes does not affect mitochondrial OXPHOS . In addition, overexpression of MRPL12 had no effect on the mitochondrial ROS, mitochondrial membrane depolarization, and caspase activity in AC-16 cardiomyocytes. Whereas RNA interference (RNAi)-mediated MRPL12 silencing remarkedly reduced mitochondrial oxidative phosphorylation in AC-16 cells without any stress. In addition, knockdown of MRPL12 increased mitochondrial membrane depolarization mitochondrial ROS and reduced maximal respiratory capacity of mitochondria without any stress. Overall, our results provide new insights into the role of MRPL12 in the pathophysiology of MI in diabetes.

Mitochondria-targeted anti-oxidant AntiOxCIN4 improved liver steatosis in Western diet-fed mice by preventing lipid accumulation due to upregulation of fatty acid oxidation, quality control mechanism and antioxidant defense systems

Non-alcoholic fatty liver disease (NAFLD) is a health concern affecting 24% of the population worldwide. Although the pathophysiologic mechanisms underlying disease are not fully clarified, mitochondrial dysfunction and oxidative stress are key players in disease progression. Consequently, efforts to develop more efficient pharmacologic strategies targeting mitochondria for NAFLD prevention/treatment are underway. The conjugation of caffeic acid anti-oxidant moiety with an alkyl linker and a triphenylphosphonium cation (TPP+), guided by structure-activity relationships, led to the development of a mitochondria-targeted anti-oxidant (AntiOxCIN4) with remarkable anti-oxidant properties. Recently, we described that AntiOxCIN4 improved mitochondrial function, upregulated anti-oxidant defense systems, and cellular quality control mechanisms (mitophagy/autophagy) via activation of the Nrf2/Keap1 pathway, preventing fatty acid-induced cell damage. Despite the data obtained, AntiOxCIN4 effects on cellular and mitochondrial energy metabolism in vivo were not studied.In the present work, we proposed that AntiOxCIN4 (2.5 mg/day/animal) may prevent non-alcoholic fatty liver (NAFL) phenotype development in a C57BL/6J mice fed with 30% high-fat, 30% high-sucrose diet for 16 weeks. HepG2 cells treated with AntiOxCIN4 (100 μM, 48 h) before the exposure to supraphysiologic free fatty acids (FFAs) (250 μM, 24 h) were used for complementary studies. AntiOxCIN4 decreased body (by 43%), liver weight (by 39%), and plasma hepatocyte damage markers in WD-fed mice. Hepatic-related parameters associated with a reduction of fat liver accumulation (by 600%) and the remodeling of fatty acyl chain composition compared with the WD-fed group were improved. Data from human HepG2 cells confirmed that a reduction of lipid droplets size and number can be a result from AntiOxCIN4-induced stimulation of fatty acid oxidation and mitochondrial OXPHOS remodeling. In WD-fed mice, AntiOxCIN4 also induced a hepatic metabolism remodeling by upregulating mitochondrial OXPHOS, anti-oxidant defense system and phospholipid membrane composition, which is mediated by the PGC-1α-SIRT3 axis. AntiOxCIN4 prevented lipid accumulation-driven autophagic flux impairment, by increasing lysosomal proteolytic capacity.AntiOxCIN4 improved NAFL phenotype of WD-fed mice, via three main mechanisms: a) increase mitochondrial function (fatty acid oxidation); b) stimulation anti-oxidant defense system (enzymatic and non-enzymatic) and; c) prevent the impairment in autophagy. Together, the findings support the potential use of AntiOxCIN4 in the prevention/treatment of NAFLD.

Characterization of mitochondrial dysfunction due to laser damage by 2-photon FLIM microscopy

Mitochondria are the central organelles in cellular bio-energetics with key roles to play in energy metabolism and cell fate decisions. Fluorescence Lifetime Imaging microscopy (FLIM) is used to track metabolic changes by following the intrinsic co-enzymes NAD(P)H and FAD, present in metabolic pathways. FLIM records-lifetimes and the relative fractions of free (unbound) and bound states of NAD(P)H and FAD are achieved by multiphoton excitation of a pulsed femto-second infra-red laser. Optimization of multiphoton laser power levels is critical to achieve sufficient photon counts for correct lifetime fitting while avoiding phototoxic effects. We have characterized two photon (2p) laser induced changes at the intra-cellular level, specifically in the mitochondria, where damage was assessed at rising 2p laser average power excitation. Our results show that NAD(P)H-a2%—the lifetime-based enzyme bound fraction, an indicator of mitochondrial OXPHOS activity is increased by rising average power, while inducing changes in the mitochondria at higher power levels, quantified by different probes. Treatment response tracked by means of NAD(P)H-a2% can be confounded by laser-induced damage producing the same effect. Our study demonstrates that 2p-laser power optimization is critical by characterizing changes in the mitochondria at increasing laser average power.

