No direct effect of creatine phosphate on the cross-bridge cycle in cardiac myofibrils

Creatine phosphate (CP) and creatine kinase (CK) are involved in the rapid resynthesis of ATP and thereby serve to stabilize ATP concentration and to maintain free ADP low inside cardiac muscle cells during contraction. Recently, it has been suggested from experiments in permeabilized multicellular preparations that CP/CK also regulate the kinetics of the actomyosin interaction (cross-bridge cycle) and may explain contractile dysfunction, for instance, during ischemia. However, the reported effects of CP/CK may be confounded by diffusion limitations in multicellular preparations in which inorganic phosphate (P_i) and ADP may significantly accumulate during contraction. To test this hypothesis, we measured force production and the rates of force development (k _ACT and k _TR) in isolated cardiac myofibrils, in which rapid concentration changes of Ca²⁺, CP/CK, and P_i were imposed using a rapid perfusion change system. The results showed that CP/CK did not influence maximum force-generating capacity, whereas P_i markedly reduced force and increased the rates of force development. No effects of CP/CK on the rates of force development were observed, consistent with the notion that CP/CK do not exert a direct effect on the actomyosin interaction.     

Mechanism of cross-bridge detachment in isometric force relaxation of skeletal and cardiac myofibrils

Skeletal and cardiac muscle relaxation is governed by the interplay between two macromolecular systems: (i) membrane bound Ca²⁺ transport proteins and (ii) sarcomeric proteins. Photolysis experiments in skinned muscle preparations and fast solution switching studies in single myofibrils offer means for isolating sarcomeric mechanisms of relaxation from those related to myoplasmic Ca²⁺ removal. Single myofibril experiments have recently shown that cross-bridge mechanics and detachment kinetics are the major determinants of the time course of relaxation. Full force decay in myofibrils occurs in two phases: a slow one followed by a rapid one. The latter is initiated by sarcomere ‘give’ and dominated by inter-sarcomere dynamics while the former occurs under nearly isometric conditions. Strong evidence has been found that the slow rate of force decay in myofibril relaxation reflects the rate at which cross-bridges leave force-generating states under isometric conditions. Dissection of chemo-mechanical transduction process in myofibrils indicate that both forward and backward transitions of cross-bridges from force-generating to non-force-generating states contribute to muscle relaxation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Theoretical determination of force-length relations of intact human skeletal muscles using the cross-bridge model

The purpose of this study was to determine force-length relations of selected human skeletal muscles, based on the theoretical foundations of the cross-bridge model and to calculate a strength curve for knee extension from these relations. Force-length relations were determined for the rectus femoris, vastus lateralis, vastus medialis, vastus intermedius and gastrocnemius muscles, using sarcomere/ fiber length data form both legs of four cadavers and sarcomere geometry data reported in the literature. It appears that the two-joint muscles investigated in this study are not able to produce force throughout their full anatomical range of motion, whereas the one-joint muscles can. The strength curve for knee extension was determined as the sum of the force-length relations of the individual knee extensor muscles and showed good agreement with experimentally obtained knee extensor strength curves.     

Effects of inorganic phosphate on cross-bridge behavior after photorelease of ATP in permeabilized cells of rat skeletal muscle

Effects of 20 mM inorganic phosphate on the cross-bridge behavior after photorelease of ATP from caged ATP was studied by X-ray diffraction in rat skinned psoas muscle fibers at 16 degrees C. In the first 30 ms after the photorelease, tension was similar in the presence and absence of phosphate. The tension development was then suppressed in the presence of phosphate. At 500 ms after the photolysis, it was lower by about 30% in the presence of phosphate. In the presence of phosphate, the intensity of the third meridional reflection from the thick filament at 1/14.4 nm(-1) increased more slowly and was 60% of the level without phosphate at 500 ms after the photolysis. The intensities of the equatorial (1,1) reflection and the actin layer-line at 1/36 nm(-1) decreased to a lower level in the presence of phosphate. These results suggest that phosphate does not affect dissociation of myosin heads from actin, but decreases the number of myosin heads in force-generating conformation and reduces tension. Phosphate may also shift the equilibrium between the detached and attached states towards the former.     

