RESUMO
Ex vivo heart perfusion (EVHP) may facilitate resuscitation of discarded donor hearts and expand the donor pool; however, a reliable means of demonstrating organ viability prior to transplantation is required. Therefore, we sought to identify metabolic and functional parameters that predict myocardial performance during EVHP. To evaluate the parameters over a broad spectrum of organ function, we obtained hearts from 9 normal pigs and 37 donation after circulatory death pigs and perfused them ex vivo. Functional parameters obtained from a left ventricular conductance catheter, oxygen consumption, coronary vascular resistance, and lactate concentration were measured, and linear regression analyses were performed to identify which parameters best correlated with myocardial performance (cardiac index: mL·min(-1)·g(-1)). Functional parameters exhibited excellent correlation with myocardial performance and demonstrated high sensitivity and specificity for identifying hearts at risk of poor post-transplant function (ejection fraction: R(2) = 0.80, sensitivity = 1.00, specificity = 0.85; stroke work: R(2) = 0.76, sensitivity = 1.00, specificity = 0.77; minimum dP/dt: R(2) = 0.74, sensitivity = 1.00, specificity = 0.54; tau: R(2) = 0.51, sensitivity = 1.00, specificity = 0.92), whereas metabolic parameters were limited in their ability to predict myocardial performance (oxygen consumption: R(2) = 0.28; coronary vascular resistance: R(2) = 0.20; lactate concentration: R(2) = 0.02). We concluded that evaluation of functional parameters provides the best assessment of myocardial performance during EVHP, which highlights the need for an EVHP device capable of assessing the donor heart in a physiologic working mode.
Assuntos
Transplante de Coração , Coração/fisiologia , Preservação de Órgãos/métodos , Perfusão/métodos , Sobrevivência de Tecidos/fisiologia , Coleta de Tecidos e Órgãos/métodos , Animais , Desenho de Equipamento , Feminino , Modelos Biológicos , Preservação de Órgãos/instrumentação , Soluções para Preservação de Órgãos , Consumo de Oxigênio/fisiologia , Perfusão/instrumentação , Sus scrofa , Coleta de Tecidos e Órgãos/instrumentaçãoRESUMO
We examined the role of redox-sensitive signal transduction mechanisms in modifying the changes in [Ca(2+)](i) produced by ouabain upon incubating adult rat cardiomyocytes with antioxidants or inhibitors of different protein kinases and monitoring alterations in fura-2 fluorescence. Ouabain increased basal [Ca(2+)](i), augmented the KCl-induced increase in [Ca(2+)](i), and promoted oxyradical production in cardiomyocytes. These actions of ouabain were attenuated by an oxyradical scavenging mixture (superoxide dismutase plus catalase), and the antioxidants (N-acetyl-L-cysteine and N-(2-mercaptoproprionyl)glycine). An inhibitor of MAP kinase (PD98059) depressed the ouabain-induced increase in [Ca(2+)], whereas inhibitors of tyrosine kinase (tyrphostin and genistein) and PI3 kinase (Wortmannin and LV294002) enhanced the ouabain-induced increase in [Ca(2+)](i). Inhibitors of protein kinase C (calphostin and bisindolylmalaimide) augmented the ouabain-induced increase in [Ca(2+)](i), whereas stimulation of protein kinase C by a phorbol ester (phorbol 12-myristate 13-acetate) depressed the action of ouabain. These results suggest that ouabain-induced inhibition of Na (+)-K(+) ATPase may alter the redox status of cardiomyocytes through the production of oxyradicals, and increase the activities of various protein kinases. Thus, these redox-sensitive signal transduction mechanisms involving different protein kinases may modify Ca(2+)-handling sites in cardiomyocytes and determine the magnitude of net increase in [Ca(2+)](i) in response to ouabain.
Assuntos
Cálcio/metabolismo , Cardiotônicos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Ouabaína/farmacologia , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , Oxirredução , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Espectrometria de FluorescênciaRESUMO
We tested whether the activation of proteolytic enzymes, calpain, and matrix metalloproteinases (MMPs) during ischemia-reperfusion (I/R) is mediated through oxidative stress. For this purpose, isolated rat hearts were subjected to a 30 min global ischemia followed by a 30 min reperfusion. Cardiac function was monitored and the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, calpain, and MMP were measured. Depression of cardiac function and Na(+)/K(+)-ATPase activity in I/R hearts was associated with increased calpain and MMP activities. These alterations owing to I/R were similar to those observed in hearts perfused with hypoxic medium, H(2)O(2) and xanthine plus xanthine oxidase. The I/R-induced changes were attenuated by ischemic preconditioning as well as by perfusing the hearts with N-acetylcysteine or mercaptopropionylglycine. Inhibition of MMP activity in hearts treated with doxycycline depressed the I/R-induced changes in cardiac function and Na(+)/K(+)-ATPase activity without affecting the calpain activation. On the other hand, inhibition of calpain activity upon treatment with leupeptin or MDL 28170 significantly reduced the MMP activity in addition to attenuating the I/R-induced alterations in cardiac function and Na(+)/K(+)-ATPase activity. These results suggest that the I/R-induced depression in Na(+)/K(+)-ATPase and cardiac function may be a consequence of the increased activities of both calpain and MMP because of oxidative stress in the heart.
