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SKELETAL MUSCLE AND EXERCISE |
Uppsala University, Department of Neuroscience,
1 Clinical Neurophysiology
3 Rehabilitation Medicine, 75185 Uppsala, Sweden
2 University of Münster, Physiology and Biophysics Unit, 48149 Münster, Germany
4 Center for Development and Health Genetics, The Pennsylvania State University, University Park, PA 16802, USA
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Abstract |
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(Received 7 July 2006;
accepted after revision 21 August 2006;
first published online 24 August 2006)
Corresponding authors W. A. Linke: Physiology and Biophysics Unit, University of Münster, Schlossplatz 5, D-48149 Münster, Germany; L. Larsson: Department of Neuroscience, Clinical Neurophysiology, Uppsala University, Ing 85, 3 tr., 751 85 Uppsala, Sweden. Email: wlinke{at}uni-muenster.de or lars.larsson{at}neurofys.uu.se
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Introduction |
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The nature of muscle hypertonia, unrelated to stretch reflex activation, is still poorly understood. Results from the few studies published to date on intrinsic muscle properties in patients with spasticity have reported shorter muscle fibres by some (Tardieu et al. 1982; Fridén & Lieber, 2003), but not by others (Lieber et al. 2003). Passive tension (PT) was found to be increased in single fibres from spastic muscle, indicating intrinsic muscle changes unrelated to reflex activation or extracellular matrix and connective tissue (Fridén & Lieber, 2003). Surprisingly, decreased PT was observed in fibre bundles from patients with spasticity and was attributed to altered extracellular matrix material (Lieber et al. 2003). These apparently contradictory results warrant follow-up studies.
When resting muscles are stretched, the PT is thought to arise from several structures, including the sarcomere protein titin, the intermediate filaments, and the extracellular connective tissue located within and surrounding the muscle belly (Wang et al. 1991, 1993; Horowits, 1992; Granzier & Wang, 1993; Linke et al. 1996; Gajdosik, 2001; Neagoe et al. 2003; Prado et al. 2005). Out of these, the collagen content, cross-linking status and isoform composition, and the titin isoform expression pattern are believed to be the major determinants of PT generation (Granzier & Wang, 1993; Linke et al. 1996; Gajdosik, 2001; Neagoe et al. 2003; Prado et al. 2005). Titin isoform size and stiffness are highly variable among fast-twitch muscles (Prado et al. 2005), whereas slow muscles tend to have long titin isoforms and low titin-based stiffness (Wang et al. 1991; Linke et al. 1996; Granzier & Labeit, 2002; Prado et al. 2005). The relative contributions from titin and collagen to total PT vary greatly among different skeletal muscles (Prado et al. 2005). Mutations in titin can cause several forms of muscular dystrophy (Hackman et al. 2002; Lange et al. 2005; Udd et al. 2005) and secondary titin changes have also been observed in skeletal muscles from patients with Duchenne muscular dystrophy (Tkatchenko et al. 2001).
The purpose of this project was to unravel the mechanisms underlying intrinsic changes in the structure and function of spastic vastus lateralis muscles from patients with spinal cord injury at the protein, myofibril, muscle cell and whole muscle levels, unrelated to stretch reflex activity. To test the hypothesis that there are structural and functional changes in spasticity intrinsic to the muscle cells, we examined PT in relation to adaptations in intracellular contractile and structural proteins (myosin, titin, desmin) and the extracellular matrix. We found increased PT at the whole-muscle and single-fibre levels in spasticity, but not at the myofibrillar level. Spastic muscles contained increased proportions of fast fibres expressing myosin heavy chain (MyHC) type IIx, and only these fibres exhibited substantially elevated PT levels. The differences in PT between spastic and control muscles were unrelated to protein expression changes of titin or desmin, but are explainable by ultrastructural remodelling within both the extracellular matrix and the muscle cells.
