Activities Of Electron Transport Chain Complexes Research Articles

Alterations of Myocardial Mitochondrial Morphology and Function in a Canine Model of Premature Ventricular Contractions-Induced Cardiomyopathy.

Changes in myocardial mitochondrial morphology and function in premature ventricular contractions (PVCs)-induced cardiomyopathy (PVCCM) remain poorly studied. Here, we investigated the effects of PVCs with different coupling intervals (CIs) on myocardial mitochondrial remodelling in a canine model of PVCCM. Twenty-one beagles underwent pacemaker implantation and were randomised into the sham (n = 7), short-coupled PVCs (SCP, n = 7), and long-coupled PVCs (LCP, n = 7) groups. Right ventricular (RV) apical bigeminy was produced for 12-week to induce PVCCM in the SCP (CI, 250 ms) and LCP (CI, 350 ms) groups. Echocardiography was performed at baseline and biweekly thereafter to evaluate cardiac function. Masson's trichrome staining measured ventricular interstitial fibrosis. The ultrastructural morphology of the myocardial mitochondria was analysed using transmission electron microscopy. Mitochondrial Ca2+ concentration, reactive oxygen species (ROS) levels, adenosine triphosphate (ATP) content, membrane potential, and electron transport chain (ETC) complex activity were measured to assess myocardial mitochondrial function. Twelve-week-PVCs led to left ventricular (LV) enlargement with systolic dysfunction, disrupted mitochondrial morphology, increased mitochondrial Ca2+ concentration and ROS levels, decreased mitochondrial ATP content and membrane potential, and impaired ETC complex activity in both the SCP and LCP groups (all p < 0.01 vs the sham group). Ventricular fibrosis was observed only in canines with LCP. Worse cardiac function and more pronounced abnormalities in mitochondrial morphology and function were observed in the LCP group than to the SCP group (all p < 0.05). We demonstrated myocardial mitochondrial abnormalities in dogs with PVCCM, characterised by abnormal mitochondrial morphology, mitochondrial Ca2+ overload, oxidative stress, and impaired mitochondrial energy metabolism. Compared to SCP, long-term LCP exposure resulted in more severe mitochondrial remodelling and cardiac dysfunction in dogs.

The interplay between electron transport chain function and iron regulatory factors influences melanin formation in Cryptococcus neoformans.

Mitochondrial functions are critical for the ability of the fungal pathogen Cryptococcus neoformans to cause disease. However, mechanistic connections between key functions such as the mitochondrial electron transport chain (ETC) and virulence factor elaboration have yet to be thoroughly characterized. Here, we observed that inhibition of ETC complex III suppressed melanin formation, a major virulence factor. This inhibition was partially overcome by defects in Cir1 or HapX, two transcription factors that regulate iron acquisition and use. In this regard, loss of Cir1 derepresses the expression of laccase genes as a potential mechanism to restore melanin, while HapX may condition melanin formation by controlling oxidative stress. We hypothesize that ETC dysfunction alters redox homeostasis to influence melanin formation. Consistent with this idea, inhibition of growth by hydrogen peroxide was exacerbated in the presence of the melanin substrate L-DOPA. In addition, loss of the mitochondrial chaperone Mrj1, which influences the activity of ETC complex III and reduces ROS accumulation, also partially overcame antimycin A inhibition of melanin. The phenotypic impact of mitochondrial dysfunction was consistent with RNA-Seq analyses of WT cells treated with antimycin A or L-DOPA, or cells lacking Cir1 that revealed influences on transcripts encoding mitochondrial functions (e.g., ETC components and proteins for Fe-S cluster assembly). Overall, these findings reveal mitochondria-nuclear communication via ROS and iron regulators to control virulence factor production in C. neoformans.IMPORTANCEThere is a growing appreciation of the importance of mitochondrial functions and iron homeostasis in the ability of fungal pathogens to sense the vertebrate host environment and cause disease. Many mitochondrial functions such as heme and iron-sulfur cluster biosynthesis, and the electron transport chain (ETC), are dependent on iron. Connections between factors that regulate iron homeostasis and mitochondrial activities are known in model yeasts and are emerging for fungal pathogens. In this study, we identified connections between iron regulatory transcription factors (e.g., Cir1 and HapX) and the activity of complex III of the ETC that influence the formation of melanin, a key virulence factor in the pathogenic fungus Cryptococcus neoformans. This fungus causes meningoencephalitis in immunocompromised people and is a major threat to the HIV/AIDS population. Thus, understanding how mitochondrial functions influence virulence may support new therapeutic approaches to combat diseases caused by C. neoformans and other fungi.