Assuntos
Calpaína/metabolismo , Metaloproteinases da Matriz/metabolismo , Traumatismo por Reperfusão Miocárdica/enzimologia , Miocárdio/enzimologia , Estresse Oxidativo , Sarcolema/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Antioxidantes/farmacologia , Calpaína/antagonistas & inibidores , Regulação para Baixo , Ativação Enzimática , Hipóxia/enzimologia , Técnicas In Vitro , Precondicionamento Isquêmico Miocárdico , Masculino , Inibidores de Metaloproteinases de Matriz , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Perfusão , Inibidores de Proteases/farmacologia , Ratos , Ratos Sprague-Dawley , Sarcolema/efeitos dos fármacos , Fatores de Tempo , Função Ventricular Esquerda , Pressão VentricularRESUMO
It has become evident that protein degradation by proteolytic enzymes, known as proteases, is partly responsible for cardiovascular dysfunction in various types of heart disease. Both extracellular and intracellular alterations in proteolytic activities are invariably seen in heart failure associated with hypertrophic cardiomyopathy, dilated cardiomyopathy, hypertensive cardiomyopathy, diabetic cardiomyopathy, and ischemic cardiomyopathy. Genetic cardiomyopathy displayed in different strains of hamsters provides a useful model for studying heart failure due to either cardiac hypertrophy or cardiac dilation. Alterations in the function of several myocardial organelles such as sarcolemma, sarcoplasmic reticulum, myofibrils, mitochondria, as well as extracellular matrix have been shown to be due to subcellular remodeling as a consequence of changes in gene expression and protein content in failing hearts from cardiomyopathic hamsters. In view of the increased activities of various proteases, including calpains and matrix metalloproteinases in the hearts of genetically determined hamsters, it is proposed that the activation of different proteases may also represent an important determinant of subcellular remodeling and cardiac dysfunction associated with genetic cardiomyopathy.
Assuntos
Cardiomiopatias/metabolismo , Modelos Animais de Doenças , Insuficiência Cardíaca/metabolismo , Organelas/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Cardiomiopatias/genética , Cricetinae , Humanos , Modelos CardiovascularesRESUMO
Na(+)/Ca(2+) exchangers (NCX) constitute a major Ca(2+) export system that facilitates the re-establishment of cytosolic Ca(2+) levels in many tissues. Ca(2+) interactions at its Ca(2+) binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na(+)/Ca(2+) exchange activity. The structure of the Ca(2+)-bound form of CBD1, the primary Ca(2+) sensor from canine NCX1, but not the Ca(2+)-free form, has been reported, although the molecular mechanism of Ca(2+) regulation remains unclear. Here, we report crystal structures for three distinct Ca(2+) binding states of CBD1 from CALX, a Na(+)/Ca(2+) exchanger found in Drosophila sensory neurons. The fully Ca(2+)-bound CALX-CBD1 structure shows that four Ca(2+) atoms bind at identical Ca(2+) binding sites as those found in NCX1 and that the partial Ca(2+) occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca(2+) binding at CBD1. The structures also predict that the primary Ca(2+) pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu(455), which coordinates the primary Ca(2+) pair, produces dramatic reductions of the regulatory Ca(2+) affinity for exchange current, whereas mutagenesis of Glu(520), which coordinates the secondary Ca(2+) pair, has much smaller effects. Furthermore, our structures indicate that Ca(2+) binding only enhances the stability of the Ca(2+) binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca(2+) regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.
