TFAM-deficient mouse skin fibroblasts - an ex vivo model of mitochondrial dysfunction

Mitochondrial dysfunction associates with several pathological processes and contributes to chronic inflammatory and ageing-related diseases. Mitochondrial transcription factor A (TFAM) plays a critical role in maintaining mtDNA integrity and function. Taking advantage of Tfam(fl/fl) UBC-Cre/ERT2+/+ mice to investigate mitochondrial dysfunction in the stromal cell component, we describe an inducible in vitro model of mitochondrial dysfunction by stable depletion of TFAM in primary mouse skin fibroblasts (SK-FBs) after 4-hydroxytamoxifen (4-OHT) administration. Tfam gene deletion caused a sustained reduction in Tfam and mtDNA-encoded mRNA in Cre(+) SK-FBs cultured for low (LP) and high (HP) passages that translated into a loss of TFAM protein. TFAM depletion led to a substantial reduction in mitochondrial respiratory chain complexes that was exacerbated in HP SK-FB cultures. The assembly patter n showed that the respiratory complexes fail to reach the respirasome in 4-OHT-treated Cre(+) SK-FBs. Functionally, mito-stress and glycolysis-stress tests showed that mitochondrial dysfunction developed after long-term 4-OHT treatment in HP Cre(+) SK-FBs and was compensated by an increase in the glycolytic capacity. Finally, expression analysis revealed that 4-OHT-treated HP Cre(+) SK-FBs showed a senescent and pro-inflammatory phenotype.

Automated summary

Mitochondrial dysfunction was induced in primary mouse skin fibroblasts through stable depletion of TFAM, causing a reduction in mitochondrial respiratory chain complexes and assembly pattern alterations. Functionally, mitochondrial dysfunction developed in high passage Cre(+) SK-FBs after long-term 4-OHT treatment, compensated by an increase in glycolytic capacity. Analysis showed a senescent and pro-inflammatory phenotype in these cells.