Contraction Of Skeletal Muscle Fibers Research Articles

A controversial issue: Can mitochondria modulate cytosolic calcium and contraction of skeletal muscle fibers?

Mitochondria are characterized by a high capacity to accumulate calcium thanks to the electrochemical gradient created by the extrusion of protons in the respiratory chain. Thereby calcium can enter crossing the inner mitochondrial membrane via MCU complex, a high-capacity, low-affinity transport mechanism. Calcium uptake serves numerous purposes, among them the regulation of three dehydrogenases of the citric cycle, apoptosis via permeability transition, and, in some cell types, modulation of cytosolic calcium transients. This Review is focused on mitochondrial calcium uptake in skeletal muscle fibers and aims to reanalyze its functional impact. In particular, we ask whether mitochondrial calcium uptake is relevant for the control of cytosolic calcium transients and therefore of contractile performance. Recent data suggest that this may be the case, at least in particular conditions, as modified expression of MCU complex subunits or of proteins involved in mitochondrial dynamics and ablation of the main cytosolic calcium buffer, parvalbumin.

Measurement of force and calcium release using mechanically skinned fibers from mammalian skeletal muscle.

The mechanically skinned (or "peeled") skeletal muscle fiber technique is a highly versatile procedure that allows controlled examination of each of the steps in the excitation-contraction (EC)-coupling sequence in skeletal muscle fibers, starting with excitation/depolarization of the transverse tubular (T)-system through to Ca2+ release from sarcoplasmic reticulum (SR) and finally force development by the contractile apparatus. It can also show the overall response of the whole EC-coupling sequence together, such as in twitch and tetanic force responses. A major advantage over intact muscle fiber preparations is that it is possible to set and rapidly manipulate the "intracellular" conditions, allowing examination of the effects of key variables (e.g., intracellular pH, ATP levels, redox state, etc.) on each individual step in EC coupling. This Cores of Reproducibility in Physiology (CORP) article describes the rationale, procedures, and experimental details of the various ways in which the mechanically skinned fiber technique is used in our laboratory to examine the physiological mechanisms controlling Ca2+ release and contraction in skeletal muscle fibers and the aberrations and dysfunction occurring with exercise and disease.

Extracellular matrix fibronectin initiates endothelium-dependent arteriolar dilatation via the heparin-binding, matricryptic RWRPK sequence of the first type III repeat of fibrillar fibronectin.

The local arteriolar dilatation produced by contraction of skeletal muscle is dependent upon multiple signalling mechanisms. In addition to the many metabolic signals that mediate this vasodilatation, we show here that the extracellular matrix protein fibronectin also contributes to the response. This vasodilatory signal requires the heparin-binding matricryptic RWRPK sequence in the first type III repeat of fibrillar fibronectin. The fibronectin-dependent component of the integrated muscle contraction-dependent arteriolar vasodilatation is coupled through an endothelial cell-dependent signalling pathway. Recent studies in contracting skeletal muscle have shown that functional vasodilatation in resistance arterioles has an endothelial cell (EC)-dependent component, and, separately have shown that the extracellular matrix protein fibronectin (FN) contributes to functional dilatation in these arterioles. Here we test the hypotheses that (i) the matricryptic heparin-binding region of the first type III repeat of fibrillar FN (FNIII1H) mediates vasodilatation, and (ii) this response is EC dependent. Engineered FN fragments with differing (defined) heparin- and integrin-binding capacities were applied directly to resistance arterioles in cremaster muscles of anaesthetized (pentobarbital sodium, 65 mg kg(-1)) mice. Both FNIII1H,8-10 and FNIII1H induced dilatations (12.2 ± 1.7 μm, n = 12 and 17.2 ± 2.4 μm, n = 14, respectively) whereas mutation of the active sequence (R(613) WRPK) of the heparin binding region significantly diminished the dilatation (3.2 ± 1.8 μm, n = 10). Contraction of skeletal muscle fibres via electrical field stimulation produced a vasodilatation (19.4 ± 1.2 μm, n = 12) that was significantly decreased (to 7.0 ± 2.7 μm, n = 7, P < 0.05) in the presence of FNIII1Peptide 6, which blocks extracellular matrix (ECM) FN and FNIII1H signalling. Furthermore, FNIII1H,8-10 and FNIII1H applied to EC-denuded arterioles failed to produce any dilatation indicating that endothelium was required for the response. Finally, FNIII1H significantly increased EC Ca(2+) (relative fluorescence 0.98 ± 0.02 in controls versus 1.12 ± 0.05, n = 17, P < 0.05). Thus, we conclude that ECM FN-dependent vasodilatation is mediated by the heparin-binding (RWRPK) sequence of FNIII1 in an EC-dependent manner. Importantly, blocking this signalling sequence decreased the dilatation to skeletal muscle contraction, indicating that there is a physiological role for this FN-dependent mechanism.

