IInvestigation of possible oxidative damage caused by formaldehyde exposure in the rat's heart and aorta tissue / Investigación de posible daño oxidativo causado por la exposición al formaldehído en el corazón de la rata y tejido de la aorta

SUMMARY: Formaldehyde (FA) is a toxic substance used frequently in the field of medicine as well as in many industrial areas. Especially people working in the field of anatomy, histology, and pathology are in high risk group because of the use of the FA. Studies showing the effects of FA on the cardiovascular system are few in number. The purpose of the present study was to investigate the effects of FA exposure, which we believe can cause oxidative stress, on the heart and aorta with various biochemical analyses. A total of 24 Wistar Albino rats were used in our study. We divided the rats into 3 groups as the Control Group (CG), the group exposed to low-dose FA (avg. 1 ppm) (DDG) Group, and the group exposed to high-dose FA (avg. 10 ppm) (YDG). At the end of the subchronic FA exposure, the blood samples, heart and aorta tissues of the rats were taken and subjected to biochemical analyses. As a result of the analyses, statistically significant differences were detected between CG (2.96?0.85 ng/mg), and HDG (2.08?0.77 ng/mg) in aortic tissues in TXNIP analysis (p<0.05). In heart tissues, significant differences were detected between CG (0.73?0.27 ng/mg) and LDG (1.13?0.22 ng/mg) (p<0.05). Statistically significant differences were also detected between CG (1.98?0.31 mM/ml) and YDG (2.43?0.31 mM/ml) in serum MDA analyses (p<0.05). It was shown that subchronic application of FA to LDG rats through inhalation had no effects on apoptosis markers in heart tissues. More studies are required to show FA toxicity and the mechanism of action of pathology on the cardiovascular system. We believe that our study will contribute to clarifying the roles of mild and subchronic exposure of FA in heart and aortic tissues in terms of oxidative stress risk.

RESUMEN: El formaldehído es una sustancia tóxica que se utiliza con frecuencia en el campo de la medicina, así como en muchas áreas industriales. Especialmente las personas que trabajan en el area de la anatomía, y patología se encuentran en el grupo de alto riesgo debido al uso de esta sustancia. Pocos son los estudios que muestran los efectos del formaldehído en el sistema cardiovascular. El propósito del presente estudio fue investigar a través de análisis bioquímicos, los efectos de la exposición a formaldehído, que podría causar estrés oxidativo, en el corazón y la aorta. Se utilizaron un total de 24 ratas Albinas Wistar. Dividimos a las ratas en 3 grupos: grupo control (GC), grupo expuesto a dosis bajas de AG (promedio 1 ppm) (DDG) y grupo expuesto a dosis altas de AG (promedio 10 ppm) (YDG). Al término de la exposición a FA subcrónica, se tomaron muestras de sangre, tejido cardíaco y aorta de las ratas y se sometieron a análisis bioquímicos. Como resultado de los análisis, se detec- taron diferencias estadísticamente significativas entre GC (2,96 ? 0,85 ng / mg) y HDG (2,08 ? 0,77 ng / mg) en los tejidos aórticos en el análisis TXNIP (p <0,05). En los tejidos cardíacos se detectaron diferencias significativas entre GC (0,73 ? 0,27 ng / mg) y LDG (1,13 ? 0,22 ng / mg) (p <0,05). También se detectaron diferencias estadísticamente significativas entre CG (1,98 ? 0,31 mM / ml) y YDG (2,43 ? 0,31 mM / ml) en los análisis de MDA en suero (p <0,05). Se demostró que la aplicación subcrónica de formaldehído a ratas LDG a través de la inhalación no tuvo efectos sobre los marcadores de apoptosis en los tejidos del corazón. Se requieren más estudios para demostrar la toxicidad de los AG y el mecanismo de acción de la patología en el sistema cardiovascular. Creemos que nuestro estudio contribuirá a aclarar las funciones de la exposición leve y subcrónica de formaldehído en los tejidos cardíacos y aórticos en términos de riesgo al estrés oxidativo.

Resistance Exercise Modulates Oxidative Stress Parameters and TNF-α Content in the Heart of Mice with Diet-Induced Obesity / Exercício Resistido Modula Parâmetros de Estresse Oxidativo e Conteúdo de TNF-α no Coração de Camundongos com Obesidade Induzida por Dieta

Abstract Background: Obesity can be characterized by low-grade chronic inflammation and is associated with an excesso production of reactive oxygen species, factors that contribute to coronary heart disease and other cardiomyopathies. Objective: To verify the effects of resistance exercise training on oxidative stress and inflammatory parameters on mice with obesity induced by a high-fat diet (HFD). Methods: 24 Swiss mice were divided into 4 groups: standard diet (SD), SD + resistance exercise (SD + RE), diet-induced obesity (DIO), DIO + RE. The animals were fed SD or HFD for 26 weeks and performed resistance exercises in the last 8 weeks of the study. The insulin tolerance test (ITT) and body weight monitoring were performed to assess the clinical parameters. Oxidative stress and inflammation parameters were evaluated in the cardiac tissue. Data were expressed by mean and standard deviation (p < 0.05). Results: The DIO group had a significant increase in reactive oxygen species levels and lipid peroxidation with reduction after exercise. Superoxide dismutase and the glutathione system showed no significant changes in DIO animals, with an increase in SD + RE. Only catalase activity decreased with both diet and exercise influence. There was an increase in tumor necrosis factor-alpha (TNF-α) in the DIO group, characterizing a possible inflammatory condition, with a decrease when exposed to resistance training (DIO+RE). Conclusion: The DIO resulted in a redox imbalance in cardiac tissue, but the RE was able to modulate these parameters, as well as to control the increase in TNF-α levels.

