RESUMEN
The left ventricular working, crystalloid-perfused heart is used extensively to evaluate basic cardiac function, pathophysiology, and pharmacology. Crystalloid-perfused hearts may be limited by oxygen delivery, as adding oxygen carriers increases myoglobin oxygenation and improves myocardial function. However, whether decreased myoglobin oxygen saturation impacts oxidative phosphorylation (OxPhos) is unresolved, since myoglobin has a much lower affinity for oxygen than cytochrome c oxidase (COX). In the present study, a laboratory-based synthesis of an affordable perfluorocarbon (PFC) emulsion was developed to increase perfusate oxygen carrying capacity without impeding optical absorbance assessments. In left ventricular working hearts, along with conventional measurements of cardiac function and metabolic rate, myoglobin oxygenation and cytochrome redox state were monitored using a novel transmural illumination approach. Hearts were perfused with Krebs-Henseleit (KH) or KH supplemented with PFC, increasing perfusate oxygen carrying capacity by 3.6-fold. In KH-perfused hearts, myoglobin was deoxygenated, consistent with cytoplasmic hypoxia, and the mitochondrial cytochromes, including COX, exhibited a high reduction state, consistent with OxPhos hypoxia. PFC perfusate increased aortic output from 76 ± 6 to 142 ± 4 ml/min and increased oxygen consumption while also increasing myoglobin oxygenation and oxidizing the mitochondrial cytochromes. These results are consistent with limited delivery of oxygen to OxPhos resulting in an adapted lower cardiac performance with KH. Consistent with this, PFCs increased myocardial oxygenation, and cardiac work was higher over a wider range of perfusate Po2. In summary, heart mitochondria are limited by oxygen delivery with KH; supplementation of KH with PFC reverses mitochondrial hypoxia and improves cardiac performance, creating a more physiological tissue oxygen delivery. NEW & NOTEWORTHY Optical absorbance spectroscopy of intrinsic chromophores reveals that the commonly used crystalloid-perfused working heart is oxygen limited for oxidative phosphorylation and associated cardiac work. Oxygen-carrying perfluorocarbons increase myocardial oxygen delivery and improve cardiac function, providing a more physiological mitochondrial redox state and emphasizing cardiac work is modulated by myocardial oxygen delivery.
Asunto(s)
Soluciones Cristaloides/farmacología , Fluorocarburos/farmacología , Corazón/efectos de los fármacos , Mitocondrias Cardíacas/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Oxígeno/metabolismo , Perfusión/métodos , Función Ventricular Izquierda/efectos de los fármacos , Animales , Soluciones Cristaloides/síntesis química , Citocromos c/metabolismo , Emulsiones , Fluorocarburos/síntesis química , Glucosa/farmacología , Corazón/fisiología , Preparación de Corazón Aislado , Mitocondrias Cardíacas/metabolismo , Mioglobina/metabolismo , Oxidación-Reducción , Fosforilación Oxidativa/efectos de los fármacos , Conejos , Trometamina/farmacologíaRESUMEN
Succinyl-CoA synthetase (SCS) is the only mitochondrial enzyme capable of ATP production via substrate level phosphorylation in the absence of oxygen, but it also plays a key role in the citric acid cycle, ketone metabolism, and heme synthesis. Inorganic phosphate (P(i)) is a signaling molecule capable of activating oxidative phosphorylation at several sites, including NADH generation and as a substrate for ATP formation. In this study, it was shown that P(i) binds the porcine heart SCS alpha-subunit (SCSalpha) in a noncovalent manner and enhances its enzymatic activity, thereby providing a new target for P(i) activation in mitochondria. Coupling 32P labeling of intact mitochondria with SDS gel electrophoresis revealed that 32P labeling of SCSalpha was enhanced in substrate-depleted mitochondria. Using mitochondrial extracts and purified bacterial SCS (BSCS), we showed that this enhanced 32P labeling resulted from a simple binding of 32P, not covalent protein phosphorylation. The ability of SCSalpha to retain its 32P throughout the SDS denaturing gel process was unique over the entire mitochondrial proteome. In vitro studies also revealed a P(i)-induced activation of SCS activity by more than 2-fold when mitochondrial extracts and purified BSCS were incubated with millimolar concentrations of P(i). Since the level of 32P binding to SCSalpha was increased in substrate-depleted mitochondria, where the matrix P(i) concentration is increased, we conclude that SCS activation by P(i) binding represents another mitochondrial target for the P(i)-induced activation of oxidative phosphorylation and anaerobic ATP production in energy-limited mitochondria.
