Mitochondrial Respiratory Parameters Research Articles

Curcumin pre-treatment may protect against mitochondrial damage in LRRK2-mutant Parkinson's disease and healthy control fibroblasts

Mitochondrial dysfunction has been proposed as one of the pathobiological underpinnings in Parkinson's disease. Environmental stressors, such as paraquat, induce mitochondrial dysfunction and promote reactive oxygen species production. Targeting oxidative stress pathways could prevent mitochondrial dysfunction and thereby halt the neurodegeneration in Parkinson's disease. Since curcumin is touted as an antioxidant and neuroprotective agent, the aim of this study was to investigate if curcumin is a suitable therapy to target mitochondrial dysfunction in Parkinson's disease using a paraquat-toxicity induced model in fibroblasts from LRRK2-mutation positive Parkinson's disease individuals and healthy controls. The fibroblasts were exposed to five treatment groups, (i) untreated, (ii) curcumin only, (iii) paraquat only, (iv) pre-curcumin group: with curcumin for 2hr followed by paraquat for 24hr and (v) post-curcumin group: with paraquat for 24hr followed by curcumin for 2hr. Mitochondrial function was determined by measuring three parameters of mitochondrial respiration (maximal respiration, ATP-associated respiration, and spare respiratory capacity) using the Seahorse XFe96 Extracellular Flux Analyzer. As expected, paraquat effectively disrupted mitochondrial function for all parameters. Pre-curcumin treatment improved maximal and ATP-associated respiration whereas, post-curcumin treatment had no effect. These findings indicate that curcumin may be most beneficial as a pre-treatment before toxin exposure, which has implications for its therapeutic use. These promising findings warrant future studies testing different curcumin dosages, exposure times and curcumin formulations in larger sample sizes of Parkinson's disease and control participants.

Do Autophagy Enhancers/ROS Scavengers Alleviate Consequences of Mild Mitochondrial Dysfunction Induced in Neuronal-Derived Cells?

Mitochondrial function is at the nexus of pathways regulating synaptic-plasticity and cellular resilience. The involvement of brain mitochondrial dysfunction along with increased reactive oxygen species (ROS) levels, accumulating mtDNA mutations, and attenuated autophagy is implicated in psychiatric and neurodegenerative diseases. We have previously modeled mild mitochondrial dysfunction assumed to occur in bipolar disorder (BPD) using exposure of human neuronal cells (SH-SY5Y) to rotenone (an inhibitor of mitochondrial-respiration complex-I) for 72 and 96 h, which exhibited up- and down-regulation of mitochondrial respiration, respectively. In this study, we aimed to find out whether autophagy enhancers (lithium, trehalose, rapamycin, and resveratrol) and/or ROS scavengers [resveratrol, N-acetylcysteine (NAC), and Mn-Tbap) can ameliorate neuronal mild mitochondrial dysfunction. Only lithium (added for the last 24/48 h of the exposure to rotenone for 72/96 h, respectively) counteracted the effect of rotenone on most of the mitochondrial respiration parameters (measured as oxygen consumption rate (OCR)). Rapamycin, resveratrol, NAC, and Mn-Tbap counteracted most of rotenone’s effects on OCR parameters after 72 h, possibly via different mechanisms, which are not necessarily related to their ROS scavenging and/or autophagy enhancement effects. The effect of lithium reversing rotenone’s effect on OCR parameters is compatible with lithium’s known positive effects on mitochondrial function and is possibly mediated via its effect on autophagy. By-and-large it may be summarized that some autophagy enhancers/ROS scavengers alleviate some rotenone-induced mild mitochondrial changes in SH-SY5Y cells.

