Original article

U.U. BOTZENHART1, V. VAAL1, I. RENTZSCH2, T. GREDES1, T. GEDRANGE1, C. KUNERT-KEIL1

CHANGES IN CAVEOLIN-1, CAVEOLIN-3 AND VASCULAR ENDOTHELIAL GROWTH FACTOR EXPRESSION AND PROTEIN CONTENT AFTER BOTULINUM TOXIN A INJECTION IN THE RIGHT MASSETER MUSCLE OF DYSTROPHIN DEFICIENT (MDX-) MICE

1Department of Orthodontics, Carl Gustav Carus Campus Technische Universitaet, Dresden, Germany; 2Department of Anaesthesiology and Intensive Care Medicine, Carl Gustav Carus Campus, Technische Universitaet, Dresden, Germany
Progressive muscle wasting, frequently associated with inflammation, muscle fibre degeneration and fibrosis, is a characteristic of DMD (Duchenne muscular dystrophy). Its most common used animal model, the mdx mouse, however can overcome muscle degeneration by regeneration processes and is for this reason not suitable to answer all scientific questions. The aim of this study was to evaluate the ability of botulinum toxin A (BTX-A) in breaking down muscle regeneration in mdx mice. For this purpose, the right masseter muscle of 100 days old mdx and healthy mice was paralyzed by a single specific intramuscular injection of BTX-A. After 21 days, right and left masseter and temporal muscles as well as tongue muscle were carefully dissected, and gene and protein expression of caveolin-1, caveolin-3 and vascular endothelial growth factor (VEGF) were determined using quantitative RT-PCR and Western blot technique. Statistics were performed using Student’s t-test and Mann Whitney U-test (significance level: P ≤ 0.05). After BTX-A injection, in both mice strains and for all three studied genes, no significant differences in mRNA amount could be detected between treated and untreated masseter muscles. A significant increase in caveolin-1, caveolin-3 and VEGF mRNA expression could only be found in the right temporal muscle of control mice compared to the left side. All three investigated proteins were more frequent to be found in dystrophic masseter muscle samples compared to the corresponding control samples, whereas significant decreased caveolin-3 protein levels could only be detected in the treated masseter versus untreated masseter muscle of controls. In contrast to previous conclusions, with this study it was not possible to prove a BTX-A-induced dystrophic phenotype in control animals, in which only the known decreases of caveolin-3 protein expression could be verified due to denervation. At the same time, however, gene and protein expression in dystrophic mice was not changed after BTX-A injection.
Key words:
botulinum toxin A, caveolin-1, caveolin-3, vascular endothelial growth factor, mdx mice, muscular dystrophy, masticatory muscles

INTRODUCTION

Plasticity of the plasma membrane is an essential property of mammalian cells and in part accomplished by specialization into differentiated membrane domains (1), a key feature of eukaryotic cells (2). Among these, the sarcolemma of skeletal muscle represents one of the most known specialized plasma membrane systems (3).

Caveolae, named from the Latin for ‘little caves’ (4), are small flask-shaped invaginations of the plasma membrane (5, 6). They are especially abundant in mechanically stressed cells (1) like adipocytes, myocytes and endothelia cells which are dysfunctional without caveolae (1, 4, 5). But they are also enriched in epithelial cells, fibroblasts, macrophages, and type 1 pneumocytes, and have also been identified ultrastructural in the peripheral nervous system (1, 5, 7).

These plasma membrane invaginations are versatile and highly integrated into cellular physiology (5), serve as membrane organizing centres (8) and sensors (2), and regulate multiple cellular pathways (1), including several mechanotransduction pathways and various transmembrane signalling events (2, 4, 9, 10). The microdomain created by caveolae is ideal for promoting cell signalling through both, localization of many different types of receptors, bioavailability of signalling molecules and the direct actions of caveolin proteins (11).

