Methodological Issue of Mitochondrial Isolation in Acute-Injury Rat Model: Asphyxia Cardiac Arrest and Resuscitation

Background: Identification of the mechanisms underlying mitochondrial dysfunction is key to understanding the pathophysiology of acute injuries such as cardiac arrest (CA); however, effective methods for measurement of mitochondrial function associated with mitochondrial isolation have been debated for a long time. This study aimed to evaluate the dysregulation of mitochondrial respiratory function after CA while testing the sampling bias that might be induced by the mitochondrial isolation method.Materials and Methods: Adult rats were subjected to 10-min asphyxia-induced CA. 30 min after resuscitation, the brain and kidney mitochondria from animals in sham and CA groups were isolated (n = 8, each). The mitochondrial quantity, expressed as protein concentration (isolation yields), was determined, and the oxygen consumption rates were measured. ADP-dependent (state-3) and ADP-limited (state-4) respiration activities were compared between the groups. Mitochondrial quantity was evaluated based on citrate synthase (CS) activity and cytochrome c concentration, measured independent of the isolation yields.Results: The state-3 respiration activity and isolation yield in the CA group were significantly lower than those in the sham group (brain, p < 0.01; kidney, p < 0.001). The CS activity was significantly lower in the CA group as compared to that in the sham group (brain, p < 0.01; kidney, p < 0.01). Cytochrome c levels in the CA group showed a similar trend (brain, p = 0.08; kidney, p = 0.25).Conclusions: CA decreased mitochondrial respiration activity and the quantity of mitochondria isolated from the tissues. Owing to the nature of fragmented or damaged mitochondrial membranes caused by acute injury, there is a potential loss of disrupted mitochondria. Thus, it is plausible that the mitochondrial function in the acute-injury model may be underestimated as this loss is not considered.

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Methodological Issue of Isolating Mitochondria in Acute Injury: A Rat Cardiac Arrest Model

10.21203/rs.3.rs-127088/v1 ◽

2020 ◽

Author(s):

Tomoaki Aoki ◽

Yu Okuma ◽

Lance Becker ◽

Kei Hayashida ◽

Koichiro Shinozaki

Keyword(s):

Cardiac Arrest ◽

Cytochrome C ◽

Sham Group ◽

Citrate Synthase ◽

Methodological Issue ◽

Sampling Bias ◽

Acute Injury ◽

Adult Rats ◽

Respiration Activity ◽

Kidney Mitochondria

Abstract Background Mitochondrial studies are key to understanding the pathophysiology of cardiac arrest (CA), however there is a potential risk of sampling bias during the mitochondrial isolation process. This study aimed to evaluate the dysregulation of mitochondrial respiratory function after CA while testing the sampling bias induced by the mitochondrial isolation method.Methods and Results Adult rats were subjected to 10-minunte asphyxia-induced CA. Thirty minutes after resuscitation, brain and kidney mitochondria from sham and CA group animals were isolated (n=8, each). The mitochondria quantity, expressed as protein concentrations (isolation yields), was determined and then oxygen consumption rates were measured. ADP-dependent (state-3) and ADP-limited (state-4) respiration activity were compared between the groups. The mitochondrial quantity was evaluated by citrate synthase (CS) activity and cytochrome c concentration measured independently from isolation yields. The state-3 respiration activity and isolation yield in the CA group declined significantly as compared to those in the sham group (brain, p < 0.01; kidney, p < 0.001). The CS activity in the CA group declined significantly as compared to that of the sham group (brain, p < 0.01; kidney, p < 0.01). Likewise, cytochrome c levels in the CA group had decreasing trends (brain, p = 0.08; kidney, p = 0.25).Conclusions CA decreased mitochondrial respiration activity and the quantity of mitochondria isolated from the tissues. Due to the nature of fragmented or damaged mitochondria membranes caused by this acute injury model, it is plausible that the mitochondrial function measured in the acute injury animal model might be underestimated.

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Schisandrin Restores the Amyloid β-Induced Impairments on Mitochondrial Function, Energy Metabolism, Biogenesis, and Dynamics in Rat Primary Hippocampal Neurons

Pharmacology ◽

10.1159/000507818 ◽

2021 ◽

pp. 1-11

Author(s):

Zhongyuan Piao ◽

Lin Song ◽

Lifen Yao ◽

Limei Zhang ◽

Yichan Lu

Keyword(s):