Resumo Fundamento: A obesidade pode ser caracterizada por uma inflamação crônica de baixo grau e está associada à produção excessiva de espécies reativas de oxigênio, fatores que contribuem para doenças coronarianas e outras cardiomiopatias. Objetivo: Verificar os efeitos do treinamento resistido sobre os parâmetros de estresse oxidativo e parâmetro inflamatório em camundongos com obesidade induzida por dieta hiperlipídica (DIO). Métodos: 24 camundongos Swiss foram divididos em 4 grupos: dieta padrão (DP), DP + exercício resistido (DP+ER), obesidade induzida por DIO, DIO + ER. Os animais foram alimentados por 26 semanas com DP ou hiperlipídica realizando treinamento resistido nas 8 semanas finais do estudo. Para avaliar parâmetros clínicos foi realizado o teste de tolerância à insulina (TTI) e monitoramento do peso corporal. No tecido cardíaco foram avaliados parâmetros de estresse oxidativo e inflamação. Dados expressos por média e desvio padrão (p < 0,05). Resultados: O grupo DIO teve um aumento significativo nos níveis espécies reativas e peroxidação lipídica com redução após o exercício. A superóxido dismutase e o sistema glutationa não demonstraram alterações importantes nos animais DIO, com elevação perante DP+ER. Somente a atividade da catalase reduziu tanto com influência da dieta como do exercício. Ocorreu um aumento do fator de necrose tumoral-alfa (TNF-α) no grupo DIO, caracterizando um possível quadro inflamatório, com redução quando expostos ao treino resistido (DIO+ER). Conclusão: A DIO ocasionou um desequilíbrio redox no tecido cardíaco, porém o ER foi capaz de modular estes parâmetros, bem como controlar o aumento do TNF-α.

Heat shock proteins: protection and potential biomarkers for ischemic injury of cardiomyocytes after surgery

Abstract The heat shock proteins are endogenous proteins with the ability to act as molecular chaperones. Methods that provide cell protection by way of some damage can positively influence the results of surgery. The present review summarizes current knowledge concerning the cardioprotective role of the heat shock proteins as occurs in heart damage, including relevant information about the stresses that regulate the expression of these proteins and their potential role as biomarkers of heart disease.

Testosterone and Voluntary Exercise, Alone or Together Increase Cardiac Activation of AKT and ERK1/2 in Diabetic Rats / Testosterona e Exercício Voluntário, Individualmente ou em Conjunto, Aumentam a Ativação Cardíaca de AKT e ERK1/2 em Ratos Diabéticos

Abstract Background: Impaired angiogenesis in cardiac tissue is a major complication of diabetes. Protein kinase B (AKT) and extracellular signal regulated kinase (ERK) signaling pathways play important role during capillary-like network formation in angiogenesis process. Objectives: To determine the effects of testosterone and voluntary exercise on levels of vascularity, phosphorylated Akt (P- AKT) and phosphorylated ERK (P-ERK) in heart tissue of diabetic and castrated diabetic rats. Methods: Type I diabetes was induced by i.p injection of 50 mg/kg of streptozotocin in animals. After 42 days of treatment with testosterone (2mg/kg/day) or voluntary exercise alone or in combination, heart tissue samples were collected and used for histological evaluation and determination of P-AKT and P-ERK levels by ELISA method. Results: Our results showed that either testosterone or exercise increased capillarity, P-AKT, and P-ERK levels in the heart of diabetic rats. Treatment of diabetic rats with testosterone and exercise had a synergistic effect on capillarity, P-AKT, and P-ERK levels in heart. Furthermore, in the castrated diabetes group, capillarity, P-AKT, and P-ERK levels significantly decreased in the heart, whereas either testosterone treatment or exercise training reversed these effects. Also, simultaneous treatment of castrated diabetic rats with testosterone and exercise had an additive effect on P-AKT and P-ERK levels. Conclusion: Our findings suggest that testosterone and exercise alone or together can increase angiogenesis in the heart of diabetic and castrated diabetic rats. The proangiogenesis effects of testosterone and exercise are associated with the enhanced activation of AKT and ERK1/2 in heart tissue.

Resumo Fundamento: Angiogênese prejudicada em tecido cardíaco é uma das principais complicações das diabetes. As vias de sinalização da proteína-quinase B (AKT) e a quinase regulada por sinal extracelular (ERK) exercem um importante papel durante a formação de uma rede similar à capilar no processo de angiogênese. Objetivos: Determinar os efeitos da testosterona e exercícios voluntários sobre os níveis de vascularidade, AKT fosforilada (P- AKT) e ERK fosforilada (P-ERK) sobre o tecido cardíaco de ratos diabéticos e castrados diabéticos. Métodos: A diabetes tipo 1 foi induzida através de injeção intraperitoneal de 50 mg/kg de estreptozotocina em animais. Após 42 dias de tratamento com testosterona (2mg/kg/dia) ou exercícios voluntários, individualmente ou em conjunto, as amostras de tecidos cardíacos foram coletadas e usadas para avaliação histológica e determinação de níveis de P-AKT e P-ERK através do método ELISA. Resultados: Os nossos resultados mostraram que a testosterona ou os exercícios aumentaram a capilaridade, os níveis de P-AKT, e P-ERK nos corações de ratos diabéticos. O tratamento de ratos diabéticos com testosterona e exercícios obteve um efeito sinérgico sobre a capilaridade, níveis de P-AKT, e P-ERK no coração. Além disto, na capilaridade do grupo diabético castrado, os níveis de P-AKT e P-ERK diminuíram significativamente no coração, ao passo que o tratamento com testosterona ou o treinamento com exercícios reverteu tais efeitos. O tratamento simultâneo de ratos diabéticos castrados com testosterona e exercícios obteve um efeito aditivo sobre os níveis de P-AKT e P-ERK. Conclusão: Nossas descobertas sugerem que a testosterona e exercícios, em conjunto ou individualmente, podem aumentar a angiogênese nos corações de ratos diabéticos e castrados diabéticos. Os efeitos favoráveis à angiogênese da testosterona e dos exercícios estão associados à ativação reforçada de AKT e ERK1/2 no tecido cardíaco.

The TcI and TcII Trypanosoma cruzi experimental infections induce distinct immune responses and cardiac fibrosis in dogs

Trypanosoma cruzi infection may be caused by different strains with distinct discrete typing units (DTUs) that can result in variable clinical forms of chronic Chagas disease. The present study evaluates the immune response and cardiac lesions in dogs experimentally infected with different T. cruzi strains with distinct DTUs, namely, the Colombian (Col) and Y strains of TcI and TcII DTU, respectively. During infection with the Col strain, increased levels of alanine aminotransferase, erythrocytes, haematocrit and haemoglobin were observed. In addition, CD8+ T-lymphocytes isolated from the peripheral blood produced higher levels of interleukin (IL)-4. The latter suggests that during the acute phase, infection with the Col strain may remain unnoticed by circulating mononuclear cells. In the chronic phase, a significant increase in the number of inflammatory cells was detected in the right atrium. Conversely, infection with the Y strain led to leucopoenia, thrombopoenia, inversion of the ratio of CD4+/CD8+ T-lymphocytes and alterations in monocyte number. The Y strain stimulated the production of interferon-γ by CD4+ and CD8+ T-lymphocytes and IL-4 by CD8+ T-cells. In the chronic phase, significant heart inflammation and fibrosis were observed, demonstrating that strains of different DTUs interact differently with the host.

