Molecular determinants of force production in human skeletal muscle fibers: effects of myosin isoform expression and cross-sectional area

Abstract

Skeletal muscle contractile performance is governed by the properties of its constituent fibers, which are, in turn, determined by the molecular interactions of the myofilament proteins. To define the molecular determinants of contractile function in humans, we measured myofilament mechanics during maximal Ca(2+)-activated and passive isometric conditions in single muscle fibers with homogenous (I and IIA) and mixed (I/IIA and IIA/X) myosin heavy chain (MHC) isoforms from healthy, young adult male (n = 5) and female (n = 7) volunteers. Fibers containing only MHC II isoforms (IIA and IIA/X) produced higher maximal Ca(2+)-activated forces over the range of cross-sectional areas (CSAs) examined than MHC I fibers, resulting in higher (24-42%) specific forces. The number and/or stiffness of the strongly bound myosin-actin cross bridges increased in the higher force-producing MHC II isoforms and, in all isoforms, better predicted force than CSA. In men and women, cross-bridge kinetics, in terms of myosin attachment time and rate of myosin force production, were independent of CSA, although women had faster (7-15%) kinetics. The relative proportion of cross bridges and/or their stiffness was reduced as fiber size increased, causing a decline in specific force. Results from our examination of molecular mechanisms across the range of physiological CSAs explain the variation in specific force among the different fiber types in human skeletal muscle, which may have relevance to understanding how various physiological and pathophysiological conditions modulate single-fiber and whole muscle contractility.

Keywords: cross bridge; mechanical properties; muscle fiber.

Copyright © 2015 the American Physiological Society.

Figures

Fig. 1.

Single skeletal muscle fiber maximum Ca2+-activated (pCa 4.5) specific force, force, and cross-sectional area (CSA) by fiber type at 15°C and 0.25 mM Pi. Left: average values, with number of fibers indicated at the base of each bar. Where fiber type effects were observed, different letters above bars identify pair-wise differences (P ≤ 0.05) between fiber types. Right: scatterplots, with each point representing an individual fiber. Lines indicate linear regressions for MHC I, IIA, and IIA/X fibers from raw data in scatterplots, with Pearson's correlation coefficients (r) = 0.705, 0.622, and 0.566, respectively, and all coefficients significant at P < 0.01. Number of fibers was inadequate to calculate linear regression of MHC I/IIA fiber force with CSA.

Fig. 2.

Single skeletal muscle fiber maximum Ca2+-activated (pCa 4.5) specific force, force, and CSA by fiber type at 25°C and 5 mM Pi. Left: average values, with number of fibers indicated at the base of each bar. Where fiber type effects were observed, different letters above bars identify pair-wise differences (P < 0.05) between fiber types. Right: scatterplots, with each point representing an individual fiber. Lines indicate linear regressions for MHC I, IIA, and IIA/X fibers, with r = 0.723, 0.617, and 0.527 and P < 0.01, P < 0.01, and P = 0.02, respectively. Number of fibers was inadequate to calculate linear regression of MHC I/IIA fiber force with CSA.

Fig. 3.

Sinusoidal analysis model parameters for maximum Ca2+-activated (pCa 4.5) fibers by fiber type at 25°C and 5 mM Pi. 2πb is the rate of myosin transition between the weakly and strongly bound states, and ton represents the average time myosin is attached to actin. Parameters B and C are proportional to the number of myosin heads strongly bound to actin and cross-bridge stiffness. A and k represent the viscoelastic magnitude and relationship between viscous and elastic modulus for the underlying stiffness of the lattice structure and the attached myosin heads in series. Average values are shown, with number of fibers indicated at the base of each bar. Where fiber type effects were observed, different letters above bars identify pair-wise differences (P < 0.05) between fiber types.

Fig. 4.

Changes in myosin-actin cross-bridge kinetics with isoform concentration in MHC I/IIA and IIA/X fibers. Increasing values on the x-axis represent percentage of the faster MHC isoform (IIA for I/IIA and IIX for IIA/X fibers). Lines indicate linear regressions for 2πb (r = 0.769, P < 0.01) and ton (r = −0.689, P < 0.01) from MHC IIA/X fibers. Linear regressions were not determined from MHC I/IIA fibers, as percent MHC values were either high or low, with no clear relationship between them.

