Maximal Oxygen Consumption Rate Research Articles

Reduced methionine synthase expression results in uracil accumulation in mitochondrial DNA and impaired oxidative capacity.

Adequate thymidylate [deoxythymidine monophosphate (dTMP) or the "T" base in DNA] levels are essential for stability of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Folate and vitamin B12 (B12) are essential cofactors in folate-mediated one-carbon metabolism (FOCM), a metabolic network which supports synthesis of nucleotides (including dTMP) and methionine. Perturbations in FOCM impair dTMP synthesis, causing misincorporation of uracil (or a "U" base) into DNA. During B12 deficiency, cellular folate accumulates as 5-methyltetrahdryfolate (5-methyl-THF), limiting nucleotide synthesis. The purpose of this study was to determine how reduced levels of the B12-dpendent enzyme methionine synthase (MTR) and dietary folate interact to affect mtDNA integrity and mitochondrial function in mouse liver. Folate accumulation, uracil levels, mtDNA content, and oxidative phosphorylation capacity were measured in male Mtr+/+ and Mtr+/- mice weaned onto either a folate-sufficient control (C) diet (2 mg/kg folic acid) or a folate-deficient (FD) diet (lacking folic acid) for 7 weeks. Mtr heterozygosity led to increased liver 5-methyl-THF levels. Mtr+/- mice consuming the C diet also exhibited a 40-fold increase in uracil in liver mtDNA. Mtr+/- mice consuming the FD diet exhibited less uracil accumulation in liver mtDNA as compared to Mtr+/+ mice consuming the FD diet. Furthermore, Mtr+/- mice exhibited 25% lower liver mtDNA content and a 20% lower maximal oxygen consumption rates. Impairments in mitochondrial FOCM are known to lead to increased uracil in mtDNA. This study demonstrates that impaired cytosolic dTMP synthesis, induced by decreased Mtr expression, also leads to increased uracil in mtDNA.

Blood immune cells from people with HIV on antiviral regimens that contain tenofovir alafenamide (TAF) and tenofovir disoproxil fumarate (TDF) have differential metabolic signatures

BackgroundMitochondria regulate immune and organ function. It is unknown whether higher intracellular drug levels observed in peripheral blood mononuclear cells (PBMCs) treated with tenofovir alafenamide (TAF) compared to tenofovir disoproxil fumarate (TDF) may alter mitochondrial function and energy production in immune cells in HIV(+) patients. MethodsCellular bioenergetics were determined in PBMCs from HIV-1(−) participants exposed to TAF versus TDF in vitro, at a comparable concentration to a clinically relevant plasma exposure. A decrease in cellular oxygen consumption rate (OCR) at baseline (basal-OCR) and under cellular stress (max-OCR) may suggest mitochondrial dysfunction. We also assessed the in vivo impact of TAF vs TDF on OCR in PBMCs from 26 people with HIV (PWH) interchanged from TDF-based to TAF-based antiretroviral therapy (ART) over a 9-month period in the setting of an open label clinical trial. The Wilcoxon and Mann Whitney tests were used for comparison of continuous variables. ResultsPBMCs from HIV-1(−) participants exposed in vitro to a concentration of 0.12–3.3 μM for TAF and TDF at 2 and 24 h, reduced basal and maximal OCR compared to vehicle control. Switch studies of antivirals (TAF vs TDF) within the same PWH showed that TAF-based ART was associated with reduced OCR compared to TDF-based ART in PBMCs. We observed that TAF-treated PBMCs selectively relied more on glucose/pyruvate supply rather than fatty acid to fuel their mitochondria. ConclusionsCompared to TDF, TAF may alter bioenergetics in immune cells from PWH in vitro and in vivo. The clinical significance in terms of the differential impact caused by TAF versus TDF on mitochondrial function and energy production in immune cells, a regulator of immune function, requires further studied in HIV, preexposure prophylaxis and hepatitis B.

GLUCOSE INTOLERANCE PREDICTS CARDIORESPIRATORY FITNESS IN A HEALTHY POPULATION

https://youtu.be/oIXKiLYDh6g BACKGROUND Aerobic capacity or maximum oxygen consumption rate (VO2max) is accepted as the single best indicator of cardiorespiratory fitness. VO2max is also an indicator of metabolic health and known as an all-cause mortality predictor. It is well established that glucose intolerance and insulin resistance in patients with diabetes is associated with decline in cardiorespiratory fitness. However, it is not clear if glucose intolerance is associated with poorer cardiorespiratory fitness in a population without diabetes. Therefore, the purpose of this study was to assess if glucose intolerance measured by oral glucose tolerance test (OGTT) is associated with cardiorespiratory fitness in a population without diabetes. METHODS Fifty one (30 males and 21 females) predominantly Mexican-American adults without diabetes (Age 26.1 ± 5.3 years; BMI 26.7 ± 4.2 kg/m2; fasting blood glucose 92.1 ± 10.0 mg/dL) from the border region of El Paso, TX participated in this study. VO2max was assessed using a standardized graded exercise test protocol on a treadmill with Parvomedics metabolic cart. Glucose tolerance was measured by a 3-hour OGTT preformed after a 12-hour overnight fast. Blood glucose measured at fasting and every 30 minutes after the ingestion of 75g of glucose drink during OGTT and glucose area under the curve was determined to access glucose tolerance. Pearson correlation analysis was performed at 0.05 significance level. RESULTS Glucose intolerance measured by glucose area under the curve was negatively correlated with VO2max (r = −0.40, p = 0.006). Additionally, blood glucose level during OGTT test at 120 min (r = −0.43, p = 0.003), 150 min (r = −0.36, p = 0.014), and 180 min (r = −0.32, p = 0.048) were also negatively associated with VO2max. However, fasting blood glucose was not associated with VO2max (r = −0.09, p = 0.56). CONCLUSION Poorer cardiorespiratory fitness is indicated by greater glucose intolerance and a greater surge of glucose level during an OGTT, but not fasting blood glucose in a population without diabetes.