Angiotensin II and Angiotensin‐(1‐7) Modulate Mitochondrial Respiration in Mouse Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) constitute bone marrow niches that support homing and quiescence of hematopoietic stem cells via paracrine functions. MSCs have the potential of differentiating into a variety of cell types and therefore have therapeutic potential in regenerative medicine. Mitochondria modulates cellular functions including paracrine functions by which MSCs modulate quiescence and differentiation of stem cells. Previous studies have provided evidence for the expression of renin-angiotensin system in MSCs. The current study evaluated the effects of Angiotensin II (Ang II) and Angiotensin-(1-7) (Ang-(1-7)) on mitochondrial respiration in mouse bone marrow MSCs. Study was carried out in cultured MSCs, passage eight or nine, and mitochondrial respiration was evaluated by using Seahorse (XFp). Expression of ACE, ACE2, AT1R, AT2R and Mas receptor were evaluated by real-time PCR and western blotting. Oxygen consumption rate (OCR) was determined by carrying out mito-stress test. For measuring OCR in real-time, the mito-stress test was carried out, which involved use of different pharmacological agents that modulate mitochondrial electron transport chain such as oligomycin (2µm), FCCP (0.5µm), rotenone/ antimycin A (0.5µm) to determine ATP-linked respiration, maximal respiration, non-mitochondrial respiration, respectively, after recording basal respiration. PCR detected mRNA transcripts of ACE, AT1R, AT2R, and Mas, but not ACE2. Mito-stress test showed dose-dependent (0.1nm, 1nm, 10nm) increase in basal, ATP-linked, maximal, and nonmitochondrial respiration by Ang-(1-7) and effects at 10nm concentration were significant (P<0.05, n=5) compared to the untreated cells (Figure 1). All these changes were blocked by A-779 (1µm). All of the mitochondrial respiratory parameters were significantly (P<0.05, n=5) decreased when only A-779 was present compared to untreated cells. Ang II (100nm) did not alter OCR in MSCs in mito-stress test (Figure 1). Proton leak was not changed by any treatments. Intriguingly, treatment with PD123391 (1µm) significantly increased basal respiration and maximal respiration compared to untreated cells (P<0.05). These effects were not observed when cells were treated with Ang II along with PD123391. Along similar lines, losartan (10µm) significantly increased the basal respiration compared to untreated MSCs (P<0.05), and this change was not observed in the presence of Ang II. This preliminary study shows that mitochondrial oxygen consumption in MSCs is differentially modulated by angiotensin peptides. Further investigations are needed to address the significance of mitochondrial modulation in the regenerative functions of MSCs by angiotensins.

Sinularin, an Anti-Cancer Agent Causing Mitochondria-Modulated Apoptosis and Cytoskeleton Disruption in Human Hepatocellular Carcinoma.

Liver cancer remains a leading cause of death, despite advances in anti-cancer therapies. To develop novel drugs, natural products are being considered as a good source for exploration. In this study, a natural product isolated from a soft coral was applied to evaluate its anti-cancer activities in hepatocellular carcinoma SK-HEP-1 cells. Sinularin was determined to have half-maximal inhibitory concentration (IC50) values of ~10 μM after 24, 48, and 72 h. The TUNEL assay and annexin V/PI staining results showed that sinularin induced DNA fragmentation and apoptosis, respectively. An investigation at the molecular level demonstrated that the expression levels of cleaved caspases 3/9 were significantly elevated at 10 μM sinularin. Mitochondrial and intracellular reactive oxygen species (ROS) levels were significantly increased following sinularin treatment, which also affected the mitochondrial membrane potential. In addition, it significantly lowered the mitochondrial respiration parameters and extracellular acidification rates at 10 μM. Further investigation showed that sinularin significantly attenuated wound healing, cell migration, and potential colony formation at 10 μM. Fluorescence microscopic observations showed that the distribution of F-actin filaments was significantly altered at 10 μM sinularin. Supported by Western blot analyses, the expression levels of AKT, p-ERK (extracellular-signal-related kinase), vimentin and VEGF were significantly down-regulated, whereas p-p38, pJNK and E-cadherin were significantly increased. Overall, at the IC50 concentration, sinularin was able to significantly affect SK-HEP-1 cells.

DRP1 haploinsufficiency attenuates cardiac ischemia/reperfusion injuries.

Mitochondrial dynamics is a possible modulator of myocardial ischemia/reperfusion injuries (IRI). We previously reported that mice partially deficient in the fusion protein OPA1 exhibited higher IRI. Therefore, we investigated whether deficiency in the fission protein DRP1 encoded by Dnm1l gene would affect IRI in Dnm1l+/- mouse. After baseline characterization of the Dnm1l+/- mice heart, using echocardiography, electron microscopy, and oxygraphy, 3-month-old Dnm1l+/- and wild type (WT) mice were exposed to myocardial ischemia/reperfusion (I/R). The ischemic area-at-risk (AAR) and area of necrosis (AN) were delimited, and the infarct size was expressed by AN/AAR. Proteins involved in mitochondrial dynamics and autophagy were analyzed before and after I/R. Mitochondrial permeability transition pore (mPTP) opening sensitivity was assessed after I/R. Heart weight and left ventricular function were not significantly different in 3-, 6- and 12-month-old Dnm1l+/- mice than in WT. The cardiac DRP1 protein expression levels were 60% lower, whereas mitochondrial area and lipid degradation were significantly higher in Dnm1l+/- mice than in WT, though mitochondrial respiratory parameters and mPTP opening did not significantly differ. Following I/R, the infarct size was significantly smaller in Dnm1l+/- mice than in WT (34.6±3.1% vs. 44.5±3.3%, respectively; p<0.05) and the autophagic markers, LC3 II and P62 were significantly increased compared to baseline condition in Dnm1l+/- mice only. Altogether, data indicates that increasing fusion by means of Dnm1l deficiency was associated with protection against IRI, without alteration in cardiac or mitochondrial functions at basal conditions. This protection mechanism due to DRP1 haploinsufficiency increases the expression of autophagic markers.

