Abstract 15731: Sildenafil Improves Mitochondrial Function in Failing Single Ventricles

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Anastacia M Garcia ◽  
Carissa A Miyano ◽  
Raleigh Joschner ◽  
Matthew Stone ◽  
Brian L Stauffer ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Mitochondrial Function ◽  
Complex I ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Mitochondrial Oxygen Consumption ◽  
Ventricular Tissue ◽  
Substrate Inhibitor ◽  
Phosphodiesterase 5 ◽  
One Way Anova

Introduction: Heart failure (HF) remains a leading cause of death and indication for transplant in single ventricle congenital heart disease (SV). However, little is known regarding the molecular mechanisms leading to HF in SV. The purpose of this study was to characterize mitochondrial function in the myocardium of failing (SVHF) and non-failing (SVNF) SV patients compared to biventricular NF controls (BVNF). Furthermore, we investigated the effect of ex vivo treatment with the phosphodiesterase-5 inhibitor (PDE5i) sildenafil on mitochondrial function. Methods: Freshly explanted ventricular tissue was saponin permeabilized and mitochondrial oxygen consumption was measured sequentially throughout the electron transport system using SUbstrate-Inhibitor-Titration (“SUIT”) protocols and an Oroboros O2k high resolution respirometer. Permeabilized ventricular tissue was treated for 40 min with sildenafil [1μM] prior to measurement of oxygen consumption. A Western blot for PDE5 was performed in isolated mitochondrial proteins from SVHF subjects ± PDE5i. Results: Compared to BVNF (n=15) and SVNF (n=6), SVHF (n=8) hearts have decreased function of Complex I and Complex I and II (A, B), and decreased maximal respiration (C), all of which improve with acute ex vivo treatment with sildenafil in SVHF (SVHF+PDE5i, n=6). Importantly, mitochondrial function is impaired in BVNF+PDE5i (n=5) and SVNF+PDE5i hearts (n=5) (A-C, one-way Anova p<0.05). PDE5 protein is expressed in SVHF mitochondria, but expression is not affected by ex vivo PDE5i treatment (D). Conclusions: Our results indicate that mitochondrial function is impaired in SVHF, PDE5 protein is expressed in SVHF mitochondria, and PDE5i improves mitochondrial function in SVHF, but may be detrimental to mitochondrial function in SVNF and BVNF. Together these data suggest that mitochondrial PDE5 is a potential therapeutic target, but that indiscriminate use of PDE5i in SV patients may not be advisable.

Effect of hyperthermia and acidosis on equine skeletal muscle mitochondrial oxygen consumption

2020 ◽  
pp. 1-10
Author(s):  
M.S. Davis ◽  
M.R. Fulton ◽  
A. Popken
Keyword(s):  
Skeletal Muscle ◽  
Oxygen Consumption ◽  
Oxidative Phosphorylation ◽  
Muscle Fatigue ◽  
Mitochondrial Function ◽  
Mitochondrial Respiration ◽  
Complex I ◽  
Mitochondrial Oxygen Consumption ◽  
Biochemical Basis ◽  
Complex Ii

The skeletal muscle of exercising horses develops pronounced hyperthermia and acidosis during strenuous or prolonged exercise, with very high tissue temperature and low pH associated with muscle fatigue or damage. The purpose of this study was to evaluate the individual effects of physiologically relevant hyperthermia and acidosis on equine skeletal muscle mitochondrial function, using ex vivo measurement of oxygen consumption to assess the function of different mitochondrial elements. Fresh triceps muscle biopsies from 6 healthy unfit Thoroughbred geldings were permeabilised to permit diffusion of small molecular weight substrates through the sarcolemma and analysed in a high resolution respirometer at 38, 40, 42, and 44 °C, and pH=7.1, 6.5, and 6.1. Oxygen consumption was measured under conditions of non-phosphorylating (leak) respiration and phosphorylating respiration through Complex I and Complex II. Data were analysed using a one-way repeated measures ANOVA and data are expressed as mean ± standard deviation. Leak respiration was ~3-fold higher at 44 °C compared to 38 °C regardless of electron source (Complex I: 22.88±3.05 vs 8.08±1.92 pmol O2/mg/s), P=0.002; Complex II: 79.14±23.72 vs 21.43±11.08 pmol O2/mg/s, P=0.022), resulting in a decrease in efficiency of oxidative phosphorylation. Acidosis had minimal effect on mitochondrial respiration at pH=6.5, but pH=6.1 resulted in a 50% decrease in mitochondrial oxygen consumption. These results suggest that skeletal muscle hyperthermia decreases the efficiency of oxidative phosphorylation through increased leak respiration, thus providing a specific biochemical basis for hyperthermia-induced muscle fatigue. The effect of myocellular acidosis on mitochondrial respiration was minimal under typical levels of acidosis, but atypically severe acidosis can lead to impairment of mitochondrial function.