Energy Metabolism ◽

Cytochrome C ◽

Mitochondrial Biogenesis ◽

Mitochondrial Function ◽

Hippocampal Neurons ◽

Citrate Synthase ◽

Mitochondrial Permeability Transition ◽

Amyloid Β ◽

Mitochondrial Functions ◽

Primary Hippocampal Neurons

Introduction: Schisandrin which is derived from Schisandra chinensis has shown multiple pharmacological effects on various diseases including Alzheimer’s disease (AD). It is demonstrated that mitochondrial dysfunction plays an essential role in the pathogenesis of neurodegenerative disorders. Objective: Our study aims to investigate the effects of schisandrin on mitochondrial functions and metabolisms in primary hippocampal neurons. Methods: In our study, rat primary hippocampal neurons were isolated and treated with indicated dose of amyloid β1–42 (Aβ1–42) oligomer to establish a cell model of AD in vitro. Schisandrin (2 μg/mL) was further subjected to test its effects on mitochondrial function, energy metabolism, mitochondrial biogenesis, and dynamics in the Aβ1–42 oligomer-treated neurons. Results and Conclusions: Our findings indicated that schisandrin significantly alleviated the Aβ1–42 oligomer-induced loss of mitochondrial membrane potential and impaired cytochrome c oxidase activity. Additionally, the opening of mitochondrial permeability transition pore and release of cytochrome c were highly restricted with schisandrin treatment. Alterations in cell viability, ATP production, citrate synthase activity, and the expressions of glycolysis-related enzymes demonstrated the relief of defective energy metabolism in Aβ-treated neurons after the treatment of schisandrin. For mitochondrial biogenesis, elevated expression of peroxisome proliferator-activated receptor γ coactivator along with promoted mitochondrial mass was found in schisandrin-treated cells. The imbalance in the cycle of fusion and fission was also remarkably restored by schisandrin. In summary, this study provides novel mechanisms for the protective effect of schisandrin on mitochondria-related functions.

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Mitochondrial Function in Oxidative and Glycolytic Bovine Skeletal Muscle Postmortem

Meat and Muscle Biology ◽

10.22175/mmb.10698 ◽

2019 ◽

Vol 3 (2) ◽

Author(s):

P. Ramos ◽

L. Bell ◽

S. Wohlgemuth ◽

T. Scheffler

Keyword(s):

Cytochrome C ◽

Oxidative Phosphorylation ◽

Outer Membrane ◽

Functional Properties ◽

Mitochondrial Function ◽

Complex I ◽

Fixed Effects ◽

Citrate Synthase ◽

Electron Flow ◽

Coupling Efficiency

ObjectivesMitochondrial function in postmortem muscle is affected by decreasing oxygenation. Functional properties relating to energy production and integrity of mitochondria may influence development of meat quality characteristics. Therefore, the objective was to evaluate changes in mitochondrial function in oxidative and glycolytic muscles during the first 24h postmortem.Materials and MethodsSteers (n = 6) of primarily Angus (80 to 100%) genetics were harvested at approximately 18.5 mo and 630 kg live weight. Samples from the longissimus lumborum (LL) and diaphragm (Dia) were collected at 1, 3, and 24h postmortem. Fresh-preserved muscle samples were permeabilized using saponin, and muscle bundles (2–4 mg) were transferred to a high-resolution oxygraph for respiration measurements (oxygen consumption rate, OCR, pmol/sec/mg of tissue). Samples were assessed in duplicate under hyperoxia. First, pyruvate and malate were added to support the TCA cycle and assess leak respiration. Then, ADP was added to support electron flow through complex I. The influence of glutamate on NADH production (complex I) was tested, followed by complex II activation by succinate. Integrity of the mitochondria outer membrane was tested with cytochrome c. Next, an uncoupler (FCCP) was added to force the electron transport system (ETS) to maximum capacity. Citrate synthase (CS) activity (nmol/min/mg tissue) was determined in frozen samples and used as a marker of mitochondria content. Subsequently, respiration data were normalized to CS activity (pmol/sec/U CS) to account for differences in mitochondria content. Coupling efficiency of oxidative phosphorylation was calculated as 1– (Leak/ADP-stimulated oxidative phosphorylation capacity). Raw and normalized OCR were analyzed in a randomized block design, with slaughter date as block and fixed effects of muscle, time, and the interaction. Time was considered a repeated measure.ResultsMuscle type affected (P = 0.0002) leak OCR, with Dia showing a higher rate than LL. After ADP was added, mitochondria from Dia exhibited higher OCR at all times tested and at all steps, with OCR being 4 times higher after FCCP addition. Mitochondrial content, evidenced by greater (P < 0.0001) CS activity in Dia, largely explained differences in OCR between muscles. After OCR was normalized to CS activity, the 1 and 3h postmortem OCR from Dia and LL were similar (P > 0.05). However, at 24h postmortem, OCR after ADP, glutamate, and FCCP additions were greater (P < 0.05) in Dia mitochondria. Time, but not muscle, affected cytochrome c response. At 1h postmortem, cytochrome c increased OCR by 6.6%, supporting that mitochondria outer membrane integrity is not compromised. However, cytochrome c response at 3h postmortem increased 52.4%, indicating outer membrane damage. Coupling efficiency is different between muscles (P = 0.005) with Dia exhibiting greater efficiency.ConclusionDespite inherent metabolic differences between the LL and Dia, mitochondria from both muscles are intact and coupled at 1h postmortem. However, by 24h postmortem, functional properties of LL mitochondria are reduced compared to Dia. Declining mitochondrial function may be associated with calcium overload, mitochondrial fragmentation, and protease activation.