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Methods |
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The spasticity in the patients selected for this study was secondary to an upper motor neuron lesion at the cervical or thoracic spinal cord levels. We excluded patients with spasticity secondary to a neurodegenerative lesion, cerebral infarction or haemorrhage to obtain a more homogeneous population. Seven male SCI subjects with complete or partial para- or tetraparesis and quadriceps muscle spasticity (SCI-S) were studied (Table 1) together with seven age- and sex-matched control subjects (CTL) with no history of neuromuscular disease. The mean age for SCI-S was 44 ± 2 years (range, 38–51) and for CTL 44 ± 2 years (range, 37–52) and the SCI-S subjects were evaluated clinically prior to the experiments using the modified 5-point Ashworth scale (Table 1) (Ashworth, 1964; Bohannon & Smith, 1987; Knutsson et al. 1997). The percutaneous conchotome method was used under local anaesthesia to obtain biopsies from the vastus lateralis muscle with the understanding and consent of the subjects. This study was conducted in accordance with the Declaration of Helsinki guidelines and full written informed consent was obtained from all subjects. The study was approved by the local ethical committee at Uppsala University.
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Heart and skeletal (psoas, soleus) muscle tissue was obtained from a Sprague-Dawley rat and a New Zealand White rabbit, respectively. Animals were anaesthetized and killed prior to muscle dissection according to approved guidelines of the institutional Animal Care and Use Committee (University of Münster). The tissue was stored at –80°C until usage for gel electrophoresis. A frozen tissue chunk of human soleus muscle (excised post mortem with institutional approval) was a kind gift of Professor R. Bittner (University of Vienna).
In vivo muscle function
Dynamic dynamometry was used to measure forces opposing passive movements of the knee and electromyography (EMG) was used to monitor activation patterns in stretched and shortened muscles. Passive isokinetic knee extensions and flexions were performed under the control of a dynamic dynamometer (Kin-Com H500, Chattanooga Corp., Chattanooga, TN, USA) as described previously (Knutsson et al. 1997). Torque and EMG in knee extensions and flexions (from 20 to 120 deg) were recorded at four different movement velocities, 15, 30, 60 and 120 deg s–1. All torque recordings were corrected for gravitation. The average torque was calculated by averaging three matching records (differences < 15%) from each movement type.
Muscle sample preparation
Fresh muscle biopsies were divided into two pieces. One piece was frozen in liquid nitrogen-chilled propane and stored at –80°C, and the other piece was immediately placed in an ice-cold relaxing solution (in mmol l–1: 100 KCl, 20 imidazole, 7 MgCl2, 2 EGTA, 4 ATP, pH 7.0; 4°C). Small bundles of 
25–50 fibres were dissected free from the muscle and tied to a glass microcapillary tube, slightly stretched, and then placed in a skinning solution (relaxing solution containing glycerol; 50 : 50 v/v) at 4°C for 24 h. Subsequently the bundles were stored at –20°C for use within 3 weeks or treated with cryoprotectant (sucrose) solution for long-term storage at –80°C as described previously (Frontera & Larsson, 1997).
Single-fibre mechanics
A single skinned muscle fibre was dissected out from the bundle and transferred to a stainless steel experimental chamber containing pCa 9.0 solution (composition in mmol l–1: 79.2 KCl, 20 imidazole, 7 EGTA, 0.02 CaCl2, 5.42 MgCl2, 14.5 creatine phosphate, 4.74 ATP). The ends of the fibre were securely attached to the arms of a motor (model 300H, Cambridge Technology Inc., Cambridge, MA, USA) and force transducer (model 403, Cambridge Technology) and the fibre was adjusted to an initial sarcomere length (SL) of 2.2 µm at 15°C as described previously (Moss et al. 1983). The chamber assembly was then placed on the stage of an inverted microscope (Olympus IX70, Tokyo, Japan) fitted with a x 40 objective and a camera (Olympus DP11). SLs were assessed from video prints (Mitsubishi P67E, Kyoto, Japan) and were taken regularly during passive force measurements in pCa 9.0 solution and during maximum activation in pCa 4.5 solution (composition in mmol l–1: 64 KCl, 20 imidazole, 7 EGTA, 7.01 CaCl2, 5.26 MgCl2, 14.5 creatine phosphate, 4.81 ATP). Before each maximum activation, the muscle fibre was exposed to a pre-activating solution containing (in mmol l–1): 79.2 KCl, 20 imidazole, 0.07 EGTA, 5.42 MgCl2, 14.5 creatine phosphate, and 4.74 ATP. The ionic strength of all solutions was adjusted to 180 mmol l–1 using potassium hydroxide, pH 7.0 at 15°C.
