myosin heavy chain
Non-Technical Summary The contractile function of human single muscle fibres is of particular importance for whole muscle contractile function. Yet, whereas ageing and short-term disuse (immobilisation) separately have been shown to impair single fibre contractile function, very little attention has been given to their combined effects. We show that 2 weeks of lower limb immobilisation reduces force and specific force (force per cross-sectional area) of both slow and fast single muscle fibres and that this occurred to a similar extent in young and old individuals. In contrast, disuse led to reduced Ca2+ sensitivity in fast fibres of young and in slow fibres of old, respectively. These results help us to better understand the underlying physiological mechanisms responsible for the deleterious effects of short-term disuse on whole muscle contractile function in both young and old.
Abstract Very little attention has been given to the combined effects of healthy ageing and short-term disuse on the contractile function of human single muscle fibres. Therefore, the present study investigated the effects of 2 weeks of lower limb cast immobilisation (i.e. disuse) on selected contractile properties of single muscle fibres (n= 378) from vastus lateralis of nine young (24 ± 1 years) and eight old (67 ± 2 years) healthy men with comparable levels of physical activity. Prior to immobilisation, MHC IIa fibres produced higher maximum Ca2+-activated force (approx. 32%) and specific force (approx. 33%) and had lower Ca2+ sensitivity than MHC I fibres (P < 0.05), with no differences between young and old. After immobilisation, the decline in single fibre force (MHC I: young 21% and old 22%; MHC IIa: young 22% and old 30%; P < 0.05) as well as specific force (MHC I: young 14% and old 13%; MHC IIa: young 18% and old 25%; P < 0.05) was more pronounced in MHC IIa fibres compared to MHC I fibres (P < 0.05), with no differences between young and old. Notably, there was a selective decrease in Ca2+ sensitivity in MHC IIa fibres of young (P < 0.05) and in MHC I fibres of old individuals (P < 0.05), respectively. In conclusion, 2 weeks of lower limb immobilisation caused greater impairments in single muscle fibre force and specific force in MHC IIa than MHC I fibres independently of age. In contrast, immobilisation-induced changes in Ca2+ sensitivity that were dependent on age and MHC isoform.
Parameters of muscle mechanical function appear to be more markedly impaired in old compared to young individuals after brief periods of disuse (7–14 days) (Deschenes et al. 2008; Hvid et al. 2010). In order to counteract the deleterious effects of short-term disuse on contractile muscle function in the elderly, it is therefore of vital importance to identify the underlying physiological mechanisms.
Bypassing all preceding activating steps, the distinct contractile function of single muscle fibres have been proposed to be important for whole muscle mechanical function (Bottinelli et al. 1999; Frontera et al. 2000; Trappe et al. 2004; Korhonen et al. 2006; Yu et al. 2007; Erskine et al. 2009; Canepari et al. 2010). Apart from dynamic measures of single muscle fibre contractile function (e.g. shortening velocity and power) (Bottinelli et al. 1999; Widrick et al. 1998, 2002; D'Antona et al. 2003; Trappe et al. 2003, 2004; Yu et al. 2007), properties such as maximal Ca2+-activated isometric force and specific force (force normalized to fibre cross-sectional area, CSA) as well as measures of Ca2+ sensitivity derived from the force–Ca2+ relationship (i.e. relative force at graded Ca2+ concentrations) also provides fundamental information about the mechanical behaviour of single muscle fibres. In addition to the involvement of other myofibrillar proteins (e.g. troponin and tropomyosin), these properties are governed by the myosin heavy chain (MHC) isoform composition of the muscle fibre, where clear distinctions have been observed between MHC I and IIa fibres (Bottinelli et al. 1999; Trappe et al. 2003, 2004; Canepari et al. 2010). Furthermore, the overall MHC isoform composition has previously been demonstrated to strongly influence parameters of whole muscle mechanical function (Harridge et al. 1996; Aagaard & Andersen, 1998; Korhonen et al. 2006; Hvid et al. 2010). However, evidence exists to suggest that the contractile properties of single muscle fibres can be altered without detectable changes in MHC isoform composition, indicating the presence of intrinsic ‘qualitative’ changes (Bottinelli et al. 1999; Canepari et al. 2010).
Viewed separately, ageing and disuse have been shown to reduce single fibre force and specific force as well as to alter Ca2+ sensitivity in both MHC I and IIa fibres (Larsson et al. 1997; Widrick et al. 1998; Frontera et al. 2000; D'Antona et al. 2003; Trappe et al. 2003, 2004; Korhonen et al. 2006; Ochala et al. 2007; Yu et al. 2007). However, very little attention has been given to the combined effects of ageing and short-term disuse on the contractile function of human single muscle fibres. To our knowledge, only one study has addressed this aspect previously, and while based on cross-sectional data, D’Antona and colleagues observed that age-related impairments in the contractile properties of single muscle fibres were amplified with long-term disuse (3.5 months of immobilisation due to total knee arthroplasty) (D'Antona et al. 2003). It is difficult, however, to ascertain whether these results reflected 3.5 months of post-surgical immobilisation or were the result of a generally reduced physical activity level (due to knee joint pain) in the years preceding the operation (Zeni & Snyder-Mackler, 2010) since the level of physical activity per se has been shown to affect the contractile properties of human single muscle fibres (D'Antona et al. 2007). Thus, knowledge is lacking about the effect of short-term disuse on single muscle fibre contractile function in old healthy individuals, and it also remains unknown whether the potential adaptive modulations differ from those occurring in young healthy individuals. Albeit data are lacking in humans, findings from animal studies indicate that ageing may be associated with an elevated susceptibility to the deleterious effects of short-term disuse on the contractile function of single muscle fibres (Thompson et al. 1998; Arora et al. 2008).
