The effect of ageing and immobilization on structure and function of human skeletal muscle fibres

Abstract

Biopsy samples were taken from vastus lateralis muscle of seven young (YO, age 30.2 +/- 2.2 years), and seven elderly (EL, age 72.7 +/- 2.3 years) subjects and two elderly subjects whose right leg had been immobilized for 3.5 months (EL-IMM, ages 70 and 75). The following main parameters were studied: (1) myosin heavy chain (MHC) isoform distribution of the samples, determined by SDS-PAGE; (2) cross-sectional area (CSA), specific force (Po/CSA) and maximum shortening velocity (Vo) of a large population (n = 593) of single skinned muscle fibres, classified on the basis of MHC isoform composition determined by SDS-PAGE; (3) actin sliding velocity (Vf) on pure myosin isoforms determined by in vitro motility assays; (4) myosin concentration in single fibres determined by quantitative SDS-PAGE. MHC isoform distribution was shifted towards fast isoforms in EL and to a larger extent in EL-IMM. In EL and, more consistently, in EL-IMM we observed a higher percentage of hybrid fibres than in YO, and noted the presence of MHC-neonatal and of unusual hybrid fibres containing more than two MHC isoforms. Po/CSA significantly decreased in type 1 and 2A fibres in the order YO EL EL-IMM. Vo of type 1 and 2A fibres was significantly lower in EL and higher in EL-IMM than in YO, i.e. immobilization more than counteracted the age-dependent decrease in Vo. The latter phenomenon was not observed for Vf. Vf on myosin 1 was lower in both EL and EL-IMM than in YO. Vf on myosin 2X was lower in EL than in YO, and a similar trend was observed for myosin 2A. Myosin concentration decreased in type 1 and 2A fibres in the order YO EL EL-IMM and was linearly related to the Po/CSA values of corresponding fibre types from the same subjects. The experiments suggest that (1) myosin concentration is a major determinant of the lower Po/CSA of single fibres in ageing and especially following immobilization and (2) ageing is associated with lower Vo of single fibres due to changes in the properties of myosin itself, whereas immobilization is associated with higher Vo in the absence of a change in myosin function.

Figures

Figure 1. Electrophoretic (SDS-PAGE) separation of myosin heavy chain (MHC) isoforms in fibre bundles and in single muscle fibres of young (YO), elderly (EL) and elderly immobilized (EL-IMM) subjects

A, SDS-PAGE analysis of MHC isoforms in muscle bundles. Vastus lateralis samples of a YO, an EL and an EL-IMM subject were loaded in lanes 1, 2, and 3 respectively. Four MHC isoforms were separated and their area of migration is indicated by the labels on the right. In lanes 1 and 2 the samples from YO and EL subjects show the three adult MHC isoforms. In lane 3 the sample from the EL-IMM subject shows, besides the three adult MHC isoforms, a fourth band. This band was identified as MHC-neonatal (MHC-neo)(see Methods). B, SDS-PAGE analysis of single muscle fibres. The areas of migration of the four MHC isoforms are indicated by the labels on the right. Lane 1: a fibre from EL containing MHC-2A (type 2A); lane 2: a fibre from EL containing MHC-1 (type 1); lane 3: a fibre from EL-IMM containing MHC-1, MHC-2A and MHC-neonatal (MHC-neo)(type 1-2-neo); lane 4: a fibre from EL-IMM containing all three adult MHC isoforms (type 1-2AX); lane 5: a fibre from EL-IMM containing all four MHC isoforms (type 1-2-neo).

Figure 2. Identification of the extra protein band in the area of migration of MHC isoforms

A, Western blot analysis of a sample from one EL-IMM subject (lane 1) and of a sample from one YO subject (lane 2). The antibody specific for all myosin isoforms (BF-49) was used. Four bands are stained in the EL-IMM sample, whereas the usual three adult MHC isoforms are stained in the sample from YO. B, a cross-cryosection of a muscle sample from one EL-IMM subject stained by the antibody for MHC-neo (NCL-MHCn). Some fibres are stained and therefore contain MHC-neonatal.

