Measure Of Mitochondrial Function Research Articles

Mitochondrial genetic variation and risk of chronic kidney disease and acute kidney injury in UK Biobank participants

Experimental models suggest an important role for mitochondrial dysfunction in the pathogenesis of chronic kidney disease (CKD) and acute kidney injury (AKI), but little is known regarding the impact of common mitochondrial genetic variation on kidney health. We sought to evaluate associations of inherited mitochondrial DNA (mtDNA) variation with risk of CKD and AKI in a large population-based cohort. We categorized UK Biobank participants who self-identified as white into eight distinct mtDNA haplotypes, which were previously identified based on their associations with phenotypes associated with mitochondrial DNA copy number, a measure of mitochondrial function. We used linear and logistic regression models to evaluate associations of these mtDNA haplotypes with estimated glomerular filtration rate by serum creatinine and cystatin C (eGFRCr-CysC, N = 362,802), prevalent (N = 416 cases) and incident (N = 405 cases) end-stage kidney disease (ESKD), AKI defined by diagnostic codes (N = 14,170 cases), and urine albumin/creatinine ratio (ACR, N = 114,662). The mean age was 57 ± 8 years and the mean eGFR was 90 ± 14 ml/min/1.73 m2. MtDNA haplotype was significantly associated with eGFR (p = 2.8E−12), but not with prevalent ESKD (p = 5.9E−2), incident ESKD (p = 0.93), AKI (p = 0.26), or urine ACR (p = 0.54). The association of mtDNA haplotype with eGFR remained significant after adjustment for diabetes mellitus and hypertension (p = 1.2E−10). When compared to the reference haplotype, mtDNA haplotypes I (β = 0.402, standard error (SE) = 0.111; p = 2.7E−4), IV (β = 0.430, SE = 0.073; p = 4.2E−9), and V (β = 0.233, SE = 0.050; p = 2.7E−6) were each associated with higher eGFR. Among self-identified white UK Biobank participants, mtDNA haplotype was associated with eGFR, but not with ESKD, AKI or albuminuria.

Stathmin-1 Regulates Hematopoietic Stem Cell Mitochondrial and Cellular Function

Mitochondrial function is important to control HSC quiescence and self-renewal. HSCs are known to have low oxidative phosphorylation (OXPHOS), and low mitochondrial reactive oxygen species, however, studies have shown that HSCs have high mitochondrial membrane potential (MMP), which supports their function (Hinge et al 2020; Mansell et al 2021). Microtubules are important for normal mitochondrial function, acting as the main pathway through which mitochondria transport occurs. Stathmin 1 (Stmn1) is a cytoplasmic phosphoprotein that regulates microtubule dynamics via the promotion of microtubule catastrophe or the sequestration of free alpha/beta-tubulin dimers. It is highly expressed in normal hematopoietic stem cells (HSCs) and neural tissues. Also known as oncoprotein 18 (OP18) or leukemia-associated protein 18 (LAP18), Stmn1 is overexpressed in multiple cancers, including both myeloid and lymphoid leukemias. Despite its robust expression in normal HSCs and its elevated expression in leukemias, little is known about Stmn1's role in either normal or malignant HSCs. Herein, we found that Stmn1 is a critical regulator of HSC mitochondrial and cellular function. To determine the role of Stmn1 in HSCs, we first assessed HSC numbers and function in Stmn1 -/-mice. We determined HSC and progenitor numbers, as well as peripheral blood lineages, in young adult Stmn1 -/-mice and WT littermates. Compared to WT, Stmn1 -/- mice have mild anemia and thrombocytopenia, however, HSC numbers are similar. Despite normal HSC numbers, Stmn1 -/-HSCs had markedly impaired repopulating activity in competitive transplantation experiments (10% vs. 60% chimerism at 24 weeks p<0.0001). Similar defects in repopulating activity were found in non-competitive transplants and competitive intra-tibial transplants (done to circumvent any potential homing defects). In addition, Stmn1 -/- HSCs formed fewer colonies when plated in methocult, and were not capable of serial replating. Finally, Stmn1 -/- mice had delayed hematopoietic recovery following a single dose of the chemotherapeutic agent 5-fluorouracil (5-FU). We next assessed the cycling and apoptosis of HSCs. Stmn1 -/- HSCs are hyper-quiescent at baseline, although they can be stimulated to cycle in response to cytokine stimulation (1.3% vs. 3.4% S/G 2/M phase at baseline and 3.4% vs. 7% S/G 2/M phase following stimulation). In addition, they have elevated levels of apoptosis compared to WT (18.5% vs. 9.90%). Consistent with its role as a microtubule destabilizer, Stmn1 expression in HSCs coincides with tubulin expression by confocal immunofluorescent microscopy, and Stmn1 -/- HSCs have decreased tubulin compared to WT HSCs. To gain further insight into their impaired function, we next performed RNA-seq on sorted Stmn1 -/- and WT HSCs. Differential pathway analysis showed alterations in genes associated with cellular metabolism, particularly mitochondrial function (OXPHOS pathway) in the Stmn1 -/- HSCs compared to WT. Indeed, measurement of mitochondrial function using the Seahorse analyzer showed that Stmn1 -/- HSCs and progenitors have impaired maximal respiratory capacity compared to WT (251.29 vs 411.84 pmol/min). In addition, Stmn1 -/- HSCs showed reduced mitochondrial membrane potential and increased mtROS as assessed by flow-based assays. Finally, the mitochondria of Stmn1 -/- HSCs showed markedly abnormal cristae morphology by TEM, consistent with their impaired function, and Stmn1 -/- HSCs have decreased mitochondria number compared to WT (medium 12.8 vs 17.3 per 2D slice cell; p<0.0250). As elevated ROS can impair HSC function, we assessed Stmn1 -/- HSCs following treatment with MitoTEMPO, a specific scavenger of mitochondrial superoxide. Treatment of Stmn1 -/- mice with 4mg/Kg of MitoTEMPO or vehicle control for two weeks resulted in improved HSC colony formation and serial replating capacity in Methocult. Together, these data identify Stmn1 as a critical regulator of HSC function and suggest a model whereby Stmn1 promotes normal microtubule dynamics and mitochondrial health in HSCs. Ongoing experiments are aimed at understanding Stmn1's role in regulating mitochondrial quality control processes such as mitophagy and fission in both normal and malignant HSCs, and in elucidating the effects of Stmn1 loss on leukemic cell growth and viability.