Functional differences in type-I fibres from two slow skeletal muscles of rabbit

The present study addressed the question of whether the slow fibres of mammalian skeletal muscle, containing the myosin heavy chain MHCI (type-I fibres), are a functionally homogeneous population. We compared various properties of Ca²⁺-activated, skinned, type-I fibres from the soleus and semitendinosus muscles of a rabbit. Soleus type-I fibres showed significantly faster kinetics of stretch activation, measured as the time-to-peak of the stretch-induced, delayed force increase, t₃, than semitendinosus fibres (1239±438 ms, n=136, vs. 1600±409 ms, n=208 respectively) (means±SD, 22 °C). Similarly, the speed of unloaded shortening at 15 °C was faster in soleus than in semitendinosus fibres [0.79±0.16 fibre lengths (FL) s^–1, n=44, vs. 0.65±0.15 FL s^–1, n=35 respectively]. The kinetics of stretch activation were more temperature sensitive in semitendinosus than in soleus fibres. Finally, the generation of steady-state isometric force was more sensitive to Ca²⁺ in semitendinosus than in soleus fibres: [pCa₅₀ (–log [Ca²⁺] for half-maximal activation) at 22 °C: 6.29±0.15, n=28, vs. 6.19±0.10, n=18 respectively]. These results suggest strongly that there is no functional homogeneity within type-I fibres of different muscles. The observed differences might reflect the existence of more than one functionally different slow myosin heavy chain isoforms or other modifications of contractile proteins.     

Transverse stiffness of myofibrils of skeletal and cardiac muscles studied by atomic force microscopy

The transverse stiffness of single myofibrils of skeletal and cardiac muscles was examined by atomic force microscopy. The microscopic images of both skeletal and cardiac myofibrils in a rigor state showed periodical striation patterns separated by Z-bands, which is characteristic of striated muscle fibers. However, sarcomere patterns were hardly distinguishable in the stiffness distributions of the relaxed myofibrils of skeletal and cardiac muscles. Myofibrils in a rigor state were significantly stiff compared with those in a relaxed state, and in each state, cardiac myofibrils were significantly stiffer compared with skeletal myofibrils. By proteolytic digestions of sarcomere components of myofibrils, it was suggested that cardiac myofibrils are laterally stiffer than skeletal myofibrils because Z-bands, connectin (titin) filament networks, and other components of sarcomere structures for the former myofibrils are stronger than those for the latter.     

Temperature dependence of the force-generating process in single fibres from frog skeletal muscle

Generation of force and shortening in striated muscle is due to the cyclic interactions of the globular portion (the head) of the myosin molecule, extending from the thick filament, with the actin filament. The work produced in each interaction is due to a conformational change (the working stroke) driven by the hydrolysis of ATP on the catalytic site of the myosin head. However, the precise mechanism and the size of the force and length step generated in one interaction are still under question. Here we reinvestigate the endothermic nature of the force-generating process by precisely determining, in tetanised intact frog muscle fibres under sarcomere length control, the effect of temperature on both isometric force and force response to length changes. We show that raising the temperature: (1) increases the force and the strain of the myosin heads attached in the isometric contraction by the same amount (∼70 %, from 2 to 17 °C); (2) increases the rate of quick force recovery following small length steps (range between −3 and 2 nm (half-sarcomere)⁻¹) with a Q ₁₀ (between 2 and 12 °C) of 1.9 (releases) and 2.3 (stretches); (3) does not affect the maximum extent of filament sliding accounted for by the working stroke in the attached heads (10 nm (half-sarcomere)⁻¹). These results indicate that in isometric conditions the structural change leading to force generation in the attached myosin heads can be modulated by temperature at the expense of the structural change responsible for the working stroke that drives filament sliding. The energy stored in the elasticity of the attached myosin heads at the plateau of the isometric tetanus increases with temperature, but even at high temperature this energy is only a fraction of the mechanical energy released by attached heads during filament sliding.     