P851: TARGETING GENE DEPENDENCIES IN MYC OVEREXPRESSING MULTIPLE MYELOMA

Background: Multiple myeloma (MM) is an incurable hematological malignancy characterized by a proliferation of clonal plasma cells in bone marrow. MM progresses from precursor stages, named monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM), to the symptomatic myeloma, MM. MYC abnormalities play a critical role in the disease progression. However, MYC is not therapeutically targetable due to its nuclear localization and the short half-life of the protein. Aims: To overcome this, we hypothesized that the proliferative advantage promoted by MYC overexpression induces differential genomic dependencies on other signaling pathways thus creates vulnerabilities that can be exploited therapeutically. Methods: We searched for genetic vulnerabilities associated with MYC overexpression by leveraging genome-scale pooled short hairpin RNA screening data for 236 cancer cell lines from Project Achilles. We generated an isogenic model of MYC overexpression (OE) in U266 cell line using EF1A-C-MYC lentiviral vector. Then, we performed RNA-seq, quantitative proteomics by Tandem Mass Tag mass spectrometry (TMT-MS) and a drug screening with ~2000 compounds. For validation, we performed pharmacological inhibition of glutamine catabolism as well as shRNA-mediated GLS1 knockdown. To determine the functional mechanisms, we used capillary electrophoresis-mass spectrometry (CE-MS) and Agilent Seahorse XF analyzer. Results: Achilles analysis revealed main dependencies associated with MYC overexpression on glutamine metabolism and specifically GLS1 (glutaminase) and SLC1A1 (glutamine transporter). Using a screening of 2000 small molecules, we further observed that inhibitors of NAD synthesis and mTORC1, which rely on the intracellular glutamine pool, had preferential effect on the proliferation of U266/MYC. Both RNA-seq and quantitative proteomics showed no significant upregulation of glutaminolysis related genes, suggesting a non-oncogenic dependency. Our validation tests confirmed this metabolic dependency, MYC OE cell lines failed to proliferate in the context of glutamine starvation and showed higher sensitivity to CB-839 and V-9302 inhibiting GLS1 and SLC1A5, respectively. Using the Seahorse analyzer, we measured the oxygen consumption rate (OCR) induced by glutamine. U266/MYC cells possess the ability to oxidize glutamine at a higher rate compared to U266/Ctrl. Additionally, higher sensitivity of U266/MYC to CB-839 was observed on both baseline and FCCP-induced OCR highlighting the role of glutamine in controlling mitochondrial OXPHOS in MYC OE cells. To understand the differential metabolic rewiring in MYC OE cells, we performed metabolomic analysis and observed higher GLS1 activity in U266/MYC presented by elevated glutamine to glutamate flux. We also identified the enriched metabolic pathways under GLS1 inhibition. Notably, CB-839 in U266/MYC results in more pronounced changes in TCA cycle and energy debt. This effect was blunted by the co-incubation with a-ketoglutarate, in a rescue experiment. Interestingly GLS1 inhibition was not limited to this effect, but extended to redox balance. CB839 significantly decreased glutathione level in U266/MYC. Furthermore, the intracellular concentrations of the glutamate-dependent amino acids were more depleted under GLS1 inhibition in U266/MYC compared to U266/Ctrl. Summary/Conclusion: Combining these observations, we were able to identify vulnerabilities to glutamine metabolism in MYC overexpressing cells. These results may lead to developing new therapeutic strategies to target MYC in clinical practice.

Targeting mitochondrial metabolism for metastatic cancer therapy.

Primary tumors evolve metabolic mechanisms favoring glycolysis for adenosine triphosphate(ATP) generation and antioxidant defenses. In contrast, metastatic cells frequently depend on mitochondrial respiration and oxidative phosphorylation (OxPhos). This reliance of metastatic cells on OxPhos can be exploited using drugs that target mitochondrial metabolism. Therefore, therapeutic agents that act via diverse mechanisms, including the activation of signaling pathways that promote the production of reactive oxygen species (ROS) and/or a reduction in antioxidant defenses may elevate oxidative stress and inhibit tumor cell survival. In this review, we will provide (1) a mechanistic analysis of function-selective extracellular signal-regulated kinase-1/2 (ERK1/2) inhibitors that inhibit cancer cells through enhanced ROS, (2) a review of the role of mitochondrial ATP synthase in redox regulation and drug resistance, (3) a rationale for inhibiting ERK signaling and mitochondrial OxPhos towardthe therapeutic goal of reducing tumor metastasis and treatment resistance. Recent reports from our laboratories using metastatic melanoma and breast cancer models have shown the preclinical efficacy of novel and rationally designed therapeutic agents that target ERK1/2 signaling and mitochondrial ATP synthase, which modulate ROS events that may prevent or treat metastatic cancer. These findings and those of others suggest that targeting a tumor's metabolic requirements and vulnerabilities may inhibit metastatic pathways and tumor growth. Approaches that exploit the ability of therapeutic agents to alter oxidative balance in tumor cells may be selective for cancer cells and may ultimately have an impact on clinical efficacy and safety. Elucidating the translational potential of metabolic targeting could lead to the discovery of new approaches for treatment of metastatic cancer.