ALTERAÇÕES DE VO2MAX OCASIONADAS PELO EXERCÍCIO AERÓBICO EM NADADORES

ABSTRACT Introduction The maximal oxygen consumption rate (VO2max) can directly reflect the body's aerobic metabolism and oxygen delivery system. This is a measure of aerobic capacity used to monitor swimmers’ levels and their strength and speed performance. Objective Verify the changes in VO2max when incorporating aerobic exercise training into swimmers’ regular training. Methods 12 volunteer swimmers were divided into two categories: strength training group and regular training group. Both groups performed a fitness test on the 200-meter track before starting the training. VO2 maximal was measured and worked out statistically to verify the results of this competition. The effects of aerobic exercise on the strength and competitive status of the athletes were investigated according to an updated literature review. Results There was a significant change in the maximal oxygen uptake of the athletes after the experimental protocol. The athletes in the strength training group increased their VO2 max compared to the regular training group. The results showed a significant difference in the VO2max index before and after strength training (P<0.05). This suggests that strength training has a significant effect on maximal oxygen consumption. Conclusion Strength training interventions significantly affect athletes’ maximal oxygen consumption intensity and exercise capacity. The aerobic exercise intervention showed evidence of improving the competitive level of athletes. Level of evidence II; Therapeutic studies -investigation of treatment outcomes.

Efficacy of exercise in patients with pulmonary fibrosis: A systematic review and meta-analysis

Pulmonary fibrosis (PF) is easily caused by a variety of factors, resulting in dyspnea, exertion and movement intolerance. This systematic review aims to synthesize evidence on exercise training during rehabilitation for PF in order to improve patients' exercise capacity, quality of life, and lung function. Retrieved from the Cochrane Library, Web of Science, PubMed, Scopus and Embase from inception until April 2022. Participants: patients with PF; Intervention measures: exercise training; Results: exercise ability, quality of life, lung function and cardiopulmonary endurance. Two reviewers independently screen the title, abstract and full text. Finally, quality evaluation and meta-analysis were conducted. In this study, 13 randomized controlled studies from 1468 articles were selected. A total of 456 patients with PF were enrolled. Compared with usual care in the control group, the 6-minute walking distance, predicted forced vital capacity, predicted forced expiratory volume at 1 second and maximal rate of oxygen consumption were increased significantly after exercise training, while there was no significant change in quality of life and predicted diffusing capacity of the lung for carbon monoxide. Exercise training can significantly improve the exercise capacity, lung function and cardiopulmonary endurance of patients with PF, but has no effect on the quality of life. Exercise training is an effective rehabilitation strategy for PF.

Effects of Statins on Fat Oxidation Improvements After Aerobic Exercise Training.

Statins blunt cardiorespiratory fitness improvements after exercise training and may affect fat oxidation adaptations to training. To determine if long-term statin use in dyslipidemic individuals restricts the improvements in fat oxidation typically observed after an intense exercise-training program. A total of 106 metabolic syndrome individuals either chronically medicated with statins (ie, statin group; n = 46) or statin naive (ie, control group; n = 60) completed a 16-week supervised high-intensity interval training program. Maximal rates of oxygen consumption (V˙O2MAX), fat oxidation (FOMAX), and the shape of the workload-fat oxidation curve were assessed before and 48 hours after training in an overnight fasted state. Starting from a similar value at baseline, both groups increased V˙O2MAX after training, but the increase was larger in the control than in the statin group (19.4% vs 12.6%; P = .013). Before training, FOMAX in the statin group was lower (0.19 ± 0.08 vs 0.23 ± 0.07 g·min-1; P = .023) and took place at a lower workload (33 ± 21 vs 37 ± 19 W; P = .015) than in the control group. After training, FOMAX improved similarly in both groups (0.06 ± 0.08; 95% CI, 0.03-0.08 g·min-1 and 0.05 ± 0.09; 95% CI, 0.03-0.07 g·min-1, for statin and control groups, respectively; (P < .001). Still, after training, FOMAX occurred at a 28% lower workload in the statin group (38 ± 26 vs 53 ± 32 W; P = .048). The V˙O2-workload slope decreased after training in both groups (both P < .001) along with reductions in the respiratory exchange ratio-workload slope. Fat oxidation increased at all workloads after training regardless of the use of statins. Long-term statin treatment is associated with blunted exercise fat oxidation before exercise training. However, statin use does not attenuate the improvements in exercise fat oxidation (FOMAX) derived from intense aerobic exercise training. This finding should encourage statin users to exercise-train to benefit from increased fat oxidation once their fitness level improves.