Validation of mitochondrial function measurements in permeabilized myocardial tissue from porcine left ventricular specimens with ischemia/reperfusion

Abstract Background Mitochondrial function is critical to myocardial ischemia/reperfusion injury and cardioprotection. The measurement of mitochondrial function in isolated mitochondria requires cardiac specimens of about 300 mg and is therefore only possible at the end of an animal experiment or during cardiosurgical interventions in humans. As an alternative, permeabilized myocardial tissue can be used for quantification of mitochondrial respiration in small myocardial specimens of about only 2-5 mg, which can be retrieved by sequential biopsies in animal experiments and during cardiac catheterization in humans. Whether or not mitochondrial dysfunction during ischemia/reperfusion is reflected to the same extent in saponin-permeabilized myocardial tissue specimens (PMT) and in isolated mitochondria is still unclear. Purpose We attempted to validate measurements of mitochondrial respiration from PMT by comparison to those from isolated mitochondria in left ventricular myocardium from anesthetized pigs undergoing coronary occlusion/reperfusion. Methods Göttingen open-chest, anesthetized minipigs (n=10) were subjected to 60 min distal left anterior descending artery occlusion and 180 min reperfusion. At the end of reperfusion, PMT and isolated mitochondria were prepared from remote and ischemic/reperfused myocardium. Baseline, adenosine diphosphate-stimulated complex I (without/with cytochrome c, to account for absence or presence of the outer mitochondrial membrane integrity) and complex IV respiration was measured at 37°C during magnetic stirring using a high-resolution respirometer. Mitochondrial respiration in PMT and isolated mitochondria was normalized to protein content and the content of the mitochondrial marker protein cytochrome c oxidase 4. Results Mitochondrial respiration measurements in PMT and isolated mitochondria correlated well (Figure 1A) and agreed well in Bland-Altman plot (Figure 1B). While baseline respiration in PMT was somewhat higher than that in isolated mitochondria (Figure 2A), mitochondrial dysfunction during ischemia/reperfusion was equally reflected in PMT and isolated mitochondria (Figure 2B & 2C). Complex IV respiration was not different between remote myocardium and ischemic/reperfused myocardium, independently of the preparation, indicating a comparable amount of viable mitochondria (Figure 2D). Conclusion Mitochondrial function measurements in PMT can substitute for that in isolated mitochondria to reflect their dysfunction in ischemia/reperfusion. Thus, mitoprotective strategies targeting ischemia/reperfusion injury in small tissue samples and also in human myocardial biopsies can be followed in PMT.Figure 1Figure 2

Assessing Platelet Mitochondrial Dysfunction in a Murine Model of Acute Acetaminophen Toxicity.

Acetaminophen (APAP) toxicity remains a significant cause of adult and pediatric liver failure in North America and Europe. Previous research has evaluated the impaired mitochondrial function associated with APAP toxicity. The primary aim of this study was to evaluate the effects of APAP toxicity on platelet mitochondrial function using platelet oxygen consumption in a murine model in vivo. Our secondary objectives were to determine the effect of 4-MP on platelet mitochondrial function and hepatic toxicity in the setting of APAP overdose, and to correlate platelet mitochondrial function with other markers of APAP toxicity. Male C57Bl/6 mice were randomized to receive APAP (300 or 500mg/kg) or vehicle followed 90minutes later by either 4-MP (50mg/kg) or vehicle via intraperitoneal injection. Mice were euthanized 0, 12, or 24hours later and platelets isolated from cardiac blood and counted. Platelet oxygen consumption (POC) was determined using a closed-system respirometer. Liver injury was assessed by measuring alanine transferase (ALT) and histological evaluation. Injection of 500mg/kg APAP led to increased POC versus pair-matched control (vehicle) (p < 0.001). Administration of 4-MP did not affect POC in control or 300mg/kg APAP mice. In mice receiving 500mg/kg APAP and 4-MP, POC decreased significantly compared to mice receiving 500mg/kg APAP alone (p < 0.01). Serum and histological analysis confirmed APAP-induced hepatic damage in mice receiving 500mg/kg APAP and these effects blunted by treatment with 4-MP. Platelet oxygen consumption as a measure of mitochondrial function may be useful as a biomarker of hepatic APAP toxicity in the setting of moderate to severe overdose. Treatment with 4-MP decreases hepatic necrosis and may mitigate the harmful effects of APAP on platelet mitochondrial function detected via POC.

