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Contractile properties in single muscle fibres from chronically overused motor units in relation to motoneuron firing properties in prior polio patients
JOURNAL OF THE
NEUROLOGICAL SCIENCES
ELSEVIER
Journal of the Neurological Sciences 132 (1995) 182-192
Contractile properties in single muscle fibres from chronically overused motor units in relation to motoneuron firing properties in prior polio patients Lars Larsson *, Xiaopeng Li, Anna Tollbtick, Lennart Grimby Departments of Clinical Neurophysiology
and Neurology, Karolinska Hospital, S-171 76 Stockholm, Sweden
Received 18 November 1994; revised 7 April 1995; accepted 27 April 1995
Abstract The relation between motoneuron firing rate in vivo and maximum velocity of unloaded shortening (V,,) and myosin isoform composition in single chemically skinned muscle fibres was investigated in chronically overused motor units. Ten patients with loss of a large proportion of the motoneuron pool due to a prior polio lesion and compensatory overuse of residual neurones were studied and compared with normal individuals. The tibialis anterior muscle (TA) was chosen and prior polio patients who used all residual TA motor units at high rates during the normal step cycle were selected. In prior polio patients, all motor units fired at approximately 40 Hz when maximum voluntary force was reached. A common firing rate of 30 Hz yielded 70-90% maximum force. In normal subjects, on the other hand, maximum TA force was reached when low threshold units fired at 25-30 Hz and high threshold units at 50 Hz. Myosin heavy chain (MHC) and light chain (MLC) isoforms were resolved by 6% and 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), respectively, and quantified densitometrically. In the whole biopsy cross-sections, types I, IL4 and IIB MHC proportions were 97, 3 and 0% in a typical prior polio patient and 65, 25 and 10% in an age- and sex-matched control subject. V,, differed significantly (p < 0.001) between type I fibres from the patient (0.54 f 0.12 ML/s) and the control subject (0.29 k 0.08 ML/s). The composition and relative contents of essential and regulatory MLC isoforms differed in single type I MHC fibres from the control subject and prior polio patient. 65% of the fibres co-expressed the fast and slow isoform of the regulatory light chain (MLC,) in the patient, while this combination was only observed in one of the control type I fibres. All prior polio fibres with a V,, higher than 0.45 ML/s, except one, co-expressed MLC,, and MLC,, and the only control fibre co-expressing the slow and fast MLC, isoform had the highest V,, (0.50 ML/s) among control fibres. On the other hand, a high relative content of MLC, was not associated with a high V,, in type I MHC fibres. It is suggested that the composition of fast and slow isoforms of MLC, has a significant modulatory influence on V,, within type I MHC fibres. This combination of MLCs and high V,, in type I MHC fibres is probably induced by chronic motor unit overuse and an altered motoneuron firing pattern. Keywords: Polio; Skeletal muscle; Motoneuron; Myosin; Shortening velocity
1. Introduction Contractile properties of adult mammalian skeletal muscle possess a remarkable capacity for adapting to altered functional demands. This is demonstrated most strikingly in the response to the radical change in contractile activity induced by cross-innervation or chronic electrical stimulation of skeletal muscle, directly or indirectly, via the motor nerve (for review see Edstrom and Grimby, 1986). The
* Corresponding author. Tel.: ( + 46-8) 729 4148; Fax: (+ 46-8) 33 99 53; e-mail:[email protected]. 0022-510X/95/$09.50 0 1995 Elsevier Science B.V. AR rights reserved SSDI 0022-510X(95)00138-7
alterations in the contractile properties are mainly related to changes in the myosin isoform composition, which in turn result from changes in the transcription of new mRNAs in response to the specific stimulus (Brownson et al., 1992). Patients who have lost a large proportion of their motoneurons and/or muscle fibres need all their residual muscle power for activities of daily living, while normal subjects have considerable reserves. In patients with critical tibialis anterior muscie (TA) weakness, due to a loss of functioning motor units and incomplete reinnervation of denervated muscle fibres, all residual motor units fire more than 10 discharges during each step cycle at 20-50 Hz. In
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normal subjects walking at comfortable speed, on the other hand, only low threshold motor units fire during the ordinary step cycle at lo-25 Hz, and high threshold units are recruited in corrective movements or during rapid locomotion, firing at 20-50 Hz (Grimby et al., 1984). Muscle specimens from patients overusing their residual TA motor units have a higher content of slow myosin than normal subjects (Borg et al., 1988; Jakobsson et al., 1988), but the maximum voluntary force at fast relative to slow speeds of foot dorsiflexion did not differ between patients overusing their residual motor units and control subjects (Tollback et al., 1992). This indicates a dissociation between mechanical and enzyme-histochemical properties, as has previously been reported in human skeletal muscle (Thorstensson et al., 1976). The interpretive value of in vivo measurements of contractile properties of human skeletal muscles is limited by factors that tend to obscure the behaviour of individual muscle fibres, but even if these difficulties were not present, multicellular preparations have the disadvantage that the roles played by different muscle fibre types in overall contractility are difficult to evaluate (Hofmann et al., 1991). The skinned fibre preparation allows investigation of the function of myofilament proteins in a cell with an intact filament lattice, but without the confounding effects related to intercellular connective tissue or protein heterogeneity between cells of multicellular preparations. The usefulness of this technique for studying relationships between contractility in the short human muscle fibre segments obtained with the percutaneous biopsy technique and the molecular protein composition of the fibre segment in normal muscle tissue has recently been documented (Larsson and Moss, 1993), and the findings provide a basis for future studies of the function of myofibrillar proteins in man. The present study was undertaken to investigate the effects of chronic overuse of residual motor units in patients with a lower motoneuron disorder, in order to improve our understanding of the influence of the voluntary pattern of activity on the motor unit. A well defined group of prior polio patients and control subjects was examined and specific interest was focused on motoneuron firing rates and the contractile properties of residual muscle fibres, together with their myosin isoform composition.
2. Materials
and methods
2.1. Material The tibialis anterior muscle (TA) was studied in 10 patients (6 women and 4 men) with prior polio in whom 24-57 years had elapsed since the acute stage of the disease. At the time of the study the patients were 46-77 years of age. The patients reported no progression of TA weakness. The findings were compared with previously
published data from normal individuals (Grimby et al., 1984; Jakobsson et al., 1988). Muscle biopsy specimens were taken from one of the female prior polio subjects (46 years) and compared with one healthy sex- and agematched control subject with no previous history of any locomotor or neurological disorder (47 years). The study was undertaken with the understanding and consent of the subjects, and was approved by the local Ethical Committee. 2.2. In vivo force measurements The force of the dorsiflexors of the foot was measured in such a way that it was not influenced by proximal leg muscles. In part of the experiment the measuring device was attached only to the foot and lower leg. In another part of the experiment it was attached to the examination bench, but could be controlled with a foot switch so that the heel was not lifted, i.e. so that tension did not derive from proximal muscles. However, the possibility of some co-contraction of plantar flexors could not be excluded. In a series of maximum voluntary contractions each subject in most attempts reached a certain force that was never exceeded, and this was designated the maximum force (for details of the experimental set-up see Grimby et al., 1984). 2.3. Single motor unit electromyographic ings
(EMG) record-
In the prior polio subjects there was no difficulty in obtaining and maintaining recordings of one single motor unit with commercial electrodes (Medelec 16829, Medelec Ltd., Woking, Surrey, UK), since the number of motor units was decreased and the fibre density within the remaining ones was increased. The risk of confusing potentials from different motor units was small, because of the characteristic shape of reinnervated potentials. When there was any doubt, a check was made to see that the potential evoked by supramaximal electrical stimulation of the peroneal nerve was identical to the voluntary potential being studied. The signals were amplified and displayed on an oscilloscope (Medelec 4239) and critical parts of the experiments were recorded on UV film at film speeds of between 10 and 100 cm/s. The surface EMG was recorded by electrodes (Medelec C162) strapped a few centimetres apart over a relatively preserved part of the TA muscle. The signals were rectified and integrated over periods of 80 ms and displayed during the following period. 2.4. Muscle biopsies and permeabilization
of fibres
Biopsy specimens were taken under local anaesthesia from the left TA in both the patients and the control subjects by means of the percutaneous conchotome method. The specimens were either frozen or placed in relaxing
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solution at 4°C. Bundles of m 50 fibres were dissected free from the samples in relaxing solution and then tied with surgical silk to glass capillary tubes at slightly stretched lengths. The bundles were chemically skinned for 24 h in relaxing solution containing 50% (v/v) glycerol at 4°C and were subsequently stored at - 20°C for up to 3 weeks before use. Other biopsy specimens were frozen in freon chilled with liquid nitrogen and stored at - 80°C. 2.5. Experimental procedure On the day of an experiment, chemically skinned single fibres were pulled free from one end of the bundle, mounted in an experimental apparatus, leaving an average fibre segment length of 2.0 f 0.6 mm and 1.9 f 0.4 mm (mean f SD) in the patient and control subject, respectively, exposed to the solution between connectors leading to a force transducer (Model 403, Cambridge Technology, Inc.) and a DC torque motor (Model 300H, Cambridge Technology, Inc.). While the fibres were in relaxing solution, sarcomere length (SL) was set to 2.75-2.85 pm by adjusting the overall segment length. This is longer than the SL recently used (2.55-2.75 pm) in a study of human muscle fibres (Larsson and Moss, 1993), which in turn was identical to the SL most commonly used in single skinned rabbit psoas fibres. However, the thin filament is longer in human quadriceps (1.30 pm) than in rabbit psoas fibres (1.12 pm), because of the species differences in the size of nebulin, i.e. the length-regulating protein of the thin filament (Kruger et al., 1991). SL was therefore increased and this is the most probable explanation for the higher specific tension observed in this study as compared with our previous observations (for details and references see Larsson and Moss, 1993). Photographs or videoprints of the fibre segments were taken with a 35-mm auto-exposure SLR camera (Contax, Yashica Kyocera GmbH, Hamburg, Germany) or a videoprinter (P71E, Mitsubishi Electric Corp., Japan). The sarcomere length, the segment width and the length of segment between the connectors were measured directly from the microscope via a TV-overlay with the aid of a digitizer connected to a microcomputer (Videoplan, Kontron Bildanalyse GmbH, Munich, Germany). The final magnification with the image analysis system on the TV screen was X 1480. Fibre SL was routinely measured in the fibres taken during maximum activation (Moss, 1979). Fibre depth was measured by recording the vertical displacement of the microscope nosepiece while focusing on the top and bottom surfaces of the fibre. Fibre cross-sectional area (CSA) was calculated from the width and depth, assuming an elliptical circumference, and was corrected for the 20% swelling that is known to occur during skinning (see Moss, 1979). Specific tension was calculated as maximum tension (P,) normalized to CSA. Relaxing and activating solutions were identical to those previously described (see Larsson and Moss, 1993) and V,,, was measured by the
slack-test procedure at 15°C (Edman, 1979; Moss et al., 1982). 2.6. Myosin heavy chain (MHC) and myosin light chain (MLC) compositions The MHC and MLC compositions of single fibres were determined by 6% and 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), respectively (for references and details see Larsson and Moss, 1993). Gels were silver-stained and subsequently scanned in a soft laser densitometer (Molecular Dynamics, Sunnyvale, CA, USA), with a high spatial resolution (50 pm pixel spacing) and 4096 optical density levels, to determine the relative contents of MHCs and MLCs. The volume integration function was used to quantify the amount of protein, and background activity was subtracted from all pixel values (ImageQuant software ~3.3, Molecular Dynamics). 2.7. Enzyme-histochemical techniques The frozen muscle samples were cut at their greatest girth perpendicular to the longitudinal axis of the muscle fibres into lo-pm thick cross-sections with a cryotome (2800 Frigocut E, Reichert-Jung GmbH, Heidelberg, Germany) at -20°C. The muscle fibres in the cross-sections were stained for myofibrillar ATPase after alkaline and acid preincubations, and classified as types I, IIA, IIB and IIC (for details see Edstrom and Larsson, 1987). The cross-sectional areas of the individual muscle fibres were measured semiautomatically on magnified black and white photographic prints of myofibrillar ATPase-stained cross-sections or directly from the microscope via a TVoverlay with the aid of a digitizing unit connected to a microcomputer (Videoplan, Kontron GmbH, Munich, Germany). The form factor was calculated according to the formula: fibre area/(?r/4 X maximum diameter X minimum diameter). The form factor is 1 for a circle or an ellipse and < 1 for irregular structures. 2.8. Statistics Means, SD and regression lines were calculated from individual values by standard procedures. A two-tailed independent t test was used for comparisons of two populations. When the two population variances were equal, i.e., when F was small, the pooled variance t test was used; otherwise, the separate-variance t test for means was used. Differences were considered significant at p < 0.05.
