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BACKGROUND: Cardioprotection and graft evaluation after ischemia-reperfusion (IR) are essential in facilitating heart transplantation with donation after circulatory death. Given the key role of mitochondria in IR, we aimed to investigate the tolerance of cardiac mitochondria to warm, global ischemia and to determine the predictive value of early reperfusion mitochondria-related parameters for post-ischemic cardiac recovery. METHODS: Isolated, working rat hearts underwent 0, 21, 24, 27, 30, or 33 minutes of warm, global ischemia, followed by 60 minutes of reperfusion. Functional recovery (developed pressureâ¯×â¯heart rate) was determined at 60 minutes of reperfusion, whereas mitochondrial integrity was measured at 10 minutes of reperfusion. RESULTS: Functional recovery at 60 minutes of reperfusion decreased with ≥ 27 minutes of ischemia vs no ischemia (nâ¯=â¯7-8/group; p < 0.01). Cytochrome c, succinate release, and mitochondrial Ca2+ content increased with ≥ 27 minutes of ischemia vs no ischemia (p < 0.05). Ischemia at ≥ 21 minutes decreased mitochondrial coupling, adenosine 5'-triphosphate content, mitochondrial Ca2+ retention capacity, and increased oxidative damage vs no ischemia (p < 0.05). Reactive oxygen species (ROS) from reverse electron transfer increased with 21 and 27 minutes of ischemia vs no ischemia and 33 minutes of ischemia (p < 0.05), whereas ROS from forward electron transfer increased only with 33 minutes of ischemia vs no ischemia (p < 0.05). Mitochondrial coupling and adenosine 5'-triphosphate content correlated positively and cytochrome c, succinate, oxidative damage, and mitochondrial Ca2+ content correlated negatively with cardiac functional recovery (p < 0.05). CONCLUSIONS: Mitochondrial dysfunction occurs with shorter periods of ischemia than cardiac dysfunction. Mitochondrial coupling, ROS emission from reverse electron transfer, and calcium retention are particularly sensitive to early reperfusion injury, reflecting potential targets for cardioprotection. Indicators of mitochondrial integrity may be of aid in evaluating suitability of donation after circulatory death grafts for transplantation.
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Mitocondrias Cardíacas/fisiología , Reperfusión Miocárdica/métodos , Isquemia Tibia/métodos , Animales , Muerte , Trasplante de Corazón , Masculino , Modelos Animales , Daño por Reperfusión Miocárdica/etiología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
BACKGROUND: Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) is gaining widespread use in the treatment of severe cardiorespiratory failure. Blood volume expansion is commonly used to increase ECMO flow (QECMO), with risk of positive fluid balance and worsening prognosis. We studied the effects of vasoconstriction on recruitment of blood volume as an alternative for increasing QECMO, based on the concepts of venous return. METHODS: In a closed chest, centrally cannulated porcine preparation (nâ=â9) in ventricular fibrillation and VA-ECMO with vented left atrium, mean systemic filling pressure (MSFP), and venous return driving pressure (VRdP) were determined in Euvolemia, during Vasoconstriction (norepinephrine 0.05, 0.125, and 0.2âµg/kg/min) and after Volume Expansion (3 boluses of 10âmL/kg Ringer's lactate). Maximum achievable QECMO was examined. RESULTS: Vasoconstriction and Volume Expansion both increased maximum achievable QECMO, delivery of oxygen (DO2), and MSFP, but right atrial pressure increased in parallel. VRdP did not change. The vascular elastance curve was shifted to the left by Vasoconstriction, with recruitment of stressed volume. It was shifted to the right by Volume Expansion with direct expansion of stressed volume. Volume Expansion decreased resistance to venous return and pump afterload. CONCLUSIONS: In a circulation completely dependent on ECMO support, maximum achievable flow directly depended on the vascular factors governing venous return-i.e., closing conditions, stressed vascular volume and the elastance and resistive properties of the vasculature. Both treatments increased maximum achievable ECMO flow at stable DO2, via increases in stressed volume by different mechanisms. Vascular resistance and pump afterload decreased with Volume Expansion.