477-P: Thalamic Mitochondrial and Neuronal Function Is Preserved in Painful Diabetic Peripheral Neuropathy—A Multimodal Magnetic Resonance Imaging Study

Aims: Previous neuroimaging studies have demonstrated cerebral structural, functional, and vascular alterations in Painful-Diabetic Peripheral Neuropathy (DPN). We present a multi-modal Magnetic Resonance Imaging (MRI) study to determine the structure, and neuronal and mitochondrial function of the thalamus in Painful-DPN. Methods: A total of 49 participants were enrolled, 38 with type 2 diabetes (9 without DPN [No-DPN]; 11 with Painless-DPN; and 18 with Painful-DPN) and 11 healthy volunteers (HV). Participants underwent detailed clinical and neurophysiological assessments, and multi-modal MR imaging of the thalami bilaterally including: Proton-Magnetic Spectroscopy (1H-MRS); 31-Phosphorus MRS (31P-MRS); and brain morphometric analysis. We calculated the N-acetyl aspartate to choline (NAA:Cho), a measure of neuronal function, and inorganic phosphate (Pi) to ATP (Pi:ATP), a measure of mitochondrial function. Results: There were no significant differences in thalamic volume between groups (ANOVA, p=0.927). There was a significant group effect in both metabolite ratios: NAA:Cho (ANOVA, p=0.013); Pi:ATP (p=0.021). The NAA:Cho was numerically the lowest in Painless-DPN, reaching significance compared with HV (p=0.001) and Painful-DPN (p=0.019). Additionally, the Pi:ATP was significantly higher in Painless-DPN compared with HV (p=0.013) and Painful-DPN (p=0.008). There was also a significant correlation between the NAA:Cho and Pi:PCr (Pearson’s r -0.336, p=0.034). Conclusion: This is the first study to determine perform cerebral 1H- and 31P-MRS in human DPN. We have demonstrated altered 1H- and 31P-MRS ratios in Painless- but not Painful-DPN, suggesting that mitochondrial dysfunction may underlie thalamic neuronal dysfunction in Painless-DPN. Conversely, in Painful-DPN, preservation of thalamic mitochondrial and neuronal function may be involved in the maintenance of neuropathic pain signaling. Disclosure G.P.Sloan: None. K.Teh: None. D.Selvarajah: None. S.Tesfaye: Advisory Panel; Bayer Inc., Wörwag Pharma GmbH &amp; Co. KG, Angelini, Speaker's Bureau; Viatris Inc., Nevro Corp., Procter &amp; Gamble, Novo Nordisk, Berlin-Chemie AG.

Mitochondrial respiration analysis in permeabilized porcine left ventricular and human right atrial specimens with ischemia-reperfusion.

Mitochondrial function is critical to myocardial ischemia-reperfusion injury and cardioprotection. The measurement of mitochondrial function in isolated mitochondria requires cardiac specimens of about 300 mg and is therefore only possible at the end of an animal experiment or during cardiosurgical interventions in humans. As an alternative, mitochondrial function can be measured in permeabilized myocardial tissue (PMT) specimens of about 2-5 mg, which are retrieved by sequential biopsies in animal experiments and during cardiac catheterization in humans. We attempted to validate measurements of mitochondrial respiration from PMT by comparison with those from isolated mitochondria of left ventricular myocardium from anesthetized pigs undergoing 60 min coronary occlusion and 180 min reperfusion. Mitochondrial respiration was normalized to the content of mitochondrial marker proteins [cytochrome-c oxidase 4 (COX4), citrate synthase, and manganese-dependent superoxide dismutase]. When normalized to COX4, mitochondrial respiration measurements in PMT and isolated mitochondria agreed well in Bland-Altman plots (bias score, -0.03 nmol/min/COX4; 95% confidence interval: 6.31 nmol/min/COX4 and -6.37 nmol/min/COX4) and correlated well (slope of 0.77 and Pearson's R of 0.87). Mitochondrial dysfunction by ischemia-reperfusion was equally reflected in PMT and isolated mitochondria (44 and 48% reduction of ADP-stimulated complex I respiration). Also in isolated human right atrial trabeculae, simulation of ischemia-reperfusion injury by exposure to 60 min hypoxia and 10 min reoxygenation reduced mitochondrial ADP-stimulated complex I respiration by 37% in PMT. In conclusion, mitochondrial function measurements in permeabilized cardiac tissue can substitute for that in isolated mitochondria to reflect mitochondrial dysfunction following ischemia-reperfusion.NEW & NOTEWORTHY Methods to quantify mitochondrial function in translationally relevant models and in human tissue are needed to improve the translation of cardioprotection to patients. Our present approach, using PMT instead of isolated mitochondria for the quantification of mitochondrial ischemia-reperfusion damage, provides a reference for further studies in translationally relevant large animal models and in human tissue, thus possibly improving the translation of cardioprotection to the benefit of patients with acute myocardial infarction.

Association of leukocyte mitochondrial DNA copy number with longitudinal C-reactive protein levels and survival in older adults: a cohort study.