Abstract 2386: Investigating cyclopamine tartrate as a potential suppressor of lung adenocarcinoma and squamous cell carcinoma

Abstract Lung cancer is the leading cause of death by cancer, and non-small cell lung cancer comprises of 85-90% of lung cancer cases. Previous studies have demonstrated the importance of heme in mitochondrial metabolism and OXPHOS. Heme is an essential molecule in the oxidative phosphorylation pathway especially since Complex II-IV all contain heme. During lung tumorigenesis there is a marked increase in heme synthesis and uptake; other protein and enzymes relating to heme were also upregulated. Heme-sequestration proteins (HeSPs) have shown high effectiveness in suppressing the development and progression of lung adenocarcinoma and squamous cell carcinoma, therefore targeting heme using CycT should yield similar results. Previous studies have indicated that cyclopamine tartrate (CycT) can effectively arrest lung tumor growth and progression. CycT does this by inhibiting the levels of proteins and enzymes involved in heme uptake, synthesis, as well as reducing heme synthesis and degradation in adenocarcinoma. We therefore aim to examine the effectiveness of CycT in both, adenocarcinoma and squamous cell carcinoma.In vitro experiments using NSCLC adenocarcinoma cell line A549 and squamous cell carcinoma HCC15 are in progression with initial results supporting the hypothesis. Cell proliferation assays have shown a marked decrease in CycT-treated groups in both cell lines. Based on colony formation assays, results indicate that CycT inhibits colony formation. Heme synthesis and degradation have also shown the effectiveness of CycT in suppressing tumorigenesis in both cell types. Oxygen consumption rates of both cell types were examined with CycT inhibiting oxygen consumption. Overall, in vitro experiments support the idea that CycT can suppress or arrest lung adenocarcinoma and squamous cell carcinoma. Citation Format: Eranda Berisha, Adnin Ashrafi, Maria Del Carmen Chacon Castro, Li Zhang. Investigating cyclopamine tartrate as a potential suppressor of lung adenocarcinoma and squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2386.

Abstract 4183: Differential modulation of tumor versus T cell oxidative phosphorylation potentiates anti-tumor immunity

Abstract We sought to determine whether various inhibitors of Oxidative Phosphorylation (OxPhos) could restore tumor infiltrating T cell functionality and longevity while compromising metabolic fitness of tumor cells. Using a combination of in vitro metabolic profiling with the Seahorse XFe96 Analyzer and in vivo tumor immunotherapy in the immunocompetent TRAMP-C2 prostate adenocarcinoma model, we determined the differential impact of multiple OxPhos inhibitors on both tumor immune metabolic fitness and functional capacity. Impact on the tumor immune microenvironment of TRAMP-C2 tumors was assessed through high parameter flow cytometry on a BD X-30. While OxPhos inhibition compromised initial T cell activation, we found that two of the inhibitors increased the glucose uptake, proliferation, and activation status of effector T cells. In contrast, the same two inhibitors decreased the mitochondrial fitness, proliferation and OxPhos metabolic capacity of TRAMP-C2 tumor cells themselves. To determine whether this selective inhibition of tumor versus T cell metabolic function could improve immunotherapy responses in vivo, we implanted TRAMP-C2 tumors and treated them with these OxPhos inhibitors with or without concomitant immune checkpoint blockade (ICB). The combination of ICB and OxPhos inhibition achieved highly significant and durable rates of tumor control and regression suggesting that these approaches are potentially synergistic. Analysis of the tumor microenvironment has identified the cellular mechanisms underlying these therapeutic effects which may provide useful biomarkers as this novel combination is translated to the clinic. Citation Format: Krithikaa Rajkumar Bhanu, Priyamvada Jayaprakash, Meghan Rice, Brittany Morrow, Joseph R. Marszalek, Jason P. Gay, Christopher P. Vellano, Benjamin R. Cowen, Dean J. Welsh, Michael A. Curran. Differential modulation of tumor versus T cell oxidative phosphorylation potentiates anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4183.

