Long-term administration of nicorandil abolishes ischemic and pharmacologic preconditioning of the human myocardium: Role of mitochondrial adenosine triphosphate–dependent potassium channels

AbstractBackground: Acute administration of mitochondrial adenosine triphosphate–dependent potassium channel openers preconditions the heart, but whether their long-term administration induces a permanent state of protection is unknown. These studies investigate the effect of long-term treatment with the mitochondrial adenosine triphosphate–dependent potassium channel opener nicorandil on the response of the human myocardium to ischemia and preconditioning. Methods: Right atrial tissue obtained from patients regularly treated with or without nicorandil (mean of 20 mg/d for 18.6 ± 2.5 months) and undergoing cardiac surgery was sliced and equilibrated for 30 minutes and then subjected to 90 minutes of simulated ischemia, followed by 120 minutes of reoxygenation. In study 1 the following groups were studied to investigate the effect of nicorandil on the susceptibility of the myocardium to ischemia and on the protective effect of ischemic and pharmacologic preconditioning: (1) aerobic control; (2) simulated ischemia and reoxygenation alone; (3) ischemic preconditioning with 5 minutes of simulated ischemia and 5 minutes of reoxygenation; and (4) phenylephrine (0.1 μmol/L) for 5 minutes and 5 minutes' washout before simulated ischemia and reoxygenation. In study 2 the following groups were studied to investigate the effect of nicorandil on the responsiveness of mitochondrial adenosine triphosphate–dependent potassium channels: (1) aerobic control; (2) simulated ischemia and reoxygenation; (3) ischemic preconditioning; (4) diazoxide (100 μmol/L) for 10 minutes before simulated ischemia and reoxygenation, and (5) 5-hydroxydecanoate (1 mmol/L) for 10 minutes before simulated ischemia and reoxygenation. In study 3 the following groups were included to investigate the effect of the long-term administration of nicorandil on the kinase pathway involved in preconditioning: (1) aerobic control; (2) simulated ischemia and reoxygenation alone; (3) ischemic preconditioning; (4) phorbol 12-myristate 13-acetate (1 μmol/L), a protein kinase C activator, for 10 minutes before simulated ischemia and reoxygenation; and (5) anisomycin (1 nmol/L), a p38 mitogen-activated protein kinase activator, for 10 minutes before simulated ischemia and reoxygenation. At the end of each protocol, the leakage of creatine kinase (in units per gram wet weight) and the reduction of 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide into insoluble formazan dye (in millimoles per gram wet weight) were measured. Results: In study 1 the leakage of creatine kinase and the reduction of 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide induced by simulated ischemia and reoxygenation were similar in the groups with or without nicorandil (creatine kinase, 3.4 ± 0.1 and 3.5 ± 0.2, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 74.6 ± 3.9 and 67.9 ± 7.3, respectively; P >.2 in each instance). Ischemic preconditioning and pharmacologic preconditioning protected the myocardium from patients without nicorandil (creatine kinase, 2.3 ± 0.1 and 2.4 ± 0.1, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 131.4 ± 4.9 and 128.4 ± 5.6, respectively; P < 0.001 vs simulated ischemia and reoxygenation alone in each instance) but not the myocardium from patients receiving nicorandil (creatine kinase, 3.3 ± 0.1 and 3.3 ± 0.2, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 89.7 ± 6.5 and 86.4 ± 5.2, respectively; P >.2 vs simulated ischemia and reoxygenation alone in each instance). In study 2 the administration of diazoxide had identical protection to that of ischemic preconditioning in the myocardium of patients not receiving nicorandil (creatine kinase, 2.1 ± 0.2 and 2.3 ± 0.1, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 141.4 ± 7.4 and 131.4 ± 4.9, respectively; P < 0.001 vs simulated ischemia and reoxygenation alone in each instance) but failed to precondition the myocardium from patients treated with nicorandil (creatine kinase, 3.3 ± 0.2 and 3.4 ± 0.1, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 90.1 ± 7.2 and 86.4 ± 5.2, respectively; P > 0.2 vs simulated ischemia and reoxygenation alone in each instance). In study 3 phorbol 12-myristate 13-acetate or anisomycin given for 10 minutes before simulated ischemia and reoxygenation afforded similar protection to that of ischemic preconditioning in the myocardium from patients with (creatine kinase, 1.5 ± 0.3 and 1.4 ± 0.1, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 147.0 ± 4.9 and 160.0 ± 16.1, respectively; P < 0.001 vs simulated ischemia and reoxygenation alone in each instance) and without nicorandil (creatine kinase, 1.7 ± 0.4 and 1.4 ± 0.2, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 160.3 ± 13.6 and 158.3 ± 11.8, respectively; P <.001 vs simulated ischemia and reoxygenation alone in each instance). Conclusion: The myocardium of patients chronically treated with nicorandil cannot be preconditioned either by ischemia or pharmacologically, and this is because of unresponsive mitochondrial adenosine triphosphate–dependent potassium channels. However, protection can be obtained by protein kinase C and p38 mitogen-activated protein kinase activation, which are downstream of mitochondrial adenosine triphosphate–dependent potassium channels in the signaling transduction pathway of preconditioning.J Thorac Cardiovasc Surg 2002;124:750-