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Arterias/patología , Oxigenación por Membrana Extracorpórea , Vasoconstricción , Venas/patología , Animales , Velocidad del Flujo Sanguíneo , Volumen Sanguíneo , Sistema Cardiovascular , Femenino , Atrios Cardíacos/patología , Insuficiencia Cardíaca/patología , Masculino , Oxígeno/metabolismo , Riesgo , Porcinos , Fibrilación VentricularRESUMEN
BACKGROUND: Reduced cellular ATP synthesis due to impaired mitochondrial function of immune cells may be a factor influencing the immune response in septic shock. We investigate changes in mitochondrial function and bioenergetics of human monocytes and lymphocyte subsets. METHODS: Thirty patients with septic shock were studied at ICU admission, after 24 and 48 hours, and after resolution of shock. Enzymatic activities of citrate synthase and mitochondrial complexes I, IV, and ATP synthase and ATP content of monocytes, T-cells and B-cells and pro-inflammatory (IL-1ß and IL-6) and anti-inflammatory (IL-10) cytokine plasma concentrations were compared to samples from 20 healthy volunteers. RESULTS: Large variations in mitochondrial enzymatic activities of immune cells of septic patients were detected. In monocytes, maximum levels of citrate synthase activity in sepsis were significantly lower when compared to controls (p = 0.021). Maximum relative enzymatic activity (ratio relative to citrate synthase activity) of complex I (p<0.001), complex IV (p = 0.017) and ATP synthase (p<0.001) were significantly higher. In T-cells, maximum levels of citrate synthase (p = 0.583) and relative complex IV (p = 0.602) activity did not differ between patients and controls, whereas levels of relative complex I (p = 0.006) and ATP synthase (p = 0.032) were significantly higher in septic patients. In B-cells of patients, maximum levels of citrate synthase activity (p = 0.004) and relative complex I (p<0.001) were significantly higher, and mean levels of relative complex IV (p = 0.042) lower than the control values, whereas relative ATP synthase activity did not differ (p = 1.0). No significant difference in cellular ATP content was detected in any cell line (p = 0.142-0.519). No significant correlations between specific cytokines and parameters of mitochondrial enzymatic activities or ATP content were observed. CONCLUSIONS: Significant changes of mitochondrial enzymatic activities occur in human peripheral blood immune cells in septic shock when compared to healthy controls. Assessed sub-types of immune cells showed differing patterns of regulation. Total ATP-content of human immune cells did not differ between patients in septic shock and healthy volunteers.
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Complejos de ATP Sintetasa/biosíntesis , Linfocitos B/metabolismo , Mitocondrias/genética , Choque Séptico/genética , Linfocitos T/metabolismo , Complejos de ATP Sintetasa/inmunología , Adenosina Trifosfato/biosíntesis , Anciano , Linfocitos B/inmunología , Linfocitos B/patología , Citrato (si)-Sintasa/genética , Femenino , Voluntarios Sanos , Humanos , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Masculino , Persona de Mediana Edad , Mitocondrias/inmunología , Mitocondrias/patología , Monocitos/inmunología , Monocitos/metabolismo , Choque Séptico/inmunología , Choque Séptico/patología , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
BACKGROUND: A decrease in blood lactate levels (Lac) >10% during the first hours of resuscitation in sepsis is associated with better outcomes, but the mechanisms are unclear. Our objective was to investigate the relationship between the time course of Lac, inflammatory response, and mitochondrial respiration during experimental sepsis. METHODS: Original data from two previously published studies were reanalyzed. In cohort 1, pigs were randomized to be resuscitated for 48 h starting at 6, 12, and 24 h, respectively, after fecal peritonitis induction (n = 8 each). Animals were categorized according to the decrease in Lac during the first 6 h of resuscitation (early if ≥10% [Lac ≥10%] or late if <10% or increased [Lac <10%]), and systemic hemodynamics, inflammatory parameters, and mitochondrial function were compared between groups. In a second group of animals with fecal peritonitis and 24 h of resuscitation (n = 16, cohort 2), abdominal regional Lac exchange was measured, and animals were categorized according to the decrease in Lac as in cohort 1. RESULTS: Overall mortality was 20% (4 of 20) in the Lac ≥10% group and 60% (12 of 20) in the Lac <10% group (p = 0.022). In cohort 1, systemic hemodynamics were similar in the Lac ≥10% (n = 13) and Lac <10% (n = 11) groups. Plasma interleukin-6 levels increased during unresuscitated sepsis and decreased during resusciation in both groups, but they were lower at study end in the Lac ≥10% group (p = 0.047). Complexes I and II maximal (state 3) and resting (state 4) isolated brain mitochondrial respiration at study end was higher in the Lac ≥10% group than in the Lac <10% group, whereas hepatic, myocardial, and skeletal muscle mitochondrial respiration was similar in both groups. In cohort 2, mesenteric, total hepatic, and renal blood flow at study end was higher in the Lac ≥10% group (n = 7) than in the Lac <10% group (n = 9), despite similar cardiac output. Hepatic lactate influx and uptake in the Lac ≥10% group were approximately 1.5 and 3 times higher, respectively, than in the Lac <10% group (p = 0.066 for both). CONCLUSIONS: A decrease in Lac >10% during early resuscitation (6 h) after abdominal sepsis is associated with lower levels of plasma interleukin-6 and improved brain but not hepatic or muscle mitochondrial respiration. Blood flow redistribution to abdominal organs in animals with early decrease in Lac concentrations increases the potential to both deliver and extract Lac.
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Inflamación/metabolismo , Ácido Láctico/análisis , Mitocondrias/metabolismo , Sepsis/complicaciones , Factores de Tiempo , Animales , Análisis de los Gases de la Sangre/métodos , Proteína C-Reactiva/análisis , Estudios de Cohortes , Modelos Animales de Enfermedad , Hemodinámica/fisiología , Interleucina-6/análisis , Interleucina-6/sangre , Ácido Láctico/sangre , Oxígeno/metabolismo , Peritonitis/complicaciones , Peritonitis/fisiopatología , Distribución Aleatoria , Resucitación/métodos , Sepsis/mortalidad , Sepsis/fisiopatología , Análisis de Supervivencia , Porcinos , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.