Caveolae are also important for vascular function and homeostasis. They critically regulate vascular reactivity and blood pressure (5) and can influence angiogenesis due to their content of vascular endothelial growth factor (VEGF) receptors (12, 13). Furthermore, in muscle cells caveolae may play a fundamental role in muscle membrane biology (14), maintenance of muscle integrity, are essential for muscle homeostasis as well as for normal muscle physiology (2, 3, 5). Due to their close proximity to stress fibres (1, 2) and their co-localisation with dystrophin (15, 16), caveolae have been implicated essential in the regulation of muscle cell function and integrity, acting as stress buffering system and protecting the muscle cell membrane from mechanical stress (1-4).

Caveolins are members of a gene family of small integral membrane proteins (14) that form the structural framework of caveolae (7, 17). So far three different main caveolin isoforms, caveolin-1, -2 and -3 (2, 4), have been characterized in mammals (1). Caveolin-1 (cav-1), is the first and best-studied (2) member of that multigene family (14, 18) and is 38% identical and 58% similar to caveolin-2 (cav-2) in humans (18). Caveolin-3 (cav-3) is most closely related to caveolin-1 based on protein sequence homology (14, 15), showing 65% identity and 85% similarity (10, 19). Cav-1 and cav-3 expression is essential and sufficient for the formation of caveolae (2, 20), whereas cav-2 expression is not required (4, 21). Cav-1 is essential for the formation of the great majority of caveolae in non-muscle tissue (20), whereas cav-3 is necessary for the biogenesis of caveolae in cardiac and skeletal muscles (22). It is well known, that cav-3 is transiently associated with T-tubules during skeletal muscle development and that its expression is required for the maturation of a highly organized T-tubule system (16). Cav-3 null mice display mild muscle fibre degeneration and show striking T-tubule system abnormalities (16) with a dilated and swollen T-tubule system running in irregular directions (22) which could lead to dysregulation of muscular calcium homeostasis (16). Increased clustering of nicotinic acetylcholine receptors at the neuromuscular junction have also been reported in cav-3 null mice skeletal muscle, which correlated with abnormal neuromuscular activity (23). Earlier studies have identified increased cav-3 expression levels in hindlimb muscles of patients with Duchenne muscular dystrophy (DMD) and mdx mice, the most common animal model of DMD (24, 25). Recently, increased levels of cav-1 and cav-3 have been reported in the masticatory muscles of 100 days old mdx mice (12). Furthermore, in muscle fibres from dystrophic chickens an increase in the density of caveolae, associated with extensive proliferation of the T-tubular system, has been reported (26, 27).

DMD is an X-linked genetic disease caused by mutations in the dystrophin gene (28-30). Dystrophin is an essential glycoprotein of the multi-subunit dystrophin glycoprotein complex (DGC) (28, 31) which links the actin cytoskeleton of the muscle fibre to the surrounding extracellular matrix (32) and protects its membrane against contraction induced damage (33, 34). Absence of dystrophin leads to disintegration of the DGC, instability of the muscle cell membrane and uncontrolled influx of calcium (29). In addition, changes in the expression of genes involved in calcium homeostasis have also been observed in dystrophic muscles (35). Cascades of molecular events, triggered by the absence of dystrophin, result in muscle degeneration (9), and progressive replacement of the affected muscle tissue with connective and adipose tissue (28). Therefore DMD patients are typically characterized by muscle damage and muscle weakness associated with inflammation and new vessel formation (12).

In order to understand the pathogenesis and also to find therapy concepts to extend lifetime and improve quality of DMD patients life, animal models are useful in determine the processes involve in this disease (29). The most frequently used and well-characterized animal model for Duchenne muscular dystrophy is the mdx (murine X-chromosomal dystrophy) mouse (6, 29, 30). In contrast, mdx is not associated with the severe muscle fibre degeneration and the large proliferation of connective tissue seen in human DMD muscle (25). Mdx mice show only a mild muscular dystrophy phenotype (25) and have a large number of degenerating as well as regenerating muscle fibres in the first 4 months of life. Afterwards the number of degenerative and necrotic fibres declines (36). The degeneration/ regeneration cycle of mdx skeletal muscle fibres usually begins 20 days after birth, reaches its peak intensity at 60 – 90 days of life (37) and at the age of 100 days the regeneration process exceeds degeneration (12). Research in the field of orthodontics (38) as well as to date unpublished date of our own research group in the mdx mouse during maximal dystrophic muscle degeneration confirm that there is a relationship between muscle dysfunction and craniofacial morphology. Unfortunately in the mdx mouse these effects disappear, thus, this mouse model cannot be used to proof these correlations in a sufficient way.