Tafazzin Deficiency Reduces Basal Insulin Secretion and Mitochondrial Function in Pancreatic Islets from Male Mice

Endocrinology ◽  
2021 ◽  
Author(s):  
Laura K Cole ◽  
Prasoon Agarwal ◽  
Christine A Doucette ◽  
Mario Fonseca ◽  
Bo Xiang ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Insulin Secretion ◽  
Pancreatic Islets ◽  
Mitochondrial Function ◽  
Ex Vivo ◽  
Cellular Respiration ◽  
Rna Seq ◽  
Β Cell ◽  
Mitochondrial Oxygen Consumption ◽  
Low Glucose

Abstract Tafazzin (TAZ) is a cardiolipin (CL) biosynthetic enzyme important for maintaining mitochondrial function. TAZ impacts both the species and content of CL in the inner mitochondrial membrane which are essential for normal cellular respiration. In pancreatic β-cells, mitochondrial function is closely associated with insulin secretion. However, the role of TAZ and CL in the secretion of insulin from pancreatic islets remains unknown. Male 4-month-old doxycycline-inducible TAZ knock-down (TAZ KD) mice and wild-type littermate controls were utilized. Immunohistochemistry was used to assess β-cell morphology in whole pancreas sections, while ex vivo insulin secretion, CL content, RNA-Seq analysis and mitochondrial oxygen consumption were measured from isolated islet preparations. Ex vivo insulin secretion under non-stimulatory low-glucose concentrations was reduced ~52% from islets isolated from TAZ KD mice. Mitochondrial oxygen consumption under low-glucose conditions was also reduced ~58% in islets from TAZ KD animals. TAZ-deficiency in pancreatic islets was associated with significant alteration in CL molecular species and elevated polyunsaturated fatty acid CL content. In addition, RNA-Seq of isolated islets showed that TAZ KD increased expression of extracellular matrix genes which are linked to pancreatic fibrosis, activated stellate cells and impaired β-cell function. These data indicate a novel role for TAZ in regulating pancreatic islet function, particularly under low-glucose conditions.

scholarly journals Cell-Permeable Succinate Rescues Mitochondrial Respiration in Cellular Models of Statin Toxicity

2021 ◽  
Vol 22 (1) ◽  
pp. 424
Author(s):  
Vlad F. Avram ◽  
Imen Chamkha ◽  
Eleonor Åsander-Frostner ◽  
Johannes K. Ehinger ◽  
Romulus Z. Timar ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Mitochondrial Dysfunction ◽  
Mitochondrial Respiration ◽  
Complex I ◽  
Ex Vivo ◽  
Coupling Efficiency ◽  
Human Platelets ◽  
Lipid Lowering ◽  
Lipid Lowering Therapy ◽  
Cellular Models

Statins are the cornerstone of lipid-lowering therapy. Although generally well tolerated, statin-associated muscle symptoms (SAMS) represent the main reason for treatment discontinuation. Mitochondrial dysfunction of complex I has been implicated in the pathophysiology of SAMS. The present study proposed to assess the concentration-dependent ex vivo effects of three statins on mitochondrial respiration in viable human platelets and to investigate whether a cell-permeable prodrug of succinate (complex II substrate) can compensate for statin-induced mitochondrial dysfunction. Mitochondrial respiration was assessed by high-resolution respirometry in human platelets, acutely exposed to statins in the presence/absence of the prodrug NV118. Statins concentration-dependently inhibited mitochondrial respiration in both intact and permeabilized cells. Further, statins caused an increase in non-ATP generating oxygen consumption (uncoupling), severely limiting the OXPHOS coupling efficiency, a measure of the ATP generating capacity. Cerivastatin (commercially withdrawn due to muscle toxicity) displayed a similar inhibitory capacity compared with the widely prescribed and tolerable atorvastatin, but did not elicit direct complex I inhibition. NV118 increased succinate-supported mitochondrial oxygen consumption in atorvastatin/cerivastatin-exposed platelets leading to normalization of coupled (ATP generating) respiration. The results acquired in isolated human platelets were validated in a limited set of experiments using atorvastatin in HepG2 cells, reinforcing the generalizability of the findings.