For each stretch experiment, SL started at 2.2 µm with 0.2 µm SL increments until 3.8 µm. After each incremental step, stress relaxation (force decay at a constant length) was permitted for 3 min before measuring resting (passive) force in pCa 9.0, and then the solution was switched to pCa 4.5 for measurement of maximum active force. Changes in force and motor position were sampled (16-bit resolution, DAP5216a, Microstar Laboratories) at 2.0 kHz using custom-made software developed in our laboratory and data were saved to computer files for later analysis. Cross-sectional fibre area (CSA) was calculated from the fibre width at each sarcomere length, assuming a circular circumference, and maximum active tension was calculated as maximum tension developed during pCa 4.5, normalized to fibre CSA. Once mechanical measurements were finished, fibres were cut free at the points of attachment, placed in denaturing sample buffer, and stored at –80°C until subsequent analysis of MyHC isoforms.
Single myofibril mechanics
Skeletal myofibrils from SCI-S and CTL vastus lateralis muscle samples were prepared as previously described (Minajeva et al. 2001, 2002). Briefly, muscle tissue was thawed and skinned in ice-cold low ionic strength buffer (in mmol l–1; 75 KCl, 10 Tris, 2 MgCl2, 2 EGTA, and 40 µg ml–1 protease inhibitor leupeptin, pH 7.1) supplemented with 0.5% Triton X-100 for a minimum of 5 h. The skinned tissue was minced and homogenized in low ionic strength buffer to separate myofibrils. Under a Zeiss Axiovert 135 microscope (Carl Zeiss, Oberkochen, Germany), a myofibril was suspended between two glass needles attached to a piezomotor (Physik-Instrumente, Karlsruhe, Germany) and a homebuilt force transducer, respectively (Minajeva et al. 2002). Data collection, motor control, and SL measurements were obtained with a PC, DAQ board, and custom-written LabView software (National Instruments, Austin, TX, USA). Force measurements were carried out at room temperature in relaxing buffer (in mmol l–1; 8 ATP, 20 imidazole, 4 EGTA, 12 magnesium propionate, 97 potassium propionate, pH 7.0) supplemented with 40 µg ml–1 leupeptin. A suppressor of active force, 2,3-butanedione monoxime (BDM) (20 mmol l–1), was also added to the relaxing buffer, although complete relaxation is observed in the absence of this drug as well (Linke et al. 1994; Mutungi & Ranatunga, 1996). The protocol consisted of stretching a myofibril stepwise from slack length by 0.2 µm sarcomere–1 step–1, and each step was completed in 1 s. Following each step the myofibril was held at a constant length for 20 s to wait for stress relaxation. Finally, the myofibril was released back to slack length. For analysis we considered the force at the end of the hold period, which represents titin-based force (Minajeva et al. 2001). Force data were related to the cross-sectional area inferred from the diameter of the specimens.
Histological analysis and electron microscopy
Frozen muscle tissue was thawed in ice-cold phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in PBS, dehydrated in an alcohol series, and embedded in Epon blocks. Semi-thin cross-sections were cut using a Reichert ultra-microtome and stained with Toluidine Blue. Images were recorded with a colour-CCD camera (Sony) under a Zeiss Axiovert 135 inverted microscope using x 5 or x 10 objectives. Fibre CSA was determined from digital images using PaintshopPro 4.12 shareware (JASC, Inc., Eden Prairie, MN, USA) by measuring the major and minor diameters of each fibre on the cross-sections and calculating the mean values for each muscle.
Contrasted thin sections were prepared for transmission electron microscopy (EM) and viewed with a Zeiss EM 900 at 80 kV (Prado et al. 2005). More than 200 EM images were recorded from six different SCI-S muscles and five different CTL muscles. Mitochondrial density and myofibrillar density were calculated on scanned micrographs using ImageJ software (NIH, Bethesda, MD, USA) by circling the area occupied by mitochondria/myofibrils within a rectangular region of interest and relating it to the total measured area of that region. At least five different micrographs per muscle were analysed and results averaged.