Stoichiometry of phosphocreatine and inorganic phosphate changes in rat skeletal muscle

The apparent T1's of phosphate metabolites were measured during and after series of twitch contractions in gastrocnemius muscles of pentobarbital-anesthetized rats by steady-state progressive saturation using spatially-selective composite pulses. There was no significant change in apparent T1's of inorganic phosphate (Pi), phosphocreatine (PCr), or the three phosphates of ATP. There was a 5-10% decrease in the sum of Pi and PCr integrals, and in the sum of the gamma- and beta- phosphates of ATP during stimulation, but no significant change in the ratio (Pi + PCr)/(gamma-ATP + beta-ATP). The results indicate that there is no selective decrease in NMR observable Pi during or after a series of muscle contractions.     

Spontaneous fluctuations in NAD-kinase activity of rabbit skeletal muscles

Spontaneous fluctuations in the time of activity of a 280–300-times purified preparation of NAD-kinase from rabbit skeletal muscles are described after its dilution. No flucturations of activity were found in an unfrozen but undiluted preparation. After preincubation of the diluted enzyme with substrates (NAD and ATP) its activity did not fluctuate.Department of Biochemistry, Biological Faculty, Moscow State University, Moscow. (Presented by Academician S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 8, pp. 957–959, August, 1976.     

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 Ca²⁺-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.     

Contractile effects of the exchange of cardiac troponin for fast skeletal troponin in rabbit psoas single myofibrils

The effects of the removal of fast skeletal troponin C (fsTnC) and its replacement by cardiac troponin C (cTnC) and the exchange of fast skeletal troponin (fsTn) for cardiac troponin (cTn) were measured in rabbit fast skeletal myofibrils. Electrophoretic analysis of myofibril suspensions indicated that replacement of fsTnC or exchange of fsTn with cTnC or cTn was about 90% complete in the protocols used. Mechanical measurements in single myofibrils, which were maximally activated by fast solution switching, showed that replacement of fsTnC with cTnC reduced the isometric tension, the rate of tension rise following a step increase in Ca²⁺ ( k _act ), and the rate of tension redevelopment following a quick release and restretch ( k _tr ), but had no effect on the kinetics of the fall in tension when the concentration of inorganic phosphate (P_i) was abruptly increased ( k _Pi(+)). These data suggest that the chimeric protein produced by cTnC replacement in fsTn alters those steps controlling the weak-to-strong crossbridge attachment transition. Inefficient signalling within the chimeric troponin may cause these changes. However, replacement of fsTn by cTn had no effect on maximal isometric tension, k _act or k _tr , suggesting that these mechanics are largely determined by the isoform of the myosin molecule. Replacement of fsTn by cTn, on the other hand, shifted the pCa₅₀ of the pCa-tension relationship from 5.70 to 6.44 and reduced the Hill coefficient from 3.3 to 1.4, suggesting that regulatory protein isoforms primarily alter Ca²⁺ sensitivity and the cooperativity of the force-generating mechanism.     

Evidence for phosphate as a mediator of functional hyperaemia in skeletal muscles

Maximal twitch contractions of fast muscles of the cat caused an increase in phosphate concentration in the venous plasma from them: this efflux was greater as the contraction frequency was increased. At any given frequency of contraction, the phosphate efflux from contracting gastrocnemius was less than that from tibialis anterior and extersor digitorum longus, which have a higher proportion of fast fibres and exhibit greater functional hyperaemia. Soleus muscles, when contracting, released hardly any additional phosphate, except in the one experiment in which the muscle exhibited a functional hyperaemia. There was thus a consistent relationship between the extent of functional hyperaemia and phosphate efflux in different muscles and within any one group of muscles. Inorganic phosphate, given close arterially as NaH2PO4, was shown to be vasodilator.NaH2PO4 was much more potent than Na2HPO4, though this did not seem due simply to the associated change of pH. The functional hyperaemia of fast muscles could be matched, qualitatively and quantitatively, by injections or infusions of NaH2PO4. The possibility is discussed that the contraction hyperaemia of fast muscles is functionally related to phosphate release into the interstitial fluid during contractions.     