Abstract 3771: Mitochondrial metabolism and racial disparity of bladder cancer

Abstract Potential differences and mechanism in metabolism among bladder cancer (BLCA) patients of diverse race or ethnicities remain largely unexplored. Even though the incidence rate of BLCA in African Americans (AA) is nearly half as that of European Americans (EA), but AA have the worst survival. We performed the transcriptomics and metabolomics profiling in ancestry verified patients from AA and EA BLCA and observed mitochondrial complex activities were uniquely enriched in AA tumors compared to EA tumors. In addition, in vitro assay demonstrated differences in mitochondrial complex protein and activity between AA and EA BLCA. We further confirmed the reprogramming of mitochondrial metabolism using in vitro 13C labeled tracers in both AA and EA BLCA. Integration of metabolomics and transcriptomics data reveals the enrichment of mTOR pathway in AA BLCA. Our findings indicate that an elevated mitochondrial oxphos activity through mTOR activation could be a factor for AA BLCA progression and provide the rationale to examine mitochondrial specific inhibitors along with mTOR inhibitors to target BLCA on subset of patients from the AA community. Citation Format: Karthik Reddy Kami Reddy, Jun Hyoung Park, Roni J. Bollag, Allison Bellman, Martha Terris, Seth P. Lerner, Leomar Y. Ballester, Yair Lotan, Benny Abraham Kaipparettu, Nagireddy Putluri. Mitochondrial metabolism and racial disparity of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3771.

Characterization of the mitochondrial GlutamyltRNAGln amidotransferase (GatCAB) as a new model for mitochondrial translation disorders.

Mitochondrial OXPHOS diseases are provoked by dysfunction of the OXPHOS system, showing heterogeneity from a genetic, biochemical and clinical perspective. This complexity is partly due to the involvement of proteins encoded in two genomes located in different cellular compartments, nuclear and mitochondrial DNA (mtDNA). mtDNA encodes part of the mtRNAs translation machinery, including 22 tRNAs. All these are charged by their corresponding aminoacyl-tRNA synthetases (ARS2), except mt-Gln-tRNAGln whose synthesis is carried out through an indirect pathway. The mt-tRNAGln is charged with glutamic acid (Glu) by a non-discriminating mitochondrial glutamyl-tRNA synthetase (EARS2), then the Glu is converted to Gln by a Glutamyl-tRNAGln amidotransferase, using free glutamine as an amide donor, yielding Gln- tRNAGln. This mitochondrial amidotransferase activity lies in the GatCAB complex, which is formed by three subunits: GatA (QSRL1), GatB (GATB), and GatC (GATC). Patients with mutations in these genes presented with severe cardiomyopathy and lactic acidosis, which underscores the importance of the GatCAB complex as an essential component in the translational machinery of mitochondrial protein synthesis. We have generated Knockout (KO) lines for all subunits using the genomic editing system CRISPR/Cas9 in HEK293T cells. We measured the levels of the GatCAB subunits and mitochondrial proteins by western blot. We studied the oxygen consumption levels using a Clark electrode and detected the GatCAB complex by Blue Native-western blotting. In all cases, KO cells show reduced levels of mitochondrial proteins, decreased oxygen consumption and an absence of the GatCAB complex compared to wildtype cells. In the QRSL1 and GATC KO cells the levels of all subunits diminish, but in GATB KO cells the levels of subunits A and C remain stable. Possibly, QRSL1 and GATC could form a stable but nonfunctional dimer, to which GATB would join. GATB has two potential ATG codons at the start of its sequence separated by 12 nucleotides. From over 90 clones analyzed, we only obtained one KO, by generating an upstream ORF, and two cell lines with very low expression of GATB. The latter present a duplicated region at the beginning of GATB which changes the reading frame from the first ATG; suggesting the downstream ATG as an alternative translation start codon for those ribosome small subunits that do not assemble on the first one, yielding a GATB protein missing 4 amino acids. Characterization of the molecular mechanisms that lead to the synthesis of mt-Gln-tRNAGln and its role in the physiology of the cell could allow us to comprehend the pathological manifestation of the defects in these genes and propose possible therapeutic avenues.

Defining mitochondrial protein functions through deep multiomic profiling.

Mitochondria are epicentres of eukaryotic metabolism and bioenergetics. Pioneering efforts in recent decades have established the core protein componentry of these organelles1 and have linked their dysfunction to more than 150 distinct disorders2,3. Still, hundreds of mitochondrial proteins lack clear functions4, and the underlying genetic basis for approximately 40% of mitochondrial disorders remains unresolved5. Here, to establish a more complete functional compendium of human mitochondrial proteins, we profiled more than 200 CRISPR-mediated HAP1 cell knockout lines using mass spectrometry-based multiomics analyses. This effort generated approximately 8.3 million distinct biomolecule measurements, providing a deep survey of the cellular responses to mitochondrial perturbations and laying a foundation for mechanistic investigations into protein function. Guided by these data, we discovered that PIGY upstreamopen reading frame (PYURF) is an S-adenosylmethionine-dependent methyltransferase chaperone that supports both complex I assembly and coenzyme Q biosynthesis and is disrupted in a previously unresolved multisystemic mitochondrial disorder. We further linked the putative zinc transporter SLC30A9 to mitochondrial ribosomes and OxPhos integrity and established RAB5IF as the second gene harbouring pathogenic variants that cause cerebrofaciothoracic dysplasia. Our data, which can be explored through the interactive online MITOMICS.app resource, suggest biological roles for many other orphan mitochondrial proteins that still lack robust functional characterization and define a rich cell signature of mitochondrial dysfunction that can support the genetic diagnosis of mitochondrial diseases.