Effect of the degree of ischaemic injury and reoxygenation time on the type of myocardial cell death in man: role of caspases

BACKGROUND: The importance of apoptosis in the injury sustained by the human myocardium during ischaemia and reoxygenation and the underlying mechanisms remain unclear. To quantify apoptosis and necrosis induced by simulated ischaemia/reoxygenation in the human atrial myocardium, free-hand sections of right atrial appendage (n = 8/group) were subjected to 90 minutes simulated ischaemia followed by 2, 8 and 24 hours reoxygenation. RESULTS: Apoptosis, as assessed by TUNEL, was greater than necrosis after 90 minutes simulated ischaemia and 2 hours reoxygenation (35.32 ± 3.22% vs 13.55 ± 1.3%; p < 0.05) but necrosis was greater than apoptosis by 24 hours reoxygenation (45.20 ± 2.75% vs 4.82 ± 0.79%; p < 0.05). Total caspase activation was similar after 90 minutes simulated ischaemia followed by 2 hours and 24 hours reoxygenation (515270 ± 99570 U vs 542940 ± 95216 U; p = NS). However, caspase-3 like activation was higher at 2 hours than at 24 hours reoxygenation (135900 ± 42200 U vs 54970 ± 19100 U; p < 0.05). Inhibition of caspase-3 by z.DEVD.fmk (70 nM) almost completely abolished apoptosis from 23.26 ± 2.854% to 0.73 ± 0.28 % (p < 0.05), without affecting necrosis. CONCLUSION: Cell death by apoptosis and necrosis in the human myocardium subjected to simulated ischaemia/reoxygenation depends on the degree of the ischaemic insult and have a different time-course with apoptosis happening early during reoxygenation and necrosis becoming more important later. Importantly, the apoptosis induced by simulated ischaemia/reoxygenation is mainly mediated by activation of caspase-3 but it does not affect necrosis

Dual role of nNOS in ischemic injury and preconditioning

<p>Abstract</p> <p>Background</p> <p>Nitric oxide (NO) is cardioprotective and a mediator of ischemic preconditioning (IP). Endothelial nitric oxide synthase (eNOS) is protective against myocardial ischemic injury and a component of IP but the role and location of neuronal nitric oxide synthase (nNOS) remains unclear. Therefore, the aims of these studies were to: (i) investigate the role of nNOS in ischemia/reoxygenation-induced injury and IP, (ii) determine whether its effect is species-dependent, and (iii) elucidate the relationship of nNOS with mitoK_ATPchannels and p38MAPK, two key components of IP transduction pathway.</p> <p>Results</p> <p>Ventricular myocardial slices from rats and wild and nNOS knockout mice, and right atrial myocardial slices from human were subjected to 90 min ischemia and 120 min reoxygenation (37°C). Specimens were randomized to receive various treatments (n = 6/group). Both the provision of exogenous NO and the inhibition of endogenous NO production significantly reduced tissue injury (creatine kinase release, cell necrosis and apoptosis), an effect that was species-independent. The cardioprotection seen with nNOS inhibition was as potent as that of IP, however, in nNOS knockout mice the cardioprotective effect of non-selective NOS (L-NAME) and selective nNOS inhibition and also that of IP was blocked while the benefit of exogenous NO remained intact. Additional studies revealed that the cardioprotection afforded by exogenous NO and by inhibition of nNOS were unaffected by the mitoK_ATPchannel blocker 5-HD, although it was abrogated by p38MAPK blocker SB203580.</p> <p>Conclusions</p> <p>nNOS plays a dual role in ischemia/reoxygenation in that its presence is necessary to afford cardioprotection by IP and its inhibition reduces myocardial ischemic injury. The role of nNOS is species-independent and exerted downstream of the mitoK_ATPchannels and upstream of p38MAPK.</p