ERK1/2 Inhibition Overcomes Resistance to Venetoclax in AML By Altering Drp1-Dependent Mitochondrial Fission and Metabolism

Background: Mutational (RAS, PTPN11 etc.) or non-mutational activation of RAS/RAF/ERK pathway is associated with primary or secondary resistance to venetoclax in AML therapy (Zhang et al., Sig Transduct Target Ther. 2022). While upregulation of alternate antiapoptotic proteins e.g. Mcl-1 has been implicated in resistance to venetoclax (Zhang et al., Sig Transduct Target Ther. 2022) role of mitochondrial dynamics (fission/fusion) in venetoclax resistance has not been well explored. Oncogenic RAS activates downstream ERK1/2 phosphorylation and mitochondrial fission through ERK1/2-mediated phosphorylation of the dynamin-like mitochondrial fission GTPase Drp1 (Nagdas et al., Cell Rep. 2019). Drp1 deletion inhibits tumorigenesis of KRAS driven pancreatic cancer, but the importance of RAS driven changes in mitochondrial dynamics in AML is poorly defined. ERK1/2 inhibition may therefore provide insights into the dependence of AML cells on altered mitochondrial dynamics and metabolism driven by oncogenic RAS. Objectives: We assessed the synergistic anti-leukemia efficacy of concomitant Bcl2 and ERK1/2 inhibition in preclinical models of venetoclax resistance and in primary patient samples (n=8). Further, we investigated the role of ERK1/2/Drp1-dependent mitochondrial fission in mediating resistance to venetoclax through altered metabolism. Results: ERK1/2 inhibition using Compound 27 (Heightman et al., J Med Chem. 2018) a close analog of ASTX029, which is currently under clinical investigation in solid tumors (NCT03520075), was highly synergistic with venetoclax at inducing apoptosis in AML cells. NRAS-mutant OCI-AML3 and THP1 cells, that are intrinsically resistant to venetoclax, were sensitized to venetoclax by ERK1/2 inhibition with a combination index (CI) of 0.008 (cell death ~90% for combination versus 20% with venetoclax alone). Synergy was also confirmed in OCI-AML2 cells with acquired resistance to venetoclax, resulting in 75% apoptosis versus 22% by venetoclax alone (CI=0.6). The combination was also effective in eliminating bulk and CD34+CD38- leukemia initiating cells (LICs) in primary AML samples (CI:0.03-0.23). Colony formation assay and single cell level CyTOF analysis using an NRAS-mutant PDX showed markedly impaired clonogenic potential (p=0.0002) and decreased expression of cMyc and CD44 in LICs in response to single agent ERK1/2 inhibition and combination treatment, indicating a potential impact on stemness. Mitochondrial reactive oxygen species (ROS) mitigation, Drp1-mediated mitochondrial fission and glycolysis are critical to maintain stemness in AML (Schimmer et al., Cell Stem Cell. 2018). As a result of ERK1/2 inhibition, pDrp1 (Ser616) levels decreased, and mitochondrial length (p<0.001) and mitochondrial ROS levels (p<0.01) were increased, suggesting impaired fission. In OCI-AML3 cells, combination treatment caused downregulation of Drp1 mRNA and decreased phosphorylation of Drp1 at Ser616. Rescue with overexpression of active phospho-mimetic form of Drp1 i.e. Drp1-Ser616Glu-Ser637Ala in OCI-AML3 cells resulted in decreased apoptosis and reversed the synergy between ERK1/2 and Bcl-2 inhibition when compared to wild type and phospho-null (Ser616Ala) Drp1 overexpression (p<0.001, Fig 1a). Of note, overexpression of Drp1-Ser616Glu-Ser637Ala phospho-mimetic led to decreased mitochondrial length (Fig 1b), suggesting enhanced fission with a distinct metabolic phenotype including reduced basal respiration, decreased maximal oxygen consumption rate (p<0.05) and ATP production (p<0.05), as well as reduced ROS production (p<0.01). Moreover, combination treatment failed to significantly induce ROS production in cells expressing the phospho-mimetic Drp1 as opposed to those expressing phospho-null or wildtype Drp1. Collectively, these results indicate that inhibition of ERK1/2/Drp1dependent mitochondrial fission and metabolic alterations may be a potential mechanism of synergistic tumor cell killing for the combination treatment. Conclusion: ERK1/2 inhibition alters Drp1 dependent mitochondrial dynamics and metabolism, resulting in mitochondrial defects and overcoming resistance to venetoclax by causing apoptosis in venetoclax resistant AML cells. These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in treatment of AML and warrant further investigation in the clinic. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 12910: Exercise-Induced Specialized Proresolving Mediators Stimulate AMPK Phosphorylation to Promote Mitochondrial Respiration in Macrophages