Abstract PR009: Investigating dynamics of the mitochondrial network in triple negative breast cancer chemotherapy resistance

Abstract Triple negative breast cancer (TNBC) is an aggressive disease with extremely limited targeted therapeutic options. Thus, standard cytotoxic chemotherapies (typically sequential and/or combined anthracyclines, taxanes, and/or platinums) remain a mainstay treatment for this subtype of breast cancer. Nearly 50% of TNBC patients harbor substantial residual cancer burden following standard chemotherapy treatment, leading to high rates of recurrence and death (PMID:28135148). Using longitudinal biopsies from orthotopic patient-derived xenograft (PDX) models and TNBC patients, we found that residual tumors following chemotherapy transitioned to a unique metabolic state characterized by high mitochondrial oxidative phosphorylation (oxphos). This metabolic state was transient, with tumors reverting to their baseline glycolysis-high phenotype when they were allowed to regrow in the absence of treatment. Using genomic sequencing and cellular barcode-mediated clonal tracking, we found this mechanism of chemoresistance arose in the absence of clonal selection, suggesting chemotherapy induced plastic (i.e., non-genomic) programs enabling cell survival following treatment. Oxphos inhibition using a specific inhibitor of electron transport chain Complex I (IACS010759; PMID:29892070) was significantly more efficacious against residual, rather than treatment-naive tumors (PMID:30996079), providing evidence that dynamic metabolic phenotypes represent targetable therapeutic vulnerabilities for TNBC. Mechanisms controlling mitochondrial bioenergetics are numerous but their roles in driving metabolic phenotypes of chemoresistant TNBC are not understood. We find that residual tumor cells following chemotherapy treatment have altered mitochondrial network morphology. Mitochondrial fission and fusion are well established regulators of mitochondrial morphology, yet their functional impacts on bioenergetic output of mitochondria is highly context dependent. Our analyses of mitochondrial structure in TNBC cells and PDX tumors reveal that while DNA-damaging chemotherapeutics (anthracyclines, platinums) increased mitochondrial elongation, microtubule-stabilizing chemotherapeutics (taxanes) increased mitochondrial fragmentation. This was accompanied by increased or decreased oxphos rates and mitochondrial content, respectively. Interestingly, these structural changes were reverted in PDX tumors that were allowed to regrow in the absence of treatment, suggesting mitochondrial adaptations are plastic in vivo. Using pharmacologic agents in TNBC cells, we found that mitochondrial fusion increased oxphos and chemoresistance, whereas mitochondrial fission decreased oxphos and chemoresistance. Further, knockdown of OPA1 or MFN2, mediators of mitochondrial inner and outer membrane fusion, respectively, diminished mitochondrial fusion, oxphos, and chemoresistance. Based on these findings, we hypothesize therapeutic targeting of mediators of mitochondrial network adaptability may be a promising strategy to overcome plastic metabolic states indued by chemotherapeutics in TNBC. Citation Format: Lily Baek, Mariah Berner, Junegoo Lee, Katherine Pendleton, Karen Wang, Emily B. Goff, James P. Barrish, Bora Lim, Michael T. Lewis, Philip L. Lorenzi, Weston Porter, Gloria V. Echeverria. Investigating dynamics of the mitochondrial network in triple negative breast cancer chemotherapy resistance [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR009.

Impaired Myocardial Mitochondrial Function in an Experimental Model of Anaphylactic Shock.