Soil organic carbon in peninsular Spain: Influence of environmental factors and spatial distribution

Soil organic carbon (SOC) stocks and their geographical distribution in peninsular Spain were estimated from a georeferenced database consisting of 12,724 surface samples (0–30 cm) and 3607 subsurface samples (30–50 cm), covering different climate, land use, elevation, parent material, soil type and soil pH. SOC density showed a high heterogeneity, with the lowest values in arid regions, where the average in topsoil ranged between 20 and 60 t C ha−1, under woody crops and forest respectively. Carbon stocks gradually increases as precipitation increases, and its variability is also dependent of other factors, fundamentally the presence/absence of active lime or active Al. In semi-arid zones, calcaric soils (pH ≈ 8.3) have higher contents of SOC than neutral to weakly acidic soils from siliceous materials. However, in humid regions, calcareous materials have undergo total or partial decarbonatation in the upper layer (pH < 4.0–7.5) and SOC stocks are markedly lower than in other materials. In forest soils it seems that a steady state (around 100–120 t C ha−1) (0–30 cm) has been reached in a wide range of precipitation, between 900 and 1700 mm; most of this carbon (about 80%) is labile-C. Soils from granitic rocks are acidic (pH 4.5–5.5) (Al buffering) and the mean SOC stock in the indicated precipitation range is between 170 and 200 t ha−1 (it is estimated that approximately 60% is stabilized as metal-C or mineral-C complexes). The highest values (190–240 t ha−1) are recorded in acidic soils derived from mafic rocks, which in these regions usually develop andic properties (around 73% is involved in stable metal-C or mineral C complexes). Finally, the SOC stored in neutral soils from serpentinized ultramafic rocks (without excess Ca or Al) is similar to that of the decarbonated soils derived from calcareous materials. In all regions, forest soils are a much more important SOC sink than live forest biomass (2–4 times higher in the upper 30 cm and 3–6 times greater in the upper 50 cm). Random Forest regression was used as modeling tool and digital mapping. Mean annual precipitation was estimated to be the most important predictor variable, followed by land use, lithology/soil type and soil pH. Model performance was calibrated by the internal RF validation and through cross-validation, and the results were similar. In topsoil, the mean error, root mean square error and R2 were −0.007% C, 1.48% C and 0.61, respectively. In the subsurface layer these indices were −0.020, 1.07 and 0.37, respectively. SOC stock for peninsular Spain was estimated at 3.33 Pg in the upper (0–30 cm) layer, and 0.85 Pg in the subsurface (30–50 cm) layer. Total SOC stock for 0–50 cm was 4.19 Pg, with a 95% confidence interval ranging between 3.33 and 5.03 PgThis work was supported by the Ministry of Science and Innovation, Spain (CGL2009, 13857: 2010-2014); and the Consellería de Educación-Xunta de Galicia, Spain (2014-GRC GI-124, 2014-2017)S

Differential Immune Response to Bioprosthetic Heart Valve Tissues in the α1,3Galactosyltransferase-Knockout Mouse Model

Anti-Gal antibodies; Bioprosthetic heart valves; Cellular immune infiltrateAnticuerpos anti-Gal; Válvulas cardíacas bioprotésicas; Infiltrado inmune celularAnticossos anti-Gal; Vàlvules cardíaques bioprotèsiques; Infiltrat immune cel·lularStructural valve deterioration (SVD) of bioprosthetic heart valves (BHVs) has great clinical and economic consequences. Notably, immunity against BHVs plays a major role in SVD, especially when implanted in young and middle-aged patients. However, the complex pathogenesis of SVD remains to be fully characterized, and analyses of commercial BHVs in standardized-preclinical settings are needed for further advancement. Here, we studied the immune response to commercial BHV tissue of bovine, porcine, and equine origin after subcutaneous implantation into adult α1,3-galactosyltransferase-knockout (Gal KO) mice. The levels of serum anti-galactose α1,3-galactose (Gal) and -non-Gal IgM and IgG antibodies were determined up to 2 months post-implantation. Based on histological analyses, all BHV tissues studied triggered distinct infiltrating cellular immune responses that related to tissue degeneration. Increased anti-Gal antibody levels were found in serum after ATS 3f and Freedom/Solo implantation but not for Crown or Hancock II grafts. Overall, there were no correlations between cellular-immunity scores and post-implantation antibodies, suggesting these are independent factors differentially affecting the outcome of distinct commercial BHVs. These findings provide further insights into the understanding of SVD immunopathogenesis and highlight the need to evaluate immune responses as a confounding factor.This research was funded by the European Union Seventh Framework Programme (FP7/2007–2013) under Grant Agreement no. 603049, TRANSLINK. This work was also supported by Ministerio de Economía y Competitividad-ISCiii (PI15/00181) and the PERIS SLT002/16/00445 funded by the Department of Health of Generalitat de Catalunya, all granted to CC and co-funded by FEDER funds, a way to build Europe. IDIBELL benefits from CERCA support. S.G.K. was partially supported by an IDIBELL summer internship. The funding agencies did not influence in any other way than by providing financial support