Inhibition of Inducible Nitric Oxide Synthase Prevents IL-1β-Induced Mitochondrial Dysfunction in Human Chondrocytes.

Interleukin (IL)-1β is an important pro-inflammatory cytokine in the progression of osteoarthritis (OA), which impairs mitochondrial function and induces the production of nitric oxide (NO) in chondrocytes. The aim was to investigate if blockade of NO production prevents IL-1β-induced mitochondrial dysfunction in chondrocytes and whether cAMP and AMP-activated protein kinase (AMPK) affects NO production and mitochondrial function. Isolated human OA chondrocytes were stimulated with IL-1β in combination with/without forskolin, L-NIL, AMPK activator or inhibitor. The release of NO, IL-6, PGE2, MMP3, and the expression of iNOS were measured by ELISA or Western blot. Parameters of mitochondrial respiration were measured using a seahorse analyzer. IL-1β significantly induced NO release and mitochondrial dysfunction. Inhibition of iNOS by L-NIL prevented IL-1β-induced NO release and mitochondrial dysfunction but not IL-1β-induced release of IL-6, PGE2, and MMP3. Enhancement of cAMP by forskolin reduced IL-1β-induced NO release and prevented IL-1β-induced mitochondrial impairment. Activation of AMPK increased IL-1β-induced NO production and the negative impact of IL-1β on mitochondrial respiration, whereas inhibition of AMPK had the opposite effects. NO is critically involved in the IL-1β-induced impairment of mitochondrial respiration in human OA chondrocytes. Increased intracellular cAMP or inhibition of AMPK prevented both IL-1β-induced NO release and mitochondrial dysfunction.

Effect of per os administration of dihydroquercetin aqueous form on energy exchange in blood lymphocytes of rats with experimental cardiomyopathy

Cardiomyopathies are among the most severe myocardial pathologies, which are characterized by resistance to therapy and high mortality due to increasing heart failure and arrhythmia. Cardiomyocyte pathological changes upon cardiomyopathies are associated with mitochondrial dysfunction, leading to excessive formation of reactive oxygen species and the development of oxidative stress. In this regard, the study of the therapeutic potential of antioxidants in cardiomyopathies, as well as the mechanisms of their action on the functioning of mitochondria, is relevant and of high practical importance. The aim of this study was to determine the effect of oral 14-day administration of dihydroquercetin in a water-soluble form (DHQWF) on the activity of the key marker of mitochondrial respiration [succinate dehydrogenase (SDH)] and the cytoplasmic marker of glycolysis [lactate dehydrogenase (LDH)] in blood lymphocytes, as well as on the serum level of lipid peroxidation (LPO) in control rats and rats with experimental cardiomyopathy. Material and methods. Adult male Wistar rats (body weight 220-240 g) were used for the study. Isoprenaline hydrochloride was used to induce cardiomyopathy (IIC) in animals (twice subcutaneous injection at a dose of 150 mg/kg body weight, with a break of 24 hours). DHQ-WF was added to the drinking water for 14 days at the dose of 15 or 30 mg/kg body weight. SDH and LDH activity in lymphocytes was measured using a highly sensitive cytobiochemical method on a blood smear according to the reduction of nitrotetrazolium blue chloride to diformazan of dark blue color. The content of malone dialdehyde (MDA) in the blood serum, heart and liver mitochondria was determined spectrophotometrically using thiobarbituric acid. Mitochondria were isolated from rat tissues by the conventional method of differential centrifugation. Mitochondrial respiration was recorded using a polarographic method. Results. Experimental cardiomyopathy in rats was accompanied by a twofold increase in blood serum MDA level, as well as by a significant increase in SDH and LDH activity in blood lymphocytes. The oral administration of DHQ-WF in cardiomyopathy at a dose of 15 mg/kg body weight led to a significant decrease in serum MDA level, but did not reduce the activity of SDH and LDH in blood lymphocytes, compared with animals with cardiomyopathy that did not receive DHQ-WF. In the control group of animals, the use of DHQ-WF at a dose of 15 mg/kg body weight significantly increased blood lymphocyte LDH activity, but did not have a statistically significant effect on SDH activity and the parameters of mitochondrial respiration and oxidative phosphorylation, the level of MDA in heart and liver mitochondria. Increasing the dose of DHQ-WF administered to 30 mg/kg had less effect on changes in these parameters in control animals. Conclusion. The data obtained indicate that in experimental cardiomyopathy in rats, the course application of DHQ-WF at a dose of 15 mg/kg of body weight acts as an effective antioxidant that prevents the development of lipid peroxidation in blood serum, and can modulate energy metabolism towards the enhancement of glycolysis in blood lymphocytes in control animals.