Systemic chronic inflammation occurs with age. The association of the leukocyte mitochondrial DNA copy number, a measure of mitochondrial function in aging, with the temporal profile of serum high-sensitivity C-reactive protein and mortality risk remains uncertain. The objectives of this study were to examine the association of the leukocyte mitochondrial DNA copy number with longitudinal high-sensitivity C-reactive protein levels and the association of the longitudinal high-sensitivity C-reactive protein levels with mortality risk. This prospective cohort study included 3928 adults aged ≥ 55years without systemic inflammation in the baseline examination of the Healthy Aging Longitudinal Study in Taiwan, which started in 2009. Each participant received leukocyte mitochondrial DNA copy number measurement using a fluorescence-based quantitative polymerase chain reaction at baseline, serum high-sensitivity C-reactive protein measurements at baseline and the follow-up examination five years later, and the ascertainment of all-cause death (until November 30, 2021). The relationships among the leukocyte mitochondrial DNA copy number, longitudinal serum high-sensitivity C-reactive protein levels, and time to all-cause mortality were examined using the joint longitudinal and survival modeling analysis. Of the 3928 participants (mean age: 69years; 2060 [52%] were women), 837 (21%) died during follow-up. In the adjusted analysis, one standard deviation lower natural log-transformed baseline leukocyte mitochondrial DNA copy number was associated with an increase of 0.05 (95% confidence interval [CI], 0.02 to 0.08) standard deviation in serum high-sensitivity C-reactive protein in subsequent years. An increase of 1 standard deviation in instantaneous high-sensitivity C-reactive protein levels was associated with a hazard ratio (HR) for all-cause mortality of 1.22 (95% CI, 1.14 to 1.30). Similar results were obtained after further adjusting for baseline high-sensitivity C-reactive protein levels (HR [95% CI], 1.27 [1.16 to 1.38]) and after excluding those with serum high-sensitivity C-reactive protein above 10mg/L (HR [95% CI], 1.21[1.11 to 1.31]) or 3mg/L (HR [95% CI], 1.19 [1.06 to 1.31]) during follow-up. A lower leukocyte mitochondrial DNA copy number was associated with persistently higher high-sensitivity C-reactive protein levels. Moreover, these higher time-varying high-sensitivity C-reactive protein levels were instantaneously associated with a higher risk of death.

The Role of Stathmin-1 in the Regulation of Hematopoietic Stem Cells

Stathmin-1 is a cytoplasmic phosphoprotein that regulates microtubule dynamics via the promotion of microtubule catastrophe or the sequestration of alpha-beta tubulin heterodimers. Also known as Oncoprotein 18 (OP18), Stathmin-1 is highly expressed in a wide variety of tumors, including high-risk myelodysplastic syndromes and acute leukemias (Machado-Neto et al, 2014). Stathmin-1 is also highly expressed in normal hematopoietic stem cells (HSCs), and its loss is associated with impaired erythroid differentiation and altered megakaryopoiesis (Ramlogan-Steel et al, 2021). While these prior studies have established a role of Stathmin-1 in normal erythro-megakaryopoiesis, its role in hematopoiesis- particularly in hematopoietic stem and progenitor cells- is not clear. Given the high expression of Stathmin-1 in HSCs and the importance of microtubule dynamics to HSC differentiation, we hypothesized that Stathmin-1 loss may impact normal HSC function. To address the role of Stathmin-1 in HSCs, we characterized HSC phenotype and function in a Stathmin-1 knockout mouse (Stmn1-/-). Under homeostatic conditions, Stmn1-/- HSC number and frequency are normal compared to wild-type (WT) mice. However, Stmn1-/- HSCs have markedly impaired function, as their bone marrow (BM) failed to engraft in competitive transplants (10% vs. 60% chimerism at 24 weeks post-transplant p<0.0001). Competitive intra-tibia transplants were then done to circumvent potential homing defects, and again Stmn1-/- BM displayed a significant engraftment defect compared to WT (10% vs. 78% chimerism at 24 weeks p<0.0001). Of note, Stmn-1-/- HSCs performed equivalently to WT HSCs in an in vivo homing assay, further suggesting their lack of engraftment is not due to homing defects. Stmn1-/- BM cells were capable of engraftment in non-competitive transplants, albeit less robustly than WT cells (42% vs. 90% p<0.01). Treatment of WT and Stmn1-/- mice with one single dose of the chemotherapeutic agent 5-fluorouracil showed that Stmn1-/-mice have slower tri-lineage recovery than their WT littermates (p<0.01 at day 15). Consistent with their reduced in vivo function, Stmn1-/- HSCs produced fewer colonies than WT HSCs when plated in Methocult. They were also not capable of serial replating, supporting that loss of Stathmin-1 is associated with impaired HSC function. Consistent with its known role in regulating microtubules, Stmn1-/- HSCs displayed altered microtubule morphology by confocal microscopy with a lack of normal nuclear invaginations compared to WT HSCs. Analysis of cycling status revealed that Stmn1-/- HSCs are more quiescent than WT HSCs at baseline (1.3% vs. 3.4% at S/G2/M phase) and in response to 24 hours of G-CSF stimulation (3.4% vs. 7% at S/G2/M phase). In addition, Stmn1-/- HSCs have higher rates of apoptosis than their WT counterparts (18.5% vs. 9.90%), suggesting that Sthamin-1 loss leads to both impaired HSC cycling and increased cell death. We next performed RNA-seq on sorted Stmn1-/- and WT HSCs to gain further mechanistic insight into their impaired function. Differential pathway analysis showed alterations in genes associated with cellular metabolism, particularly mitochondrial function, and cell differentiation in the Stmn1-/- HSCs compared to WT controls. Consistent with these results, measurement of mitochondrial function (oxygen consumption rate) using the Seahorse analyzer showed that Stmn1-/- HSCs and progenitors have impaired maximal respiratory capacity compared to WT. Together, these data suggest a model whereby Stathmin-1 regulates microtubule dynamics in HSCs to promote optimal mitochondrial function to support cellular cycling, survival and self-renewal.