The more marked impairment in muscle mechanical function observed in old compared to young individuals in response to short-term disuse (Hvid et al. 2010), might have involved age-related changes in single muscle fibre contractile properties. Therefore, the intent of this study was to investigate the effects of 2 weeks of lower limb immobilisation (i.e. disuse) on the contractile function of vastus lateralis single muscle fibres obtained from young and old healthy men with comparable levels of physical activity. Assessment of contractile properties comprised maximal Ca2+-activated force and specific force, force–Ca2+ relationship (including activation threshold, Ca2+ sensitivity, slope steepness of the force–Ca2+ curve), as well as Sr2+ sensitivity and differential sensitivity (i.e. Ca2+– Sr2+ sensitivity). We hypothesised that single muscle fibre contractile function would be more severely affected in old compared to young subjects following immobilisation.
This study was approved by the ethical committee of Copenhagen and Frederiksberg in accordance with the Declaration of Helsinki (KF01-322606). All subjects were informed of the risks associated with the investigation and provided written, informed consent prior to their participation in the study.
Seventeen healthy men, eight old (66.6 ± 1.5 years, 180.0 ± 2.5 cm, 90.5 ± 3.4 kg) and nine young (23.7 ± 0.6 years, 180.0 ± 1.8 cm, 75.7 ± 2.9 kg) volunteered to participate in the study. These subjects were part of a larger study that investigated the physiological and muscle mechanical effects of short-term immobilisation in young and old healthy men (Suetta et al. 2009; Hvid et al. 2010). None of the subjects had previous records of acute or chronic illness or took any medication affecting skeletal muscle anatomy, physiology or function. Furthermore, none had previously been engaged in systematic resistance training. The level of physical activity was assessed using a questionnaire estimating the amount of occupational and recreational activities (Saltin & Grimby, 1968), and showed no differences between old and young individuals (4.8 ± 1.5 vs. 4.5 ± 1.1 h week−1, respectively). Hence, the influence of age-related differences in physical activity as a confounding variable was minimized, to ensure that the results primarily reflected the effect of ageing per se.
The disuse protocol has previously been described in detail (Suetta et al. 2009), but in brief this was accomplished by 2 weeks of randomized unilateral lower limb cast immobilisation. Using a lightweight cast (X-lite, Allard, USA) enclosing the entire leg from just below the groin to just above the malleoli, the knee joint was set at an angle of 30 deg (0 deg is full extension). This joint angle was chosen to avoid ground contact in the immobilised leg during ambulatory and postural activities. Following careful instruction, subjects were required to perform all ambulatory activities using crutches, and were explicitly informed to refrain from any kind of weight-bearing activities (i.e. ground contact) and to avoid volitional muscle contractions in the immobilised leg. In order to avoid iatrogenic, muscular and vascular complications, daily contact was kept with all subjects throughout the immobilisation period. To reduce the risk of venous thrombosis, subjects were instructed to perform unloaded ankle plantar and dorsal flexion several times a day with the foot of the immobilised leg.
Muscle biopsy sampling and analysis
Muscle samples were obtained from the mid-part of the vastus lateralis muscle using needle biopsy technique with suction (Bergstrøm, 1962). Samples were obtained approximately 1 week prior to the immobilisation period (Pre) as well as immediately after removal of the whole-leg cast (Post). Efforts were made to extract tissue from the same depth and vicinity of location.
While kept cold on an ice-cooled Petri dish, each muscle sample was divided into two parts. The first part, used for single fibre analysis (see below), was quickly placed into storage (glycerol) solution and stored for 24 h at 4°C and subsequently at –20°C until the day of analysis. The second part, used for whole muscle (homogenate) MHC analysis (see below), was weighed and manually homogenized in 1:10 volumes (weight to volume ratio) of ice-cold buffer (in mm: 300 sucrose, 1 EDTA, 10 NaN3, 40 Tris-base and 40 l-histidine, pH 7.8) at 0°C in a 1 ml glass homogenizer using a glass pestle (Kontes Glass Industry, Vineland, NJ, USA). All homogenates were frozen in liquid nitrogen and stored at –80°C until the day of analysis.
Single muscle fibre analysis
Preparation and cross-sectional area Single muscle fibres were selected and analysed using blinded systematic randomisation. Hence, analysis was alternated between young and old subjects, and with fibres obtained from the same subject analysed at the same time (i.e. Pre followed by Post measurements or vice versa). On the day of analysis, a small muscle bundle (approx. 40 fibres) was blotted and transferred into cold paraffin oil (0–5°C). Under a stereomicroscope (Stemi 2000-C, Zeiss, Germany), single muscle fibres were carefully isolated using fine-precision forceps (No. 5, Dumont, Switzerland). Fibres were randomly selected from different sections of the muscle bundle. A loop of surgical silk (Genzyme, MA, USA) was attached to each end of an isolated fibre, and small metal pins were used to carefully stretch and fix the fibre at a length where its curved appearance disappeared (within ±1% of the measured slack length, see below). Using a digital camera (Canon, Powershot A80, Japan) placed on top of the stereomicroscope through a conversion lens adapter (Canon, LA-DC583, Japan), an image was taken and stored for later analysis of single fibre diameter. Cross-sectional area (CSA) was calculated from the mean of three diameter measurements along the fibre using iTEM software (version 5.0, Olympus, Germany), by assuming a circular shaped fibre. No corrections were made for fibre swelling.
