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Doctoral Thesis
DOI
Document
Author
Full name
Larissa de Oliveira Okama
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2018
Supervisor
Committee
Sobreira, Claudia Ferreira da Rosa (President)
Alberici, Luciane Carla
Nucci, Anamarli
Serafini, Luciano Neder
Sverzut, Ana Claudia Mattiello
Title in Portuguese
Avaliação da função mitocondrial muscular e sua repercussão na capacidade funcional nos pacientes com distrofia muscular de Duchenne
Keywords in Portuguese
Cadeia respiratória
Capacidade funcional
Coenzima Q10
Distrofia muscular de Duchenne
Mitocôndria
Abstract in Portuguese
A distrofia muscular de Duchenne (DMD) é uma doença hereditária, degenerativa e progressiva dos músculos esqueléticos. É causada pela ausência da proteína distrofina e caracterizada pela perda progressiva da força muscular e deterioração da capacidade funcional. Alterações na regulação da homeostase do cálcio, proteólise e alterações metabólicas, especialmente mitocondriais, são parte da patogênese da doença. A coenzima Q (CoQ10), potente antioxidante que participa da atividade da cadeia respiratória, tem sido utilizada em ensaios clínicos, entretanto, não há estudos que evidencie seu comprometimento na DMD. O objetivo deste estudo foi avaliar a CoQ10 e a da atividade da cadeia respiratória em fragmentos de biópsia muscular de pacientes com DMD e sua correlação com parâmetros clínicos e a capacidade funcional. O estudo constitui de uma etapa retrospectiva, onde foram analisadas 22 biópsias musculares de pacientes com DMD, e outra prospectiva, onde foram avaliados dez pacientes com DMD. Dez pacientes controles foram utilizados nas duas etapas do estudo. A concentração da CoQ10 foi realizada através da técnica de cromatografia líquida de alta performance de fase reversa. As atividades das enzimas da cadeia respiratória foram medidas através de técnicas espectrofotométricas. A capacidade funcional foi mensurada através das escalas Medida da Função Motora (MFM) e Escala de Avaliação para deambulantes North Star (NSAA), e dos testes cronometrados: tempo para percorrer 10 metros (T10), tempo para realizar a manobra de Gowers (TGowers) e teste da caminhada dos 6 minutos (TC6min). Fase retrospectiva: a média de idade dos pacientes com DMD foi de 6,9 anos, (DP ±2,4) e controles de 8 anos, (DP ±2,69). A dosagem média de CoQ10 nos fragmentos de pacientes com DMD foi de 8,6 µg/g de tecido (DP ±3,9) e nos fragmentos dos controles foi de 31,6 µg/g de tecido (DP ±6,9). A média da área ocupada por fibras musculares nos pacientes com DMD foi de 27,3% (DP ±14,2%) e nos controles foi de 89,2% (DP ±3,3%). Evidenciou-se alta correlação entre aquantidade de CoQ10 e a área relativa ocupada por fibras musculares (r= 0,767 e p= 0,016). As atividades dos complexos enzimáticos da cadeia respiratória dos pacientes com DMD não demonstraram deficiência. Já o resultado do ensaio conjunto dos complexos II+III, encontra-se significativamente reduzido nos pacientes com DMD. Etapa prospectiva: a média de idade dos pacientes com DMD foi de 6,5 anos, (DP ±2,4). A dosagem média de CoQ10 nos fragmentos de pacientes com DMD foi de 12,6 µg/g de tecido (DP ±5,1). A média da área ocupada por fibras musculares nos pacientes com DMD foi de 40,3% (DP ±20,4%). Houve alta correlação entre a quantidade de CoQ10 e a área relativa ocupada por fibras musculares (r= 0,690 e p= 0,058). A correlação da dosagem da CoQ10 com os instrumentos de avaliação da capacidade funcional foi alta com o TGowers e moderada com MFM total e dimensões 1 e 2, NSAA, T10 e TC6min. Em relação à área relativa de fibras musculares, houve moderada correlação com a dimensão 1 da MFM e com o TGowers. Não houve correlação da CoQ10 e da área relativa ocupada por fibras musculares com os parâmetros clínicos: idade no momento da biópsia, idade do início dos sintomas e tempo de evolução da doença. No presente estudo, concluímos que existe uma deficiência secundária de CoQ10 em pacientes com DMD, a qual contribui para entender a fisiopatologia da doença e com grande relevância para as propostas terapêuticas.