Optimization of measurement of mitochondrial electron transport activity in postmortem human brain samples and measurement of susceptibility to rotenone and 4-hydroxynonenal inhibition

Mitochondrial function is required to meet the energetic and metabolic requirements of the brain. Abnormalities in mitochondrial function, due to genetic or developmental factors, mitochondrial toxins, aging or insufficient mitochondrial quality control contribute to neurological and psychiatric diseases. Studying bioenergetics from postmortem human tissues has been challenging due to the diverse range of human genetics, health conditions, sex, age, and postmortem interval. Furthermore, fresh tissues that were in the past required for assessment of mitochondrial respiratory function were rarely available. Recent studies established protocols to use in bioenergetic analyses from frozen tissues using animal models and cell cultures. In this study we optimized these methods to determine the activities of mitochondrial electron transport in postmortem human brain. Further we demonstrate how these samples can be used to assess the susceptibility to the mitochondrial toxin rotenone and exposure to the reactive lipid species 4-hydroxynonenal. The establishment of such an approach will significantly impact translational studies of human diseases by allowing measurement of mitochondrial function in human tissue repositories.

Multiplexing Seahorse XFe24 and ImageXpress® Nano Platforms for Comprehensive Evaluation of Mitochondrial Bioenergetic Profile and Neuronal Morphology.

The measurement of mitochondrial function has become imperative to understand and characterize diseases characterized by bioenergetic alterations. The advancement of automation and application of high-throughput technologies has propelled ourunderstanding of biological complexity and facilitated drug discovery. Seahorse extracellular flux (XFe) technology measures changes in dissolved oxygen and proton concentration in cell culture media, providing kinetic measurements of oxidative phosphorylation and glycolytic metabolism. ImageXpress® Nano is an automated fluorescent microscope with the ability to perform high-content, fast, and robust imaging in multi-well formats. In this chapter, we present a comprehensive protocol to multiplex the Seahorse XFe24 analyzer withthe ImageXpress® Nano high content imaging microscope to provide a comprehensive yet rigorous profile of bioenergetics and its correlation to neuronal functionand morphology.

Targeting Mitochondria in Diabetes.

Type 2 diabetes (T2D), one of the most prevalent noncommunicable diseases, is often preceded by insulin resistance (IR), which underlies the inability of tissues to respond to insulin and leads to disturbed metabolic homeostasis. Mitochondria, as a central player in the cellular energy metabolism, are involved in the mechanisms of IR and T2D. Mitochondrial function is affected by insulin resistance in different tissues, among which skeletal muscle and liver have the highest impact on whole-body glucose homeostasis. This review focuses on human studies that assess mitochondrial function in liver, muscle and blood cells in the context of T2D. Furthermore, different interventions targeting mitochondria in IR and T2D are listed, with a selection of studies using respirometry as a measure of mitochondrial function, for better data comparison. Altogether, mitochondrial respiratory capacity appears to be a metabolic indicator since it decreases as the disease progresses but increases after lifestyle (exercise) and pharmacological interventions, together with the improvement in metabolic health. Finally, novel therapeutics developed to target mitochondria have potential for a more integrative therapeutic approach, treating both causative and secondary defects of diabetes.

Association between mitochondrial function measured by 31P magnetic resonance spectroscopy and physical performance in older people with functional impairment

AbstractBackgroundMitochondrial dysfunction is a potential therapeutic target to improve skeletal muscle function, but the contribution of mitochondrial dysfunction to impaired skeletal muscle performance in older people remains unclear. The aim of this analysis was to test the association between measures of skeletal muscle mitochondrial function and physical performance in older people.MethodsWe analysed data from the Allopurinol in Functional Impairment trial. Participants aged 65 and over, who were unable to walk 400 m in 6 min, underwent 31P magnetic resonance spectroscopy of the calf after exercise at baseline and at 20 weeks follow up. The phosphocreatine recovery half‐life time (t1/2) was derived as a measure of mitochondrial function. Participants undertook the 6‐min walk test and the Short Physical Performance Battery. Muscle mass measured using the Akern 101 bio‐impedance analysis system. Bivariate correlations and multivariable regression analyses were conducted to determine associations between t1/2 and baseline factors.ResultsOne hundred and seventeen participants underwent baseline 31P magnetic resonance spectroscopy, mean age 80.4 years (SD 6.0); 56 (48%) were female. Mean 6‐min walk was 291 m (SD 80), mean SPPB score was 8.4 (SD 1.9); t1/2 correlated significantly with Short Physical Performance Battery score (r = 0.22, P = 0.02) but not with 6‐min walk distance (r = 0.10, P = 0.29). In multivariable linear regression, muscle mass and total body weight, but not t1/2, were independently associated with Short Physical Performance Battery score and with 6‐min walk distance. Change in t1/2 was not significantly associated with change in Short Physical Performance Battery score (r = 0.03, P = 0.79) or with change in 6‐min walk distance (r = −0.11, P = 0.28).ConclusionsMuscle mass, but not phosphocreatine recovery time, was consistently associated with Short Physical Performance Battery score and 6‐min walk distance in older people with functional impairment.