Botulinum toxin A (BTX-A), a neurotoxin produced by Clostridium botulinum (39), inhibits the release of acetylcholine by binding to the presynaptic cholinergic nerve terminals, cleaving SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and shut down the synaptic vesicle cycle (39). Inhibition of acetylcholine neurotransmitter release causes chemical denervation and paralysis of the striated muscle (40, 41). As muscle paralysis is also a consequence of DMD, BTX-A toxin might also be able to trigger dystrophic features in dystrophic muscle tissue.

Since it is known that cav-3 continuously disappeared at the neuromuscular junction after permanent denervation of the extensor digitorium longus muscle (42) as well as cav-1 after axotomy of the rat sciatic nerve (7, 17), and VEGF can be used as an indicator/marker for muscle regeneration in DMD (43), the aim of the study was to investigate changes of cav-1, cav-3 and VEGF expression in healthy and mdx masticatory muscles after a single specific intramuscular BTX-A injection in the right masseter muscle of 100 days old healthy and dystrophic mice. With this method it should be elucidated if this drug can break down muscle regeneration and can be successfully used to prologue dystrophic features in mdx masticatory muscles, thus making this mouse model comparable to the human disease and accessible for research in the craniofacial region.

A recently published study on masticatory muscles of 100 days old mdx mice has indicated changes of MyHC expression after a single specific intramuscular injection in the right masseter muscle of healthy mice, whereas the dystrophic muscles however did not react on BTX-A injection (44). Due to this fact, we hypothesized that 21 days after a single intramuscular injection of BTX-A, muscle regeneration would be induced in healthy mice, while dystrophic muscles were assumed to react in an opposite way, indicating a sustained effect of dystrophy.

MATERIAL AND METHODS

Animals and experimental protocol

Mice of both genders, same age and body mass (100 days old, 30g) of the inbred strain C57BL-10ScSn (control, n = 8) and C57BL/Dmdy (mdx) (test group, n = 6) severed as laboratory animals. Experimental protocol was approved by the Laboratory Animal Research Committee of Saxony with the number: 24-9168.11-1/2013-46. As described recently for chemo-denervation, mice were temporally anaesthetized by an intraperitoneal injection of a mixture of ketamine (10%; Ceva, Tiergesundheit GmbH, Duesseldorf, Germany) and Rompun® (2%; Bayer, HealthCare AG, Leverkusen, Germany). Paralysis of the superficial and deep venter of the right masseter muscle was induced by a single specific intramuscular injection of 0.025 ml BTX-A (Botox®, Allergan®, Irvine, California, USA; 1.25 IU/0.1 ml in physiologic NaCl-solution). Previous studies on masticatory muscles of 100 days old mdx mice using BTX-A in the same way, have indicated a sufficient chemo-denervation and muscle atrophy by using this method (44).

Post-injection care included the verification of paralysis, usually evident 3 days after injection by tooth chattering and refusal of solid food, and the regular control of body mass up to the end of the experiments. During the first 7 post-injection days soft food was offered additionally. According to the international guidelines for animal protection, after 21 days mice were euthanized using an overdose of isoflurane. Immediately, right and left masseter muscle, corresponding to the superficial and (in parts) the deep masseter muscle (45), right and left temporal muscle, corresponding to the medial temporal muscle (45), and the tongue muscle, including the internal tongue muscles, were carefully dissected by the same trained operator, shock frozen in liquid nitrogen (–173°C) and up to further processing stored at –80°C.