Local control of blood flow during active hyperaemia: what kinds of integration are important?

The focus of this review is on local mechanisms modifying arteriolar resistance to match blood flow to metabolism. In skeletal muscle many local mediators are known, including K(+) , nitric oxide (NO), purines and prostaglandins. Each accounts for about 30% of the response; it is widely held that these act redundantly: this concept awaits systematic testing. Understanding signal integration also requires consideration of microvascular network morphology in relation to local communication pathways between endothelial and smooth muscle cells (which are critical for many local responses, including dilatation to skeletal muscle contraction) and in relation to the spread of vasodilator signals up- and downstream throughout the network. Mechanisms mediating the spread of dilatation from local to remote sites have been well studied using acetylcholine (ACh), but remote dilatations to contraction of skeletal muscle fibres also occur. Importantly, these mechanisms clearly differ from those initiated by ACh, but much remains undefined. Furthermore, capillaries contribute to metabolic dilatation as they dilate arterioles directly upstream in response to vasoactive agents or contraction of adjacent muscle fibres. Given the dispersed arrangement of motor units, precise matching of flow to metabolism is not attainable unless signals are initiated only by 'active' capillaries. As motor units are recruited, signals that direct blood flow towards these active fibres will eventually be supported by local and spreading responses in the arterioles associated with those fibres. Thus, mechanisms of integration of vasodilator signalling across elements of the microvasculature remain an important area of focus for new studies.

Chloride conductance in the transverse tubular system of rat skeletal muscle fibres: importance in excitation–contraction coupling and fatigue

Contraction in skeletal muscle fibres is governed by excitation of the transverse-tubular (t-) system, but the properties of the t-system and their importance in normal excitability are not well defined. Here we investigate the properties of the t-system chloride conductance using rat skinned muscle fibres in which the sarcolemma has been mechanically removed but the normal excitation-contraction coupling mechanism kept functional. When the t-system chloride conductance was eliminated, either by removal of all Cl(-) or by block of the chloride channels with 9-anthracene carboxylic acid (9-AC) or by treating muscles with phorbol 12,13-dibutyrate, there was a marked reduction in the threshold electric field intensity required to elicit a t-system action potential (AP) and twitch response. Calculations of the t-system chloride conductance indicated that it constitutes a large proportion of the total chloride conductance observed in intact fibres. Blocking the chloride conductance increased the size of the twitch response and was indicative that Cl(-) normally carries part of the repolarizing current across the t-system membrane on each AP. Block of the t-system chloride conductance also reduced tetanic force responses at higher frequency stimulation (100 Hz) and greatly reduced twitch responses in the period shortly after a brief tetanus, owing to rapid loss of t-system excitability during the AP train. Blocking activity of the Na(+)-K(+) pump in the t-system membrane caused loss of excitability owing to K(+) build-up in the sealed t-system, and this occurred approximately 3-4 times faster when the chloride conductance was blocked. These findings show that the t-system chloride conductance plays a vital role during normal activity by countering the effects of K(+) accumulation in the t-system and maintaining muscle excitability.

Alterations of the proton‐T2 time in relaxed skeletal muscle induced by passive extremity flexions

To demonstrate reciprocal changes of the apparent proton-T2 time in the biceps and triceps due to passive contraction and extension of the muscle fibers. The contraction state of the upper arm muscles of six healthy volunteers was passively changed by alternating the forearm position between the straight-arm position and an elbow flexion of 90 degrees. The relaxation of the muscle during passive contraction and extension was measured with the use of muscle electromyography (EMG) experiments. Spin-echo (SE) MRI with increasing echo times (TEs) of 12-90 msec was used to acquire the averaged signal decay of the segmented biceps and triceps. The apparent T2 was deduced using monoexponential least-square fitting. The median T2 alterations in biceps and triceps among all volunteers were found to be 1.2 and -1.3 msec in the straight and bent forearm positions, respectively. The confidence intervals (0.5 to 1.7 msec in biceps, and -2.6 to -1.1 msec in triceps) clearly indicate that proton-T2 in MR images is significantly (P < 0.05) prolonged with muscle contraction. The observed increase of the proton-T2 time was correlated with a passive contraction of skeletal muscle fibers. This passive effect can be attributed to changes in the intracellular water mobility corresponding to the well-known "active" T2 increase that occurs after stimulation of muscle.

Remote arteriolar dilations in response to muscle contraction under capillaries.