Abstract 355: Effect of Poloxamer 188 on Rat Brain Isolated Mitochondria After Exposure to Hydrogen Peroxide

Circulation ◽  
2019 ◽  
Vol 140 (Suppl_2) ◽  
Author(s):  
Johannes A Pille ◽  
Michele M Salzman ◽  
Anna A Sonju ◽  
Felicia P Lotze ◽  
Josephine E Hees ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Oxygen Consumption ◽  
Mitochondrial Function ◽  
Complex I ◽  
Room Temperature ◽  
In Vitro Model ◽  
Atp Synthesis ◽  
Complex Ii ◽  
Vitro Model

Introduction: In a pig model of myocardial infarction (MI), intracoronary delivered Poloxamer (P) 188 significantly reduces ischemia/reperfusion (IR) injury when given immediately upon reperfusion, with improved mitochondrial function as a predominant effect. As mitochondria are heavily damaged during IR, a direct effect of P188 on mitochondria may lead to better therapy options during reperfusion. To show not only a similar reduction of IR injury by P188 in the brain, but also a direct P188 effect on mitochondria, we established an in-vitro model of IR that consists of damaging isolated rat brain mitochondria with hydrogen peroxide (H 2 O 2 ), one component of ischemia, then applying P188, and analyzing mitochondrial function. Methods: Male Sprague-Dawley rat brains were removed, and the mitochondria isolated by differential centrifugation and Percoll gradients, then kept on ice to slow their bioenergetics prior to any experimental treatments. Mitochondria were exposed to 200 μM H 2 O 2 for 10 min at room temperature with slight agitation; controls received no H 2 O 2 . Samples were then diluted ½ with buffer ± P188 (250 μM after dilution) to simulate reperfusion and treatment, and kept at room temperature for 10 further minutes. ATP synthesis was measured in a luminometer using a luciferase enzymatic assay. Oxygen consumption was measured by closed cell respirometry with an oxygen meter. In both assays, Complex I and Complex II were examined; Complex I substrates glutamate and malate, Complex II substrate succinate plus the Complex I inhibitor rotenone. Statistics: Data are expressed as mean ± SEM. One-Way ANOVA, SNK-Test; Kruskal-Wallis-Test; α=0.05, * vs control. Results: In both Complex I and II, mitochondrial function was significantly impaired by H 2 O 2 , with ATP synthesis affected more at Complex I and oxygen consumption affected more at Complex II. Addition of P188 did not provide any significant improvement in mitochondrial function. Conclusions: Although P188 significantly reduced IR injury when given during reperfusion in a pig model of MI, it does not appear to provide direct protection to mitochondria in this in-vitro model. Whether the exposure to H 2 O 2 causes the appropriate injury for P188 to become effective remains to be elucidated.

scholarly journals The Cardiolipin Transacylase Tafazzin Regulates Basal Insulin Secretion and Mitochondrial Function in Pancreatic Islets from Mice

2021 ◽  
Author(s):  
Laura K. Cole ◽  
Prasoon Agarwal ◽  
Christine Doucette ◽  
Mario Fonseca ◽  
Bo Xiang ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Pancreatic Islets ◽  
Mitochondrial Function ◽  
Cell Function ◽  
Ex Vivo ◽  
Rna Seq ◽  
Β Cell ◽  
Mitochondrial Oxygen Consumption ◽  
Β Cell Function ◽  
Low Glucose

