Human TFAM peptide

Cold exposure during cycling and recovery enhances PGC-la transcription, but aspects of mitophagy and a more intense cold exposure without recovery occurring in the cold have not been explored. Purpose: Determine the expression of genes related to mitochondrial biogenesis and mitophagy following an acute cycling bout at a temperature below freezing compared to that of room temperature. Methods: Eleven male participants cycled at 65% W,.. for 1 hat 2 C and 20 C and then recovered at room temperature for 6 h. A muscle biopsy was taken from the vastus lateralis before exercise, 3 h, and 6 h post-exercise for gene expression analysis. Results: Exercising heart rate and skin temperature were lower in the cold (p < 0.001; p = 0.004), while core temperature was higher (p = 0.016). Temperature had no effect on gene expression (p > 0.05). BNIP3 and BNIP3L mRNA were not influenced by exercise (p = 0.329; p 0.233). PGC-la and VEGF were higher after cycling (p < 0.001), but the extent of PGC-la upregulation was reduced 6 h post-exercise (p 0.006). TFAM increased 6 h post-exercise (p = 0.001). NRF2, ERRa, PINK1, and PARK2 decreased 3h post-exercise (p 0.035; p = 0.005; p = 0.002; p = 0.001), but this downregulation was diminished after 6 h of recovery (p = 0.017; p 0.006; p = 0.043; p = 0.047). NRF1 was marginally attenuated with exercise (p = 0.001). Conclusions: Exercise induced alterations in gene expression for mitochondrial biogenesis and mitophagy, but these effects were independent of temperature.

The mitochondrial transcription factor A (TFAM) is a mitochondrial DNA (mtDNA) binding protein essential for the initiation of transcription and genome maintenance. Recently it was demonstrated that the primary role of TFAM is to maintain the integrity of mtDNA and that it is a key regulator of mtDNA copy number. It was also shown that TFAM plays a central role in the mtDNA stress-mediated inflammatory response. In our study, we proposed to evaluate the possibility of editing the TFAM gene by CRISPR/Cas9 technology in bovine fibroblasts, as TFAM regulates the replication specificity of mtDNA. We further attempted to maintain these cells in culture post edition in a medium supplemented with uridine and pyruvate to mimic Rho zero cells that are capable of surviving without mtDNA, because it is known that the TFAM gene is lethal in knockout mice and chicken. Moreover, we evaluated the effects of TFAM modification on mtDNA copy number. The CRISPR gRNA was designed to target exon 1 of the bovine TFAM gene and subsequently cloned. Fibroblasts were transfected with Cas9 and control plasmids. After 24 h of transfection, cells were analyzed by flow cytometry to evaluate the efficiency of transfection. The site directed-mutation frequency was assessed by T7 endonuclease assay, and cell clones were analyzed for mtDNA copy number by Sanger DNA sequencing. We achieved transfection efficiency of 51.3%. We selected 23 successfully transformed clones for further analysis, and seven of these exhibited directed mutations at the CRISPR/Cas9 targeted site. Moreover, we also found a decrease in mtDNA copy number in the gene edited clones compared to that in the controls. These TFAM gene mutant cells were viable in culture when supplemented with uridine and pyruvate. We conclude that this CRISPR/Cas9 design was efficient, resulting in seven heterozygous mutant clones and opening up the possibility to use these mutant cell lines as a model system to elucidate the role of TFAM in the maintenance of mtDNA integrity.