Assuntos
Antiporters/química , Antiporters/metabolismo , Cálcio/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Sódio/metabolismo , Animais , Antiporters/genética , Sítios de Ligação , Cristalografia , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Domínios e Motivos de Interação entre Proteínas/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Células Receptoras Sensoriais/fisiologiaRESUMO
Chemotactic movement of myofibroblasts is recognized as a common means for their sequestration to the site of tissue injury. Following myocardial infarction (MI), recruitment of cardiac myofibroblasts to the infarct scar is a critical step in wound healing. Contractile myofibroblasts express embryonic smooth muscle myosin, α-smooth muscle actin, as well as collagens I and III. We examined the effects of cardiotrophin-1 (CT-1) in the induction of primary rat ventricular myofibroblast motility. Changes in membrane potential (E(m)) and Ca(2+) entry were studied to reveal the mechanisms for induction of myofibroblast migration. CT-1-induced cardiac myofibroblast cell migration, which was attenuated through the inhibition of JAK2 (25 µM AG490), and myosin light chain kinase (20 µM ML-7). Inhibition of K(+) channels (1 mM tetraethylammonium or 100 µM 4-aminopyridine) and nonselective cation channels by 10 µM gadolinium (Gd(3+)) significantly reduced migration in the presence of CT-1. CT-1 treatment caused a significant increase in myosin light chain phosphorylation, which could be inhibited by incubation in Ca(2+)-free conditions or by application of AG490, ML-7, and W7 (100 µM; calmodulin inhibitor). Monitoring myofibroblast membrane potential with potentiometric fluorescent DiBAC(4)(3) dye revealed a biphasic response to CT-1 consisting of an initial depolarization followed by hyperpolarization. Increased intracellular Ca(2+), as assessed by fluo 3, occurred immediately after membrane depolarization and attenuated at the time of maximal hyperpolarization. CT-1 exerts chemotactic effects via multiple parallel signaling modalities in ventricular myofibroblasts, including changes in membrane potential, alterations in intracellular calcium, and activation of a number of intracellular signaling pathways. Further study is warranted to determine the precise role of K(+) currents in this process.
Assuntos
Quimiotaxia , Citocinas/metabolismo , Miofibroblastos/enzimologia , Quinase de Cadeia Leve de Miosina/metabolismo , Análise de Variância , Animais , Cálcio/metabolismo , Calmodulina/antagonistas & inibidores , Calmodulina/metabolismo , Miosinas Cardíacas/metabolismo , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Gadolínio/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/enzimologia , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Masculino , Potenciais da Membrana , Miofibroblastos/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Fosforilação , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Fatores de TempoRESUMO
OBJECTIVE: Dietary intake of omega-3 polyunsaturated fatty acids (PUFA) like alpha-linolenic acid (ALA) is antiarrhythmic and cardioprotective. PUFA may also be beneficial in hypertension. Altered Na(+)-Ca(2+) exchanger (NCX) activity has been implicated in arrhythmias, hypertension and heart failure and may be a target for PUFA. Thus, we tested the effects of ALA and other distinct fatty acids on the cardiac (NCX1.1) and vascular (NCX1.3) NCX isoforms. METHODS: HEK293 cells stably expressing NCX isoforms were ramped from +60 to -100 mV (over 1600 ms) in the absence and presence of 25 microM oleic acid (OA, omega-9), linoleic acid (LA, omega-6), ALA (omega-3), or eicosapentaenoic acid (EPA, omega-3). NiCl(2) (5 mM) was used to inhibit and therefore identify the NCX current. The effect of 25 microM ALA on NCX1.1 and NCX1.3 activity was also assessed in adult rat ventricular cardiomyocytes and rabbit aortic vascular smooth muscle cells (VSMC) by measuring [Ca(2+)](i) following substitution of [Na(+)](o) with Li(+). RESULTS: Application of Ni(2+) had no effect in non-transfected cells. ALA and EPA (25 microM) reduced the Ni(2+)-sensitive forward NCX1.1 current (at -100 mV) by 64% and reverse current (at +60 mV) by 57%, and inhibited the Ni(2+)-sensitive NCX1.3 forward and reverse currents by 79% and 76%, respectively. Neither OA nor LA (25 microM) affected the NCX1.1 currents, but both partially inhibited the forward and reverse mode NCX1.3 currents. Inhibition of NCX1.3 by ALA occurred at a much lower IC(50) ( approximately 19 nM) than for NCX1.1 ( approximately 120 nM). In cardiomyocytes and VSMC, ALA significantly reduced the Li(+)-induced rise in intracellular [Ca(2+)]. CONCLUSIONS: NCX1.3 is more sensitive to inhibition by ALA than NCX1.1. In addition, only omega-3 PUFA inhibits NCX1.1, but several classes of fatty acids inhibit NCX1.3. The differential sensitivity of NCX isoforms to fatty acids may have important implications as therapeutic approaches for hypertension, heart failure and arrhythmias.