Increased O-GlcNAcylation by Upregulation of Mitochondrial O-GlcNAc Transferase (mOGT) Inhibits the Activity of Respiratory Chain Complexes and Controls Cellular Bioenergetics.

O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible post-translational modification involved in the regulation of cytosolic, nuclear, and mitochondrial proteins. The interplay between O-GlcNAcylation and phosphorylation is critical to control signaling pathways and maintain cellular homeostasis. The addition of O-GlcNAc moieties to target proteins is catalyzed by O-linked N-acetylglucosamine transferase (OGT). Of the three splice variants of OGT described, one is destined for the mitochondria (mOGT). Although the effects of O-GlcNAcylation on the biology of normal and cancer cells are well documented, the role of mOGT remains poorly understood. In this manuscript, the effects of mOGT on mitochondrial protein phosphorylation, electron transport chain (ETC) complex activity, and the expression of VDAC porins were investigated. We performed studies using normal and breast cancer cells with upregulated mOGT or its catalytically inactive mutant. Proteomic approaches included the isolation of O-GlcNAc-modified proteins of the electron transport chain, followed by their analysis using mass spectrometry. We found that mitochondrial OGT regulates the activity of complexes I-V of the respiratory chain and identified a group of 19 ETC components as mOGT substrates in mammary cells. Furthermore, we observed that the upregulation of mOGT inhibited the interaction of VDAC1 with hexokinase II. Our results suggest that the deregulation of mOGT reprograms cellular energy metabolism via interaction with and O-GlcNAcylation of proteins involved in ATP production in mitochondria and its exchange between mitochondria and the cytosol.

Impact of ozone therapy on mouse liver mitochondrial function and antioxidant system

Ozone therapy's efficacy might stem from the regulated and mild oxidative stress resulting from ozone's interactions with various biological elements. The present work aimed to characterize the hepatic mitochondrial response to ozone treatment and its relationship with the antioxidant system response. Two groups of mice were used: one control group and another injected intraperitoneally with an O3/O2 mixture (80 ml/kg) for 5 days. Mitochondrial respiration supported by different substrates was significantly inhibited, as well as complexes I and II/III, but not complex IV. The analysis of the electron transport chain complex activity showed significant inhibitions in complexes I and II/III but not in complex IV. These inhibitions can prevent mitochondrial reactive oxygen species (ROS) production. Additionally, there was a decline in glutathione content, unaccompanied by a rise in its oxidized form. The ozone-treated groups showed a significant increase in the activity of superoxide dismutase and glutathione peroxidase, while catalase and glutathione reductase experienced no significant alterations. Adenine nucleotides increased in the ozone group, but only the increase in adenosine diphosphate is significant, so the cell's energy charge is unaffected. This study shows that mitochondria may play a crucial role in ozone treatment. However, it also highlights the need for further studies to understand the molecular mechanism.

Glutamine sustains energy metabolism and alleviates liver injury in burn sepsis by promoting the assembly of mitochondrial HSP60-HSP10 complex via SIRT4 dependent protein deacetylation

ABSTRACT Burns and burn sepsis, characterized by persistent and profound hypercatabolism, cause energy metabolism dysfunction that worsens organ injury and systemic disorders. Glutamine (Gln) is a key nutrient that remarkably replenishes energy metabolism in burn and sepsis patients, but its exact roles beyond substrate supply is unclear. In this study, we demonstrated that Gln alleviated liver injury by sustaining energy supply and restoring redox balance. Meanwhile, Gln also rescued the dysfunctional mitochondrial electron transport chain (ETC) complexes, improved ATP production, reduced oxidative stress, and protected hepatocytes from burn sepsis injury. Mechanistically, we revealed that Gln could activate SIRT4 by upregulating its protein synthesis and increasing the level of Nicotinamide adenine dinucleotide (NAD+), a co-enzyme that sustains the activity of SIRT4. This, in turn, reduced the acetylation of shock protein (HSP) 60 to facilitate the assembly of the HSP60-HSP10 complex, which maintains the activity of ETC complex II and III and thus sustain ATP generation and reduce reactive oxygen species release. Overall, our study uncovers a previously unknown pharmacological mechanism involving the regulation of HSP60-HSP10 assembly by which Gln recovers mitochondrial complex activity, sustains cellular energy metabolism and exerts a hepato-protective role in burn sepsis.