3. Results Long-term recordings have shown that TA is mainly used during locomotion in man (Grimby, 1986). The prior
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polio patients were selected as being able to walk with a normal pattern using all residual TA power. Normal subjects, on the other hand, use no more than 50% of their TA power when they are walking at comfortable speeds (Jakobsson et al., 1988). The overuse of residual muscle fibres in prior polio has been reported to cause hypertrophy and fast to slow myosin isoform switching (Borg et al., 1988). 3.1. Voluntary firing rate and force Motor units recruited at zero tension were called “low threshold motor units” (LMUs). When prior polio subjects slowly increased the voluntary drive to maximum LMUs started tonic firing at I 10 Hz and reached 2 50 Hz (Fig. 1). A firing rate of < 15 Hz caused little force. The steepest force increase occurred at 18-20 Hz. At these firing rates, the patients had difficulty in maintaining smooth regulation of tension. Force increased till the firing rate reached 40 Hz. The foot dorsiflexion force was plotted against the firing rate of an LMU and against the integrated surface EMG in each of the prior polio patients. The patients had visual feedback at 5, 10, 20, 30, 50, 70 and 90% of the maximal residual force and maintained each force for some seconds (Fig. 1 illustrates a more rapid increase of force to save space). The force-frequency curve had a sigmoid shape (Fig. 2 left). The curve of force plotted against the integrated surface EMG had a similar sigmoid shape (Fig. 2 right). Simultaneous recordings of one LMU and one or more other motor units showed that additional motor units were recruited till the LMU had reached a firing rate of 20 Hz. The newly recruited motor units started tonic firing at a lower rate than the LMU had reached before its recruitment. However, when the new motor unit was recruited,
Fig. 2. Relation between foot dorsiflexion force and firing rate of a TA low threshold motor unit (left) and relation between tension and surface EMG (right) in the 10 prior polio patients. Bars denote SD between patients.
the firing rate of the LMU decreased (Broman et al., 1984). The difference in firing rate between simultaneously firing motor units rarely exceeded 25% of the LMU firing rate. During increased voluntary drive the differences in firing rate decreased and all motor units fired at the same rate at 2 30 Hz. To get quantitative information on the relative firing of residual motor units, the integrated surface EMG reflecting recruitment and firing rates of a large number of motor units was compared with the firing of an LMU in each of the prior polio subjects. The global EMG was not systematically measurable when LMUs fired at 10 Hz, indicating that the number of LMUs was small. The global EMG increased more rapidly than the LMU firing rate between 15 and 25 Hz, as a sign of major recruitment. The global EMG was only about doubled when the LMU firing rate increased by 25-50 Hz, indicating that all motor units were recruited and had increased their firing rate in parallel. < 40 Hz was not compatible with maximum tension of the non-fatigued muscle, and a common firing rate of 30
Fig. 1. Firing rate of TA low threshold motor unit (upper trace) foot dorsiflexion force (solid line) and TA surface EMG integrated over periods of 70 ms and displayed during the following period (broken line). Thin line denotes zero tension and integrated EMG. Time bar = 1 s.
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Table 1 Proportions (o/o) and cross-sectional areas (CSA) of muscle fibre types classified according to enzyme-histochemical the control subject and prior polio patient. Values are means + SD. Type IIB
Type IL4
Type1
stainings for myofibrillar
ATPase in
Type IIC
Control
% 66.6
CSA (pm’) 4590+1090
% 26.8
CSA (pm*) 6330flSOO
% 6.6
CSA (pm*) 5070+1450
% 0
CSA (grn*) -
Prior polio
96.7
8600+3560
1.5
694Ok2700
0
-
1.8
1130 + 610
from the patient than from the control subject (0.90 f 0.07,
Hz gave rise to 70-90% of the maximum force. Increases from 40 to 50 Hz, on the other hand, caused no or little increase in force.
12= 200). 3.3. Myosin isoform composition
3.2. Enzyme-histochemical, morphometric and histopathological observations
MHCs were identified as types I, IL4 and IIB according to the order of migration on 6% SDS-PAGE in TA crosssections, as well as in 51 TA single fibre segments from the patient (n = 25) and the control subject (n = 26). In the control subject, the proportions of types I, IL4 and IIB MHCs were 65, 25 and lo%, respectively. In the patient, type I MHC was the only protein observed in the MHC region of the gel. However, a faint type IL4 MHC band was observed (less than 3% of the total MHC content) when the gel was overloaded with protein. Enzymehistochemical fibre type classifications and electrophoretic separations of MHCs were thus in agreement. In the prior polio patient, all single fibre segments expressed only type
In conformity with previous reports (Borg et al., 19881, a large majority of the muscle fibres in TA from the prior polio patient were classified as type I and these fibres were generally of large size (Table 1). Mild histopathological abnormalities such as central nuclei together with signs of “ fibre-splitting” were observed in the muscle specimen from the prior polio patient, whereas these abnormalities were not observed in the control subject. The number of angulated fibres was greater in the patient compared with the control subject. That is, the calculated form factor was lower (p < 0.001) in type I fibres (0.84 f 0.09, 12= 200)
1 Actin
-
MyLC3
-
2
3
4
5
6
7
6
9
10
11
Fig. 3. Electrophoretic protein analysis of single fibre segments from TA muscles. MLCs were resolved on 12% SDS-PAGE and only the low molecular weight region of the gel is shown. Lanes l-4 and 9-11, single fibres from the prior polio patient; lanes 5-8, single fibres from the control subject. One fibre is co-expressing type IIA and IIB MHC isoforms (lane 6), the other fibres are expressing type I MHC. Two fibres are co-expressing the fast and the slow isoform of the regulatory light chain (lanes 1 and 11). The abbreviations are the same as those used in the text.
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I MHCs even if the gels were overloaded with muscle fibre protein. In the control subject, three fibres co-expressed type IIA and IIB MHCs in different proportions, type IIB MHCs ranging between 44 and 88%, and the remaining fibres expressed only type I MHCs. Essential (MLC, and MLC,) and regulatory (MLC,) light chains were observed in all 51 fibres. MLC, and MLC, exist in a slow and a fast isoform (MLC,, is expressed in two isoforms-MLC,,, and MLC,,,-which are not separated with the present gel system), while MLC, only exists in a fast isoform. The essential and the regulatory MLCs are expressed in a stoichiometric 1:l relationship, but the essential/regulatory MLC ratio deviated from the expected value, probably due (1) to differential staining intensity of the light chains with silver when compared with Coomassie blue (Moss et al., 1982; Hofmann et al., 1990) and/or (2) to comigration of regulatory protein isoforms together with MLC, (Bottinelli et al., 1994). However, the MLC ratio did not differ between fibres from the control (1.9 + 0.5) subject and patient (1.9 + 0.5). Co-expression of MLC, and MLC,, is rarely observed in type I fibres from normal muscle and this combination was only found in 1 of 25 control fibres. In the prior polio patient, on the other hand, 17 of 26 fibres co-expressed
187
MLC,, and ML&,, although the MLC,, content was low in all fibres, varying between 1 and 14% (6 f 4%) of the regulatory light chain content (Fig. 3). All type I fibres except one fibre from the patient expressed the fast MLC, and the average content of MLC, was higher (p < 0.01) in the control subject than in the patient when expressed as percent of the essential (MLCi,+r + MLC,; 7.9 f 2.9% vs. 5.9 f 2.2%. MLCr,+r; 8.7 f 2.5% vs. 6.3 + 2.6%) or regulatory light chains (MLC2s+f; 16.5 f 7.4% vs. 11.2 + 4.0%).