Simple SummaryAnaphylactic shock (AS) is the most severe allergic hypersensitivity reaction. In the early stage of shock, AS was associated with hemodynamic impairments: severe failure in arterial blood pressure and cardiac dysfunction. Mitochondrial disorders are associated with a high incidence of acute or chronic cardiac dysfunctions. The aim of the study was to evaluate mitochondrial involvement in cardiac dysfunction at the early stage of AS. In control (CON) and sensitized Brown Norway rats, anaphylactic shock (AS) was induced with ovalbumin iv bolus injection. Aortic abdominal blood flow (ABF), mean blood arterial pressure (MAP), and lactatemia were assessed during 15 min. The myocardial mitochondrial defect in AS was assessed by mitochondrial respiration, oxidative stress production, total superoxide dismutase activity (SODs), and oxidative damage. An ultrastructural evaluation of myocardial mitochondria was also performed using electronic microscopy. AS was associated with a rapid and dramatic decrease in MAP, ABF, and severe hyperlactatemia 15 min after AS induction. Non-phosphorylating mitochondrial respiration and OXPHOS states involving mitochondrial complex II were significantly impaired in the AS group. Oxidative stress was observed with a significant increase in SODs activity after AS induction. Moreover, in the AS group we observed an increase in oxidative damage via lipid peroxidation. No modifications of the mitochondrial ultrastructure were shown at the early stage of AS. In this experimental model, AS was associated with a rapid and significant myocardial mitochondrial dysfunction with oxidative damage that could explain cardiac anaphylaxis dysfunction.Anaphylactic shock (AS) is associated with a profound vasodilation and cardiac dysfunction. The cellular mechanisms underlying AS-related cardiac dysfunction are unknown. We hypothesized that myocardial mitochondrial dysfunction may be associated with AS cardiac dysfunction. In controls and sensitized Brown Norway rats, shock was induced by ovalbumin i.v bolus, and abdominal aortic blood flow (ABF), systemic mean arterial pressure (MAP), and lactatemia were measured for 15 min. Myocardial mitochondrial function was assessed with the evaluation of mitochondrial respiration, oxidative stress production by reactive oxygen species (ROS), reactive nitrogen species (RNS), and the measurement of superoxide dismutases (SODs) activity. Oxidative damage was assessed by lipid peroxidation. The mitochondrial ultrastructure was assessed using transmission electronic microscopy. AS was associated with a dramatic drop in ABF and MAP combined with a severe hyperlactatemia 15 min after shock induction. CI-linked substrate state (197 ± 21 vs. 144 ± 21 pmol/s/mg, p < 0.05), OXPHOS activity by complexes I and II (411 ± 47 vs. 246 ± 33 pmol/s/mg, p < 0.05), and OXPHOS activity through complex II (316 ± 40 vs. 203 ± 28 pmol/s/mg, p < 0.05) were significantly impaired. ROS and RNS production was not significantly increased, but SODs activity was significantly higher in the AS group (11.15 ± 1.02 vs. 15.50 ± 1.40 U/mL/mg protein, p = 0.02). Finally, cardiac lipid peroxidation was significantly increased in the AS group (8.50 ± 0.67 vs. 12.17 ± 1.44 µM/mg protein, p < 0.05). No obvious changes were observed in the mitochondrial ultrastructure between CON and AS groups. Our experimental model of AS results in rapid and deleterious hemodynamic effects and was associated with a myocardial mitochondrial dysfunction with oxidative damage and without mitochondrial ultrastructural injury.

Combinatorial GxGxE CRISPR screen identifies SLC25A39 in mitochondrial glutathione transport linking iron homeostasis to OXPHOS.

The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes. The family is highly redundant and their transport activities coupled to metabolic state. Here, we use a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states — glucose, galactose, OXPHOS inhibition, and absence of pyruvate — designed to unmask the inter-dependence of these genes. In total, we screen 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recover 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrates that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells is genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. GxG analysis highlights a buffering interaction between the iron transporter SLC25A37 (A37) and the poorly characterized SLC25A39 (A39). Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis identify A39 as critical for mitochondrial glutathione (GSH) import. Functional studies reveal that A39-mediated glutathione homeostasis and A37-mediated mitochondrial iron uptake operate jointly to support mitochondrial OXPHOS. Our work underscores the value of studying family-wide genetic interactions across different metabolic environments.

Substrate‐Dependent Differential Regulation of Mitochondrial Bioenergetics and ROS emission in the Heart and Kidney Cortex and Outer Medulla

RationaleThe kinetics and efficiency of mitochondrial oxidative phosphorylation (OxPhos) is determined by the availability and utilization of particular respiratory substrates. Although the heart and kidney represent the two major energy consuming organs in the body, the substrate dependency of their mitochondrial OxPhos has not been characterized for these two organs within the same species and same individual animal.MethodThe effects of different substrate combinations on the kinetics and efficiency of OxPhos were characterized in mitochondria isolated from the heart, renal cortex, and renal outer medulla (OM) of adult Sprague‐Dawley rats. The substrate combinations included pyruvate+malate (PM), glutamate+malate (GM), palmitoyl‐carnitine+malate (PCM), alpha‐ketoglutarate+malate (AM), and succinate±rotenone (Suc±Rot). Mitochondrial bioenergetics (O2 consumption and membrane potential) and H2O2 emission were studied using two protocols: addition of a fixed [ADP] and sequential additions of incremental [ADP] in the presence of different substrates to determine how these substrates influence mitochondrial functional markers in each of these tissues.ResultsResults show that the kinetics and efficiency of mitochondrial OxPhos are highly dependent on the substrates used and this dependency is tissue‐specific. Heart mitochondria showed higher respiratory rates and OxPhos efficiencies for all substrates used in comparison to kidney mitochondria. Renal cortex mitochondrial respiratory rates were higher than OM mitochondria, but OM mitochondria OxPhos efficiencies were higher than cortex mitochondria. It was also found that heart mitochondria do not synthesize ATP efficiently from ADP with Suc in the absence of Rot (blocker of complex I). This may be due to (i) accumulation of oxaloacetate (OAA) and/or (ii) occurrence of reverse electron transfer (RET). In contrast, cortex and OM mitochondria achieved the same respiratory rates with Suc±Rot suggesting that efficient conversion of ADP to ATP by kidney mitochondria can occur in the presence of Suc. Similar differences were observed in mitochondrial membrane potential. However, differences in H2O2 emission in the presence of Suc±Rot were observed in heart mitochondria and to a lesser extent in OM mitochondria, but not in cortex mitochondria. The bioenergetics and H2O2 emission data observed with Suc±Rot indicate that OAA accumulation and RET play a more prominent regulatory role in heart mitochondria than in kidney mitochondria.ConclusionThis study provided novel quantitative data demonstrating that the choice of respiratory substrates affects mitochondrial bioenergetics and H2O2 emission in a tissue‐specific manner.

