Passive mechanical properties and related proteins change with botulinum neurotoxin A injection of normal skeletal muscle

Abstract

Summary The effects of botulinum neurotoxin A on the passive mechanical properties of skeletal muscle have not been investigated, but may have significant impact in the treatment of neuromuscular disorders including spasticity. Single fiber and fiber bundle passive mechanical testing was performed on rat muscles treated with botulinum neurotoxin A. Myosin heavy chain and titin composition of single fibers was determined by gel electrophoresis. Muscle collagen content was determined using a hydroxyproline assay. Neurotoxin-treated single fiber passive elastic modulus was reduced compared to control fibers (53.00 kPa versus 63.43 kPa). Fiber stiffness and slack sarcomere length were also reduced compared to control fibers and myosin heavy chain composition shifted from faster to slower isoforms. Average titin molecular weight increased 1.77% after treatment. Fiber bundle passive elastic modulus increased following treatment (168.83 kPa versus 75.14 kPa). Bundle stiffness also increased while collagen content per mass of muscle tissue increased 38%. Injection of botulinum neurotoxin A produces an effect on the passive mechanical properties of normal muscle that is opposite to the changes observed in spastic muscles.

Effects of botulinum toxin type A on non‐injected bi‐articular muscle include a narrower length range of force exertion and increased passive force

“…Increased passive forces suggest more specifically that stiffness of the ECM may have increased. Such elevated ECM stiffness was reported recently for isolated rat muscle fiber bundles after BTX‐A injection . The increased intramuscular connective tissue content shown in the BTX group may explain this finding.…”

“…On the other hand, our findings were obtained only 5 days after BTX‐A injection. Even so, similar effects on passive mechanical properties, as described by Thacker et al ., were obtained 1 month after injection, suggesting they remain over time. These investigators also indicated that the effects of BTX‐A on normal and spastic muscle may differ.…”

Skeletal muscle fibrosis and stiffness increase after rotator cuff tendon injury and neuromuscular compromise in a rat model

“…25 Homogenized protein solution was resuspended to 0.125 μg μl −1 protein (BCA protein assay, Pierce, Rockford, IL, USA) in a sample buffer consisting of 100 mmol l −1 DTT, 2% SDS, 80 mmol l −1 Tris-base pH 6.8, 10% glycerol and 0.01% w/v Bromophenol Blue. Samples were boiled (2 min) and stored at 80°C.…”

Assessing the immediate impact of botulinum toxin injection on impedance of spastic muscle

“…Subsequent to the intramuscular BoNT-A injection, alterations in the physiological and mechanical properties of muscle have been reported in various histological, mechanical, and medical imaging studies [7–11]. These modifications included decreases of muscle mass or muscle fiber cross-sectional area, a selective loss of fast-twitch muscle fibers, and changes in muscle fiber composition, sarcomere structure, and contractile proteins [10, 12, 13]. In electrophysiological studies following a BoNT-A injection, indications of progressive muscle denervation and reinnervation in the treated spastic muscles were shown through changes in compound muscle action potentials and increased muscle fiber jitter and density [3, 14].…”