Protein isoform determination and quantification
Myosin heavy chain isoforms. These were determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Each single fibre, dissolved in sample buffer, was loaded onto a 6% SDS-PAGE gel and run at 120 V for 24 h at 10°C as described previously (Larsson & Moss, 1993). Gels were subsequently silver stained and MyHC isoforms were determined.
Titin isoforms.
These were detected on agarose-strengthened 2.0% polyacrylamide gels with a Laemmli buffer system, as described (Opitz et al. 2004; Prado et al. 2005). Briefly, frozen muscle was homogenized, centrifuged and solubilized. Some gel lanes were loaded with a mix of human muscle and rabbit psoas muscle expressing two titin isoforms of 3.4 and 3.3 MDa at a ratio of 30 : 70% (Prado et al. 2005); the psoas bands could be used as internal standards. Alternate lanes on a gel were loaded with psoas muscle alone and human (CTL and SCI-S) muscle alone. All gels were also loaded with rabbit soleus (titin size, 3600 kDa) and some gels with rat heart (major titin, 3000 kDa) and human soleus (titin size, 3700 kDa) to obtain additional size markers. Human soleus tissue was kindly provided by Dr R. Bittner (University of Vienna, Austria). Attempts were made to load all lanes with equal amounts of solubilized protein after spectrophotometric analysis using the Bradford method. To determine the titin : MyHC protein ratio, we used 2.8% SDS-PAGE (Minajeva et al. 2002). Protein bands were visualized with Coomassie Brilliant Blue and gels were digitized by multiple scanning using a CanoScan 9900F scanner. Densitometry and molecular mass measurements were performed using TotalLab software (Phoretix, Newcastle, UK). A linear relationship between log molecular mass and migration distance was assumed.
Desmin concentrations. These were quantified by the enzyme-linked immunosorbent assay (ELISA) method as previously described (Berggard et al. 2003). Briefly, denatured, individual skinned fibres, or muscle sections, from SCI-S and CTL vastus lateralis were diluted in 50 mmol l–1 carbonate–bicarbonate buffer (pH 9.0) and placed on the bottom of a microtitrate plate. The primary antibody recognizing desmin (1 : 20; Sigma-Aldrich, Schnelldorf, Germany) was followed by an alkaline phosphatase-conjugated secondary antibody (1 : 350; chicken anti-rabbit, Santa Cruz Bioscience, Heidelberg, Germany). The plates were analysed in an ELISA reader (iEMS Reader MF, Labsystems, Finland) at optical density 405/490 nM and the desmin concentration in each fibre was related to its total protein content as determined by a commercially available protein quantification kit (NanoOrange, N-6666, Molecular Probes, Leiden, The Netherlands).
Data analysis
All data are presented as means ± standard error of the mean (S.E.M.). Statistical analyses of the data were performed using either a repeated measures analysis of variance (ANOVA) or Student's unpaired t tests when appropriate, except for the comparison of MyHC isoforms determined from single fibres where the non-parametric Mann-Whitney rank sum test was used. P values < 0.05 were taken as indicating significant differences.
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Results |
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Torque recordings during passive lengthening of proximal leg muscles at four constant speeds of movement between 15 and 120 deg s–1 were collected from patients with spasticity and control subjects using an isokinetic dynamometer (Fig. 1). On average, torque opposing the movement, along with EMG activity both in lengthened and shortened muscles, was increased for extensions and flexions at the higher speeds of movement in the SCI-S group (Fig. 1A and B). Control subjects showed no resistance to the movement and no or very low levels of muscle activation during passive movements of the limb, independent of knee angle, i.e. muscle length (data not shown). However, resistance and muscle activation varied substantially between the SCI-S patients (compare Fig. 1A with Fig. 1B). Furthermore, a significant increase in quadriceps PT was observed in some SCI-S patients at long muscle lengths (Fig. 1C), but never in control subjects at similar muscle lengths (not shown). The increase in PT at long muscle lengths in SCI-S was unrelated to speed of movement and muscle activation.
