Chronic heart failure decreases cross-bridge kinetics in single skeletal muscle fibres from humans

Abstract

Skeletal muscle function is impaired in heart failure patients due, in part, to loss of myofibrillar protein content, in particular myosin. In the present study, we utilized small-amplitude sinusoidal analysis for the first time in single human skeletal muscle fibres to measure muscle mechanics, including cross-bridge kinetics, to determine if heart failure further impairs contractile performance by altering myofibrillar protein function. Patients with chronic heart failure (n = 9) and controls (n = 6) were recruited of similar age and physical activity to diminish the potentially confounding effects of ageing and muscle disuse. Patients showed decreased cross-bridge kinetics in myosin heavy chain (MHC) I and IIA fibres, partially due to increased myosin attachment time (t(on)). The increased t(on) compensated for myosin protein loss previously found in heart failure patients by increasing the fraction of the total cycle time myosin is bound to actin, resulting in a similar number of strongly bound cross-bridges in patients and controls. Accordingly, isometric tension did not differ between patients and controls in MHC I or IIA fibres. Patients also had decreased calcium sensitivity in MHC IIA fibres and alterations in the viscoelastic properties of the lattice structure of MHC I and IIA fibres. Collectively, these results show that heart failure alters skeletal muscle contraction at the level of the myosin-actin cross-bridge, leading to changes in muscle mechanics which could contribute to impaired muscle function. Additionally, we uncovered a unique kinetic property of MHC I fibres, a potential indication of two distinct populations of cross-bridges, which may have important physiological consequences.

Figure 1. The six-parameter model used to fit the Nyquist plots produced by sinusoidal analysis

A, the Nyquist plot of a Ca²⁺-activated (pCa 4.5) MHC I fibre, where each open circle represents one of the 48 oscillation frequencies (0.25 to 200 Hz) performed during sinusoidal analysis, provides information on the mechanical properties (elastic and viscous moduli) of the muscle fibre and its components. The sinusoidal analysis results (open circles) are well-characterized by the continuous line, calculated using a six-parameter model whose equation is in the text. B, the six model parameters, which can be related to cross-bridge function and myofilament structural properties, are paired into three different processes: the linear A-process (continuous line, described by parameters A and k), the semicircular B-process (dash–dot line, described by parameters B and b) and the C-process (dashed line, described by parameters C and c). As a negative viscous modulus indicates positive work production, the B-process is work producing and the A- and C-processes are work absorbing. The B and C parameters, displayed as arrows the length of the diameter of the semicircular B- and C-processes, are magnitudes (N mm⁻²) proportional to the number of strongly bound cross-bridges and cross-bridge stiffness. The parameters b and c indicate the frequencies (Hz) at which the B- and C-processes exhibit viscous modulus values that are the most negative (or largest oscillatory work production) and positive (or largest oscillatory work absorption), respectively. Importantly, 2πb and 2πc are characteristic rates (s⁻¹) related to cross-bridge kinetics, with (2πc)⁻¹ being equivalent to myosin attachment time (t_on). The A-process represents the viscoelastic properties of the underlying lattice structure as well as strongly bound cross-bridges. The parameter A, represented by an arrow, indicates a magnitude (N mm⁻²) and k (a unitless exponent) is the angle at which the A-process lies relative to the x-axis.

Figure 2. Representative MHC and MLC gels from single human skeletal muscle fibres

Silver-stained SDS-PAGE gels show separation of MHC (A) and MLC (B) isoforms. MSF, multiple single fibres.

Figure 3. Representative Nyquist plots for Ca²⁺-activated (pCa 4.5) single human skeletal muscle fibres and cardiac strips

A, an MHC I human skeletal muscle fibre at 25°C and 5 mm P_i with a muscle length perturbation amplitude of 0.05% produces a notch (labelled at 2.75 Hz), although no notch is observed when the perturbation amplitude is increased to 0.125%. B, a human cardiac muscle strip (male, 67 years old with coronary heart disease and hypertension) at 37°C and muscle length amplitude oscillation of 0.125% produces a notch (labelled at 4.5 Hz). C, an MHC IIA human skeletal muscle fibre at 25°C and 5 mm P_i with a muscle length perturbation amplitude of 0.05% does not produce a notch. Sinusoidal length oscillations were applied from 0.25 to 200 Hz for each fibre type.

