Mitochondrial Respiratory Activity Research Articles

Strobilurin X acts as an anticancer drug by inhibiting protein synthesis and suppressing mitochondrial respiratory chain activity

PurposeStrobilurins act as antifungal agents by inhibiting the mitochondrial respiratory chain. The cytotoxic activity of strobilurins, focusing on its anticancer activities, has been reported. However, the mechanisms involved in these activities remain unclear.MethodsThe cytotoxic effects of strobilurin X isolated from the mycelium of Mucidula. venosolamellata were examined in human cancer cell lines (A549 and HeLa) and normal fibroblasts (WI-38).ResultsStrobilurin X significantly decreased the viability of A549 and HeLa cells compared to that in the WI-38 cells after 48 h of exposure. The EC50 values for cytotoxicity in the A549, HeLa, and WI-38 cells were 3.4, 5.4, and 16.8 μg/mL, respectively. Strobilurin X inhibited the mitochondrial respiratory chain and enhanced the release of lactate in the A549 cells. The IC50 value of strobilurin X against the mitochondrial respiratory chain complex III activity was 139.8 ng/mL. The cytotoxicity induced by strobilurin X was not completely rescued after adding uridine, methyl pyruvate, or N-acetyl cysteine. Furthermore, pharmacological approaches demonstrated that strobilurin X failed to modulate the mitogen-activated protein kinase family and phosphoinositide 3-kinase-Akt pathways; alternatively, it suppressed protein synthesis independent of uridine.ConclusionStrobilurin X induced cytotoxicity by blocking the mitochondrial respiratory chain and suppressing protein synthesis. These findings may aid in the development of novel anticancer drugs using strobilurins.

Mitochondrial respiration is lower in the intrauterine growth-restricted fetal sheep heart.

Fetuses affected by intrauterine growth restriction have an increased risk of developing heart disease and failure in adulthood. Compared with controls, late gestation intrauterine growth-restricted (IUGR) fetal sheep have fewer binucleated cardiomyocytes, reflecting a more immature heart, which may reduce mitochondrial capacity to oxidize substrates. We hypothesized that the late gestation IUGR fetal heart has a lower capacity for mitochondrial oxidative phosphorylation. Left (LV) and right (RV) ventricles from IUGR and control (CON) fetal sheep at 90% gestation were harvested. Mitochondrial respiration (states 1-3, LeakOmy, and maximal respiration) in response to carbohydrates and lipids, citrate synthase (CS) activity, protein expression levels of mitochondrial oxidative phosphorylation complexes (CI-CV), and mRNA expression levels of mitochondrial biosynthesis regulators were measured. The carbohydrate and lipid state 3 respiration rates were lower in IUGR than CON, and CS activity was lower in IUGR LV than CON LV. However, relative CII and CV protein levels were higher in IUGR than CON; CV expression level was higher in IUGR than CON. Genes involved in lipid metabolism had lower expression in IUGR than CON. In addition, the LV and RV demonstrated distinct differences in oxygen flux and gene expression levels, which were independent from CON and IUGR status. Low mitochondrial respiration and CS activity in the IUGR heart compared with CON are consistent with delayed cardiomyocyte maturation, and CII and CV protein expression levels may be upregulated to support ATP production. These insights will provide a better understanding of fetal heart development in an adverse in utero environment. KEY POINTS: Growth-restricted fetuses have a higher risk of developing and dying from cardiovascular diseases in adulthood. Mitochondria are the main supplier of energy for the heart. As the heart matures, the substrate preference of the mitochondria switches from carbohydrates to lipids. We used a sheep model of intrauterine growth restriction to study the capacity of the mitochondria in the heart to produce energy using either carbohydrate or lipid substrates by measuring how much oxygen was consumed. Our data show that the mitochondria respiration levels in the growth-restricted fetal heart were lower than in the normally growing fetuses, and the expression levels of genes involved in lipid metabolism were also lower. Differences between the right and left ventricles that are independent of the fetal growth restriction condition were identified. These results indicate an impaired metabolic maturation of the growth-restricted fetal heart associated with a decreased capacity to oxidize lipids postnatally.

10-(6-Plastoquinonyl) decyltriphenylphosphonium imparts anti-colitogenic protection through recovery of mitochondrial dysfunction in ulcerated murine colon: Implications in ulcerative colitis