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Passive tension was increased in single skinned skeletal muscle fibres from SCI-S subjects compared with controls (ANOVA, P < 0.01; Fig. 2A). However, individual variability among fibres within the SCI-S group was large (Fig. 2B). Notably, the subject with the highest in vivo PT at long quadriceps muscle lengths (SCI-S7; Fig. 1C) also had the highest PT at the single-fibre level (SCI-S7; Fig. 2B). No difference in slack SL was found between the two groups. Moreover, maximum active force normalized to muscle fibre cross-sectional area (measured at SL 2.8 µm) was not different between the SCI-S (328 ± 27 mN mm–2) and CTL (302 ± 24 mN mm–2) groups, although the CSA of the single skinned fibres at SL 2.8 µm was significantly lower (P < 0.01) in the SCI-S (4390 ± 410 µm2) than the CTL (9550 ± 570 µm2) group. To account for overestimation of single-fibre CSA due to the well-known effect of swelling of skinned muscle samples, we also measured the fibre CSA on tissue sections and obtained mean values of 3120 ± 829 µm2 for SCI-S and 6584 ± 1297 µm2 for CTL muscles (Fig. 3, inset). Thus, the fibres from spastic muscles were confirmed to be atrophic. Further, there was a much larger variability in fibre diameters in spastic than in control muscles (data not shown), which was also reflected in a large variability in fibre CSA among SCI-S muscles (Fig. 3).
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Passive force measurements were performed on non-activated isolated myofibrils obtained from four SCI-S and three CTL patients. We used those SCI-S muscles that showed the highest PT increase at the single-fibre level (SCI-S7, 3, 4 and 5; see Fig. 2B). However, passive tension during stepwise stretch from SL 1.8 to 3.6 µm was not different between myofibrils from the SCI-S and CTL groups (Fig. 5). Pooled results for the individual muscles from the two groups are shown in Fig. 5B, indicating that none of the variability seen at the whole-muscle and single-fibre levels remains at the myofibrillar level.
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Gel electrophoretic studies were performed on muscle homogenates as well as single fibres to establish the MyHC isoform compositions. For muscle homogenates, a representative gel is shown in Fig. 6A. Densitometry results for muscle homogenate gels demonstrated that the SCI-S group exhibits a significantly higher proportion of fast MyHC isoforms than the CTL group (P < 0.05; Fig. 6B). Similar results were obtained at the single-fibre level (Fig. 6C); the difference between SCI-S fibres and control fibres, as determined by the non-parametric Mann-Whitney rank sum test, again was statistically significant (P < 0.01).
Skeletal muscles contain titin isoforms varying in size between approximately 3.3 and at least 3.7 MDa (Freiburg et al. 2000; Prado et al. 2005). The titin isoform size of human vastus lateralis muscles was found to be similar (3.7 MDa) to that of the human soleus muscle, for which the titin sequence is known (Freiburg et al. 2000) (Fig. 7B). To obtain reliable titin size values, control and SCI-S vastus lateralis muscle tissue was mixed with rabbit psoas, which expresses two titin isoforms at 3300 and 3400 kDa (Prado et al. 2005), and the rabbit psoas titin bands were used as internal size markers (Fig. 7C and D). However, in none of the seven SCI-S muscles was a difference in titin isoform size detectable in comparison to controls (Fig. 7). The intermuscle variability in titin size was between 3660 and 3700 kDa and the mean of seven means per group reached 3682 ± 11 kDa for the spastic muscles and 3680 ± 9 kDa for the control muscles (Fig. 7E). Moreover, there was no difference with regard to the stoichiometry between titin and myosin for SCI-S and CTL muscles determined from 2.8% SDS-PAGE gels (data not shown): the titin : MyHC ratio (not corrected for molecular weight) was 0.20 ± 0.03 and 0.22 ± 0.06, respectively.
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Histological analyses (not shown) and electron micrographs (Fig. 8) prepared from vastus lateralis biopsies of SCI-S subjects demonstrated extensive remodelling compared to CTL muscles. These changes included (see Fig. 8A): (i) a distinct increase in collagen content and connective tissue, (ii) the presence of lipid droplets, and (iii) replacement of myofibrils by amorphous, dot-like material. Moreover, in accordance with the increased proportion of fast-twitch fibres, the average mitochondrial density in SCI-S muscles was significantly lower than in CTL muscles, 4.5 ± 0.5% versus 11.0 ± 0.8% (Fig. 8Bc). The average myofibrillar volume fraction however, remained unchanged (80.5 ± 2.9% in control muscles; 78.6 ± 3.1% in spastic muscles) which was probably related to the (observed) relative abundance of amorphous material inside the myofibres of spastic patients.