RNA-extraction and reverse transcription

The same tissue samples and RNA extracts which had been recently dissected and isolated (44) were used for the following protocol: about 20 – 30g of each tissue sample was homogenized under constant cooling using liquid nitrogen and Trizol (QIAzol Lysis Reagent QIAGEN, Hilden, Germany). Total RNA was isolated using the RNeasy® Lipid Tissue Mini Kit (QIAGEN, Hilden, Germany) following manufacturer´s instructions. Total RNA (400 ng) was transcribed reversely using innuScript Reverse Transcriptase, inNucleotide Mix and Random primer as well as the TOptical cycler (Analytik Jena AG, Jena, Germany).

qRT-PCR (quantitative real-time polymerase chain reaction)

Gene expression and quantification of cav-1, cav-3 and VEGF mRNA amount in the extracted muscle samples was performed as described previously (12, 44) by qRT-PCR using specific TaqMan PCR primers and probes purchased from PE Applied Biosystems® (Weiterstadt, Germany; Taq-Man® Assays: cav-1: Mm00483057_m1, cav-3: Mm01182632_m1, VEGF: Mm01281449_m1), innuMix qPCR Master Mix Probe (Analytik Jena AG), RNase-free water and the TOptical cycler (Analytik Jena AG). Values of gene copies, calculated using standard curves, were given in relation to those of 18S rRNA (Eucaryotic 18S rRNA Endogenous control: 4310893E; PE Applied Biosystems®). Experiments for each sample were performed twice and a ‘non-template control’ with water was carried out for validation of the results.

Western blot

Protein isolation from each muscle sample, electrophoresis and protein transfer to the western blot membrane was performed as described earlier (44). Blots were incubated with specific antibodies directed against cav-1 (clon N20; polyclonal, Santa Cruz Biotechnology, Heidelberg, Germany; dilution 1:1000 in PBS containing 5% bovine serum albumin (BSA)), cav-3 (monoclonal, BD Biosciences, Heidelberg, Germany; dilution 1:400 in PBS containing 5% BSA) or VEGF (ab46154, polyclonal, Abcam, Berlin, Germany; dilution 1:1000 in PBS containing 5% BSA) overnight at 4°C followed by horseradish peroxidase (HRP)-conjugate goat anti-mouse or anti-rabbit immunoglobulins (Dako, Hamburg, Germany) at a dilution of 1:5000.

Visualization of bound primary antibodies was performed using an enhanced chemiluminescence system (WesternBright Chemilumineszenz Substrat Quantum, Advansta Inc., Menlo Park, U.S.A.). Monoclonal anti-glyceraldehyde-phosphate dehydrogenase (GAPDH) antibody (clone 6C5; 1:1000; Millpore, Billerica, Massachusettes, U.S.A.), at an incubation time of 2 hours at room temperature severed as loading control to verify protein content on each gel.

GelScan 5.2 software (Serva, Heidelberg, Germany) was used for quantitative analysis of protein bands of cav-1 (22 kDa), cav-3 (18 kDa), VEGF (43 kDa) and GAPDH (35 kDa). Results are given as MOD (mean optical density ± S.E.M.) in all cases for n = 3 muscle samples (different animals) and two independent experiments.

Statistical analysis

To verify differences in mRNA expression and protein content of cav-1, cav-3 and VEGF in the analysed masticatory muscle samples of 100 days old mdx and control mice, statistical analysis was performed using SigmaStat software version 3.5 (Systat Software Inc., San Jose, California, U.S.A.). Mann Whitney U-test or in case of normal distribution unpaired t-test was used (significance level: P ≤ 0.05).

RESULTS

Quantification of mRNA amounts

For all three studied genes no significant differences in the mRNA amount were detected between treated and untreated masseter muscle 21 days after BTX-A injection. This was demonstrated in both, controls as well as mdx mice. In contrast, a significant increase in cav-1, cav-3 and VEGF mRNA expression was detected in the right temporal muscle of control mice compared to the left side, whereas no differences were found in dystrophic mice. Furthermore, significant differences were detected between healthy and dystrophic mice, e.g. in the right temporal muscle for cav-1 and in the tongue for cav-3 (Table 1).