390 Micronutrients, One-Carbon Metabolism, and Epigenetics: Potential Developmental and Production Outcomes

Abstract One-carbon metabolism is the network of biochemical pathways in which methyl groups are transferred from one compound to another for methylation processes. Expansion of the core pathway connects one-carbon metabolism to polyamine synthesis, nucleotide synthesis, redox metabolism, and the citric acid cycle. One-carbon metabolites (OCM) are methyl donors and cofactors which play key roles in the one-carbon metabolism pathway and include B-vitamins (choline, vitamin B12, vitamin B6, riboflavin, and folate), minerals (cobalt and sulfur) and amino acids (methionine, serine, and glycine). One-carbon metabolites are fundamental methyl donors for epigenetic modifications. Immediately post-fertilization, the embryonic genome undergoes epigenetic remodeling, and is the time when cell division is greatest (cell divisions/total cell number). At this time OCM supplementation may have its greatest impact on programming of offspring development, growth and postnatal performance, due to established metabolic roles in epigenetics (methyl transfer), growth (polyamine and nucleotide synthesis), and energetics. Limited data are available, however, which directly investigates the developmental effects of OCM supplementation in ruminants. In dairy cows, OCM supplementation in late gestation increased calf birth weight, nutrient sensing pathway activation, and offspring performance through the pre-weaning period. Methyl deficient diets during the pre-conception period in ewes altered the offspring hepatic methylome at 90 days of gestation, as well as the body composition and insulin tolerance of ram lambs at 22-mo of age. Bovine embryonic fibroblasts cultured in vitro with increasing OCM had greater growth rates and mitochondrial respiration parameters. Additional research into the area of one-carbon metabolism and the roles that OCM supplementation may play on postnatal function will provide new knowledge that could lead to altered management practices and increased efficiency of beef cattle. USDA is an equal opportunity provider and employer.

Участь кальційзалежних калієвих каналів великої провідності в модуляції параметрів дихання мітохондрій міокарда докозагексаєновою кислотою

Omega-3 polyunsaturated fatty acids (PUFA) provide protection against myocardial damage in ischemia-reperfusion. However, the mechanisms that provide cardioprotection are not fully understood. In this study, we investigated the effect of docosahexaenoic acid (DHA), a member of omega -3 PUFA, on mitochondrial respiration parameters and the role of mitochondrial calcium-dependent potassium channels of large conductance (ВКСа) in the implementation of these effects. Using the patch-clamp method, it was shown that functional ВКСа channels are expressed in the inner mitochondrial membrane of cardiac cells and their activity increases with the addition of DHA. We investigated the role of mitochondrial ВКСа channels in the regulation of mitochondrial respiratory processes. In experiments with isolated mitochondria from rat hearts, we showed that DHA prevented an increase in the respiratory rate of mitochondria in the V4 state and a decrease in the respiratory control elicited by addition of 10 μM Ca2+. Qualitatively the same effect was caused by NS1619, the ВКСа opener. In the presence of 10 μM Ca2+, the ВКСа channel inhibitor paxilin (1 μM) prevented the protective effect of DHA and NS1619 on the parameters of respiratory control. We conclude that mitochondrial ВКСа channels are involved in the implementation of the effects of DHA on mitochondrial respiration.

Insulin Promotes Mitochondrial Respiration and Survival through PI3K/AKT/GSK3 Pathway in Human Embryonic Stem Cells.

SummaryInsulin is an essential growth factor for the survival and self-renewal of human embryonic stem cells (hESCs). Although it is best known as the principal hormone promoting glycolysis in somatic cells, insulin's roles in hESC energy metabolism remain unclear. In this report, we demonstrate that insulin is essential to sustain hESC mitochondrial respiration that is rapidly decreased upon insulin removal. Insulin-dependent mitochondrial respiration is stem cell specific, and mainly relies on pyruvate and glutamine, while glucose suppresses excessive oxidative phosphorylation. Pharmacologic and genetic manipulations reveal that continuous insulin signal sustains mitochondrial respiration through PI3K/AKT activation and downstream GSK3 inhibition. We further show that insulin acts through GSK3 inhibition to suppress caspase activation and rescue cell survival. This study uncovers a critical role of the AKT/GSK3 pathway in the regulation of mitochondrial respiration and cell survival, highlighting insulin as an essential factor for accurate assessment of mitochondrial respiration in hESCs.