The antioxidant and anti-inflammatory activities of avasopasem manganese in age-associated, cisplatin-induced renal injury

PurposeCisplatin contributes to acute kidney injury (AKI) and chronic kidney disease (CKD) that occurs with greater frequency and severity in older patients. Age-associated cisplatin sensitivity in human fibroblasts involves increased mitochondrial superoxide produced by older donor cells. Experimental designYoung and old C57BL/6 J murine models of cisplatin-induced AKI and CKD were treated with the SOD mimetic avasopasem manganese to investigate the potential antioxidant and anti-inflammatory effects. Adverse event reporting from a phase 2 and a phase 3 randomized clinical trial (NCT02508389 and NCT03689712) conducted in patients treated with cisplatin and AVA was determined to have established the incidence and severity of AKI. ResultsCisplatin-induced AKI and CKD occurred in all mice, however, was more pronounced in older mice. AVA reduced cisplatin-induced mortality, AKI, and CKD, in older animals. AVA also alleviated cisplatin-induced alterations in mitochondrial electron transport chain (ETC) complex activities and NADPH Oxidase 4 (NOX4) and inhibited the increased levels of the inflammation markers, TNFα, IL1, ICAM-1, and VCAM-1. Analysis of age-stratified subjects treated with cisplatin from clinical trials (NCT02508389, NCT03689712) also supported that the incidence of AKI increased with age and AVA reduced age-associated therapy-induced adverse events (AE), including hypomagnesemia, increased creatinine, and AKI. ConclusionsOlder mice and humans are more susceptible to cisplatin-induced kidney injury, and treatment with AVA mitigates age-associated damage. Mitochondrial ETC and NOX4 activities represent sources of superoxide production contributing to cisplatin-induced kidney injury, and pro-inflammatory cytokine production and endothelial dysfunction may also be increased by superoxide formation.

Mitochondrial hyperfusion induces metabolic remodeling in lung endothelial cells by modifying the activities of electron transport chain complexes I and III

ObjectivePulmonary hypertension (PH) is a progressive disease with vascular remodeling as a critical structural alteration. We have previously shown that metabolic reprogramming is an early initiating mechanism in animal models of PH. This metabolic dysregulation has been linked to remodeling the mitochondrial network to favor fission. However, whether the mitochondrial fission/fusion balance underlies the metabolic reprogramming found early in PH development is unknown. MethodsUtilizing a rat early model of PH, in conjunction with cultured pulmonary endothelial cells (PECs), we utilized metabolic flux assays, Seahorse Bioassays, measurements of electron transport chain (ETC) complex activity, fluorescent microscopy, and molecular approaches to investigate the link between the disruption of mitochondrial dynamics and the early metabolic changes that occur in PH. ResultsWe observed increased fusion mediators, including Mfn1, Mfn2, and Opa1, and unchanged fission mediators, including Drp1 and Fis1, in a two-week monocrotaline-induced PH animal model (early-stage PH). We were able to establish a connection between increases in fusion mediator Mfn1 and metabolic reprogramming. Using an adenoviral expression system to enhance Mfn1 levels in pulmonary endothelial cells and utilizing 13C-glucose labeled substrate, we found increased production of 13C lactate and decreased TCA cycle metabolites, revealing a Warburg phenotype. The use of a 13C5-glutamine substrate showed evidence that hyperfusion also induces oxidative carboxylation. The increase in glycolysis was linked to increased hypoxia-inducible factor 1α (HIF-1α) protein levels secondary to the disruption of cellular bioenergetics and higher levels of mitochondrial reactive oxygen species (mt-ROS). The elevation in mt-ROS correlated with attenuated ETC complexes I and III activities. Utilizing a mitochondrial-targeted antioxidant to suppress mt-ROS, limited HIF-1α protein levels, which reduced cellular glycolysis and reestablished mitochondrial membrane potential. ConclusionsOur data connects mitochondrial fusion-mediated mt-ROS to the Warburg phenotype in early-stage PH development.

PSV-10 Effects of Rumen-Bypass Protein Supplement on Growth Performance, Hepatic Mitochondrial Function and Immune Gene Expression of Beef Steers with Divergent Residual Feed Intake Phenotype