Table 1. Messenger RNA expression of cav-1, cav-3 and VEGF in BTX-A treated and untreated muscle tissue. Means ± standard deviations for controls (n = 8) and mdx mice (n = 6); Mann Whitney U-test; *P ≤ 0.05 treated versus untreated masseter muscle; #P ≤ 0.05 control versus mdx. Significant values are indicated by bolt lettering.
Table 1

Quantification of protein amounts

Using Western blot analysis specific protein bands were detected for cav-1, cav-3, VEGF and GAPDH, respectively (Fig. 1). After quantitative analysis, for the expression of cav-1 the following significant differences were detected: a 2.8 fold increase in both the right, as well as in the left dystrophic masseter compared to each masseter of the control animals (Fig. 2A). In the case of cav-3, a 2.3 fold decreased level was found in the treated masseter compared to the untreated masseter (mean ± standard error; treated versus untreated control masseter; 0.65 ± 0.14 versus 0.29 ± 0.06; P = 0.041). A similar result was also seen in dystrophic mice, but this was not statistical significant. Furthermore, doubling of cav-3 expression was visible in dystrophic masseter in comparison to control mice (Fig. 2B). For VEGF, increased protein expression was found in mdx mice in the right and left masseter, respectively, whereas in the tongue muscle decreased protein levels were measured. There were also considerable differences between the right and left temporal muscle in mdx mice (mean ± standard error; right temporal versus left temporal; 0.81 ± 0.07 versus 0.47 ± 0.06; P = 0.02; Fig. 2C).

Figure 1 Fig. 1. Detection of cav-1, cav-3 and VEGF in masseter muscle. Representative Western blots of the right (injected) and left (non-injected) masseter muscle of control (C57Bl) and mdx mice. A monoclonal antibody was used to detect GAPDH serving as an internal control.
Figure 2 Fig. 2.Quantitative analysis of cav-1, cav-3 and VEGF Western blots in masseter, temporal and tongue muscle of mdx and control mice 21 days after BTX-A injection. Protein bands attributed to cav-1, cav-3 and VEGF were evaluated using GelScan 5.2 Software (Serva, Germany). Mean optical densities (MOD) ± S.E.M. of control and mdx mice are given in all cases for n = 3 muscle samples (different animals) and two independent experiments (*P ≤ 0.05 control versus mdx; #P ≤ 0.05 right versus left side).

DISCUSSION

In this study for the first time we demonstrated changes in the expression of cav-1, cav-3 and VEGF after injection of BTX-A in the right masseter muscle of mice. Previous studies on masticatory muscles of 100 days old mdx mice have already indicated changes of MyHC expression after a single specific intramuscular injection in the right masseter muscle of healthy mice compared to mdx mice, simulating a dystrophic phenotype in healthy mice, whereas the dystrophic muscles however did not react on BTX-A injection (44).

BTX-A is a potent neurotoxin, produced as a fermentation product by the gram-positive anaerobic spore-forming bacterium Clostridium botulinum (39). An intramuscular injection of BTX-A leads to immediately diffusion of the toxin into the muscle within few centimetres of the needle tip (46) and can selectively be applied for therapeutic purpose to locally induce chemo-denervation. Today therapeutic use of this neurotoxin has been implemented as an alternative treatment modality in many medical, orofacial and dental conditions including cervical dystonia, hyperhidrosis, strabismus and blepharospasm as well as temporomandibular joint disorders, bruxism and pathologic clenching, associated headache, mandibular spasm and facial pain, and it has also been successfully used to treat masseter hypertrophy (47-50). Its indications are rapidly expanding. BTX-A permanently and specifically binds to the cholinergic motor end plate preventing presynaptic release of acetylcholine at the neuromuscular junction causing local paralysis which is followed by atrophy of the affected muscle due to their inactivity (51-53). These effects have been proofed in humans and animals (40, 41, 44, 54).