Prophylactic effect of Mucuna pruriens Linn (velvet bean) seed extract against experimental Naja sputatrix envenomation: Gene expression studies.

Mucuna pruriens is widely used in traditional medicine for treatments of various diseases. In certain region of Nigeria, the seed is used as oral prophylactics for snakebite. Rats pretreated with the aqueous extract from M. pruriens seed (MPE) were protected against the lethal effects of Naja sputatrix (Javan spitting cobra) venom [Tan et al., J Ethnopharmacol, 123 (2009) 356]. The pretreatment also protected against venom-induced histopathological changes in rat heart. To contribute to the understanding of the mechanism of cardio-protective action, the present study examined the effects of MPE-pretreatment on gene expression profile of rat heart as well as effect of MPE-pretreatment on N. sputatrix venom-induced gene expression alterations in rat heart. The gene expression profiles were examined by microarray analysis and verified by real time PCR. The results showed that pretreatment with MPE caused 50 genes in the rat heart substantially up-regulated of which 19 were related to immune responses, 7 were related to energy production and metabolism. The up-regulation of genes related to energy metabolism probably plays a role in maintaining the viability of the heart. Four other genes that were up-regulated (alpha synuclein, natriuretic peptide precursor, calsequestrin and triadin) were involved in the maintenance of homeostasis of the heart or maintaining its viability, thereby contributing to the direct protective action. The results demonstrated that protective effect of MPE pretreatment against snake venom poisoning may involve a direct action on the heart.

The impact of gender, age and tissues in vitro on estimating postmortem interval by FTIR spectroscopy / 法医学杂志

To investigate the influence of such individual factors as gender, age and tissues in vitro to the postmortem interval (PMI) by the Fourier transform infrared (FTIR) spectrometer in animal experiments. SD rats were classified into male and female groups, different age groups (21-day, 42-day and 63-day group), and tissues in vitro and in vivo groups. The rats were sacrificed by cervical dislocation, whose bodies were kept in a controlled environmental chamber set at (20+/-2) degrees C and 50% humidity. The liver, kidney, spleen, myocardium, brain, lung and skeletal muscle tissues were collected for measurement from time zero to 48 h postmortem. With the change of PMI, no obvious changes were found in the main FTIR absorbance peaks and their ratios at different time points. All the experimental groups showed no significant changes when compared with the controls. The gender, age and tissues in vitro were not found to be contributing factors in the estimation of PMI via FTIR spectroscopy.

Effect of renal denervation procedure on left ventricular hypertrophy of hypertensive rats and its mechanisms / Efeito da denervação renal na hipertrofia do ventrículo esquerdo de ratos hipertensos e seu mecanismo

PURPOSE: To investigate the effect of renal denervation (RDN) on the blood pressure, left ventricular hypertrophy and myocardial expression of TLR4/NF-κB in spontaneously hypertensive rats (SHR). METHODS: A total of 36 SHR were randomly assigned into control group (D0), RDN group (D) and sham group (S). 12 WKY rats of same age served as controls (WKY group). Rats in the D0 and WKY groups were sacrificed, but rats in the D and S group were sacrificed at one week and six weeks after surgery. The heart was collected and the left ventricle weighted followed by calculation of left ventricular mass index (LVMI). RESULTS: In the D0 group, the blood pressure, LVMI and protein expression of TLR4, NF-κB, TNF-α and IL-6 in the myocardium were markedly higher than that in the WKY group (p<0.05). In the D1 and D2 group, the LVMI, NE and protein expression of TLR4, NF-κB, TNF-α and IL-6 in the myocardium were significantly reduced (p<0.05). CONCLUSION: Renal denervation can significantly delay the progression of left ventricular hypertrophy in spontaneously hypertensive rats, which may be attributed to the not only the suppression of sympathetic activity and attenuation of pressure load but the improvement of myocardial immuno-inflammation.

OBJETIVO: Investigar o efeito da denervação renal na pressão sanguínea, na hipertrofia do ventrículo esquerdo e a expressão miocárdica de TLR4/NF-kB em ratos espontaneamente hipertensos. MÉTODOS: Trinta e seis SHR ratos foram aleatoriamente distribuídos em grupo controle, grupo denervação renal (D) e grupo sham(S). 12 WKY ratos de mesma idade serviram de controle. Os ratos controles foram sacrificados, mas os ratos com denervação renal e sham foram sacrificados uma semana e seis semanas após a cirurgia. O coração foi retirado e o ventrículo esquerdo pesado seguido pelo cálculo da massa ventricular (LVMI). RESULTADOS: No grupo DO, a pressão sanguínea, LVMI e a expressão proteica de TLR4, NF-κB, TNF-α e IL-6, no miocárdio foram marcadamente maiores do que o grupo WKY (p<0,05). Nos grupos D1 e D2, o LVMI, NE e a expressão proteica de TLR4, NF-κB, TNF-α e IL-6 no miocárdio foi significantemente reduzido (p<0,05). CONCLUSÃO: A denervação renal pode significantemente retardar a progressão da hipertrofia ventricular esquerda em ratos espontaneamente hipertensos, o que pode ser atribuído não apenas pela supressão da atividade simpática e atenuação da pressão, mas pela melhora na imunoinflamação miocárdica.

