It is known that muscles that lack KATP channel activity generate much greater unstimulated [Ca2+]i and force than normal muscles during fatigue. The increase in unstimulated force in KATP channel deficient muscles is abolished by a partial inhibition of L-type Ca2+ channels, suggesting that it is due to a Ca2+ influx through L-type Ca2+ channels and a subsequent increased myoplasmic Ca2+. However, there is also evidence that the increase in resting force is abolished by NAC, a ROS scavenger. The objective of this study was to reconcile these observations by studying the hypothesis that “the increase in resting [Ca2+]i during fatigue in KATP channel deficient muscles starts with an excess Ca2+ influx through L-type Ca2+ channels, followed by an excess ROS production that causes a further increase in resting [Ca2+]i”. To test the hypothesis, single FDB fibres were fatigued with one tetanic contraction/sec for 180 sec. KATP channel deficient fibres were obtained i) by exposing wild type muscle fibers to glibenclamide, a KATP channel blocker and ii) by using fibres from Kir6.2-/- mice, which are null mice for the Kir6.2 gene that encodes for the protein forming the channel pore. Verapamil, a L-type Ca2+ channel blocker, applied at 1 μM, significantly reduced resting [Ca2+]i during fatigue in glibenclamide-exposed wild type fibres. NAC (1 mM) also reduced resting [Ca2+]i in glibenclamide-exposed muscles. The results suggest that the increase in resting [Ca2+]i during fatigue in KATP channel deficient FDB fibres is due to an influx through L-type Ca2+ channels, and an excess ROS production.

Made available in DSpace on 2014-07-29T15:07:48Z (GMT). No. of bitstreams: 1 dissetacao renata.pdf: 569516 bytes, checksum: 1593b15e04d7cc7d31a23f1ca9feef36 (MD5) Previous issue date: 2008-12-16 / The technology of animal origin products increase considerably, and the meat with its commercial importance is the main focus in the technologic development, searching for less costs production, major regiment and mainly better quality of the final product, however, some aspects are not totally clear, among them it finds the electrical stimulation effect and the freezing temperature influencing in the meat quality. The aim of this research was to evaluate the electrical stimulation effect and the freezing speed, about aspects that influences the meat quality. Twenty Nellore bovines non gelding bred were slaughter, around 24 months, kept in feedlot. After bleeding, half animals of each group were electrically stimulated. The carcass were submitted to one of the freezing treatment, conventional and slowly. During the freezing it were registered the curves of temperature reduction and the carcass pH reduction. After the opening of the cameras, the animals were taken to the room where the bones were taken out and then strip loin (longissimus dorsi muscle) of 2.5 cm of thickness approximately, and strip loin steaks with 1.0 cm close to. After 30 minutes it were evaluated the steak color using the portable colorímetro in the structure CIE L*, a* and b*. The steaks were vacuum packed and kept in the stock camera in 0oC for 24 hours. After this period it was analyzed for cooking loss, shear force and length sarcomere. There was (p<0.05) effect of the cooling and the electrical stimulation method in the sarcomere length measure. The sample slowly freezing without electrical stimulation showed biggest lengths sarcomere than the ones freezing by the conventional method, however, in the conventional freezing with the electrical stimulation, the samples stimulated had biggest length sarcomere than the samples of carcass no electrically stimulated. There was effect (p<0.05) of the freezing method in the shear force. The sample freezing slowly had smaller force of shear force considering the same way of freezing. There was not effect (p>0.05) of the freezing method and the electrical stimulation use in the loss cooking and in the values of luminosity L*, a*, b* in the strip loin samples. / A tecnologia aplicada aos produtos de