ABSTRACTObjectiveTafazzin (TAZ) is a cardiolipin (CL) biosynthetic enzyme important for maintaining mitochondrial function. TAZ impacts both the species and content of CL in the inner mitochondrial membrane which are essential for normal cellular respiration. In pancreatic β-cells, mitochondrial function is closely associated with insulin secretion. However, the role of TAZ and CL in the secretion of insulin from pancreatic islets remains unknown.MethodsMale 4-month-old doxycycline-inducible TAZ knock-down (TAZ KD) mice and wild-type littermate controls were utilized. Immunohistochemistry was used to assess β-cell morphology in whole pancreas sections, while ex vivo insulin secretion, CL content, RNA-Seq analysis and mitochondrial oxygen consumption were measured from isolated islet preparations.ResultsEx vivo insulin secretion under non-stimulatory low-glucose concentrations was reduced ∼52% from islets isolated from TAZ KD mice. Mitochondrial oxygen consumption under low-glucose conditions was also reduced ∼58% in islets from TAZ KD animals. TAZ-deficiency in pancreatic islets was associated with significant alteration in CL molecular species and reduced oxidized CL content. In addition, RNA-Seq of isolated islets showed that TAZ KD increased expression of extracellular matrix genes which are linked to pancreatic fibrosis, activated stellate cells and impaired β-cell function.ConclusionThese data indicate a novel role for TAZ in regulating normal β-cell function, particularly under low-glucose conditions.

scholarly journals A Multivariate Metabolomics Method for Estimating Platelet Mitochondrial Oxygen Consumption Rates in Patients with Sepsis

Metabolites ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 139
Author(s):  
Marc R. McCann ◽  
Cora E. McHugh ◽  
Maggie Kirby ◽  
Theodore S. Jennaro ◽  
Alan E. Jones ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Mitochondrial Function ◽  
Regression Models ◽  
Consumption Rate ◽  
P Value ◽  
Linear Regression Models ◽  
Mitochondrial Oxygen Consumption ◽  
Non Invasive ◽  
Consumption Rates ◽  
Multiple Linear Regression Models

Background: Sepsis-induced alterations in mitochondrial function contribute to organ dysfunction and mortality. Measuring mitochondrial function in vital organs is neither feasible nor practical, highlighting the need for non-invasive approaches. Mitochondrial function may be reflected in the concentrations of metabolites found in platelets and whole blood (WB) samples. We proposed to use these as alternates to indirectly estimate platelet mitochondrial oxygen consumption rate (mOCR) in sepsis patients. Methods: We determined the relationships between platelet mOCR and metabolites in both platelets and WB, as measured by quantitative 1H-NMR metabolomics. The associations were identified by building multiple linear regression models with stepwise forward-backward variable selection. We considered the models to be significant with an ANOVA test (p-value ≤ 0.05) and a positive predicted-R2. Results: The differences in adjusted-R2 and ANOVA p-values (platelet adj-R2: 0.836 (0.0003), 0.711 (0.0004) vs. WB adj-R2: 0.428 (0.0079)) from the significant models indicate the platelet models were more associated with platelet mOCR. Conclusions: Our data suggest there are groups of metabolites in WB (leucine, acetylcarnitine) and platelets (creatine, ADP, glucose, taurine) that are associated with platelet mOCR. Thus, WB and platelet metabolites could be used to estimate platelet mOCR.

scholarly journals Mitochondrial connexin 43 impacts on respiratory complex I activity and mitochondrial oxygen consumption

2012 ◽  
Vol 16 (8) ◽  
pp. 1649-1655 ◽  
Author(s):  
Kerstin Boengler ◽  
Marisol Ruiz-Meana ◽  
Sabine Gent ◽  
Elvira Ungefug ◽  
Daniel Soetkamp ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Complex I ◽  
Connexin 43 ◽  
Mitochondrial Oxygen Consumption ◽  
Respiratory Complex ◽  
Respiratory Complex I ◽  
Complex I Activity

scholarly journals Dramatic changes in mitochondrial substrate use at critically high temperatures: a comparative study using Drosophila

2021 ◽  
Vol 224 (6) ◽  
pp. jeb240960
Author(s):  
Lisa Bjerregaard Jørgensen ◽  
Johannes Overgaard ◽  
Florence Hunter-Manseau ◽  
Nicolas Pichaud
Keyword(s):  
High Temperatures ◽  
Heat Tolerance ◽  
Mitochondrial Function ◽  
Complex I ◽  
Mitochondrial Oxygen Consumption ◽  
Alternative Substrates ◽  
Atp Production ◽  
C Complex ◽  
Titration Protocol ◽  
Catalytic Capacity