Assuntos
Músculo Liso Vascular/metabolismo , Miocárdio/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Ácido alfa-Linolênico/farmacologia , Análise de Variância , Animais , Aorta , Western Blotting/métodos , Linhagem Celular , Células Cultivadas , Ácido Eicosapentaenoico/farmacologia , Humanos , Ácido Linoleico/farmacologia , Níquel/farmacologia , Ácido Oleico/farmacologia , Técnicas de Patch-Clamp , Isoformas de Proteínas/metabolismo , Coelhos , Ratos , Ratos Sprague-Dawley , TransfecçãoRESUMO
BACKGROUND: Normothermic ex vivo heart perfusion (EVHP) has been shown to improve the preservation of hearts donated after circulatory arrest and to facilitate clinical successful transplantation. Steroids are added to the perfusate solution in current clinical EVHP protocols; however, the impact of this approach on donor heart preservation has not been previously investigated. We sought to determine the impact of steroids on the inflammatory response and development of myocardial edema during EVHP. METHODS: Thirteen pigs were anesthetized, mechanical ventilation was discontinued, and a hypoxemic cardiac arrest ensued. A 15-minute warm-ischemic standoff period was observed, and then hearts were resuscitated with a cardioplegic solution. Donor hearts were then perfused ex vivo in a normothermic beating state for 6 hours with 500 mg of methylprednisolone (steroid: n = 5) or without (control: n = 8). RESULTS: The addition of steroids to the perfusate solution reduced the generation of proinflammatory cytokines (interleukin-6, -8, -1ß, and tumor necrosis factor-α) and the development of myocardial edema during EVHP (percentage of weight gain: control = 26% ± 7% versus steroid = 16% ± 10%, p = 0.049). Electron microscopy suggested less endothelial cell edema in the steroid group (injury score: control = 1.8 ± 0.2 versus steroid = 1.2 ± 0.2, p = 0.06), whereas perfusate troponin-I (control = 11.9 ± 1.9 ng/mL versus steroid = 9.5 ± 2.4 ng/mL, p = 0.448) and myocardial function were comparable between the groups. CONCLUSIONS: The addition of methylprednisolone to the perfusion solution minimizes the generation of proinflammatory cytokines and development of myocardial edema during normothermic ex vivo perfusion of hearts donated after circulatory arrest.
Assuntos
Soluções Cardioplégicas/farmacologia , Edema Cardíaco/prevenção & controle , Metilprednisolona/farmacologia , Preservação de Órgãos/métodos , Animais , Modelos Animais de Doenças , Sobrevivência de Enxerto , Parada Cardíaca , Transplante de Coração/métodos , Humanos , Distribuição Aleatória , Valores de Referência , Sensibilidade e Especificidade , SuínosRESUMO
BACKGROUND: Hearts donated after circulatory death may represent an additional donor source. The influx of sodium and calcium ions across the sarcolemma play a central role in the pathogenesis of ischemia-reperfusion injury; however, this process may be inhibited if the initial reperfusion solution is rendered hypocalcemic and acidic. We sought to determine the calcium concentration and pH of the initial reperfusion solution that yielded optimal functional recovery of hearts donated after circulatory death during ex vivo heart perfusion. METHODS: Pigs were anesthetized, mechanical ventilation was discontinued, and a 15-minute standoff period was observed after circulatory arrest. Hearts were reperfused with a normothermic cardioplegia of varying calcium concentrations (part 1 [50 µmol/L, n = 4; 220 µmol/L, n = 9; 500 µmol/L, n = 4; and 1,250 µmol/L, n = 5]) and pH (part 2 [7.9, n = 5; 7.4, n = 9; 6.9, n = 8; and 6.4, n = 6]). Myocardial function was then assessed in a physiologic working model 1 hour after initiation of normothermic ex vivo heart perfusion. RESULTS: The calcium concentration and pH of the cardioplegic solution affected the development of myocardial edema (part 1: 50 µmol/L = 5.8% ± 0.9%; 220 µmol/L = 4.3% ± 0.4%; 500 µmol/L = 7.0% ± 0.6%; and 1,250 µmol/L = 6.6% ± 0.8% weight gain, p = 0.015; part 2: 7.9 = 3.6% ± 0.4%, 7.4 = 4.3% ± 0.4%, 6.9 = 3.7% ± 0.6%, and 6.4 = 6.4% ± 1.3% weight gain, p = 0.056) and the recovery of myocardial function (cardiac index part 1: 50 µmol/L = 2.6 ± 0.6; 220 µmol/L = 6.0 ± 0.8; 500 µmol/L = 2.3 ± 0.5; and 1,250 µmol/L = 1.9 ± 0.6 mL · m-1 · g-1, p < 0.001; part 2: 7.9 = 1.5 ± 0.7; 7.4 = 6.0 ± 0.8; 6.9 = 8.4 ± 1.8; and 6.4 = 3.1 ± 0.8 mL · m-1 · g-1, p = 0.003) during ex vivo heart perfusion. CONCLUSIONS: Initial reperfusion of hearts donated after circulatory death with a hypocalcemic and moderately acidic cardioplegia minimizes edema and optimizes functional recovery during subsequent ex vivo heart perfusion.