AimsTo elucidate the impact of 10-(6-plastoquinonyl) decyltriphenylphosphonium (SkQ1) as an anti-colitogenic agent for maintenance of colon epithelial tract in ulcerated mice through recovery of mitochondrial dysfunction and mitochondrial stress by virtue of its free radical scavenging properties. Main methodsDSS induced ulcerated BALB/c mice were treated with SkQ1 for 14 days @ 30 nmol/kg/body wt./day/mice. Post-treatment, isolated colonic mitochondria were utilized for spectrophotometric and spectrofluorometric biochemical analysis of various mitochondrial functional variables including individual mitochondrial respiratory enzyme complexes. Confocal microscopy was utilized for measuring mitochondrial membrane potential in vivo. ELISA technique was adapted for measuring colonic nitrite and 3–nitrotyrosine (3-NT) content. Finally in vitro cell line study was carried out to substantiate in vivo findings and elucidate the involvement of free radicals in UC using antioxidant/free radical scavenging regimen. Key findingsTreatment with SkQ1 in vivo reduced histopathological severity of colitis, induced recovery of mitochondrial respiratory complex activities and associated functional variables, improved oxidative stress indices and normalized mitochondrial cardiolipin content. Importantly, SkQ1 lowered nitrite concentration and 3–nitrotyrosine formation in vivo. In vitro SkQ1 restored mitochondrial functions wherein the efficacy of SkQ1 proved equal or better compared to SOD and DMSO indicating predominant involvement of O2− and OH in UC. However, NO and ONOO− also seemed to play a secondary role as MEG and L-NAME provided lesser protection as compared to SOD and DMSO. SignificanceSkQ1 can be considered as a potent anti-colitogenic agent by virtue of its free radical scavenging properties in treating UC.

AMPK regulates immature boar Sertoli cell proliferation through affecting CDK4/Cyclin D3 pathway and mitochondrial function

Sertoli cell (SC) proliferation plays an important role in sperm production and quality; however, the regulatory mechanism of SC proliferation is not well understood. This study investigated the role of adenosine monophosphate-activated protein kinase (AMPK) in the regulation of immature boar SC activity. Cell counting kit-8, Seahorse XFe96, mitochondrial respiratory enzyme-related assay kits, and transmission electron microscopy were used to detect SC proliferative viability, oxygen consumption rate (OCR), mitochondrial respiratory enzyme activity, and the ultrastructure of primary cultured SCs in vitro from the testes of 21-day-old boars. A dual luciferase reporter assay was performed to determine the miRNA-mRNA target interaction. Western blotting was used to analyze cell proliferation-related protein expression of p38, p21, proliferating cell nuclear antigen (PCNA), Cyclin-dependent kinase 4 (CDK4), Cyclin D3, and phosphorylated retinoblastoma protein (Rb). Each experiment had a completely randomized design, with three replicates in each experiment. The results showed that the AMPK inhibitor (Compound C, 20 μM-24 h) increased cell proliferation viability, ATP production, and maximal respiration of SCs by 0.64-, 0.12-, and 0.08-fold (p < 0.05), respectively; increased the SC protein expression of PCNA, CDK4, Cyclin D3, and p-Rb by 0.13-, 0.09-, 0.88-, and 0.12-fold (p < 0.05), respectively; and decreased the SC protein expression of p38 and p21 by 0.36- and 0.27-fold (p < 0.05), respectively. The AMPK agonist AICAR (2 mM-6 h) significantly inhibited SC ultrastructure, OCR, mitochondrial respiratory enzyme activity, and cell proliferation-related protein levels. AMPK was validated to be a target gene of miR-1285 based on the result in which the miR-1285 mimic inhibited the luciferase activity of wild-type AMPK by 0.54-fold (p < 0.001). MiR-1285 mimic promoted the OCR of SCs, with 0.45-, 0.15-, 0.21-, and 0.30-fold (p < 0.01) increases in ATP production, basal and maximal respiration, and spare capacity, respectively. MiR-1285 mimic increased the mitochondrial respiratory enzyme activity of SCs, with 0.63-, 0.70-, and 0.97-fold (p < 0.01) increases in NADH-Q oxidoreductase, cytochrome c oxidase, and ATP synthase, respectively. Moreover, the miR-1285 mimic increased the protein expression of PCNA, CDK4, Cyclin D3, and p-Rb by 0.24-, 0.30-, 0.22-, and 0.13-fold (p < 0.05), respectively, and reduced the protein expression of p38 and p21 by 0.58- and 0.66-fold (p < 0.001). MiR-1285 inhibitor showed opposite effects on the above indicators and induced numerous autophagosomes and large lipid droplets in SCs. A high dose of estradiol (10 μM-6 h, showed a promotion of AMPK activation in a previous study) significantly inhibited SC ultrastructure, mitochondrial function, and proliferation-related pathways, while these adverse effects were weakened by Compound C treatment or miR-1285 mimic transfection. Our findings suggest that the activation and inhibition of AMPK induced by specific drugs or synthesized targeted miRNA fragments could regulate immature boar SC proliferative activity by influencing the CDK4/Cyclin D3 pathway and mitochondrial function; this helps to provide a basis for the prevention and treatment of male sterility in clinical practice.