origem animal avança acentuadamente, e a carne com sua importância comercial, é o principal foco desses avanços tecnológicos, procurando reduzir custos, aumentar rendimentos e principalmente melhor a qualidade do produto final. Entretanto alguns aspectos não estão totalmente esclarecidos, dentre eles encontra-se o efeito da estimulação elétrica e do resfriamento sobre a na qualidade da carne. O objetivo desta pesquisa foi avaliar o efeito da estimulação elétrica e da velocidade de resfriamento, sobre parâmetros qualitativos e quantitativos ( pH, perda por cozimento, força de cisalhamento, comprimento de sarcômero e cor ) de carnes. Foram utilizados 20 bovinos inteiros, com idade média de 24 meses, terminados em confinamento sendo animais da raça Nelore. Após o abate e a sangria, metade das carcaças foram estimulados eletricamente, destas cinco foram submetidas ao tratamento de resfriamento convencional e cinco ao resfriamento lento sendo o mesmo procedimento aplicada as não estimuladas. Durante o resfriamento foram registradas a redução de temperatura e redução do pH das carcaças. Após 48 horas do abate as carcaças foram desossadas e em seguida foram retirados bifes de contrafilé de 2,5 cm de espessura aproximadamente, e bifes com 1,0 cm aproximadamente. Após 30 minutos foi avaliada a cor dos bifes utilizando-se colorímetro portátil no esquema CIE L*, a* e b*. Os bifes foram embalados a vácuo e permaneceram na câmara de estocagem a 0oC por 24 horas. Após este período foram analisadas para com relação a perdas de peso por cozimento, força de cisalhamento e comprimento de sarcômero. As amostras resfriadas lentamente sem o uso da estimulação elétrica apresentaram maiores comprimento de sarcômero (p<0,05) que as amostras resfriadas pelo método convencional sem estimulação elétrica, no entanto no resfriamento convencional com o uso da estimulação elétrica, as amostras estimuladas possuíram maiores comprimentos de sarcômero (p<0,05) que as amostras das carcaças não estimuladas eletricamente. Amostras resfriadas lentamente apresentaram menores forças de cisalhamento (p<0,05) que as amostras resfriadas pelo método convencional. As amostras estimuladas eletricamente apresentaram menores forças de cisalhamento (p<0,05) que as amostras não estimuladas. Não houve efeito (p>0,05) do método de resfriamento e do uso da estimulação elétrica na perda por cozimento e nos parâmetros de cor L*, a*, b* nas amostras de contrafilé.

It is known that muscles that lack KATP channel activity generate much greater unstimulated [Ca2+]i and force than normal muscles during fatigue. The increase in unstimulated force in KATP channel deficient muscles is abolished by a partial inhibition of L-type Ca2+ channels, suggesting that it is due to a Ca2+ influx through L-type Ca2+ channels and a subsequent increased myoplasmic Ca2+. However, there is also evidence that the increase in resting force is abolished by NAC, a ROS scavenger. The objective of this study was to reconcile these observations by studying the hypothesis that “the increase in resting [Ca2+]i during fatigue in KATP channel deficient muscles starts with an excess Ca2+ influx through L-type Ca2+ channels, followed by an excess ROS production that causes a further increase in resting [Ca2+]i”. To test the hypothesis, single FDB fibres were fatigued with one tetanic contraction/sec for 180 sec. KATP channel deficient fibres were obtained i) by exposing wild type muscle fibers to glibenclamide, a KATP channel blocker and ii) by using fibres from Kir6.2-/- mice, which are null mice for the Kir6.2 gene that encodes for the protein forming the channel pore. Verapamil, a L-type Ca2+ channel blocker, applied at 1 μM, significantly reduced resting [Ca2+]i during fatigue in glibenclamide-exposed wild type fibres. NAC (1 mM) also reduced resting [Ca2+]i in glibenclamide-exposed muscles. The results suggest that the increase in resting [Ca2+]i during fatigue in KATP channel deficient FDB fibres is due to an influx through L-type Ca2+ channels, and an excess ROS production.