During the manual dissection of the chemically skinned fibre bundles, no signs of fibre branching were observed in the control subject. In the patient with the prior poliomyelitis, on the other hand, branched fibres were frequently seen during dissection (Fig. 4). The proportion of branched fibres ranged between 10 and 42% of all fibres per bundle (9-12 fibres per bundle were branched), except for one bundle (17 fibres), where there were no signs of fibre branching. It was noted that the fibres were generally smaller (6000 + 580 pm’, range 5200-6600 prn2) in the bundle with no signs of fibre branching than in those bundles where branched fibres were frequently observed pm2). Some (8360f2630 pm2, range 4370-12560 branched fibres were divided into two or three different
Fig. 4. Microphotographs of four different branched chemically skinned fibre segments from percutaneous TA muscle biopsy specimens from the prior polio patient. Bar = 100 Fm.
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parts and subsequently analysed for their MHC and MLC compositions. Identical myosin isoform compositions were observed in the different branches from the same fibre. 3.4. Contractile properties in single tibialis anterior MUSde fibres Vmax was determined in the 44 single fibres which were analysed for their myosin isoform composition. However,
a V,,, value was included in subsequent analyses only when linear regressions included four or more data points, and data were discarded if r for the fitted line was less than 0.97 or if SL during isometric tension development changed by more than 0.10 pm compared with SL while relaxed (Moss, 1979). A total of 19 fibres in the control subject and 14 fibres in the patient fulfilled these criteria for acceptance and were included in the following analyses (Fig. 5).
Fig. 5. Photomicrographs of a single chemically skinned fibre from the tibialis anterior muscle of a prior polio patient; (a) in relaxing solution and (b) during maximum activation. The length of the fibre exposed to solution between connectors was 2.1 mm and the cross-sectional area was 8750 pm*. A well maintained cross-striation pattern and sarcomere length was observed during activation. Bar = 100 pm.
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0.0L
0.01
0
10
20 YyLC3/YyLc2
30
0
40
5
10
15
189
20
YyLCB/YyLC 1
Fig. 6. Maximum shortening velocity (V,,,) in relation to MLC isoform composition in type I MHC fibres from a control subject (open symbols) and prior polio patient (filled symbols). MLC, content is expressed as a fraction of MLC, or MLC,. x-axis: %; y-axis: V,,, , ML/s.
fast MLC, isoform had the highest V,,, (0.50 ML/s) among control fibres and all prior polio fibres, except one, with a V,,, higher than 0.45 ML/s co-expressed MLC,, and MLC,,. Further, among the muscle fibres from the prior polio patient, V,,, was significantly higher (p < 0.05) in fibres co-expressing the fast and the slow MLC, isoform (0.57 k 0.13 ML/s) than in those expressing only MLC,, (0.42 f 0.08 ML/s). The average MLC, content was significantly higher in the control fibres than in the fibres from the patient, but there was no correlation between V,,, and MLC, content in either control or prior polio fibres (Fig. 6). The content of MLC, in relation to the essential or regulatory MLC content did not differ between muscle fibres co-expressing the fast and slow isoform of MLC, in the prior polio patient, and it therefore appears to be an unlikely factor in explaining V,,, differences within type I MHC fibres. Maximum force normalised to cross-sectional fibre area (specific tension) was significantly lower (p < 0.01) in type I fibres from the prior polio patient than in those from the control subject (Table 2). These differences in specific tensions are probably related to the different diffusion distances between large and small skinned muscle fibres rather than an impaired muscle fibre force-generating capacity in the prior polio patient. That is, there is a larger
In the control subject, V,,, ranged between 0.18 and 1.64 ML/s. In the three fibres co-expressing type IIA and IIB MHCs, V,,, ranged from 1.13 to 1.64 ML/s. In the other fibres expressing only one MHC, the slow type I MHC, V,,, ranged between 0.18 and 0.50 ML/s (0.29 f 0.08 ML/s). These values conform with the maximum shortening velocities recently found in human type I fibres from the soleus (0.32 + 0.18, IZ = 27) and vastus lateralis muscles (0.33 f 0.12, it = 41) as well as in fibres co-expressing type IIA and IIB MHCs in the vastus lateralis muscle (1.38 &- 0.75, n = 23) (Larsson and Moss, 1993). In the prior polio patient, V,,, in the type I fibres ranged between 0.33 and 0.73 ML/s (0.54 f 0.12 ML/s, n = 14) and were thus significantly (p < 0.001) faster than the type I fibres from the control TA muscle (Fig. 6, Table 2) as well as when compared with the V,,, data in type I fibres from human soleus (p < 0.001) and vastus lateralis (p < 0.001) muscles in normal young control subjects (Larsson and Moss, 1993). V,,, in these type I fibres is intermediate between that in type I and IIA fibres classified according to their MHC isoform composition (Larsson and Moss, 1993). 65% of the fibres co-expressed MLC,, and MLC,, in the prior polio patient, while only one of the control type I fibres co-expressed these isoforms. The control fibre co-expressing the slow and the
Table 2 Maximum velocity of shortening (V,,,), specific tension, resting tension (per cent of maximum tension) and cross-sectional area (CSA) in type I and HA/B muscle fibres, classified according to the myosin heavy chain (MHC) composition, from a control subject (C) and post-polio (PP) patient. Values are means f SD and the number of fibres is given within parentheses. MHC
Specific tension (N/cm* )
V,,, (ML/s) C
Type1
0.29kO.08
Type &4/B
1.36 kO.21 (3)
(16)
* * *
PP
C
0.54 + 0.12 (14) -
31.7k7.8 (16) 29.9k4.4 (3)
* *
Resting tension (%) CSA ( pm*)
PP
C
PP
C
24.6k.5.5 (14) -
3*1 (16) 3*1 (3)
3+1 (14) -
2710+670 (16) 326Ok560 (3)
Differences between control and post-polio fibres are indicated (* * p < 0.01; * * * p < 0.001).
PP * * *
8630+2230 (14) -
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diffusion gradient from the periphery to the center of the fibre in the extremely large fibres in the prior polio patient than in the smaller control fibres which may influence the concentration of the activating calcium and/or tension suppressing inorganic phosphate.
4. Discussion The major observations in this study are the differences between control subjects and prior polio patients in motoneuron firing properties and maximum velocities of unloaded shortening in muscle fibres expressing the slow (type I> myosin heavy chain isoform. Further, myosin isoform data indicate that the composition of regulatory myosin light chain isoforms has a modulatory influence on the shortening velocity within type I MHC fibres. 4.1. Motoneuron firing rate and muscle force In normal subjects, a differentiated TA motoneuron firing rate is observed during voluntary activation. During slowly increasing isometric voluntary contraction, motoneurons recruited before 25% of the maximum force has been reached start tonic firing at about 10 Hz and fire at about 30 Hz at maximum force. Motoneurons recruited after 50% maximum force, on the other hand, have a minimum firing rate of 20 Hz and a maximum rate of up to 60 Hz. Normally about three-fourths of the TA muscle fibres are type I and one-fourth type II, and the whole type I population appears to be recruited at 30-40% of maximum force. It was concluded that during normal sustained contraction, type I motor units have a firing rate of about lo-30 Hz, while type II motor units have higher minimum and maximum rates (for review see Edstrom and Grimby, 1986). In the prior polio patients the maximum TA force was about 25% of the normal force, the number of muscle fibres per motor unit was greater than normal because of collateral sprouting, and the force per muscle fibre was increased because of hypertrophy. Thus, only a small proportion of the motoneurons remained. A large selective loss of motoneurons would result in similar firing properties of residual ones and a large random loss in different proportions of motoneuron types in different patients if there were no adaptive processes. The present study shows that the minimum firing properties of residual motoneurons were differentiated as in normal subjects and that the variability between patients was small indicating that residual motoneurons or their neural drive adapt to the functional demands so that their properties become similar in similarly paralysed patients. In prior polio all residual TA motor units reached 2 50 Hz during sustained maximum voluntary effort. The low residual tension in prior polio might result in low proprioceptive inhibition and some luxury rate. However, in prior
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polio all motor units fired at 40 Hz when the maximum force was barely maintained, and a common firing rate of 30 Hz gave rise to force of only 70-90% of maximum. Since only a few per cent of the muscle fibres were type II, it is not surprising that an increase of the stimulation rate from 40 to 50 Hz caused no significant increase in tension. However, the small proportion of type II fibres cannot have been responsible for the large increase in force recorded when the common stimulation rate increased from 30 to 40 Hz. A large proportion of type I muscle fibres in prior polio must have a higher fusion rate than normal type I fibres. In normal subjects, the force-frequency curve of each single motor unit has a sigmoid shape, whereas the relation between the force and EMG of a whole muscle is linear (see review by Lenman, 19811, indicating that normal motor units reach the steep part of the individual forcefrequency curve at different levels of contraction. In our prior polio patients, on the other hand, the curve obtained by plotting the force of the whole muscle against the firing rate of low threshold motor units and the curve obtained by plotting force against integrated surface EMG both had a sigmoid shape, indicating that a large proportion of the motor units reached the steep part of the force-frequency curve at the same time, possibly because of similar contractile properties (cf. below). 4.2. Contractile properties and myosin isoform composition Contraction of muscle fibres is often characterized in terms of the maximum shortening velocity (V,,,), since in muscles from many species V,,, is proportional to the actin-activated myosin ATPase activity (for review see Larsson and Moss, 1993). The head portion of myosin (subfragment-l) contains the ATPase activity and each myosin molecule consists of six subunits, which may be variably expressed in individual fibres: two myosin heavy chains (MHCS) with molecular weights of about 200 kDa, and four myosin light chains (MLCs) of 16-25 kDa (see Larsson and Moss, 1993). Close relationships between Vmax and MHC have been demonstrated in single fibres from crustacean, amphibian, avian and mammalian skeletal muscles, including man (Larsson and Moss, 1993). However, there is considerable variation in V,,, in muscle fibres which may be due to the presence of undetected MHC subtypes and/or variable ratios of MLCs, although experimental evidence is inconsistent on this point (Larsson and Moss, 1993). The large variability in V,,, is especially pronounced within muscle fibres expressing subtypes of type II MHCs (see Larsson and Moss, 19931, although the present results also demonstrate a substantial variability within type I MHC fibres. In the past years, much interest has been paid to the study of MLCs and their possible role in determining the kinetics of the actin-myosin interaction. In smooth muscle,
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phosphorylation of the regulatory light chain results in a marked increase in actin-activated Mg-ATPase activity, but it has no effect on myosin ATPase activity in rat and rabbit striated muscle (see Sweeney et al., 1993). During maturation of mammalian fast-twitch muscle, V,,, and myosin ATPase activity increase in parallel with the reciprocal changes in the MLC,, and MLC, contents (see Moss et al., 1990). In this study the MLC, content was significantly higher in the control type I fibres than in fibres from the prior polio subject but there was no significant correlation between V,,, and MLC, content in human type I fibres from either control or prior polio TA muscle, and Vmax was significantly lower in the control fibres in spite of a higher MLC, content. Thus, the present data conform with our previous observations in skinned human fibres (Larsson and Moss, 19931, i.e. that a high MLC, content is not necessarily associated with a high V,,, in human skeletal muscle fibres, and additional work is required before conclusions regarding the effects of essential light chains on V,,, can be drawn in man. It is interesting to note that Billeter et al. (1981) observed higher MLC, contents in fibres from human skeletal muscles with signs of disuse atrophy. The lower MLC, content in prior polio patients may accordingly be related to the chronic overuse of the slow-twitch motor units. Co-expression of MLC,, and MLC,, is rarely observed in type I fibres from normal muscle (Billeter et al., 1981; Larsson and Moss, 19931, but the majority of the fibres in the prior polio patient co-expressed MLC, and MLC,,. Co-expression of fast and slow isoforms of the regulatory light chain by single fibres is also frequently observed in patients with muscular dystrophies (Salviati et al., 1986). The present results indicate that V,,, was modulated by the isoform composition of the regulatory light chains, i.e. type I MHC fibres co-expressing MLC, and MLC,, had higher shortening velocities than those expressing only MLC,, . These data are analogous with the observation that in human type IIA and IIB MHC fibres, fibres expressing MLC,, and MLC,, were slower than those expressing only MLC,, (Larsson and Moss, 1993). The mechanism underlying the modulatory influence of the regulatory light chain isoform composition on V,,, is not known, but it may involve a change in the enzymatic properties of myosin (Hofmann et al., 1990) or the mechanical rigidity of the alpha helical portion of myosin subfragment 1 (see Rayment et al., 1993). Another possibility that has to be considered is the expression of MHC isoforms that remain undetected with the gel system used in this study, and the recent finding by Hughes et al. (1993) of three different slow MHC isoforms in mammalian skeletal muscle is of specific interest in this context. It cannot be ruled out that the differences in V,,, between type I fibres in control and prior polio muscle may be attributable to transitions within these different slow MHC isoforms and that the combinations of MLCs observed in this study are primarily related to the type of slow MHC expressed in the fibre.