Acyl-CoA synthetase 4 modulates mitochondrial function in breast cancer cells

Mitochondria are dynamic organelles that respond to cellular stress through changes in global mass, interconnection, and subcellular location. As mitochondria play an important role in tumor development and progression, alterations in energy metabolism allow tumor cells to survive and spread even in challenging conditions. Alterations in mitochondrial bioenergetics have been recently proposed as a hallmark of cancer, and positive regulation of lipid metabolism constitutes one of the most common metabolic changes observed in tumor cells. Acyl-CoA synthetase 4 (ACSL4) is an enzyme catalyzing the activation of long chain polyunsaturated fatty acids with a strong substrate preference for arachidonic acid (AA). High ACSL4 expression has been related to aggressive cancer phenotypes, including breast cancer, and its overexpression has been shown to positively regulate the mammalian Target of Rapamycin (mTOR) pathway, involved in the regulation of mitochondrial metabolism genes. However, little is known about the role of ACSL4 in the regulation of mitochondrial function and metabolism in cancer cells. In this context, our objective was to study whether mitochondrial function and metabolism, processes usually altered in tumors, are modulated by ACSL4 in breast cancer cells. Using ACSL4 overexpression in MCF-7 cells, we demonstrate that this enzyme can increase the mRNA and protein levels of essential mitochondrial regulatory proteins such as nuclear respiratory factor 1 (NRF-1), voltage-dependent anion channel 1 (VDAC1) and respiratory chain Complex III. Furthermore, respiratory parameters analysis revealed an increase in oxygen consumption rate (OCR) and in spare respiratory capacity (SRC), among others. ACSL4 knockdown in MDA-MB-231 cells led to the decrease in OCR and in SCR, supporting the role of ACSL4 in the regulation of mitochondrial bioenergetics. Moreover, ACSL4 overexpression induced an increase in glycolytic function, in keeping with an increase in mitochondrial respiratory activity. Finally, there was a decrease in mitochondrial mass detected in cells that overexpressed ACSL4, while the knockdown of ACSL4 expression in MDA-MB-231 cells showed the opposite effect. Altogether, these results unveil the role of ACSL4 in mitochondrial function and metabolism and expand the knowledge of ACSL4 participation in pathological processes such as breast cancer.

Mitochondrial citrate transporters Ctp1-Yhm2 and respiratory chain: A coordinated functional connection in Saccharomyces cerevisiae metabolism

The mitochondrial inner membrane contains some hydrophobic proteins that mediate the exchange of metabolites between the mitochondrial matrix and the cytosol. Ctp1 and Yhm2 are two carrier proteins in the yeast Saccharomyces cerevisiae responsible for the transport of citrate, a tricarboxylate involved in several metabolic pathways.Since these proteins also contribute to respiratory metabolism, in this study we investigated for the first time whether changes in citrate transport can affect the structural organization and functional properties of respiratory complexes.Through experiments in yeast mutant cells in which the gene encoding Ctp1 or Yhm2 was deleted, we found that in the absence of either mitochondrial citrate transporter, mitochondrial respiration was impaired. Structural analysis of the respiratory complexes III and IV revealed different expression levels of the catalytic and supernumerary subunits in the Δctp1 and Δyhm2 strains. In addition, Δyhm2 mitochondria appeared to be more sensitive than Δctp1 to the oxidative damage.Our results provide the first evidence for a coordinated modulation of mitochondrial citrate transport and respiratory chain activity in S. cerevisiae metabolism.

Oxidative stress in the remaining muscle after volumetric muscle loss contributes to impaired mitochondrial bioenergetics and is attenuated by SS-31

Volumetric muscle loss (VML) injury results in the non-recoverable loss of muscle mass and function. Our previous studies indicate mitochondrial dysfunction in the remaining muscle fibers after VML injury, marked by changes in mitochondrial respiration, membrane potential, and enzyme activities. In particular, mitochondrial membrane potential is hyperpolarized during the first 14-days post-injury, a condition that could lead to the production of reactive oxygen species (ROS). When ROS emissions exceed the antioxidant buffering capacity, cellular damage can ensue. The primary objective of this study was to determine ROS production, ROS emission, and antioxidant buffering capacity of remaining muscle fibers after VML injury in the first month after injury. The secondary objective of this study was to determine if a mitochondrial antioxidant (SS-31) could mitigate mitochondrial dysfunction after VML injury. We hypothesized that VML injury will alter the redox status of the remaining muscle. Study 1: Male C57BL/6J mice (n=40) were randomized to Uninjured or VML-injured cohorts. ROS (H2O2) emission/production and endogenous antioxidant buffering capacity were analyzed fluorometrically during live muscle fiber respiration (Oroboros O2k) at 3-, 7-, 14-, 21-, and 30-days post-injury. Study 2: Male C57BL/6J mice (n=32) were randomized into two groups: VML and VML+SS-31. SS-31 is a mitochondrial-targeted peptide with ROS-scavenging properties. VML+SS-31 received SS-31 (8mg/kg/day) for 14 days, and the VML received equal volume saline injections. At 14- and 28-days post-VML, mitochondrial respiration and electron conductance, ROS emission, and antioxidant buffering capacity were assessed in permeabilized gastrocnemius muscle fibers from the muscle remaining after the initial injury. Group differences were detected through ANOVA and unpaired t-test. ROS emission and antioxidant buffering capacity were different between VML-injured and Uninjured mice through 14-days post-injury, specifically at 3-, 7-, and 14-days, mitochondrial ROS emission was significantly higher (+28%, p&lt;0.001), and antioxidant buffering capacity was less (-28%, p=0.002) in VML mice compared to Uninjured mice. Two weeks of SS-31 treatment lead to greater muscle fiber respiration and electron conductance (p≤0.008) at both 14- and 28-days post-VML. Importantly, at 14-days post-injury, mitochondrial ROS emission was 71% lower in VML+SS-31 than VML. At both 14- and 28-days post-injury, antioxidant buffering capacity was 12% greater in VML+SS-31 (p≤0.003) compared to VML. Oxidative stress is a feature of early VML pathophysiology marked by greater ROS emission and less antioxidant buffering capacity. Attenuating oxidative stress during the first two-weeks post-VML injury improves mitochondrial respiration. W81XWH-20-1-0885 and R01AR078903 to SG and JC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of Elamipretide on the Vitrification of Mouse Ovarian Tissue by Freezing.