SFRP2 expression in rabbit myogenic progenitor cells and in adult skeletal muscles

Satellite cells derived from fast- and slow-twitch muscles have different properties in culture. We have used the differential display technique to retrieve genes differentially expressed in fast- and slow-twitch muscle satellite cell cultures. Amongst these genes we have identified, cloned, sequenced and studied the expression in muscle of rabbit secreted frizzled related protein 2 (SFRP2) mRNA, whose importance in cell fate determination has been well documented. It has been shown that SFRP2 is widely expressed in the developing embryo but its expression in the adult is much more restricted. We show that primary cultures of satellite cells from adult rabbit fast- and slow-twitch muscles strongly and differentially express SFRP2 mRNA. Embryonic rabbit muscle cell primary cultures also strongly express SFRP2 mRNA whereas the myoblast C2.7 cell line shows little expression. We also studied SFRP2 mRNA expression in growing, regenerating and denervated muscles. Embryonic rabbit muscles express SFRP2 mRNA but this rapidly falls off after birth. In adult rabbit muscles SFRP2 mRNA is detected within 1 day of either muscle damage or denervation. Thereafter the SFRP2 mRNA expression profiles are different for fast- and slow-twitch muscle. The function of SFRP2 in muscle is unknown but its putative activity as a Wnt antagonist and its precocious expression after muscle damage suggest a role in satellite cell activation.This revised version was published online in September 2005 with corrections to the Cover Date.     

Alterations in neuromuscular junction morphology during fast-to-slow transformation of rabbit skeletal muscles

Summary Chronic low frequency stimulation of motor nerves results in transformation of muscle fibre phenotype from fast- to slow-twitch. We examined the light and electron microscopic structure of neuromuscular junctions in normally fast twitch muscles, tibialis anterior and extensor digitorum longus of rabbit after 3 weeks of stimulation to determine whether synaptic structure is also modified during fibre type transformation. Neuromuscular junctions of stimulated and unstimulated (control) tibialis anterior and extensor digitorum longus muscles and unstimulated slow twitch soleus muscle were visualized with rhodamine-conjugated -bungarotoxin. Video light microscopic images of neuromuscular junctions were digitized to allow quantification of their surface areas, perimeters, lengths and widths. Three weeks of stimulation resulted in a decrease in the maximal velocity of muscle fibre shortening and augmentation of mitochondrial volume in fast muscles, demonstrating the efficacy of the stimulation protocol employed in altering muscle fibre phenotype. Neuromuscular junctions of control tibialis anterior and extensor digitorum longus are thin, compact, and continuous, with complex branching patterns. In contrast, those of slow-twitch soleus are thicker and discontinuous. Neuromuscular junctions in control tibialis anterior and extensor digitorum longus are larger than those in soleus. Three weeks of stimulation causes a marked decrease in the size of neuromuscular junctions in tibialis anterior and extensor digitorum longus, as reflected in the significant reduction in neuromuscular junction surface area, length and width. Electron microscopy of these junctions suggests that secondary postsynaptic folds in stimulated muscles are more closely spaced. Also, axon terminals of stimulated muscles appear to contain more densely packed synaptic vesicles and mitochondria than controls. Decreases in neuromuscular junction dimensions can be partly explained by muscle fibre atrophy. However, the decrease in neuromuscular junction size is proportionately greater than that of muscle fibre diameter in both muscles, indicating that factors other than fibre atrophy may contribute to the reduced neuromuscular junction size in stimulated muscles. Neuromuscular junctions of stimulated tibialis anterior and extensor digitorum longus muscles exhibit some features characteristic of normal soleus neuromuscular junctions, indicating structural adaptations consistent with the altered muscle fibre phenotype. On the other hand, neuromuscular junctions of 3 week stimulated tibialis anterior and extensor digitorum longus and their synaptic branches remain as thin and continuous as those of unstimulated controls, suggesting that the transformation of neuromuscular junctions towards a morphology characteristic of slow muscle, is only partial. These results demonstrate that an altered pattern of impulse activity causes significant synaptic remodelling in adult rabbit skeletal muscles.