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Mitocondrias Musculares/química , Músculo Esquelético/citología , Animales , Biopsia , Células Cultivadas , Centrifugación , Modelos Animales , Músculo Esquelético/metabolismo , PorcinosRESUMEN
A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.
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Respiración de la Célula/fisiología , Células Hep G2/metabolismo , Mitocondrias Hepáticas/fisiología , Consumo de Oxígeno/fisiología , Células Cultivadas , Humanos , Fosforilación OxidativaRESUMEN
BACKGROUND: Remifentanil interferes with hepatic mitochondrial function. The aim of the present study was to evaluate whether hepatic mitochondrial function is affected by fentanyl, a more widely used opioid than remifentanil. METHODS: Human hepatoma HepG2 cells were exposed to fentanyl or pretreated with naloxone (an opioid receptor antagonist) or 5-hydroxydecanoate (5-HD, an inhibitor of mitochondrial adenosine triphosphate (ATP)-sensitive potassium [mitoKATP] channels), followed by incubation with fentanyl. Mitochondrial function and metabolism were then analyzed. RESULTS: Fentanyl marginally reduced maximal mitochondrial complex-specific respiration rates using exogenous substrates (decrease in medians: 11%-18%; P = 0.003-0.001) but did not affect basal cellular respiration rates (P = 0.834). The effect on stimulated respiration was prevented by preincubation with naloxone or 5-HD. Fentanyl reduced cellular ATP content in a dose-dependent manner (P < 0.001), an effect that was not significantly prevented by 5-HD and not explained by increased total ATPase concentration. However, in vitro ATPase activity of recombinant human permeability glycoprotein (an ATP-dependent drug efflux transporter) was significantly stimulated by fentanyl (P = 0.004). CONCLUSIONS: Our data suggest that fentanyl reduces stimulated mitochondrial respiration of cultured human hepatocytes by a mechanism that is blocked by a mitoKATP channel antagonist. Increased energy requirements for fentanyl efflux transport may offer an explanation for the substantial decrease in cellular ATP concentration.
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Analgésicos Opioides/farmacología , Fentanilo/farmacología , Mitocondrias Hepáticas/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/agonistas , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Respiración de la Célula/efectos de los fármacos , Ácidos Decanoicos/farmacología , Relación Dosis-Respuesta a Droga , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Metabolismo Energético/efectos de los fármacos , Células Hep G2 , Humanos , Hidroxiácidos/farmacología , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/ultraestructura , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Consumo de Oxígeno/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismoRESUMEN
OBJECTIVES: Exposure to high altitudes is associated with oxidative cellular damage due to the increased level of reactive oxygen and nitrogen species and altered activity of antioxidant systems. Subjects were submitted to prolonged hypoxia, to evaluate changes in mitochondrial enzyme activities of monocytes and their attenuation by supplementation with antioxidants. METHODS: Twelve subjects were randomly assigned to receive antioxidant supplements or placebo prior to and during an expedition to Pik Lenin (7145 m). Monocytes were isolated from blood samples to determine the activity of mitochondrial enzymes cytochrome c oxidase and citrate synthase at 490 m (baseline) and at the altitudes of 3550 m, 4590 m, and 5530 m. RESULTS: An increase in citrate synthase activity at all altitudes levels was observed. Hypoxia induced an increase in the activity of cytochrome c oxidase only at 4590 m. Neither citrate synthase activity nor cytochrome c oxidase activity differed between the subjects receiving antioxidant supplements and those receiving placebo. CONCLUSIONS: Hypoxia leads to an increase in citrate synthase activity of monocyte mitochondria as a marker of mitochondrial mass, which is not modified by antioxidant supplementation. The increase in mitochondrial mass may represent a compensatory mechanism to preserve oxidative phosphorylation of monocytes at high altitudes.
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Antioxidantes/uso terapéutico , Hipoxia/tratamiento farmacológico , Hipoxia/enzimología , Mitocondrias/enzimología , Monocitos/enzimología , Adolescente , Adulto , Anciano , Presión Atmosférica , Femenino , Humanos , Hipoxia/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Masculino , Persona de Mediana Edad , Estrés Oxidativo/efectos de los fármacos , Adulto JovenRESUMEN
INTRODUCTION: Results on mitochondrial dysfunction in sepsis are controversial. We aimed to assess effects of LPS at wide dose and time ranges on hepatocytes and isolated skeletal muscle mitochondria. METHODS: Human hepatocellular carcinoma cells (HepG2) were exposed to placebo or LPS (0.1, 1, and 10 µg/mL) for 4, 8, 16, and 24 hours and primary human hepatocytes to 1 µg/mL LPS or placebo (4, 8, and 16 hours). Mitochondria from porcine skeletal muscle samples were exposed to increasing doses of LPS (0.1-100 µg/mg) for 2 and 4 hours. Respiration rates of intact and permeabilized cells and isolated mitochondria were measured by high-resolution respirometry. RESULTS: In HepG2 cells, LPS reduced mitochondrial membrane potential and cellular ATP content but did not modify basal respiration. Stimulated complex II respiration was reduced time-dependently using 1 µg/mL LPS. In primary human hepatocytes, stimulated mitochondrial complex II respiration was reduced time-dependently using 1 µg/mL LPS. In isolated porcine skeletal muscle mitochondria, stimulated respiration decreased at high doses (50 and 100 µg/mL LPS). CONCLUSION: LPS reduced cellular ATP content of HepG2 cells, most likely as a result of the induced decrease in membrane potential. LPS decreased cellular and isolated mitochondrial respiration in a time-dependent, dose-dependent and complex-dependent manner.