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4.3. Muscle fibre branching The number of branched fibres was significantly increased in the prior polio patient, especially in bundles where extreme fibre hypertrophy was observed. However, the mechanism of muscle fibre branching is still poorly understood and it remains to be determined whether branching in muscle hypertrophy and regeneration has the same aetiology (Blaivas and Carlson, 1991). Although the cause of the increased number of branched extremely hypertrophic muscle fibres in our prior polio patient cannot be determined from the present data, the identical myosin isoform composition in the different branches from the same fibre strongly support the view that all branches are innervated by the same motoneuron. 4.4. Conclusion In conclusion, the contractile properties of a muscle fibre are largely dependent on its long-term use. The TA muscle is mainly used for locomotion. Normally, low threshold motor units are used during walking at a comfortable speed, and high threshold motor units are recruited mainly during rapid locomotion and corrective movements. A corresponding differentiation of the muscle fibres seems logical. In our prior polio patients residual muscle fibres were largely used in an all or none manner. Residual TA power was fully utilised at each step cycle, but the number of step cycles per day was low. Thus, there seems to be no need for any differentiation of residual muscle fibres and perhaps nothing to incite it. It is suggested that the absence in prior polio not only of type II fibres but also of the slowest type I is due to a transition caused by their intermediate use.
Acknowledgements This study was supported by grants from the Swedish Medical Research Council (8651), the Wallenberg Foundation, the Swedish Work Environment Fund, the Bergwall Foundation, the NHR Foundation and the RTP to L.L and grants from the Swedish Medical Research Council (4749) and the RTP to L.G. We are grateful to Ms. Ulrika Mliller for performing the SDS-PAGE.
References Billeter, R., Heizmann, C.W., Howald, H. and Jenny, E. (1981) Analysis of myosin light and heavy chain types in single human skeletal muscle fibers. Eur. J. Biochem., 116: 389-395. Blaivas, M. and Carlson, B.M. (1991) Muscle fiber branching - difference between grafts in old and young rats. Mech. Ageing Dev., 60: 43-53. Borg, K., Borg, J., Edstrom, L. and Grimby, L. (1988) Effects of excessive use of remaining muscle fibres in prior polio and LV lesion. Muscle Nerve, 11: 1219-1230.
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Bottinelli, R., Betto, R., Schiaffino, S. and Reggiani, C. (1994) Unloaded shortening velocity and myosin heavy and alkali light chain isoform composition in rat skeletal muscle fibres. .I. Physiol., 478: 342-349. Broman, H., De Luca, C.J. and Mambrito, B. (1984) Motor unit recruitment and firing rates interaction in the control of human muscles. Brain Res., 337: 311-319. Brownson, C., Little, P., Jarvis, J.C. and Salmons, S. (1992) Reciprocal changes in myosin isoform mRNAs of rabbit skeletal muscle in response to the initiation and cessation of chronic electrical stimulation Muscle Nerve, 15: 694-700. Edman, K.A.P. (1979) The velocity of unloaded shortening and its relation to sarcomere length and isometric force in vertebrate muscle fibres. J. Physiol., 291: 143-150. Edstrdm, L. and Grimby, L. (1986) Effects of exercise on the motor unit. Muscle Nerve, 9: 104-126. Edstrom, L. and Larsson, L. (1987) Effects of age on muscle fibre characteristics of fast- and slow-twitch single motor units in the rat. J. Physiol., 392: 129-145. Grimby, L. (1986) Single motor units discharge during voluntary contraction and locomotion. In: N.L. Jones (Ed.), Human Muscle Power Human Kinetics, Publishers Inc., Chicago, IL, pp. 111-130. Grimby, L., Holm, K. and Sjdstriim, L. (1984) Abnormal use of remaining motor units during locomotion in peroneal palsy. Muscle Nerve, 7: 327-331. Hofmann, P.A., Hartzell, H.C. Moss, R.L. (1991) Alterations in Ca*+ sensitive tension due to partial extraction of C-protein from rat skinned cardiac myocytes and rabbit skeletal muscle fibers. J. Gen. Physiol., 97: 1141-1163. Hofmann, P.A., Metzger, J.M., Greaser, M.L. and Moss, R.L. (1990) Effects of partial extraction of light chain 2 on the Ca2+ sensitivities of isometric tension, stiffness and velocity of shortening in skinned fibers. J. Gen. Physiol., 95: 477-498. Hughes, S.H., Cho, M., Karsch-Mizrachi, I., Travis, M., Silberstein, L., Leinwand, L.L. and Blau, H. (1993) Three slow myosin heavy chains sequentially expressed in developing mammalian skeletal muscle. Dev. Biol., 158: 183-199. Jakobsson, F., Borg, J., Edstriim, L. and Grimby, L. (1988) Use of motor units in relation to muscle fibre type and size in man. Muscle Nerve, 11: 1211-1218.
Kruger, M., Wright, J. and Wang, K. (1991) Nebulin as a length regulator of thin filaments of vertebrate skeletal muscles: correlation of thin filament length, nebulin size and epitope profile. J. Cell Biol., 115: 97-107. Larsson, L. and Moss, R.L. 1993. Maximal velocity of unloaded shortening in relation to myosin heavy and light chain isoform composition in human skeletal muscles. J. Physiol., 472: 595-614. Lenman, J.A.R. (1981) Integration and analysis of the electromyogram and related techniques. In: J. Walton (Ed.), Disorders of Voluntary Muscle, Churchill Livingstone, New York, pp. 976-1007. Moss, R.L. (1979) Sarcomere length-tension relations in frog skinned muscle fibres during calcium activation at short lengths. J. Physiol., 292: 177-192. Moss, R.L., Giulian, G.G. and Greaser, M.L. (1982) Physiological effects accompanying the removal of myosin LC, from skinned skeletal muscle fibers. J. Biol. Chem., 257: 8588-8591. Moss, R.L., Reiser, P., Greaser, M.L. and Eddinger, T. (1990) Varied expression of myosin alkali light chains is associated with altered speed of contraction in rabbit fast-twitch skeletal muscles. In: D. Pette (Ed.), The Dynamic State of Muscle Fibers, Walter de Gruyter, Berlin, New York, pp. 355-368. Rayment, I., Rypniewski, W.R., Schmidt-Base, K., Smith, R., Tomchick, D.R., Benning, M.M., Winkelmann, D.A., Wesenberg, G. and Holden, H.M. (1993) Three-dimensional structure of myosin subfragment-1: a molecular motor. Science, 261: 50-58. Salviati, G., Betto, R., Danieli-Betto, D., Biasia, E., Serena, M., Mini, M. and Scarlato, G. (1986) Myosin light chains and muscle pathology. Neurology, 36: 693-697. Sweeney, H.L., Bowman, B.F. and Stull, J.T. (1993) Myosin light chain phosphorylation in vertebrate striated muscle: regulation and function. Am. J. Physiol., 33: C1085-C1095. Thorstensson, A., Grimby, G. and Karlsson, J. (1976) Force-velocity relations and fiber composition in human knee extensor muscles. J. Appl. Physiol., 40: 12-16. Tollback, A., Knutsson, E., Borg, J., Borg, K. and Jakobsson, F. (1992) Torque-velocity relation and muscle fibre characteristics of foot dorsiflexors after long-term overuse of residual muscle fibres due to prior polio or L5 root lesion. Stand. J. Rehab. Med., 24: 151-156.