Advanced glycation end-products disrupt brain microvascular endothelial cell barrier: The role of mitochondria and oxidative stress

During diabetes mellitus, advanced glycation end-products (AGEs) are major contributors to the development of alterations in cerebral capillaries, leading to the disruption of the blood-brain barrier (BBB). Consequently, this is often associated with an amplified oxidative stress response in microvascular endothelial cells. As a model to mimic brain microvasculature, the bEnd.3 endothelial cell line was used to investigate cell barrier function. Cells were exposed to native bovine serum albumin (BSA) or modified BSA (BSA-AGEs). In the presence or absence of the antioxidant compound, N-acetyl-cysteine, cell permeability was assessed by FITC-dextran exclusion, intracellular free radical formation was monitored with H2DCF-DA probe, and mitochondrial respiratory and redox parameters were analyzed. We report that, in the absence of alterations in cell viability, BSA-AGEs contribute to an increase in endothelial cell barrier permeability and a marked and prolonged oxidative stress response. Decreased mitochondrial oxygen consumption was associated with these alterations and may contribute to reactive oxygen species production. These results suggest the need for further research to explore therapeutic interventions to restore mitochondrial functionality in microvascular endothelial cells to improve brain homeostasis in pathological complications associated with glycation.

Modulation of mitochondrial respiration rate and calcium-induced swelling by new cromakalim analogues

A cellular model of cardiomyocytes (H9c2 cell line) and mitochondria isolated from mouse liver were used to understand the drug action of BPDZ490 and BPDZ711, two benzopyran analogues of the reference potassium channel opener cromakalim, on mitochondrial respiratory parameters and swelling, by comparing their effects with those of the parent compound cromakalim. For these three compounds, the oxygen consumption rate (OCR) was determined by high-resolution respirometry (HRR) and their impact on adenosine triphosphate (ATP) production and calcium-induced mitochondrial swelling was investigated. Cromakalim did not modify neither the OCR of H9c2 cells and the ATP production nor the Ca-induced swelling. By contrast, the cromakalim analogue BPDZ490 (1) induced a strong increase of OCR, while the other benzopyran analogue BPDZ711 (2) caused a marked slowdown. For both compounds, 1 displayed a biphasic behavior while 2 still showed an inhibitory effect. Both compounds 1 and 2 were also found to decrease the ATP synthesis, with pronounced effect for 2, while cromakalim remained without effect. Overall, these results indicate that cromakalim, as parent molecule, does not induce per se any direct effect on mitochondrial respiratory function neither on whole cells nor on isolated mitochondria whereas both benzopyran analogues 1 and 2 display totally opposite behavior profiles, suggesting that compound 1, by increasing the maximal respiration capacity, might behave as a mild uncoupling agent and compound 2 is taken as an inhibitor of the mitochondrial electron-transfer chain.

Tenofovir alafenamide does not inhibit mitochondrial function and cholesterol biosynthesis in human T lymphoblastoid cell line

In clinical trials, the concentration of tenofovir diphosphate (TFV-DP) in peripheral mononuclear cells was 4 to 5-fold higher in individuals treated with tenofovir alafenamide (TAF) compared to individuals treated with tenofovir disoproxil fumarate (TDF). We hypothesized that the higher intracellular accumulation of TFV-DP could cause mitochondrial toxicity from either polymerase gamma (Pol-γ)-dependent or Pol-γ-independent mechanism(s). To test this hypothesis, we cultured human T lymphoblastoid cell line (CEM cells) for up to 12 days with TAF or TDF (multiplicities of Cmax) to investigate the effects on mitochondrial function and respiration, and cholesterol biosynthesis. Both TAF and TDF treatments had no significant effect on cell growth, mitochondrial potential (ΔΨ), production of reactive oxygen species (ROS), and mitochondrial respiratory parameters. TAF had no statistically significant effect on expression of Pol-γ mRNA, mitochondria DNA (mtDNA) content, expression of proteins of the electron transport chain (ETC), and key genes of cholesterol biosynthesis. TDF had significant reduction in mtDNA content at 8xCmax, and statistically significant reduction in mRNA expression of squalene epoxidase (SQLE). Our findings do not support our hypothesis that the higher intracellular accumulation of TFV-DP in cells treated with TAF could cause mitochondrial dysfunction. In conclusion, our findings add to the emerging data that TAF may have a low potential for causing mitochondrial toxicity in HIV-infected individuals on TAF-containing regimens.

Mitochondrial spare respiratory capacity: Mechanisms, regulation, and significance in non-transformed and cancer cells.