Asunto(s)
Mitocondrias Cardíacas/metabolismo , Proteínas Mitocondriales/metabolismo , Fosfatos/metabolismo , Subunidades de Proteína/metabolismo , Succinato-CoA Ligasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Electroforesis en Gel Bidimensional , Activación Enzimática , Guanosina Trifosfato/metabolismo , Concentración de Iones de Hidrógeno , Proteínas Mitocondriales/química , Fosfatos/química , Unión Proteica , Desnaturalización Proteica , Subunidades de Proteína/química , Proteómica/métodos , Dodecil Sulfato de Sodio/química , Succinato-CoA Ligasas/química , Tensoactivos/química , PorcinosRESUMEN
With the use of iTRAQ labeling and mass spectrometry on an LTQ-Orbitrap with HCD capability, we assessed relative changes in protein phosphorylation in the mitochondria upon physiological perturbation. As a reference reaction, we monitored the well-characterized regulation of pyruvate dehydrogenase (PDH) activity via phosphorylation/dephosphorylation by pyruvate dehydrogenase kinase/pyruvate dehydrogenase phosphatase in response to dichloroacetate, de-energization and Ca2+. Relative quantification of phosphopeptides of PDH-E1alpha subunit from porcine heart revealed dephosphorylation at three serine sites (Ser231, Ser292 and Ser299). Dephosphorylation at Ser292 (i.e., the inhibitory site) with DCA correlated with an activation of PDH activity as previously reported, consistent with our de-energization data. Calcium also dephosphorylated (i.e., activated) PDH, thus, confirming calcium activation of PDP. With this approach, we successfully monitored other phosphorylation sites of mitochondrial proteins including adenine nucleotide translocase, malate dehydrogenase and mitochondrial creatine kinase. Among them, four proteins exhibited phosphorylation changes with these physiological stimuli: (1) BCKDH-E1alpha subunit increased phosphorylation at Ser337 with DCA and de-energization; (2) apoptosis-inducing factor phosphorylation was elevated at Ser345 with calcium; (3) ATP synthase F1 complex alpha subunit and (4) mitofilin dephosphorylated at Ser65 and Ser264 upon de-energization. This screening validated the iTRAQ/HCD technology as a method for functional quantitation of mitochondrial protein phosphorylation as well as providing insight into the regulation of mitochondria via phosphorylation.
Asunto(s)
Espectrometría de Masas/métodos , Mitocondrias Cardíacas/metabolismo , Proteínas Mitocondriales/análisis , Fosfoproteínas/análisis , Proteómica/métodos , Animales , Calcio/farmacología , Ácido Dicloroacético/farmacología , Electroforesis en Gel Bidimensional , Marcaje Isotópico , Proteínas Mitocondriales/metabolismo , Fosfopéptidos/análisis , Fosfopéptidos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Piruvato Deshidrogenasa (Lipoamida)-Fosfatasa/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , PorcinosRESUMEN
The effect of neighbouring plants on crop root system architecture may directly interfere with water and nutrient acquisition, yet this important and interesting aspect of competition remains poorly understood. Here, the effect of the weed blackgrass (Alopecurus myosuroides Huds.) on wheat (Triticum aestivum L.) roots was tested, since a low density of this species (25 plants m-2) can lead to a 10% decrease in wheat yield and herbicide resistance is problematic. We used a simplified growth system based on gelled medium, to grow wheat alongside a neighbour, either another wheat plant, a blackgrass or Brachypodium dystachion individual (a model grass). A detailed analysis of wheat seminal root system architecture showed that the presence of a neighbour principally affected the root length, rather than number or diameter under a high nutrient regime. In particular, the length of first order lateral roots decreased significantly in the presence of blackgrass and Brachypodium. However, this effect was not noted when wheat plants were grown in low nutrient conditions. This suggests that wheat may be less sensitive to the presence of blackgrass when grown in low nutrient conditions. In addition, nutrient availability to the neighbour did not modulate the neighbour effect on wheat root architecture.
Asunto(s)
Brachypodium/crecimiento & desarrollo , Poaceae/crecimiento & desarrollo , Triticum/crecimiento & desarrollo , Biomasa , Ecosistema , Modelos Biológicos , Raíces de Plantas/anatomía & histología , Raíces de Plantas/crecimiento & desarrollo , Triticum/anatomía & histologíaRESUMEN
The role of Ca(2+) as a cytosolic signaling molecule between porcine cardiac sarcoplasmic reticulum (SR) ATPase and mitochondrial ATP production was evaluated in vitro. The Ca(2+) sensitivity of these processes was determined individually and in a reconstituted system with SR and mitochondria in a 0.5:1 protein-to-cytochrome aa(3) ratio. The half-maximal concentration (K(1/2)) of SR ATPase was 335 nM Ca(2+). The ATP synthesis dependence was similar with a K(1/2) of 243 nM for dehydrogenases and 114 nM for overall ATP production. In the reconstituted system, Ca(2+) increased thapsigargin-sensitive ATP production (maximum approximately 5-fold) with minimal changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH). NADH concentration remained stable despite graded increases in NADH turnover induced over a wide range of Ca(2+) concentrations (0 to approximately 500 nM). These data are consistent with a balanced activation of SR ATPase and mitochondrial ATP synthesis by Ca(2+) that contributes to a homeostasis of energy metabolism metabolites. It is suggested that this balanced activation by cytosolic Ca(2+) is partially responsible for the minimal alteration in energy metabolism intermediates that occurs with changes in cardiac workload in vivo.