Carlson et al. could proof a continuous disappearance of cav-3 at the neuromuscular junction (NMJ) after permanent denervation of the extensor digitorum longus muscle of 120 days old rats (42). After denervation of muscle, postsynaptic components of the NMJ begin to undergo dissolution (42), the axon terminals degenerate within one or two days, and by three days the postsynaptic apparatus already shows significant degenerative changes, in particular a reduction in the height and complexity of the postsynaptic folds (55, 56). Concomitant the pattern and intensity of cav-3 staining of the NMJ rapidly becomes less distinct, begins to break up as early as three days, by 10 days little staining remains, and after that time the concentration of cav-3 at the NMJ remains obscure (42). With BTX-A induced chemo-denervation same effects have to be expected. Hence, it is not surprising that in our study cav-3 protein levels decreased after BTX-A injection in the right masseter muscle, whereas a significance could only be observed for the injected masseter muscle of healthy mice compared to the non-injection side.

A reduction of cav-3 at the neuromuscular junction has also been described with age-related changes in that structure (57, 58) underlying considerable remodelling (42). Kawabe et al. noted a downregulation of cav-3 expression in muscle from young to older adults as well as upregulation from neonates to young adults (59), and Carlson et al. could also proof a remarkably less intense staining for cav-3 at the NMJ in neonates compared to adults (42). Concerning the formation of new nerve sprouts during the process of reinnervation, after BTX-induced denervation, at first, less intense cav-3 appearance is therefore to be expected in these newly formed junctions (42). However upregulation of cav-3 mRNA in the right masseter muscle, which had to be expected as a sign of ongoing muscle regeneration and maturing of newly formed neuromuscular junctions could neither be proofed for healthy nor for mdx mice.

Another explanation for the reduced cav-3 expression levels might possibly be the selected investigation period of 21 days, which might have been too short to proof any effect following reinnervation. As the NMJ (neuromuscular junction) is one of the few anatomical structures whose regeneration capacity has been retained through evolution (60, 61), motor axon terminal can be restored by inducing a degeneration/ regeneration process (62). In short term, BTX results in terminal sprouting of the motor nerves (63-65). Considering the rate of muscular activity recovery with time, Morbiato and co-workers have provided a long duration of action for BTX-A serotype (66) reaching up to 6 month activity until muscle reinnervation occurs. The mean duration in most human long-term studies is approximately 3 month (67-69). In rats and mice, although functional recovery is 3 – 4 times faster than in humans (70-72). At the cellular level 3 to 4 weeks after a single injection of BTX-A in mice, there is sprouting of new processes along the nerve axon, with formation of multiple synapses with the muscle and upregulation of the muscle nicotinic receptors. Subsequently the neuronal sprouts undergo regression and the original synaptic connection is restored with restoration of the original neuromuscular junction (73). In rodents, nerve terminal regeneration and functional reinnervation has been reported to be fully restored within 28 – 30 days (62, 74). Another study on different mouse hindlimb muscles indicated a period of 35 ± 2 days for complete recovery verified by DAS (digit abduction scoring) assay (66). As indicated by Morbiato et al. (66), muscle recovery can be measured by different methods all of which leading to different reports of time intervals for full regeneration, making it difficult to catch the optimal time point for research. In contrast to the findings of complete functional reinnervation shown by DAS assays (62, 74), at the same time point demonstrated by electrophysiological recordings, recovery was not complete, on average counting only 50% (62). These observations are in accordance with de Paiva et al. who found that the original terminals in nerves, innervating sternomastoid muscles, were able to generate new sprouts and elicit muscle twitch 28 days after BTX-A injection (75). Twenty-one and 35 days after BTX-A injection only 7% and 11% nerve sprouting could be observed in mice EDL (extensor digitorum longus) muscle by confocal microscopy, respectively (66), so that muscles are weak but no longer paralyzed (62).

It has also been reported that intrinsic differences between skeletal muscle types alter their responsiveness to and recovery from the effects of BTX injection, so that the time course of this nerve sprouting is muscle-dependent (76). From other studies in dystrophic mice, masticatory muscle had been proofed to be different regarding histologic structure (77), MyHC composition (78) and calcium homeostasis (35). In our recent study investigating the fibre type composition 21 days after BTX-A injection in mdx and healthy mice, in the same tissue samples we used here, we found no changes in fibre type composition in the mdx mice triggered by BTX-A injection compared to healthy mice, indicating that the adaptation process after denervation might be different in dystrophic muscles (44).

