Downhill running induced DNA damage enhances mitochondrial membrane permeability by facilitating ER-mitochondria signaling

Abstract

To observe whether downhill running can lead to DNA damage in skeletal muscle cells and changes in mitochondrial membrane permeability and to explore whether the DNA damage caused by downhill running can lead to changes in mitochondrial membrane permeability by regulating the components of the endoplasmic reticulum mitochondrial coupling structure (MAM). A total of 48 male adult Sprague-Dawley rats were randomly divided into a control group (C, n = 8) and a motor group (E, n = 40). Rats in Group E were further divided into 0 h (E0), 12 h (E12), 24 h (E24), 48 h (E48) and 72 h (E72) after prescribed exercise, with 8 rats in each group. At each time point, flounder muscle was collected under general anaesthesia. The DNA oxidative damage marker 8-hydroxydeoxyguanosine (8-OHdG) was detected by immunofluorescence. The expression levels of the DNA damage-related protein p53 in the nucleus and the EI24 protein and reep1 protein in whole cells were detected by Western blot. The colocalization coefficients of the endoplasmic reticulum protein EI24 and the mitochondrial protein Vdac2 were determined by immunofluorescence double staining, and the concentration of Ca²⁺ in skeletal muscle mitochondria was detected by a fluorescent probe. Finally, the opening of the mitochondrial membrane permeability transition pore (mPTP) was detected by immunofluorescence. Twelve hours after downhill running, the mitochondrial membrane permeability of the mPTP opened the most (P < 0.05), the content of 8-OHdG in skeletal muscle peaked (P < 0.05), and the levels of the regulatory protein p53, mitochondrial Ca²⁺, and the EI24 and reep1 proteins peaked (P < 0.01). Moreover, the colocalization coefficients of EI24 and Vdac2 and the Mandes coefficients of the two proteins increased first and then recovered 72 h after exercise (P < 0.05). (1) Downhill running can lead to DNA damage in skeletal muscle cells, overload of mitochondrial Ca²⁺ and large opening of membrane permeability transformation pores. (2) The DNA damage caused by downhill running may result in p53 promoting the transcriptional activation of reep1 and EI24, enhancing the interaction between EI24 and Vdac2, and then leading to an increase in Ca²⁺ in skeletal muscle mitochondria and the opening of membrane permeability transition pores.

Keywords: DNA damage; Downhill running; MAM; Mitochondrial membrane permeability transition pore; Skeletal muscle.