Emerging epidemiological data suggests that exercise imparts its CVD risk-reducing effects in part through improvements in systemic inflammation. The mechanisms underpinning the salutary effects of physical activity, however, remain incompletely understood. We recently reported that exercise enhances the production of specialized proresolving mediators (SPMs), which act as agonist of inflammation resolution through actions on macrophages (Mϕ). In this study, we find that wheel running accelerated the metabolism of proinflammatory lipid mediators LTB 4 and PGD 2 into their respective metabolites 20-OH-LTB 4 (Sed 27.14±5.88 vs Exe 70.40±10.82 pg/mL; P&lt;0.05; n =3-4) and PGJ 2 (Sed 92.63±16.08 vs Exe 227.90±30.23 pg/mL; P&lt;0.05; n =3-4), resulting in an increase in the ratio of proresolving-to-proinflammatory lipid mediator levels (RvE1+RvD1:leukotrienes; Sed 0.19±0.02 vs Exe 0.32±0.90; P&lt;0.05; n =3-4). These findings were associated with enhanced mitochondrial metabolism in Mϕ, as evidenced by increased basal (Sed 0.12±0.06 vs Exe 1.53±0.07; P&lt;0.05; n =3-4), maximum (Sed 9.20±0.30 vs Exe 12.02±0.82; P&lt;0.05; n =3-4), and ATP-linked oxygen consumption rate (Sed 1.25±0.06 vs Exe 1.47±0.09 pmol/min/μg protein; P&lt;0.05; n =3-4). We also find that SPMs RvD1, RvD2, RvE1 or MaR1 stimulate basal (Ctrl 6.11±0.1 vs RvD1 7.76±0.6 vs RvD2 9.48±0.9 vs RvE1 7.20±0.5 vs MaR1 8.73±0.9 pmol/min/μg protein; P&lt;0.05; n =3) and ATP-linked respiration (Ctrl 2.83±0.2 vs RvD1 4.50±0.3 vs RvD2 4.39±0.2 vs RvE1 4.71±0.3 vs MaR1 4.20±0.2 pmol/min/μg protein; P&lt;0.05; n =3). We find that an increase in basal (Ctrl 1.80±0.13 vs RvD1 2.43±0.19 pmol/min/μg protein; P&lt;0.05; n =3-5) and ATP-linked respiration (Ctrl 1.44±0.10 vs RvD1 1.70±0.15 pmol/min/μg protein; P&lt;0.05; n =3-5) with RvD1 treatment occurs concomitantly with AMPK phosphorylation (Ctrl 1.00±0.07 vs RvD1 1.40±0.02 pAMPK/AMPK fold change; P&lt;0.05; n =3) and inhibiting the kinase activity of AMPK with Compound C abrogates SPM-induced mitochondrial respiration. Taken together, these data suggest that exercise-enhanced SPM production stimulates mitochondrial metabolism in Mϕ via AMPK, which may contribute to exercise-induced amelioration of chronic inflammation.

OR09-5 mTOR Signaling is Associated with Regulation of Mitochondrial Respiration in Thyroid Cancer

Abstract Background Thyroid cancers (TC) are divided into BRAF-like and RAS-like tumors based on their molecular signature. The mTOR (mammalian target of rapamycin) signaling pathway is commonly activated in RAS-like tumors. Oncogene-driven signaling pathways are involved in the regulation of intracellular metabolism, including glycolysis and oxidative phosphorylation (OXPHOS). There is inadequate information on the role of mTOR signaling in the regulation of TC metabolism. The purpose of this study was to determine the association between mTOR signaling and mitochondrial respiration in TC. Methods We conducted an in vitro study using 4 TC cell lines (2 BRAF-like, 2 RAS-like) characterized by variable activation of mTOR signaling pathway, as documented by immunoblotting to quantify a ratio of p-mTOR/mTOR. OXPHOS was measured through oxygen consumption rate (OCR) of TC cells by the Seahorse XF Cell Mito Stress Test Assay. Standard MTT assay was performed to evaluate cell proliferation. A specific mTOR inhibitor (rapamycin) as well as an mTOR activator (leucine) were used for functional studies. The association between OCR and p-mTOR/mTOR was calculated by Pearson correlation coefficient (r). The one-way Anova or Kruskal-Wallis test were used for between group comparisons, as appropriate. P-values of ≤ 0.05 were considered statistically significant. Results The baseline activation of mTOR signaling in TC cells correlated with baseline (r=0.75, p=0.04) and maximum OCR (r=0.91, p=0.01). In addition, the RAS-like cell lines with the highest activation of mTOR showed considerable response after inhibition of mTOR with rapamycin by inhibiting baseline OCR (FTC133: 6320 ± 1531 vs 3368 ± 651 pmol/min/mg, p=0.01; THJ29T: 4484 ± 514 vs 3067 ± 620 pmol/min/mg, p=0.047), while the TC cell lines with minimal baseline mTOR activation did not show change in mTOR phosphorylation status nor basal OCR after rapamycin treatment (TPC1: 1817 ± 201 vs 1974 ± 336 pmol/min/mg, p&amp;gt;0.99, BCPAP: 1051 ± 162 vs 1130 ± 228 pmol/min/mg, p&amp;gt;0.99). Treatment with rapamycin significantly reduced cell proliferation rates in all examined cells. On the other hand, treatment with the mTOR activator leucine increased both mTOR phosphorylation and basal OCR in TC cell lines characterized by minimal baseline mTOR activation (TPC1: 1778 ± 280 vs 2372 ± 249 pmol/min/mg, p=0.004, BCPAP: 1581 ± 603 vs 2064 ± 560 pmol/min/mg, p=0.04), but did not significantly increase OCR further in cell lines with high baseline mTOR activation and OXPHOS rate (FTC133: 4512 ± 683 vs 5355 ± 870 pmol/min/mg, p=0.27; THJ29T: 3881 ± 880 vs 4764 ± 1273 pmol/min/mg, p=0.14). Conclusion mTOR signaling is associated with the regulation of mitochondrial respiration as its inhibition decreases OXPHOS rate, while its activation results in increased mitochondrial respiration. The change in mitochondrial respiration might be one of the mechanisms of the growth inhibition caused by medications targeting mTOR signaling in TC. Presentation: Saturday, June 11, 2022 12:30 p.m. - 12:45 p.m.