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Endotoxinas/farmacología , Hepatocitos/metabolismo , Mitocondrias Musculares/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Respiración de la Célula/efectos de los fármacos , Separación Celular , Supervivencia Celular/efectos de los fármacos , Células Hep G2 , Hepatocitos/efectos de los fármacos , Humanos , Lipopolisacáridos/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Musculares/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sus scrofa , Factores de TiempoRESUMEN
INTRODUCTION: Sepsis-induced myopathy and critical illness myopathy (CIM) are possible causes of muscle weakness in intensive care patients. They have been attributed to muscle membrane dysfunction. The aim of this study was to investigate membrane properties in the early stage of experimental sepsis by evaluating muscle excitability. METHODS: In total, 20 anesthetized and mechanically ventilated pigs were randomized to either faecal peritonitis (n = 10) or to non-septic controls (n = 10). Resuscitation with fluids and vasoactive drugs was started 3 hours after peritonitis induction. Muscle membrane properties were investigated by measuring muscle velocity recovery cycles before induction of peritonitis as well as 6, 18 and 27 hours thereafter. Muscle relative refractory period (MRRP) and early supernormality (ESN) were assessed. RESULTS: Peritonitis lasting 27 hours was associated with an increase of MRRP by 28% from 2.38 ± 0.18 ms (mean ± SD) to 3.47 ± 1.79 ms (P <0.01) and a decrease of ESN by 31% from 9.64 ± 2.82% to 6.50 ± 2.64% (P <0.01). ESN reduction was already apparent 6 hours after induction of peritonitis. Values in controls did not show any significant alterations. CONCLUSIONS: Muscle membrane abnormalities consistent with membrane depolarization and/or sodium channel inactivation occurred within 6 hours of peritonitis induction. This indicates that changes that have been described in established sepsis-induced myopathy and/or CIM start early in the course of sepsis. Muscle excitability testing facilitates evaluation of the time course of these changes.
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Debilidad Muscular/etiología , Músculo Esquelético/patología , Peritonitis/patología , Sepsis/complicaciones , Animales , Biomarcadores/sangre , Western Blotting , Enfermedad Crítica , Modelos Animales de Enfermedad , Frecuencia Cardíaca/fisiología , Debilidad Muscular/patología , Debilidad Muscular/fisiopatología , Músculo Esquelético/fisiopatología , Conducción Nerviosa/fisiología , Peritonitis/fisiopatología , Respiración Artificial/efectos adversos , Volumen Sistólico , Porcinos , Factores de Tiempo , Nervio Cubital/fisiologíaRESUMEN
Acute reduction of portal vein blood flow (Qpv) increases hepatic arterial perfusion (Qha) [the hepatic arterial buffer response (HABR)]. Angiotensin II (AT-II) reduces Qpv, but its effect on HABR is not known. We explored interactions of AT-II and enalapril with hepatic blood flow regulation. Twenty healthy anesthetized pigs were randomized to receive AT-II (n = 8) from 5 to 61 ng/kg per min, enalapril (n = 8) from 3 to 24 µg/kg per h, or saline (n = 4). HABR was assessed by occluding portal vein and expressed as 1) ratio between changes in Qha and Qpv, 2) hepatic arterial conductance (Cha). AT-II infusion increased mean arterial blood pressure from 74 (66-77) mmHg to 116 (109-130) mmHg (median, IQR; P < 0.0001) and decreased cardiac output, Qpv, and renal artery flow (-24%, -28% and -45%, respectively). The fraction of cardiac output of Qha, carotid, and femoral flows increased. With enalapril, blood pressure decreased, whereas cardiac output was maintained with flow redistribution favoring hepatic and renal arteries. In AT-II group, dQha/dQpv increased from 0.06 (0.03, 0.17) to 0.24 (0.13, 0.31) (P = 0.002), but Cha during acute portal vein occlusion decreased from 4.3 (1.6, 6.6) to 2.9 (1.2, 3.7) ml/mmHg (P = 0.003). Both variables remained unchanged in the enalapril group and in controls. AT-II infusion reduces portal flow in parallel with cardiac output and induces a dose-dependent redistribution of flow, favoring brain, hepatic artery, and peripheral tissues at the expense of renal perfusion. During HABR, AT-II decreases Cha but increases Qha compensation, likely as result of increased hepatic arterial perfusion pressure. Enalapril had no effect on HABR.
