RESUMO
Heart failure remains a major public-health problem with an increase in the number of patients worsening from this disease. Despite current medical therapy, the condition still has a poor prognosis. Heart failure is complex but mitochondrial dysfunction seems to be an important target to improve cardiac function directly. Our goal was to analyze the effects of MitoQ (100⯵M in drinking water) on the development and progression of heart failure induced by pressure overload after 14 weeks. The main findings are that pressure overload-induced heart failure in rats decreased cardiac function in vivo that was not altered by MitoQ. However, we observed a reduction in right ventricular hypertrophy and lung congestion in heart failure animals treated with MitoQ. Heart failure also decreased total mitochondrial protein content, mitochondrial membrane potential in the intermyofibrillar mitochondria. MitoQ restored membrane potential in IFM but did not restore mitochondrial protein content. These alterations are associated with the impairment of basal and stimulated mitochondrial respiration in IFM and SSM induced by heart failure. Moreover, MitoQ restored mitochondrial respiration in heart failure induced by pressure overload. We also detected higher levels of hydrogen peroxide production in heart failure and MitoQ restored the increase in ROS production. MitoQ was also able to improve mitochondrial calcium retention capacity, mainly in the SSM whereas in the IFM we observed a small alteration. In summary, MitoQ improves mitochondrial dysfunction in heart failure induced by pressure overload, by decreasing hydrogen peroxide formation, improving mitochondrial respiration and improving mPTP opening.
Assuntos
Antioxidantes/farmacologia , Insuficiência Cardíaca/fisiopatologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Compostos Organofosforados/farmacologia , Ubiquinona/análogos & derivados , Animais , Modelos Animais de Doenças , Mitocôndrias/efeitos dos fármacos , Ratos , Ubiquinona/farmacologiaRESUMO
xtracorporeal membrane oxygenation (ECMO) is used in cardiopulmonary resuscitation (CPR) of refractory cardiac arrest. We used a 2×2 study design to compare ECMO versus CPR and epinephrine versus placebo in a porcine model of ischemic refractory ventricular fibrillation (VF). Pigs underwent 5 minutes of untreated VF, 10 minutes of CPR, and were randomized to receive epinephrine versus placebo for another 35 minutes. Animals were further randomized to LAD reperfusion at minute 45 with ongoing CPR versus veno-arterial ECMO cannulation at minute 45 of CPR and subsequent LAD reperfusion. Four-hour survival was improved with ECMO while epinephrine showed no effect.
RESUMO
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.
Assuntos
Encéfalo/irrigação sanguínea , Reanimação Cardiopulmonar/métodos , Pós-Condicionamento Isquêmico/métodos , Mitocôndrias/fisiologia , Parada Cardíaca Extra-Hospitalar/terapia , Animais , Encéfalo/fisiologia , Modelos Animais de Doenças , Coração/fisiopatologia , Mitocôndrias Cardíacas/fisiologia , Parada Cardíaca Extra-Hospitalar/fisiopatologia , Distribuição Aleatória , Suínos , Fibrilação VentricularRESUMO
BACKGROUND: Poloxamer 188 (P188) is a nonionic triblock copolymer believed to prevent cellular injury after ischemia and reperfusion. OBJECTIVES: This study compared intracoronary infusion of P188 immediately after reperfusion with delayed infusion through a peripheral intravenous catheter in a porcine model of ST segment elevation myocardial infarction (STEMI). Cellular and mitochondrial injury were assessed. METHODS: STEMI was induced in 55 pigs using 45 minutes of endovascular coronary artery occlusion. Pigs were then randomized to four groups: control, immediate intracoronary (IC) P188, delayed peripheral P188, and polyethylene glycol (PEG) infusion. Heart tissue was collected after 4 hours of reperfusion. Assessment of mitochondrial function or infarct size was performed. RESULTS: Mitochondrial yield improved significantly with IC P188 treatment compared to control animals (0.25% vs. 0.13%) suggesting improved mitochondrial morphology and survival. Mitochondrial respiration and calcium retention were also significantly improved with immediate IC P188 compared to controls (complex I RCI: 7.4 vs. 3.7 and calcium retention (nmol): 1152 vs. 386). This benefit was only observed with activation of complex I of the mitochondrial respiratory chain suggesting a specific impact of ischemia and reperfusion on this complex. Infarct size and serum troponin I were significantly reduced by immediate IC P188 infusion (infarct size: 13.9% vs. 41.1% and troponin I (µg/L): 19.2 vs. 77.4 µg/L). Delayed P188 and PEG infusion did not provide a significant benefit. CONCLUSIONS: Intracoronary infusion of P188 immediately upon reperfusion significantly reduces cellular and mitochondrial injury after ischemia and reperfusion in this clinically relevant porcine model of STEMI. The timing and route of delivery were critical to achieve the benefit.
RESUMO
Vascular smooth muscle cells (VSMCs) play important roles in cardiovascular disorders and biology. Outlined in this paper is a step-by-step procedure for isolating aortic VSMCs from adult C57BL6J male mice by enzymatic digestion of the aorta using collagenase. The plating, culturing, and subculturing of the isolated cells are discussed in detail along with techniques to characterize VSMC phenotype by gene expression and immunofluorescence. Traction force microscopy was used to characterize contractility of single subcultured VSMCs at baseline.