Abstract We examined the effects of rumen-bypass protein supplement on growth performance, hepatic mitochondrial Electron Transport Chain (ETC) activities, and mRNA expression of innate and adaptive immune genes of beef steers with divergent residual feed intake (RFI) phenotype. Thirty-nine crossbred beef steers (average BW = 492 ± 36 kg) were used in a generalized randomized block design with a 2 × 2 factorial arrangement of treatments for a 42-d experimental period. The factors evaluated were 1) RFI classification [low-RFI (-2.12kg/d) vs. high-RFI (2.02kg/d)], and 2) feed additive: rumen-bypass protein supplement (RCP; 227 g· steer-1· day-1) vs. Control diet (CON; 0 g/d), resulting in four treatments: Low-RFI-CON (n = 10), low-RFI-RCP (n = 9), high-RFI-CON (n = 10), and high-RFI-RCP (n = 10). The rumen-bypass protein supplement (84% CP) is a blend of hydrolyzed feather meal, porcine blood meal, and DL-methionine hydroxy analogue. The beef steers were stratified by BW and randomly assigned to treatments and housed in four pens equipped with two GrowSafe feed bunks each to measure individual DMI. Body weight was measured every 7 days. Liver tissue was sampled on d 42 from all animals using a liver biopsy needle for mRNA expression analysis of 84 immune genes and analysis of mitochondrial ETC activities of complexes I – V. No effects of supplemental feed additive or RFI × feed additive interactions were observed (P &amp;gt; 0.05) for ADG and DMI. Compared with high-RFI, low-RFI beef steers tended to have less DMI (13.6 vs 12.9 kg/d; P = 0.09) but ADG was similar for the two RFI groups. Feed additive × RFI interactions were observed (P ≤ 0.05) for mitochondrial activities of Complexes IV, V, and mRNA expressions of some immune genes. Supplemental RCP increased Complex IV (39.2 nmol/min/mg) activity and reduced that of Complex V (94.1 nmol· min-1· mg-1) in high-RFI beef steers whereas the activity of Complex IV (13.1 nmol· min-1· mg-1) was reduced and that of Complex V (120 nmol· min-1· mg-1) was increased in low-RFI beef steers. Supplemental RCP increased (P &amp;lt; 0.05) the mRNA expression of TLR2 [fold change (FC) = 1.17 vs 0.98], TLR3 (FC = 1.49 vs 0.97), NFKB1 (FC = 1.02 vs 0.90), and IL23A (FC = 1.35 vs 1.07) in high-RFI beef steers but the expression of these genes were not altered in low-RFI beef steers. In conclusion, RCP supplementation did not affect the growth performance and the observed effects on hepatic immune gene expression and activities of mitochondrial ETC complexes are dependent on the RFI phenotype of the beef steers. Further research should explore the mechanism responsible for the interactions between RCP supplementation and RFI status.

Antimicrobial mitochondrial reactive oxygen species induction by lung epithelial immunometabolic modulation.

Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infection an urgent need. Manipulating the lungs' intrinsic host defenses by therapeutic delivery of certain pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. Here we show that antimicrobial ROS are induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODN) with mitochondrial voltage-dependent anion channel 1 (VDAC1). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), increases mitochondrial membrane potential (ΔΨm), differentially modulates ETC complex activities and consequently results in leak of electrons from ETC complex III and superoxide formation. The ODN-induced mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a therapeutic metabolic manipulation strategy to broadly protect against pneumonia without reliance on antibiotics.

Protective effect of grape seed extract and exercise training on tissues toxicities in doxorubicin-treated healthy rat

Objective The aim of the present study was to investigate the effects of Grape seed extract (GSE) and exercise training on Doxorubicin (Doxo)-induced cardio, hepato and myo toxicities in healthy rats. Methods Thirty male Wistar rats were randomly divided into five groups and daily treated by intraperitoneal route during two months either with ethanol 10% (Control); Doxo (1.5 mg/kg); Doxo + exercise (1.5 mg/kg + swimming exercise for 30 min twice a week); Doxo + GSE (1.5 mg/kg + GSE 2.5 g/kg); Doxo + GSE + exercise (1.5 mg/kg + GSE 2.5 g/kg + swimming exercise for 30 min twice a week). At the end of the treatment, tissues were collected and processed for the determination of oxidative stress (OS), intracellular mediators, energy fuelling biomarkers, carbohydrate metabolism parameters and muscle histopathology. Results Doxo provoked OS characterised by an increased lipoperoxidation (LPO) and protein carbonylation and decreased antioxidant enzyme activities. Doxo also affected intracellular mediators, disturbed carbohydrate metabolism and energy fuelling in skeletal muscle as assessed by down-regulated Electron Transport Chain (ETC) complex activities leading in fine to altered skeletal muscle structure and function. Conclusion Almost all Doxo-induced disturbances were partially corrected with GSE and exercise on their own and more efficiently with the combined treatment (GSE + exercise).

Different Effects of SSRIs, Bupropion, and Trazodone on Mitochondrial Functions and Monoamine Oxidase Isoform Activity.