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Angiotensina II/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Enalapril/farmacología , Hemodinámica/efectos de los fármacos , Arteria Hepática/efectos de los fármacos , Circulación Hepática/efectos de los fármacos , Animales , Presión Arterial/efectos de los fármacos , Gasto Cardíaco/efectos de los fármacos , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/fisiología , Circulación Cerebrovascular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Arteria Hepática/fisiopatología , Masculino , Vena Porta/efectos de los fármacos , Vena Porta/fisiología , Arteria Renal/efectos de los fármacos , Arteria Renal/fisiología , Circulación Renal/efectos de los fármacos , Porcinos , Factores de TiempoRESUMEN
OBJECTIVES: To compare effects of norepinephrine and angiotensin II in experimental sepsis on hemodynamics, organ function, and mitochondrial respiration. DESIGN: Randomized, controlled, study. SETTING: University experimental laboratory. SUBJECTS: Twenty-eight anesthetized, mechanically ventilated pigs. INTERVENTIONS: Sixteen pigs were randomized to receive after 12 hours of fecal peritonitis fluid resuscitation and either norepinephrine (group NE; n = 8) or angiotensin II (group AT-II; n = 8) for 48 hours. A separate group (n = 8), treated with enalapril for 1 week before peritonitis and until study end, received fluids and norepinephrine (group E). The blood pressure dose-response to angiotensin II was evaluated in additional four nonseptic pigs. MEASUREMENTS AND MAIN RESULTS: Blood pressure target (75-85 mm Hg) was reached in both NE and AT-II, and cardiac output increased similarly (NE: from 64 mL/kg/min [60-79 mL/kg/min] to 139 mL/kg/min [126-157 mL/kg/min]; AT-II from 79 mL/kg/min [65-86 mL/kg/min] to 145 mL/kg/min [126-147 mL/kg/min]; median, interquartile range). Renal plasma flow, prevalence of acute kidney injury, inflammation and coagulation patterns, and mitochondrial respiration did not differ between NE and AT-II. In group E, blood pressure targets were not achieved (mean arterial pressure at study end: NE: 81 mm Hg [76-85 mm Hg]; AT-II: 80 mm Hg [77-84 mm Hg]; E: 53 mm Hg [49-66 mm Hg], p = 0.002, compared to NE), whereas skeletal muscle adenosine triphosphate concentrations were increased. During resuscitation one animal died in groups AT-II and E. CONCLUSIONS: Angiotensin II reversed sepsis-induced hypotension with systemic and regional hemodynamic effects similar to those of norepinephrine. Inhibition of angiotensin-converting enzyme before sepsis worsened the hypotension but enhanced skeletal muscle adenosine triphosphate. Modifying the renin-angiotensin system in sepsis should be further evaluated.
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Angiotensina II/farmacología , Hipotensión/tratamiento farmacológico , Norepinefrina/farmacología , Peritonitis/complicaciones , Choque Séptico/tratamiento farmacológico , Vasoconstrictores/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Gasto Cardíaco/efectos de los fármacos , Respiración de la Célula/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Fluidoterapia , Hemodinámica/efectos de los fármacos , Hipotensión/complicaciones , Masculino , Mitocondrias/fisiología , Distribución Aleatoria , Sistema Renina-Angiotensina/efectos de los fármacos , Choque Séptico/etiología , PorcinosRESUMEN
BACKGROUND: Changes in hepatosplanchnic lactate exchange are likely to contribute to hyperlactatemia in sepsis. We hypothesized that septic and cardiogenic shock have different effects on hepatosplanchnic lactate exchange and its contribution to hyperlactatemia. MATERIALS AND METHODS: 24 anesthetized pigs were randomized to fecal peritonitis (P), cardiac tamponade (CT), and to controls (n = 8 per group). Oxygen transport and lactate exchange were calculated during 24 hours. RESULTS: While hepatic lactate influx increased in P and in CT, hepatic lactate uptake remained unchanged in P and decreased in CT. Hepatic lactate efflux contributed 20% (P) and 33% (CT), respectively, to whole body venous efflux. Despite maintained hepatic arterial blood flow, hepatic oxygen extraction did not increase in CT. CONCLUSIONS: Whole body venous lactate efflux is of similar magnitude in hyperdynamic sepsis and in cardiogenic shock. Although jejunal mucosal pCO2 gradients are increased, enhanced lactate production from other tissues is more relevant to the increased arterial lactate. Nevertheless, the liver fails to increase hepatic lactate extraction in response to rising hepatic lactate influx, despite maintained hepatic oxygen consumption. In cardiac tamponade, regional, extrasplanchnic lactate production is accompanied by hepatic failure to increase oxygen extraction and net hepatic lactate output, despite maintained hepatic arterial perfusion.
