Effect of botulinum toxin A‐induced paralysis and exercise training on mechanosensing and signalling gene expression in juvenile rat gastrocnemius muscle

Abstract

Intramuscular injections of the paralytic botulinum neurotoxin A (Btx) and physical exercise are used in the treatment of chronic spasticity in children with cerebral palsy. We tested whether Btx-induced paralysis and/or exercise training would have differential effects on the expression of mechanosensing and signalling genes implicated in the adaptive remodelling of skeletal muscle. Juvenile (29-day-old) male rats were injected with Btx or saline (NoBtx) into the right gastrocnemius and housed in standard cages (NoEx) or with running wheels (Ex), for 3 weeks (n = 6 per group). The mRNA expression of nine sarcomere-associated genes in the medial gastrocnemius was then determined by quantitative reverse transcriptase-polymerase chain reaction. The Btx-injected muscles weighed 50% less than NoBtx muscles, but Ex had no effect on the wet mass of Btx or NoBtx muscles. Atrogenic MuRF1, sarcomeric Titin and myogenic MyoD were upregulated (2-fold) with the elimination of contractile activity in Btx muscle. Expression of CARP, Ankrd2 and MLP was increased with mechanical stimuli associated with Btx (5-to 10-fold) or Ex (2-to 4-fold). Expression of CARP and Ankrd2 increased synergistically in Btx-Ex muscle (≥20-fold), indicating that these genes may be sensitive to passive stretch of the sarcomeric I-band region of titin to which their proteins bind. Tcap, Myopalladin and Atrogin1 were not, or were no longer responsive to the altered mechanical stimuli after 3 weeks of Btx or Ex. The expression of Ankrd2, CARP and MLP may thus be enhanced by passive stretch within the Btx-paralysed and/or exercising gastrocnemius and contribute to adaptations, other than muscle mass, in juvenile rats.

Botulinum toxin‐induced paralysis leads to slower myosin heavy chain isoform composition and reduced titin content in juvenile rat gastrocnemius muscle

“…A reduction in the relative content of titin has been found in different models that induce atrophy such as hindlimb unloading, 15 hibernation, 38 and surgical denervation, 4 indicating that titin is more susceptible to degradation than the MHCs. However, in a previous study we showed that titin gene expression was upregulated in response to botulinum toxin-induced paralysis, 37 suggesting instead that the concentration of titin protein may increase in this model.…”

“…Thirty male Sprague-Dawley rats (including animals described in Velders et al 37 ) at 4 weeks of age were randomly allocated to five groups: (1) Baseline: sacrificed at the beginning of the study (n ϭ 6); (2) Con: received a saline injection in the right gastrocnemius muscle and housed individually in standard cages (n ϭ 6); (3) Ex: received a saline injection in the right gastrocnemius muscle and housed individually with access to a running wheel (n ϭ 6); (4) Btx: received a botulinum toxin injection (described below) in the right gastrocnemius muscle and housed individually in standard cages (n ϭ 6); and (5) Btx-Ex: received a botulinum toxin injection in the right gastrocnemius muscle and housed individually with access to a running wheel (n ϭ 6). Running distance and velocity were recorded continuously.…”

“…This dose was similar to that previously shown to be effective in inducing functional deficits (contractile properties) after injection in adult rat gastrocnemius muscles 10,11 and that we have applied since in juvenile rats. 37 Paralysis is visually evident in the Btx-A-injected limb the next day. The ankle appears more dorsiflexed when the rat is sitting or moving around the cage, and there is an absence or reduced toe-spread reflex response upon rapid lowering of the rat held by its tail.…”

Ouabain exacerbates botulinum neurotoxin-induced muscle paralysis via progression of muscle atrophy in mice

“…These two genes are up-regulated with a progression of muscle atrophy (Gomes et al, 2001) and the increase in mRNA expression levels of the genes is used as markers of muscle atrophy (Glass, 2005;Velders et al, 2008). We tested mRNA levels of the genes in BoNT/Ainjected gastrocnemius muscles with or without an intraperitoneal injection of ouabain.…”

How Does Botulinum Toxin Injection and Physiotherapy Complement Each Other in Cerebral Palsy?

“…It was reported that in some animals, BoNT/A injections made to nonspastic muscles prevented normal growth of muscle and caused progressive and persistent atrophy of muscle. In another study, it was reported that atrophy caused by BoNT/A injection was not reversed by exercise training [80,81]. There are studies reporting that injections can cause decrease in muscle strength in children with CP; nevertheless, it was reported that there was 4-5% decrease in muscle volume by injections made to gastrocnemius muscle in 15 children with CP, and this was not as dramatic as in animal experiments [73,82].…”