Morfologia dos cardiomiócitos e quantificação do colágeno no miocárdio de ratas tratadas com isoflavonas ou estrogênios / Cardiomyocytes morphology and collagen quantification in the myocardium of female rats treated with isoflavones or estrogens

OBJETIVOS: Avaliar a morfologia dos cardiomiócitos e quantificar o colágeno presente no miocárdio de ratas tratadas com extrato concentrado de soja ou 17β-estradiol (E2). MÉTODOS: Vinte e oito ratas foram divididas em quatro grupos: GCtrl - fase de estro; GOvx - ovariectomizadas (Ovx); GIso - Ovx tratadas com extrato de soja (150 mg/kg, por dia); GE2 - Ovx tratadas com E2 (10 µg/kg, por dia). As drogas e o veículo (0,2 mL de propilenoglicol) foram administrados após 30 dias da realização da ovariectomia, por 30 dias consecutivos. No último dia os animais foram anestesiados, o coração retirado, mergulhado em formaldeído a 10%, e fragmentos dos ventrículos submetidos a processamento histológico, sendo os cortes corados pela hematoxilina e eosina ou pelo picrosirius-red. As análises histomorfométricas (contagem, volume nuclear e quantificação do colágeno) foram realizadas em microscópio de luz e software AxioVision Rel. 4.2, sendo o colágeno determinado pelo programa Imagelab 2000. Os dados foram submetidos ao teste de ANOVA complementado pelo teste de Tukey (p<0,05). RESULTADOS: Notamos maior quantidade de núcleos de cardiomiócitos nos animais dos grupos Ovx e Iso do que no GE2 e GCtrl (GOvx=121,7±20,2=GIso=92,8±15,4>GE2=70,5±14,8=GCtrl=66,3±9,6; p<0,05), sendo o volume nuclear maior nos animais do grupo Ctrl e E2 (GE2=35,7±4,8=GCtrl=29,9±3,6>GIso=26,5±4,5=GOvx=22,4±2,9; p<0,05). Com relação ao colágeno notamos maior concentração no grupo Ovx (GOvx=5,4±0,1>GCtrl=4,0±0,1=GIso=4,4±0,08=GE2=4,3±0,5; p<0,05). CONCLUSÕES: Os estrogênios previnem a diminuição do volume nuclear dos cardiomiócitos e a deposição de colágeno entre as fibras musculares cardíacas. Já a administração de isoflavonas previne somente a deposição de colágeno, o que pode preservar as propriedades mecânicas das fibras cardíacas.

PURPOSES: To evaluate the histomorphometry of cardiomyocytes and collagen present in the myocardium of rats treated with a concentrated extract of soy or 17β-estradiol (E2). METHODS: Twenty-eight rats were divided into four groups: GCtrl - estrus phase; GOvx - ovariectomized (Ovx) and receiving vehicle; GIso - Ovx and treated with soy extract (150 mg/kg per day); GE2 - Ovx and treated with E2 (10 µg/kg per day). The drugs and vehicle (0.2 mL propylene glycol) were administered for 30 consecutive days after ovariectomy. On the last day the animals were anesthetized, the hearts removed, submerged in 10% formaldehyde and fragments of the ventricles underwent histological procedures, and the sections were stained with hematoxylin and eosin or picrosirius-red. Histomorphometric analysis (number and volume of nuclei and quantification of collagen) was performed under a light microscope with AxioVision Rel. 4.2 software, and collagen fibers were quantified using IMAGELAB-2000 software. Data were submitted to ANOVA followed by the Tukey test (p<0.05). RESULTS: We observed a higher number of cardiomyocyte nuclei in animals of the Ovx and Iso groups than in GE2 and GCtrl animals (GOvx=121.7±20.2=GIso=92.8±15.4>GE2=70.5±14,8=GCtrl=66.3±9.6; p <0.05), while the nuclear volume was greater in the Ctrl and E2 groups (GE2=35.7±4.8 GCtrl=29.9±3.6=>GIso=26.5±4.5=GOvx=22.4±2.9; p <0.05). Collagen concentration was higher in the Ovx group (GOvx=5.4±0.1>GCtrl=4.0±0.1=GIso=4.4±0.08=GE2=4.3±0.5; p <0.05). CONCLUSIONS: Estrogen may prevent the reduction of the nuclear volume of cardiomyocytes and collagen deposition between heart muscle fibers, while the administration of isoflavones only prevents the deposition of collagen, which can preserve the mechanical properties of cardiac fibers.

S100B protein expression in the heart of deceased individuals by overdose: a new forensic marker?

OBJECTIVE: The evaluation of S100B protein expression in the human heart and its correlation with drug-related death. METHOD: Left ventricular samples were collected from 74 serial forensic autopsies (15 overdose-related deaths; 59 non-overdose-related deaths) from 2007 to 2010. Tissue sections from each sample were immunostained for S100B protein by a commercial antibody. RESULTS: The S100B protein was detected in the heart samples of all 15 cases of drug-related deaths; S100B immunoreactivity was mainly observed in the cytoplasm of cardiomyocytes and as globular deposits in the interstitial spaces. No reactivity or weak reactivity was found in the cardiomyocytes of the 59 subjects who died of other causes. CONCLUSION: Our preliminary data show that the S100B protein accumulates in injured cardiomyocytes during drug-related sudden death. Given the near absence of S100B protein in the heart of subjects who died from causes other than drug overdose, S100B immunopositivity may be used as a new ancillary screening tool for the postmortem diagnosis of overdose-related cardiac death.

Quantification of left ventricular myocardial collagen system in children, young adults, and the elderly / Cuantificación del sistema de colágeno del ventrículo izquierdo del miocardio en niños, adultos jóvenes y ancianos

Studies on the collagen system of the human myocardium are still limited compared to those on small laboratory animals. The aim of this work was to observe the collagen tissue of the myocardium of the human heart as a function of age. The types of collagen, as well as the density of collagen tissue and the diameter of collagen fibrils, were examined. Fragments of the left ventricular wall from 15 hearts, 5 from children, 5 from young adults, and 5 from elderly individuals, were analyzed by using the Picrosirius-polarization method and by transmission electron microscopy (TEM). The results showed the presence of collagen type III and collagen type I, both in the endomysium and perimysium of the 3 groups studied. Measurements of collagen content in myocardial tissue displayed that both endomysial and perimysial collagen increase in number and thickness in the adult and elderly. These histochemical results coincided with the observations obtained with the electron microscope in showing an increase in the number of collagen fibrils with a large diameter in the adult and elderly hearts. The present results on cardiac collagen may be important for assessing the pathogenesis of several cardiopathies in the hearts of children, young adults, and the elderly.