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Abnormal Platelet Mitochondrial Function in Patients Affected by Migraine With and Without Aura

Cephalalgia ◽

10.1046/j.1468-2982.1994.1401021.x ◽

1994 ◽

Vol 14 (1) ◽

pp. 21-23 ◽

Cited By ~ 54

Author(s):

S Sangiorgi ◽

M Mochi ◽

R Riva ◽

P Cortelli ◽

L Monari ◽

...

Keyword(s):

Cytochrome C ◽

Migraine With Aura ◽

Mitochondrial Function ◽

Nadh Dehydrogenase ◽

Citrate Synthase ◽

Reductase Activity ◽

Mitochondrial Enzymes ◽

Healthy Control ◽

Patient Groups ◽

Nadh Cytochrome

To investigate energy metabolism in migraine, we determined platelet mitochondrial enzyme activities in 40 patients with migraine with aura and in 40 patients with migraine without aura during attack-free intervals and in 24 healthy control subjects. NADH-dehydrogenase, citrate synthase and cytochrome-c-oxidase activities in both patient groups were significantly lower than in controls ( p < 0.01), while NADH-cytochrome-c-reductase activity was reduced only in migraine with aura ( p < 0.01). No alteration in succinate-dehydrogenase was observed. Monoamine-oxidase activity differed between sexes (p < 0.05) but within each sex group no difference was observed between patients and controls. We hypothesize that the defect in mitochondrial enzymes observed indicates a systemic impairment of mitochondrial function in migraine patients.

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Atorvastatin impairs liver mitochondrial function in obese Göttingen Minipigs but heart and skeletal muscle are not affected

Scientific Reports ◽

10.1038/s41598-021-81846-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Liselotte Bruun Christiansen ◽

Tine Lovsø Dohlmann ◽

Trine Pagh Ludvigsen ◽

Ewa Parfieniuk ◽

Michal Ciborowski ◽

...

Keyword(s):

Skeletal Muscle ◽

Mitochondrial Function ◽

Citrate Synthase ◽

Obese Group ◽

Control Group ◽

Dependent Manner ◽

Respiratory Capacity ◽

Protein Carbonyl Content ◽

Functional Changes ◽

Mitochondrial Respiratory

AbstractStatins lower the risk of cardiovascular events but have been associated with mitochondrial functional changes in a tissue-dependent manner. We investigated tissue-specific modifications of mitochondrial function in liver, heart and skeletal muscle mediated by chronic statin therapy in a Göttingen Minipig model. We hypothesized that statins enhance the mitochondrial function in heart but impair skeletal muscle and liver mitochondria. Mitochondrial respiratory capacities, citrate synthase activity, coenzyme Q10 concentrations and protein carbonyl content (PCC) were analyzed in samples of liver, heart and skeletal muscle from three groups of Göttingen Minipigs: a lean control group (CON, n = 6), an obese group (HFD, n = 7) and an obese group treated with atorvastatin for 28 weeks (HFD + ATO, n = 7). Atorvastatin concentrations were analyzed in each of the three tissues and in plasma from the Göttingen Minipigs. In treated minipigs, atorvastatin was detected in the liver and in plasma. A significant reduction in complex I + II-supported mitochondrial respiratory capacity was seen in liver of HFD + ATO compared to HFD (P = 0.022). Opposite directed but insignificant modifications of mitochondrial respiratory capacity were seen in heart versus skeletal muscle in HFD + ATO compared to the HFD group. In heart muscle, the HFD + ATO had significantly higher PCC compared to the HFD group (P = 0.0323). In the HFD group relative to CON, liver mitochondrial respiration decreased whereas in skeletal muscle, respiration increased but these changes were insignificant when normalizing for mitochondrial content. Oral atorvastatin treatment in Göttingen Minipigs is associated with a reduced mitochondrial respiratory capacity in the liver that may be linked to increased content of atorvastatin in this organ.