Smoking Status Impacts Mitochondrial Function in Peripheral Arterial Disease Patient-Derived Mesenchymal Stem Cells

INTRODUCTION: Patient mesenchymal stem cells (MSCs) present a new regenerative medicine tool to treat peripheral arterial disease (PAD). Mitochondrial function, i.e., bioenergetics, and turnover via mitophagy are key regulators of “stemness” and may impact regenerative potential of MSCs. Patient smoking status is known to impact MSC proliferation, but the effect on mitochondrial function is unknown. We hypothesize that smoking status impacts mitophagy and mitochondrial bioenergetics in MSCs. METHODS: Non-PAD MSCs were purchased from ATCC. PAD MSCs were obtained after major amputation under our IRB-approved project, Learning from Failure. Mitochondria were stained with Mitotracker Green FM and lysosomes with Lysotracker Deep Red for co-localization in live cells using confocal microscopy. Co-localization was quantified using FIJI software and reported using Spearman’s Rank correlation coefficient. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse XFe96 Analyzer. RESULTS: Two PAD patient lines were from smokers and two from non-smokers. Mitophagy (co-localization of mitochondria with lysosomes) did not differ significantly by smoking status in PAD MSCs. PAD MSCs also did not differ in mitophagy when compared with the non-PAD control. Maximal mitochondrial respiration (maximal OCR) differed significantly among the smoking PAD, non-smoking PAD, and non-PAD control MSCs (p < .0001). Glycolytic capacity (ECAR) also differed significantly between groups (p < .0001) (Figure). CONCLUSION: Active smoking status affected mitochondrial bioenergetics in patient MSCs, suggesting a potential impact on “stemness.” Further characterization of the impact of smoking on regenerative capacity of patient MSCs will inform optimal development of stem cell-based therapies to treat PAD.Figure

Mitochondrial oxidative stress contributes to diastolic dysfunction through impaired mitochondrial dynamics

Diastolic dysfunction (DD) underlies heart failure with preserved ejection fraction (HFpEF), a clinical syndrome associated with aging that is becoming more prevalent. Despite extensive clinical studies, no effective treatment exists for HFpEF. Recent findings suggest that oxidative stress contributes to the pathophysiology of DD, but molecular mechanisms underpinning redox-sensitive cardiac remodeling in DD remain obscure. Using transgenic mice with mitochondria-targeted NOX4 overexpression (Nox4TG618) as a model, we demonstrate that NOX4-dependent mitochondrial oxidative stress induces DD in mice as measured by increased E/E′, isovolumic relaxation time, Tau Glantz and reduced dP/dtmin while EF is preserved. In Nox4TG618 mice, fragmentation of cardiomyocyte mitochondria, increased DRP1 phosphorylation, decreased expression of MFN2, and a higher percentage of apoptotic cells in the myocardium are associated with lower ATP-driven and maximal mitochondrial oxygen consumption rates, a decrease in respiratory reserve, and a decrease in citrate synthase and Complex I activities. Transgenic mice have an increased concentration of TGFβ and osteopontin in LV lysates, as well as MCP-1 in plasma, which correlates with a higher percentage of LV myocardial periostin- and ACTA2-positive cells compared with wild-type mice. Accordingly, the levels of ECM as measured by Picrosirius Red staining as well as interstitial deposition of collagen I are elevated in the myocardium of Nox4TG618 mice. The LV tissue of Nox4TG618 mice also exhibited increased ICaL current, calpain 2 expression, and altered/disrupted Z-disc structure. As it pertains to human pathology, similar changes were found in samples of LV from patients with DD. Finally, treatment with GKT137831, a specific NOX1 and NOX4 inhibitor, or overexpression of mCAT attenuated myocardial fibrosis and prevented DD in the Nox4TG618 mice.Together, our results indicate that mitochondrial oxidative stress contributes to DD by causing mitochondrial dysfunction, impaired mitochondrial dynamics, increased synthesis of pro-inflammatory and pro-fibrotic cytokines, activation of fibroblasts, and the accumulation of extracellular matrix, which leads to interstitial fibrosis and passive stiffness of the myocardium.Further, mitochondrial oxidative stress increases cardiomyocyte Ca2+ influx, which worsens CM relaxation and raises the LV filling pressure in conjunction with structural proteolytic damage.

A Comparative Analysis On Aerobically- And Anaerobically-trained Individuals’ Skeletal Muscle Oxygen Saturation