Los estudios sobre el colágeno del miocardio humano son aún escasos en comparación con los hechos en pequeños animales de laboratorio. El objetivo de este trabajo fue cuantificar el tejido colágeno del miocardio del corazón humano en función de la edad. Se estudiaron los tipos de colágeno, su densidad y el diámetro de las fibrillas de colágeno. Para esto se utilizaron fragmentos de la pared del ventrículo izquierdo de 15 corazones, cinco de niños, cinco de adultos jóvenes y 5 de personas de edad avanzada. Las muestras se analizaron mediante el método de Picrosirius-polarización y por microscopía electrónica de transmisión (MET). Los resultados mostraron la presencia de colágeno tipo III y de tipo I, tanto en el endomisio como en el perimisio de los tres grupos estudiados. Además, aumenta el colágeno tanto en el endomisio como en el perimísio, así como su número y grosor a medida que aumenta la edad. Los resultados histoquímicos coincidieron con las observaciones obtenidas con el microscopio electrónico, en las que se observa un aumento en el número de fibrillas de colágeno de gran diámetro en los corazones de los adultos y los ancianos. Estos resultados podrían ser importantes para la evaluación de la patogénesis de varias cardiopatías en los corazones de niños, jóvenes y ancianos.

Papel da titina na modulação da função cardíaca e suas implicações fisiopatológicas / The role of titin in the modulation of cardiac function and its pathophysiological implications / Papel de la Titina en la Modulación de la Función Cardíaca y sus Implicaciones Fisiopatológicas

A titina é uma proteína sarcomérica gigante que se estende desde a linha Z até a linha M. Em razão de sua localização, representa um importante sensor biomecânico com um papel fundamental na manutenção da integridade estrutural do sarcômero. A titina funciona como uma "mola bidirecional" que regula o comprimento sarcomérico e realiza ajustes adequados da tensão passiva sempre que o comprimento varia. Dessa forma, não só determina a rigidez ventricular e a função diastólica, como também influencia a função cardíaca sistólica, modulando o mecanismo de Frank-Starling. O miocárdio expressa duas isoformas dessa macromolécula: a N2B, mais rígida, e a isoforma N2BA, mais complacente. As alterações na expressão relativa das duas isoformas da titina ou alterações do seu estado de fosforilação têm sido implicadas na fisiopatologia de várias doenças como a insuficiência cardíaca diastólica, a cardiomiopatia dilatada, a cardiomiopatia isquêmica e a estenose aórtica. Neste artigo pretende-se descrever sumariamente a estrutura e localização da titina, a sua relação com diferentes cardiomiopatias, e compreender de que forma as alterações dessa macromolécula influenciam a fisiopatologia da insuficiência cardíaca diastólica, salientando o potencial terapêutico da manipulação dessa macromolécula.

Titin is a giant sarcomeric protein that extends from the Z-line to the M-line. Due to its location, it represents an important biomechanical sensor, which has a crucial role in the maintenance of the sarcomere structural integrity. Titin works as a "bidireactional spring" that regulates the sarcomeric length and performs adequate adjustments of passive tension whenever the length varies. Therefore, it determines not only ventricular rigidity and diastolic function, but also systolic cardiac function, modulating the Frank-Starling mechanism. The myocardium expresses two isoforms of this macromolecule: the N2B, more rigid and the isoform N2BA, more compliant. The alterations in the relative expression of the two titin isoforms or alterations in their state of phosphorylation have been implicated in the pathophysiology of several diseases, such as diastolic heart failure, dilated cardiomyopathy, ischemic cardiomyopathy and aortic stenosis. The aim of this study is to describe, in brief, the structure and location of titin, its association with different cardiomyopathies and understand how alterations in this macromolecule influence the pathophysiology of diastolic heart failure, emphasizing the therapeutic potential of the manipulation of this macromolecule.

La titina es una proteína sarcomérica gigante que se extiende desde la línea Z hasta la línea M. En razón de su ubicación, representa un importante sensor biomecánico con un papel fundamental en la manutención de la integridad estructural del sarcómero. La titina funciona como un "resorte bidireccional" que regula el largo sarcomérico y realiza ajustes adecuados de la tensión pasiva siempre que ese largo varía. De esa forma, no sólo determina la rigidez ventricular y la función diastólica, sino también influye en la función cardíaca sistólica, modulando el mecanismo de Frank-Starling. El miocardio expresa dos isoformas de esa macromolécula: la N2B, más rígida, y la isoforma N2BA, más complaciente. Las alteraciones en la expresión relativa de las dos isoformas de la titina o alteraciones de su estado de fosforilación han sido implicadas en la fisiopatología de varias enfermedades como la insuficiencia cardíaca diastólica, la cardiomiopatía dilatada, la cardiomiopatía isquémica y la estenosis aórtica. Este artículo pretende describir sumariamente la estructura y ubicación de la titina, su relación con diferentes cardiomiopatías, y comprender de qué forma las alteraciones de esa macromolécula influyen en la fisiopatología de la insuficiencia cardíaca diastólica, destacando el potencial terapéutico de la manipulación de esa macromolécula.

Ativação adrenérgica intramiocárdica na cardiomiopatia chagásica e doença arterial coronariana / Intramyocardial adrenergic activation in chagasic cardiomyopathy and coronary artery disease / Activación adrenérgica intramiocárdica en la cardiomiopatía chagásica y enfermedad arterial coronaria

FUNDAMENTO: A norepinefrina miocárdica está alterada na disfunção ventricular esquerda. Em pacientes com cardiomiopatia chagásica (CC), essa questão ainda não foi discutida. OBJETIVO: Determinar o nível de norepinefrina (NE) miocárdica em pacientes com CC e compará-la em pacientes com doença arterial coronariana (DAC) e relacionar NE miocárdica com a fração de ejeção do ventrículo esquerdo (FEVE). MÉTODOS: Estudamos 39 pacientes com CC, divididos em grupo 1: 21 indivíduos com FEVE normal e grupo 2: 18 com FEVE diminuída. Dezessete pacientes com DAC foram divididos em grupo 3: 12 indivíduos com FEVE normal e grupo 4: 5 indivíduos com FEVE diminuída. Ecocardiografia bidimensional foi usada para medir a FEVE. A NE miocárdica foi determinada através de Cromatografia Líquida de Alta Eficiência (HPLC). RESULTADOS: A NE miocárdica na CC com e sem disfunção ventricular foi 1,3±1,3 e 6,1±4,2 pg/μg de proteína não-colagenosa, respectivamente (p<0,0001); na DAC com e sem disfunção ventricular, foi 3,3±3,0 e 9,8±4,2 pg/μg de proteína não-colagenosa, respectivamente (p<0,0001). Uma correlação positive foi observada entre a FEVE e a concentração de NE miocárdica em pacientes com CC (p<0,01; r = 0,57) e também naqueles com DAC (p<0,01; r=0,69). Uma diferença significante foi demonstrada entre as concentrações de NE em pacientes com FEVE normal (grupos 1 e 3; p = 0,0182), mas nenhuma diferença foi observada em pacientes com FEVE diminuída (grupos 2 e 4; p = 0,1467). CONCLUSÃO: Pacientes com CC e fração de ejeção global normal apresentam uma denervação cardíaca precoce, quando comparados à pacientes com doença arterial coronariana.