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Melatonin improves neurological outcomes and preserves hippocampal mitochondrial function in a rat model of cardiac arrest

PLoS ONE ◽

10.1371/journal.pone.0207098 ◽

2018 ◽

Vol 13 (11) ◽

pp. e0207098 ◽

Cited By ~ 2

Author(s):

Linghui Yang ◽

Jing Wang ◽

Yan Deng ◽

Cansheng Gong ◽

Qin Li ◽

...

Keyword(s):

Cardiac Arrest ◽

Rat Model ◽

Mitochondrial Function ◽

Neurological Outcomes

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Abstract 2630: Evidence of Early Heart Mitochondrial Dysfunction after Resuscitation from Cardiac Arrest

Circulation ◽

10.1161/circ.116.suppl_16.ii_583 ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Natalia A Riobo ◽

Fei Han ◽

Tong Da ◽

Lance B Becker

Keyword(s):

Cardiac Arrest ◽

Cytochrome C ◽

Mitochondrial Dysfunction ◽

Complex I ◽

Atp Synthesis ◽

Spontaneous Circulation ◽

Metabolic Dysfunction ◽

Amplex Red ◽

Production Of Hydrogen ◽

Complex I Activity

Background: Reperfusion injury post-resuscitation is associated with metabolic dysfunction and free radicals production. It has been hypothesized that mitochondrial permeability changes induced by calcium overload underlies this uncoupling of respiration from ATP synthesis and initiates cytochrome c-dependent apoptotic cascades. Our goal is to evaluate the intrinsic status of the mitochondrial electron transfer chain, i.e. irreversible modifications, in resuscitated animals independently of transient changes in calcium and permeability. Methods: Female mice were subjected to 8 min cardiac arrest by KCl injection followed by mechanical ventilation and CPR until return of spontaneous circulation (resuscitation rate: 86%, 24 h survival: 40%) under continuous blood pressure and temperature monitoring. Animals were divided in 4 groups: SHAM (instrumented, no cardiac arrest), CA (8 min cardiac arrest), R30 (30 min post-resuscitation), and R60 (60 min post-resuscitation). Heart mitochondria were immediately isolated and physically disrupted to dissipate ionic gradients in the presence of a calcium chelator. Production of hydrogen peroxide (H 2 O 2 ) by Complex I and III was determined fluorometrically with the Amplex Red-HRP system and the activities of Complex I, II, and the Complex I-III and II-III segments by spectrophotometric techniques using appropriate substrates and inhibitors. Cytochrome c content and the COX IV subunit (as a loading control) were analyzed by western blot and densitometry. Results: A 40% increase in H 2 O 2 production by Complex I and III was evident after just 8 min of cardiac arrest (ECA group, p<0.05), which was followed by a progressive reduction in Complex I activity (ECA>R30>R60), resulting in a relative doubling of electron leak. In contrast, Complex II and II-III activities and cytochrome c content remained unaffected at all times evaluated. Conclusions: Using an animal model that closely mimics resuscitation in humans, we have found signs of early mitochondrial dysfunction independently of transient changes in permeability and calcium. These changes are consistent with increased ROS production at expense of impaired oxidative phosphorylation.

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Abstract 24: Bnip3 Provokes ROS Production and Maladaptive Autophagy by Displacing Uncoupling Protein3 (UCP3) From Cytochrome c Oxidase of Respiration Chain Complex in Cardiotoxicity

Circulation Research ◽

10.1161/res.117.suppl_1.24 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Rimpy Dhingra ◽

Victoria Margulets ◽

Davinder Jassal ◽

Gerald Dorn II ◽

Lorrie A. Kirshenbaum

Keyword(s):