Skeletal muscle oxygen saturation (SmO2) is an important measure of how well muscles uptake oxygen from the blood and has been shown to correlate to exercise load. Maximal rate of oxygen consumption (VO2max) has been used to measure cardiorespiratory fitness which influences how much oxygen is being carried in the blood. What is not fully elucidated is if there is a difference in SmO2 and VO2max between aerobically trained individuals (runners) and anaerobically trained individuals (CrossFitters). Purpose: To determine if CrossFit-trained individuals and runners have differences in SmO2 during and after VO2 max test. Methods: Seventeen Crossfitters (n = 12 male; n = 5 female, Age 28.6 ± 7.6 years, VO2max 43.6 ± 7.2 ml/kg/min) and 18 endurance runners (n = 10 male; n = 8 female, Age 43.1 ± 12.3 years, VO2max 41.1 ± 7.6 ml/kg/min) completed a single VO2 max ramp protocol treadmill test while wearing a MOXY Sensor to assess SmO2. The MOXY Sensor was placed on the lateral-posterior belly of the gastrocnemius. The high, average, and low measures of SmO2 were obtained at baseline, during exercise, and post-exercise. In addition, total hemoglobin (THb) was also measured at all time points. The data was analyzed using a paired sample t-test to compare SmO2 measures, VO2max, THb, and time to recovery (secs). In addition, a linear regression was performed to show differences between groups. All analyses were performed using SAS (v.9.3). Results: There was a significant difference between the highest point of baseline SmO2 data between groups (p = 0.0330) with Crossfit individuals having a higher baseline SMO2. However, no significant difference was found for VO2max, THb, and time to recovery between groups. Additionally, we controlled for age and gender by running a linear regression. For gender, we did not find any statistical significance; however, we found that age had a significant effect on VO2 max (p = 0.0358). Conclusion: Analysis of the data indicated that aerobically-trained and anaerobically-trained individuals did not have a significant difference in SmO2 during the VO2 max test. Due to our disproportionate gender and age sample size, future research is needed in order to determine the full extent of whether there is a significant difference in SmO2 between anaerobically- and aerobically-trained individuals.

Pharmacology Management in Improving Exercise Capacity of Patients with Fontan Circulation: A Systematic Review and Meta-analysis.

The Fontan procedure is currently the mainstay therapy for single functional ventricles. However, with prolonged follow-up duration, various complications have been observed that seriously influence the quality of life of patients. The aim of this meta-analysis is to compare the effectiveness of pharmacologic agents in improving exercise capacity in patients with Fontan circulation. This meta-analysis was conducted according to the Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) statement and has been registered in the International Prospective Register for Systematic Reviews database with registration no. 282352. Quality assessments of the included studies were assessed using the Strengthening Reporting of Observational Studies in Epidemiology (STROBE) statement. Twelve studies met the predetermined inclusion criteria and were included in this metaanalysis. This meta-analysis found that treatment with bosentan significantly improved New York Heart Association Functional Class (NYHA FC) in Fontan patients (standard mean difference - 0.59, 95% CI -0.94 - -0.25; P=0.0008; I2 = 1%). However, the use of bosentan (P=0.66) and sildenafil (P=0.13) did not show a significant improvement in the maximum rate of oxygen consumption (VO2 max). This meta-analysis shows that people with Fontan circulation may benefit from using bosentan as it decreases postexercise heart rate and improves NYHA FC and 6-minute walking test results. Therefore, indirectly improving exercise capacity. Nonetheless, considerable work is required to strengthen our knowledge in improving the exercise capacity of Fontan patients.

Skeletal Muscle Cells Derived From Old Donors Show Altered Mitochondrial Morphology And Reduced Respiratory Capacity

With age, skeletal muscle mitochondria lose their oxidative capacity and their ability to respond to energy demand. These phenomena can lead to a reduction in skeletal muscle oxygen consumption, atrophy, and increased risk of developing age-related diseases such as sarcopenia. Whether age-derived changes in mitochondrial function correspond to structural changes in the mitochondrial reticulum remains unknown. PURPOSE: Investigate age-related changes in mitochondrial morphology and function using primary skeletal muscle cells derived from healthy young and old men. METHODS: Primary skeletal muscle progenitor cells (SkM) derived from the Rectus abdominis muscle of healthy active 18-19-year-old men (SkM Young), and 66-69-year-old men (SkM Old) were obtained from Cook MyoSite Inc. (Pittsburgh, PA). The mitochondrial network was analyzed in live cells using confocal microscopy. Oxygen Consumption Rate (OCR) was measured in intact cells using extracellular flux assays and a Seahorse analyzer (Agilent Technologies; Santa Clara, CA). RESULTS: Primary cells derived from SkM Young revealed a higher Basal and Maximal OCR compared to SkM Old (Basal: 23.20 ± 1.49 vs. 11.34 ± 2.31; Maximal: 41.50 ± 4.72 vs. 18.36 ± 1.65 pmol/min/protein). SkM Young also had higher ATP production and Spare Respiratory Capacity (ATP: 19.08 ± 1.21 vs. 9.19 ± 2.10; SRC: 18.30 ± 3.47 vs. 7.03 ± 1.29 pmol/min/protein). No differences were revealed in morphology (Individuals: 37.80 ± 23.18 vs. 46.67 ± 22.43 counts; Networks: 10.20 ± 2.48 vs. 15.33 ± 5.75 counts; Mean Branches per Network: 15.52 ± 6.41 vs. 11.27 ± 3.67 counts). CONCLUSIONS: These preliminary results show primary skeletal muscle progenitor cells derived from healthy young donors have higher respiration, produce more ATP, and have a greater capacity to adapt to energy demands when compared to SkM derived from Old donors. Further analyses can give us an insight into human skeletal muscle-derived cellular physiological capacity. Technology to observe human muscle mitochondrial dysfunction in vitro helps us understand the effects of aging on skeletal muscle mitochondria and whole-body declines of resting and exercise metabolic rates. Additional studies will be required on aging individuals to determine if predicted decrements in energy coupling efficiency occur with aging in vivo.