NEUROLOGICAL SCIENCES
ELSEVIER
Journal of the Neurological Sciences 132 (1995) 182-192
Contractile properties in single muscle fibres from chronically overused motor units in relation to motoneuron firing properties in prior polio patients Lars Larsson *, Xiaopeng Li, Anna Tollbtick, Lennart Grimby Departments of Clinical Neurophysiology
and Neurology, Karolinska Hospital, S-171 76 Stockholm, Sweden
Received 18 November 1994; revised 7 April 1995; accepted 27 April 1995
Abstract The relation between motoneuron firing rate in vivo and maximum velocity of unloaded shortening (V,,) and myosin isoform composition in single chemically skinned muscle fibres was investigated in chronically overused motor units. Ten patients with loss of a large proportion of the motoneuron pool due to a prior polio lesion and compensatory overuse of residual neurones were studied and compared with normal individuals. The tibialis anterior muscle (TA) was chosen and prior polio patients who used all residual TA motor units at high rates during the normal step cycle were selected. In prior polio patients, all motor units fired at approximately 40 Hz when maximum voluntary force was reached. A common firing rate of 30 Hz yielded 70-90% maximum force. In normal subjects, on the other hand, maximum TA force was reached when low threshold units fired at 25-30 Hz and high threshold units at 50 Hz. Myosin heavy chain (MHC) and light chain (MLC) isoforms were resolved by 6% and 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), respectively, and quantified densitometrically. In the whole biopsy cross-sections, types I, IL4 and IIB MHC proportions were 97, 3 and 0% in a typical prior polio patient and 65, 25 and 10% in an age- and sex-matched control subject. V,, differed significantly (p < 0.001) between type I fibres from the patient (0.54 f 0.12 ML/s) and the control subject (0.29 k 0.08 ML/s). The composition and relative contents of essential and regulatory MLC isoforms differed in single type I MHC fibres from the control subject and prior polio patient. 65% of the fibres co-expressed the fast and slow isoform of the regulatory light chain (MLC,) in the patient, while this combination was only observed in one of the control type I fibres. All prior polio fibres with a V,, higher than 0.45 ML/s, except one, co-expressed MLC,, and MLC,, and the only control fibre co-expressing the slow and fast MLC, isoform had the highest V,, (0.50 ML/s) among control fibres. On the other hand, a high relative content of MLC, was not associated with a high V,, in type I MHC fibres. It is suggested that the composition of fast and slow isoforms of MLC, has a significant modulatory influence on V,, within type I MHC fibres. This combination of MLCs and high V,, in type I MHC fibres is probably induced by chronic motor unit overuse and an altered motoneuron firing pattern. Keywords: Polio; Skeletal muscle; Motoneuron; Myosin; Shortening velocity
1. Introduction Contractile properties of adult mammalian skeletal muscle possess a remarkable capacity for adapting to altered functional demands. This is demonstrated most strikingly in the response to the radical change in contractile activity induced by cross-innervation or chronic electrical stimulation of skeletal muscle, directly or indirectly, via the motor nerve (for review see Edstrom and Grimby, 1986). The
* Corresponding author. Tel.: ( + 46-8) 729 4148; Fax: (+ 46-8) 33 99 53; e-mail:[email protected]. 0022-510X/95/$09.50 0 1995 Elsevier Science B.V. AR rights reserved SSDI 0022-510X(95)00138-7
alterations in the contractile properties are mainly related to changes in the myosin isoform composition, which in turn result from changes in the transcription of new mRNAs in response to the specific stimulus (Brownson et al., 1992). Patients who have lost a large proportion of their motoneurons and/or muscle fibres need all their residual muscle power for activities of daily living, while normal subjects have considerable reserves. In patients with critical tibialis anterior muscie (TA) weakness, due to a loss of functioning motor units and incomplete reinnervation of denervated muscle fibres, all residual motor units fire more than 10 discharges during each step cycle at 20-50 Hz. In
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normal subjects walking at comfortable speed, on the other hand, only low threshold motor units fire during the ordinary step cycle at lo-25 Hz, and high threshold units are recruited in corrective movements or during rapid locomotion, firing at 20-50 Hz (Grimby et al., 1984). Muscle specimens from patients overusing their residual TA motor units have a higher content of slow myosin than normal subjects (Borg et al., 1988; Jakobsson et al., 1988), but the maximum voluntary force at fast relative to slow speeds of foot dorsiflexion did not differ between patients overusing their residual motor units and control subjects (Tollback et al., 1992). This indicates a dissociation between mechanical and enzyme-histochemical properties, as has previously been reported in human skeletal muscle (Thorstensson et al., 1976). The interpretive value of in vivo measurements of contractile properties of human skeletal muscles is limited by factors that tend to obscure the behaviour of individual muscle fibres, but even if these difficulties were not present, multicellular preparations have the disadvantage that the roles played by different muscle fibre types in overall contractility are difficult to evaluate (Hofmann et al., 1991). The skinned fibre preparation allows investigation of the function of myofilament proteins in a cell with an intact filament lattice, but without the confounding effects related to intercellular connective tissue or protein heterogeneity between cells of multicellular preparations. The usefulness of this technique for studying relationships between contractility in the short human muscle fibre segments obtained with the percutaneous biopsy technique and the molecular protein composition of the fibre segment in normal muscle tissue has recently been documented (Larsson and Moss, 1993), and the findings provide a basis for future studies of the function of myofibrillar proteins in man. The present study was undertaken to investigate the effects of chronic overuse of residual motor units in patients with a lower motoneuron disorder, in order to improve our understanding of the influence of the voluntary pattern of activity on the motor unit. A well defined group of prior polio patients and control subjects was examined and specific interest was focused on motoneuron firing rates and the contractile properties of residual muscle fibres, together with their myosin isoform composition.
2. Materials
and methods
2.1. Material The tibialis anterior muscle (TA) was studied in 10 patients (6 women and 4 men) with prior polio in whom 24-57 years had elapsed since the acute stage of the disease. At the time of the study the patients were 46-77 years of age. The patients reported no progression of TA weakness. The findings were compared with previously
published data from normal individuals (Grimby et al., 1984; Jakobsson et al., 1988). Muscle biopsy specimens were taken from one of the female prior polio subjects (46 years) and compared with one healthy sex- and agematched control subject with no previous history of any locomotor or neurological disorder (47 years). The study was undertaken with the understanding and consent of the subjects, and was approved by the local Ethical Committee. 2.2. In vivo force measurements The force of the dorsiflexors of the foot was measured in such a way that it was not influenced by proximal leg muscles. In part of the experiment the measuring device was attached only to the foot and lower leg. In another part of the experiment it was attached to the examination bench, but could be controlled with a foot switch so that the heel was not lifted, i.e. so that tension did not derive from proximal muscles. However, the possibility of some co-contraction of plantar flexors could not be excluded. In a series of maximum voluntary contractions each subject in most attempts reached a certain force that was never exceeded, and this was designated the maximum force (for details of the experimental set-up see Grimby et al., 1984). 2.3. Single motor unit electromyographic ings
(EMG) record-
In the prior polio subjects there was no difficulty in obtaining and maintaining recordings of one single motor unit with commercial electrodes (Medelec 16829, Medelec Ltd., Woking, Surrey, UK), since the number of motor units was decreased and the fibre density within the remaining ones was increased. The risk of confusing potentials from different motor units was small, because of the characteristic shape of reinnervated potentials. When there was any doubt, a check was made to see that the potential evoked by supramaximal electrical stimulation of the peroneal nerve was identical to the voluntary potential being studied. The signals were amplified and displayed on an oscilloscope (Medelec 4239) and critical parts of the experiments were recorded on UV film at film speeds of between 10 and 100 cm/s. The surface EMG was recorded by electrodes (Medelec C162) strapped a few centimetres apart over a relatively preserved part of the TA muscle. The signals were rectified and integrated over periods of 80 ms and displayed during the following period. 2.4. Muscle biopsies and permeabilization
of fibres
Biopsy specimens were taken under local anaesthesia from the left TA in both the patients and the control subjects by means of the percutaneous conchotome method. The specimens were either frozen or placed in relaxing
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solution at 4°C. Bundles of m 50 fibres were dissected free from the samples in relaxing solution and then tied with surgical silk to glass capillary tubes at slightly stretched lengths. The bundles were chemically skinned for 24 h in relaxing solution containing 50% (v/v) glycerol at 4°C and were subsequently stored at - 20°C for up to 3 weeks before use. Other biopsy specimens were frozen in freon chilled with liquid nitrogen and stored at - 80°C. 2.5. Experimental procedure On the day of an experiment, chemically skinned single fibres were pulled free from one end of the bundle, mounted in an experimental apparatus, leaving an average fibre segment length of 2.0 f 0.6 mm and 1.9 f 0.4 mm (mean f SD) in the patient and control subject, respectively, exposed to the solution between connectors leading to a force transducer (Model 403, Cambridge Technology, Inc.) and a DC torque motor (Model 300H, Cambridge Technology, Inc.). While the fibres were in relaxing solution, sarcomere length (SL) was set to 2.75-2.85 pm by adjusting the overall segment length. This is longer than the SL recently used (2.55-2.75 pm) in a study of human muscle fibres (Larsson and Moss, 1993), which in turn was identical to the SL most commonly used in single skinned rabbit psoas fibres. However, the thin filament is longer in human quadriceps (1.30 pm) than in rabbit psoas fibres (1.12 pm), because of the species differences in the size of nebulin, i.e. the length-regulating protein of the thin filament (Kruger et al., 1991). SL was therefore increased and this is the most probable explanation for the higher specific tension observed in this study as compared with our previous observations (for details and references see Larsson and Moss, 1993). Photographs or videoprints of the fibre segments were taken with a 35-mm auto-exposure SLR camera (Contax, Yashica Kyocera GmbH, Hamburg, Germany) or a videoprinter (P71E, Mitsubishi Electric Corp., Japan). The sarcomere length, the segment width and the length of segment between the connectors were measured directly from the microscope via a TV-overlay with the aid of a digitizer connected to a microcomputer (Videoplan, Kontron Bildanalyse GmbH, Munich, Germany). The final magnification with the image analysis system on the TV screen was X 1480. Fibre SL was routinely measured in the fibres taken during maximum activation (Moss, 1979). Fibre depth was measured by recording the vertical displacement of the microscope nosepiece while focusing on the top and bottom surfaces of the fibre. Fibre cross-sectional area (CSA) was calculated from the width and depth, assuming an elliptical circumference, and was corrected for the 20% swelling that is known to occur during skinning (see Moss, 1979). Specific tension was calculated as maximum tension (P,) normalized to CSA. Relaxing and activating solutions were identical to those previously described (see Larsson and Moss, 1993) and V,,, was measured by the
slack-test procedure at 15°C (Edman, 1979; Moss et al., 1982). 2.6. Myosin heavy chain (MHC) and myosin light chain (MLC) compositions The MHC and MLC compositions of single fibres were determined by 6% and 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), respectively (for references and details see Larsson and Moss, 1993). Gels were silver-stained and subsequently scanned in a soft laser densitometer (Molecular Dynamics, Sunnyvale, CA, USA), with a high spatial resolution (50 pm pixel spacing) and 4096 optical density levels, to determine the relative contents of MHCs and MLCs. The volume integration function was used to quantify the amount of protein, and background activity was subtracted from all pixel values (ImageQuant software ~3.3, Molecular Dynamics). 2.7. Enzyme-histochemical techniques The frozen muscle samples were cut at their greatest girth perpendicular to the longitudinal axis of the muscle fibres into lo-pm thick cross-sections with a cryotome (2800 Frigocut E, Reichert-Jung GmbH, Heidelberg, Germany) at -20°C. The muscle fibres in the cross-sections were stained for myofibrillar ATPase after alkaline and acid preincubations, and classified as types I, IIA, IIB and IIC (for details see Edstrom and Larsson, 1987). The cross-sectional areas of the individual muscle fibres were measured semiautomatically on magnified black and white photographic prints of myofibrillar ATPase-stained cross-sections or directly from the microscope via a TVoverlay with the aid of a digitizing unit connected to a microcomputer (Videoplan, Kontron GmbH, Munich, Germany). The form factor was calculated according to the formula: fibre area/(?r/4 X maximum diameter X minimum diameter). The form factor is 1 for a circle or an ellipse and < 1 for irregular structures. 2.8. Statistics Means, SD and regression lines were calculated from individual values by standard procedures. A two-tailed independent t test was used for comparisons of two populations. When the two population variances were equal, i.e., when F was small, the pooled variance t test was used; otherwise, the separate-variance t test for means was used. Differences were considered significant at p < 0.05.