Solutions The storage solution contained (in mm): 5 EGTA, 2 Na2+-ATP, 2 MgCl2, 150 potassium propionate, and 50% vol/vol glycerol. Solutions having different concentrations of free Ca2+ were made by mixing three stock solutions as previously published (Nielsen et al. 2007). The standard K-HDTA intracellular solution contained (in mm): 50 HDTA2− (Fluka, Buchs, Switzerland) as the impermeant anion, 8 Na2+-ATP, 10 Na2+-CrP, 36 Na+, 127 K+, 8.5 total Mg2+ and 90 Hepes. Two solutions similar to the K-HDTA solution were made with all HDTA replaced by 50 mm EGTA (pCa > 9.0, relaxing solution) or 50 mM Ca-EGTA (pCa ≈ 4.5, maximal activating solution). The Ca2+ dependence of force production by the contractile apparatus was examined by exposing a fibre segment to a sequence of solutions of progressively higher free [Ca2+], which were made by mixing the HDTA-based solution and appropriate EGTA/Ca-EGTA solutions as previously described (Stephenson & Williams, 1981), with the total [EGTA] set at 10 mm (i.e. 8 parts of HDTA-based solution to 2 parts EGTA-based solution) to ensure strong Ca2+ buffering. A series of seven solutions with various pCa (–log[Ca2+]) values were used: pCa 9.0 (relaxing solution, 100% EGTA), pCa 6.7, 6.4, 6.2, 5.9, 5.7 and 4.5 (maximal activating solution, 100% Ca-EGTA). A similar series was made with Sr2+ instead of Ca2+, using a Sr 40/50 stock solution which contained (in mm): 90 Hepes, 8.5 MgO, 50 EGTA, 40 SrCO3, 8 Na2+-ATP and 10 Na2+-CrP. Solutions of progressively higher free [Sr2+] were made by mixing relaxing solution (100% EGTA) and Sr 40/50 solutions in appropriate volumes (Stephenson & Williams, 1981). A series of seven solutions with various pSr (–log[Sr2+]) values were used: pSr 9.0 (relaxing solution, 100% EGTA), pSr 6.3, 5.6, 5.0, 4.8, 4.5 and 4.0 (submaximal and maximal activating solutions: 100% mix of EGTA and Sr 40/50). All solutions had an osmolality of 298 ± 8 mosmol.l−1, a pH of 7.10 ± 0.01 and a calculated free [Mg2+] of 1 mm (Lamb & Stephenson, 1994). The pCa of solutions (for pCa < 7.2) was measured with a Ca2+-sensitive electrode (Orion Research, Cambridge, MA, USA) (Nielsen et al. 2007). Chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA), unless otherwise stated.
Properties of contractile function After the preparation procedures the isolated muscle fibre was mounted between a force transducer (AE801, Memscap, France) fixed to a micromanipulator and a rigid metal pin under a dissecting microscope (Nikon, Japan) with an integrated eyepiece graticule for length measurements (Nielsen et al. 2007). Using this setup, the fibre could rapidly be transferred between chambers containing relaxing or activating solution (Stephenson & Williams, 1981; Lynch et al. 1993, 1994). Following mounting, the fibre was ‘washed’ for ∼15 s in relaxing solution and subsequently skinned chemically for ∼60 s in relaxing solution with 1% Triton X-100 added. Together with the storage solution, the latter process dissolves all membrane structures, leaving an intact and freely accessible contractile apparatus. After skinning, the fibre were immersed in relaxing solution and carefully adjusted to slack length (100%), defined as the length where a force rise could be visually identified (<0.015 mN for all analysed fibres), corresponding to the point where the curved appearance of the slack fibre disappeared. Knot-to-knot distance (slack length, 1.76 ± 0.02 mm for all analysed fibres, mean ± SEM) was measured in this condition, after which the fibre was adjusted to 120% slack length to ensure optimal conditions for cross-bridge interaction and held for 120 s to ensure plateauing in passive force. The fibre was then immersed in the different solutions in order to obtain maximal Ca2+-activated force (pCa 4.5), force–Ca2+ relationship (pCa 9.0 → 4.5), as well as force–Sr2+ relationship (pSr 9.0 → 4.0) (Fig. 1). In addition, a second and third maximal Ca2+-activated force was carried out again after both force–Ca2+ and force–Sr2+ relationship, respectively (Fig. 1). If fibres broke or if maximal Ca2+-activated force differed by more than 15% between successive recordings (first vs. third or first vs. second in case fibres broke during force–Sr2+ relationship), the measurements were discarded. All measurements were carried out at room temperature (22.1 ± 0.0°C for all analysed fibres, mean ± SEM).
The signals from the force transducer were sampled at 1000 Hz, and stored on a PC for later analysis using custom-made software (LabView 8.0, National Instruments, TX, USA) (Nielsen et al. 2007). All transducers used for measurements were calibrated using standardized weights (3.8–142.9 mg, r2= 0.99). Force responses were calculated as the difference in force when immersed in relaxing solution compared to that of submaximal or maximal activating solution (at 120% slack length). Maximal Ca2+-activated force was expressed in absolute units (mN) and as specific force (force normalized to fibre CSA, kN m−2). Using non-linear curve fitting (GraphPad Prism 4.0, GraphPad Software Inc., CA, USA), the force–Ca2+ relationship was determined by plotting relative force, calculated by normalizing each submaximal force to the maximal force, as a function of pCa. Based on the Hill equation, activation threshold (defined as concentration of Ca2+ needed to elicit 10% of maximal force, pCa10), Ca2+ sensitivity (defined as concentration of Ca2+ needed to elicit 50% of maximal force, pCa50), as well as Hill coefficient (representing slope of the relationship, n) were derived as previously described (Lynch et al. 1993, 1994). Sr2+ sensitivity (defined as the concentration of Sr2+ needed to elicit 50% of maximal force, pSr50) was the only parameter derived from the force–Sr2+ relationship. Based on the differential sensitivity to Ca2+ and Sr2+ (i.e. pCa50– pSr50, Δ50) it is possible to type a fibre, i.e. relating it to a measure of contractile function (Lynch et al. 1994). In addition to yielding information of the functional diversity of human single muscle fibres, this measure was included in our analysis to serve as a verification of (and alternative to) the electrophoretic analysis.