In hamster cremaster muscle, it has been shown previously that contraction of skeletal muscle fibers underlying small groups of capillaries (modules) induces dilations that are proportional to metabolic rate in the two arteriolar generations upstream of the stimulated capillaries (Berg BR, Cohen KD, and Sarelius IH. Am J Physiol Heart Circ Physiol 272: H2693-H2700, 1997). These remote dilations were hypothesized to be transmitted via gap junctions and not perivascular nerves. In the present study, halothane (0.07%) blocked dilation in the module inflow arteriole, and dilation in the second arteriolar generation upstream, the branch arteriole, was blocked by both 600 mosM sucrose and halothane but not tetrodotoxin (2 microM). Dilations in both arterioles were not blocked by the gap junction uncoupler 18-beta-glycyrrhetinic acid (40 microM), and 80 mM KCl did not block dilation of the module inflow arteriole. These data implicate a gap junctional-mediated pathway insensitive to 18-beta-glycyrrhetinic acid in dilating the two arterioles upstream of the capillary module during "remote" muscle contraction. Dilation in the branch arteriole, but not the module inflow arteriole, was attenuated by 100 microM N(omega)-nitro-L-arginine. Thus selective contraction of muscle fibers underneath capillaries results in dilations in the upstream arterioles that have characteristics consistent with a signal that is transmitted along the vessel wall through gap junctions, i.e., a conducted vasodilation. The observed insensitivities to 18-beta-glycyrrhetinic acid, to KCl, and to N(omega)-nitro-L-arginine suggest, however, that there are multiple signaling pathways by which remote dilations can be initiated in these microvessels.

Mechanical and structural properties underlying contraction of skeletal muscle fibers after partial 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide cross-linking

We show prolonged contraction of permeabilized muscle fibers of the frog during which structural order, as judged from low-angle x-ray diffraction, was preserved by means of partial cross-linking of the fibers using the zero-length cross-linker 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide. Ten to twenty percent of the myosin cross-bridges were cross-linked, allowing the remaining 80-90% to cycle and generate force. These fibers displayed a well-preserved sarcomeric order and mechanical characteristics similar to those of intact muscle fibers. The intensity of the brightest meridional reflection at 14.5 nm, resulting from the projection of cross-bridges evenly spaced along the myofilament length, decreased by 60% as a relaxed fiber was deprived of ATP and entered the rigor state. Upon activation of a rigorized fiber by the addition of ATP, the intensity of this reflection returned to 97% of the relaxed value, suggesting that the overall orientation of cross-bridges in the active muscle was more perpendicular to the filament axis than in rigor. Following a small-amplitude length step applied to the active fibers, the reflection intensity decreased for both releases and stretches. In rigor, however, a small stretch increased the amplitude of the reflection by 35%. These findings show the close link between cross-bridge orientation and tension changes.

Altered kinetics of contraction in skeletal muscle fibers containing a mutant myosin regulatory light chain with reduced divalent cation binding

We examined the kinetic properties of rabbit skinned skeletal muscle fibers in which the endogenous myosin regulatory light chain (RLC) was partially replaced with a mutant RLC (D47A) containing a point mutation within the Ca2+/Mg2+ binding site that severely reduced its affinity for divalent cations. We found that when approximately 50% of the endogenous RLC was replaced by the mutant, maximum tension declined to approximately 60% of control and the rate constant of active tension redevelopment (ktr) after mechanical disruption of cross-bridges was reduced to approximately 70% of control. This reduction in ktr was not an indirect effect on kinetics due to a reduced number of strongly bound myosin heads, because when the strongly binding cross-bridge analog N-ethylmaleimide-modified myosin subfragment1 (NEM-S1) was added to the fibers, there was no effect upon maximum ktr. Fiber stiffness declined after D47A exchange in a manner indicative of a decrease in the number of strongly bound cross-bridges, suggesting that the force per cross-bridge was not significantly affected by the presence of D47A RLC. In contrast to the effects on ktr, the rate of tension relaxation in steadily activated fibers after flash photolysis of the Ca2+ chelator diazo-2 increased by nearly twofold after D47A exchange. We conclude that the incorporation of the nondivalent cation-binding mutant of myosin RLC decreases the proportion of cycling cross-bridges in a force-generating state by decreasing the rate of formation of force-generating bridges and increasing the rate of detachment. These results suggest that divalent cation binding to myosin RLC plays an important role in modulating the kinetics of cross-bridge attachment and detachment.