3. Results Long-term recordings have shown that TA is mainly used during locomotion in man (Grimby, 1986). The prior
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polio patients were selected as being able to walk with a normal pattern using all residual TA power. Normal subjects, on the other hand, use no more than 50% of their TA power when they are walking at comfortable speeds (Jakobsson et al., 1988). The overuse of residual muscle fibres in prior polio has been reported to cause hypertrophy and fast to slow myosin isoform switching (Borg et al., 1988). 3.1. Voluntary firing rate and force Motor units recruited at zero tension were called “low threshold motor units” (LMUs). When prior polio subjects slowly increased the voluntary drive to maximum LMUs started tonic firing at I 10 Hz and reached 2 50 Hz (Fig. 1). A firing rate of < 15 Hz caused little force. The steepest force increase occurred at 18-20 Hz. At these firing rates, the patients had difficulty in maintaining smooth regulation of tension. Force increased till the firing rate reached 40 Hz. The foot dorsiflexion force was plotted against the firing rate of an LMU and against the integrated surface EMG in each of the prior polio patients. The patients had visual feedback at 5, 10, 20, 30, 50, 70 and 90% of the maximal residual force and maintained each force for some seconds (Fig. 1 illustrates a more rapid increase of force to save space). The force-frequency curve had a sigmoid shape (Fig. 2 left). The curve of force plotted against the integrated surface EMG had a similar sigmoid shape (Fig. 2 right). Simultaneous recordings of one LMU and one or more other motor units showed that additional motor units were recruited till the LMU had reached a firing rate of 20 Hz. The newly recruited motor units started tonic firing at a lower rate than the LMU had reached before its recruitment. However, when the new motor unit was recruited,
Fig. 2. Relation between foot dorsiflexion force and firing rate of a TA low threshold motor unit (left) and relation between tension and surface EMG (right) in the 10 prior polio patients. Bars denote SD between patients.
the firing rate of the LMU decreased (Broman et al., 1984). The difference in firing rate between simultaneously firing motor units rarely exceeded 25% of the LMU firing rate. During increased voluntary drive the differences in firing rate decreased and all motor units fired at the same rate at 2 30 Hz. To get quantitative information on the relative firing of residual motor units, the integrated surface EMG reflecting recruitment and firing rates of a large number of motor units was compared with the firing of an LMU in each of the prior polio subjects. The global EMG was not systematically measurable when LMUs fired at 10 Hz, indicating that the number of LMUs was small. The global EMG increased more rapidly than the LMU firing rate between 15 and 25 Hz, as a sign of major recruitment. The global EMG was only about doubled when the LMU firing rate increased by 25-50 Hz, indicating that all motor units were recruited and had increased their firing rate in parallel. < 40 Hz was not compatible with maximum tension of the non-fatigued muscle, and a common firing rate of 30
Fig. 1. Firing rate of TA low threshold motor unit (upper trace) foot dorsiflexion force (solid line) and TA surface EMG integrated over periods of 70 ms and displayed during the following period (broken line). Thin line denotes zero tension and integrated EMG. Time bar = 1 s.
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Table 1 Proportions (o/o) and cross-sectional areas (CSA) of muscle fibre types classified according to enzyme-histochemical the control subject and prior polio patient. Values are means + SD. Type IIB
Type IL4
Type1
stainings for myofibrillar
ATPase in
Type IIC
Control
% 66.6
CSA (pm’) 4590+1090
% 26.8
CSA (pm*) 6330flSOO
% 6.6
CSA (pm*) 5070+1450
% 0
CSA (grn*) -
Prior polio
96.7
8600+3560
1.5
694Ok2700
0
-
1.8
1130 + 610
from the patient than from the control subject (0.90 f 0.07,
Hz gave rise to 70-90% of the maximum force. Increases from 40 to 50 Hz, on the other hand, caused no or little increase in force.
12= 200). 3.3. Myosin isoform composition
3.2. Enzyme-histochemical, morphometric and histopathological observations
MHCs were identified as types I, IL4 and IIB according to the order of migration on 6% SDS-PAGE in TA crosssections, as well as in 51 TA single fibre segments from the patient (n = 25) and the control subject (n = 26). In the control subject, the proportions of types I, IL4 and IIB MHCs were 65, 25 and lo%, respectively. In the patient, type I MHC was the only protein observed in the MHC region of the gel. However, a faint type IL4 MHC band was observed (less than 3% of the total MHC content) when the gel was overloaded with protein. Enzymehistochemical fibre type classifications and electrophoretic separations of MHCs were thus in agreement. In the prior polio patient, all single fibre segments expressed only type
In conformity with previous reports (Borg et al., 19881, a large majority of the muscle fibres in TA from the prior polio patient were classified as type I and these fibres were generally of large size (Table 1). Mild histopathological abnormalities such as central nuclei together with signs of “ fibre-splitting” were observed in the muscle specimen from the prior polio patient, whereas these abnormalities were not observed in the control subject. The number of angulated fibres was greater in the patient compared with the control subject. That is, the calculated form factor was lower (p < 0.001) in type I fibres (0.84 f 0.09, 12= 200)
1 Actin
-
MyLC3
-
2
3
4
5
6
7
6
9
10
11
Fig. 3. Electrophoretic protein analysis of single fibre segments from TA muscles. MLCs were resolved on 12% SDS-PAGE and only the low molecular weight region of the gel is shown. Lanes l-4 and 9-11, single fibres from the prior polio patient; lanes 5-8, single fibres from the control subject. One fibre is co-expressing type IIA and IIB MHC isoforms (lane 6), the other fibres are expressing type I MHC. Two fibres are co-expressing the fast and the slow isoform of the regulatory light chain (lanes 1 and 11). The abbreviations are the same as those used in the text.
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I MHCs even if the gels were overloaded with muscle fibre protein. In the control subject, three fibres co-expressed type IIA and IIB MHCs in different proportions, type IIB MHCs ranging between 44 and 88%, and the remaining fibres expressed only type I MHCs. Essential (MLC, and MLC,) and regulatory (MLC,) light chains were observed in all 51 fibres. MLC, and MLC, exist in a slow and a fast isoform (MLC,, is expressed in two isoforms-MLC,,, and MLC,,,-which are not separated with the present gel system), while MLC, only exists in a fast isoform. The essential and the regulatory MLCs are expressed in a stoichiometric 1:l relationship, but the essential/regulatory MLC ratio deviated from the expected value, probably due (1) to differential staining intensity of the light chains with silver when compared with Coomassie blue (Moss et al., 1982; Hofmann et al., 1990) and/or (2) to comigration of regulatory protein isoforms together with MLC, (Bottinelli et al., 1994). However, the MLC ratio did not differ between fibres from the control (1.9 + 0.5) subject and patient (1.9 + 0.5). Co-expression of MLC, and MLC,, is rarely observed in type I fibres from normal muscle and this combination was only found in 1 of 25 control fibres. In the prior polio patient, on the other hand, 17 of 26 fibres co-expressed
187
MLC,, and ML&,, although the MLC,, content was low in all fibres, varying between 1 and 14% (6 f 4%) of the regulatory light chain content (Fig. 3). All type I fibres except one fibre from the patient expressed the fast MLC, and the average content of MLC, was higher (p < 0.01) in the control subject than in the patient when expressed as percent of the essential (MLCi,+r + MLC,; 7.9 f 2.9% vs. 5.9 f 2.2%. MLCr,+r; 8.7 f 2.5% vs. 6.3 + 2.6%) or regulatory light chains (MLC2s+f; 16.5 f 7.4% vs. 11.2 + 4.0%).