Mitochondrial metabolism must constantly adapt to stress conditions in order to maintain bioenergetic levels related to cellular functions. This absence of proper adaptation can be seen in a wide array of conditions, including cancer. Metabolic adaptation calls on mitochondrial function and draws on the mitochondrial reserve to meet increasing needs. Among mitochondrial respiratory parameters, the spare respiratory capacity (SRC) represents a particularly robust functional parameter to evaluate mitochondrial reserve. We provide an overview of potential SRC mechanisms and regulation with a focus on its particular significance in cancer cells.

Mitochondrial function parameters as a tool for tailored drug treatment of an individual with psychosis: a proof of concept study

Pharmacological treatment of mental disorders is currently decided based on "trial and error" strategy. Mitochondrial multifaceted dysfunction is assumed to be a major factor in the pathophysiology and treatment of schizophrenia (SZ) and bipolar disorder (BD). This study aimed to explore the feasibility of using a profile of mitochondrial function parameters as a tool to predict the optimal drug for an individual patient (personalized medicine). Healthy controls (n = 40), SZ (n = 48) and BD (n = 27) patients were recruited. Mental and global state of the subjects, six mitochondrial respiration parameters and 14 mitochondrial function-related proteins were assessed in fresh lymphocytes following in-vitro or in-vivo treatment with five antipsychotic drugs and two mood-stabilizers. In healthy controls, hierarchal clustering shows a drug-specific effect profile on the different mitochondrial parameters following in-vitro exposure. Similar changes were observed in untreated SZ and BD patients with psychosis. Following a month of treatment of the latter patients, only responders showed a significant correlation between drug-induced in-vitro effect (prior to in-vivo treatment) and short-term in-vivo treatment effect for 45% of the parameters. Long- but not short-term psychotropic treatment normalized mitochondria-related parameters in patients with psychosis. Taken together, these data substantiate mitochondria as a target for psychotropic drugs and provide a proof of concept for selective mitochondrial function-related parameters as a predictive tool for an optimized psychotropic treatment in a given patient. This, however, needs to be repeated with an expanded sample size and additional mitochondria related parameters.

Cardiometabolic risk factors are associated with immune cell mitochondrial respiration in humans.

Modifiable cardiometabolic risk factors induce the release of proinflammatory cytokines and reactive oxygen species from circulating peripheral blood mononuclear cells (PBMCs), resulting in increased cardiovascular disease risk and compromised immune health. These changes may be driven by metabolic reprogramming of PBMCs, resulting in reduced mitochondrial respiration; however, this has not been fully tested. We aimed to determine the independent associations between cardiometabolic risk factors including BMI, blood pressure, fasting glucose, and plasma lipids with mitochondrial respiration in PBMCs isolated from generally healthy individuals (n = 21) across the adult lifespan (12 men/9 women; age, 56 ± 21 yr; age range, 22-78 yr; body mass index, 27.9 ± 5.7 kg/m2; blood pressure, 123 ± 16/72 ± 10 mmHg; glucose, 90 ± 14 mg/dL; low-density lipoprotein cholesterol (LDL-C), 111 ± 22 mg/dL; and high-density lipoprotein cholesterol (HDL-C), 62 ± 16 mg/dL). PBMCs were isolated from whole blood by density-dependent centrifugation and used to assess mitochondrial function by respirometry. Primary outcomes included basal and maximal oxygen consumption rate (OCR), which were subsequently used to determine spare respiratory capacity and OCR metabolic potential. After we corrected for systolic blood pressure (SBP), diastolic blood pressure (DBP), and blood glucose, LDL-C was negatively associated with maximal respiration (r = -0.56, P = 0.016), spare respiratory capacity (r = -0.58, P = 0.012), and OCR metabolic potential (r = -0.71, P = 0.0011). In addition, SBP was negatively associated with OCR metabolic potential (r = -0.62, P = 0.0056) after we corrected for DBP, blood glucose, and LDL-C. These data suggest a link between blood cholesterol, SBP, and mitochondrial health that may provide insight into how cardiometabolic risk factors contribute to impaired immune cell function.NEW & NOTEWORTHY Independent of other cardiometabolic risk factors, low-density lipoprotein cholesterol, and systolic blood pressure were found to be negatively associated with several parameters of mitochondrial respiration in peripheral blood mononuclear cells of healthy adults. These data suggest that low-density lipoprotein cholesterol and systolic blood pressure may induce metabolic reprogramming of immune cells, contributing to increased cardiovascular disease risk and impaired immune health.

Acute Myocardial Infarction Reduces Respiration in Rat Cardiac Fibers, despite Adipose Tissue Mesenchymal Stromal Cell Transplant.