Assuntos
Técnicas de Cultura de Células/normas , Separação Celular/normas , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Actinas/genética , Actinas/metabolismo , Animais , Aorta/citologia , Aorta/metabolismo , Aorta/fisiologia , Biomarcadores/metabolismo , Proliferação de Células , Separação Celular/métodos , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fenótipo , VasoconstriçãoRESUMO
We recently developed a method to measure mitochondrial proteome dynamics with heavy water ((2)H2O)-based metabolic labeling and high resolution mass spectrometry. We reported the half-lives and synthesis rates of several proteins in the two cardiac mitochondrial subpopulations, subsarcolemmal and interfibrillar (SSM and IFM), in Sprague Dawley rats. In the present study, we tested the hypothesis that the mitochondrial protein synthesis rate is reduced in heart failure, with possible differential changes in SSM versus IFM. Six to seven week old male Sprague Dawley rats underwent transverse aortic constriction (TAC) and developed moderate heart failure after 22weeks. Heart failure and sham rats of the same age received heavy water (5% in drinking water) for up to 80days. Cardiac SSM and IFM were isolated from both groups and the proteins were separated by 1D gel electrophoresis. Heart failure reduced protein content and increased the turnover rate of several proteins involved in fatty acid oxidation, electron transport chain and ATP synthesis, while it decreased the turnover of other proteins, including pyruvate dehydrogenase subunit in IFM, but not in SSM. Because of these bidirectional changes, the average overall half-life of proteins was not altered by heart failure in both SSM and IFM. The kinetic measurements of individual mitochondrial proteins presented in this study may contribute to a better understanding of the mechanisms responsible for mitochondrial alterations in the failing heart.
Assuntos
Óxido de Deutério/metabolismo , Insuficiência Cardíaca/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/biossíntese , Biossíntese de Proteínas , Proteoma/metabolismo , Animais , Peso Corporal , Respiração Celular , Citrato (si)-Sintase/metabolismo , Meia-Vida , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Masculino , Tamanho do Órgão , Oxirredução , Pressão , Estabilidade Proteica , Ratos Sprague-Dawley , Sarcolema/metabolismoRESUMO
PURPOSE: Supplementation with the n3 polyunsaturated fatty acid docosahexaenoic acid (DHA) is beneficial in heart failure patients, however the mechanisms are unclear. DHA is incorporated into membrane phospholipids, which may prevent mitochondrial dysfunction. Thus we assessed the effects of DHA supplementation on cardiac mitochondria and the development of heart failure caused by aortic pressure overload. METHODS: Pathological cardiac hypertrophy was generated in rats by thoracic aortic constriction. Animals were fed either a standard diet or were supplemented with DHA (2.3 % of energy intake). RESULTS: After 14 weeks, heart failure was evident by left ventricular hypertrophy and chamber enlargement compared to shams. Left ventricle fractional shortening was unaffected by DHA treatment in sham animals (44.1 ± 1.6 % vs. 43.5 ± 2.2 % for standard diet and DHA, respectively), and decreased with heart failure in both treatment groups, but to a lesser extent in DHA treated animals (34.9 ± 1.7 %) than with the standard diet (29.7 ± 1.5 %, P < 0.03). DHA supplementation increased DHA content in mitochondrial phospholipids and decreased membrane viscosity. Myocardial mitochondrial oxidative capacity was decreased by heart failure and unaffected by DHA. DHA treatment enhanced Ca(2+) uptake by subsarcolemmal mitochondria in both sham and heart failure groups. Further, DHA lessened Ca(2+)-induced mitochondria swelling, an index of permeability transition, in heart failure animals. Heart failure increased hydrogen peroxide-induced mitochondrial permeability transition compared to sham, which was partially attenuated in interfibrillar mitochondria by treatment with DHA. CONCLUSIONS: DHA decreased mitochondrial membrane viscosity and accelerated Ca(2+) uptake, and attenuated susceptibility to mitochondrial permeability transition and development of left ventricular dysfunction.
Assuntos
Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/uso terapêutico , Insuficiência Cardíaca/tratamento farmacológico , Disfunção Ventricular Esquerda/tratamento farmacológico , Animais , Ácido Araquidônico/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/fisiopatologia , Masculino , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/fisiologia , Fosfolipídeos/metabolismo , Pressão , Ratos , Ratos Sprague-Dawley , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
Traditional proteomics provides static assessment of protein content, but not synthetic rates. Recently, proteome dynamics with heavy water ((2)H2O) was introduced, where (2)H labels amino acids that are incorporated into proteins, and the synthesis rate of individual proteins is calculated using mass isotopomer distribution analysis. We refine this approach with a novel algorithm and rigorous selection criteria that improve the accuracy and precision of the calculation of synthesis rates and use it to measure protein kinetics in spatially distinct cardiac mitochondrial subpopulations. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated from adult rats, which were given (2)H2O in the drinking water for up to 60 days. Plasma (2)H2O and myocardial (2)H-enrichment of amino acids were stable throughout the experimental protocol. Multiple tryptic peptides were identified from 28 proteins in both SSM and IFM and showed a time-dependent increase in heavy mass isotopomers that was consistent within a given protein. Mitochondrial protein synthesis was relatively slow (average half-life of 30 days, 2.4% per day). Although the synthesis rates for individual proteins were correlated between IFM and SSM (R(2) = 0.84; P < 0.0001), values in IFM were 15% less than SSM (P < 0.001). In conclusion, administration of (2)H2O results in stable enrichment of the cardiac precursor amino acid pool, with the use of refined analytical and computational methods coupled with cell fractionation one can measure synthesis rates for cardiac proteins in subcellular compartments in vivo, and protein synthesis is slower in mitochondria located among the myofibrils than in the subsarcolemmal region.