During the manual dissection of the chemically skinned fibre bundles, no signs of fibre branching were observed in the control subject. In the patient with the prior poliomyelitis, on the other hand, branched fibres were frequently seen during dissection (Fig. 4). The proportion of branched fibres ranged between 10 and 42% of all fibres per bundle (9-12 fibres per bundle were branched), except for one bundle (17 fibres), where there were no signs of fibre branching. It was noted that the fibres were generally smaller (6000 + 580 pm’, range 5200-6600 prn2) in the bundle with no signs of fibre branching than in those bundles where branched fibres were frequently observed pm2). Some (8360f2630 pm2, range 4370-12560 branched fibres were divided into two or three different
Fig. 4. Microphotographs of four different branched chemically skinned fibre segments from percutaneous TA muscle biopsy specimens from the prior polio patient. Bar = 100 Fm.
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parts and subsequently analysed for their MHC and MLC compositions. Identical myosin isoform compositions were observed in the different branches from the same fibre. 3.4. Contractile properties in single tibialis anterior MUSde fibres Vmax was determined in the 44 single fibres which were analysed for their myosin isoform composition. However,
a V,,, value was included in subsequent analyses only when linear regressions included four or more data points, and data were discarded if r for the fitted line was less than 0.97 or if SL during isometric tension development changed by more than 0.10 pm compared with SL while relaxed (Moss, 1979). A total of 19 fibres in the control subject and 14 fibres in the patient fulfilled these criteria for acceptance and were included in the following analyses (Fig. 5).
Fig. 5. Photomicrographs of a single chemically skinned fibre from the tibialis anterior muscle of a prior polio patient; (a) in relaxing solution and (b) during maximum activation. The length of the fibre exposed to solution between connectors was 2.1 mm and the cross-sectional area was 8750 pm*. A well maintained cross-striation pattern and sarcomere length was observed during activation. Bar = 100 pm.
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et al./Journal
0.0L
0.01
0
10
20 YyLC3/YyLc2
30
0
40
5
10
15
189
20
YyLCB/YyLC 1
Fig. 6. Maximum shortening velocity (V,,,) in relation to MLC isoform composition in type I MHC fibres from a control subject (open symbols) and prior polio patient (filled symbols). MLC, content is expressed as a fraction of MLC, or MLC,. x-axis: %; y-axis: V,,, , ML/s.
fast MLC, isoform had the highest V,,, (0.50 ML/s) among control fibres and all prior polio fibres, except one, with a V,,, higher than 0.45 ML/s co-expressed MLC,, and MLC,,. Further, among the muscle fibres from the prior polio patient, V,,, was significantly higher (p < 0.05) in fibres co-expressing the fast and the slow MLC, isoform (0.57 k 0.13 ML/s) than in those expressing only MLC,, (0.42 f 0.08 ML/s). The average MLC, content was significantly higher in the control fibres than in the fibres from the patient, but there was no correlation between V,,, and MLC, content in either control or prior polio fibres (Fig. 6). The content of MLC, in relation to the essential or regulatory MLC content did not differ between muscle fibres co-expressing the fast and slow isoform of MLC, in the prior polio patient, and it therefore appears to be an unlikely factor in explaining V,,, differences within type I MHC fibres. Maximum force normalised to cross-sectional fibre area (specific tension) was significantly lower (p < 0.01) in type I fibres from the prior polio patient than in those from the control subject (Table 2). These differences in specific tensions are probably related to the different diffusion distances between large and small skinned muscle fibres rather than an impaired muscle fibre force-generating capacity in the prior polio patient. That is, there is a larger
In the control subject, V,,, ranged between 0.18 and 1.64 ML/s. In the three fibres co-expressing type IIA and IIB MHCs, V,,, ranged from 1.13 to 1.64 ML/s. In the other fibres expressing only one MHC, the slow type I MHC, V,,, ranged between 0.18 and 0.50 ML/s (0.29 f 0.08 ML/s). These values conform with the maximum shortening velocities recently found in human type I fibres from the soleus (0.32 + 0.18, IZ = 27) and vastus lateralis muscles (0.33 f 0.12, it = 41) as well as in fibres co-expressing type IIA and IIB MHCs in the vastus lateralis muscle (1.38 &- 0.75, n = 23) (Larsson and Moss, 1993). In the prior polio patient, V,,, in the type I fibres ranged between 0.33 and 0.73 ML/s (0.54 f 0.12 ML/s, n = 14) and were thus significantly (p < 0.001) faster than the type I fibres from the control TA muscle (Fig. 6, Table 2) as well as when compared with the V,,, data in type I fibres from human soleus (p < 0.001) and vastus lateralis (p < 0.001) muscles in normal young control subjects (Larsson and Moss, 1993). V,,, in these type I fibres is intermediate between that in type I and IIA fibres classified according to their MHC isoform composition (Larsson and Moss, 1993). 65% of the fibres co-expressed MLC,, and MLC,, in the prior polio patient, while only one of the control type I fibres co-expressed these isoforms. The control fibre co-expressing the slow and the
Table 2 Maximum velocity of shortening (V,,,), specific tension, resting tension (per cent of maximum tension) and cross-sectional area (CSA) in type I and HA/B muscle fibres, classified according to the myosin heavy chain (MHC) composition, from a control subject (C) and post-polio (PP) patient. Values are means f SD and the number of fibres is given within parentheses. MHC
Specific tension (N/cm* )
V,,, (ML/s) C
Type1
0.29kO.08
Type &4/B
1.36 kO.21 (3)
(16)
* * *
PP
C
0.54 + 0.12 (14) -
31.7k7.8 (16) 29.9k4.4 (3)
* *
Resting tension (%) CSA ( pm*)
PP
C
PP
C
24.6k.5.5 (14) -
3*1 (16) 3*1 (3)
3+1 (14) -
2710+670 (16) 326Ok560 (3)
Differences between control and post-polio fibres are indicated (* * p < 0.01; * * * p < 0.001).
PP * * *
8630+2230 (14) -
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diffusion gradient from the periphery to the center of the fibre in the extremely large fibres in the prior polio patient than in the smaller control fibres which may influence the concentration of the activating calcium and/or tension suppressing inorganic phosphate.
4. Discussion The major observations in this study are the differences between control subjects and prior polio patients in motoneuron firing properties and maximum velocities of unloaded shortening in muscle fibres expressing the slow (type I> myosin heavy chain isoform. Further, myosin isoform data indicate that the composition of regulatory myosin light chain isoforms has a modulatory influence on the shortening velocity within type I MHC fibres. 4.1. Motoneuron firing rate and muscle force In normal subjects, a differentiated TA motoneuron firing rate is observed during voluntary activation. During slowly increasing isometric voluntary contraction, motoneurons recruited before 25% of the maximum force has been reached start tonic firing at about 10 Hz and fire at about 30 Hz at maximum force. Motoneurons recruited after 50% maximum force, on the other hand, have a minimum firing rate of 20 Hz and a maximum rate of up to 60 Hz. Normally about three-fourths of the TA muscle fibres are type I and one-fourth type II, and the whole type I population appears to be recruited at 30-40% of maximum force. It was concluded that during normal sustained contraction, type I motor units have a firing rate of about lo-30 Hz, while type II motor units have higher minimum and maximum rates (for review see Edstrom and Grimby, 1986). In the prior polio patients the maximum TA force was about 25% of the normal force, the number of muscle fibres per motor unit was greater than normal because of collateral sprouting, and the force per muscle fibre was increased because of hypertrophy. Thus, only a small proportion of the motoneurons remained. A large selective loss of motoneurons would result in similar firing properties of residual ones and a large random loss in different proportions of motoneuron types in different patients if there were no adaptive processes. The present study shows that the minimum firing properties of residual motoneurons were differentiated as in normal subjects and that the variability between patients was small indicating that residual motoneurons or their neural drive adapt to the functional demands so that their properties become similar in similarly paralysed patients. In prior polio all residual TA motor units reached 2 50 Hz during sustained maximum voluntary effort. The low residual tension in prior polio might result in low proprioceptive inhibition and some luxury rate. However, in prior
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polio all motor units fired at 40 Hz when the maximum force was barely maintained, and a common firing rate of 30 Hz gave rise to force of only 70-90% of maximum. Since only a few per cent of the muscle fibres were type II, it is not surprising that an increase of the stimulation rate from 40 to 50 Hz caused no significant increase in tension. However, the small proportion of type II fibres cannot have been responsible for the large increase in force recorded when the common stimulation rate increased from 30 to 40 Hz. A large proportion of type I muscle fibres in prior polio must have a higher fusion rate than normal type I fibres. In normal subjects, the force-frequency curve of each single motor unit has a sigmoid shape, whereas the relation between the force and EMG of a whole muscle is linear (see review by Lenman, 19811, indicating that normal motor units reach the steep part of the individual forcefrequency curve at different levels of contraction. In our prior polio patients, on the other hand, the curve obtained by plotting the force of the whole muscle against the firing rate of low threshold motor units and the curve obtained by plotting force against integrated surface EMG both had a sigmoid shape, indicating that a large proportion of the motor units reached the steep part of the force-frequency curve at the same time, possibly because of similar contractile properties (cf. below). 4.2. Contractile properties and myosin isoform composition Contraction of muscle fibres is often characterized in terms of the maximum shortening velocity (V,,,), since in muscles from many species V,,, is proportional to the actin-activated myosin ATPase activity (for review see Larsson and Moss, 1993). The head portion of myosin (subfragment-l) contains the ATPase activity and each myosin molecule consists of six subunits, which may be variably expressed in individual fibres: two myosin heavy chains (MHCS) with molecular weights of about 200 kDa, and four myosin light chains (MLCs) of 16-25 kDa (see Larsson and Moss, 1993). Close relationships between Vmax and MHC have been demonstrated in single fibres from crustacean, amphibian, avian and mammalian skeletal muscles, including man (Larsson and Moss, 1993). However, there is considerable variation in V,,, in muscle fibres which may be due to the presence of undetected MHC subtypes and/or variable ratios of MLCs, although experimental evidence is inconsistent on this point (Larsson and Moss, 1993). The large variability in V,,, is especially pronounced within muscle fibres expressing subtypes of type II MHCs (see Larsson and Moss, 19931, although the present results also demonstrate a substantial variability within type I MHC fibres. In the past years, much interest has been paid to the study of MLCs and their possible role in determining the kinetics of the actin-myosin interaction. In smooth muscle,
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phosphorylation of the regulatory light chain results in a marked increase in actin-activated Mg-ATPase activity, but it has no effect on myosin ATPase activity in rat and rabbit striated muscle (see Sweeney et al., 1993). During maturation of mammalian fast-twitch muscle, V,,, and myosin ATPase activity increase in parallel with the reciprocal changes in the MLC,, and MLC, contents (see Moss et al., 1990). In this study the MLC, content was significantly higher in the control type I fibres than in fibres from the prior polio subject but there was no significant correlation between V,,, and MLC, content in human type I fibres from either control or prior polio TA muscle, and Vmax was significantly lower in the control fibres in spite of a higher MLC, content. Thus, the present data conform with our previous observations in skinned human fibres (Larsson and Moss, 19931, i.e. that a high MLC, content is not necessarily associated with a high V,,, in human skeletal muscle fibres, and additional work is required before conclusions regarding the effects of essential light chains on V,,, can be drawn in man. It is interesting to note that Billeter et al. (1981) observed higher MLC, contents in fibres from human skeletal muscles with signs of disuse atrophy. The lower MLC, content in prior polio patients may accordingly be related to the chronic overuse of the slow-twitch motor units. Co-expression of MLC,, and MLC,, is rarely observed in type I fibres from normal muscle (Billeter et al., 1981; Larsson and Moss, 19931, but the majority of the fibres in the prior polio patient co-expressed MLC, and MLC,,. Co-expression of fast and slow isoforms of the regulatory light chain by single fibres is also frequently observed in patients with muscular dystrophies (Salviati et al., 1986). The present results indicate that V,,, was modulated by the isoform composition of the regulatory light chains, i.e. type I MHC fibres co-expressing MLC, and MLC,, had higher shortening velocities than those expressing only MLC,, . These data are analogous with the observation that in human type IIA and IIB MHC fibres, fibres expressing MLC,, and MLC,, were slower than those expressing only MLC,, (Larsson and Moss, 1993). The mechanism underlying the modulatory influence of the regulatory light chain isoform composition on V,,, is not known, but it may involve a change in the enzymatic properties of myosin (Hofmann et al., 1990) or the mechanical rigidity of the alpha helical portion of myosin subfragment 1 (see Rayment et al., 1993). Another possibility that has to be considered is the expression of MHC isoforms that remain undetected with the gel system used in this study, and the recent finding by Hughes et al. (1993) of three different slow MHC isoforms in mammalian skeletal muscle is of specific interest in this context. It cannot be ruled out that the differences in V,,, between type I fibres in control and prior polio muscle may be attributable to transitions within these different slow MHC isoforms and that the combinations of MLCs observed in this study are primarily related to the type of slow MHC expressed in the fibre.