Mitochondrial GRK2 is a Novel Regulator of Cardiac Energetics

G protein‐coupled receptor (GPCR) kinase 2 (GRK2) is highly expressed in the heart, where during injury or heart failure (HF), both its levels and activity increase. GRK2 is canonically studied in the context of GPCR phosphorylation; however, noncanonical activities of GRK2 have emerged and it is now appreciated that GRK2 has a large non‐GPCR interactome. For example, in cardiac myocytes, GRK2 translocates from the cytosol to mitochondria (mtGRK2) following oxidative stress or ischemia injury, and this pool of mtGRK2 is associated with negative effects on metabolism and also induces myocyte cell death. However, the mechanisms by which mtGRK2 contributes to cardiac dysfunction and HF are not fully understood. We hypothesized that mtGRK2 could have novel substrates and phosphorylate proteins involved in mitochondrial bioenergetics, thus contributing to our previously established post‐injury phenotype. Stress‐induced mitochondrial translocation of cytosolic GRK2 was validated in cell and animal models and the mtGRK2 interactome was identified using liquid chromatography‐mass spectroscopy (LCMS). Proteomics analysis identified mtGRK2 interacting proteins which were involved in mitochondrial dysfunction, bioenergetics, and OXPHOS, particularly complexes I, II, IV and V of the electron transport chain (ETC). Specifically, mtGRK2 interactions with Complex V (ATP synthase) subunits were particularly increased following stress. We established that mtGRK2 phosphorylates ATP synthase on the F1 catalytic barrel, which is critical for oxidative phosphorylation and ATP production. We have also determined that alterations in either the levels or activity of GRK2 appear to alter ATP synthase enzymatic activity in vitro. Excitingly, in vivo data suggest that reducing levels of GRK2 in a mouse model of myocardial infarction prevents the post‐injury reduction in ATP synthesis. We are currently assessing the ability of the SSRI drug paroxetine, a GRK2 inhibitor, to preserve mitochondrial bioenergetics in a transgenic GRK2 mouse model. Thus, phosphorylation of the ATP synthesis machinery by mtGRK2 may contribute to the impaired mitochondrial phenotype observed in injured or failing heartssuch as reduced fatty acid metabolism and substrate utilization. These data uncover a druggable, novel target for rescuing cardiac function in patients with injured and/or failing hearts.

Metabolic reprogramming by ex vivo glutamine inhibition endows CAR-T cells with less-differentiated phenotype and persistent antitumor activity

The inadequate in vivo persistence of chimeric antigen receptor (CAR)-modified T cells has been shown to lead to poor therapeutic efficacy and disease recurrence. In vivo persistence is associated with the differentiation subsets infused, with less differentiated TN or TCM conferring superior renewal capacity and antitumor immunity compared to TEM or TEFF. However, ex vivo expanded CAR-T cells exhibit phenotypic heterogeneity with majority of TEM or TEFF subsets and very low populations of TN and TCM. The transition of differentiation subsets is closely correlated with T cell metabolism fitness. Effector T cell differentiation from TN or TCM requires glutamine uptake and metabolism. Using a CD19-specific CAR, we demonstrated that glutamine inhibition by adding the glutamine antagonist 6-Diazo-5-oxo-l-norleucine (DON) into the culture endows CAR-T cells with enhanced mitochondrial OXPHOS utilizing fatty acids and reduced glycolytic activity, and retains more TN or TCM subsets. DON- pretreated CAR-T cells exhibited stronger cytotoxic lysis in vitro and more robust elimination of tumor burdens in vivo. This study suggests that glutamine inhibition ex vivo would be a potential approach for modulating metabolism and differentiation state to improve the efficacy of CAR-T cell therapy.

Skeletal muscle mitoribosomal defects are linked to low bone mass caused by bone marrow inflammation in male mice.