ABSTRACTEctotherm thermal tolerance is critical to species distribution, but at present the physiological underpinnings of heat tolerance remain poorly understood. Mitochondrial function is perturbed at critically high temperatures in some ectotherms, including insects, suggesting that heat tolerance of these animals is linked to failure of oxidative phosphorylation (OXPHOS) and/or ATP production. To test this hypothesis, we measured mitochondrial oxygen consumption rate in six Drosophila species with different heat tolerance using high-resolution respirometry. Using a substrate–uncoupler–inhibitor titration protocol, we examined specific steps of the electron transport system to study how temperatures below, bracketing and above organismal heat limits affect mitochondrial function and substrate oxidation. At benign temperatures (19 and 30°C), complex I-supported respiration (CI-OXPHOS) was the most significant contributor to maximal OXPHOS. At higher temperatures (34, 38, 42 and 46°C), CI-OXPHOS decreased considerably, ultimately to very low levels at 42 and 46°C. The enzymatic catalytic capacity of complex I was intact across all temperatures and accordingly the decreased CI-OXPHOS is unlikely to be caused directly by hyperthermic denaturation/inactivation of complex I. Despite the reduction in CI-OXPHOS, maximal OXPHOS capacity was maintained in all species, through oxidation of alternative substrates – proline, succinate and, particularly, glycerol-3-phosphate – suggesting important mitochondrial flexibility at temperatures exceeding the organismal heat limit. Interestingly, this failure of CI-OXPHOS and compensatory oxidation of alternative substrates occurred at temperatures that correlated with species heat tolerance, such that heat-tolerant species could defend ‘normal’ mitochondrial function at higher temperatures than sensitive species. Future studies should investigate why CI-OXPHOS is perturbed and how this potentially affects ATP production rates.

Abstract 330: No Direct Mitochondrial Protection Against Ischemia Reperfusion Injury in Rat Isolated Hearts by P188 Postconditioning

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_4) ◽  
Author(s):  
Josephine Eskaf ◽  
Luise J Meyer ◽  
William J Cleveland ◽  
Zhu Li ◽  
Matthias L Riess
Keyword(s):  
Oxygen Consumption ◽  
Mitochondrial Function ◽  
Ischemia Reperfusion Injury ◽  
Ex Vivo ◽  
Ischemia Reperfusion ◽  
Atp Synthesis ◽  
Protective Effects ◽  
Sprague Dawley ◽  
Retention Capacity

Introduction: Myocardial infarction and cardiac arrest lead to ischemia-reperfusion (IR) injury in the heart. Timely reperfusion through percutaneous coronary intervention and cardiopulmonary resuscitation, respectively, reduces ischemia but also exacerbates myocardial injury. Maintaining mitochondrial function is crucial in maintaining cardiomyocyte function in IR injury. Poloxamer 188 (P188) is a triblock copolymer that has shown protective effects in in-vitro, ex-vivo and in-vivo myocardial IR models. P188 is thought to improve cellular and mitochondrial function during IR by stabilizing membranes. Hypothesis: P188 postconditioning has direct protective effects on mitochondrial function as assessed by ATP synthesis, oxygen consumption and calcium retention capacity (CRC). Methods: After approval by the local authorities, hearts of 42 adult male Sprague-Dawley rats were isolated and perfused ex-vivo with oxygenated Krebs Buffer (KB) for 20 min before 30 min of no-flow ischemia. Hearts were reperfused for 10 min with KB. Cardiac mitochondria were isolated with 1 mM P188 vs 1 mM polyethylene glycol (PEG) vs vehicle by differential centrifugation. Mitochondrial function was assessed for complex I and II substrates of the respiratory chain. Statistics: Kruskal-Wallis with Dunn’s posthoc testing; alpha=0.05. Results: Mitochondrial function decreased significantly after ischemia and showed improvement with reperfusion. P188 did not result in significant improvements in mitochondrial ATP synthesis, oxygen consumption and CRC function after IR, and neither did PEG. Conclusions: P188 does not have a direct protective effect on mitochondria in this model. This might be owed to the fact that no additional damage could be observed after reperfusion which is the type of injury targeted by P188 post-conditioning.

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