Mitochondrial dysfunction is involved in the pathophysiology of psychiatric and neurodegenerative disorders and can be used as a modulator and/or predictor of treatment responsiveness. Understanding the mitochondrial effects of antidepressants is important to connect mitochondria with their therapeutic and/or adverse effects. Pig brain-isolated mitochondria were used to evaluate antidepressant-induced changes in the activity of electron transport chain (ETC) complexes, monoamine oxidase (MAO), mitochondrial respiratory rate, and ATP. Bupropion, escitalopram, fluvoxamine, sertraline, paroxetine, and trazodone were tested. All tested antidepressants showed significant inhibition of complex I and IV activities at high concentrations (50 and 100 µmol/L); complex II + III activity was reduced by all antidepressants except bupropion. Complex I-linked respiration was reduced by escitalopram >> trazodone >> sertraline. Complex II-linked respiration was reduced only by bupropion. Significant positive correlations were confirmed between complex I-linked respiration and the activities of individual ETC complexes. MAO activity was inhibited by all tested antidepressants, with SSRIs causing a greater effect than trazodone and bupropion. The results indicate a probable association between the adverse effects of high doses of antidepressants and drug-induced changes in the activity of ETC complexes and the respiratory rate of mitochondria. In contrast, MAO inhibition could be linked to the antidepressant, procognitive, and neuroprotective effects of the tested antidepressants.

One-year reductions in cisplatin-related chronic kidney disease (CKD) in patients with head and neck (HNC) cancer treated with avasopasem manganese: A prespecified analysis from the phase 3 ROMAN trial.

6066 Background: Cisplatin-induced acute kidney injury (AKI), acute kidney disease (AKD), and chronic kidney disease (CKD) are associated with increased production of reactive oxygen species, notably superoxide, in conjunction with alterations in mitochondrial electron transport chain complex activities in tubular epithelial cells (Mapuskar et al. Antioxidants 2021;10:1329). These pathologies can be targeted and thereby inhibited by increasing superoxide dismutase activity, which converts superoxide to hydrogen peroxide. The investigational new drug avasopasem manganese (AVA), a selective dismutase mimetic, ameliorated cisplatin-induced kidney injury in mice (Mapuskar et al. Redox Biol 2019;20:98). Preclinical results also demonstrate that AVA does not interfere with cisplatin anticancer activity (Mohanty et al. Cancer Res 2018;78[13_suppl]:Abstract 2929). Consistent with this, a retrospective analysis of 1- and 2-year kidney function of a limited subset of patients treated in a randomized, placebo (PBO)-controlled phase 2b trial of AVA to reduce severe oral mucositis (SOM) from radiotherapy and cisplatin (CRT) suggested that AVA prevented or reduced cisplatin-related CKD compared to PBO (Steinbach et al. J Clin Oncol 2020;38[15_suppl]:Abstract 12071). Methods: The ROMAN phase 3 trial (NCT03689712; ITT n = 407) of CRT in combination with AVA or PBO demonstrated improved incidence, duration, and onset of SOM by AVA (n = 241) vs PBO (n = 166) (Anderson et al. J Clin Oncol 2022;40[16_suppl]:Abstract 6005). In ROMAN, prospectively defined follow-up of serum creatinine (sCr) and estimated glomerular filtration rates (eGFR) were assessed every 3 months for 1 year following 7 weeks of CRT. Results: At 1-year follow-up, AVA was associated with significant improvements in preservation of eGFR ( P= 0.0008) and sCr ( P= 0.006) levels vs PBO. Grade 3+ CKD (eGFR &lt; 60 mL/min) results through 12 months show 10% AVA vs 20% PBO (relative risk 0.55, P= 0.0043). Regarding cisplatin schedule, patients receiving Q3W therapy show grade 3+ CKD of 12% AVA vs 22% PBO; patients receiving QW therapy show AVA 9% vs PBO 19%. During treatment, nominal reduction of the incidence of AKI adverse events (0.8% AVA vs 3.6% PBO) was also observed. Conclusions: At 1-year follow-up, AVA appears to reduce cisplatin-related CKD in the study population and may also reduce cisplatin-related AKI during treatment. These results carry significance beyond CRT for HNC, potentially impacting platinum-containing regimens in other cancers. Clinical trial information: NCT03689712 .

Cu2−x Se nanoparticles suppress cell proliferation and migration in hepatocellular carcinoma by impairing mitochondrial respiration

Cu2−x Se nanoparticles (Cu2−x Se NPs) as a new therapeutic drug platform is widely used in disease treatment due to their strong near-infrared optical absorption. In recent years, with their continuous expansion of applications in different fields, their own biological effects have received increasing attention. However, little is known about the effect of Cu2−x Se NPs on cancer cell. In this research, we found that Cu2−x Se NPs inhibited proliferation of HepG2 cells (IC50: 15.91 μM) and SMMC-7721 cells (IC50: 43.15 μM) and they mainly induced cell cycle arrest at the G2/M phase. Moreover, Cu2−x Se NPs inhibited HepG2 and SMMC-7721 cell migration and lamellopodia formation. Further studies indicated that Cu2−x Se NPs impaired mitochondrial respiration by inhibiting electron transport chain complex activity, thus reducing adenosine triphosphate levels. The insufficient energy supply subsequently impaired actin cytoskeleton assembly, ultimately inhibiting HepG2 and SMMC-7721 cell proliferation and migration. These findings suggest that Cu2−x Se NPs may have potentially antitumor activity, which might provide new insights of NPs into specific cancer treatment.