Assuntos
Cálcio/metabolismo , Soluções Cardioplégicas/farmacologia , Parada Cardíaca Induzida/métodos , Transplante de Coração , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Obtenção de Tecidos e Órgãos , Animais , Modelos Animais de Doenças , Concentração de Íons de Hidrogênio , Traumatismo por Reperfusão Miocárdica/prevenção & controle , SuínosRESUMO
The cardiac Na+-Ca2+ exchanger (NCX) plays an essential role in regulating Ca2+ under physiological and pathophysiological conditions. In its forward mode of operation, which predominates under physiological conditions, it extrudes the Ca2+ that enters the cardiac myocyte on a beat-to-beat basis. During ischemia and reperfusion, increased intracellular Na+ leads to a decrease in Ca2+ efflux and enhanced Ca2+ influx via the NCX, potentially leading to Ca2+ overload, which is one of the major pathophysiological mechanisms for ischemia-reperfusion injury. Novel NCX inhibitors discovered in recent years have shown great promise in attenuating ischemia-reperfusion injury.
Assuntos
Traumatismo por Reperfusão Miocárdica/prevenção & controle , Trocador de Sódio e Cálcio/antagonistas & inibidores , Tioureia/análogos & derivados , Compostos de Anilina/farmacologia , Animais , Antiarrítmicos/farmacologia , Cardiotônicos/farmacologia , Guanidinas/farmacologia , Humanos , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Éteres Fenílicos/farmacologia , Trocador de Sódio e Cálcio/fisiologia , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Sulfonas/farmacologia , Tioureia/farmacologiaRESUMO
BACKGROUND: Ex vivo heart perfusion (EVHP) provides the opportunity to resuscitate unused donor organs and facilitates assessments of myocardial function that are required to demonstrate organ viability before transplantation. We sought to evaluate the effect of different oxygen carriers on the preservation of myocardial function during EVHP. METHODS: Twenty-seven pig hearts were perfused ex vivo in a normothermic beating state for 6 hours and transitioned into working mode for assessments after 1 (T1), 3 (T3), and 5 (T5) hours. Hearts were allocated to 4 groups according to the perfusate composition. Red blood cell concentrate (RBC, n = 6), whole blood (RBC+Plasma, n = 6), an acellular hemoglobin-based oxygen carrier (HBOC, n = 8), or HBOC plus plasma (HBOC+Plasma, n = 7) were added to STEEN Solution (XVIVO Perfusion, Goteborg, Sweden) to achieve a perfusate hemoglobin concentration of 40 g/liter. RESULTS: The perfusate composition affected the preservation of systolic (T5 dP/dtmax: RBC+Plasma = 903 ± 99, RBC = 771 ± 77, HBOC+Plasma = 691 ± 82, HBOC = 563 ± 52 mm Hg/sec; p = 0.047) and diastolic (T5 dP/dtmin: RBC+Plasma = -574 ± 48, RBC = -492 ± 63, HBOC+Plasma = -326 ± 32, HBOC = -268 ± 22 mm Hg/sec; p < 0.001) function, and the development of myocardial edema (weight gain: RBC+Plasma = 6.6 ± 0.9, RBC = 6.6 ± 1.2, HBOC+Plasma = 9.8 ± 1.7, HBOC = 16.3 ± 1.9 g/hour; p < 0.001) during EVHP. RBC+Plasma hearts exhibited less histologic evidence of myocyte damage (injury score: RBC+Plasma = 0.0 ± 0.0, RBC = 0.8 ± 0.3, HBOC+Plasma = 2.6 ± 0.2, HBOC = 1.75 ± 0.4; p < 0.001) and less troponin-I release (troponin-I fold-change T1-T5: RBC+Plasma = 7.0 ± 1.7, RBC = 13.1 ± 1.6, HBOC+Plasma = 20.5 ± 1.1, HBOC = 16.7 ± 5.8; p < 0.001). Oxidative stress was minimized by the addition of plasma to RBC and HBOC hearts (oxidized phosphatidylcholine compound fold-change T1-T5: RBC+Plasma = 1.83 ± 0.20 vs RBC = 2.31 ± 0.20, p < 0.001; HBOC+Plasma = 1.23 ± 0.17 vs HBOC = 2.80 ± 0.28, p < 0.001). CONCLUSIONS: A whole blood-based perfusate (RBC+Plasma) minimizes injury and provides superior preservation of myocardial function during EVHP. The beneficial effect of plasma on the preservation of myocardial function requires further investigation.
