# Cell-Penetrating Botulinum Neurotoxin Type A Proteins Alleviate Skeletal Muscle Hypertrophy with Associated Alterations of Mitochondrial Homeostasis

## TL;DR

This study shows that modified botulinum toxin can reduce muscle size and alter mitochondrial function in rat muscles.

## Contribution

The study introduces CPP-fused BoNT/A1 as a novel approach to enhance muscle-reducing effects and mitochondrial changes.

## Key findings

- CPP-rBoNT/A1 induced greater muscle atrophy compared to unmodified rBoNT/A1.
- ZFP-fused rBoNT/A1 caused the highest muscle atrophy and increased type I muscle fibers.
- rBoNT/A1 altered mitochondrial structure and function, affecting biogenesis and mitophagy.

## Abstract

Skeletal muscle is the largest metabolic demanding organ in human body. Alterations of skeletal muscle in shape and size significantly affect its biological functions. Botulinum neurotoxin type A1 (BoNT/A1) has been successfully used in clinics to treat masseter, trapezius and gastrocnemius hypertrophy. Here, we used a healthy rat-based skeletal muscle hypertrophy model to evaluate the muscle-reducing activity of recombinant BoNT/A1 (rBoNT/A1) with genetically fused cell-penetrating peptides (CPPs), which was previously reported to increase the cellular uptake of BoNT/A1. Analyses of treated muscle sections using hematoxylin–eosin and immunofluorescence staining showed that both wild-type rBoNT/A1 without modification (WT-rBoNT/A1) and rBoNT/A1 with CPP fusion (CPP-rBoNT/A1) could induce myocomma atrophy and altered gastrocnemius muscle fiber proportions as a result of denervation and reinnervation. Importantly, rBoNT/A1 with the fusion of a specific CPP, zinc finger protein (ZFP), resulted in the highest degree of muscle atrophy and greatest increase in the ratio of type I muscle fibers over type II fibers. An examination of gastrocnemius muscle cells at the subcellular levels using TEM staining revealed swelled mitochondria and diminished mitochondrial crista upon rBoNT/A1 administration. Transcriptomic RNA sequencing (RNA-Seq) analysis followed by RT-qPCR validation showed that rBoNT/A1 treatment also caused changes in mitochondrial biogenesis and mitophagy. Collectively, our results demonstrated that rBoNT/A1 proteins could alleviate skeletal muscle hypertrophy, with associated alterations of mitochondrial homeostasis.