BACKGROUND: Myocardial norepinephrine is altered in left ventricular impairment. In patients with Chagas' cardiomyopathy (CC), this issue has not been addressed. OBJECTIVE: To determine the level of myocardial norepinephrine in patients with CC and compare it in patients with coronary artery disease, and to relate myocardial norepinephrine to left ventricular ejection fraction (LVEF). METHODS: We studied 39 patients with CC, divided into group 1: 21 individuals with normal LVEF and group 2: 18 individuals with decreased LVEF. Seventeen patients with coronary artery disease were divided into group 3: 12 individuals with normal LVEF and group 4: 5 individuals with decreased LVEF. Two-dimensional echocardiography was used to measure LVEF. Myocardial norepinephrine was determined by high-performance liquid chromatography. RESULTS: Myocardial norepinephrine in CC with and without ventricular dysfunction was 1.3±1.3 and 6.1±4.2 pg/μg noncollagen protein, respectively (p<0.0001); in coronary artery disease with and without ventricular dysfunction, it was 3.3±3.0 and 9.8±4.2 pgμg noncollagen protein, respectively (p<0.0001). A positive correlation was found between LVEF and myocardial norepinephrine concentration in the patients with Chagas' cardiomyopathy (p<0.01, r = 0.57) and also in those with coronary artery disease (p<0.01, r=0.69). A significant difference was demonstrated between norepinephrine concentrations in patients with normal LVEF (groups 1 and 3; p = 0.0182), but no difference was found in patients with decreased LVEF (groups 2 and 4; p = 0.1467). CONCLUSION: In patients with Chagas' cardiomyopathy and normal global ejection fraction there is an early cardiac denervation, when compared to coronary artery disease patients.

FUNDAMENTO: La norepinefrina miocárdica está alterada en la disfunción ventricular izquierda. En pacientes con cardiomiopatía chagásica (CC), esa cuestión aun no fue discutida. OBJETIVO: Determinar el nivel de norepinefrina (NE) miocárdica en pacientes con CC y compararla en pacientes con enfermedad arterial coronaria (EAC) y relacionar NE miocárdica con la fracción de eyección del ventrículo izquierdo (FEVI). MÉTODOS: 39 pacientes con CC, divididos en grupo 1: 21 individuos con FEVI normal y grupo 2: 18 con FEVI disminuida. Diecisiete pacientes con EAC fueron divididos en grupo 3: 12 individuos con FEVI normal y grupo 4: 5 individuos con FEVI disminuida. Ecocardiografía bidimensional fue usada para medir la FEVI. La NE miocárdica fue determinada a través de Cromatografía Líquida de Alta Eficiencia (HPLC). RESULTADOS: La NE miocárdica en la CC con y sin disfunción ventricular fue 1,3±1,3 y 6,1±4,2 pg/µg de proteína no colagenosa, respectivamente (p<0,0001); en la EAC con y sin disfunción ventricular, fue 3,3±3,0 y 9,8±4,2 pg/µg de proteína no colagenosa, respectivamente (p<0,0001). Una correlación positiva fue observada entre la FEVI y la concentración de NE miocárdica en pacientes con CC (p<0,01; r=0,57) y también en aquellos con EAC (p<0,01; r=0,69). Una diferencia significativa fue demostrada entre las concentraciones de NE en pacientes con FEVI normal (grupos 1 y 3; p = 0,0182), pero ninguna diferencia fue observada en pacientes con FEVI disminuida (grupos 2 y 4; p = 0,1467). CONCLUSIONES: Pacientes con CC y fracción de eyección global normal presentan una denervación cardíaca precoz, cuando son comparados a pacientes con enfermedad arterial coronaria.

Experimental induction of steatosis in different tissues after the ingestion of a carbohydrate-rich diet: effect on the liver, on the heart and on indicators of oxidation / Esteatose experimental em diferentes tecidos induzida pela ingestão de dieta rica em carboidratos: efeito sobre o fígado, coração e indicadores do estresse oxidativo

CONTEXT: The western dietary pattern is characterized by a high calorie intake with a high proportion of simple sugars. This diet is associated with comorbidities such as hepatic fat deposition and is possibly related to non-alcoholic fatty liver disease. OBJECTIVE: To evaluate the capacity of a hyperglucidic diet to induce steatosis in adult male Wistar rats. After the administration of a carbohydrate-rich diet, we also evaluated the presence of hepatic and cardiac steatosis and the levels of intrinsic antioxidants in the liver. METHODS: Forty-six eutrophic adult male Wistar rats were used and 10 of them were chosen, at random, to serve as controls, while the remaining ones formed the experimental group. Control animals received the standard ration offered by the animal house and the experimental group received the hyperglucidic diet. The diets were offered for 21 days and, at the end of this period, tissue samples were collected for analysis of indicators of oxidative stress (malondialdehyde, and reduced glutathione) and of vitamin E. The animals were then sacrificed by decapitation and their viscera were removed for analysis of liver and heart fat. RESULTS: The hyperglucidic diet used induced hepatic fat deposition, with lipid vacuoles being detected in 83 percent of the livers analyzed by histology. No lipid vacuoles were observed in the heart. Malondialdehyde and reduced glutathione levels remained unchanged when the animals were submitted to the hyperglucidic diet, probably because there was no liver development of fibrosis or inflammation. In contrast, the levels of vitamin E (antioxidant) were reduced, as confirmed in the literature for steatotic animals. CONCLUSION: The hyperglucidic diet induced hepatic steatosis. In the heart there was an increase in fat content, although no histological changes were observed. These alterations cannot be explained by the presence of malondialdehyde or reduced glutathione (indicators of oxidation), since the values were similar in the groups studied. However, a significant reduction of vitamin E was observed in the experimental group.