BackgroundMitochondrial oxidative phosphorylation (OxPhos) is a critical regulator of skeletal muscle mass and function. Although muscle atrophy due to mitochondrial dysfunction is closely associated with bone loss, the biological characteristics of the relationship between muscle and bone remain obscure. We showed that muscle atrophy caused by skeletal muscle‐specific CR6‐interacting factor 1 knockout (MKO) modulates the bone marrow (BM) inflammatory response, leading to low bone mass.MethodsMKO mice with lower muscle OxPhos were fed a normal chow or high‐fat diet and then evaluated for muscle mass and function, and bone mineral density. Immunophenotyping of BM immune cells was also performed. BM transcriptomic analysis was used to identify key factors regulating bone mass in MKO mice. To determine the effects of BM‐derived CXCL12 (C–X–C motif chemokine ligand 12) on regulation of bone homeostasis, a variety of BM niche‐resident cells were treated with recombinant CXCL12. Vastus lateralis muscle and BM immune cell samples from 14 patients with hip fracture were investigated to examine the association between muscle function and BM inflammation.ResultsMKO mice exhibited significant reductions in both muscle mass and expression of OxPhos subunits but increased transcription of mitochondrial stress response‐related genes in the extensor digitorum longus (P < 0.01). MKO mice showed a decline in grip strength and a higher drop rate in the wire hanging test (P < 0.01). Micro‐computed tomography and von Kossa staining revealed that MKO mice developed a low mass phenotype in cortical and trabecular bone (P < 0.01). Transcriptomic analysis of the BM revealed that mitochondrial stress responses in skeletal muscles induce an inflammatory response and adipogenesis in the BM and that the CXCL12–CXCR4 (C–X–C chemokine receptor 4) axis is important for T‐cell homing to the BM. Antagonism of CXCR4 attenuated BM inflammation and increased bone mass in MKO mice. In humans, patients with low body mass index (BMI = 17.2 ± 0.42 kg/m2) harboured a larger population of proinflammatory and cytotoxic senescent T‐cells in the BMI (P < 0.05) and showed reduced expression of OxPhos subunits in the vastus lateralis, compared with controls with a normal BMI (23.7 ± 0.88 kg/m2) (P < 0.01).ConclusionsDefects in muscle mitochondrial OxPhos promote BM inflammation in mice, leading to decreased bone mass. Muscle mitochondrial dysfunction is linked to BM inflammatory cytokine secretion via the CXCL12–CXCR4 signalling axis, which is critical for inducing low bone mass.

Effects of Mitochondrial Dynamics in the Pathophysiology of Obesity.

Obesity has become an urgent and serious public health challenge with an overwhelming increase over the decades worldwide. The rate of obese children and adolescents has recently accelerated, especially in China. Obesity is closely related to unbalanced cellular energy metabolism. Mitochondria, as the main organelles of energy metabolism, play an important role in the pathophysiology of obesity. Recent researches have revealed that mitochondrial dynamics with constant fission and fusion, can alter mitochondrial structure, organelle connections, ROS production, neuronal activity, and OXPHOS system as well as adipose tissue thermogenesis, which ultimately lead to obesity. In this review, we will update the latest findings about mitochondrial fission/fusion related GTPase proteins and discuss the effects of mitochondrial dynamics in the pathophysiology of obesity.

Antitumor Activity of a Mitochondrial-Targeted HSP90 Inhibitor in Gliomas.

To investigate the antitumor activity of a mitochondrial-localized HSP90 inhibitor, Gamitrinib, in multiple glioma models, and to elucidate the antitumor mechanisms of Gamitrinib in gliomas. A broad panel of primary and temozolomide (TMZ)-resistant human glioma cell lines were screened by cell viability assays, flow cytometry, and crystal violet assays to investigate the therapeutic efficacy of Gamitrinib. Seahorse assays were used to measure the mitochondrial respiration of glioma cells. Integrated analyses of RNA sequencing (RNAseq) and reverse phase protein array (RPPA) data were performed to reveal the potential antitumor mechanisms of Gamitrinib. Neurospheres, patient-derived organoids (PDO), cell line-derived xenografts (CDX), and patient-derived xenografts (PDX) models were generated to further evaluate the therapeutic efficacy of Gamitrinib. Gamitrinib inhibited cell proliferation and induced cell apoptosis and death in 17 primary glioma cell lines, 6 TMZ-resistant glioma cell lines, 4 neurospheres, and 3 PDOs. Importantly, Gamitrinib significantly delayed the tumor growth and improved survival of mice in both CDX and PDX models in which tumors were either subcutaneously or intracranially implanted. Integrated computational analyses of RNAseq and RPPA data revealed that Gamitrinib exhibited its antitumor activity via (i) suppressing mitochondrial biogenesis, OXPHOS, and cell-cycle progression and (ii) activating the energy-sensing AMP-activated kinase, DNA damage, and stress response. These preclinical findings established the therapeutic role of Gamitrinib in gliomas and revealed the inhibition of mitochondrial biogenesis and tumor bioenergetics as the primary antitumor mechanisms in gliomas.