Figure 3. Quantification of myosin concentration in single muscle fibres

A, an example of the procedure used to ensure precise determination of myosin content in single fibres. The graph shows the relation between the brightness-area product (BAP) and the MHC content, expressed in μg, of the bands of the gel in the inset on the right. Six samples of 0.5, 1.0, 2.0, 3.0, 3.5, and 4.0 μg of a myosin standard were loaded in lanes 1-6, respectively. The BAPs of such bands were plotted in the graph (▪) and were used to build a standard curve (continuous line). The slope of the standard curve is very significantly different from zero (P < 0.0001, r2 = 0.996). Two segments of the same fibre of equal length were loaded in lanes 7 and 8 and their BAPs were plotted in the graph with open circles. Two segments of the same fibre of different length (2 and 4 mm) were loaded in lanes 9 and 11 and their BAPs were plotted in the graph with open squares. Two segments of the same fibre of different lengths (3 and 6 mm) were loaded in lanes 10 and 12 and their BAPs were plotted in the graph with open triangles. B, an example of the procedure used to determine myosin content in single fibres. Five samples of 0.5, 1.0, 2.5, 3.0, and 4.0 μg of the myosin standard were loaded from left to right in the gel in the inset (lanes indicated with St) and used to build a standard curve (▪) in the graph. The standard curve had a slope very significantly different from zero (P < 0.0001, r2 = 0.998). Segments of single fibres from YO (*), EL (▿), EL-IMM ( ⋄) were loaded in lanes 1-6 and their BAPs plotted in the graph to determine myosin content using the standard curve.

Figure 4. Myosin heavy chain (MHC) isoform distribution and fibre type distribution in young (YO), elderly (EL) and elderly immobilized (EL-IMM) subjects

A, MHC isoform distribution determined from biopsy samples of the three groups of subjects by SDS-PAGE separation and subsequent densitometric analysis of MHC bands. The height of each vertical bar represents the mean values (± S.E.M.). B, fibre type distribution in a population of single muscle fibres (n = 730) identified on the basis of MHC isoform composition determined by SDS-PAGE. Hybrid fibre types containing more than two MHCs were found (1-2AX, 1-2-neo). In the group 1-2-neo the fibres containing MHC-neo together with two or three adult MHCs were grouped. • Significantly different from all the other subject groups, ••significantly different from EL, P < 0.05.

Figure 5. Cross-sectional area (CSA; A) and specific force (Po/CSA; B) of the single muscle fibres used for mechanical experiments from young (YO), elderly (EL) and elderly immobilized (EL-IMM) subjects

CSA is expressed in μm2 and Po/CSA in kN m2. The height of the bars represents the mean values (± S.E.M.). The numbers of fibres studied per type and per group were: 143 (YO), 128 (EL) and 28 (EL-IMM) for type 1; 20 (YO), 32 (EL) and 13 (EL-IMM) for type 1-2A; 75 (YO), 64 (EL) and 21 (EL-IMM) for type 2A; 19 (YO), 8 (EL) and 20 (EL-IMM) for type 2XA; 9 (YO), 5 (EL) and 8 (EL-IMM) for type 2X. • Significantly different from all the other subject groups, •• significantly different from YO, P < 0.05.

Figure 6. Maximum shortening velocity (Vo) of single muscle fibres (A) and actin sliding velocity (Vf) on pure myosin isoforms extracted from single muscle fibres (B) of young (YO), elderly (EL) and elderly immobilized (EL-IMM) subjects

Vo is expressed in segment length per second (L s−1) and Vf in μm s−1. Vo was determined at 12 °C and Vf at 25 °C. The numbers of fibres analysed for Vo per type and per group are the same as those reported in Fig. 5. The numbers of samples studied for Vf per myosin isoform and per group were: 20 (YO), 30 (EL) and 6 (EL-IMM) for myosin 1; 15 (YO), 20 (EL) and 12 (EL-IMM) for myosin 2A; 6 (YO), 8 (EL) for myosin 2X. The height of the bars represents mean values (± S.E.M.). • Significantly different from all the other subject groups, •• significantly different from YO, P < 0.05.

Figure 7. Myosin concentration of type 1 and 2A single muscle fibres of YO, EL, and EL-IMM subjects plotted versus mean Po/CSA values of type 1 and 2A fibres from the same subjects

The numbers of fibres used for determination of myosin concentration per fibre type and per group are reported in the text. The number of fibres plotted for Po/CSA per type and per group were: 60 (YO), 49 (EL) and 28 (EL-IMM) for type 1; 45 (YO), 28 (EL) and 21 (EL-IMM) for type 2A. Error bars are ± S.E.M. The slope of the regression line for both type 1 (0.185 ± 0.006, P = 0.023) and type 2A fibres (0.314±.041, P = 0.028) was statistically different from zero,(P < 0.05). Open symbols indicate type 1 fibres, filled symbols type 2A fibres. Circles, fibres from YO subjects; squares: fibres from EL subjects; triangles, fibres from EL-IMM subjects.