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

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.

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

2021 ◽  
Vol 8 ◽  
Author(s):  
Tomoaki Aoki ◽  
Yu Okuma ◽  
Lance B. Becker ◽  
Kei Hayashida ◽  
Koichiro Shinozaki
Keyword(s):  
Cardiac Arrest ◽  
Cytochrome C ◽  
Mitochondrial Function ◽  
Sham Group ◽  
Citrate Synthase ◽  
Methodological Issue ◽  
Acute Injury ◽  
Adult Rats ◽  
Respiration Activity ◽  
Kidney Mitochondria

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 &lt; 0.01; kidney, p &lt; 0.001). The CS activity was significantly lower in the CA group as compared to that in the sham group (brain, p &lt; 0.01; kidney, p &lt; 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.

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

Pharmacology ◽  
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.

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

Circulation ◽  
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.

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

Circulation ◽  
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.

scholarly journals Perinatal maturation of rat kidney mitochondria

1995 ◽  
Vol 305 (2) ◽  
pp. 675-680 ◽  
Author(s):  
B Prieur ◽  
L Cordeau-Lossouarn ◽  
A Rotig ◽  
J Bismuth ◽  
J P Geloso ◽  
...  
Keyword(s):  
Respiratory Chain ◽  
Atp Synthase ◽  
Nuclear Dna ◽  
Citrate Synthase ◽  
De Novo ◽  
Adenine Nucleotide ◽  
Rat Kidney ◽  
Oxidative Capacity ◽  
Respiratory Chain Complexes ◽  
Kidney Mitochondria

In the rat kidney, NaK-ATPase activity increased between days 19 and 20 of gestation (+50%) and between 1 and 24 h after birth (+20%), requiring an increased energy supply. In order to determine whether mitochondrial changes were involved, renal mitochondrial development was investigated from day 19 of gestation to 1 day after birth. Slot-blot analyses of mitochondrial-DNA/nuclear-DNA ratio and determination of citrate synthase activity showed a doubling in the mitochondrial pool between days 19 and 20 of gestation. In isolated mitochondria, oxygen consumption remained unchanged between days 19 and 20 of gestation, and then it was enhanced between days 20 and 21 of gestation (+70%) and between 1 and 24 h after birth (+50%). We also focused on one of the respiratory-chain complexes, ATP synthase, and measured its activity and content during the perinatal period. We demonstrated increases in both activity and content of ATP synthase between days 20 and 21 of gestation and between 1 and 24 h after birth, thus suggesting that changes in ATP synthase activity are ascribed to an increase in the mitochondrial density of ATP synthase complexes. Moreover, the mitochondrial ATP/ADP ratio only increased between 1 and 24 h (+90%), indicating a critical step in the renal respiratory-chain maturation at that time. We therefore conclude that the postnatal enhancement of renal mitochondrial oxidative capacity might depend on protein synthesis de novo and on changes in the adenine nucleotide concentrations.

scholarly journals Increased cytochrome c in rat cerebrospinal fluid after cardiac arrest and its effects on hypoxic neuronal survival

Resuscitation ◽  
2012 ◽  
Vol 83 (12) ◽  
pp. 1491-1496 ◽  
Author(s):  
Hao Liu ◽  
Syana M. Sarnaik ◽  
Mioara D. Manole ◽  
Yaming Chen ◽  
Sunita N. Shinde ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Cardiac Arrest ◽  
Cytochrome C ◽  
Neuronal Survival

Regrowth of atrophied skeletal muscle in adult rats after ending immobilization

1978 ◽  
Vol 44 (2) ◽  
pp. 225-230 ◽  
Author(s):  
F. W. Booth
Keyword(s):  
Skeletal Muscle ◽  
Body Weight ◽  
Adult Female ◽  
Total Protein ◽  
Recovery Time ◽  
Time Course ◽  
Citrate Synthase ◽  
Female Rats ◽  
Adult Rats ◽  
Wet Weight

The recovery time course of muscle atrophied by immobilization was followed after removal of hindlimb casts from adult female rats. Increases of only 9% in body weight, 4% in gastrocnemius weight, and 10% in soleus weight occurred in controls during the 78-day duration of the experiment. There were no increases in the amounts of total protein or of citrate synthase activities in gastrocnemius or soleus during the first 3 days after removal of hindlimb casts; thereafter, there were increases in these paramters. Citrate synthase activities per mg of gastrocnemius protein were significantly higher at the 16th and 50th day of recovery. No significant differences for citrate synthase activity per mg of soleus occurred during recovery. Until the 50th day of recovery, no significant differences for total protein in soleus and for total protein and wet weight of gastrocnemius were observed between control and recovery values. However, the wet weight of the soleus returned rapidly during recovery and was not significantly different from control during recovery.

scholarly journals Exercise Training-Induced PPARβ Increases PGC-1α Protein Stability and Improves Insulin-Induced Glucose Uptake in Rodent Muscles

Nutrients ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 652 ◽  
Author(s):  
Ju-Sik Park ◽  
John O. Holloszy ◽  
Kijin Kim ◽  
Jin-Ho Koh
Keyword(s):  
Cytochrome C ◽  
Exercise Training ◽  
Protein Stability ◽  
Glucose Uptake ◽  
Citrate Synthase ◽  
Peroxisome Proliferator ◽  
Mitochondrial Enzymes ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Peroxisome Proliferator Activated Receptor