42 Is Mitochondrial Function Measured by 31P Magnetic Resonance Spectroscopy Associated with Physical Performance in Older People with Functional Impairment?

Abstract Background Mitochondrial dysfunction has been proposed as a therapeutic target to improve muscle strength and endurance, but the contribution that mitochondrial dysfunction makes to impaired skeletal muscle performance in older people remains unclear. We studied the relationship between phosphocreatine recovery rate (a measure of skeletal muscle mitochondrial function) and physical performance in older people. Methods We analysed data from the Allopurinol in Functional Impairment (ALFIE) trial. Participants aged 65 and over, who were unable to walk 400 m in six minutes, underwent 31P magnetic resonance spectroscopy of the calf after exercise at baseline and at 20 weeks follow up. The phosphocreatine recovery half-life time (t-half) was derived as a measure of mitochondrial function. Participants also undertook the 6-minute walk distance, the Short Physical Performance Battery test (SPPB), and had muscle mass measured using bio-impedance analysis. Bivariate correlations and multivariable regression analyses were conducted to determine associations between t-half and baseline factors. Results One hundred and seventeen people underwent baseline 31P magnetic resonance spectroscopy, mean age 80.4 years (SD 6.0); 56 (48%) were female. Mean 6-minute walk was 291 m (SD 80) and mean SPPB score was 8.4 (SD 1.9). T-half was significantly correlated with SPPB score (r = 0.22, p = 0.02) but not with 6-minute walk distance (r = 0.10, p = 0.29). In multivariable linear regression, muscle mass and weight, but not t-half, were independently associated with SPPB score and with 6-minute walk distance. The change in t-half between baseline and 20 weeks was not significantly associated with the change in SPPB (r = 0.03, p = 0.79) or with the change in 6-minute walk distance (r = −0.11, p = 0.28). Conclusion Muscle mass, but not phosphocreatine recovery time, was associated with Short Physical Performance Battery score and 6-minute walk distance in this cohort of older people with functional impairment.

Mitochondrial Dysfunction in Alzheimer's Disease: A Biomarker of the Future?

Alzheimer’s disease (AD) is the most common cause of dementia worldwide and is characterised pathologically by the accumulation of amyloid beta and tau protein aggregates. Currently, there are no approved disease modifying therapies for clearance of either of these proteins from the brain of people with AD. As well as abnormalities in protein aggregation, other pathological changes are seen in this condition. The function of mitochondria in both the nervous system and rest of the body is altered early in this disease, and both amyloid and tau have detrimental effects on mitochondrial function. In this review article, we describe how the function and structure of mitochondria change in AD. This review summarises current imaging techniques that use surrogate markers of mitochondrial function in both research and clinical practice, but also how mitochondrial functions such as ATP production, calcium homeostasis, mitophagy and reactive oxygen species production are affected in AD mitochondria. The evidence reviewed suggests that the measurement of mitochondrial function may be developed into a future biomarker for early AD. Further work with larger cohorts of patients is needed before mitochondrial functional biomarkers are ready for clinical use.

Effect of Mitochondrial Antioxidant (Mito-TEMPO) on Burn-Induced Cardiac Dysfunction

Imbalance of oxidants/antioxidants results in heart failure, contributing to mortality after burn injury. Cardiac mitochondria are a prime source of reactive oxygen species (ROS), and a mitochondrial-specific antioxidant may improve burn-induced cardiomyopathy. We hypothesize that the mitochondrial-specific antioxidant, Triphenylphosphonium chloride (Mito-TEMPO), could protect cardiac function after burn. Male rats had a 60% total body surface area (TBSA) scald burn injury and were treated with/without Mito-TEMPO (7 mg/kg-1, intraperitoneal) and harvested at 24 hours post-burn. Echocardiography (ECHO) was used for measurement of heart function. Masson Trichrome and hematoxylin and eosin (H & E) staining were used for cardiac fibrosis and immune response. Qualitative polymerase chain reaction (qPCR) was used for mitochondrial DNA replication and gene expression. Burn-induced cardiac dysfunction, fibrosis, and mitochondrial damage were assessed by measurement of mitochondrial function, DNA replication, and DNA-encoded electron transport chain-related gene expression. Mito-TEMPO partially improved the abnormal parameters. Burn-induced cardiac dysfunction was associated with crosstalk between the NFE2L2-ARE pathway, PDE5A-PKG pathway, PARP1-POLG-mtDNA replication pathway, and mitochondrial SIRT signaling. Mito-TEMPO reversed burn-induced cardiac dysfunction by rescuing cardiac mitochondrial dysfunction. Mitochondria-targeted antioxidants may be an effective therapy for burn-induced cardiac dysfunction.

Skeletal Muscle Mitochondrial Dysfunction Is Present in Patients with CKD before Initiation of Maintenance Hemodialysis.