MHC isoform analysis in whole muscle homogenate and single fibres
The MHC isoform composition of whole muscle homogenates was analysed using gel electrophoresis as previously described (Danieli et al. 1986), using a modified protocol for human muscle samples (Andersen & Aagaard, 2000). Briefly, muscle homogenate (80 μl) was mixed with 200 μl of sample-buffer (10% glycerol, 5% 2-mercaptoethanol and 2.3% SDS, 62.5 mm Tris and 0.2% bromophenolblue at pH 6.8), boiled in a water bath at 100°C for 3 min and loaded (10–40 μl) on a SDS-PAGE gel (6% polyacrylamide (100:1 acrylamid:bisacrylamid), 30% glycerol, 67.5 mm tris-base, 0.4% SDS and 0.1 m glycine). Gels were run at 80 V for at least 42 h at 4°C and MHC bands were visualized with Coomassie staining. Subsequently, all gels were scanned (Linoscan 1400 scanner, Heidelberg, Germany) and MHC bands quantified densitometrically (Phoretix 1D, nonlinear, Newcastle, UK). MHC II was identified by Western blotting using a monoclonal antibody (Sigma M 4276) and the Xcell IITM protocol (Invitrogen, Carlsbad, CA, USA).
Following measurements of contractile function, each single fibre was placed in an Eppendorf tube containing 20 μl of sample-buffer (see above), boiled for 3 min, and stored at –80C° until further analysis of the myosin heavy chain (MHC) composition by SDS-PAGE (Danieli et al. 1986). Fibres were run on an 8% polyacrylamide gel for 42 h as previously described (Andersen & Aagaard, 2000). Subsequently, gels were silver stained using a commercial kit (Amresham Bioscienes AB, Uppsala, Sweden). MHC I, IIa, IIx or mixed isoforms (I/IIa, IIa/IIx) were determined by comparing protein band migration to a standard myosin extract run in one or more lanes on the gel (Nielsen et al. 2007). Figure 2 shows a representative MHC gel analysis.
Statistical analysis on the contractile function of human single muscle fibres was performed only on pure MHC I and IIa fibres. This was carried out using a linear mixed model (STATA 10.1, StataCorp, College Station, TX, USA). If data were not normally distributed, appropriate transformations were carried out prior to analysis (including log, square-root, inverse of square-root and inverse of cubic). Properties of contractile function were analysed with Subject ID and MHC isoform as random effects and with Group (Young, Old), Time (Pre, Post) and MHC isoform (I, IIa) as fixed effects. Whole muscle (homogenate) MHC isoform composition were analysed with Subject ID as random effect and with Group (Young, Old) and Time (Pre, Post) as fixed effects. Data are presented as mean ± SEM. The level of statistical significance was P < 0.05.
Whole muscle homogenate MHC isoform composition
Prior to immobilisation, MHC isoform composition did not differ between young and old. Furthermore, immobilisation did not lead to any changes in MHC I, IIa and IIx composition in either young or old (Table 1). Overall, whole muscle homogenate MHC isoform composition in both young and old corresponded with previously reported (Harridge et al. 1996; D'Antona et al. 2007).
|%||Young men||Old men|
|MHC I||52 ± 4||55 ± 6||60 ± 5||54 ± 4|
|MHC IIa||46 ± 5||43 ± 6||37 ± 6||42 ± 3|
|MHC IIx||2 ± 1||2 ± 1||2 ± 1||4 ± 1|
Single muscle fibre number and MHC isoforms
A total of 378 single muscle fibres were successfully analysed, corresponding to approximately 11 fibres per subject per time point. Since the number of fibres expressing pure MHC IIx or mixed isoforms were too limited (approx. 10%) to provide any meaningful statistical comparisons, results are only given for pure MHC I and IIa fibres. Due to the methodological approach (Fig. 1), different numbers of MHC I and IIa fibres are presented with different measures, i.e. CSA, force and specific force (n= 337), force–Ca2+ relationship (n= 252), and force–Sr2+ relationship (n= 221).
Single muscle fibre CSA, force and specific force
Prior to immobilisation, CSA did not differ between young and old individuals or between MCH I and IIa (Fig. 3A). Following immobilisation, MHC IIa CSA decreased (P < 0.05) in young (4% from 6458 ± 300 to 6215 ± 318 μm2) and old (10% from 7587 ± 348 to 6626 ± 340 μm2). In contrast, MHC I CSA remained unchanged in young and only tended (P= 0.094) to decrease in old (13% from 6922 ± 609 to 6213 ± 576 μm2) (Fig. 3A).