The importance of ovarian cortical cryopreservation in fertility preservation is receiving increasing attention from reproductive specialists, and mitochondrial dysfunction is an important cause of reduced ovarian tissue cryopreservation. Elamipretide (SS-31) is a novel mitochondria-targeted antioxidant. However, whether it has a protective effect on mouse ovarian tissue cryopreservation remains to be studied. In this study, we examined follicular morphology and viability, mitochondrial function and oxidative stress levels, apoptosis, and culture in vitro after vitrification cryoresuscitation operation by treating ovarian tissues with SS-31 in cryoprotectant resuscitation solution. At the end of the experiment, the addition of 100 μmol/L SS-31 significantly improved follicle quality and oocyte maturation rate in vitro (p < 0.05) and significantly reduced apoptosis (p < 0.05) and oxidative stress levels (superoxide dismutase, catalase, malondialdehyde, p < 0.05). Meanwhile, mitochondrial respiratory chain complex enzyme activity, mtDNA copy number (p < 0.05), and adenosine triphosphate (p < 0.05) content were significantly increased in the 100 μmol/L SS-31-treated group. In addition, the mRNA expression levels of mitochondrial energy metabolism- and biosynthesis-related genes (STRT1, PGC-1a, PPAR-a, TFAM, p < 0.05) were markedly upregulated (p < 0.05) in the 100 μmol/L SS-31 group. In conclusion, SS-31 improved the cryopreservation of ovarian tissues, and 100 μmol/L SS-31 was found to be the most effective.

Tetrahedral framework nucleic acids’ role in facilitating chronic diabetic wound repair via the endoplasmic reticulum-mitochondrial pathway

Under diabetic circumstances, keratinocytes are constantly exposed to hyperglycaemic micromilieu, leading to disrupted differentiation and increased chronic wound susceptibility during to sustained endoplasmic reticulum (ER) stress (ERS) and mitochondrial dysfunction during hyperglycaemia. As mitochondrial respiratory chain activity is a primary generator of intracellular reactive oxygen species (ROS), we postulated that tFNAs could potentially boost mitochondrial function and energy metabolism in the presence of oxidative stress. To this end, we first explored the ER-mitochondrial (ER-MITO) axis-associated intracellular transportation of tetrahedral framework nucleic acids (tFNAs). tFNAs are transported to the ER and mitochondria via caveolae-mediated endocytosis, thereby regulating the ER-MITO axis. Additionally, tFNAs reduce ERS by downregulating PERK-ATF 4-CHOP, thereby stabilising mitochondrial dynamics, improving energy metabolism, maintaining membrane potential polarisation, and scavenging generated reactive oxygen species to counteract oxidative stress during hyperglycaemia. Consequently, tFNAs restore cell proliferation, migration, and differentiation and enhance chronic wound healing and skin graft survival. Furthermore, tFNAs repaired the diabetic skin mechanical barrier, underscoring their versatility of ER-MITO axis modulation. Overall, this study highlights the importance of tFNAs in ER-MITO axis modulation and a novel therapeutic approach for chronic diabetic wound healing.

Exploring the Interplay between Mitochondrial DNA and Lifestyle Factors in the Pathogenesis of Psychiatric Disorders