Patients with CKD suffer from frailty and sarcopenia, which is associated with higher morbidity and mortality. Skeletal muscle mitochondria are important for physical function and could be a target to prevent frailty and sarcopenia. In this study, we tested the hypothesis that mitochondrial dysfunction is associated with the severity of CKD. We also evaluated the interaction between mitochondrial function and coexisting comorbidities, such as impaired physical performance, intermuscular adipose tissue infiltration, inflammation, and oxidative stress. Sixty-three participants were studied, including controls (n=21), patients with CKD not on maintenance hemodialysis (CKD 3-5; n=20), and patients on maintenance hemodialysis (n=22). We evaluated in vivo knee extensors mitochondrial function using 31P magnetic resonance spectroscopy to obtain the phosphocreatine recovery time constant, a measure of mitochondrial function. We measured physical performance using the 6-minute walk test, intermuscular adipose tissue infiltration with magnetic resonance imaging, and markers of inflammation and oxidative stress in plasma. In skeletal muscle biopsies from a select number of patients on maintenance hemodialysis, we also measured markers of mitochondrial dynamics (fusion and fission). We found a prolonged phosphocreatine recovery constant in patients on maintenance hemodialysis (53.3 [43.4-70.1] seconds, median [interquartile range]) and patients with CKD not on maintenance hemodialysis (41.5 [35.4-49.1] seconds) compared with controls (38.9 [32.5-46.0] seconds; P=0.001 among groups). Mitochondrial dysfunction was associated with poor physical performance (r=0.62; P=0.001), greater intermuscular adipose tissue (r=0.44; P=0.001), and increased markers of inflammation and oxidative stress (r=0.60; P=0.001). We found mitochondrial fragmentation and increased content of dynamin-related protein 1, a marker of mitochondrial fission, in skeletal muscles from patients on maintenance hemodialysis (0.86 [0.48-1.35] arbitrary units (A.U.), median [interquartile range]) compared with controls (0.60 [0.24-0.75] A.U.). Mitochondrial dysfunction is due to multifactorial etiologies and presents prior to the initiation of maintenance hemodialysis, including in patients with CKD stages 3-5.

MO045MITOCHONDRIAL DYSFUNCTION AND MUSCLE ENERGETICS IN CKD PATIENTS

Abstract Background and Aims Patients with chronic kidney disease (CKD) suffer from frailty and sarcopenia. Skeletal muscle mitochondria are important for physical function and could be a target to prevent frailty and sarcopenia. Method We tested the hypothesis that mitochondrial function worsens with the progression of CKD. We evaluated the interaction between mitochondrial function and co-existing comorbidities such as impaired physical performance, intermuscular adipose tissue (IMAT) infiltration, inflammation, and oxidative stress. We evaluated in-vivo thigh mitochondrial function using 31-phosphorus magnetic resonance spectroscopy to obtain the phosphocreatine (PCr) recovery constant, a measure of mitochondrial function. We measured physical performance using the six-minute walk test, IMAT infiltration and markers of inflammation in plasma. Results Sixty-three participants were studied including controls (n=21), patients with CKD not on maintenance hemodialysis (MHD; n=20), and patients on MHD (n=22). We found a prolonged PCr recovery constant in patients on MHD (53.3 (43.4, 70.1) seconds) and with CKD not on MHD (46.3 (40,0, 49.9) seconds) compared to controls (34.2 (28.8, 43.7) seconds) (p&amp;lt;0.001 between groups), Figure 1A-C. Mitochondrial dysfunction was associated with poor physical performance, greater IMAT, and increased markers of inflammation Figure 2A-C. Conclusion Mitochondrial function worsens with the progression of CKD and correlates with physical function, IMAT, inflammation, and oxidative stress. These data suggest that therapeutic approaches targeted at mitochondrial dysfunction and dynamics could prevent or treat frailty and sarcopenia in patients CKD.

98 Effects of maternal age on oxygen consumption of oocytes and invitro-produced equine embryos

Mitochondrial replication is arrested during early cleavage stages, leaving the embryo dependent on maternally derived mitochondria for oxidative phosphorylation. Numbers of mitochondrial DNA (mtDNA) are used as indicators of functional mitochondria; however, direct comparisons for mtDNA and oxygen consumption rate (OCR) have not been performed for horses. The objectives of this study were to compare equine oocyte mtDNA copy numbers with a measure of mitochondrial function (OCR) and to determine whether maternal age of the oocyte donor impacts OCR of early-stage embryos. We hypothesised that (1) OCR in oocytes is not directly associated with mitochondrial numbers and (2) aerobic metabolism (OCR) is lower in early embryos from old than from young mares. Mares ages 6-13 years (Young, n=7) and=20 years (Old, n=8) were used as oocyte donors. Oocytes were collected from dominant follicles (=35mm) during oestrus and at 16±2h after induction of follicular maturation. Recovered oocytes were incubated in tissue culture medium 199 with 10% fetal calf serum, 25mgmL−1 of gentamicin, and 0.2mM pyruvate for 26±2h. Metaphase II oocytes (Young, n=14; Old, n=15) were fertilized by intracytoplasmic sperm injection (ICSI) using frozen-thawed sperm from one stallion. Presumptive zygotes were cultured in global medium (LifeGlobal Group). Other oocytes and early embryos were used for OCR. A microchamber containing an electrochemical sensor was used to measure OCR from individual oocytes (Young, n=9; Old, n=14) and early embryos (Young, n=8; Old, n=10). After analyses, oocytes were snap frozen, and mtDNA was later quantified by qPCR. Metabolic assays of embryos that cleaved were performed at Day 2 after ICSI. After the assay, embryos were placed back to culture until blastocyst formation at Day 7 or 8. Two-tailed Student's t-tests were used for OCR and mtDNA comparisons, and Fisher's exact tests were used to compare development rates. We found that OCR was higher (P=0.007) for oocytes from Young (mean±s.e.m.: 1.8±0.2) than from Old (1.3±0.1 fmol s−1). However, mtDNA numbers were not different (P=0.3) for Young (5.6±0.4×105) and Old (6.2±0.4×105). Cleavage rates were similar (P=0.6) between Young (11 out of 14, 79%) and Old (13 of 15, 87%). Day 2 embryos from Young had higher basal OCR compared with Old (3.8±0.1 and 3.2±0.2 fmol s−1, respectively; P=0.05). Blastocyst rates per cleaved oocytes were similar for Young (5 of 11, 45%) and Old (4 of 13, 31%; P=0.7). Lower OCR was observed in oocytes and early embryos from Old, which indicates that mitochondrial metabolic function is reduced for mitochondria originating in the oocytes of Old compared with Young. Use of mtDNA was not indicative of mitochondrial metabolic function. Although sample numbers were limited, cleavage and blastocyst development were not significantly different between Young and Old. Further developmental competence was not determined, although the compromised metabolic capacity of oocytes and embryos from old mares could ultimately contribute to lower fertility outcomes.

