Qualitatively different cross-bridge attachments in fast and slow muscle fiber types

Contractile properties differ between skeletal, cardiac and smooth muscles as well as between various skeletal muscle fiber types. This functional diversity is thought to be mainly related to different speeds of myosin head pulling cycles, with the molecular mechanism of force generation being essentially the same. In this study, force-generating attachments of myosin heads were investigated by applying small perturbations of myosin head pulling cycles in stepwise stretch experiments on skeletal muscle fibers of different type. Slow fibers (frog tonic and rat slow-twitch) exhibited only a ‘slow-type’ of myosin head attachment over the entire activation range, while fast fibers (frog and rat fast-twitch) displayed a ‘slow-type’ of myosin head attachment at low levels of activation, and an up to 30-times faster type at high levels of activation. These observations indicate that there are qualitative differences between the mechanisms of myosin head attachment in slow and fast vertebrate skeletal muscle fibers

Myosin heavy chain isoform composition and stretch activation kinetics in single fibres of Xenopus laevis iliofibularis muscle

Skeletal muscle is composed of specialized fibre types that enable it to fulfil complex and variable functional needs. Muscle fibres of Xenopus laevis, a frog formerly classified as a toad, were the first to be typed based on a combination of physiological, morphological, histochemical and biochemical characteristics. Currently the most widely accepted criterion for muscle fibre typing is the myosin heavy chain (MHC) isoform composition because it is assumed that variations of this protein are the most important contributors to functional diversity. Yet this criterion has not been used for classification of Xenopus fibres due to the lack of an effective protocol for MHC isoform analysis. In the present study we aimed to resolve and visualize electrophoretically the MHC isoforms expressed in the iliofibularis muscle of Xenopus laevis, to define their functional identity and to classify the fibres based on their MHC isoform composition. Using a SDS-PAGE protocol that proved successful with mammalian muscle MHC isoforms, we were able to detect five MHC isoforms in Xenopus iliofibularis muscle. The kinetics of stretch-induced force transients (stretch activation) produced by a fibre was strongly correlated with its MHC isoform content indicating that the five MHC isoforms confer different kinetics characteristics. Hybrid fibre types containing two MHC isoforms exhibited stretch activation kinetics parameters that were intermediate between those of the corresponding pure fibre types. These results clearly show that the MHC isoforms expressed in Xenopus muscle are functionally different thereby validating the idea that MHC isoform composition is the most reliable criterion for vertebrate skeletal muscle fibre type classification. Thus, our results lay the foundation for the unequivocal classification of the muscle fibres in the Xenopus iliofibularis muscle and for gaining further insights into skeletal muscle fibre diversity

Elementary Steps of the Cross-Bridge Cycle in Fast-Twitch Fiber Types from Rabbit Skeletal Muscles

To understand the molecular mechanism underlying the diversity of mammalian skeletal muscle fibers, the elementary steps of the cross-bridge cycle were investigated in three fast-twitch fiber types from rabbit limb muscles. Skinned fibers were maximally Ca(2+)-activated at 20°C and the effects of MgATP, phosphate (P, P(i)), and MgADP were studied on three exponential processes by sinusoidal analysis. The fiber types (IIA, IID, and IIB) were determined by analyzing the myosin heavy-chain isoforms after mechanical experiments using high-resolution SDS-PAGE. The results were consistent with the following cross-bridge scheme: [Image: see text] where A is actin, M is myosin, D is MgADP, and S is MgATP. All states except for those in brackets are strongly bound states. All rate constants of elementary steps (k(2), 198–526 s(−1); k(−2), 51–328 s(−1); k(4), 13.6–143 s(−1); k(−4), 13.6–81 s(−1)) were progressively larger in the order of type IIA, type IID, and type IIB fibers. The rate constants of a transition from a weakly bound state to a strongly bound state (k(−2), k(4)) varied more among fiber types than their reversals (k(2), k(−4)). The equilibrium constants K(1) (MgATP affinity) and K(2) (=k(2)/k(−2), ATP isomerization) were progressively less in the order IIA, IID, and IIB. K(4) (=k(4)/k(−4), force generation) and K(5) (P(i) affinity) were larger in IIB than IIA and IID fibers. K(1) showed the largest variation indicating that the myosin head binds MgATP more tightly in the order IIA (8.7 mM(−1)), IID (4.9 mM(−1)), and IIB (0.84 mM(−1)). Similarly, the MgADP affinity (K(0)) was larger in type IID fibers than in type IIB fibers

Dependence of cross-bridge kinetics on myosin light chain isoforms in rabbit and rat skeletal muscle fibres

Cross-bridge kinetics underlying stretch-induced force transients was studied in fibres with different myosin light chain (MLC) isoforms from skeletal muscles of rabbit and rat. The force transients were induced by stepwise stretches (< 0.3% of fibre length) applied on maximally Ca(2+)-activated skinned fibres. Fast fibre types IIB, IID (or IIX) and IIA and the slow fibre type I containing the myosin heavy chain isoforms MHC-IIb, MHC-IId (or MHC-IIx), MHC-IIa and MHC-I, respectively, were investigated. The MLC isoform content varied within fibre types. Fast fibre types contained the fast regulatory MLC isoform MLC2f and different proportions of the fast alkali MLC isoforms MLC1f and MLC3f. Type I fibres contained the slow regulatory MLC isoform MLC2s and the slow alkali MLC isoform MLC1s. Slow MLC isoforms were also present in several type IIA fibres. The kinetics of force transients differed by a factor of about 30 between fibre types (order from fastest to slowest kinetics: IIB > IID > IIA ≫ I). The kinetics of the force transients was not dependent on the relative content of MLC1f and MLC3f. Type IIA fibres containing fast and slow MLC isoforms were about 1.2 times slower than type IIA fibres containing only fast MLC isoforms. We conclude that while the cross-bridge kinetics is mainly determined by the MHC isoforms present, it is affected by fast and slow MLC isoforms but not by the relative content of MLC1f and MLC3f. Thus, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the cross-bridge kinetics

Tension Recovery in Permeabilized Rat Soleus Muscle Fibers after Rapid Shortening and Restretch

Permeabilized rat soleus muscle fibers were subjected to rapid shortening/restretch protocols (20% muscle length, 20 ms duration) in solutions with pCa values ranging from 6.5 to 4.5. Force redeveloped after each restretch but temporarily exceeded the steady-state isometric tension reaching a maximum value ∼2.5 s after relengthening. The relative size of the overshoot was <5% in pCa 6.5 and pCa 4.5 solutions but equaled 17% ± 4% at pCa 6.0 (approximately half-maximal Ca(2+) activation). Muscle stiffness was estimated during pCa 6.0 activations by imposing length steps at different time intervals after repeated shortening/restretch perturbations. Relative stiffness and relative tension were correlated (p < 0.001) during recovery, suggesting that tension overshoots reflect a temporary increase in the number of attached cross-bridges. Rates of tension recovery (k(tr)) correlated (p < 0.001) with the relative residual force prevailing immediately after restretch. Force also recovered to the isometric value more quickly at 5.7 ≤ pCa ≤ 5.9 than at pCa 4.5 (ANOVA, p < 0.05). These results show that k(tr) measurements underestimate the rate of isometric force development during submaximal Ca(2+) activations and suggest that the rate of tension recovery is limited primarily by the availability of actin binding sites