Microarrays, Enzymatic Assays, and MALDI-MS for Determining Specific Alterations to Mitochondrial Electron Transport Chain Activity, ROS Formation, and Lipid Composition in a Monkey Model of Parkinson’s Disease

Multiple evidences suggest that mitochondrial dysfunction is implicated in the pathogenesis of Parkinson's disease via the selective cell death of dopaminergic neurons, such as that which occurs after prolonged exposure to the mitochondrial electron transport chain (ETC) complex I inhibitor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrine (MPTP). However, the effects of chronic MPTP on the ETC complexes and on enzymes of lipid metabolism have not yet been thoroughly determined. To face these questions, the enzymatic activities of ETC complexes and the lipidomic profile of MPTP-treated non-human primate samples were determined using cell membrane microarrays from different brain areas and tissues. MPTP treatment induced an increase in complex II activity in the olfactory bulb, putamen, caudate, and substantia nigra, where a decrease in complex IV activity was observed. The lipidomic profile was also altered in these areas, with a reduction in the phosphatidylserine (38:1) content being especially relevant. Thus, MPTP treatment not only modulates ETC enzymes, but also seems to alter other mitochondrial enzymes that regulate the lipid metabolism. Moreover, these results show that a combination of cell membrane microarrays, enzymatic assays, and MALDI-MS provides a powerful tool for identifying and validating new therapeutic targets that might accelerate the drug discovery process.

Cardiomyocyte-specific PCSK9 deficiency compromises mitochondrial bioenergetics and heart function.

Pro-protein convertase subtilisin-kexin type 9 (PCSK9), which is expressed mainly in the liver and at low levels in the heart, regulates cholesterol levels by directing low-density lipoprotein receptors to degradation. Studies to determine the role of PCSK9 in the heart are complicated by the close link between cardiac function and systemic lipid metabolism. Here, we sought to elucidate the function of PCSK9 specifically in the heart by generating and analysing mice with cardiomyocyte-specific Pcsk9 deficiency (CM-Pcsk9-/- mice) and by silencing Pcsk9 acutely in a cell culture model of adult cardiomyocyte-like cells. Mice with cardiomyocyte-specific deletion of Pcsk9 had reduced contractile capacity, impaired cardiac function, and left ventricular dilatation at 28 weeks of age and died prematurely. Transcriptomic analyses revealed alterations of signalling pathways linked to cardiomyopathy and energy metabolism in hearts from CM-Pcsk9-/- mice vs. wild-type littermates. In agreement, levels of genes and proteins involved in mitochondrial metabolism were reduced in CM-Pcsk9-/- hearts. By using a Seahorse flux analyser, we showed that mitochondrial but not glycolytic function was impaired in cardiomyocytes from CM-Pcsk9-/- mice. We further showed that assembly and activity of electron transport chain (ETC) complexes were altered in isolated mitochondria from CM-Pcsk9-/- mice. Circulating lipid levels were unchanged in CM-Pcsk9-/- mice, but the lipid composition of mitochondrial membranes was altered. In addition, cardiomyocytes from CM-Pcsk9-/- mice had an increased number of mitochondria-endoplasmic reticulum contacts and alterations in the morphology of cristae, the physical location of the ETC complexes. We also showed that acute Pcsk9 silencing in adult cardiomyocyte-like cells reduced the activity of ETC complexes and impaired mitochondrial metabolism. PCSK9, despite its low expression in cardiomyocytes, contributes to cardiac metabolic function, and PCSK9 deficiency in cardiomyocytes is linked to cardiomyopathy, impaired heart function, and compromised energy production.

Abstract P6-11-10: Investigating the role of mitochondrial protein translation in the metabolic adaptation of chemoresistant triple negative breast cancer