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Taponamiento Cardíaco/complicaciones , Heces , Lactatos/sangre , Peritonitis/complicaciones , Vísceras/patología , Animales , Dióxido de Carbono/metabolismo , Taponamiento Cardíaco/sangre , Taponamiento Cardíaco/fisiopatología , Hemodinámica , Mucosa Intestinal/irrigación sanguínea , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Hígado/irrigación sanguínea , Hígado/metabolismo , Hígado/patología , Hígado/fisiopatología , Oxígeno/metabolismo , Presión Parcial , Peritonitis/sangre , Peritonitis/fisiopatología , Flujo Sanguíneo Regional , Sus scrofa , Vísceras/irrigación sanguíneaRESUMEN
The circulating, endocrine renin-angiotensin system (RAS) is important to circulatory homeostasis, while ubiquitous tissue and cellular RAS play diverse roles, including metabolic regulation. Indeed, inhibition of RAS is associated with improved cellular oxidative capacity. Recently it has been suggested that an intra-mitochondrial RAS directly impacts on metabolism. Here we sought to rigorously explore this hypothesis. Radiolabelled ligand-binding and unbiased proteomic approaches were applied to purified mitochondrial sub-fractions from rat liver, and the impact of AngII on mitochondrial function assessed. Whilst high-affinity AngII binding sites were found in the mitochondria-associated membrane (MAM) fraction, no RAS components could be detected in purified mitochondria. Moreover, AngII had no effect on the function of isolated mitochondria at physiologically relevant concentrations. We thus found no evidence of endogenous mitochondrial AngII production, and conclude that the effects of AngII on cellular energy metabolism are not mediated through its direct binding to mitochondrial targets.
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Angiotensina II/farmacología , Angiotensina II/farmacocinética , Mitocondrias Hepáticas/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Sistema Renina-Angiotensina/fisiología , Animales , Células Cultivadas , Mitocondrias Hepáticas/efectos de los fármacos , Ratas , Sistema Renina-Angiotensina/efectos de los fármacosRESUMEN
BACKGROUND: The relevance of mitochondrial dysfunction as to pathogenesis of multiple organ dysfunction and failure in sepsis is controversial. This focused review evaluates the evidence for impaired mitochondrial function in sepsis. DESIGN: Review of original studies in experimental sepsis animal models and clinical studies on mitochondrial function in sepsis. In vitro studies solely on cells and tissues were excluded. PubMed was searched for articles published between 1964 and July 2012. RESULTS: Data from animal experiments (rodents and pigs) and from clinical studies of septic critically ill patients and human volunteers were included. A clear pattern of sepsis-related changes in mitochondrial function is missing in all species. The wide range of sepsis models, length of experiments, presence or absence of fluid resuscitation and methods to measure mitochondrial function may contribute to the contradictory findings. A consistent finding was the high variability of mitochondrial function also in control conditions and between organs. CONCLUSION: Mitochondrial function in sepsis is highly variable, organ specific and changes over the course of sepsis. Patients who will die from sepsis may be more affected than survivors. Nevertheless, the current data from mostly young and otherwise healthy animals does not support the view that mitochondrial dysfunction is the general denominator for multiple organ failure in severe sepsis and septic shock. Whether this is true if underlying comorbidities are present, especially in older patients, should be addressed in further studies.
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Mitocondrias/fisiología , Insuficiencia Multiorgánica/fisiopatología , Sepsis/fisiopatología , Animales , Humanos , Modelos Animales , Insuficiencia Multiorgánica/complicaciones , Choque Séptico/complicaciones , Choque Séptico/fisiopatologíaRESUMEN
INTRODUCTION: The objective of this study was to evaluate the effects of two different mean arterial blood pressure (MAP) targets on needs for resuscitation, organ dysfunction, mitochondrial respiration and inflammatory response in a long-term model of fecal peritonitis. METHODS: Twenty-four anesthetized and mechanically ventilated pigs were randomly assigned (n = 8/group) to a septic control group (septic-CG) without resuscitation until death or one of two groups with resuscitation performed after 12 hours of untreated sepsis for 48 hours, targeting MAP 50-60 mmHg (low-MAP) or 75-85 mmHg (high-MAP). RESULTS: MAP at the end of resuscitation was 56 ± 13 mmHg (mean ± SD) and 76 ± 17 mmHg respectively, for low-MAP and high-MAP groups. One animal each in high- and low-MAP groups, and all animals in septic-CG died (median survival time: 21.8 hours, inter-quartile range: 16.3-27.5 hours). Norepinephrine was administered to all animals of the high-MAP group (0.38 (0.21-0.56) mcg/kg/min), and to three animals of the low-MAP group (0.00 (0.00-0.25) mcg/kg/min; P = 0.009). The high-MAP group had a more positive fluid balance (3.3 ± 1.0 mL/kg/h vs. 2.3 ± 0.7 mL/kg/h; P = 0.001). Inflammatory markers, skeletal muscle ATP content and hemodynamics other than MAP did not differ between low- and high-MAP groups. The incidence of acute kidney injury (AKI) after 12 hours of untreated sepsis was, respectively for low- and high-MAP groups, 50% (4/8) and 38% (3/8), and in the end of the study 57% (4/7) and 0% (P = 0.026). In septic-CG, maximal isolated skeletal muscle mitochondrial Complex I, State 3 respiration increased from 1357 ± 149 pmol/s/mg to 1822 ± 385 pmol/s/mg, (P = 0.020). In high- and low-MAP groups, permeabilized skeletal muscle fibers Complex IV-state 3 respiration increased during resuscitation (P = 0.003). CONCLUSIONS: The MAP targets during resuscitation did not alter the inflammatory response, nor affected skeletal muscle ATP content and mitochondrial respiration. While targeting a lower MAP was associated with increased incidence of AKI, targeting a higher MAP resulted in increased net positive fluid balance and vasopressor load during resuscitation. The long-term effects of different MAP targets need to be evaluated in further studies.