CONTEXTO: O padrão alimentar ocidental é caracterizado pela ingestão de dieta rica em açúcares simples. Esta alimentação é associada com comorbidades como, por exemplo, deposição de gordura no fígado e possivelmente relacionada com a esteatose hepática não-alcoólica. OBJETIVO: Avaliar a capacidade de uma alimentação hiperglicídica induzir esteatose em ratos Wistar adultos. Após administração de uma dieta rica em hidratos de carbono, foi avaliada a presença de esteatose hepática cardíaca e a presença de antioxidantes no fígado. MÉTODOS: Quarenta e seis ratos Wistar adultos eutróficos foram utilizados no experimento. Destes, 10 animais escolhidos por meio de sorteio simples (ao acaso) foram considerados controles e os demais pertencentes ao grupo experimental. Os animais controles receberam, durante todo experimento dieta usual do biotério. Os animais do grupo experimental, durante 21 dias, receberam dieta com 70 por cento de sacarose. Ao final os animais foram sacrificados por decapitação e suas vísceras (fígado e coração) analisada quanto ao teor de gordura. As amostras de tecido hepático foram também analisadas quanto ao teor de antioxidantes (malondialdehido e glutationa reduzida) e vitamina E. RESULTADOS: A dieta hiperglicídica induziu a deposição de gordura no fígado, sendo os vacúolos lipídicos detectados em 83 por cento das amostras no fígado (histologia). No coração foi detectado bioquimicamente aumento do percentual de gordura, sem a detecção de vacúolos lipídico por histologia. Os teores de malondialdehido e glutationa reduzida não foram diferentes entre os animais dos grupos controle e experimental. Por outro lado, os valores de vitamina E, no grupo experimental, foram significativamente inferiores ao do grupo controle. CONCLUSÃO: A dieta hiperglicídica induziu ao esteatose hepática. No coração houve maior deposição de lípides, embora a histologia não tenha mostrado alterações. Esta deposição, tanto no coração como no fígado, não pode ser explicada pelos indicadores de oxidação utilizados. No entanto, foram observados baixos níveis de vitamina E, que pode estar associada a esta indução de esteatose, principalmente, hepática.

Determinaciones de niveles de creatina y lípidos mediante espectroscopia por resonancia magnética en miocardio de pacientes con insuficiencia cardiaca no isquémica / Myocardial lipids and creatine measured by magnetic resonance spectroscopy among patients with heart failure

Background: Heart failure (HF) is characterized, among other features, by the development of alterations in myocardial energy metabolism, involving a decrease in glucose utilization and increased free fatty acid uptake by cardiomyocytes, associated with decreased deposits of high-energy phosphates (creatine phosphate/ creatine transporter). Magnetic resonance (MR) imaging allows a direct and noninvasive assessment of myocardial metabolites. Aim: To measure myocardial creatine and lipids by MR spectroscopy among patients with HF. Material and Methods: Cardiac MR spectroscopy (1.5 Tesla) with Hydrogen antenna and single voxel acquisition was performed in fve patients with non-ischemic heart failure, aged 58 ± 9.7 years, (60 percent males) and 5 healthy volunteers matched for age and sex. We analyzed the signals of creatine (Cr), lipids (L) and water (W) in the interventricular septum, establishing the water/lipid (W/L) and water/creatine (W/Cr) index to normalize the values obtained. Results: Among patients, left ventricular ejection fraction was 32 ± 6.9 percent, 60 percent were in functional capacity II, 60 percent had hypertension and one was diabetic. Spectroscopic curves showed a depletion of total Cr, evidenced by the W/ Cr index, among patients with heart failure, when compared with healthy controls (1.46 ± 1.21 and 5.96 ± 2.25 respectively, p < 0,05). Differences in myocardial lipid content, measured as the W/L index, were not significant (5.06 ± 2.66 and 1.80 ± 1.62 respectively, p = 0.08). Conclusions: Among patients with heart failure of non-ischemic etiology, there is a depletion of creatine levels measured by MR spectroscopy.

Tissue expression and subcellular localization of Mipu1, a novel myocardial ischemia-related gene

Myocardial ischemic preconditioning up-regulated protein 1 (Mipu1), a novel zinc finger protein, was originally cloned using bioinformatic analysis and 5' RACE technology of rat heart after a transient myocardial ischemia/reperfusion procedure in our laboratory. In order to investigate the functions of Mipu1, the recombinant prokaryotic expression vector pQE31-Mipu1 was constructed and transformed into Escherichia coli M15(pREP4), and Mipu1-6His fusion protein was expressed and purified. The identity of the purified protein was confirmed by mass spectrometry. The molecular mass of the Mipu1 protein was 70.03779 kDa. The fusion protein was intracutaneously injected to immunize New Zealand rabbits to produce a polyclonal antibody. The antibody titer was approximately 1:16,000. The antibody was tested by Western blotting for specificity and sensitivity. Using the antibody, it was found that Mipu1 was highly expressed in the heart and brain of rats and was localized in the nucleus of H9c2 myogenic cells. The present study lays the foundation for further study of the biological functions of Mipu1.

Collagen content, but not the ratios of collagen type III/I mRNAs, differs among hypertensive, alcoholic, and idiopathic dilated cardiomyopathy

Cardiac interstitial fibrosis may contribute to ventricular dysfunction and the prognosis of patients with dilated cardiomyopathy. The objective of the present study was to determine if total myocardial collagen content and collagen type III/I (III/I ratio) mRNAs differ in hypertensive, alcoholic, and idiopathic dilated cardiomyopathy subjects. Echocardiography and exercise cardiopulmonary testing were performed in patients with idiopathic (N = 22), hypertensive (N = 12), and alcoholic (N = 11) dilated cardiomyopathy. Morphometric analysis of collagen was performed in fragments obtained by endomyocardial biopsy with picrosirius red staining. The collagen III/I ratio was determined by reverse transcription polymerase chain reaction. Samples of controls (N = 10) were obtained from autopsy. Echocardiographic variables and maximal oxygen uptake were not different among dilated cardiomyopathy groups. Collagen was higher in all dilated cardiomyopathy groups (idiopathic, hypertensive and alcoholic, 7.36 ± 1.09 percent) versus controls (1.12 ± 0.18 percent), P < 0.05. Collagen was lower in idiopathic dilated cardiomyopathy (4.97 ± 0.83 percent) than hypertensive (8.50 ± 1.11 percent) and alcoholic (10.77 ± 2.09 percent) samples (P < 0.005 for both). The collagen III/I ratio in all samples from dilated cardiomyopathy patients was higher compared to that in controls (0.29 ± 0.04, P < 0.05) but was the same in the samples from idiopathic (0.77 ± 0.07), hypertensive (0.75 ± 0.07), and alcoholic (0.81 ± 0.16) dilated cardiomyopathy groups. Because of the different physical properties of the types of collagen, the higher III/I ratio may contribute to progressive ventricular dilation and dysfunction in dilated cardiomyopathy patients.

