# Tauroursodeoxycholic acid-induced increase in ectopic muscle mineralization occurs exclusively in dystrophic muscles and is independent of endoplasmic reticulum stress

## TL;DR

Tauroursodeoxycholic acid unexpectedly increases muscle calcification in dystrophic muscles, but not in healthy ones, and this effect is unrelated to endoplasmic reticulum stress.

## Contribution

The study reveals that TUDCA induces calcification in dystrophic muscles independently of ER stress, challenging its protective role in muscular dystrophy.

## Key findings

- TUDCA increased calcification in dystrophic muscles but not in dystrophin-positive muscles.
- TUDCA did not reduce ER-stress markers or affect calcification-related proteins in dystrophic muscles.
- The calcification effect of TUDCA is specific to dystrophin-deficient muscles and unrelated to ER stress or calcification proteins.

## Abstract

Calcification of dystrophic skeletal muscles was described previously and attributed, among others, to ER-stress, elevated phosphate concentration and chronic inflammation. Tauroursodeoxycholic acid (TUDCA) is considered an artificial chaperone protecting cells against ER-stress thus could prevent an ectopic mineralisation of soft tissues. Because an enhanced ER-stress is a feature of dystrophic muscles and it promotes soft tissue mineralisation we hypothesised that TUDCA treatment should reduce mineral deposits in dystrophic skeletal muscles, and tested this concept using two mouse models of DMD. Four-week old mdx, mdxβetageo and w/t mice were administered TUDCA in drinking water for 4 weeks. At 8 weeks, following tissue-clearing and calcium minerals staining with alizarin, mineralisation was evaluated using whole body scanning. Additionally, isolated skeletal muscles were analysed by Western blotting for ER-stress and calcification markers, and using various microscopic methods. Enzymatic activity of alkaline phosphatase was also assayed. Unexpectedly, TUDCA enhanced calcification of dystrophic but not dystrophin-positive muscles. TUDCA did not affect the elevated ER-stress markers found in dystrophic muscles nor impact pro-calcifying proteins RUNX2, Osterix and BMP2/4, which were also overexpressed in dystrophic muscles. The alkaline phosphatase levels, which were reduced in dystrophic muscles, were not affected by this treatment. The increase in ectopic calcification in dystrophic muscles induced by TUDCA is specific to muscles lacking dystrophin. This effect is not linked to the alleviation of ER stress or the overexpression of proteins directly involved in calcium mineral accumulation.

The online version contains supplementary material available at 10.1038/s41598-025-21534-0.