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BACKGROUND - Out-of-hospital cardiac arrest (CA) is a prevalent medical crisis resulting in severe injury to the heart and brain and an overall survival of less than 10%. Mitochondrial dysfunction is predicted to be a key determinant of poor outcomes following prolonged CA. However, the onset and severity of mitochondrial dysfunction during CA and cardiopulmonary resuscitation (CPR) is not fully understood. Ischemic postconditioning (IPC), controlled pauses during the initiation of CPR, has been shown to improve cardiac function and neurologically favorable outcomes after 15min of CA. We tested the hypothesis that mitochondrial dysfunction develops during prolonged CA and can be rescued with IPC during CPR (IPC-CPR).
METHODS - A total of 63 swine were randomized to no ischemia (Naïve), 19min of ventricular fibrillation (VF) CA without CPR (Untreated VF), or 15min of CA with 4min of reperfusion with either standard CPR (S-CPR) or IPC-CPR. Mitochondria were isolated from the heart and brain to quantify respiration, rate of ATP synthesis, and calcium retention capacity (CRC). Reactive oxygen species (ROS) production was quantified from fresh frozen heart and brain tissue.
RESULTS - Compared to Naïve, Untreated VF induced cardiac and brain ROS overproduction concurrent with decreased mitochondrial respiratory coupling and CRC, as well as decreased cardiac ATP synthesis. Compared to Untreated VF, S-CPR attenuated brain ROS overproduction but had no other effect on mitochondrial function in the heart or brain. Compared to Untreated VF, IPC-CPR improved cardiac mitochondrial respiratory coupling and rate of ATP synthesis, and decreased ROS overproduction in the heart and brain.
CONCLUSIONS - Fifteen minutes of VF CA results in diminished mitochondrial respiration, ATP synthesis, CRC, and increased ROS production in the heart and brain. IPC-CPR attenuates cardiac mitochondrial dysfunction caused by prolonged VF CA after only 4min of reperfusion, suggesting that IPC-CPR is an effective intervention to reduce cardiac injury. However, reperfusion with both CPR methods had limited effect on mitochondrial function in the brain, emphasizing an important physiological divergence in post-arrest recovery between those two vital organs.
Copyright © 2017 Elsevier B.V. All rights reserved.
Abstract Studies in experimental models suggest that n-3 polyunsaturated fatty acids (PUFAs) improve metabolic and anti-inflammatory/antioxidant capacity of the heart, although the mechanisms are unclear and translational evidence is lacking. In this study, patients ingested a moderately high dose of n-3 PUFAs (3.4 g/day eicosapentaenoic (EPA) and doxosahexaenoic acid (DHA) ethyl-esters) for a period of 2-3 weeks before having elective cardiac surgery. Blood was obtained before treatment and at the time of surgery, and myocardial tissue from the right atrium was also dissected during surgery. Blood EPA levels increased and myocardial tissue EPA and DHA levels were significantly higher in n-3 PUFA-treated patients compared with untreated, standard-of-care control patients. Interestingly, n-3 PUFA patients had greater nuclear transactivation of peroxisome proliferator-activated receptor-γ (PPARγ), fatty acid metabolic gene expression, and enhanced mitochondrial respiration supported by palmitoyl-carnitine in the atrial myocardium, despite no difference in mitochondrial content. Myocardial tissue from n-3 PUFA patients also displayed greater expression and activity of key antioxidant/anti-inflammatory enzymes. These findings lead to our hypothesis that PPARγ activation is a mechanism by which fish oil n-3 PUFAs enhance mitochondrial fatty acid oxidation and antioxidant capacity in human atrial myocardium, and that this preoperative therapeutic regimen may be optimal for mitigating oxidative/inflammatory stress associated with cardiac surgery.
A constant provision of ATP is of necessity for cardiac contraction. As the heart progresses toward failure following a myocardial infarction (MI), it undergoes metabolic alterations that have the potential to compromise the ability to meet energetic demands. This study evaluated the efficacy of mesenchymal stem cell (MSC) transplantation into the infarcted heart to minimize impairments in the metabolic processes that contribute to energy provision. Seven and twenty-eight days following the MI and MSC transplantation, MSC administration minimized cardiac systolic dysfunction. Hyperinsulinemic-euglycemic clamps, coupled with 2-[(14)C]deoxyglucose administration, were employed to assess systemic insulin sensitivity and tissue-specific, insulin-mediated glucose uptake 36 days following the MI in the conscious, unrestrained, C57BL/6 mouse. The improved systolic performance in MSC-treated mice was associated with a preservation of in vivo insulin-stimulated cardiac glucose uptake. Conserved glucose uptake in the heart was linked to the ability of the MSC treatment to diminish the decline in insulin signaling as assessed by Akt phosphorylation. The MSC treatment also sustained mitochondrial content, ADP-stimulated oxygen flux, and mitochondrial oxidative phosphorylation efficiency in the heart. Maintenance of mitochondrial function and density was accompanied by preserved peroxisome proliferator-activated receptor-γ coactivator-1α, a master regulator of mitochondrial biogenesis. These studies provide insight into mechanisms of action that lead to an enhanced energetic state in the infarcted heart following MSC transplantation that may assist in energy provision and dampen cardiac dysfunction.