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Discussion |
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Patient heterogeneity
A detailed understanding of the neural and muscular mechanisms underlying the muscle hypertonus in patients with spasticity is of vital importance for the design of treatment, as some patients fail to respond to conventional treatment, e.g. botulinum toxin injections. In patients with spasticity secondary to an upper motor neuron lesion, increased PT at the whole-muscle level is a common feature (Tardieu et al. 1982; Sinkjaer et al. 1993; Mirbagheri et al. 2001). Indeed, some of the SCI-S patients studied here showed increased resistance to stretching of the quadriceps muscle, although other patients did not. The increased resistance to stretch, when present, was not of the same origin in different patients. In some, it was velocity dependent, increasing with increasing velocity and most likely due to exaggerated stretch reflexes. In others, it was muscle-length dependent and unrelated to muscle activity, increasing only at the end of the movement range, probably due to structural alterations. This large variability in in vivo PT of SCI-S patients is consistent with previous observations (Knutsson et al. 1997). However, we were not able to identify any clinical parameter underlying the inter-patient variability in PT, i.e. PT did not correlate with the duration of spasticity, the spinal level of the traumatic injury, age of subjects, or medication.
MyHC isoform expression and fibre-type dependence of PT increase in spasticity
Recent work has shown that increased resistance to stretch and high PT in patients with spasticity also persist at the single-fibre level (Fridén & Lieber, 2003; Lieber et al. 2004). Our results on skinned single fibres agree with these findings. Although no earlier study has related increases in PT to specific MyHC isoforms, previous work on patients with SCI (but unknown spasticity levels) demonstrated an increase in fast MyHC isoforms within a year of the injury (reviewed in Talmadge, 2000). Elsewhere, the muscles of SCI-S patients were found to have a higher percentage of fast MyHC isoforms than those of control subjects (Fridén & Lieber, 2003). However, there are conflicting reports as to whether there is a general MyHC isoform switch in response to spasticity, which may be related to the fact that different muscles were studied from controls and spastic patients, and that individuals in the two groups were usually of different age (reviewed in Lieber et al. 2004). In the present work we avoided these shortcomings and observed a significant increase in MyHC-IIx isoform expression in the SCI-S group. This fibre-type shift in spastic muscles occurred in addition to fibre atrophy, confirming previous observations. Our results thus support the notion of a shift toward fast MyHC isoforms most likely as a consequence of chronic decreased use of the spastic muscles.
A novel finding of this work is that PT in the SCI-S patients increased in a MyHC isoform-dependent manner. There was an almost 10-fold increase in PT in spastic muscle fibres expressing the type IIx isoform compared with those expressing type I MyHC (Fig. 4). In contrast, single fibres containing type IIx MyHC showed no elevated PT in muscles of healthy athletes, suggesting that higher PT of type IIx fibres in SCI-S patients represents an adaptation to the upper motor neuron lesion. In summary, the elevated PT only in fast-type fibres, together with an increased proportion of fast-type IIx fibres, contribute to the increased PT in SCI-S patients at the whole-muscle level.
Known passive tension-generating proteins in the fibres are unaltered in spastic muscles
We hypothesized that increased PT in spastic muscles is due to changes in titin isoforms since the PT of muscle fibres is closely related to titin isoform size (Wang et al. 1991; Horowits, 1992; Linke et al. 1996; Prado et al. 2005). Titin isoform changes have been reported, e.g. in end-stage failing human hearts (Neagoe et al. 2002; Makarenko et al. 2004). Here the titin in human vastus lateralis muscles was compared to the titin bands in rabbit psoas muscles serving as internal markers to obtain a high reliability of the titin size estimates (Prado et al. 2005). However, no statistically significant changes in titin isoform size were found between SCI-S and CTL. Similarly, spastic and control muscles exhibited no difference in the titin : MyHC ratio. Consistent with these results, PT was similar in the two groups at the isolated myofibril level. Notably, the values of PT in isolated myofibrils (Fig. 5), particularly at high SLs, were similar to those of slow skinned fibres, but lower than in fast skinned fibres (Fig. 4). Therefore the possibility exists that all SCI-S myofibrils studied mechanically were from slow fibres. However, our finding that there is no change in titin isoform size in spasticity patients (Fig. 7) supports our conclusion that myofibrillar PT, which has been shown to be higher, the smaller the titin isoform (Prado et al. 2005), remains unaltered in SCI-S muscles. Taken together these data suggest that the spasticity in SCI-S patients is not associated with a change in titin expression and titin-based stiffness.