Adipose-derived mesenchymal stromal cell (AD-MSC) administration improves cardiac function after acute myocardial infarction (AMI). Although the mechanisms underlying this effect remain to be elucidated, the reversal of the mitochondrial dysfunction may be associated with AMI recovery. Here, we analyzed the alterations in the respiratory capacity of cardiomyocytes in the infarcted zone (IZ) and the border zone (BZ) and evaluated if mitochondrial function improved in cardiomyocytes after AD-MSC transplantation. Female rats were subjected to AMI by permanent left anterior descending coronary (LAD) ligation and were then treated with AD-MSCs or PBS in the border zone (BZ). Cardiac fibers were analyzed 24 hours (necrotic phase) and 8 days (fibrotic phase) after AMI for mitochondrial respiration, citrate synthase (CS) activity, F0F1-ATPase activity, and transmission electron microscopy (TEM). High-resolution respirometry of permeabilized cardiac fibers showed that AMI reduced numerous mitochondrial respiration parameters in cardiac tissue, including phosphorylating and nonphosphorylating conditions, respiration coupled to ATP synthesis, and maximal respiratory capacity. CS decreased in IZ and BZ at the necrotic phase, whereas it recovered in BZ and continued to drop in IZ over time when compared to Sham. Exogenous cytochrome c doubled respiration at the necrotic phase in IZ. F0F1-ATPase activity decreased in the BZ and, to more extent, in IZ in both phases. Transmission electron microscopy showed disorganized mitochondrial cristae structure, which was more accentuated in IZ but also important in BZ. All these alterations in mitochondrial respiration were still present in the group treated with AD-MSC. In conclusion, AMI led to mitochondrial dysfunction with oxidative phosphorylation disorders, and AD-MSC improved CS temporarily but was not able to avoid alterations in mitochondria function over time.

Mechanistic studies of cytotoxic activity of the mesoionic compound MIH 2.4Bl in MCF-7 breast cancer cells.

In the present study, the cytotoxic effects of a 1,3-thiazolium-5-thiolate derivative of a mesoionic compound, MIH 2.4Bl, were assessed in the MCF-7 breast cancer cell line. The cytotoxic effects of MIH 2.4Bl were determined using a crystal violet assay. Using a dose-response curve, the IC50 value of MIH 2.4Bl was determined to be 45.8±0.8 µM. Additionally, the effects of MIH 2.4Bl on mitochondrial respiration were characterized using oxygen consumption rate analysis. Treating MCF-7 cells with increasing concentrations of MIH 2.4Bl resulted in a significant reduction in all mitochondrial respiratory parameters compared with the control cells, indicative of an overall decrease in mitochondrial membrane potential. The induction of autophagy by MIH 2.4Bl was also examined by measuring changes in the expression of protein markers of autophagy. As shown by western blot analysis, treatment of MCF-7 cells with MIH 2.4Bl resulted in increased protein expression levels of Beclin-1 and ATG5, as well as an increase in the microtubule-associated protein 1A/1B light chain 3B (LC3B)-II to LC3B-I ratio compared with the control cells. Microarray analysis of changes in gene expression following MIH 2.4Bl treatment demonstrated 3,659 genes exhibited a fold-change ≥2. Among these genes, 779 were up-regulated, and 2,880 were down-regulated in cells treated with MIH 2.4Bl compared with the control cells. Based on the identity of the transcripts and fold-change of expression, six genes were selected for verification by reverse transcription-quantitative (RT-q)PCR; activating transcription factor 3, acidic repeat-containing protein, heparin-binding EGF-like growth factor, regulator of G-protein signaling 2, Dickkopf WNT signaling pathway inhibitor 1 and adhesion molecule with Ig like domain 2. The results of RT-qPCR analysis of RNA isolated from control and MIH 2.4Bl treated cells were consistent with the expression changes identified by microarray analysis. Together, these results suggest that MIH 2.4Bl may be a promising candidate for treating breast cancer and warrants further in vitro and in vivo investigation.

Impact of \u03b2-glycerophosphate on the bioenergetic profile of vascular smooth muscle cells