The objectives of this study were to investigate the interaction of mitochondrial DNA (mtDNA) and lifestyle factors in the development of psychiatric disorders and to gain greater insight into their pathogenesis and comorbidity. We analyzed data from approximately 150,000 individuals from the UK Biobank. Mitochondrial gene-by-environment interaction studies (mtGEIS) were performed to assess the relationships between mtDNA and psychiatric disorders, such as anxiety, depression, and self-harm. These disorders were defined using diagnostic and severity indicators derived from the General Anxiety Disorder (GAD-7) and Patient Health Questionnaire (PHQ-9). Smoking and drinking behaviors were characterized based on UK Biobank criteria. For the mtGEIS, logistic and linear regression models from PLINK 2.0 were employed, accounting for covariates like age, sex, PC1-10, Townsend Deprivation Index (TDI), and educational attainment. We also conducted sex-stratified analyses to detect any gender-specific effects. Our findings highlighted significant associations between mtDNA and three psychiatric disorders. Moreover, the interplay between mtDNA and lifestyle factors showed significant associations with psychiatric disorders (all P values &lt; 0.05). Specifically, two mutant loci, T5004C (BAnx_self=−0.0026, BDep_self=−0.0024, BSelf−harm=−0.0018) and G9123A (BAnx_self=−0.0030, BDep_self=−0.0024, BSelf−harm=−0.0017), were found to reduce the risk of three disorders after interacting with alcohol. Sex-specific differences were also observed. In summary, the expression of mitochondrial genes could be modulated by lifestyle factors like smoking and drinking, potentially affecting psychiatric disorders. These habits might influence mitochondrial respiratory chain activity and the replication and transcriptional regulation of mitochondrial genes, culminating in changes in mitochondrial functionality and subsequently psychiatric disorders.

MORN2 regulates the morphology and energy metabolism of mitochondria and is required for male fertility in mice

BackgroundMitochondria produce adenosine triphosphate through respiratory activities to power sperm differentiation and motility, and decreased mitochondrial respiratory activity can result in poor sperm motility and asthenospermia. The mitochondrial sheath is a component of the mid-piece of the sperm flagellum, and dysfunction of the sheath can reduce sperm motility and cause male infertility. The membrane occupation and recognition nexus-motif protein 2 (MORN2) is testis enriched in mice, and the MORN motif was reported to play a role in the regulation of bioelectrical signal homeostasis in cardiomyocytes.MethodsWe generated Morn2–/– mice using CRISPR/Cas9 and evaluated the potential functions of MORN2 in spermiogenesis through histological analysis, fertility examination, RT-PCR, CASA, immunofluorescence, TUNEL, electron microscopy analysis, mitochondrial energy metabolism analysis, etc.ResultsThe Morn2–/– mice were infertile, and their sperm showed severe motility defects. Morn2–/– sperm also had abnormal morphology characterized by bent heads, aberrant mitochondrial sheath formation, lower mitochondrial membrane potential, higher levels of reactive oxygen species, and decreased mitochondrial respiratory activity.ConclusionsOur study demonstrates that MORN2 is essential for male fertility and indicates that MORN2 functions in mitochondrial sheath formation and regulates mitochondrial respiratory activity.

L-Citrulline Ameliorates Iron Metabolism and Mitochondrial Quality Control via Activating AMPK Pathway in Intestine and Improves Microbiota in Mice with Iron Overload.

Oxidative stress caused by iron overload tends to result in intestinal mucosal barrier dysfunction and intestinalmicrobiotaimbalance. As a neutral and nonprotein amino acid, L-Citrulline (L-cit) has been implicated in antioxidant and mitochondrial amelioration properties. This study investigates whether L-cit can alleviate iron overload-induced intestinal injury and explores the underlying mechanisms. C57BL/6J mice are intraperitoneally injected with iron dextran, then gavaged with different dose of L-cit for 2 weeks. L-cit treatment significantly alleviates intestine pathological injury, oxidative stress, ATP level, and mitochondrial respiratory chain complex activities, accompanied by ameliorating mitochondrial quality control. L-cit-mediated protection is associated with the upregulation of Glutathione Peroxidase 4 (GPX4) expression, inhibition Nuclear Receptor Coactivator 4 (NCOA4)-mediated ferritinophagy and ferroptosis, and improvement of gut microbiota. To investigate the underlying molecular mechanisms, Intestinal Porcine Epithelial Cell line-J2 (IPEC-J2) cells are treated with L-cit or AMP-activated Protein Kinase (AMPK) inhibitor. AMPK signaling has been activated by L-cit. Notably, Compound C abolishes L-cit's protection on intestinal barrier, mitochondrial function, and antioxidative capacity in IPEC-J2 cells. L-cit may restrain ferritinophagy and ferroptosis to regulate iron metabolism, and induce AMPK pathway activation, which contributes to exert antioxidation, ameliorate iron metabolism and mitochondrial quality control, and improve intestinal microbiota. L-cit is a promising therapeutic strategy for iron overload-induced intestinal injury.

Pyruvate dehydrogenase kinase 2 knockdown restores the ability of amyotrophic lateral sclerosis-linked SOD1G93A rat astrocytes to support motor neuron survival by increasing mitochondrial respiration.

Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron (MN) degeneration. Various studies using cellular and animal models of ALS indicate that there is a complex interplay between MN and neighboring non-neuronal cells, such as astrocytes, resulting in noncell autonomous neurodegeneration. Astrocytes in ALS exhibit a lower ability to support MN survival than nondisease-associated ones, which is strongly correlated with low-mitochondrial respiratory activity. Indeed, pharmacological inhibition of pyruvate dehydrogenase kinase (PDK) led to an increase in the mitochondrial oxidative phosphorylation pathway as the primary source of cell energy in SOD1G93A astrocytes and restored the survival of MN. Among the four PDK isoforms, PDK2 is ubiquitously expressed in astrocytes and presents low expression levels in neurons. Herein, we hypothesize whether selective knockdown of PDK2 in astrocytes may increase mitochondrial activity and, in turn, reduce SOD1G93A-associated toxicity. To assess this, cultured neonatal SOD1G93A rat astrocytes were incubated with specific PDK2 siRNA. This treatment resulted in a reduction of the enzyme expression with a concomitant decrease in the phosphorylation rate of the pyruvate dehydrogenase complex. In addition, PDK2-silenced SOD1G93A astrocytes exhibited restored mitochondrial bioenergetics parameters, adopting a more complex mitochondrial network. This treatment also decreased lipid droplet content in SOD1G93A astrocytes, suggesting a switch in energetic metabolism. Significantly, PDK2 knockdown increased the ability of SOD1G93A astrocytes to support MN survival, further supporting the major role of astrocyte mitochondrial respiratory activity in astrocyte-MN interactions. These results suggest that PDK2 silencing could be a cell-specific therapeutic tool to slow the progression of ALS.

JinChan YiShen TongLuo Formula ameliorate mitochondrial dysfunction and apoptosis in diabetic nephropathy through the HIF-1α-PINK1-Parkin pathway

Ethnopharmacological relevanceThe JinChan YiShen TongLuo (JCYSTL) formula, a traditional Chinese medicine (TCM), has been used clinically for decades to treat diabetic nephropathy (DN). TCM believes that the core pathogenesis of DN is “kidney deficiency and collateral obstruction," and JCYSTL has the effect of “tonifying kidney and clearing collateral," thus alleviating the damage to kidney structure and function caused by diabetes. From the perspective of modern medicine, mitochondrial damage is an important factor in DN pathogenesis. Our study suggests that the regulation of mitophagy and mitochondrial function by JCYSTL may be one of the internal mechanisms underlying its good clinical efficacy. Aim of the studyThis study aimed to investigate the mechanisms underlying the renoprotective effects of JCYSTL. Materials and methodsUnilateral nephrectomy combined with low-dose streptozotocin intraperitoneally injected in a DN rat model and high glucose (HG) plus hypoxia-induced HK-2 cells were used to explore the effects of JCYSTL on the HIF-1α/mitophagy pathway, mitochondrial function and apoptosis. ResultsJCYSTL treatment significantly decreased albuminuria, serum creatinine, blood urea nitrogen, and uric acid levels and increased creatinine clearance levels in DN rats. In vitro, medicated serum containing JCYSTL formula increased mitochondrial membrane potential (MMP); improved activities of mitochondrial respiratory chain complexes I, III, and IV; decreased the apoptotic cell percentage and apoptotic protein Bax expression; and increased anti-apoptotic protein Bcl-2 expression in HG/hypoxia-induced HK-2 cells. The treatment group exhibited increased accumulation of PINK1, Parkin, and LC3-II and reduced P62 levels in HG/hypoxia-induced HK-2 cells, whereas in PINK1 knockdown HK-2 cells, JCYSTL did not improve the HG/hypoxia-induced changes in Parkin, LC3-II, and P62. When mitophagy was impaired by PINK1 knockdown, the inhibitory effect of JCYSTL on Bax and its promoting effect on MMP and Bcl-2 disappeared. The JCYSTL-treated group displayed significantly higher HIF-1α expression than the model group in vivo, which was comparable to the effects of FG-4592 in DN rats. PINK1 knockdown did not affect HIF-1α accumulation in JCYSTL-treated HK-2 cells exposed to HG/hypoxia. Both JCYSTL and FG-4592 ameliorated mitochondrial morphological abnormalities and reduced the mitochondrial respiratory chain complex activity in the renal tubules of DN rats. Mitochondrial apoptosis signals in DN rats, such as increased Bax and Caspase-3 expression and apoptosis ratio, were weakened by JCYSTL or FG-4592 administration. ConclusionThis study demonstrates that the JCYSTL formula activates PINK1/Parkin-mediated mitophagy by stabilizing HIF-1α to protect renal tubules from mitochondrial dysfunction and apoptosis in diabetic conditions, presenting a promising therapy for the treatment of DN.

