[Expression of a defect in the respiratory chain in cultured human cells]. / Espressione dei deficit della catena respiratoria nelle colture cellulari umane.

Large scale deletions of mitochondrial DNA (mtDNA) or altered inter-genomic regulation in skeletal muscle have been demonstrated in patients with mitochondrial encephalomyopathies due to Cytochrome C oxidase (COx) deficiency. We have analyzed by Southern blotting and Polymerase Chain Reaction (PCR) the mtDNA in primary muscle cultures (myoblast-myotube stages and at clonal densities) and in fibrogenic subclones obtained from 9 patients with partial COx deficiency who had in their muscle biopsy a subpopulation of mtDNA showing deletions of variable size (between 2.1 and 6.5 Kb). Only in the cultures from one patient, southern analysis revealed in myoblasts and myotubes a mtDNA almost identical to that found in the original muscle biopsy and persistence of deletion in muscle cells grown at clonal densities. The deletion was detectable in fibrogenic lineage only by PCR amplification. The deleted mtDNA molecules were detectable in myogenic or fibrogenic cultures from other patients only by PCR amplification. The different amounts of deleted mtDNA in the various tissues could be due either to an unequal distribution of the altered mtDNA during embryogenesis with amplification of deleted molecules in myogenic lineage or could result from negative selection against the altered mtDNA in rapidly proliferating cells, such as fibroblasts.

CK-MM PGAM-MM G6PD and am biochemical markers of functional innervated cultured human muscle fibers.

RESULTS AND CONCLUSIONS: The biochemical analyses of the total enzymatic activities of CK. PGAM in innervated cultures with advanced morphological maturation showed a progressive increase up to 60 days after innervation, fetal isozyme patterns of CK-BB and PGAM-BB at early stage of innervation and almost complete transition of CK and PGAM from BB to MM isozymes in more advanced stage of innervation (Figs 1, 2). Time course of G6PD activity in parallel cultures showed a higher activity in early stages of myogenesis(myoblastmyotube: 121.4+/-10 nM/min/mg prot) and in early phase of innervation (30 days after innervation: 109.66+/-26.10 nM/min/mg prot) with a decline of activity in advanced stage of innervation (60 days after innervation: 82.33+/-36.55 nM/min/mg prot) A progressive increase of AM activity in mid (30 days after innervation: 1623.66+/-10.96 pM/min/mg prot) and late stage of innervation (45 days after innervation: 2150.33+/-568.27 pM/min/mg prot) in comparison with aneural (536+/-107.39 pM/min/mg prot) was also found our study suggests these enzymes as biochemical markers of functional innervation of cultured human muscle fibers.