Lineage Tracing of Cardiac Explant Derived Cells

AIMS: Cultured cardiac explants produce a heterogeneous population of cells including a distinctive population of refractile cells described here as small round cardiac explant derived cells (EDCs). The aim of this study was to explore the source, morphology and cardiogenic potential of EDCs. METHODS: Transgenic MLC2v-Cre/ZEG, and actin-eGFP mice were used for lineage-tracing of EDCs in vitro and in vivo. C57B16 mice were used as cell transplant recipients of EDCs from transgenic hearts, as well as for the general characterisation of EDCs. The activation of cardiac-specific markers were analysed by: immunohistochemistry with bright field and immunofluorescent microscopy, electron microscopy, PCR and RT-PCR. Functional engraftment of transplanted cells was further investigated with calcium transient studies. RESULTS: Production of EDCs was highly dependent on the retention of blood-derived cells or factors in the cultured explants. These cells shared some characteristics of cardiac myocytes in vitro and survived engraftment in the adult heart in vivo. However, EDCs failed to differentiate into functional cardiac myocytes in vivo as demonstrated by the absence of stimulation-evoked intracellular calcium transients following transplantation into the peri-infarct zone. CONCLUSIONS: This study highlights that positive identification based upon one parameter alone such as morphology or immunofluorescene is not adequate to identify the source, fate and function of adult cardiac explant derived cells

Carbono orgânico nos solos do norte de Espanha (Galiza, Astúrias, Cantabria e País Basco)

