Oxidative stress and metabolic reprogramming: study of pharmacological targets in cellular models of mithocondrial disease.

Oxidative stress is a well known factor involved in cancer, neurodegeneration, cardiovascular diseases and aging. ROS and radical oxygen species are among the principal molecules causing cell damage, at the same time ROS generation is a physiological event, strictly controlled by defence mechanisms, also possibly by cell remodelling. Understanding if a metabolic reprogramming following oxidative stress might be phenotyped would be of relevance to target new drugs to prevent and/or to slow the progression of these different pathologies. Mutations in human mitochondrial DNA (mtDNA) cause a large variety of multi-system disorders, whose main unifying feature is the altered energy homeostasis also reducing cell viability. The aim of this project is to define the response to oxidative stress in cell model of a mitochondrial disease, such as mitochondrial isolated cardiomyopathy (MIC). With this purpose as the first step of our project was to collected cells deriving from umbilical veins or cutaneous biopsy or muscular cells from healthy donors or patients with mitochondrial cardiomiopathy (MIC). Fibroblasts and miofibroblasts cell lines were obtained from 4 MIC affected patients with two different mtDNA mutations, as well as well fibroblasts from 7 controls. These cell lines were used to generate cytoplasmatic cybrids by fusion of enucleated fibroblast with osteosarcoma-derived (143BTK-) cell lines deprived of mitochondria, according to King and Attardi (1). This cell model gives the opportunity to investigate the influence of mitDNA polymorphism without the influence of the donor nuclear genome. For all cell line used in this study the complete mtDNA sequence, the homoplasmy of the specific mtDNA mutation and the haplotype were determined. Cells with oxphos defects show difficulties to grow and undergo apoptotic cell death in glucose-free galactose medium. We measured the response of cybrids, viability and ROS generation, in wild type and MIC cybrids after exposure to glucose-free-galactose medium. Results indicated a marked decrease in cell number of mutant cybrids incubated in galactose medium and an increase in ROS levels as compared to controls. Our findings suggest cell models of mitochondrial diseases and tools of molecular biology and metabolic flux analysis of interest for pharmacological studies aimed to possibly manipulate the redox balance/unbalance signal as related to cell phenotype. King and Attardi. (1996) Methods Enzymol. 264:304-13