La Enfermedad Pulmonar Obstructiva Crónica (EPOC), presenta signos y síntomas sistémicos que se han venido explorando desde hace algún tiempo. La presente Tesis Doctoral estudia la disfunción que presentan los músculos respiratorios y periféricos, específicamente el cuádriceps de los pacientes con EPOC. Esta disfunción muscular afecta las actividades de la vida diaria, la tolerancia al ejercicio, limita la calidad de vida y disminuye la expectativa de vida de estos pacientes. Una de las posibles causas de disfunción muscular en los pacientes con EPOC puede ser la pérdida de masa muscular; por eso nos dedicamos a evaluar, los datos antropométricos de los pacientes con EPOC que asistieron durante 2 años a nuestro Laboratorio de Función Respiratoria del Hospital del Mar de Barcelona. La prevalencia de bajo peso en pacientes con EPOC en un hospital con predominante población mediterránea con, índice de masa corporal (IMC) por debajo de 20 Kg/m2, fue de tan sólo 6,6%, cifra que se reducía al 3,1% si el dintel escogido era de 18 Kg/m2. Estas cifras van claramente en oposición a la prevalencia referida en el mundo anglosajón y del norte-centro europeo, que muestra una prevalencia de bajo peso en pacientes con EPOC que oscila entre el 25 y el 35%. Teniendo en cuentas estos datos de baja prevalencia de bajo peso en nuestros pacientes con EPOC, no se explica la alta prevalencia de disfunción muscular en ellos. Por ello evaluamos variables de función muscular en pacientes con EPOC grave, específicamente del músculo cuádriceps y encontramos que los pacientes con EPOC tenían una disminución tanto de la fuerza muscular (43%), como de la resistencia muscular (77%), al compararlos con controles del mismo grupo etáreo.Tratando de profundizar en la causa de la disfunción muscular periférica en los pacientes con EPOC, hicimos a continuación otro estudio donde evaluamos el daño sarcoplásmico y sarcomérico mediante inmunohistoquímica y microscopía electrónica. Este trabajo demostró por primera vez que los pacientes con EPOC presentan mayor daño a nivel del músculo cuádriceps que los controles sanos.Debido a que en la EPOC los músculos periféricos, específicamente los de las extremidades inferiores, pudieran estar afectados por el sedentarismo, decidimos estudiar otras causas de disfunción muscular en un grupo de músculos en que este factor se hallara ausente, como son los músculos respiratorios. Estos mantienen su nivel de actividad normal o incluso aumentada. Para ello tomamos un músculo como el intercostal externo, que es fácilmente accesible a la biopsia, siguiendo un modelo mínimamente invasivo descrito por nuestro grupo. Como en trabajos precedentes ya habíamos valorado el daño sarcomérico, el estrés oxidativo o la actividad enzimática, en esta ocasión el trabajo se centró en la evaluación de la actividad inflamatoria. En él se ha demostrado que las citocinas proinflamatorias TNF-&#945; e IL-6 se encuentran aumentadas en los músculos intercostales externos de los pacientes con EPOC al compararlo con los controles sanos. / Chronic Obstructive Pulmonary Disease (COPD), shows systemic sign and symptoms that have been studied for some time. The present Doctoral Thesis studies the dysfunction shown by the respiratory and peripheral muscles, specifically the quadriceps muscle of patients with COPD. This muscle dysfunction affects the activities of daily living, tolerance to exercise, limits quality of life and diminishes life expectancy of these patients. One of the possible causes of muscle dysfunction in the patients with COPD could be the loss of muscle mass; for that reason we evaluated, the anthropometrics data of the patients with COPD that attended during 2 years our Respiratory Function Laboratory, Hospital del Mar of Barcelona. The prevalence of low weight in patients with COPD in a hospital with a predominantly Mediterranean population with an Body Mass Index (BMI) below 20 Kg/m2, was only 6.6%, a figure that was reduced to 3.1% if the chosen threshold was 18 Kg/m2. These numbers starkly contrast to the prevalence in Northern Europe, with a low weight in patients with COPD raging from 25 to 35%. Considering these data of low prevalence of low weight in our patients with COPD, the high prevalence of muscle dysfunction is not explained. We evaluated variables of muscle function in patients with severe COPD, specifically the quadriceps muscle and we found that the patients with COPD had a decreased muscle strength (43%), and muscle endurance (77%), when comparing with healthy age matched. To study the cause of the peripheral muscle dysfunction in patients with COPD, we began another study where we assessed sarcoplasmic and sarcomeric damage by immunohystochemical methods and electronic microscopy. This work demonstrated for the first time that patients with COPD show greater muscle injury at the quadriceps muscle level that healthy age matched controls.Because in COPD, peripheral muscles, specifically those of the lower limbs, could be affected by sedentarism, we decided to study other causes of muscle dysfunction in a muscle group in which this factor was absent, as they are the respiratory muscles. These maintain their level of normal activity or activity is even increased. For this we chose a respiratory muscle like the external intercostal muscle, that is easily accessible by biopsy, following a minumum invasive model described by our group. As in preceding works of our group we evaluated the sarcomeric damage, oxidative stress or the enzymatic activity, the present work was focused in the inflammatory activity evaluation. We demonstrated that proinflammatory cytokines such as TNF-&#945; and IL-6 are increased in the external intercostal muscles of patients with COPD when comparing with healthy age matched controls.