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Presión Arterial/fisiología , Hemodinámica/fisiología , Riñón/fisiología , Sepsis/metabolismo , Sepsis/fisiopatología , Vasoconstrictores , Equilibrio Hidroelectrolítico/fisiología , Animales , Presión Arterial/efectos de los fármacos , Femenino , Hemodinámica/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Riñón/efectos de los fármacos , Masculino , Distribución Aleatoria , Resucitación/métodos , Sepsis/terapia , Porcinos , Vasoconstrictores/farmacología , Vasoconstrictores/uso terapéuticoRESUMEN
During sepsis, liver dysfunction is common, and failure of mitochondria to effectively couple oxygen consumption with energy production has been described. In addition to sepsis, pharmacological agents used to treat septic patients may contribute to mitochondrial dysfunction. This study addressed the hypothesis that remifentanil interacts with hepatic mitochondrial oxygen consumption. The human hepatoma cell line HepG2 and their isolated mitochondria were exposed to remifentanil, with or without further exposure to tumor necrosis factor-α (TNF-α). Mitochondrial oxygen consumption was measured by high-resolution respirometry, Caspase-3 protein levels by Western blotting, and cytokine levels by ELISA. Inhibitory κBα (IκBα) phosphorylation, measurement of the cellular ATP content and mitochondrial membrane potential in intact cells were analysed using commercial ELISA kits. Maximal cellular respiration increased after one hour of incubation with remifentanil, and phosphorylation of IκBα occurred, denoting stimulation of nuclear factor κB (NF-κB). The effect on cellular respiration was not present at 2, 4, 8 or 16 hours of incubation. Remifentanil increased the isolated mitochondrial respiratory control ratio of complex-I-dependent respiration without interfering with maximal respiration. Preincubation with the opioid receptor antagonist naloxone prevented a remifentanil-induced increase in cellular respiration. Remifentanil at 10× higher concentrations than therapeutic reduced mitochondrial membrane potential and ATP content without uncoupling oxygen consumption and basal respiration levels. TNF-α exposure reduced respiration of complex-I, -II and -IV, an effect which was prevented by prior remifentanil incubation. Furthermore, prior remifentanil incubation prevented TNF-α-induced IL-6 release of HepG2 cells, and attenuated fragmentation of pro-caspase-3 into cleaved active caspase 3 (an early marker of apoptosis). Our data suggest that remifentanil increases cellular respiration of human hepatocytes and prevents TNF-α-induced mitochondrial dysfunction. The results were not explained by uncoupling of mitochondrial respiration.
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Hepatocitos/efectos de los fármacos , Hipnóticos y Sedantes/farmacología , Mitocondrias/efectos de los fármacos , Piperidinas/farmacología , Adenosina Trifosfato/metabolismo , Caspasa 3/metabolismo , Respiración de la Célula/efectos de los fármacos , Respiración de la Célula/fisiología , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Complejo I de Transporte de Electrón/metabolismo , Complejo II de Transporte de Electrones/antagonistas & inhibidores , Complejo II de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Complejo IV de Transporte de Electrones/metabolismo , Células Hep G2 , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Quinasa I-kappa B/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Fosforilación , Remifentanilo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
OBJECTIVE: Early treatment in sepsis may improve outcome. The aim of this study was to evaluate how the delay in starting resuscitation influences the severity of sepsis and the treatment needed to achieve hemodynamic stability. DESIGN: Prospective, randomized, controlled experimental study. SETTING: Experimental laboratory in a university hospital. SUBJECTS: Thirty-two anesthetized and mechanically ventilated pigs. INTERVENTIONS: Pigs were randomly assigned (n=8 per group) to a nonseptic control group or one of three groups in which fecal peritonitis (peritoneal instillation of 2 g/kg autologous feces) was induced, and a 48-hr period of protocolized resuscitation started 6 (ΔT-6 hrs), 12 (ΔT-12 hrs), or 24 (ΔT-24 hrs) hrs later. The aim of this study was to evaluate the impact of delays in resuscitation on disease severity, need for resuscitation, and the development of sepsis-associated organ and mitochondrial dysfunction. MEASUREMENTS AND MAIN RESULTS: Any delay in starting resuscitation was associated with progressive signs of hypovolemia and increased plasma levels of interleukin-6 and tumor necrosis factor-α prior to resuscitation. Delaying resuscitation increased cumulative net fluid balances (2.1±0.5 mL/kg/hr, 2.8±0.7 mL/kg/hr, and 3.2±1.5 mL/kg/hr, respectively, for groups ΔT-6 hrs, ΔT-12 hrs, and ΔT-24 hrs; p<.01) and norepinephrine requirements during the 48-hr resuscitation protocol (0.02±0.04 µg/kg/min, 0.06±0.09 µg/kg/min, and 0.13±0.15 µg/kg/min; p=.059), decreased maximal brain mitochondrial complex II respiration (p=.048), and tended to increase mortality (p=.08). Muscle tissue adenosine triphosphate decreased in all groups (p<.01), with lowest values at the end in groups ΔT-12 hrs and ΔT-24 hrs. CONCLUSIONS: Increasing the delay between sepsis initiation and resuscitation increases disease severity, need for resuscitation, and sepsis-associated brain mitochondrial dysfunction. Our results support the concept of a critical window of opportunity in sepsis resuscitation.