In our study at protein level in healthy mice significant lower cav-3 values could be found on the injection- compared to the non-injection side, which could presumably be traced back to BTX-A effect. A similar result was also seen in dystrophic mice, but this was not statistical significant. Though in mdx mice a comparable effect could be ascertained, the same dosage of BTX-A in dystrophic masseter muscle did not induced such a great reduction in cav-3 levels as shown in controls. This could be explained by the fact that dystrophic muscles usually express high cav-3 levels (24, 25). Mdx mice, like DMD patients, show upregulation of cav-3 as a consequence of downregulation of dystrophin and dystrophin-associated glycoproteins (16). Recently, increased levels of cav-3 have been reported in the masticatory muscles of 100 days old mdx mice (12) and in our study increased cav-3 levels were also detected in BTX-A treated dystrophic masseter muscle compared to paralyzed masseter muscle of controls. Tight regulation of cav-3 expression appears essential for maintaining normal muscle homeostasis (79). It is also known that exercise induced stress may induce cav-3 expression due to its mechano-protective role in muscles and furthermore exercises, such as fast force (80, 81) or durable training (81, 82) may change muscle performance (81, 83). Pihut et al. evaluated the degree of occlusal forces after BTX-A injection in the masseter muscle of humans suffering from functional disorders due to masseter hyperfunction and could proof a significant and constant reduction of occlusal forces 10 days after drug administration (50). Hence, increased cav-3 levels found in the left masseter muscle of healthy mice compared to its right counterpart might also be explained by exposure to higher functional stress and compensation for the functional loss of the paralysed right masseter muscle.

In vascular smooth muscle cells (5) cav-1 and cav-3 are usually co-expressed (15), whereas in contrast to cav-1, cav-3 is predominantly found in skeletal muscle tissue caveolae (22). Due to the fact that we used the whole muscle tissue and analysed the included vessels too, we cannot differentiate between protein expression in these both tissue types, and raised cav-3 levels found in the left masseter muscles might also be explained by exercise induced increase of vasculature (84) and caveolae. As indicated by our results, cav-1 protein expression in left and right masseter muscles of healthy and mdx mice were not affected by BTX-A effect. In contrast to healthy mice, mdx mice showed a significant increased cav-1 expression in all tested masticatory muscles which is in accordance with former reports (12) and can be explained by cellular effects to overcome dystrophy. In DMD, muscle usually is affected by functional ischemia (33), and increased cav-1 expression may indicate either the active process of adaptation or the already successful regeneration and may not be induced by BTX-A effect. In contrast, by BTX-A, a reduction of cav-1 expression due to denervation was suspected as already been shown by Mikol et al. after axotomy of the rat sciatic nerve (7, 17). Furthermore, VEGF protein level was significantly increased in both, treated and untreated dystrophic masseter muscles and may indicate a high regenerative activity in this tissue. Usually, as shown previously (12), the expression of VEGF mRNA in dystrophic masticatory muscles of 100 days old mdx mice is not altered. In this study, at mRNA level, no significant changes between controls and mdx mice as well as treated and untreated muscles could be detected. Thus, the results obtained here are in agreement with earlier studies. As no data on the protein content of VEGF in untreated dystrophic muscles were determined in the work mentioned above, unfortunately, only speculations can be made. It is well known that exercise can increase muscle VEGF mRNA expression (85, 86) and DMD patients usually have limited physical activity, which can explain the lower level of VEGF in these patients (43). Furthermore, VEGF has a direct neuroprotective potential, preventing neuronal cell death from ischemia and promoting neurogenesis in vitro and in vivo (87, 88). Thus it can also be used as a marker for muscle regeneration after denervation.