Measurement of Respiration Rate in Live Caenorhabditis elegans

Mitochondrial function and dysfunction are at the core of aging and involved in many age-dependent diseases. Rate of oxygen consumption is a measure of mitochondrial function and energy production rate. The nematode Caenorhabditis elegans (C. elegans) offers an opportunity to study "living" mitochondria without the need for mitochondrial extraction, purification and associated artifacts. Oxygen consumption rate (OCR) is traditionally measured using single-chamber Clark electrodes with or without the addition of metabolic modulators. More recently, multi-well oxygen electrodes with automated injection system have been developed to enable rapid measurement of OCR under different conditions. Here, we describe a detailed protocol that we have adapted from existing protocols to measure coupled and uncoupled mitochondrial respiration (with and without metabolic modulators) in live respiring nematodes using a Seahorse XFe96 extracellular flux analyzer. We present details on our protocol, including preparation of nematode culture, use of metabolic modulators, execution of Seahorse XF assay as well as post-experimental data analysis. As a reference, we provide results of a series of experiments in which the metabolic activity of N2 wild-type nematodes was compared to N2 nematode treated with paraquat, a compound that generates reactive oxygen species (ROS), thus causing oxidative damage and mitochondrial dysfunction. These data illustrate the kind of insights that can be obtained even using a low number of nematodes (10 animals only per well).

167 Basal and maximal oxygen consumption of oocytes from young and old mares

Mitochondria play a critical role in oocyte developmental competence by providing energy to the oocyte through oxidative phosphorylation. We hypothesised that oxygen consumption, a measure of mitochondrial function, would be lower in the oocytes of old versus young mares as a potential mechanism of reduced developmental competence associated with equine aging. To test this hypothesis, we used a novel electrochemical sensor device to compare basal and maximal oxygen consumption rates of oocytes obtained from mares aged 6 to 12 years (Young, n=8 from 7 mares) and=20 years (Old, n=12 from 8 mares). Cumulus-oocyte complexes (COC) were collected by transvaginal, ultrasound-guided follicular aspirations of dominant follicles (35mm) during oestrus and at 16±2h after induction of follicular maturation (2000IU of hCG and 0.75mg of deslorelin acetate). Recovered COC were incubated in medium [TCM-199; Life Technologies, Grand Island, NY, USA) with 10% fetal calf serum, 25mg mL−1 gentamicin, and 0.2mM pyruvate] at 38.5°C in 5% CO2 and air for 26±2h. After maturation, COC were stripped of cumulus cells and checked for the presence of an extruded polar body before electrochemical measurements using amperometry. A microchamber with a 3-electrode system (working, counter, and reference electrodes) was filled with 120µL of medium (G-MOPS™, Vitrolife, Englewood, CO, USA) and overlaid with 120µL of paraffin oil (OVOIL™, Vitrolife). The baseline current was measured before adding the oocyte through the oil layer and over the centre of the working electrode. Oxygen consumption was monitored as the linear decrease in the oxygen reduction current over time, which was converted to femtomoles of O2 consumed per second (fmol s−1) using a calibration curve for the sensor. Basal (nonstimulated) oxygen consumption was measured for 5min, followed by assessment of maximal noncoupled respiratory capacity obtained by titrating 1 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 8-min intervals. The highest value observed during CCCP titrations was considered the maximal oxygen consumption. Two-tailed Student’s t-tests were used to analyse data. Oocytes from young mares had higher basal oxygen consumption compared with old mares, respectively (mean±s.e.m., 1.74±0.18 and 1.27±0.09 fmol s−1; P=0.04) as well as higher maximal oxygen consumption (2.67±0.19 and 2.05±0.12 fmol s−1; P=0.016). Mitochondrial efficiency (maximal/basal oxygen consumption) was not different for Young and Old, respectively (1.62±0.13 and 1.65±0.07; P=0.9). In summary, we used a novel electrochemical sensor device to measure basal and maximal oxygen consumption of oocytes and to confirm our hypothesis that aging impairs mitochondrial respiratory capacity in equine oocytes, which may contribute to age-associated changes in equine fertility.