Assuntos
Eritrócitos , Transplante de Coração , Ventrículos do Coração/efeitos dos fármacos , Miocárdio , Soluções para Preservação de Órgãos/farmacologia , Perfusão/métodos , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Diástole , Modelos Animais de Doenças , Circulação Extracorpórea , Feminino , Insuficiência Cardíaca/cirurgia , Suínos , SístoleRESUMO
Ionic regulation of Na(+)/Ca(2+) exchange describes the secondary modulating effects exerted on exchange activity by the transport substrates Na(+) and Ca(2+). These effects have been extensively characterized for the cardiac Na(+)/Ca(2+) exchanger, NCX1.1, primarily by the giant excised patch-clamp technique. Moreover, several studies have provided functional evidence for ionic regulation of Na(+)/Ca(2+) exchange activity in intact cellular systems. Through structure-function analyses, important protein domains involved in these regulatory processes have been identified. However, despite major progress in characterizing ionic regulation at the functional and molecular levels, the physiological importance of these processes remains unknown. In this study, we have examined Na(+)/Ca(2+) exchange activity for three members of the NCX1 family, namely NCX1.1, NCX1.3, and NCX1.4. These exchangers were expressed in Xenopus laevis oocytes and were characterized using the giant excised patch-clamp technique. We show that these three splice variants exhibit considerable differences in the kinetic features of their ionic regulatory profiles. Information of this type is beginning to provide insight into the physiological basis for tissue-specific expression of alternatively spliced Na(+)/Ca(2+) exchangers.
Assuntos
Oócitos/fisiologia , Trocador de Sódio e Cálcio/fisiologia , Processamento Alternativo , Animais , Encéfalo/fisiologia , Cálcio/fisiologia , DNA Complementar , Feminino , Variação Genética , Homeostase , Rim/fisiologia , Potenciais da Membrana , Especificidade de Órgãos , Técnicas de Patch-Clamp , Isoformas de Proteínas/fisiologia , Proteínas Recombinantes/metabolismo , Sódio/fisiologia , Trocador de Sódio e Cálcio/genética , Xenopus laevisRESUMO
Various procedures such as angioplasty, thrombolytic therapy, coronary bypass surgery, and cardiac transplantation are invariably associated with ischemia-reperfusion (I/R) injury. Impaired recovery of cardiac function due to I/R injury is considered to be a consequence of the occurrence of both oxidative stress and intracellular Ca(2+)-overload in the myocardium. These changes in the ischemic myocardium appear to activate both extracellular and intracellular proteases which are responsible for the cleavage of extracellular matrix and subcellular structures involved in the maintenance of cardiac function. It is thus intended to discuss the actions of I/R injury on several proteases, with a focus on calpain, matrix metalloproteinases, and cathepsins as well as their role in inducing alterations both inside and outside the cardiomyocytes. In addition, modifications of subcellular organelles such as myofibrils, sarcoplasmic reticulum and sarcolemma as well as extracellular matrix, and the potential regulatory effects of endogenous inhibitors on protease activities are identified. Both extracellular and intracellular proteolytic activities appear to be imperative in determining the true extent of I/R injury and their inhibition seems to be of critical importance for improving the recovery of cardiac function. Thus, both extracellular and intracellular proteases may serve as potential targets for the development of cardioprotective interventions for reducing damage to the heart and retarding the development of contractile dysfunction caused by I/R injury.