AIMS - Angiotensin II (AngII)-induced superoxide (O2(•-)) production by the NADPH oxidases and mitochondria has been implicated in the pathogenesis of endothelial dysfunction and hypertension. In this work, we investigated the specific molecular mechanisms responsible for the stimulation of mitochondrial O2(•-) and its downstream targets using cultured human aortic endothelial cells and a mouse model of AngII-induced hypertension.
RESULTS - Western blot analysis showed that Nox2 and Nox4 were present in the cytoplasm but not in the mitochondria. Depletion of Nox2, but not Nox1, Nox4, or Nox5, using siRNA inhibits AngII-induced O2(•-) production in both mitochondria and cytoplasm. Nox2 depletion in gp91phox knockout mice inhibited AngII-induced cellular and mitochondrial O2(•-) and attenuated hypertension. Inhibition of mitochondrial reverse electron transfer with malonate, malate, or rotenone attenuated AngII-induced cytoplasmic and mitochondrial O2(•-) production. Inhibition of the mitochondrial ATP-sensitive potassium channel (mitoK(+)ATP) with 5-hydroxydecanoic acid or specific PKCɛ peptide antagonist (EAVSLKPT) reduced AngII-induced H2O2 in isolated mitochondria and diminished cytoplasmic O2(•-). The mitoK(+)ATP agonist diazoxide increased mitochondrial O2(•-), cytoplasmic c-Src phosphorylation and cytoplasmic O2(•-) suggesting feed-forward regulation of cellular O2(•-) by mitochondrial reactive oxygen species (ROS). Treatment of AngII-infused mice with malate reduced blood pressure and enhanced the antihypertensive effect of mitoTEMPO. Mitochondria-targeted H2O2 scavenger mitoEbselen attenuated redox-dependent c-Src and inhibited AngII-induced cellular O2(•-), diminished aortic H2O2, and reduced blood pressure in hypertensive mice.
INNOVATION AND CONCLUSIONS - These studies show that Nox2 stimulates mitochondrial ROS by activating reverse electron transfer and both mitochondrial O2(•-) and reverse electron transfer may represent new pharmacological targets for the treatment of hypertension.
BACKGROUND - This study aimed to evaluate the efficacy of mesenchymal stem cell (MSC) transplantation to mitigate abnormalities in cardiac-specific and systemic metabolism mediated by a combination of a myocardial infarction and diet-induced insulin resistance.
METHODS - C57BL/6 mice were high-fat fed for eight weeks prior to induction of a myocardial infarction via chronic ligation of the left anterior descending coronary artery. MSCs were administered directly after myocardial infarction induction through a single intramyocardial injection. Echocardiography was performed prior to the myocardial infarction as well as seven and 28 days post-myocardial infarction. Hyperinsulinemic-euglycemic clamps coupled with 2-[14C]deoxyglucose were employed 36 days post-myocardial infarction (13 weeks of high-fat feeding) to assess systemic insulin sensitivity and insulin-mediated, tissue-specific glucose uptake in the conscious, unrestrained mouse. High-resolution respirometry was utilized to evaluate cardiac mitochondrial function in saponin-permeabilized cardiac fibers.
RESULTS - MSC administration minimized the decline in ejection fraction following the myocardial infarction. The greater systolic function in MSC-treated mice was associated with increased in vivo cardiac glucose uptake and enhanced mitochondrial oxidative phosphorylation efficiency. MSC therapy promoted reductions in fasting arterial glucose and fatty acid concentrations. Additionally, glucose uptake in peripheral tissues including skeletal muscle and adipose tissue was elevated in MSC-treated mice. Enhanced glucose uptake in these tissues was associated with improved insulin signalling as assessed by Akt phosphorylation and prevention of a decline in GLUT4 often associated with high-fat feeding.
CONCLUSIONS - These studies provide insight into the utility of MSC transplantation as a metabolic therapy that extends beyond the heart exerting beneficial systemic effects on insulin action.