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Resucitación/métodos , Sepsis/fisiopatología , Sepsis/terapia , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Fluidoterapia , Hemodinámica , Masculino , Peritonitis/mortalidad , Peritonitis/fisiopatología , Peritonitis/terapia , Estudios Prospectivos , Distribución Aleatoria , Sepsis/mortalidad , Índice de Severidad de la Enfermedad , Porcinos , Factores de TiempoRESUMEN
BACKGROUND: Effects of androgens on angiogenesis are controversial. Hypoxia-inducible factor (HIF)-1α promotes expression of vascular endothelial growth factor (VEGF) that stimulates angiogenesis. PURPOSE: This study investigates whether androgens stabilize HIF-1α in endothelial cells, and androgen depletion decreases VEGF concentrations and skin flap survival. MATERIALS AND METHODS: Male human umbilical vein endothelial cells (HUVECs) were exposed to dihydrotestosterone (DHT) and HIF-1α expression was measured. In male Wistar rats, standardized proximally based random pattern dorsal skin flaps (3 × 9 cm) were raised 4 weeks after orchiectomy and sham operation, respectively (n = 10, each). Flap VEGF concentrations (immunohistochemistry), perfusion (Laser Doppler), and viability (digital planimetry) were measured. RESULTS: DHT induced HIF-1α expression in HUVECs. Androgen depletion induced decreased VEGF expression (P = 0.003), flap perfusion (P < 0.05), and survival (44.4% ± 5.2%) compared to controls (35.5% ± 4.5%; P = 0.003). CONCLUSION: In vitro, androgens may stimulate HIF-1α under normoxic conditions. In rats, androgen depletion decrease VEGF expression and flap survival.
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Andrógenos/fisiología , Dihidrotestosterona , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Isquemia , Neovascularización Fisiológica/fisiología , Colgajos Quirúrgicos/irrigación sanguínea , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inmunohistoquímica , Flujometría por Láser-Doppler , Masculino , Orquiectomía , Ratas , Ratas Wistar , Colgajos Quirúrgicos/fisiología , Cicatrización de Heridas/fisiologíaRESUMEN
This work was motivated by the incomplete characterization of the role of vascular endothelial growth factor-A (VEGF-A) in the stressed heart in consideration of upcoming cancer treatment options challenging the natural VEGF balance in the myocardium. We tested, if the cytotoxic cancer therapy doxorubicin (Doxo) or the anti-angiogenic therapy sunitinib alters viability and VEGF signaling in primary cardiac microvascular endothelial cells (CMEC) and adult rat ventricular myocytes (ARVM). ARVM were isolated and cultured in serum-free medium. CMEC were isolated from the left ventricle and used in the second passage. Viability was measured by LDH-release and by MTT-assay, cellular respiration by high-resolution oxymetry. VEGF-A release was measured using a rat specific VEGF-A ELISA-kit. CMEC were characterized by marker proteins including CD31, von Willebrand factor, smooth muscle actin and desmin. Both Doxo and sunitinib led to a dose-dependent reduction of cell viability. Sunitinib treatment caused a significant reduction of complex I and II-dependent respiration in cardiomyocytes and the loss of mitochondrial membrane potential in CMEC. Endothelial cells up-regulated VEGF-A release after peroxide or Doxo treatment. Doxo induced HIF-1α stabilization and upregulation at clinically relevant concentrations of the cancer therapy. VEGF-A release was abrogated by the inhibition of the Erk1/2 or the MAPKp38 pathway. ARVM did not answer to Doxo-induced stress conditions by the release of VEGF-A as observed in CMEC. VEGF receptor 2 amounts were reduced by Doxo and by sunitinib in a dose-dependent manner in both CMEC and ARVM. In conclusion, these data suggest that cancer therapy with anthracyclines modulates VEGF-A release and its cellular receptors in CMEC and ARVM, and therefore alters paracrine signaling in the myocardium.