Comparing Cardiopulmonary Functions of National Race Walkers by Walking and Running during Graded Exercise Testing

PURPOSE: This study aimed to identify a sport-specific assessment for elite race-walking athletes by comparing physiological responses between walking and running graded exercise test (GXT) protocols.METHODS: Six elite race-walking athletes completed both walking and running protocols in a randomized crossover design with 1 week interval between trials. Maximal oxygen consumption (VO&lt;sub&gt;2&lt;/sub&gt;max), ventilation (VE), and heart rate (HR) were measured throughout and after each trial, and blood lactate levels were collected during the recovery period.RESULTS: There were significant differences in VO&lt;sub&gt;2&lt;/sub&gt;max, VE, and HR values between the walking and running protocols (&lt;i&gt;p&lt;/i&gt;&lt;.001). During the recovery period, VO&lt;sub&gt;2&lt;/sub&gt;max, VE, HR, and blood lactate levels were significantly different in each measurement (&lt;i&gt;p&lt;/i&gt;&lt;.001); however, there was no difference between the walking and running protocols.CONCLUSIONS: Based on these results, both the walking and running GXT protocols were inappropriate for assessing elite racewalking athletes. This study suggests that a supplement GXT protocol is required to specifically assess elite race-walking athletes.

Skeletal muscle blood flow during exercise is reduced in a rat model of pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is characterized by exercise intolerance. Muscle blood flow may be reduced during exercise in PAH; however, this has not been directly measured. Therefore, we investigated blood flow during exercise in a rat model of monocrotaline (MCT)-induced pulmonary hypertension (PH). Male Sprague-Dawley rats (∼200 g) were injected with 60 mg/kg MCT (MCT, n = 23) and vehicle control (saline; CON, n = 16). Maximal rate of oxygen consumption (V̇o2max) and voluntary running were measured before PH induction. Right ventricle (RV) morphology and function were assessed via echocardiography and invasive hemodynamic measures. Treadmill running at 50% V̇o2max was performed by a subgroup of rats (MCT, n = 8; CON, n = 7). Injection of fluorescent microspheres determined muscle blood flow via photo spectroscopy. MCT demonstrated a severe phenotype via RV hypertrophy (Fulton index, 0.61 vs. 0.31; P < 0.001), high RV systolic pressure (51.5 vs. 22.4 mmHg; P < 0.001), and lower V̇o2max (53.2 vs. 71.8 mL·min-1·kg-1; P < 0.0001) compared with CON. Two-way ANOVA revealed exercising skeletal muscle blood flow relative to power output was reduced in MCT compared with CON (P < 0.001), and plasma lactate was increased in MCT (10.8 vs. 4.5 mmol/L; P = 0.002). Significant relationships between skeletal blood flow and blood lactate during exercise were observed for individual muscles (r = -0.58 to -0.74; P < 0.05). No differences in capillarization were identified. Skeletal muscle blood flow is significantly reduced in experimental PH. Reduced blood flow during exercise may be, at least in part, consequent to reduced exercise intensity in PH. This adds further evidence of peripheral muscle dysfunction and exercise intolerance in PAH.

Variable effects of omaveloxolone (RTA408) on primary fibroblasts with mitochondrial defects.

Omaveloxolone (RTA408) is a second-generation oleanane triterpenoid Nrf2 inducer with antioxidant and anti-inflammatory properties and was reported to improve mitochondrial bioenergetics. It is currently being tested in medical trials for Friedrich ataxia, a genetic, multi-organ disease involving mitochondrial dysfunction. Thus, omaveloxolone could potentially be beneficial for additional disorders involving mitochondrial dysfunction. To this end, we investigated its effect on primary fibroblasts derived from patients with mitochondrial complex I deficiency, mitochondrial cytochrome oxidase deficiency, and two recessive forms of Parkinson’s disease. Patients and control cells were incubated in the presence or absence of 50 nM omaveloxolone for 72 h prior to measurements. Generally, growth on galactose medium and ATP production were unaltered. Mitochondrial membrane potential was slightly but significantly decreased, while reactive oxygen species (ROS) production was variably decreased. Mitochondrial mass and mitochondrial DNA (mtDNA) contents were significantly increased in the patient’s cells. These results were partially confirmed by the results of oxygen consumption studies which disclosed increased maximal oxygen consumption rates in most cells and increased energy status in all treated cells. Further investigation is required to explore the precise effect of omaveloxolone on mitochondrial function in disease.

Pharmaceutical Agents for Contractile-Metabolic Dysfunction After Volumetric Muscle Loss.