Cell Death ◽

Cytochrome C ◽

Cytochrome C Oxidase ◽

Cardiac Myocytes ◽

Mitochondrial Function ◽

Uncoupling Protein ◽

Mitochondrial Morphology ◽

Necrotic Cell Death ◽

Ros Production ◽

Necrotic Cell

Doxorubicin is known for its cardiotoxic effects and inducing cardiac failure, however, the underlying mechanisms remain cryptic. Earlier we established the inducible - death protein, Bcl-2-like Nineteen- Kilodalton- Interacting - Protein 3 (Bnip3) to be crucial for disrupting mitochondrial function and inducing cell death of cardiac myocytes. Whether Bnip3 underlies cardiotoxic effects of doxorubicin toxicity is unknown. Herein we demonstrate a novel signaling pathway that functionally links activation and preferential mitochondrial targeting of Bnip3 to the cardiotoxic properties of doxorubicin. Perturbations to mitochondria including increased calcium loading, ROS, loss of αΨm and mPTP opening were observed in cardiac myocytes treated with doxorubicin. In mitochondria, Bnip3 forms strong association with Cytochrome c oxidase subunit1 (COX1) of respiratory chain and displaces uncoupling protein 3 (UCP3) resulting in increased ROS production, decline in maximal and reserved respiration capacity and cell viability. Impaired mitochondrial function was accompanied by an accumulated increase in autophagosomes and necrosis demonstrated by increase release of LDH, cTnT and loss of nuclear High Mobility Group Protein 1 (HMGB-1) immunoreactivity. Interestingly, pharmacological or genetic inhibition of autophagy with 3-methyl adenine (3-MA), or Atg7 knock-down suppressed necrotic cell death induced by doxorubicin. Loss of function of Bnip3 restored UCP3-COX complexes, mitochondrial respiratory integrity and abrogated necrotic cell death induced by doxorubicin. Mice germ-line deficient for Bnip3 were resistant to doxorubicin cardiotoxicity displaying normal mitochondrial morphology, cardiac function and survival rates comparable to vehicle treated mice. The findings of the present study demonstrate that doxorubicin provokes maladaptive autophagy and necrotic cell death of ventricular myocytes that is mutually dependent and obligatorily linked to Bnip3.

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Abstract 294: Alterations in Cerebral Mitochondrial Dynamics and Mitochondrial Mass After Successful Cardiopulmonary Resuscitation in a Porcine Model of Pediatric Cardiac Arrest

Circulation ◽

10.1161/circ.138.suppl_2.294 ◽

2018 ◽

Vol 138 (Suppl_2) ◽

Author(s):

Kumaran Senthil ◽

Marco M Hefti ◽

Michael Karlsson ◽

Ryan W Morgan ◽

Johannes Ehinger ◽

...

Keyword(s):

Cardiac Arrest ◽

Protein Expression ◽

Citrate Synthase ◽

Mitochondrial Dynamics ◽

Coronary Perfusion Pressure ◽

Short Chain ◽

Coronary Perfusion ◽

Mitochondrial Mass ◽

Convergence Point ◽

Neurologic Function

Introduction: A critical convergence point for neurologic injury following cardiac arrest is cerebral mitochondrial (Mt) dysfunction, regulated by Mt dynamics (balance of fusion and fission). Both fusion and fission are important to Mt homeostasis under normal conditions; however, under stress fission can decrease Mt mass and signal apoptosis, while fusion promotes oxidative phosphorylation efficiency. Our group has previously shown hemodynamic directed CPR (HD-CPR) improves Mt bioenergetics compared to AHA depth-guided CPR (DG-CPR), which correlated with better neurologic function. Hypothesis: Compared to DG-CPR, HD-CPR will result in cerebral cortical protein expression consistent with Mt fusion and preservation of mitochondrial mass 24 hours post-ROSC. Methods: One-month-old piglets were designated into three groups: 1) DG-CPR (n=5); 2) HD-CPR (n=5; goal SBP 90 mmHg, goal coronary perfusion pressure 20mmHg); 3) Shams (n=7). Groups 1 and 2 underwent 7 minutes of asphyxia, VF, 10-20 min of CPR, and post-ROSC therapies to maintain hemodynamic, oxygenation and ventilation goals. The primary outcomes were immunoblot quantification of cortical proteins for fusion (Opa-1, Opa-1 long to short chain ratio, MFN-2), fission (DRP-1), and citrate synthase activity for Mt mass. Groups shown as median and IQR and analyzed by Kruskal-Wallis, then Dunn’s multiple comparisons. Results: HD-CPR subjects had increased total Opa-1 expression compared to sham (1.52 [1.02,1.69] vs. 0.47 [0.34,0.51], p=0.001). Opa-1 long to short chain ratio was higher in HD-CPR than both sham (0.64 [0.46,0.92] vs. 0.28 [0.25,0.36], p=0.025) and DG-CPR (0.26 [0.26,0.31], p=0.018). There were no differences in MFN-2 or DRP-1 expression between groups. Mt mass was lower in DG-CPR than sham (11.02 [10.15,12.29] vs. 13.9 [12.35,15.63], p=0.029), but preserved in HD-CPR. Discussion: Piglets treated with HD-CPR exhibited protein expression consistent with Mt fusion, as evidenced by upregulation of total Opa-1, elevated Opa-1 long to short chain ratio, and preservation of Mt mass compared to standard AHA depth guided CPR strategy. We speculate that neurotherapeutics that target Mt dynamics could further improve brain Mt bioenergetics and neurologic function after pediatric CA.

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