Whereas maximal Ca2+-activated force of MHC I and IIa were similar between young and old prior to immobilisation, MHC I demonstrated lower force (P < 0.05) than MHC IIa in both young (38%, 0.50 ± 0.03 vs. 0.80 ± 0.05 mN) and old (26%, 0.59 ± 0.04 vs. 0.79 ± 0.08 mN) (Fig. 3B). Following immobilisation, force decreased (P < 0.05) in both MHC I and IIa in young (MHC I: 21% to 0.40 ± 0.02 mN; MHC IIa: 22% to 0.63 ± 0.05 mN) and old (MHC I: 22% to 0.45 ± 0.04 mN; MHC IIa: 30% to 0.55 ± 0.07 mN) (Fig. 3B). The observed decrease in MHC IIa force were larger than that observed in MHC I, both in young and old (P < 0.05) (Fig. 3B).
Comparable trends were observed when maximal Ca2+-activated force was normalized to fibre CSA. Specifically, specific force did not differ between young and old prior to immobilisation, and MHC I demonstrated lower (P < 0.05) specific force than MHC IIa both in young (32%, 80.9 ± 4.3 vs. 118.8 ± 6.0 kN m−2) and old (34%, 77.0 ± 3.1 vs. 117.4 ± 7.9 kN m−2) (Fig. 3C). Specific force decreased (P < 0.05) with immobilisation in both MHC I and IIa of young (MHC I: 14% to 69.2 ± 4.0 kN m−2; MHC IIa: 18% to 96.9.0 ± 5.1 kN m−2) and old (MHC I: 13% to 67.2 ± 3.7 kN m−2; MHC IIa: 25% to 88.0 ± 6.9 kN m−2) (Fig. 3C), with MHC IIa demonstrating larger decreases than MHC I (P < 0.05) (Fig. 3C).
Single muscle fibre force–Ca2+ relationship, force–Sr2+ relationship and differential sensitivity
Prior to immobilisation, Ca2+ activation threshold, Ca2+ (and Sr2+) sensitivity, differential sensitivity, and steepness of the force–Ca2+ relationship were similar in young and old individuals (Table 2, Fig. 4). However, MHC I was characterised by a lower activation threshold, a lower differential sensitivity, and a less steep slope of the force–Ca2+ curve than MHC IIa (young: 30%, 3.35 ± 0.20 vs. 4.80 ± 0.25; old: 32%, 3.17 ± 0.16 vs. 4.67 ± 0.47), along with an elevated Ca2+ and Sr2+ sensitivity (P < 0.05) (Table 2, Fig. 4). Hence, MHC I fibres required less free Ca2+ to reach 10% and 50% relative force, respectively, both in young (29 and 9%, respectively) and old (51 and 34%, respectively) compared to MHC IIa fibres (P < 0.05) (data not shown).
|MHC I||Young men||Old men|
|Pre (n= 40)||Post (n= 35)||Pre (n= 44)||Post (n= 34)|
|pCa10||6.23 ± 0.04||6.30 ± 0.07||6.31 ± 0.05||6.22 ± 0.04a|
|pCa50||5.90 ± 0.03||5.92 ± 0.04||5.96 ± 0.04||5.93 ± 0.03a|
|n||3.35 ± 0.20||3.15 ± 0.26||3.17 ± 0.16||3.58 ± 0.19a|
|Pre (n= 33)||Post (n= 33)||Pre (n= 41)||Post (n= 32)|
|pSr50||6.27 ± 0.03||6.27 ± 0.03||6.34 ± 0.01||6.32 ± 0.02a,e|
|Δ50||−0.37 ± 0.02||−0.36 ± 0.02||−0.38 ± 0.03||−0.38 ± 0.01a|
|MHC IIa||Pre (n= 40)||Post (n= 28)||Pre (n= 15)||Post (n= 16)|
|pCa10||6.08 ± 0.03b||6.01 ± 0.04a,b||6.00 ± 0.06b||5.92 ± 0.06b|
|pCa50||5.86 ± 0.02b||5.79 ± 0.02a,b||5.78 ± 0.05b||5.73 ± 0.05b|
|n||4.80 ± 0.25b||5.77 ± 0.63a,b||4.67 ± 0.47b||7.36 ± 1.26b|
|Pre (n= 36)||Post (n= 21)||Pre (n= 13)||Post (n= 12)|
|pSr50||5.72 ± 0.02b||5.69 ± 0.03a,b||5.67 ± 0.04b||5.53 ± 0.10b,e|
|Δ50||0.14 ± 0.02b||0.12 ± 0.02b||0.14 ± 0.03b||0.26 ± 0.07b,d|
In young subjects, MHC IIa (but not MHC I) showed decreased Ca2+ and Sr2+ sensitivity following immobilisation, accompanied by elevated Ca2+ activation threshold and an increase in steepness of the force–Ca2+ curve (20% to 5.77 ± 0.63) (P < 0.05) (Table 2 and Fig. 4). Consequently, the amount of free Ca2+ required to reach 10% and 50% relative force, increased in MHC IIa (18 and 17%, respectively, P < 0.05) (data not shown). In contrast, old MHC I fibres (but not MHC IIa) showed decreased Ca2+ and Sr2+ sensitivity following immobilisation, accompanied by elevated Ca2+ activation threshold and an increase in steepness of the force–Ca2+ curve (13% to 3.58 ± 0.19) (P < 0.05) (Table 2, Fig. 4). Thus, the amount of free Ca2+ required to reach 10% and 50% relative force, increased in MHC I (21 and 7%, respectively, P < 0.05) (data not shown). The observed changes in Sr2+ sensitivity in MHC I and IIa were greater in old than in young (P < 0.05) (Table 2).
Fibre typing by differential sensitivity
Single muscle fibres (young and old pooled) having differential sensitivity values below zero were identified as MHC I fibres by gel electrophoresis with a specificity of 93% (n= 144 fibres), and those having differential sensitivity values above zero were identified as MHC II fibres (IIa, IIa/IIx, IIx) with a specificity of 97% (n= 105 fibres).