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4.3. Muscle fibre branching The number of branched fibres was significantly increased in the prior polio patient, especially in bundles where extreme fibre hypertrophy was observed. However, the mechanism of muscle fibre branching is still poorly understood and it remains to be determined whether branching in muscle hypertrophy and regeneration has the same aetiology (Blaivas and Carlson, 1991). Although the cause of the increased number of branched extremely hypertrophic muscle fibres in our prior polio patient cannot be determined from the present data, the identical myosin isoform composition in the different branches from the same fibre strongly support the view that all branches are innervated by the same motoneuron. 4.4. Conclusion In conclusion, the contractile properties of a muscle fibre are largely dependent on its long-term use. The TA muscle is mainly used for locomotion. Normally, low threshold motor units are used during walking at a comfortable speed, and high threshold motor units are recruited mainly during rapid locomotion and corrective movements. A corresponding differentiation of the muscle fibres seems logical. In our prior polio patients residual muscle fibres were largely used in an all or none manner. Residual TA power was fully utilised at each step cycle, but the number of step cycles per day was low. Thus, there seems to be no need for any differentiation of residual muscle fibres and perhaps nothing to incite it. It is suggested that the absence in prior polio not only of type II fibres but also of the slowest type I is due to a transition caused by their intermediate use.
Acknowledgements This study was supported by grants from the Swedish Medical Research Council (8651), the Wallenberg Foundation, the Swedish Work Environment Fund, the Bergwall Foundation, the NHR Foundation and the RTP to L.L and grants from the Swedish Medical Research Council (4749) and the RTP to L.G. We are grateful to Ms. Ulrika Mliller for performing the SDS-PAGE.
References Billeter, R., Heizmann, C.W., Howald, H. and Jenny, E. (1981) Analysis of myosin light and heavy chain types in single human skeletal muscle fibers. Eur. J. Biochem., 116: 389-395. Blaivas, M. and Carlson, B.M. (1991) Muscle fiber branching - difference between grafts in old and young rats. Mech. Ageing Dev., 60: 43-53. Borg, K., Borg, J., Edstrom, L. and Grimby, L. (1988) Effects of excessive use of remaining muscle fibres in prior polio and LV lesion. Muscle Nerve, 11: 1219-1230.
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Bottinelli, R., Betto, R., Schiaffino, S. and Reggiani, C. (1994) Unloaded shortening velocity and myosin heavy and alkali light chain isoform composition in rat skeletal muscle fibres. .I. Physiol., 478: 342-349. Broman, H., De Luca, C.J. and Mambrito, B. (1984) Motor unit recruitment and firing rates interaction in the control of human muscles. Brain Res., 337: 311-319. Brownson, C., Little, P., Jarvis, J.C. and Salmons, S. (1992) Reciprocal changes in myosin isoform mRNAs of rabbit skeletal muscle in response to the initiation and cessation of chronic electrical stimulation Muscle Nerve, 15: 694-700. Edman, K.A.P. (1979) The velocity of unloaded shortening and its relation to sarcomere length and isometric force in vertebrate muscle fibres. J. Physiol., 291: 143-150. Edstrdm, L. and Grimby, L. (1986) Effects of exercise on the motor unit. Muscle Nerve, 9: 104-126. Edstrom, L. and Larsson, L. (1987) Effects of age on muscle fibre characteristics of fast- and slow-twitch single motor units in the rat. J. Physiol., 392: 129-145. Grimby, L. (1986) Single motor units discharge during voluntary contraction and locomotion. In: N.L. Jones (Ed.), Human Muscle Power Human Kinetics, Publishers Inc., Chicago, IL, pp. 111-130. Grimby, L., Holm, K. and Sjdstriim, L. (1984) Abnormal use of remaining motor units during locomotion in peroneal palsy. Muscle Nerve, 7: 327-331. Hofmann, P.A., Hartzell, H.C. Moss, R.L. (1991) Alterations in Ca*+ sensitive tension due to partial extraction of C-protein from rat skinned cardiac myocytes and rabbit skeletal muscle fibers. J. Gen. Physiol., 97: 1141-1163. Hofmann, P.A., Metzger, J.M., Greaser, M.L. and Moss, R.L. (1990) Effects of partial extraction of light chain 2 on the Ca2+ sensitivities of isometric tension, stiffness and velocity of shortening in skinned fibers. J. Gen. Physiol., 95: 477-498. Hughes, S.H., Cho, M., Karsch-Mizrachi, I., Travis, M., Silberstein, L., Leinwand, L.L. and Blau, H. (1993) Three slow myosin heavy chains sequentially expressed in developing mammalian skeletal muscle. Dev. Biol., 158: 183-199. Jakobsson, F., Borg, J., Edstriim, L. and Grimby, L. (1988) Use of motor units in relation to muscle fibre type and size in man. Muscle Nerve, 11: 1211-1218.
Kruger, M., Wright, J. and Wang, K. (1991) Nebulin as a length regulator of thin filaments of vertebrate skeletal muscles: correlation of thin filament length, nebulin size and epitope profile. J. Cell Biol., 115: 97-107. Larsson, L. and Moss, R.L. 1993. Maximal velocity of unloaded shortening in relation to myosin heavy and light chain isoform composition in human skeletal muscles. J. Physiol., 472: 595-614. Lenman, J.A.R. (1981) Integration and analysis of the electromyogram and related techniques. In: J. Walton (Ed.), Disorders of Voluntary Muscle, Churchill Livingstone, New York, pp. 976-1007. Moss, R.L. (1979) Sarcomere length-tension relations in frog skinned muscle fibres during calcium activation at short lengths. J. Physiol., 292: 177-192. Moss, R.L., Giulian, G.G. and Greaser, M.L. (1982) Physiological effects accompanying the removal of myosin LC, from skinned skeletal muscle fibers. J. Biol. Chem., 257: 8588-8591. Moss, R.L., Reiser, P., Greaser, M.L. and Eddinger, T. (1990) Varied expression of myosin alkali light chains is associated with altered speed of contraction in rabbit fast-twitch skeletal muscles. In: D. Pette (Ed.), The Dynamic State of Muscle Fibers, Walter de Gruyter, Berlin, New York, pp. 355-368. Rayment, I., Rypniewski, W.R., Schmidt-Base, K., Smith, R., Tomchick, D.R., Benning, M.M., Winkelmann, D.A., Wesenberg, G. and Holden, H.M. (1993) Three-dimensional structure of myosin subfragment-1: a molecular motor. Science, 261: 50-58. Salviati, G., Betto, R., Danieli-Betto, D., Biasia, E., Serena, M., Mini, M. and Scarlato, G. (1986) Myosin light chains and muscle pathology. Neurology, 36: 693-697. Sweeney, H.L., Bowman, B.F. and Stull, J.T. (1993) Myosin light chain phosphorylation in vertebrate striated muscle: regulation and function. Am. J. Physiol., 33: C1085-C1095. Thorstensson, A., Grimby, G. and Karlsson, J. (1976) Force-velocity relations and fiber composition in human knee extensor muscles. J. Appl. Physiol., 40: 12-16. Tollback, A., Knutsson, E., Borg, J., Borg, K. and Jakobsson, F. (1992) Torque-velocity relation and muscle fibre characteristics of foot dorsiflexors after long-term overuse of residual muscle fibres due to prior polio or L5 root lesion. Stand. J. Rehab. Med., 24: 151-156.