Assuntos
Calpaína/metabolismo , Catepsinas/metabolismo , Metaloproteinases da Matriz/metabolismo , Traumatismo por Reperfusão Miocárdica/enzimologia , Animais , Proteínas da Matriz Extracelular/metabolismo , Humanos , Espaço Intracelular/metabolismo , Lisossomos/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologiaRESUMO
BACKGROUND: Ex vivo heart perfusion (EVHP) has been proposed as a means to facilitate the resuscitation of donor hearts after cardiocirculatory death (DCD) and increase the donor pool. However, the current approach to clinical EVHP may exacerbate myocardial injury and impair function after transplant. Therefore, we sought to determine if a cardioprotective EVHP strategy that eliminates myocardial exposure to hypothermic hyperkalemia cardioplegia and minimizes cold ischemia could facilitate successful DCD heart transplantation. METHODS: Anesthetized pigs sustained a hypoxic cardiac arrest and a 15-minute warm ischemic standoff period. Strategy 1 hearts (S1, n = 9) underwent initial reperfusion with a cold hyperkalemic cardioplegia, normothermic EVHP, and transplantation after a cold hyperkalemic cardioplegic arrest (current EVHP strategy). Strategy 2 hearts (S2, n = 8) underwent initial reperfusion with a tepid adenosine-lidocaine cardioplegia, normothermic EVHP, and transplantation with continuous myocardial perfusion (cardioprotective EVHP strategy). RESULTS: At completion of EVHP, S2 hearts exhibited less weight gain (9.7 ± 6.7 [S2] vs 21.2 ± 6.7 [S1] g/hour, p = 0.008) and less troponin-I release into the coronary sinus effluent (4.2 ± 1.3 [S2] vs 6.3 ± 1.5 [S1] ng/ml; p = 0.014). Mass spectrometry analysis of oxidized pleural in post-transplant myocardium revealed less oxidative stress in S2 hearts. At 30 minutes after wean from cardiopulmonary bypass, post-transplant systolic (pre-load recruitable stroke work: 33.5 ± 1.3 [S2] vs 19.7 ± 10.9 [S1], p = 0.043) and diastolic (isovolumic relaxation constant: 42.9 ± 6.7 [S2] vs 65.2 ± 21.1 [S1], p = 0.020) function were superior in S2 hearts. CONCLUSION: In this experimental model of DCD, an EVHP strategy using initial reperfusion with a tepid adenosine-lidocaine cardioplegia and continuous myocardial perfusion minimizes myocardial injury and improves short-term post-transplant function compared with the current EVHP strategy using cold hyperkalemic cardioplegia before organ procurement and transplantation.
Assuntos
Adenosina/uso terapêutico , Parada Cardíaca Induzida , Transplante de Coração , Lidocaína/uso terapêutico , Preservação de Órgãos/métodos , Animais , Morte , Feminino , Perfusão , SuínosRESUMO
µ-Calpain is a Ca(2+)-activated protease abundant in mammalian tissues. Here, we examined the effects of µ-calpain on three alternatively spliced variants of NCX1 using the giant, excised patch technique. Membrane patches from Xenopus oocytes expressing either heart (NCX1.1), kidney (NCX1.3), or brain (NCX1.4) variants of NCX1 were exposed to µ-calpain and their Na(+)-dependent (I(1)) and Ca(2+)-dependent (I(2)) regulatory phenotypes were assessed. For these exchangers, I(1) inactivation is evident as a Na(+)(i)-dependent decay of peak outward currents whereas I(2) regulation manifests as outward current activation by micromolar Ca(2+)(i) concentrations. Notably, with NCX1.1 and NCX1.4 but not in NCX1.3, higher Ca(2+)(i) levels alleviate I(1) inactivation. Our results show that (i) µ-calpain selectively ablates Ca(2+)-dependent (I(2)) regulation leading to a constitutive activation of exchange current, (ii) µ-calpain has much smaller effects on Na(+)-dependent (I(1)) regulation, produced by a slight destabilization of the I(1) state, and (iii) Ca(2+)-dependent regulation (I(2)) and Ca(2+)-mediated alleviation of I(1) appear to be functionally distinct mechanisms, the latter of which is left largely intact after µ-calpain treatment. The ability of µ-calpain to selectively and constitutively activate Na(+)-Ca(2+) exchange currents may have important pathophysiological implications in tissue where these splice variants are expressed.