Within the physiological SL range (2.0–4.0 µm) titin is not the sole source of PT (Wang et al. 1993; Neagoe et al. 2003; Prado et al. 2005), as collagen fibres and also intermediate filaments (desmin) become important contributors to PT at longer SLs (Granzier & Wang, 1993; Wang et al. 1993). At these longer SLs we found the greatest spasticity-related increase in PT of single fibres. The desmin content has also been observed to differ between rat muscles in response to denervation; the protein decreased in the slow-twitch soleus muscle but increased in the fast-twitch gastrocnemius (Boudriau et al. 1996). Elsewhere, the PT of desmin-deficient mice reportedly increased (Anderson et al. 2001), decreased (Shah et al. 2004), or remained unchanged (Anderson et al. 2002). Our quantitative desmin measurements did not reveal differences in relative desmin content between SCI-S and CTL muscles. Further, there was no correlation between relative desmin content and MyHC isoform despite the higher PT in muscle cells from the SCI-S group expressing IIx MyHC or a combination of IIa and IIx isoforms. In summary, the intracellular proteins commonly thought to contribute to PT generation in striated muscle were unaltered in the vastus lateralis from spasticity patients, thus leaving the PT of the myofibrils unaffected.
Cellular mechanisms of passive stiffening in spasticity
In the absence of changes in titin and desmin expression and in titin-based stiffness, the increased PT in single fibres from SCI patients with spasticity appears to be somehow associated with the shift toward the faster MyHC isoforms (IIx alone or in combination with IIa MyHC). Consistent with the higher frequency of fast-type fibres, muscles from the SCI-S group showed decreased mitochondrial volume density, but there was no concomitant increase in the myofibrillar volume fraction of spastic muscle fibres (and no change in active tension), as some myofibrils were apparently replaced by amorphous material. If this material were stiffer than the myofibrils, its presence could add to the increased PT in spastic fibres. Further along this line, we speculate that the myofibrils in fast-type fibres of spastic muscles may be particularly vulnerable to replacement by such amorphous material, which would be an explanation for the elevated PT in these fibres. Fast-type fibres appear to be more susceptible than slow fibres to external force-induced microtraumatic damage, even in healthy subjects (Fridén et al. 1983) and this predisposition may be aggravated in spastic muscles, perhaps leading to increased remodelling inside the fast fibres. Finally, Fridén & Lieber (2003) suggested that the collagen content could be increased even at the single ‘demembranated’ fibre level where theoretically no collagen should remain, and this might explain increased PT. Whether there were collagen remnants in our study was not determined, but the fact that we found MyHC isoform-specific changes in PT speaks against some generally increased presence of collagen around the muscle fibres.
In conclusion, although the primary lesion in spinal cord-injured patients with spasticity is neural in origin, profound secondary changes occur in the musculature itself at the protein, single-fibre and whole-muscle levels. The ultrastructural remodelling in spastic myocytes includes expression shifts toward increased proportions of the fast MyHC-IIx isoform, in association with elevated passive tension only in these fast-type fibres, although titin size and titin-based stiffness remain unaltered. Our results extend current knowledge about muscle hypertonia and spinal cord lesions as a prerequisite for future intervention and rehabilitation strategies for patients with an upper motor neuron lesion.
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Acknowledgements |
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Author's present address
M. C. Olsson: Department of Sports & Exercise Science, Malmö University, 205 06 Malmö, Sweden.
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; control subjects, 
. B, high variability in PT among SCI-S subjects. Symbols represent PT measurements from individual SCI-S muscles (see key in figure). Inset: low variability in PT among control subjects, symbols represent individual CTL muscles. Data points are means ±
S.E.M.
