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

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.

Keywords: botulinum neurotoxin type A; cell-penetrating peptides; hypertrophy; mitochondrial homeostasis; skeletal muscle.

Figure 1

Characterization of rBoNT/A1 proteins in rats. (A) Structural organization of rBoNT/A1 constructs. TAT, a widely used cell-penetrating peptide derived from the transactivator of transcription (TAT) protein of human immunodeficiency virus (HIV) with the sequence of GRKKRRQRRRPQ. ZFP₃, three tandem repeats of engineered Zif268 C₂H₂ zinc finger proteins with cell-penetrating activity. The full sequences of recombinant BoNT/A1 proteins can be found in Table S1. (B) Determination of the IMLD₅₀. The doses below those indicated by red lines cause no death (100% survival), and the corresponding colored lines are overlapped with the black lines. (C) Determination of the IMED₅₀ values of BoNT/A1 proteins using DAS assay. The DAS values at day 2 after rBoNT/A1 injection were used for calculating IMED₅₀. The short lines indicate the mean DAS values of the four replicates at each dose point. In this study, the IMED₅₀ is defined as the amount of proteins, in intramuscular active units (ImU), that lead to 50% of rats (in terms of per kilogram body weight) exhibiting a DAS value of 2 or above. The conversion between ImU and mass (in nanograms) of each sample is shown in parentheses. The ImU of each BoNT/A1 protein is determined in a previous study [38] as the amount of proteins, in the unit of nanograms, that results in 50% death of mice administrated with intramuscularly injected samples. (D) Summary of the IMLD₅₀, IMED₅₀ and safety margin of each BoNT/A1 protein. Safety margin is defined by the ratio of IMLD₅₀ values over IMED₅₀ values. Source data are provided as a Source Data file.

Figure 2

The effects of rBoNT/A1 treatment on rat body weight and gastrocnemius muscle wet weight. (A) Schematic diagram showing experimental procedures. (B–E) Analysis of changes in body weight and gastrocnemius muscle wet weight in mice with injections of Botox (B), WT-rBoNT/A1 (C), TAT-rBoNT/A1 (D) and ZFP-rBoNT/A1 (E) on right hind limbs. For (B–E), the data are from three biological replicates and are presented as mean ± standard deviation (SD). The significant difference is analyzed using two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Figure 3

The effects of rBoNT/A1 on muscle cell morphology and muscle fiber organization. (A) Representative images of HE staining of rBoNT/A1-treated gastrocnemius muscles at 4, 6, 8 and 12 weeks after injection, respectively. There are six rats in each group. The colors are blue for muscle cell nuclei and pink for cytoplasm. Scale bar, 100 μm. (B) Quantification of the area of nuclei per unit area. (C) Quantification of the ratio of muscle cells over intercellular space. For (B,C), the data are from three biological replicates and are shown as mean ± SD. The significant difference between mock and rBoNT/A1-treated groups is determined using two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Figure 4

Evaluation of the chemodenervating effects of rBoNT/A1 treatment on gastrocnemius muscles in rats. (A) Representative images of immunofluorescence staining for type I slow-twitch fibers (green) and type II fast-twitch fibers (red). Scale bar, 200 μm. (B) Quantification of the ratio of type I fibers over type II fibers in rBoNT/A1-treated gastrocnemius muscles at 8 weeks after injection. The data are from three biological replicates and are shown as mean ± SD. The significant difference between mock and rBoNT/A1-treated groups is determined using two-tailed unpaired Student’s t test. Insert, analysis of the significant difference between WT-rBoNT/A1 and TAT-rBoNT/A1 or ZFP-rBoNT/A1 using two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Figure 5

The ultrastructural characteristics of rat gastrocnemius after rBoNT/A1 injection. (A) Arrangement of myofibrils along A-I bands in the mock group. (B) Arrangement of Z-lines in the mock group. (C) Presentation of the two distinct morphologies of mitochondria, including visible, oval and fragmented mitochondria and elongated and fused mitochondria. (D) The broken and damaged myofibrils with dissolved or vanished A-I bands in rBoNT/A1-treated groups. (E) The twisted, dissolved or vanished Z-lines in rBoNT/A1-treated groups. (F) Increased quantity of mitochondria in rBoNT/A1-treated groups. (G) The enlarged and swelled mitochondria in rBoNT/A1-treated groups. (H) The appearance of vacuolation in mitochondria in rBoNT/A1-treated groups. (I) The intensified staining of mitochondrial matrix in rBoNT/A1-treated groups. (J) The disorganized, shortened or vanished cristae of the mitochondria in rBoNT/A1-treated groups. (K) The occurrence of glycogen deposition in rBoNT/A1-treated groups. Scale bars: (A,B,D–F) 5 μm; (C,G–J) 200 nm.

Figure 6

Semi-quantification of rBoNT/A1-induced degeneration of myofibrils, mitochondria and lysosomes. The degree of degeneration is scored from 0 to 5, dictating small to large degeneration (0 = no degeneration; 1 = mild; 2 = moderate; 3 = severe; 4 = extensive; 5 = complete degeneration). There are four rats in each group, and the data are shown as mean ± SD. The significant difference between mock and rBoNT/A1 treatment groups is determined using two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Figure 7

Differential expression analysis of our RNA-seq. (A) Venn diagram of the DEGs in the three-protein treatment. The percentage refers to the union set of all DEGs in the three groups. (B–D) GO analysis of DEGs in the categories of biological processes (BP), cell components (CC) and molecular functions (MF). In the bar graph, GO terms in BP, CC and MF are ranked by the number of enriched genes.

Figure 8

RT-qPCR verification of DEGs related to mitochondrial biogenesis and mitophagy. (A) The expression of mitochondrial biogenesis-related genes Atp5po, Atp5f1c, Cox5 and Cox10. Gapdh is the internal control. (B) The expression of mitophagy-related genes Bax, Atg5, Beclin1, and Map1lc3. The data are presented as mean ± SD (n = 3) from three biological replicates. The significant difference between test and mock is analyzed using two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.