Fig. 5.

Relationship between sinusoidal analysis model parameters (2πb, ton, and CCSA) and CSA for various fiber types at 25°C, pCa 4.5, and 5 mM Pi. Data points represent results from an individual fiber. Lines indicate linear regressions for MHC I, IIA, and IIA/X with Pearson's correlation coefficient and statistical significance. Myosin-actin cross-bridge kinetics (2πb and ton) did not change with CSA, as their slopes were not different from zero, except for ton in MHC I fibers (r = −0.284, P < 0.01). CCSA, proportional to the number of myosin heads strongly bound to actin and cross-bridge stiffness, increased with CSA (r = 0.560, 0.506, and 0.296; P < 0.01, P < 0.01, and P = 0.22 in I, IIA, and IIA/X, respectively). Number of fibers was inadequate to calculate linear regression of MHC I/IIA fibers with CSA.

Fig. 6.

Relationship between sinusoidal analysis model parameters (ACSA and k) and CSA for various fiber types at 25°C, pCa 4.5, and 5 mM Pi. Data points represent results from an individual fiber. Lines indicate linear regressions for MHC I, IIA, and IIA/X, respectively. ACSA and k represent the viscoelastic magnitude and relationship between viscous and elastic modulus for the underlying stiffness of the lattice structure and the attached myosin heads in series. ACSA increased with CSA (r = 0.292, 0.262, and 0.450; P < 0.01, P = 0.02, and P = 0.05 in I, IIA, and IIA/X, respectively), but k had a slope different from zero only in MHC I fibers (r = 0.179, P = 0.08). Number of fibers was inadequate to calculate linear regression of MHC I/IIA fibers with CSA.

Fig. 7.

Relationship between cross-bridge kinetics (2πb and ton) and CSA for men and women in MHC I and IIA fibers at 25°C, pCa 4.5, and 5 mM Pi. Data points represent results from an individual fiber. Lines indicate linear regressions. Myosin-actin cross-bridge kinetics (2πb and ton) did not change with CSA, as their slopes were not different from zero.

Fig. 8.

Relationship between force and CCSA, which is proportional to the number of myosin heads strongly bound to actin and cross-bridge stiffness, at 25°C, pCa 4.5, and 5 mM Pi. CCSA explains more of the variance in the force data (55, 61, and 68% in I, IIA, and IIA/X, respectively) than CSA or the other sinusoidal analysis parameters (Table 2). Data points represent results from an individual fiber. Lines indicate linear regressions for MHC I, IIA, and IIA/X, with Pearson's correlation coefficient and statistical significance in Table 2.

Fig. 9.

Isometric specific force, as well as elastic and viscous modulus responses, under relaxed conditions (pCa 8) with 2,3-butanedione monoxime (BDM), which places myosin heads in a weakly bound state. Number of fibers is indicated at the base of each bar. *Significant difference (P < 0.05) between MHC I and IIA fibers, as the number of hybrid fiber types (I/IIA and IIA/X) was inadequate to include in the analysis.

Fig. 10.

Relationship of isometric specific force, force/CCSA, and C to CSA for various fiber types at 25°C, pCa 4.5, and 5 mM Pi. Each data point represents an individual fiber. Lines indicate linear regressions for MHC I, IIA, and IIA/X, respectively, with Pearson's correlation coefficient and statistical significance. Isometric specific force (r = −0.527, −0.533, and −0.501; P < 0.01, P < 0.01, and P = 0.03 in I, IIA, and IIA/X, respectively) and C (r = −0.521, −0.541, and −0.513; P < 0.01, P < 0.01, and P = 0.03) decreased with CSA. Force/CCSA did not change with CSA, as their slopes were not different from zero. Number of fibers was inadequate to calculate linear regression of MHC I/IIA fibers with CSA.