Assuntos
Processamento Alternativo/fisiologia , Encéfalo/metabolismo , Calpaína/metabolismo , Rim/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Calpaína/genética , Cães , Proteínas Musculares/genética , Proteínas do Tecido Nervoso/genética , Especificidade de Órgãos/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Trocador de Sódio e Cálcio/genética , Xenopus laevisRESUMO
OBJECTIVE: Low plasma high-density lipoprotein cholesterol (HDL-C) concentration is associated with the metabolic syndrome (MetS) and increased prevalence of cardiovascular disease (CVD). Animal and human studies report infusion of apolipoprotein A-1 (apoA-1) can reduce endothelial dysfunction, and/or induce regression of atherosclerosis. However, the direct mechanisms underlying the vascular benefits of either apoA-1 or HDL-C remain unclear. In this study, we assessed the ability of reconstituted HDL (rHDL) to improve vascular complications of MetS, including left ventricular (LV)-hypertrophy, arterial cholesterol deposition and myocardial lesion development. METHODS AND RESULTS: Obese insulin resistant (IR) JCR:LA-cp rats were infused with rHDL (0.4 mg/kg) over 3 days before assessing cardiac function (Echocardiography) at days 7 and 50 post-infusion, as well as haematoxylin and eosin staining of myocardial lesions at day 50. Acute ex vivo arterial cholesterol deposition was assessed with acute infusion of rHDL ex-vivo. Infusion of rHDL partially corrected abnormal diastolic compliance (18%; *p<0.05) and improved parameters of cardiac function in IR rats. Further, acute rHDL infusion in carotid vessels reduced remnant lipoprotein associated-cholesterol deposition (30-86%; **p<0.01) ex vivo in IR and male Wistar rats and reduced (41%; *p<0.05) the frequency of early-stage myocardial lesions in IR rats. CONCLUSION: Short-term infusion of rHDL may beneficially reduce chronic vascular sequelae of MetS, including temporary improvement in LV-dysfunction, acute reduction of acute arterial cholesterol deposition and the development of early-stage myocardial lesions in the JCR:LA-cp rat.
Assuntos
Apolipoproteína A-I/administração & dosagem , Artérias Carótidas/efeitos dos fármacos , Doenças das Artérias Carótidas/tratamento farmacológico , Colesterol/metabolismo , Resistência à Insulina , Lipoproteínas HDL/administração & dosagem , Síndrome Metabólica/tratamento farmacológico , Miocárdio/patologia , Disfunção Ventricular Esquerda/tratamento farmacológico , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Doenças das Artérias Carótidas/etiologia , Doenças das Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/patologia , Modelos Animais de Doenças , Ecocardiografia Doppler , Humanos , Infusões Parenterais , Masculino , Síndrome Metabólica/etiologia , Síndrome Metabólica/metabolismo , Síndrome Metabólica/patologia , Miocárdio/metabolismo , Obesidade/complicações , Ratos , Ratos Wistar , Fatores de Tempo , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologiaAssuntos
Antiporters , Proteínas de Transporte/fisiologia , Drosophila/fisiologia , Trocador de Sódio e Cálcio/antagonistas & inibidores , Tioureia/análogos & derivados , Tioureia/farmacologia , Animais , Antiarrítmicos/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Drosophila/efeitos dos fármacos , Proteínas de Drosophila/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologiaAssuntos
Coração/fisiologia , Contração Miocárdica/fisiologia , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/fisiologia , Animais , Cinética , Mamíferos , Modelos Moleculares , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Termodinâmica , VertebradosAssuntos
Encéfalo/metabolismo , Rim/metabolismo , Miocárdio/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/fisiologia , Processamento Alternativo , Animais , Variação Genética , Cinética , Potenciais da Membrana/fisiologia , Oócitos/fisiologia , Técnicas de Patch-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Transporte Proteico , Proteínas Recombinantes/metabolismo , XenopusRESUMO
Na(+)/Ca(2+) exchangers (NCXs) promote the extrusion of intracellular Ca(2+) to terminate numerous Ca(2+)-mediated signaling processes. Ca(2+) interaction at two Ca(2+) binding domains (CBDs; CBD1 and CBD2) is important for tight regulation of the exchange activity. Diverse Ca(2+) regulatory properties have been reported with several NCX isoforms; whether the regulatory diversity of NCXs is related to structural differences of the pair of CBDs is presently unknown. Here, we reported the crystal structure of CBD2 from the Drosophila melanogaster exchanger CALX1.1. We show that the CALX1.1-CBD2 is an immunoglobulin-like structure, similar to mammalian NCX1-CBD2, but the predicted Ca(2+) interaction region of CALX1.1-CBD2 is arranged in a manner that precludes Ca(2+) binding. The carboxylate residues that coordinate two Ca(2+) in the NCX1-CBD1 structure are neutralized by two Lys residues in CALX1.1-CBD2. This structural observation was further confirmed by isothermal titration calorimetry. The CALX1.1-CBD2 structure also clearly shows the alternative splicing region forming two adjacent helices perpendicular to CBD2. Our results provide structural evidence that the diversity of Ca(2+) regulatory properties of NCX proteins can be achieved by (1) local structure rearrangement of Ca(2+) binding site to change Ca(2+) binding properties of CBD2 and (2) alternative splicing variation altering the protein domain-domain conformation to modulate the Ca(2+) regulatory behavior.