Interestingly an increase in VEGF protein expression was found in treated and untreated dystrophic masseter muscles with no statistical differences between the sides, which might be explained on the one hand by regenerative effects after BTX-A injection in the right masseter muscle, and on the other hand by an adaptation to higher functional stress in the left masseter muscle due to inactivity of the right one. Nevertheless, in healthy mice no effects on VEGF expression could be found in treated and untreated masseter muscle, what points to the fact that increased VEGF levels found in mdx muscles were not related to BTX-effect. It is well known that VEGF can be used as marker for muscle regeneration in DMD and give a reflection of the severity of DMD pathology (43). In contrast to dystrophic mice, healthy mice did not show any increase of VEGF expression in masseter and temporal muscle, which might possibility be related to a better ability of healthy muscles to compensate the BTX-A induced functional loss. Maybe in healthy mice dystrophin deficiency did not had to be compensated by upregulation of the investigated genes, e.g. VEGF, due to the fact that in contrast to dystrophic muscles no deficits in vascularisation are present. With increased protein levels of cav-1 and VEGF, improved vascularization in these muscles has to be expected, may be improving the functional ischemia which is known to be present in DMD (33). In earlier studies we demonstrated an increase in the amount of blood vessels in areas with small regenerating muscle fibres and CD45 positive cells (12). Both, the localization pattern of cav-1 and CD45 showed a significant correlation in all tested muscle specimens of both, control and mdx mice (12). In this regard, it is important to note that the treatment with BTX-A resulted in a significant decrease in muscle weight (44) and consequently in a significant decrease of the mean fibre diameter of the right masseter muscle in both, control and mdx mice. The reduction of the fibre diameter was approximately 22% in controls and 35% in mdx mice compared to the untreated masseter muscle (89). Several studies on humans have revealed that BTX-A injection into the masseter muscles resulted in a sustained reduction of masseter hyperactivity (52, 90) concomitant with a reduction in masseter muscle size (maximum reduction 35.4%) (52). The pharmaceutical application to induce muscle atrophy has recently also been demonstrated by the suppression of heat shock proteins (HSPs) which directly caused muscle atrophy (91).

At mRNA level statistical significant changes in cav-1, cav-3 and VEGF expression could be found in the right temporal muscle of healthy mice compared to the left and for cav-1 compared to mdx mice. Increased VEGF mRNA and protein levels could also be found in the right dystrophic temporal muscle compared to the left. We assumed that in control mice the right temporal muscle compensated for the functional loss of the right masseter muscle, so that, due to exercise induced stress, all studied genes were upregulated in these mice, whereas in mdx mice no further upregulation could be found. Compensatory effects of masticatory muscles, in particular of the temporal muscle, could also be detected in the pig after paralysis of the right masseter muscle following BTX-A injection (41).

In summary, typical effects of BTX-A induced chemo-denervation accompanied by reduction of cav-3 expression could only be found in injected masseter muscle of controls, but failed to induce any other changes in these mice. At the same time, however, it was confirmed that BTX-A had no effect on gene and protein expression in dystrophic mice, whereas mRNA in the right temporal muscle of healthy mice were upregulated for all studied genes, indicating a compensatory mechanisms for the functional deficit induced by BTX-A injection.

In dystrophic mice a high regenerative activity was seen in both, the treated and untreated masseter muscle indicated by increased cav-1 and cav-3 protein expression concomitant with increased VEGF protein levels, which are usually not to be found in dystrophic masticatory muscles of this age.

Due to the fact that these effects could not be seen at mRNA level, it would be interesting and relevant to investigate further periods to fully elucidate the long-term effect of BTX-A on the regenerative capacity of dystrophic muscles also in view of reinnervation which usually occurs after a certain time span and might change muscle organization.

Acknowledgements: We like to thank Micaela Krause for excellent technical assistance and the team of the Experimental Centre of the Technische Universitaet Dresden for the excellent support of the experimental animals.

Source of funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interests: None declared.

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R e c e i v e d : February 27, 2017
A c c e p t e d : April 26, 2017
Author’s address: Dr. Ute Ulrike Botzenhart, 74 Fetscherstrasse, Haus 28, D-01307 Dresden, Germany. E-mail: Ute.Botzenhart@uniklinikum-dresden.de