Abstract BACKGROUND: Nearly 50% of patients with triple negative breast cancer (TNBC) treated with neoadjuvant chemotherapy (NACT) retain residual tumors resulting in high rates of metastatic relapse and poor overall survival. Residual tumors surviving NACT (Adriamycin plus cyclophosphamide; AC) were found to undergo a metabolic transition to heightened mitochondrial oxidative phosphorylation (oxphos; PMID: 30996079). Pharmacologic inhibition of mitochondrial electron transport chain (ETC) complex I with IACS-010759 (PMID: 29892070) had enhanced efficacy in residual, rather than treatment-naïve, tumors of orthotopic patient-derived xenograft (PDX) models. Our analyses of mitochondrial structure and function in human TNBC cell lines revealed differing adaptations in residual cells surviving treatment with conventional NACT agents. While DNA-damaging chemotherapies (e.g.Adriamycin, carboplatin) induced mitochondrial fusion and oxphos, taxanes (e.g.paclitaxel, docetaxel) induced mitochondrial fragmentation and reduced oxphos (Baek et al., Biorxiv Doi 10.1101/2022.02.25.481996). The mechanistic basis of these mitochondrial adaptations is not yet understood. The mitochondrial ETC consists of 92 proteins, 13 of which are encoded in the mitochondrial genome (mtDNA) and translated by the mitoribosome, while the remaining are encoded by the nuclear genome (nDNA), translated by the cytoribosome, and inserted into the inner mitochondrial membrane by accessory proteins, namely Oxidase (Cytochrome C) Assembly 1-Like (OXA1L). Disruption of OXA1L in mammalian cells has been shown to affect the levels and activity of ETC complexes I, III, IV, and V, and thus diminish oxphos. We aim to determine whether mitochondrial translation and OXA1L activity represent therapeutic vulnerabilities to overcome pro-survival metabolic adaptations in chemoresistant TNBC thereby augmenting treatment response. METHODS: Weare evaluating the effects of conventional TNBC chemotherapies singly, and in standard combinations, on mitochondrial translation and ETC formation in human TNBC cells and PDX models(PIM001-P, WHIM14, BCM15116) using metabolomic and proteomic profiling. To perturb these processes genetically, we knocked down (KD) OXA1Lwith siRNA. We are complementing these studies pharmacologically using conventional antibiotics, such as tigecycline, as previous studies showed they inhibit mitochondrial translation in breast and other cancers (PMID: 25625193). These studies will reveal whether OXA1L and mitochondrial translation are required for metabolic adaption and chemotherapy resistance of residual TNBC cells. PDX preclinical trials based on our published residual tumor testing schema (PMID: 30996079), will reveal whether the sequential combination of NACT followed by tigecycline can effectively perturb residual tumor relapse. RESULTS: Proteomic profiling of longitudinally harvested PDX tumors demonstrates substantial disruption of mitochondria-and nuclear-encoded ETC components in residual vs. treatment-naïve tumors. Interestingly, these patterns are distinct between different chemotherapy treatments, with an increase of ETC components in carboplatin-treated residual tumors compared to a decrease in docetaxel-treated residual tumors. Western blot analyses of human cell lines show OXA1LKD perturbs levels of both nuclear-and mitochondria-encoded ETC components. Preliminary findings suggest OXA1LKD increases sensitivity to chemotherapies in human TNBC cell lines. Finally, tigecycline effectively inhibits TNBC cell growth. We next will evaluate whether residual cells not killed by conventional chemotherapies have enhanced tigecycline susceptibility. CONCLUSION: These data suggest targeting mitochondrial translation may be a promising approach to overcome pro-survival metabolic adaptations in residual TNBC cells not killed by conventional chemotherapies. Citation Format: Mariah J. Berner, Lily Baek, Junegoo Lee, Philip L. Lorenzi, Mei Leng, Alexander B. Saltzman, Anna Malovannaya, Lacey E. Dobrolecki, Christina Sallas, Michael T. Lewis, Gloria V. Echeverria. Investigating the role of mitochondrial protein translation in the metabolic adaptation of chemoresistant triple negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-10.

High-Fat Diet Augments the Effect of Alcohol on Skeletal Muscle Mitochondrial Dysfunction in Mice.

Previous studies have shown that chronic heavy alcohol consumption and consumption of a high-fat (HF) diet can independently contribute to skeletal muscle oxidative stress and mitochondrial dysfunction, yet the concurrent effect of these risk factors remains unclear. We aimed to assess the effect of alcohol and different dietary compositions on mitochondrial activity and oxidative stress markers. Male and female mice were randomized to an alcohol (EtOH)-free HF diet, a HF + EtOH diet, or a low-Fat (LF) + EtOH diet for 6 weeks. At the end of the study, electron transport chain complex activity and expression as well as antioxidant activity and expression, were measured in skeletal muscles. Complex I and III activity were diminished in muscles of mice fed a HF + EtOH diet relative to the EtOH-free HF diet. Lipid peroxidation was elevated, and antioxidant activity was diminished, in muscles of mice fed a HF + EtOH diet as well. Consumption of a HF diet may exacerbate the negative effects of alcohol on skeletal muscle mitochondrial health and oxidative stress.

Observations that suggest a contribution of altered dermal papilla mitochondrial function to androgenetic alopecia.