Diabetes increases oxidant stress and doubles the risk of dying after myocardial infarction, but the mechanisms underlying increased mortality are unknown. Mice with streptozotocin-induced diabetes developed profound heart rate slowing and doubled mortality compared with controls after myocardial infarction. Oxidized Ca(2+)/calmodulin-dependent protein kinase II (ox-CaMKII) was significantly increased in pacemaker tissues from diabetic patients compared with that in nondiabetic patients after myocardial infarction. Streptozotocin-treated mice had increased pacemaker cell ox-CaMKII and apoptosis, which were further enhanced by myocardial infarction. We developed a knockin mouse model of oxidation-resistant CaMKIIδ (MM-VV), the isoform associated with cardiovascular disease. Streptozotocin-treated MM-VV mice and WT mice infused with MitoTEMPO, a mitochondrial targeted antioxidant, expressed significantly less ox-CaMKII, exhibited increased pacemaker cell survival, maintained normal heart rates, and were resistant to diabetes-attributable mortality after myocardial infarction. Our findings suggest that activation of a mitochondrial/ox-CaMKII pathway contributes to increased sudden death in diabetic patients after myocardial infarction.
Studies in humans and in mice have highlighted the importance of short telomeres and impaired mitochondrial function in driving age-related functional decline in the heart. Although telomere and mitochondrial dysfunction have been viewed mainly in isolation, recent studies in telomerase-deficient mice have provided evidence for an intimate link between these two processes. Telomere dysfunction induces a profound p53-dependent repression of the master regulators of mitochondrial biogenesis and function, peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α and PGC-1β in the heart, which leads to bioenergetic compromise due to impaired oxidative phosphorylation and ATP generation. This telomere-p53-PGC mitochondrial/metabolic axis integrates many factors linked to heart aging including increased DNA damage, p53 activation, mitochondrial, and metabolic dysfunction and provides a molecular basis of how dysfunctional telomeres can compromise cardiomyocytes and stem cell compartments in the heart to precipitate cardiac aging.
Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis. The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture. This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.
The finding that upon neuronal activation glutamate is transported postsynaptically from synaptic clefts and increased lactate availability for neurons suggest that brain mitochondria (BM) utilize a mixture of substrates, namely pyruvate, glutamate, and the tricarboxylic acid cycle metabolites. We studied how glutamate affected oxidative phosphorylation and reactive oxygen species (ROS) production in rat BM oxidizing pyruvate + malate or succinate. Simultaneous oxidation of glutamate + pyruvate + malate increased state 3 and uncoupled respiration by 52 and 71%, respectively. The state 4 ROS generation increased 100% over BM oxidizing pyruvate + malate and 900% over that of BM oxidizing glutamate + malate. Up to 70% of ROS generation was associated with reverse electron transport. These effects of pyruvate + glutamate + malate were observed only with BM and not with liver or heart mitochondria. The effects of glutamate + pyruvate on succinate-supported respiration and ROS generation were not organ-specific and depended only on whether mitochondria were isolated with or without bovine serum albumin. With the non-bovine serum albumin brain and heart mitochondria oxidizing succinate, the addition of pyruvate and glutamate abrogated inhibition of Complex II by oxaloacetate. We conclude that (i) during neuronal activation, simultaneous oxidation of glutamate + pyruvate temporarily enhances neuronal mitochondrial ATP production, and (ii) intrinsic inhibition of Complex II by oxaloacetate is an inherent mechanism that protects against ROS generation during reverse electron transport.
Exposure to an adverse intrauterine environment is recognized as an important risk factor for the development of cardiovascular disease later in life. Although oxidative stress has been proposed as a mechanism for the fetal programming phenotype, the role of mitochondrial O(2)(*-) (superoxide radical) production has not been explored. To determine whether mitochondrial ROS (reactive oxygen species) production is altered by in utero programming, pregnant ewes were given a 48-h dexamethasone (dexamethasone-exposed, 0.28 mg.kg(-1) of body weight.day(-1)) or saline (control) infusion at 27-28 days gestation (term=145 days). Intact left ventricular mitochondria and freeze-thaw mitochondrial membranes were studied from offspring at 4-months of age. AmplexRed was used to measure H(2)O(2) production. Activities of the antioxidant enzymes Mn-SOD (manganese superoxide dismutase), GPx (glutathione peroxidase) and catalase were measured. Compared with controls, a significant increase in Complex I H(2)O(2) production was found in intact mitochondria from dexamethasone-exposed animals. The treatment differences in Complex I-driven H(2)O(2) production were not seen in mitochondrial membranes. Consistent changes in H(2)O(2) production from Complex III in programmed animals were not found. Despite the increase in H(2)O(2) production in intact mitochondria from programmed animals, dexamethasone exposure significantly increased mitochondrial catalase activity, whereas Mn-SOD and GPx activities were unchanged. The results of the present study point to an increase in the rate of release of H(2)O(2) from programmed mitochondria despite an increase in catalase activity. Greater mitochondrial H(2)O(2) release into the cell may play a role in the development of adult disease following exposure to an adverse intrauterine environment.