Volumetric muscle loss (VML) injuries represent a majority of military service member casualties and are common in civilian populations following blunt and/or penetrating traumas. Characterized as a skeletal muscle injury with permanent functional impairments, there is currently no standard for rehabilitation, leading to lifelong disability. Toward developing rehabilitative strategies, previous research demonstrates that the remaining muscle after a VML injury lacks similar levels of plasticity or adaptability as healthy, uninjured skeletal muscle. This may be due, in part, to impaired innervation and vascularization of the remaining muscle, as well as disrupted molecular signaling cascades commonly associated with muscle adaptation. The primary objective of this study was to assess the ability of four pharmacological agents with a strong record of modulating muscle contractile and metabolic function to improve functional deficits in a murine model of VML injury. Male C57BL/6 mice underwent a 15% multimuscle VML injury of the posterior hindlimb and were randomized into drug treatment groups (formoterol [FOR], 5-aminoimidazole-4-carboxamide riboside [AICAR], pioglitazone [PIO], or sildenafil [SIL]) or untreated VML group. At the end of 60 days, the injury model was first validated by comparison to age-matched injury-naive mice. Untreated VML mice had 22% less gastrocnemius muscle mass, 36% less peak-isometric torque, and 27% less maximal mitochondrial oxygen consumption rate compared to uninjured mice (p < 0.01). Experimental drug groups were, then, compared to VML untreated, and there was minimal evidence of efficacy for AICAR, PIO, or SIL in improving contractile and metabolic functional outcomes. However, FOR-treated VML mice had 18% greater peak isometric torque (p < 0.01) and permeabilized muscle fibers had 36% greater State III mitochondrial oxygen consumption rate (p < 0.01) compared to VML untreated mice, suggesting an overall improvement in muscle condition. There was minimal evidence that these benefits came from greater mitochondrial biogenesis and/or mitochondrial complex protein content, but could be due to greater enzyme activity levels for complex I and complex II. These findings suggest that FOR treatment is candidate to pair with a rehabilitative approach to maximize functional improvements in VML-injured muscle. Impact statement Volumetric muscle loss (VML) injuries result in deficiencies in strength and mobility, which have a severe impact on patient quality of life. Despite breakthroughs in tissue engineering, there are currently no treatments available that can restore function to the affected limb. Our data show that treatment of VML injuries with clinically available and FDA-approved formoterol (FOR), a beta-agonist, significantly improves strength and metabolism of VML-injured muscle. FOR is therefore a promising candidate for combined therapeutic approaches (i.e., regenerative rehabilitation) such as pairing FOR with structured rehabilitation or cell-seeded biomaterials as it may provide greater functional improvements than either strategy alone.

Ozone high dose therapy (OHT) improves mitochondrial bioenergetics in peripheral blood mononuclear cells

BackgroundThe worldwide increasing number of people with chronic diseases is pushing conventional therapy to its limits. The so-called Major AutoHaemo Therapy (MAH) has been used in many practices for years. Despite suspicions, especially the 10-passes ozone-high-dosis Therapy (OHT) has shown substantial benefits in chronic ailments. However, knowledge of scientifically based effects of high ozone concentrations are still rare. The present investigation focussed on verifying whether OHT may be linked to a beneficial effect on mitochondrial bioenergetics which can be expressed as a bioenergetic health index (BHI).MethodsWe report on six patients which received OHT for preventive purposes twice within one week. The BHI in peripheral blood mononuclear cells (PBMC) is calculated from parameters of a cellular mitochondrial function assay, which gives insights into different aspects of mitochondrial function: 1) Basal oxygen consumption rate (OCR); 2) ATP-linked OCR and proton leak; 3) Maximal OCR and reserve capacity; 4) Non-mitochondrial OCR.ResultsThe results clearly show that the bioenergetic health index in PBMC improves significantly after just 2 OHT applications over a period of 1 week. The overall improvement of the BHI is based primarily on a significant increase in the reserve capacity and the maximum respiration of the mitochondria. The increase in non-mitochondrial oxygen consumption, which has a negative impact on the BHI value, is indicative for the Nrf-2 dependent activation of antioxidant and detoxifying enzymes activated through OHT.ConclusionThese data demonstrate for the first time the beneficial effect of OHT on mitochondrial parameters. Thus, the results of this study suggest that OHT could be a safe and effective therapeutic option alone or as integrative and complementary support for pharmacological therapy in a variety of chronic and acute diseases where mitochondrial dysfunction plays a central role.

Cardiolipin nanodisks confer protection against doxorubicin-induced mitochondrial dysfunction

Doxorubicin (DOX) is an aqueous soluble anthracycline therapeutic widely used in cancer treatment. Although DOX anti-cancer activity is dose-dependent, increased dosage enhances the risk of cardiotoxicity. Despite intensive investigation, the molecular basis of this undesirable side effect has yet to be established. In addition to serving as a DNA intercalation agent, DOX is known to bind to the signature mitochondrial phospholipid, cardiolipin (CL). Consistent with this, DOX associates with aqueous soluble nanoparticles, termed nanodisks (ND), comprised solely of CL and an apolipoprotein scaffold. Fluorescence microscopy analysis revealed that DOX uptake, and targeting to the nucleus of cultured hepatocarcinoma (HepG2) or breast cancer (MCF7) cells, was unaffected by its association with CL-ND. Subsequent studies revealed that free DOX and DOX-CL-ND were equivalent in terms of growth inhibition activity in both cell lines. By contrast, in studies with H9C2 cardiomyocytes, DOX-CL-ND induced a lesser concentration-dependent decline in cell viability than free DOX. Whereas incubation of H9C2 cardiomyocytes with free DOX caused a steep decline in maximal oxygen consumption rate, DOX-CL-ND treated cells were largely unaffected. The data indicate that association of DOX with CL-ND does not diminish its cancer cell growth inhibition activity yet confers protection to cardiomyocytes from DOX-induced effects on aerobic respiration. This study illustrates that interaction with CL plays a role in DOX-induced mitochondrial dysfunction and suggests CL-ND provide a tool for investigating the mechanistic basis of DOX-induced cardiotoxicity.