Abstract B070: Myc synthetic lethality as a therapeutic strategy for resistance: Insights into the targetability of mitochondrial complex I in Myc-high TNBC

Abstract Patients with advanced triple-negative breast cancer (TNBC) frequently develop chemoresistance, emphasizing an urgent need for novel therapeutic strategies able to eradicate treatment resistant tumor cells. Recent studies have highlighted MYC as a common oncogenic alteration and a driver of stem-like TNBC phenotype, which commonly features high mitochondrial respiratory activity instated through OXPHOS system and chemoresistance. Interestingly, emerging evidence suggests that residual tumors from chemo treatment remain susceptible to OXPHOS inhibition through inhibition of respiratory complex I. Our previous studies have revealed a specific MYC-dependent and tumor suppressive synthetic lethal action for combination of metformin and BH3 mimetics in cells, ex vivo and in vivo models of aggressive breast cancer (PMID: 30728358). Metformin is a systemic and local metabolic modulator, which acts as a weak complex I inhibitor and promoter of AMPK activation. Here, we have explored the role of MYC in OXPHOS regulation, to address the question if metformin specifically and lethally targets MYC-modulated OXPHOS in MYC-high TNBC. We found that a selective c-MYC inhibitor (EN4) leads to a significant decrease in complex I activity despite only a modest drop in complex I mass in endogenously MYC-high TNBC cell lines. Conversely, engineered MYC overexpression in these cells upregulated complex I activity. Our current work explores transcriptomic, proteomic, and functional approaches to the mechanisms through which MYC regulates complex I activity, structure, and dynamics, ultimately hoping to define a biological rationale for therapeutic targeting of OXPHOS in Myc-high TNBC. Citation Format: Antti O Hiltunen, Linda J Id, Juha Klefström. Myc synthetic lethality as a therapeutic strategy for resistance: Insights into the targetability of mitochondrial complex I in Myc-high TNBC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B070.

Functional resilience: An active oxidative phosphorylation system prevails amid foreign proteins in holoparasitic plants

Mitonuclear incompatibility results from a breakdown of the coordinated function between co-evolved genes located in nuclear and mitochondrial compartments. Horizontal Gene Transfer (HGT), involving the acquisition of genes from unrelated species, can trigger mitonuclear incompatibilities when foreign gene products interact with native ones, particularly in multisubunit complexes. Recent findings highlighted rampant HGT in the mitochondrial genomes of holoparasitic plants of the genus Lophophytum (Balanophoraceae). In Lophophytum, some mitochondrial genes involved in the Oxidative Phosphorylation (OXPHOS) system were acquired from their legume hosts, unlike the nuclear-encoded OXPHOS subunits, which remain native. This unique configuration of a doubly chimeric OXPHOS, combining native nuclear-encoded subunits with both foreign and native mitochondrial-encoded subunits, raises questions regarding the potential effects of the interactions between native and foreign proteins on mitochondrial respiration activity in Lophophytum. We examined the mitochondrial ultrastructure, evaluated protein expression via Western blots, and analyzed cellular respiration through oxygen consumption rates and adenylate content in these holoparasitic plants. Surprisingly, our results revealed no disruption of the OXPHOS machinery or activity in Lophophytum despite the functional replacement of several native protein subunits by foreign homologs. Furthermore, there was no apparent impact on the OXPHOS system given their parasitic lifestyle and complete loss of photosynthesis.

Ex Vivo 13C-Metabolic Flux Analysis of Porcine Circulating Immune Cells Reveals Cell Type-Specific Metabolic Patterns and Sex Differences in the Pentose Phosphate Pathway.

In general, females present with stronger immune responses than males, but scarce data are available on sex-specific differences in immunometabolism. In this study, we characterized porcine peripheral blood mononuclear cell (PBMC) and granulocyte energy metabolism using a Bayesian 13C-metabolic flux analysis, which allowed precise determination of the glycolytic, pentose phosphate pathway (PPP), and tricarboxylic acid cycle (TCA) fluxes, together with an assessment of the superoxide anion radical (O2•-) production and mitochondrial O2 consumption. A principal component analysis allowed for identifying the cell type-specific patterns of metabolic plasticity. PBMCs displayed higher TCA cycle activity, especially glutamine-derived aspartate biosynthesis, which was directly related to mitochondrial respiratory activity and inversely related to O2•- production. In contrast, the granulocytes mainly utilized glucose via glycolysis, which was coupled to oxidative PPP utilization and O2•- production rates. The granulocytes of the males had higher oxidative PPP fluxes compared to the females, while the PBMCs of the females displayed higher non-oxidative PPP fluxes compared to the males associated with the T helper cell (CD3+CD4+) subpopulation of PBMCs. The observed sex-specific differences were not directly attributable to sex steroid plasma levels, but we detected an inverse correlation between testosterone and aldosterone plasma levels and showed that aldosterone levels were related with non-oxidative PPP fluxes of both cell types.