The present study investigated the combined effect of ageing and short-term disuse on the contractile properties of human single muscle fibres for the first time. The main findings were that 2 weeks of lower limb immobilisation (i.e. disuse) elicited a greater decrease in vastus lateralis single muscle fibre force and specific force of MHC IIa fibres than MHC I fibres, independently of age. In contrast, the present data revealed that disuse-induced changes in characteristics of the force–Ca2+ relationship that were dependent on age, with young individuals selectively demonstrating changes in MHC IIa fibres and old individuals selectively in MHC I fibres. Thus, the initial study hypothesis that single fibre contractile function would be more affected in old compared to young individuals could not be fully supported by the present data.
Effects of ageing
Ageing is associated with a progressive impairment in whole muscle mechanical function (Aagaard et al. 2010), which has been suggested to rely at least in part on changes in the contractile properties of single muscle fibres (Frontera et al. 2000; Korhonen et al. 2006; Yu et al. 2007; Canepari et al. 2010). However, the present study observed no differences between young and old healthy men with comparable levels of physical activity in MHC I and IIa single fibre CSA, force or specific force. Although the maintained specific force (i.e. unaltered intrinsic ‘quality’) conflicts with some previous findings of an age-related decline in specific force (Larsson et al. 1997; Frontera et al. 2000; D'Antona et al. 2003; Ochala et al. 2007; Yu et al. 2007), this disparity is most probably explained by differences in subject characteristics. Importantly, detailed information about the level of physical activity has been lacking in most studies, regardless of this parameter having essential influence on the contractile function of single muscle fibres (D'Antona et al. 2007). Notably, the present findings of maintained specific force with ageing are supported by previous cross-sectional studies comparing young and old individuals having similar levels of physical activity (Trappe et al. 2003; Korhonen et al. 2006). One should also bear in mind that the findings of the present study are restricted to the age of the included old subjects (67 ± 2 years). Nevertheless, even in very old individuals (>80 years) it seems that the isometric contractile function of single muscle fibres is preserved, and that the age-related decline in knee extensor muscle mechanical function is due to other factors such as a reduction in quadriceps muscle CSA (Trappe et al. 2003; Frontera et al. 2008; Raue et al. 2009).
Three main mechanisms could be responsible for the previous reports of an age-related decline in single fibre specific force, i.e. a reduced number of cross-bridges (indicated by a lower concentration of myosin), and/or a reduced force produced per cross-bridge (both in the low- and strong-binding state), and/or a lower fraction of myosin heads in the strong-binding force-generating state (Larsson et al. 1997; Lowe et al. 2001; D'Antona et al. 2003; Zhong et al. 2006; Haus et al. 2007b; Prochniewicz et al. 2007). Furthermore, ageing is also known to be associated with modifications and/or reduced concentrations of other cytoskeletal proteins important for contractile function (Bloch & Gonzalez-Serratos, 2003; Chopard et al. 2005; Haus et al. 2007b). In the present study, however, the maintained specific force in single fibres from old individuals suggests that the above mechanisms mainly become initiated as a result of progressively reduced levels (and/or altered patterns) of physical activity rather than occurring in consequence of ageing per se. In support of this notion, the myosin concentration in muscles or single fibres from both human and animals, as well as the fraction of myosin heads in the strong-binding state of fast (semimembranosus, type II) single fibres in rats, has been shown to be either unaltered with ageing, or not reduced before reaching a very high age (Trappe et al. 2003; Thompson et al. 2006; Zhong et al. 2006; Haus et al. 2007b; Prochniewicz et al. 2007).
To our knowledge, no previous investigations have examined the influence of ageing on the force–Ca2+ (and force–Sr2+) relationship of human single muscle fibres. Although such data were previously reported for old individuals, the absence of a young reference group made it difficult to deduce the effect of ageing (Godard et al. 2002). The present findings showing that ageing did not affect the force–Ca2+ (or force–Sr2+) relationship are therefore novel within this area of research. This is also supported by the limited animal data showing no age-related changes in the force–Ca2+ (or force–Sr2+) relationship of slow (SOL, type I) fibres, and if any, only minor changes in fast (EDL, type II) fibres (Eddinger et al. 1986; Lynch et al. 1993; Plant & Lynch, 2001). Since the appearance of the force–Ca2+ (and force–Sr2+) relationship curve is mainly governed by the expression of troponin (Tn) and tropomyosin (Tm) isoforms as well as that of regulatory myosin light chains (MLCs) (MacIntosh, 2003), we assume that these proteins were not influenced in our old subjects.
Effects of MHC isoform composition
The previous observations of marked distinctions in the contractile properties of single fibres between MHC I and IIa (Lynch et al. 1994; Bottinelli et al. 1999; Canepari et al. 2010) were confirmed by the present data. For instance, the 37% higher specific force in MHC IIa compared to MHC I fibres of our young and old individuals were in accordance with the previously reported range of 9–50% (Larsson et al. 1997; Bottinelli et al. 1999; Frontera et al. 2000; D'Antona et al. 2003; Trappe et al. 2003, 2004). Although it has been suggested that myosin represents a greater fraction of the total protein content in human MHC IIa vs. I fibres (Carroll et al. 2004), the majority of evidence indicates that myosin concentration is similar between human muscle fibres expressing different MHC isoforms, and between slow and fast animal muscles (D'Antona et al. 2003; Chopard et al. 2005; Thompson et al. 2006; Borina et al. 2010). Collectively, these data indicate that the reduced specific force in MHC I vs. IIa fibres may predominantly be caused by a reduced force produced per attached cross-bridge and/or due to a lower fraction of myosin heads in the strong-binding force-generating state, which is partly supported by recent human single muscle fibre data (Li & Larsson, 2010).
In the present study the force–Ca2+ (and force–Sr2+) relationship were found to differ markedly between MHC I and IIa fibres of both young and old individuals. Specifically, MHC I fibres were more sensitive to Ca2+ (and Sr2+) than MHC IIa fibres, and showed a less steep slope (indicating a lower degree of co-operativity between the involved regulatory proteins), in accordance with previous findings in young and old (Lynch et al. 1993, 1994; Widrick et al. 1998; Yamashita-Goto et al. 2001; Bastide et al. 2002; Godard et al. 2002; Mounier et al. 2009). These fibre-type-related disparities may rely on differences in Tn, Tm and regulatory MLC isoform expression, which has been shown to modulate actin–myosin interactions (Bastide et al. 2002; MacIntosh, 2003; Mounier et al. 2009).
The above analysis enabled us to distinguish single muscle fibres by their differential sensitivity (i.e. pCa50– pSr50, Δ50). Apart from providing information about the functional diversity of human single muscle fibres in relation to their contractile properties (Bottinelli et al. 1999; Canepari et al. 2010), this measure (Δ50) has previously been put forward as a means of classifying fibre types (Lynch et al. 1994). Nevertheless, concurrent assessments of differential sensitivity and MHC isoform composition have not previously been carried out in human single muscle fibres from old individuals, or from the human vastus lateralis muscle in general. The present data (fibres from young and old pooled) showed that differential sensitivity (Δ50) may be used in human single muscle fibres as an alternative to gel electrophoresis for fibre type classification into the two main MHC isoforms (I vs. II).
Effects of immobilisation
The combined effect of healthy ageing and short-term immobilisation (i.e. disuse) on the contractile function of human single muscle fibres has not previously been investigated. The present study demonstrated that single fibre force and specific force was equally affected in old and young individuals following 2 weeks of lower limb immobilisation. D’Antona and colleagues compared the contractile function of single fibres obtained from old individuals exposed to long-term disuse (n= 2) with those from an age-matched group of healthy individuals (n=7), and observed considerable reductions in MHC I and IIa CSA (approx. 38 an 16%, respectively) and specific force (approx. 42 and 30%, respectively) (D'Antona et al. 2003). Considering the longer period of disuse (on average 3.5 months due to total knee arthroplasty), these changes were in good agreement with the smaller relative changes observed in our healthy old subjects after only 2 weeks of immobilisation. An exception to this trend was observed for specific force in MHC IIa fibres, which decreased by approx. 30% in both studies.
In healthy young individuals, disparate findings have been reported about the effects of disuse on single fibre CSA and contractile function (Larsson et al. 1996; Widrick et al. 1998, 2002). Whereas MHC I fibres generally seems to be more affected than MHC IIa fibres with disuse (Trappe et al. 2004; Fitts et al. 2007), this was not observed in the present study. This may be explained by the different periods and models of disuse used in the respective studies (e.g. bed rest, cast immobilisation, unilateral lower limb suspension) (Larsson et al. 1996; Widrick et al. 1998, 2002; Trappe et al. 2004; Fitts et al. 2007) as well as by the differences in mechanical loading/behaviour of the investigated muscles, e.g. postural (soleus, gastrocnemius) compared to non-postural (vastus lateralis) (Widrick et al. 2002; Fitts et al. 2007; Haus et al. 2007a). In support of the present findings, 37 days of disuse (i.e. bed rest) led to reduced specific force of vastus lateralis MHC I (1.2% per day, total 45%) and IIa fibres (1.1% per day, total 42%) (Larsson et al. 1996), which paralleled that observed in our young subjects (1.0 and 1.3% per day in MHC I and IIa fibres, respectively). Furthermore, data from animal studies have shown that MHC IIa fibres (gastrocnemius) of aged animals were more affected than MHC I fibres by 7 days of hindlimb suspension (Sandmann et al. 1998), thus supporting the observations in our old subjects (Fig. 3B and C). Although we can only speculate whether our findings also apply for very old individuals (≥80 years), they may respond to a lesser extent based on their low adaptive responses following resistance training (Slivka et al. 2008; Raue et al. 2009).
The observed decline induced by disuse in single fibre specific force for both young and old subjects may be due to a reduced number of cross-bridges (indicated by a lower concentration of myosin), a lower force produced per cross-bridge (both in the low- and strong-binding state), and/or a lower fraction of myosin heads in the strong-binding force-generating state (Larsson et al. 1996; Riley et al. 1998; D'Antona et al. 2003; Zhong et al. 2006; Udaka et al. 2008). Markedly lower myosin concentration have been observed in MHC I and IIa single fibres from young individuals following 35 days of disuse (Borina et al. 2010), as well as in long-term unloaded old individuals compared to age-matched individuals (MHC I: 160 vs. 89 μm and MHC IIa: 147 vs. 101 μm, respectively) (D'Antona et al. 2003). In contrast, myosin concentration in the vastus lateralis muscle of young individuals remained unaltered (approx. 47 μg (mg wet muscle weight)−1) following 35 and 90 days of disuse, suggesting that only the absolute amount of myosin (and other myofibrillar proteins) had decreased (since quadriceps muscle atrophied markedly) (Haus et al. 2007a). A recent study showed that approximately one third of the reduction in force of fast (semimembranosus, type II) fibres following 3 weeks of hindlimb suspension in both young and old rats could be attributed to a lower fraction of myosin heads in the strong-binding state (Zhong et al. 2006). Furthermore, a loss of thin filaments which has been shown in some but not all disuse studies may potentially reduce the number of recruited cross-bridges, while increased lattice spacing could lead to suboptimal myosin–actin interactions, resulting in reduced force produced per cross-bridge (Riley et al. 1998; Udaka et al. 2008). It remains unknown if these factors interact in an age-dependent manner.
As an important observation in the present study, 2 weeks of immobilisation led to alterations in the force–Ca2+ (and force–Sr2+) relationship which were dependent on age, with young subjects preferentially demonstrating changes in MHC IIa fibres and old subjects preferentially in MHC I fibres. No previous disuse studies have investigated this aspect in ageing humans or animals. In young individuals, there have been observations of both a rightward shift (decreased Ca2+ sensitivity) and a leftward shift in the force–Ca2+ relationship curve (increased Ca2+ sensitivity) of MHC I fibres from soleus and vastus lateralis following 17–60 days of disuse (Widrick et al. 1998; Yamashita-Goto et al. 2001; Fitts et al. 2007; Mounier et al. 2009), leaving our observations in between these. Evidence from disuse in rats supports our data, since MCH IIa fibres appeared to be more affected in young animals (Bastide et al. 2002). At present, we can only speculative about the mechanisms responsible for the present findings of age-dependent effects in the force–Ca2+ relationship. Disuse has been shown to induce a ‘slow-to-fast’ transition in the Tn isoforms C (domain that binds Ca2+ ions) and T (domain that binds Tn to Tm) (Bastide et al. 2002; Mounier et al. 2009). As this causes a rightward shift in the force–Ca2+ relationship curve, it may be speculated that ageing affects the degree of adaptability of these protein isoforms following disuse in a manner that makes MHC I fibres more susceptible to changes than MHC IIa fibres, as presently seen in our old subjects. An increase in lattice spacing due to a loss of thin filaments (i.e. actin, titin and other proteins responsible for maintaining the cytoskeletal integrity) has been shown following disuse (Riley et al. 1998; Udaka et al. 2008). However, when dextran was added to restore lattice spacing to the level observed prior to 6 weeks of hindlimb suspension in rats, Ca2+ sensitivity became fully restored in slow (SOL, type I) fibres (Udaka et al. 2008). Hence, the possibility exists that lattice spacing increased dominantly in MHC I fibres of our old individuals and dominantly in MHC IIa fibres of our young individuals following 2 weeks of immobilisation.
Adding to the methodological challenges of assessing single fibre CSA and hence to determine specific force (Larsson et al. 1997), especially in elderly individuals that may have a higher prevalence of irregular or ‘flattened’-shaped fibres (Andersen, 2003), the extent of radial swelling of single fibres due to the skinning procedure is also of importance. It is generally accepted that the single fibre diameter increases by approximately 20% due to swelling (corresponding to a 44% increase in CSA) (Elzinga et al. 1989; Olsson et al. 2006). However, if the rigidity of the contractile apparatus (or cytoskeleton) is compromised, for instance due to altered concentrations of cytoskeletal proteins, this could affect the extent of swelling (Blaauw et al. 2009). We therefore compared the CSA of our analysed single fibres with the mean CSA of fibres in cryosections from the same muscle biopsy samples (Hvid et al. 2010). This analysis showed that the extent of swelling in single fibre CSA corresponded to 21.5 ± 2.5% (data not shown) with no differences observed between young and old, between MHC I and IIa, or between Pre and Post. Hence, the present changes in single fibre specific tension observed following immobilisation and the concurrent effect of ageing (or lack hereof) could not be explained by differential alterations in single muscle fibre swelling. Concerning the presented values of single fibre specific force shown in Fig. 3C, these increased by a factor of 1/(1 – 0.215) when using the corrected CSA values, thus making them comparable to previously reported values (in absolute terms) of human vastus lateralis fibres (e.g. Trappe et al. 2003, 2004; Slivka et al. 2008).
Based on the findings of the present study where young (24 ± 1 years) and old (67 ± 2 years) men with similar levels of physical activity were compared, ageing did not seem to negatively affect the contractile properties of human single muscle fibres. In contrast, marked impairments in single fibre contractile properties were observed following short-term disuse in both young and old individuals. Specifically, 2 weeks of lower limb cast immobilisation caused greater impairments in MHC IIa than in MHC I single fibre force and specific force, respectively, independently of age. In contrast, the observed disuse-induced changes in the characteristics of the force–Ca2+ relationship were dependent on age, with a selective decrease in Ca2+ sensitivity in MHC IIa fibres of young and in MHC I fibres of old individuals. Thus, the present data suggest that short-term disuse leads to impaired contractile function of human single muscle fibres, and that this process is not affected by ageing per se.
Single fibre experiments were performed at the Institute of Sports Science and Clinical Biomechanics, University of Southern Denmark, Denmark. The contributions of the authors were as follows: conception and design of the immobilisation study: C.S. and M.K.; conception and design of the single fibre analysis: L.G.H. and N.Ø.; collection, analysis and interpretation of data: L.G.H., N.Ø., P.A. and C.S.; drafting the article or revising it critically for important intellectual content: L.G.H., N.Ø., P.A., M.K. and C.S. All authors approved the final version.
We like to thank Lab Technician Chris Christensen for valuable assistance with the gel electrophoresis analyses. We also thank the experimental subjects for providing muscle tissue, making this study possible. The study was supported by grants from the Lundbeck Foundation, the Danish National Research Council (Medical Sciences: FSS), the Danish Rheumatology Association, Faculty of Health Sciences, University of Copenhagen, and The Danish Ministry of Culture.