Title in English
Assessment of mitochondrial function in muscle and its relation to functional capacity in patients with Duchenne muscular dystrophy
Keywords in English
Coenzyme Q10
Duchenne muscular dystrophy
Functional capacity
Mitochondria
Respiratory chain
Abstract in English
Duchenne muscular dystrophy (DMD) is a hereditary, degenerative and progressive skeletal muscles disease. It is caused by the absence of the protein dystrophin and characterized by progressive loss of muscle strength and deterioration of functional capacity. Alterations in the regulation of calcium homeostasis, proteolysis and metabolic abnormalities, especially mitochondrial dysfunction, are part of the pathogenesis of the disease. Coenzyme Q (CoQ10), a potent antioxidant that participates in respiratory chain activity, has been used in clinical trials, however, there are no studies showing its involvement in DMD. The purpose of this study was to investigate CoQ10 content and respiratory chain activity in muscle biopsy of patients with DMD and its correlation with clinical parameters and functional capacity. The study consisted of a retrospective phase, in which 22 muscle biopsies from patients with DMD were analyzed, and a prospective phase, where ten patients with DMD were evaluated. The same control group of ten patients were used in the two phases of the study. The concentration of CoQ10 was measured using the reverse phase high performance liquid chromatography technique. Activities of the respiratory chain enzymes were measured by spectrophotometry. The functional capacity was evaluated using the Motor Function Measurement (MFM) and North Star Ambulatory Assessment (NSAA) and the following timed tests: to run 10 meters (T10), to perform the Gowers maneuver (TGowers) and the 6-minute walk test (6MWT). Retrospective phase: the mean age of patients with DMD was 6.9 years (SD ± 2.4) and of controls was 8 years (SD ± 2.69). The mean CoQ10 content in fragments from patients with DMD was 8.6 ?g / g tissue (DP ± 3.9) and in fragments from controls was 31.6 ?g / g tissue (DP ± 6.9). The mean area occupied by muscle fibers in patients with DMD was 27.3% (SD ± 14.2%) and in controls was 89.2% (SD ± 3.3%). There was a high correlation between the amount of CoQ10 and the relative area occupied by muscle fibers (r= 0.767 and p= 0.016). The activities of respiratory chain enzymes from patients with DMD were not deficient. On the other hand, the results of the combined analysis of complexes II + III were significantly reduced in patients with DMD. Prospective phase: the mean age of patients with DMD was 6.5 years (SD ± 2.4). The mean CoQ10 content in fragments from patients with DMD was 12.6 ?g / g tissue (SD ± 5.1). The mean area occupied by muscle fibers in patients with DMD was 40.3% (SD ± 20.4%). There was a high correlation between the amount of CoQ10 and the relative area occupied by muscle fibers (r= 0.690 and p= 0.058). The correlation between the amount of CoQ10 and the functional capacity assessment instruments was high forTGowers and moderate for MFM total and dimensions 1 and 2, NSAA, T10 andd TC6min. Regarding the relative area of muscle fibers, there was a moderate correlation with MFM dimension 1 (standing position and transfers) and TGowers. There was no correlation between CoQ10 and relative area occupied by muscle fibers with clinical parameters: age at time of biopsy, age of onset of symptoms and time of disease progression. In the present study, we conclude that there is a secondary deficiency of CoQ10 in patients with DMD, which contributes for the understanding of its physiopathology and is relevant for therapy.
 
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2019-08-15
 
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