Androgenetic alopecia (AGA) is a prevalent hair loss condition in males that develops due to the influence of androgens and genetic predisposition. With the aim of elucidating genes involved in AGA pathogenesis, we modelled AGA with three-dimensional culture of keratinocyte-surrounded dermal papilla (DP) cells. We co-cultured immortalised balding and non-balding human DP cells (DPCs) derived from male AGA patients with epidermal keratinocyte (NHEK) using multi-interfacial polyelectrolyte complexation technique. We observed up-regulated mitochondria-related gene expression in balding compared with non-balding DP aggregates which indicated altered mitochondria metabolism. Further observation of significantly reduced electron transport chain complex activity (complexes I, IV and V), ATP levels and ability to uptake metabolites for ATP generation demonstrated compromised mitochondria function in balding DPC. Balding DP was also found to be under significantly higher oxidative stress than non-balding DP. Our experiments suggest that application of antioxidants lowers oxidative stress levels and improves metabolite uptake in balding DPC. We postulate that the observed up-regulation of mitochondria-related genes in balding DP aggregates resulted from an over-compensatory effort to rescue decreased mitochondrial function in balding DP through the attempted production of new functional mitochondria. In all, our three-dimensional co-culturing revealed mitochondrial dysfunction in balding DPC, suggesting a metabolic component in the aetiology of AGA.

Blocked O-GlcNAc cycling alters mitochondrial morphology, function, and mass

O-GlcNAcylation is a prevalent form of glycosylation that regulates proteins within the cytosol, nucleus, and mitochondria. The O-GlcNAc modification can affect protein cellular localization, function, and signaling interactions. The specific impact of O-GlcNAcylation on mitochondrial morphology and function has been elusive. In this manuscript, the role of O-GlcNAcylation on mitochondrial fission, oxidative phosphorylation (Oxphos), and the activity of electron transport chain (ETC) complexes were evaluated. In a cellular environment with hyper O-GlcNAcylation due to the deletion of O-GlcNAcase (OGA), mitochondria showed a dramatic reduction in size and a corresponding increase in number and total mitochondrial mass. Because of the increased mitochondrial content, OGA knockout cells exhibited comparable coupled mitochondrial Oxphos and ATP levels when compared to WT cells. However, we observed reduced protein levels for complex I and II when comparing normalized mitochondrial content and reduced linked activity for complexes I and III when examining individual ETC complex activities. In assessing mitochondrial fission, we observed increased amounts of O-GlcNAcylated dynamin-related protein 1 (Drp1) in cells genetically null for OGA and in glioblastoma cells. Individual regions of Drp1 were evaluated for O-GlcNAc modifications, and we found that this post-translational modification (PTM) was not limited to the previously characterized residues in the variable domain (VD). Additional modification sites are predicted in the GTPase domain, which may influence enzyme activity. Collectively, these results highlight the impact of O-GlcNAcylation on mitochondrial dynamics and ETC function and mimic the changes that may occur during glucose toxicity from hyperglycemia.

Ginsenoside Rh2 stimulates the production of mitochondrial reactive oxygen species and induces apoptosis of cervical cancer cells by inhibiting mitochondrial electron transfer chain complex.

Ginsenoside Rh2 (G-Rh2) is a monomeric compound that extracted from ginseng and possesses anti-cancer activities both in vitro and in vivo. Previously, we reported that G-Rh2 induces apoptosis in HeLa cervical cancer cells and that the process was related to reactive oxygen species (ROS) accumulation and mitochondrial dysfunction. However, the upstream mechanisms of G-Rh2, along with its cellular targets, remain to be elucidated. In the present study, the Cell Counting Kit-8 assay, flow cytometry and Hoechst staining revealed that G-Rh2 significantly inhibited cell viability and induced apoptosis of cervical cancer cells. However, G-Rh2 was demonstrated to be non-toxic to End1/e6e7 cells. JC-1, rhodamine 123 staining, oxidative phosphorylation and glycolysis capacity assays demonstrated that G-Rh2 exposure caused an immediate decrease in mitochondrial transmembrane potential due to its inhibition of mitochondrial oxidative phosphorylation, as well as glycolysis, both of which reduced cellular ATP production. Western blotting and electron transport chain (ETC) activity assays revealed that G-Rh2 significantly inhibited the activity of ETC complexes I, III and V. Overexpression of ETC complex III partially significantly restored mitochondrial ROS and inhibited the apoptosis of cervical cancer cells induced by G-Rh2. The predicted results of binding energy in molecular docking, confirmed that G-Rh2 was highly likely to induce mitochondrial ROS production and promote cell apoptosis by targeting the ETC complex, especially for ETC complex III. Taken together, the present results revealed the potential anti-cervical cancer activity of G-Rh2 and provide direct evidence for the contribution of impaired ETC complex activity to cervical cancer cell death.