The soil organic carbon content was analyzed in more than 7 000 soil samples under different land uses, climates and lithologies from northern Spain (Galicia, Asturias, Cantábria y País Vasco). GIS maps (1:50 000) were made of the % SOC and SOC stocks. The % SOC varies according to land use (higher in forest and scrub soils and lower in agricultural soils) and climate, and there is a highly significant correlation between SOC content and mean annual precipitation. There are significant differences between the soils of Galicia/Western Asturias (GAw) and those of the rest of the study area (Central and Eastern Asturias, Cantabria and País Vasco) (AceCV), although these are neighbouring regions. In forest and/or scrub soils with a udic soil moisture regime, in GAw, the SOC is usually > 7% and the average stocks 260 t ha -1 (0-30 cm), and >340 t ha-1 (0-50 cm) in soils with thick organic matter rich horizons (> 40 cm); these values greatly exceed the average contents observed in forest soils from temperate zones. Under similar conditions of vegetation and climate in soils of AceCV the SOC average is 3% and the mean stocks 90-100 t ha-1 (0-30 cm). The andic character of acid forest soils in GAw and the formation of C-Al,Fe complexes are pointed out as the SOC stabilization mechanism, in contrast to the neutral and calcareous soils that predominate in AceCV, where the main species of OC are easily biodegradable.Se analiza el contenido de carbono orgánico (CO) en más de 7.000 muestras de suelos del norte de España (Galicia, Asturias, Cantábria y País Vasco) bajo diferentes tipos de ocupación, condiciones climáticas y litología, y se elaboran mapas SIG (1:50 000) del porcentaje y stock de carbono en los suelos. El porcentaje de CO varía de acuerdo al uso del suelo (mayor en suelos forestales y con matorral, y menor en suelos de cultivo) y al clima, reconociéndose una correlación altamente significativa entre el porcentaje de CO y la precipitación media anual. En cualquier caso, aún tratándose de regiones próximas, se establecen diferencias importantes entre los suelos de Galicia-oeste de Asturias (G/Ao ) y los del resto del área de estudio (Asturias central y oriental, Cantabria y País Vasco) (AceCV). En suelos forestales y/o con matorral y régimen de humedad údico, en G/Ao , el porcentaje de CO es habitualmente > 7% y el stock medio 260 tC ha-1 (0-30 cm) y puede ser > 340 tC ha-1 (0-50 cm), teniendo en cuenta la abundancia de suelos con rasgos cumúlicos y horizontes humíferos con > 40 cm de espesor; los valores obtenidos superan ampliamente los contenidos medios señalados por diferentes autores para suelos forestales de áreas templadas. En similares condiciones de vegetación y clima, el contenido medio de CO en suelos de (AceCV) es de 3% y el stock medio 90-100 tC ha-1 (0-30 cm). El carácter ándico de los suelos forestales ácidos de G/Ao, y la formación de complejos C-Al,Fe se señala como mecanismo de estabilización del CO, en contraste con los suelos neutros y calcáreos que predominan en AceCV, en los que la especie principal es el CO fácilmente biodegradable.Analisou-se o teor de carbono orgânico en mais de 7.000 amostras de solo do norte de Espanha (Galiza, Astúrias, Cantábria e País Basco), sob diferentes usos, condições climáticas e litologias, e procedeu-se ao mapeamento SIG (1:50 000) da percentagem e stock de carbono nos solos. A percentagem de CO varia de acordo com o uso do solo (maior em solos de floresta e mata e menor em solos agrícolas) e condições climáticas, observando-se uma correlação altamente significativa entre a percentagem de CO e a precipitação média anual. Em qualquer caso, mesmo para regiões vizinhas, registam-se diferenças significativas entre os solos da Galiza-oeste e das Astúrias (G/Ao) e o resto da área em estudo (Astúrias central e oriental, Cantabria e País Basco) (AceCV). Em solos de floresta e/ou de mata e regime de humidade údico, em G/Ao, a percentagem de CO é geralmente > 7% e o stock médio 260 t C ha-1(0-30 cm), e > 340 t C ha-1 (0-50 cm) em solos com espessos horizontes ricos em matéria orgânica (> 40 cm); estes valores excedem largamente os teores médios observados em solos florestais de zonas temperadas. Em condições semelhantes de vegetação e clima, em solos de AceCV, o teor médio de CO é de 3% e o stock médio de 90-100 t C ha-1 (0-30 cm). O carácter andico dos solos florestais ácidos da Galiza, e a formação do complexos C-Al,Fe são apontados como o mecanismo de estabilização do CO, em contraste com os solos neutros e calcários prevalecentes em AceCV, onde a espécie predominante é o CO facilmente biodegradável.This study was supported by the Spanish Ministry of Science and Innovation, National Plan I+D, through project CGL2009-13857S

Response of the human myocardium to ischemic injury and preconditioning: The role of cardiac and comorbid conditions, medical treatment, and basal redox status

Vàlvula aòrtica; Isquèmia; MiocardiVálvula aórtica; Isquemia; MiocardioAortic valve; Ischemia; MyocardiumBackground The diseased human myocardium is highly susceptible to ischemia/reoxygenation (I/R)-induced injury but its response to protective interventions such as ischemic preconditioning (IPreC) is unclear. Cardiac and other pre-existing clinical conditions as well as previous or ongoing medical treatment may influence the myocardial response to I/R injury and protection. This study investigated the effect of both on myocardial susceptibility to I/R-induced injury and the protective effects of IPreC. Methods and results Atrial myocardium from cardiac surgery patients (n = 300) was assigned to one of three groups: aerobic control, I/R alone, and IPreC. Lactate dehydrogenase leakage, as a marker of cell injury, and cell viability were measured. The basal redox status was determined in samples from 90 patients. The response to I/R varied widely. Myocardium from patients with aortic valve disease was the most susceptible to injury whereas myocardium from dyslipidemia patients was the least susceptible. Tissue from females was better protected than tissue from males. Myocardium from patients with mitral valve disease was the least responsive to IPreC. The basal redox status was altered in the myocardium from patients with mitral and aortic valve disease. Conclusions The response of the myocardium to I/R and IPreC is highly variable and influenced by the underlying cardiac pathology, dyslipidemia, sex, and the basal redox status. These results should be taken into account in the design of future clinical studies on the prevention of I/R injury and protection.This study was supported by the Instituto de Salud Carlos III (FIS) [grant number 12/00119]