Physiological properties of human diaphragm muscle fibres and the effect of chronic obstructive pulmonary disease

Abstract

The contractile and actomyosin ATPase properties of single fibres were examined in human diaphragm muscle obtained from patients with and without chronic obstructive pulmonary disease (COPD). Costal diaphragm biopsies were taken from five patients without evidence of COPD and from 11 age-matched individuals with varying degrees of the disease. Our aim was to establish whether changes in contractile properties of COPD diaphragm could be fully explained by the previously documented shift towards a greater proportion of type I myosin heavy chain isoform in COPD. The relative proportion of type I diaphragm fibres from non-COPD and COPD patients was measured by gel electrophoresis, and was negatively correlated with FEV(1) over the full range of values investigated. There was also significant atrophy of the type I fibre population in COPD diaphragms. Isometric tension was similar among the fibre types and between the COPD and non-COPD patients. The intrinsic energetic properties of diaphragm fibres were examined by monitoring the time-resolved actomyosin ATPase activity in COPD and non-COPD fibres that produced similar isometric forces. The isometric ATPase rate in COPD fibres was reduced to 50% of the rate in non-COPD fibres; hence, the cost of isometric contraction in type I and type IIA COPD fibres was reduced to between one-third and one-half of the tension cost calculated for non-COPD fibres. The rate of force development in type I COPD fibres was reduced to 50% of the rate seen in non-COPD type-I fibres. No difference in the rate of ATP consumption between COPD and non-COPD fibres was evident during isovelocity shortening. These data extend previous findings showing that aspects of breathing mechanics during progressive COPD are associated with remodelling of the diaphragm fibre-type distribution; on top of the increase in type I fibres there are fibre-specific reductions in force development rate (type I fibres) and ATPase rate that are consistent with the impairment of cross-bridge cycling kinetics.

Figure 1. Diaphragm fibre-type remodelling correlates with pulmonary functions in COPD patients

A and B, relationship between the proportion of slow, type I fibres versus FEV₁ (A) and FRC (B). Each pulmonary function measurement is expressed as a percentage of the predicted normal value. The filled circles were derived from densitometry of MHC isoforms on SDS-PAGE gels using muscle homogenates of non-COPD patients, whilst the open circles represent data from muscle homogenates of COPD patients (Moore et al. 2006). The relative proportion of type I fibres was determined for most (but not all) subjects detailed in Table 1; hence, electrophoretic data are reported for some additional subjects for whom anthropometric data are not documented in Table 1. The triangles in A and B show data derived from mATPase stained sections (open triangles show data from COPD patient diaphragm, whilst filled triangles show results obtained from non-COPD patients). The data derived from SDS-PAGE in each panel were fitted with a Microsoft Excel exponential trend line (in B, this was fitted only to the COPD data, because no correlation between FRC and the proportion of type I fibres was apparent in the control data), along with the correlation coefficient. C and D, staining of mATPase in sections of costal diaphragm revealed the increased proportion of type I fibres in COPD patients. C shows a section from a non-COPD individual in the highest spirometric quartile (area of the type I fibre highlighted in red is 3703 μm²), and D shows a COPD patient in the lowest spirometric quartile. Scale bar = 100 μm.

Figure 2. Typical records of the contractions of a fast fibre segment (left) and a slow fibre segment (right)

A and B, tension records. The laser flash at time 0.2 s resulted in the photolytic release of ATP from NPE-caged ATP followed by tension rise. When tension had nearly reached a plateau (and ATPase activity had reached a quasi-steady state, see C and D) the fibre segment was allowed to shorten at a constant velocity (E and F), and tension decreased to a lower level. After the shortening period tension redeveloped, and a new plateau was reached. C and D, fluorescence signal, which, after calibration, provides a measure of P_i released by the hydrolysis of ATP. A fast initial phase was followed by a quasi-steady state period. When the fibre was allowed to shorten, the gradient of the signal increased. However, following the end of the shortening period, the rate of P_i release fell below that observed during the preshortening period. Saturation of the signal is seen (C) when total P_i released approached 1.2 mm.E and F, motor output, i.e. the signal indicating the change in the length of the fibre segment.

Figure 3. Functional characterization of the fibre groups

A–D, mechanical and energetic parameters of isometric contraction. The bar graphs show the average values of isometric tension (A; n = 102), half-time for force development (expressed as the time taken for force to reach half its maximal value following photolytic release of ATP; t_1/2) (B; n = 102), the steady rate of ATP release, measured from the rate of fluorescence increase of the phosphate binding protein (C; n = 66) and cost of force production (the rate of ATP hydrolysis divided by isometric force) (D; n = 66) for the three fibre groups (type I, IIA and IIX) at 20°C. Filled bars show data from non-COPD subjects (n = 34), whilst open bars show data obtained from COPD patients (n = 68). n values are shown above each bar. *P < 0.05 different from non-COPD group. **P < 0.01 different from non-COPD group.

Figure 4. Force–velocity curves

A, F–V curves for type IIA control (n = 11; R² = 0.61) and COPD (n = 34; R² = 0.70) fibres. B, F–V curves for type I control (n = 6; R² = 0.81) and COPD (n = 18; R² = 0.88) fibres. Each data point represents a different fibre. Data points were interpolated using eqn (1) (Methods), with best fits obtained using Microsoft Excel's Solver, the parameters of which are reported in Table 3. Curves for non-COPD fibres are plotted with continuous lines, whilst COPD fibre curves are illustrated by dashed lines.

Figure 5. Energy utilization during isovelocity shortening

The ATPase rates of COPD (open symbols) and non-COPD (filled symbols) type I (n = 7 and 5, respectively) and type IIA (n = 6 and 9, respectively) fibres at various velocities of shortening are shown. Triangles show data from type I fibres, whilst data from type IIA fibres are shown by circles.