Obesity induces upregulation of genes involved in myocardial Ca2+ handling

Obesity is a complex multifactorial disorder that is often associated with cardiovascular diseases. Research on experimental models has suggested that cardiac dysfunction in obesity might be related to alterations in myocardial intracellular calcium (Ca2+) handling. However, information about the expression of Ca2+-related genes that lead to this abnormality is scarce. We evaluated the effects of obesity induced by a high-fat diet in the expression of Ca2+-related genes, focusing the L-type Ca2+ channel (Cacna1c), sarcolemmal Na+/Ca2+ exchanger (NCX), sarcoplasmic reticulum Ca2+ ATPase (SERCA2a), ryanodine receptor (RyR2), and phospholamban (PLB) mRNA in rat myocardium. Male 30-day-old Wistar rats were fed a standard (control) or high-fat diet (obese) for 15 weeks. Obesity was defined as increased percent of body fat in carcass. The mRNA expression of Ca2+-related genes in the left ventricle was measured by RT-PCR. Compared with control rats, the obese rats had increased percent of body fat, area under the curve for glucose, and leptin and insulin plasma concentrations. Obesity also caused an increase in the levels of SERCA2a, RyR2 and PLB mRNA (P < 0.05) but did not modify the mRNA levels of Cacna1c and NCX. These findings show that obesity induced by high-fat diet causes cardiac upregulation of Ca2+ transport_related genes in the sarcoplasmic reticulum.

Influências de dietas ricas em ácidos graxos saturados e insaturados sobre o miocárdio de ratos / Influences of rich in saturated and unsaturated fatty acids diets in rat myocardium

OBJETIVOS: O estudo avaliou a influência de dietas ricas em ácidos graxos saturados (AGS) e ácidos graxos insaturados (AGI) sobre a função mecânica, a morfologia e o estresse oxidativo do miocárdio de ratos. MÉTODOS: Ratos Wistar com 60 dias de idade foram alimentados com dieta padrão (n = 8) ou dietas ricas em AGS (n = 8) ou AGI (n = 8) durante 60 dias. A função mecânica foi avaliada em músculo papilar isolado do ventrículo esquerdo (VE) por meio de contrações isométrica e isotônica, em condição basal (1,25 mM de cálcio), após elevação da concentração extracelular de cálcio para 5,2 mM e estimulação beta-adrenérgica com isoproterenol 1,0 µM. Fragmentos do VE foram usados para estudo de estresse oxidativo e microscopias óptica e eletrônica. RESULTADOS: As dietas suplementadas com AGS e AGI não alteraram a função mecânica do músculo cardíaco. Entretanto, ambas provocaram estresse oxidativo, com aumento do hidroperóxido de lipídio e redução da concentração de superóxido dismutase. A dieta AGI diminuiu a expressão da catalase e a AGS reduziu a quantidade de glutationa peroxidase miocárdica. Ambas as dietas promoveram discretas alterações morfológicas visualizadas ultra-estruturalmente, como depósitos lipídicos e lesões das membranas celulares. CONCLUSÃO: Os resultados sugerem que dietas enriquecidas com AGS e AGI não acarretam alteração da função mecânica do músculo cardíaco isolado, mas causam discretas lesões estruturais e estresse oxidativo no miocárdio.

OBJECTIVES: To study the influence of saturated (SFA) and unsaturated fatty acid (UFA) rich diets on mechanical function, morphology and oxidative stress in rat myocardium. METHODS: Male, 60-day-old Wistar rats were fed a control (n=8), a SFA (n=8), or a UFA-rich diet (n=8) for sixty days. Mechanical function was studied in isolated left ventricle papillary muscle under isometric and isotonic contractions, in basal conditions (1.25mM calcium chloride) and after 5.2mM calcium chloride and beta-adrenergic stimuli with 1.0µM isoproterenol. Left ventricle fragments were used to study oxidative stress and morphology under light and electron microscopy. RESULTS: SFA and UFA-rich diets did not change myocardium mechanical function. Both diets caused oxidative stress, with high lipid hydroperoxide and low superoxide-dismutase concentrations. UFA rich diet decreased catalase expression and SFA rich diet decreased the amount of myocardial glutathione-peroxidase. Both diets promoted light ultrastructural injuries such as lipid deposits and cell membrane injuries. CONCLUSION: Results suggest that SFA and UFA rich diets do not alter isolated muscle mechanical function, but promote light myocardial morphological injuries and oxidative stress.

Proteomic inventory of myocardial proteins from patients with chronic Chagas' cardiomyopathy

Chronic Chagas' disease cardiomyopathy (CCC) is an often fatal outcome of Trypanosoma cruzi infection, with a poorer prognosis than other cardiomyopathies. CCC is refractory to heart failure treatments, and is the major indication of heart transplantation in Latin America. A diffuse myocarditis, plus intense myocardial hypertrophy, damage and fibrosis, in the presence of very few T. cruzi forms, are the histopathological hallmarks of CCC. To gain a better understanding of the pathophysiology of CCC, we analyzed the protein profile in the affected CCC myocardium. Homogenates from left ventricular myocardial samples of end-stage CCC hearts explanted during heart transplantation were subjected to two-dimensional electrophoresis with Coomassie blue staining; protein identification was performed by MALDI-ToF mass spectrometry and peptide mass fingerprinting. The identification of selected proteins was confirmed by immunoblotting. We demonstrated that 246 proteins matched in gels from two CCC patients. They corresponded to 112 distinct proteins. Along with structural/contractile and metabolism proteins, we also identified proteins involved in apoptosis (caspase 8, caspase 2), immune system (T cell receptor ß chain, granzyme A, HLA class I) and stress processes (heat shock proteins, superoxide dismutases, and other oxidative stress proteins). Proteins involved in cell signaling and transcriptional factors were also identified. The identification of caspases and oxidative stress proteins suggests the occurrence of active apoptosis and significant oxidative stress in CCC myocardium. These results generated an inventory of myocardial proteins in CCC that should contribute to the generation of hypothesis-driven experiments designed on the basis of the classes of proteins identified here.