Figure 4. Fibre type differences in single human skeletal muscle fibre kinetic parameters

2πb (A) and t_on (B) for Ca²⁺-activated (pCa 4.5) skeletal muscle fibres expressing single MHC isoforms where controls (MHC I, n = 86; MHC IIA, n = 28) and heart failure patients (MHC I, n = 80; MHC IIA, n = 32; MHC IIX, n = 1) were combined. The number of fibres is indicated at the base of each bar. Conditions: 25°C and 5 mm P_i. Muscle length perturbation amplitude = 0.05%. Bar graphs represent mean ± s.e.m. **P < 0.01 compared to MHC I.

Figure 5. Elastic and viscous moduli response for Ca²⁺-activated (pCa 4.5) MHC I and IIA fibres in heart failure and controls

Elastic and viscous moduli for MHC I (A and B) and MHC IIA (C and D) single human skeletal muscle fibres across muscle oscillation frequencies for controls (MHC I, n = 87; MHC IIA, n = 28) and heart failure patients (MHC I, n = 85; MHC IIA, n = 32). The notch, which occurs between 1–4 Hz in MHC I fibres and is easily identified in individual traces (Fig. 3A), is less noticeable when data are presented as group averages, due to the fact that the notch occurs at a variety of frequencies. Conditions: 25°C and 5 mm P_i. Muscle length perturbation amplitude = 0.05%. Values represent mean ± s.e.m. *P < 0.05.

Figure 6. Sinusoidal analysis model parameter response for Ca²⁺-activated (pCa 4.5) MHC I fibres in heart failure and controls

A- (panels A and B), B- (panels C and D) and C-process (panels E and F) parameters for Ca²⁺-activated single MHC I skeletal muscle fibres for controls (n = 86) and heart failure patients (n = 80). Conditions: 25°C and 5 mm P_i. Muscle length perturbation amplitude = 0.05%. Bar graphs represent mean ± s.e.m. **P < 0.01.

Figure 7. Sinusoidal analysis model parameter response for Ca²⁺-activated (pCa 4.5) MHC IIA fibres in heart failure and controls

A- (panels A and B), B- (panels C and D) and C-process (panels E and F) parameters for Ca²⁺-activated single MHC IIA skeletal muscle fibres for controls (n = 28) and heart failure patients (n = 32). Conditions: 25°C and 5 mm P_i. Muscle length perturbation amplitude = 0.05%. Bar graphs represent mean ± s.e.m. **P < 0.01.

Figure 8. Relative MLC isoform distribution and relationship to t_on from MHC IIA fibres in heart failure and controls

Relative MLC isoform distribution (A) and relationship between t_on and MLC 3f/2f ratio (B) in single MHC IIA skeletal muscle fibres from controls (n = 13) and heart failure patients (n = 24). Regression line equation is t_on = (1.22 * MLC 3f/2f ratio) + 15.58 and has a P = 0.76. Bar graphs represent mean ± s.e.m.

Figure 9. Tension–pCa relationship for MHC I and IIA fibres in heart failure and controls

Single skeletal muscle fibre isometric tension changes with Ca²⁺ concentration for MHC I (A) and MHC IIA (B) fibres from controls (MHC I, n = 20; MHC IIA, n = 11) and heart failure patients (MHC I, n = 22; MHC IIA, n = 9). Mean values for MHC I (pCa₅₀: control: 5.74 ± 0.03 vs. HF: 5.69 ± 0.02; n: control: 2.98 ± 0.14 vs. HF: 2.64 ± 0.18) and MHC IIA (pCa₅₀: control: 5.84 ± 0.02 vs. HF: 5.75 ± 0.02; n: control: 3.32 ± 0.14 vs. HF: 3.75 ± 0.26) fibres. Conditions: 15°C and 0.25 mm P_i. Values represent mean ± s.e.m. **P < 0.01.

Figure 10. Ca²⁺-activated (pCa 4.5) tension for MHC I and IIA fibres in heart failure and controls

Single skeletal muscle fibre Ca²⁺-activated (pCa 4.5) tensions for MHC I and IIA fibres at 25°C and 5 mm P_i (A) and at 15°C and 0.25 mm P_i (B) in controls and heart failure patients. The number of fibres is indicated at the base of each bar. Bar graphs represent mean ± s.e.m. **P < 0.01.