42]Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines

Abstract

Publisher Summary This chapter discusses the methods of assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines. Investigation of oxidative phosphorylation (OX-PHOS) in mitochondrial diseases has traditionally focused on the muscle biopsy. Skeletal muscle biopsy remains a valuable resource for biochemical studies as it is an easily accessed tissue and milligram quantities of mitochondria can be isolated for a wide range of OX-PHOS investigations. However, in mitochondrial diseases, the postmitotic muscle fibers commonly show secondary OX-PHOS defects that may hinder investigations of primary defects. Transformed cell lines expressing OX-PHOS defects provide a powerful model system for further genetic and biochemical characterization of nuclear and mitochondrial DNA (mtDNA) mutants and the design and testing of therapeutic approaches. The approach to OX-PHOS investigation combines the identification of patients with OX-PHOS defects using more sensitive muscle biopsy studies followed by further biochemical characterization of primary defects in Epstein–Barr virus-transformed lymphoblasts. The chapter presents novel methods for the production of transmitochondrial cybrids using lymphoblastoid and osteosarcoma ρO cells as recipients. Suspension enucleation of cells combined with electrofusion allows the use of any cell type, including lymphoblasts, as mitochondrial donors in fusions with either lymphoblastoid or osteosarcoma ρo cells.