In chronic kidney disease, hyperphosphatemia is a key pathological factor promoting medial vascular calcification, a common complication associated with cardiovascular events and mortality. This active pathophysiological process involves osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs) via complex intracellular mechanisms that are still incompletely understood. Little is known about the effects of phosphate on the bioenergetic profile of VSMCs during the onset of this process. Therefore, the present study explored the effects of the phosphate donor β-glycerophosphate on cellular bioenergetics of VSMCs. Mitochondrial and glycolytic functions were determined utilizing extracellular flux analysis in primary human aortic VSMCs following exposure to β-glycerophosphate. In VSMCs, β-glycerophosphate increased basal respiration, mitochondrial ATP production as well as proton leak and decreased spare respiratory capacity and coupling efficiency, but did not modify non-mitochondrial or maximal respiration. β-Glycerophosphate-treated VSMCs had higher ability to increase mitochondrial glutamine and long-chain fatty acid usage as oxidation substrates to meet their energy demand. β-Glycerophosphate did not modify glycolytic function or basal and glycolytic proton efflux rate. In contrast, β-glycerophosphate increased non-glycolytic acidification. β-Glycerophosphate-treated VSMCs had a more oxidative and less glycolytic phenotype, but a reduced ability to respond to stressed conditions via mitochondrial respiration. Moreover, compounds targeting components of mitochondrial respiration modulated β-glycerophosphate-induced oxidative stress, osteo-/chondrogenic signalling and mineralization of VSMCs. In conclusion, β-glycerophosphate modifies key parameters of mitochondrial function and cellular bioenergetics in VSMCs that may contribute to the onset of phenotypical transdifferentiation and calcification. These observations advance the understanding of the role of energy metabolism in VSMC physiology and pathophysiology of vascular calcification during hyperphosphatemia.Key messagesβ-Glycerophosphate modifies key parameters of mitochondrial respiration in VSMCs.β-Glycerophosphate induces changes in mitochondrial fuel choice in VSMCs.β-Glycerophosphate promotes a more oxidative and less glycolytic phenotype of VSMCs.β-Glycerophosphate triggers mitochondrial-dependent oxidative stress in VSMCs.Bioenergetics impact β-glycerophosphate-induced VSMC calcification.

Energy Deficit Phenotype of Cerebral Microvascular Network in the Aged Brains

ObjectiveAging is associated with an increased risk of neurodegenerative diseases and stroke. Impaired microvascular function was proposed to be the connecting link, but the underlying mechanisms are unclear. Microvasculature is critical for cerebrovascular blood flow, neurovascular coupling, and nutrient transport, and blood‐brain‐barrier. Maintaining these energy‐demanding functions by cerebral microvasculature needs optimal functioning of energy pathways like glycolysis and mitochondrial respiration. Characterization of cellular energy metabolism in the young and aged microvasculature was essential to understand the aging‐induced cerebrovascular pathologies.MethodsWe isolated the microvessels from the brains from young (3 months) and aged mice (24 months). Microvessels were isolated by a combination of filtration (40μm – 300μm) and gradient centrifugation. Microvessels were plated on Seahorse XFe24 culture plates. Basal and compensatory glycolysis, and proton efflux rate (PER) were measured from extracellular acidification rates (ECAR) using glycolysis rate kit. Similarly, mitochondrial respiratory parameters were determined by the measurements from the oxygen consumption rate (OCR), and the fuel dependency of the microvasculature was measured using mitofuel kit.ResultsAged brain microvasculature showed 26% (73.87±7.6 vs. 100.1±2.9 picomoles of H+/min/μg protein, p&lt;0.01, n=7–9 mice/phenotype) decrease in basal glycolysis when compared with the microvessels from the young mouse brain. Compensatory glycolysis is decreased by 28.3% (121.0±7.0 vs 168.9±13.4 picomoles of H+/min/μg protein, p&lt;0.004, n=7–9 mice/phenotype) in the aged mouse brain microvessels compared to the young mouse brain vessels, whereas the basal proton efflux is decreased by 35.3% (105.2±6.3 vs 162.8±5.9 picomoles of H+/min/μg protein, p&lt;0.0001, n=7–9 mice/phenotype). Microvessels from the aged mouse brain had 36% lower mitochondrial respiration compared to the young mouse brain microvasculature (64.0±10.4 vs 100.0±7.6 picomoles of O2/min/μg protein, p&lt;0.01, n=7–9 mice/phenotype). Glucose (65.6±0.2%, n=4 mice) is the primary fuel preference by the mitochondria in the young mouse brain microvessels followed by glutamine (32.9±0.5%) and fatty acids (27.9±3.0%). Aged microvasculature exhibited unaltered fuel preferences concerning glucose and fatty acids, whereas glutamine utilization is increased by 58% (54.1±2.9% vs 34.3% vs 3.57, n=3 mice) when compared to the young mouse brain microvessels.ConclusionsWe conclude that aged brain microvasculature exhibits compromised energy metabolism including both glycolysis and the mitochondrial respiration. We further conclude that mitochondrial utilization of glutamine as energy fuel is increased in the aged brain microvasculature. The energy deficit phenotype of the brain microcirculatory network may contribute to the aging‐associated cerebrovascular complications.Support or Funding InformationNational Institute of Health: National Institute of General Medical Sciences and National Institute of Neurological Disorders and Stroke (Katakam: R01NS094834) and National Institute on Aging (Mostany: AG047296).