Mechanisms of pattern generation in co-cultures of embryonic spinal cord and skeletal muscle

Spontaneous output patterns of embryonic spinal cord slices in vitro were investigated in order to study the formation of pattern-generating networks. Patterns of spontaneous contractions of skeletal muscle fibers were recorded in co-cultures of embryonic rat spinal cord, dorsal root ganglia and skeletal muscle. A part of these contractions was shown to be driven by spinal circuits. These neuron-driven activity patterns changed from random to rhythmic when the inhibitory synapses in the spinal cord were blocked by strychnine, bicuculline or both. Rhythmic patterns consisted of bursts of activity (tetanic contractions) followed by periods of relaxation. The transition from random to rhythmic patterns occurred during a period of heavily increased rate of activity. Presynaptic inhibition was not involved critically in the generation of rhythmic patterns. Such patterns were, however, modulated through muscarinic and α-adrenergic receptors. Neither NMDA nor glutamate nor its uptake blocker dihydrokainate induced rhythmic patterns of contraction, although NMDA in the presence of low magnesium increased moderately the rate of random activity. In order to study the size of pattern-generating networks, parts of the spinal cord slices were sectioned during rhythmic activity. Tangential cuts at the lateral or dorsal side of the slices reduced either the rate or the duration of the bursts or both. Sagittal cuts suppressed the activity almost totally. These findings suggest that the pattern generators in the slices consist of excitatory networks covering the entire slice, and that these networks reverberate following spontaneous activity of some distributed elements.

Quantification of axial forces in rectum-anal canal of the cat.

A newly developed probe was used to measure in vivo axial forces in the rectum-anal canal of the anesthetized cat. Spontaneous contractions of the smooth muscle of the internal anal sphincter were recorded, as were neurally evoked contractions of striated muscle of the external anal sphincter. Bilateral electrical stimulation (1-10 V, 1-5 Hz, 0.05 ms duration) of motor axons in pudendal nerves elicited two responses. One was synchronous phasic contractions of skeletal muscle fibers of the external anal sphincter that were not abolished by atropine but were by gallamine trithiodide. They occurred at short latencies (1-2 ms) and were mediated through low-threshold (1-3 V, 0.05 ms duration) efferent axons in the pudendal nerves. Contraction times ranged from 45 to 60 ms, and contraction duration ranged from 100 to 160 ms. The second response was a progressive elevation in tone of the anal canal due to contractions either of the smooth muscle of the rectum and/or that of the internal anal sphincter. The elevation in smooth muscle tone concomitant with pudendal nerve stimulation may be due to reflex activation of cholinergic neural pathways, since the response was abolished by atropine.

Direct observations of muscle arterioles and venules following contraction of skeletal muscle fibres in the rat.

Direct observations have been made of responses of individual arterioles and venules of rat spinotrapezius muscle to contraction of the skeletal muscle fibres. Stimuli of 4-6 V intensity, 0.1 ms duration, delivered via a micro-electrode inserted into the spinotrapezius, evoked contraction of a small bundle of skeletal muscle fibres, followed by vasodilatation which was limited to all those arterioles and venules which crossed or ran alongside activated muscle fibres. Since venules outside the region of contraction, but supplied by dilating arterioles, were not passively distended by the attendant rise in intravascular pressure, it is concluded that both the arterioles and venules dilated actively in response to muscle contraction. All arterioles responded to a single twitch contraction, the terminal arterioles (7-13 micron i.d.) showing the largest increase in diameter. Collecting venules (9-18 micron i.d.) responded to just two twitches in 1 s and larger venules to five twitches in 1 s. When twitch contractions were continuously evoked for 10 s, the responses in individual arterioles and venules were graded with twitch frequency, the fastest and largest response occurring at 6-8 Hz. Tetanic contraction, at 40 Hz for 1 s, produced faster responses in all vessels, a maximum 55% increase from resting internal diameter being attained in only 8 s in some terminal arterioles. In all vessels the responses to tetanic contraction were equal to the maximal dilatation induced by papaverine. These results, in contrast with conclusions drawn from indirect estimates of venous responses, show that venules, like arterioles, dilate actively in response to muscle contraction. Venule dilatation may reduce the rise in capillary hydrostatic pressure, thereby limiting the outward filtration of fluid.

Are cardiac muscle cells 'skinned' by EGTA or EDTA?

KNOWLEDGE of the processes involved in the contraction of skeletal muscle fibres has been greatly advanced by study of the ‘skinned’ fibre preparation. Here the sarcolemma is peeled off, giving access to the intracellular organelles unhampered by an intervening cell membrane. The small size of cardiac muscle cells had seemed to preclude the use of this technique, and so interest has been shown in a ‘chemical’ method for ‘skinning’ heart cells, which involves exposing the muscle to EGTA and EDTA (ref. 1). However, this paper questions the effectiveness of the EDTA/EGTA skinning methods, reporting that tension production in fully treated cells is [Na]o dependent, and that the cells can support resting and action potentials in low [K]o-‘skinning’ media. It is concluded that both the high Ca-sensitivity and low membrane potential of EGTA/EDTA-treated cells occur without radical impairment of the sarcolemmal permeability barrier. An alternative explanation for the high sensitivity to extracellular calcium is presented which involves the Ca–Na exchange mechanism.