Acetyl-CoA-driven respiration in frozen muscle contributes to the diagnosis of mitochondrial disease

Autor(es)

Zuccolotto-dos-Reis, Felippe Henrique ^[1] ; Andriao Escarso, Silvia Helena ^[1] ; Araujo, Jackeline Souza ^[2] ; Espreafico, Enilza Maria ^[2] ; Alberici, Luciane Carla ^[3] ; da Rosa Sobreira, Claudia Ferreira ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Neurosci, Div Neurol, Sao Paulo - Brazil

^[2] Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Cell & Mol Biol, Sao Paulo - Brazil

^[3] Univ Sao Paulo, Sch Pharmaceut Sci Ribeirao Preto, Dept BioMol Sci, Ribeirao Preto - Brazil

• Tipo de documento: Artigo Científico
• Fonte: EUROPEAN JOURNAL OF CLINICAL INVESTIGATION; v. 51, n. 9 SEP 2021.
• Citações Web of Science: 0

Resumo

Background Freezing human biopsies is common in clinical practice for storage. However, this technique disrupts mitochondrial membranes, hampering further analyses of respiratory function. To contribute to laboratorial diagnosis of mitochondrial diseases, this study sought to develop a respirometry approach using O2k (Oroboros Ins.) to measure the whole electron transport chain (ETC) activity in homogenates of frozen skeletal muscle biopsies. Patients and Methods We enrolled 16 patients submitted to muscle biopsy in the process of routine diagnostic investigation: four with mitochondrial disease and severe mitochondrial dysfunction; seven with exercise intolerance and multiple deletions of mitochondrial DNA, presenting mild to moderate mitochondrial dysfunction; five without mitochondrial disease, as controls. Whole homogenates of muscle fragments were prepared using grinder-type equipment. O-2 consumption rates were normalized using citrate synthase activity. Results Transmission electron microscopy confirmed mitochondrial membrane discontinuation, indicating increased permeability of mitochondrial membranes in homogenates from frozen biopsies. O-2 consumption rates in the presence of acetyl-CoA lead to maximum respiratory rates sensitive to rotenone, malonate and antimycin. This protocol of acetyl-CoA-driven respiration (ACoAR), applied in whole homogenates of frozen muscle, was sensitive enough to identify ETC abnormality, even in patients with mild to moderate mitochondrial dysfunction. We demonstrated adequate repeatability of ACoAR and found significant correlation between O-2 consumption rates and enzyme activity assays of individual ETC complexes. Conclusions We present preliminary data on a simple, low cost and reliable procedure to measure respiratory function in whole homogenates of frozen skeletal muscle biopsies, contributing to diagnosis of mitochondrial diseases in humans. (AU)

• Processo FAPESP: 17/04372-0 - DNA mitocondrial: mecanismos de manutenção de sua estabilidade e impacto em doenças
• Beneficiário: Nadja Cristhina de Souza Pinto
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 16/23509-4 - Identificação de miocinas neuroprotetoras liberadas pelo músculo esquelético humano em contração de baixa e alta intensidades: papel da bioenergética mitocondrial e do estresse oxidativo
• Beneficiário: Luciane Carla Alberici
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 16/10862-8 - Estudo do papel da miosina-Va nos processos da dinâmica mitocondrial e mitofagia
• Beneficiário: Jackeline Souza Araújo
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto