Preparation. Single muscle fibres were dissected from fresh muscle biopsies obtained from elderly women undergoing orthopaedic surgery for total hip replacement or repair of a fractured neck of femur. The women's ages ranged from 66 to 90 years (n = 7). Each woman's activity profile was obtained from interview and medical records data prior to the scheduled surgery. Care was taken to select only women with an active lifestyle prior to surgery. Informed written consent was obtained from each woman and human ethics approval was obtained from both LaTrobe University and Austin Hospital, Melbourne, Australia, Human Research Ethics Committees.
The muscle sample was dissected by an orthopaedic surgeon from the vastus lateralis muscle in elderly women undergoing orthopaedic surgery. The muscle biopsy was obtained within 5–15 mins of the commencement of the surgery. The muscle biopsy was blotted thoroughly on Whatman's filter paper (No 1) immediately upon its dissection from the vastus lateralis muscle to remove any excess interstitial fluid, then placed in a jar of cold paraffin oil at 20C. The muscle biopsy was then placed in a thermos flask containing ice and transferred immediately to the laboratory.
The dissection of the muscle fibres from the muscle biopsy was generally commenced within 60 mins of its removal from the vastus lateralis muscle. The muscle biopsy was transferred from the cold paraffin into a Petri dish containing cold paraffin. Lynch et al. (1993) showed that this biopsy procedure had no effect on the activation characteristics of human skeletal muscle biopsy samples. Dissection of muscle fibres took place under oil. The muscle fibre was mechanically skinned (Stephenson and Williams, 1981) and tied at one end with silk thread and the other end was mounted between a pair of fixed forceps and attached to a force transducer (801 SensoNor Horten, Norway). Once a single muscle fibre had been dissected the remainder of the biopsy sample was stored under cold paraffin at 20C for up to 6 hours during which further fibres were dissected out.
The length of the fibre was adjusted such that the preparation was just taut, and the diameter of the skinned muscle fibres was measured under oil. The sarcomere length (SL) of the skinned muscle fibres was then measured by laser diffraction (mean SL 2.71+/-0.04 µm; Stephenson and Williams, 1981).
Solutions. Solutions were prepared according to standard procedures described by Stephenson and Williams (1981). The composition of the solutions is given in Table 1. Following mounting of the single skinned muscle fibre it was lowered into a relaxing solution (solution A Table 1) containing EGTA (50 mM) and allowed to equilibrate for 5 minutes. Before activation the fibre was immersed in a preactivating solution containing HDTA (solution D Table 1) to facilitate a rapid [Ca2+] rise (Moisescu & Thieleczek, 1978) when the fibre was placed in the Ca2+ activating solutions (solution B Table 1) after which it was returned to the relaxing solution (solution A Table 1). This procedure was repeated for activation of muscle fibres in Sr2+ solutions (solution C Table 1; Fig. 1). All experiments were performed at room temperature (22 +/- 1˚C).
Activation properties. Assessment of the effects of Ca2+ and Sr2+ activation was achieved by construction of force-pCa and force-pSr curves for the contractile responses of each individual fibre. The steady state tension developed in each solution was expressed as a percentage of the maximum tension developed in the sequence (Fig. 1). The curves were thus generated using a modified form of the Hill equation
Table 1
Chemical composition of stock solutions of relaxing solution (Solution A), calcium activating solution (Solution B), strontium activating solution (Solution C) and a preactivating solution (Solution D). All solutions contained (mM): K+ 117, Na+ 36. The pH was 7.10 ± 0.01 at 220C in all solutions
Chemicals | Solution A mM | Solution B mM | Solution C mM | Solution D mM |
HEPES | 60 | 60 | 60 | 60 |
EGTA | 50 | 50 | 50 | 0.1 |
HDTA | — | — | — | 49.1 |
Mg total | 10.3 | 8.12 | 8.5 | 8.5 |
Ca total | — | 49.5 | — | — |
Sr total | — | — | 40 | — |
NaN3 | 1 | 1 | 1 | 1 |
ATP | 8 | 8 | 8 | 8 |
CP | 10 | 10 | 10 | 10 |
Mg2+ | 1 | 1 | 1 | 1 |
using GraphPad Prism Software (GraphPad Software Inc., 5755 Oberlin Dr # 110 San
Diego, Ca 92121). The modified Hill equation is: Relative tension (%) = 100/(1+([Ca50]/[Ca2+])n) where n is the Hill co-efficient for Ca2+ and [Ca50] is the calcium concentration required for half-maximal tension activation. The same equation was used for the Sr2+ activation curves. The following activation properties were measured from the force activation curves generated for each muscle fibre from the two age groups, when all data points fell within 5% of the fitted curves: Ca2+ and Sr2+ threshold for contraction (pCa10 and pSr10, corresponding to 10% maximum force), sensitivity to Ca2+ and Sr2+ (pCa50 and pSr50, corresponding to 50% maximum force) and related differential sensitivity (pCa50-pSr50) and steepness of the activation curves (Hill co-efficient: nCa and nSr).
As described by Bortolotto et al. 2000, the Sr2+- data points for some fibres could not be well fitted by simple Hill-curves (i.e. not all data points fell within 5% of the best fitted Hill-curve). In such instances, the Sr2+-data points were well fitted by a biphasic curve generated by the following equation: Relative tension (%) = α/(1+([Sr501]/[Sr2+])n1 + β/(1+([Sr502]/[Sr2+])n2, where α and β represent the percentage of the two phases (α + β = 100%) and Sr501, Sr502 are the strontium concentrations corresponding to the half-maximal activation of the two phases.
Fibre classification. The Sr2+-dependent activation properties of individual muscle fibres permits unequivocal classification of fibres in three groups: type I (slow-twitch expressing TnC slow (cardiac) isoform and MHC I), type II (fast-twitch, expressing TnC fast isoform and MHC II isoforms) and hybrid (type I/ type II, expressing TnC fast/TnC slow and MHC I/MHC II isoforms) (O’Connell et al., 2004; Lamboley et al., 2013). This classification is based on the much higher force sensitivity to Sr2+ of fibres expressing the TnC slow isoform (and MHC I), than the TnC fast isoform (and MHC II isoforms) (Bortolotto et al., 2000; O’Connell et al., 2004; Lamboley et al., 2013). The hybrid fibres are characterised by biphasic force-pSr curves and express both the slow and fast TnC isoforms and a combination of MHC I and II isoforms (Bortolotto et al., 2000, O’Connell et al., 2004). Moreover, the Sr+-activation curves of hybrid fibres permit direct estimation of the fraction of TnC isoforms (MHC I/MHC II isoforms) present in the fibre (O’Connell et al., 2004) from the percentage ratio (α/β) of the two phases of the Sr2+-activation curve.
Force oscillations of myofibrillar origin. All slow-twitch (type I) fibres and some fast-twitch (type II) and hybrid fibres displayed spontaneous force oscillations of myofibrillar origin (FOMO) at submaximal levels of force activation. The highly Ca2+- and Sr2+-buffered activation solutions used in this study (containing 50 mM EGTA) eliminates the possibility that the force oscillations were in any way caused by oscillations in Ca2+ or Sr2+ concentration. Indeed in previous studies, we have shown that these oscillations are maintained even after treatment of the fibres with detergent to disrupt and extract all membrane compartments (Stephenson & Williams, 1981). Moreover, we have shown that such force oscillations at submaximal levels of activation are caused by myosin interactions with the actin filaments (Smith & Stephenson, 2009).
Maximum Ca 2+ activated specific force. Skinned muscle fibres swell when exposed to relaxing solutions and the amount of swelling depends on the sarcomere length, being larger at longer sarcomeres. Therefore, when measuring the maximum Ca2+-activated specific force it makes sense to express the maximum Ca2+-activated force developed by the fibre per unit cross-sectional area before the fibre swells. The maximum Ca2+ activated specific force was, therefore, calculated only for fibres where the fibre diameter was measured in oil, as described by Fink et al., (1990) after mechanical skinning and before the skinned fibre was exposed to solutions. The fibre cross-sectional area was calculated assuming it to be circular.
Statistics. The data analysed were from muscle biopsies obtained from 7 elderly women. In total, 28 muscle fibre segments were examined. Results were analysed with a one-way analysis of variance (ANOVA, Hewlett Packard statistical program) and/or Student t-test where appropriate, according to levels of significance. The Mann Whitney rank order test was applied when the sample size was small (n < 6) and the variance quite marked (SEM > 4% mean; Witte, 1993). Results were considered to be significant when P≤0.05.