Metabolic and Phenotypic Characterization of Human Skin Fibroblasts After Forcing Oxidative Capacity

Abstract

Human skin fibroblasts present technical advantages for the study of mitochondrial-induced toxicity, because those cells can be isolated from patients by lowly invasive methods and present specific cumulative cellular damage and mutations of particular conditions. Several drugs lead to organ toxicity, with some of these drugs having been already withdrawn from the market. Frequently, drug-induced toxicity is attributed to mitochondrial liabilities. One of the approaches to identify drug-induced mitochondrial toxicity is using glucose-free/galactose/glutamine/pyruvate-containing cell culture media that force cells to be more dependent on oxidative phosphorylation for energy production. However, the effects of this modified culture medium itself on the mitochondrial phenotype of human skin fibroblasts have not been explored in detail. Our objective was to assess the mitochondrial biology of human skin fibroblasts under standard or modified culture conditions so that system can be validated and used in a more reliable way to disclose mitochondrial liabilities of drug candidates or intrinsic metabolic differences in fibroblasts. Our results showed that forcing mitochondrial remodeling in human skin fibroblasts increased oxygen consumption rate, ATP levels, and mitochondria-related transcripts and proteins. Moreover, the metabolic remodeling increased cytotoxicity of mitochondrial poisons. In general, no alterations in gene expression related with differentiation status were observed in human skin fibroblasts, with exception of increased paxilin gene expression. Not only the current work highlights the importance of using human skin primary cells to study drug-induced mitochondrial toxicity, it also reinforces the use of this tool to detect specific mitochondrial defects in skin fibroblasts from patients.

Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants

“…To make cell lines more dependent on mitochondrial metabolism, especially the pharmaceutical industry has developed the approach of reducing glucose availability in test systems like HepG2 hepatoma cells (Blomme and Will 2016;Kamalian et al 2015;Marroquin et al 2007;Will and Dykens 2014), mouse embryonal and primary human fibroblasts (Pereira et al 2012(Pereira et al , 2018, leukemia K562 cells (Swiss et al 2013), or skeletal muscle cells (Dott et al 2014).…”

Mitochondrial and metabolic remodeling in human skin fibroblasts in response to glucose availability

“…Here, we aimed to optimize a protocol for mitochondrial health studies in human skin fibroblasts that is compatible with their short lifespan, since previous studies used adaptation protocols requiring most of the replicative lifespan of the cells 12 , shortening the window of opportunity for experiments. Thus, we wanted to reduce as much as possible the period of cellular adaptation to media of different composition, although not compromising the detection of mitochondrial toxicity was promoted.…”

“…Previous studies described increased gene expression of several mtDNA-encoded OXPHOS subunits after adaptation to OXPHOSm 12 and also in protein levels of some subunits 12,33 . Our results show that adaptation to OXPHOSm promoted significant increases in the expression of the mitochondrial-encoded CYB (complex III), COX3 (complex IV) and ATP6 (complex V) genes (Figure 4), as well as in nuclear DNA-encoded NDUFA9 (complex I) and ATP5G1 (complex V) genes (Figure 4), when compared to the expression of these genes in HGm-grown cells.…”

“…Commercial primary human fibroblast cell lines can grow in OXPHOSm 12 . However, primary skin fibroblasts have a short lifespan in culture and protocols that involve adaptation to media containing galactose are usually long, taking most of these cells' available lifespan 12 .…”

“…Hence, this approach is useful for diagnosing, characterizing, and developing treatments for mitochondrial deficiencies and toxicities 10, 11 . Commercial primary human fibroblast cell lines can grow in OXPHOSm 12 . However, primary skin fibroblasts have a short life-span in culture and protocols that involve adaptation to media containing galactose are usually long, taking most of these cells’ available lifespan 12 .…”