# Botulinum toxin-induced masseter muscle atrophy is associated with impaired autophagic flux without signs of apoptosis in mice

## TL;DR

Botulinum toxin causes masseter muscle atrophy in mice by disrupting autophagy, not through cell death.

## Contribution

The study reveals that BoNTA-induced muscle atrophy is linked to impaired autophagy, not apoptosis.

## Key findings

- No signs of apoptosis were observed in BoNTA-injected masseter muscles.
- Autophagy markers like LC3, p62, and BAG3 accumulated in BoNTA-treated muscles.
- LC3 accumulation correlated with reduced masseter mass after BoNTA injection.

## Abstract

Botulinum toxin type A (BoNTA) injection into the masseter muscle is widely used for clinical and esthetic purposes. Masseter muscle atrophy is a secondary effect of transient neuromuscular blockade induced by BoNTA. While muscle atrophy has been linked to enhanced ubiquitin-proteasome system activity, leading to increased protein degradation, the role of other catabolic pathways, such as apoptosis and autophagy, remains understudied. In the present study, we evaluated these cellular processes in a mice model of unilateral injection of BoNTA in the masseter muscle, and its relationship with muscle atrophy. Changes in neither molecular markers of apoptosis (cleaved caspase-3, cleaved PARP) nor DNA fragmentation were observed in BoNTA-injected muscles. Conversely, a significant accumulation of the autophagy markers microtubule-associated proteins 1 A/1B light chain 3B (LC3), sequestosome 1 (SQSTM1/p62), and BCL2-associated athanogene 3 (BAG3), along with a reduction in muscle fiber diameter, was observed at 7 days post-BoNTA. These changes were not affected by autophagic flux blockade with chloroquine. Interestingly, LC3 accumulation positively correlates with masseter mass reduction induced by BoNTA. These findings suggest that BoNTA disrupts skeletal muscle homeostasis, promoting atrophy through impaired autophagic activity. Our results not only shed light on the mechanism of BoNTA-induced muscle atrophy but also has broader implications for understanding and potentially treating a range of muscle wasting disorders.