This study aimed to investigate the long-term effects of training intervention and resting on protein expression and stability of peroxisome proliferator-activated receptor β/δ (PPARβ), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), glucose transporter type 4 (GLUT4), and mitochondrial proteins, and determine whether glucose homeostasis can be regulated through stable expression of these proteins after training. Rats swam daily for 3, 6, 9, 14, or 28 days, and then allowed to rest for 5 days post-training. Protein and mRNA levels were measured in the skeletal muscles of these rats. PPARβ was overexpressed and knocked down in myotubes in the skeletal muscle to investigate the effects of swimming training on various signaling cascades of PGC-1α transcription, insulin signaling, and glucose uptake. Exercise training (Ext) upregulated PPARβ, PGC-1α, GLUT4, and mitochondrial enzymes, including NADH-ubiquinone oxidoreductase (NUO), cytochrome c oxidase subunit I (COX1), citrate synthase (CS), and cytochrome c (Cyto C) in a time-dependent manner and promoted the protein stability of PPARβ, PGC-1α, GLUT4, NUO, CS, and Cyto C, such that they were significantly upregulated 5 days after training cessation. PPARβ overexpression increased the PGC-1α protein levels post-translation and improved insulin-induced signaling responsiveness and glucose uptake. The present results indicate that Ext promotes the protein stability of key mitochondria enzymes GLUT4, PGC-1α, and PPARβ even after Ext cessation.

Mitochondrial DNA replication and transcription are dissociated during embryonic cardiac hypertrophy

1991 ◽  
Vol 261 (6) ◽  
pp. C1091-C1098 ◽  
Author(s):  
J. M. Kennedy ◽  
S. R. Lobacz ◽  
S. W. Kelley
Keyword(s):  
Mitochondrial Dna ◽  
Cytochrome C ◽  
Cardiac Hypertrophy ◽  
Cytochrome C Oxidase ◽  
Oxidase Activity ◽  
Citrate Synthase ◽  
Incubation Temperature ◽  
Mitochondrial Gene ◽  
Gene Replication ◽  
Mitochondrial Dna Copy Number

Cardiac hypertrophy was produced in embryonic chicks by decreasing the incubation temperature from 38 degrees C to 32 degrees C on day 11. Increases in ventricular protein, RNA, and DNA support the cardiac enlargement. Cytochrome-c oxidase activity and citrate synthase activity were depressed in hypothermic ventricles by 63% and 56%, respectively. No significant differences were seen in enzyme activities in pectoralis muscles. The involvement of mitochondrial gene replication and transcription was evaluated using a cDNA clone for the mitochondrially encoded subunit III of cytochrome-c oxidase (CO III). Quantitative slot-blot analysis demonstrated that the relative CO III mRNA concentration was reduced in hypothermic ventricles. In contrast, the relative mitochondrial DNA concentration was increased in hypothermic ventricles. Taken together, these data indicate that a hypothermia-induced decrease in cytochrome-c oxidase activity is associated with a decrease in CO III mRNA, which is not coupled to a decrease in the mitochondrial DNA copy number. This dissociation of mitochondrial gene replication and transcription may provide a useful model for examining the regulation of mitochondrial biogenesis.

scholarly journals The Responses of Tissues from the Brain, Heart, Kidney, and Liver to Resuscitation following Prolonged Cardiac Arrest by Examining Mitochondrial Respiration in Rats

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Junhwan Kim ◽  
José Paul Perales Villarroel ◽  
Wei Zhang ◽  
Tai Yin ◽  
Koichiro Shinozaki ◽  
...  
Keyword(s):  
Cardiac Arrest ◽  
Mitochondrial Respiration ◽  
Ischemia Reperfusion ◽  
Liver Mitochondria ◽  
Brain Mitochondria ◽  
Heart Mitochondria ◽  
Whole Body ◽  
Sprague Dawley ◽  
Respiration Activity ◽  
The Brain

Cardiac arrest induces whole-body ischemia, which causes damage to multiple organs. Understanding how each organ responds to ischemia/reperfusion is important to develop better resuscitation strategies. Because direct measurement of organ function is not practicable in most animal models, we attempt to use mitochondrial respiration to test efficacy of resuscitation on the brain, heart, kidney, and liver following prolonged cardiac arrest. Male Sprague-Dawley rats are subjected to asphyxia-induced cardiac arrest for 30 min or 45 min, or 30 min cardiac arrest followed by 60 min cardiopulmonary bypass resuscitation. Mitochondria are isolated from brain, heart, kidney, and liver tissues and examined for respiration activity. Following cardiac arrest, a time-dependent decrease in state-3 respiration is observed in mitochondria from all four tissues. Following 60 min resuscitation, the respiration activity of brain mitochondria varies greatly in different animals. The activity after resuscitation remains the same in heart mitochondria and significantly increases in kidney and liver mitochondria. The result shows that inhibition of state-3 respiration is a good marker to evaluate the efficacy of resuscitation for each organ. The resulting state-3 respiration of brain and heart mitochondria following resuscitation reenforces the need for developing better strategies to resuscitate these critical organs following prolonged cardiac arrest.

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