GLUD1 inhibits hepatocellular carcinoma progression via ROS-mediated p38/JNK MAPK pathway activation and mitochondrial apoptosis

Glutamate dehydrogenase 1 (GLUD1) is an important enzyme in glutamine metabolism. Previously, we found GLUD1 was down-regulated in tumor tissues of hepatocellular carcinoma (HCC) patients by proteomics study. To explore its role in the progression of HCC, the expressional level of GLUD1 was firstly examined and presented as that both the protein and mRNA levels were down-regulated in tumor tissues compared to the normal liver tissues. GLUD1 overexpression significantly inhibited HCC cells proliferation, migration, invasion and tumor growth both in vitro and in vivo, while GLUD1 knocking-down promoted HCC progression. Metabolomics study of GLUD1 overexpressing and control HCC cells showed that 129 differentially expressed metabolites were identified, which mainly included amino acids, bases, and phospholipids. Moreover, metabolites in mitochondrial oxidative phosphorylation system (OXPHOS) were differentially expressed in GLUD1 overexpressing cells. Mechanistic studies showed that GLUD1 overexpression enhanced mitochondrial respiration activity and reactive oxygen species (ROS) production. Excessive ROS lead to mitochondrial apoptosis that was characterized by increased expression levels of p53, Cytochrome C, Bax, Caspase 3 and decreased expression level of Bcl-2. Furthermore, we found that the p38/JNK MAPK pathway was activated in GLUD1 overexpressing cells. N-acetylcysteine (NAC) treatment eliminated cellular ROS and blocked p38/JNK MAPK pathway activation, as well as cell apoptosis induced by GLUD1 overexpression. Taken together, our findings suggest that GLUD1 inhibits HCC progression through regulating cellular metabolism and oxidative stress state, and provide that ROS generation and p38/JNK MAPK pathway activation as promising methods for HCC treatment.

Critical contribution of mitochondria in the development of cardiomyopathy linked to desmin mutation.

Beyond the observed alterations in cellular structure and mitochondria, the mechanisms linking rare genetic mutations to the development of heart failure in patients affected by desmin mutations remain unclear due in part, to the lack of relevant human cardiomyocyte models. To shed light on the role of mitochondria in these mechanisms, we investigated cardiomyocytes derived from human induced pluripotent stem cells carrying the heterozygous DESE439K mutation that were either isolated from a patient or generated by gene editing. To increase physiological relevance, cardiomyocytes were either cultured on an anisotropic micropatterned surface to obtain elongated and aligned cardiomyocytes, or as a cardiac spheroid to create a micro-tissue. Moreover, when applicable, results from cardiomyocytes were confirmed with heart biopsies of suddenly died patient of the same family harboring DESE439K mutation, and post-mortem heart samples from five control healthy donors. The heterozygous DESE439K mutation leads to dramatic changes in the overall cytoarchitecture of cardiomyocytes, including cell size and morphology. Most importantly, mutant cardiomyocytes display altered mitochondrial architecture, mitochondrial respiratory capacity and metabolic activity reminiscent of defects observed in patient's heart tissue. Finally, to challenge the pathological mechanism, we transferred normal mitochondria inside the mutant cardiomyocytes and demonstrated that this treatment was able to restore mitochondrial and contractile functions of cardiomyocytes. This work highlights the deleterious effects ofDESE439Kmutation, demonstrates the crucial role of mitochondrial abnormalities in the pathophysiology of desmin-related cardiomyopathy, and opens up new potential therapeutic perspectives for this disease.

Comparison of HepaRG and HepG2 cell lines to model mitochondrial respiratory adaptations in non‑alcoholic fatty liver disease.

Although some clinical studies have reported increased mitochondrial respiration in patients with fatty liver and early non‑alcoholic steatohepatitis (NASH), there is a lack of invitro models of non‑alcoholic fatty liver disease (NAFLD) with similar findings. Despite being the most commonly used immortalized cell line for invitro models of NAFLD, HepG2 cells exposed to free fatty acids (FFAs) exhibit a decreased mitochondrial respiration. On the other hand, the use of HepaRG cells to study mitochondrial respiratory changes following exposure to FFAs has not yet been fully explored. Therefore, the present study aimed to assess cellular energy metabolism, particularly mitochondrial respiration, and lipotoxicity in FFA‑treated HepaRG and HepG2 cells. HepaRG and HepG2 cells were exposed to FFAs, followed by comparative analyses that examained cellular metabolism, mitochondrial respiratory enzyme activities, mitochondrial morphology, lipotoxicity, the mRNA expression of selected genes and triacylglycerol (TAG) accumulation. FFAs stimulated mitochondrial respiration and glycolysis in HepaRG cells, but not in HepG2 cells. Stimulated complex I, II‑driven respiration and β‑oxidation were linked to increased complex I and II activities in FFA‑treated HepaRG cells, but not in FFA‑treated HepG2 cells. Exposure to FFAs disrupted mitochondrial morphology in both HepaRG and HepG2 cells. Lipotoxicity was induced to a greater extent in FFA‑treated HepaRG cells than in FFA‑treated HepG2 cells. TAG accumulation was less prominent in HepaRG cells than in HepG2 cells. On the whole, the present study demonstrates that stimulated mitochondrial respiration is associated with lipotoxicity in FFA‑treated HepaRG cells, but not in FFA‑treated HepG2 cells. These findings suggest that HepaRG cells are more suitable for assessing mitochondrial respiratory adaptations in the developed invitro model of early‑stage NASH.