Fatigue-induced changes in muscle fiber action potentials estimated by wavelet analysis

We aimed to investigate fatigue-induced changes in the spectral parameters of slow (SMF) and fast fatigable muscle fiber (FMF) action potentials using discrete wavelet (DWT) and fast Fourier (FFT) transforms. Intracellular potentials were recorded during repetitive stimulation of isolated muscle fibers immersed in Ca²⁺-enriched medium, while extracellular potentials were obtained from muscle fibers pre-exposed to electromagnetic microwaves (MMW, 2.45 GHz, 20 mW/cm²). The changes in the frequency distribution of the action potentials during the period of uninterrupted fiber activity were used as criteria for fatigue assessment. The wavelet coefficients’ changes in the calculated frequency scales demonstrated a contribution of the increased [Ca²⁺]₀ to an earlier compression of the frequency spectrum towards lower ranges. Root mean square (RMS) analysis of the wavelet coefficients calculated from SMF potentials showed a reduction of the higher frequencies (scale 1) by 90% in elevated [Ca²⁺]₀ vs. 55% in controls and an increase of low frequencies (scale 5) by 323% vs. 187%, respectively. For FMF potentials a decrease of 71% vs. 59% for high frequencies (scale 1, elevated [Ca²⁺]₀ vs. control) and an increase of 386% vs. 295% in scale 5, respectively, were observed. MMW pre-exposure resulted in increased muscle fiber resistance to fatigue. The fatigue-induced decrease of potential high frequencies (SMF: 59% vs. 96%, MMW vs. control; FMF: 30% vs. 92%, respectively), and the increase of low frequencies (SMF: 200% vs. 207%, MMW vs. control; FMF: 93% vs. 314%, respectively) were significantly smaller and delayed in exposed muscle fibers. Data from RMS analysis indicate that DWT provides a reliable method for estimation of muscle fatigue onset and progression.     

Pain-induced changes in cervical muscle activation do not affect muscle fatigability during sustained isometric contraction

This study investigated whether pain-induced changes in cervical muscle activation affect myoelectric manifestations of cervical muscle fatigue. Surface EMG signals were detected from the sternocleidomastoid and splenius capitis muscles bilaterally from 14 healthy subjects during 20-s cervical flexion contractions at 25% of the maximal force. Measurements were performed before and after the injection of 0.5 ml of hypertonic (painful) or isotonic (control) saline into either the sternocleidomastoid or splenius capitis in two experimental sessions. EMG average rectified value and mean power spectral frequency were estimated throughout the sustained contraction. Sternocleidomastoid or splenius capitis muscle pain resulted in lower sternocleidomastoid EMG average rectified value on the side of pain (P < 0.01). However, changes over time of sternocleidomastoid EMG average rectified value and mean frequency (myoelectric manifestations of fatigue) during sustained flexion were not changed during muscle pain. These results demonstrate that pain-induced modifications of cervical muscle activity do not change myoelectric manifestations of fatigue. This finding has implications for interpreting the mechanisms underlying greater cervical muscle fatigue in people with neck pain disorders.     

Formation and maintenance of tubular membrane projections require mechanical force, but their elongation and shortening do not require additional force

Living cells develop their own characteristic shapes depending on their physiological functions, and their morphologies are based on the mechanical characteristics of the cytoskeleton and of membranes. To investigate the role of lipid membranes in morphogenesis, we constructed a simple system that can manipulate liposomes and measure the forces required to transform their shapes. Two polystyrene beads (1 microm in diameter) were encapsulated in giant liposomes and were manipulated using double-beam laser tweezers. Without any specific interaction between the lipid membrane and beads, mechanical forces could be applied to the liposome membrane from the inside. Spherical liposomes transformed into a lemon shape with increasing tension, and tubular membrane projections were subsequently generated in the tips at either end. This process is similar to the liposomal transformation caused by elongation of encapsulated cytoskeletons. In the elongation stage of lemon-shaped liposomes, the force required for the transformation became larger as the end-to-end length increased. Just before the tubular membrane was generated, the force reached the maximum strength (approximately 11 pN). However, immediately after the tubular membrane developed, the force suddenly decreased and was maintained at a constant strength (approximately 4 pN) that was independent of further tube elongation or shortening, even though there was no excess membrane reservoir as occurs in living cells. When the tube length was shortened to approximately 2 microm, the liposome reversed to a lemon shape and the force temporarily increased (to approximately 7 pN). These results indicate that the simple application of mechanical force is sufficient to form a protrusion in a membrane, that a critical force and length is needed to form and to maintain the protrusion, and suggest that the lipid bilayer itself has the ability to buffer the membrane tension.     

Muscle Z-band ultrastructure: titin Z-repeats and Z-band periodicities do not match

Vertebrate muscle Z-bands show zig-zag densities due to different sets of alpha-actinin cross-links between anti-parallel actin molecules. Their axial extent varies with muscle and fibre type: approximately 50 nm in fast and approximately 100 nm in cardiac and slow muscles, corresponding to the number of alpha-actinin cross-links present. Fish white (fast) muscle Z-bands have two sets of alpha-actinin links, mammalian slow muscle Z-bands have six. The modular structure of the approximately 3 MDa protein titin that spans from M-band to Z-band correlates with the axial structure of the sarcomere; it may form the template for myofibril assembly. The Z-band-located amino-terminal 80 kDa of titin includes 45 residue repeating modules (Z-repeats) that are expressed differentially; heart, slow and fast muscles have seven, four to six and two to four Z-repeats, respectively. Gautel et al. proposed a Z-band model in which each Z-repeat links to one level of alpha-actinin cross-links, requiring that the axial extent of a Z-repeat is the same as the axial separation of alpha-actinin layers, of which there are two in every actin crossover repeat. The span of a Z-repeat in vitro is estimated by Atkinson et al. to be 12 nm or less; much less than half the normal vertebrate muscle actin crossover length of 36 nm. Different actin-binding proteins can change this length; it is reduced markedly by cofilin binding, or can increase to 38.5 nm in the abnormally large nemaline myopathy Z-band. Here, we tested whether in normal vertebrate Z-bands there is a marked reduction in crossover repeat so that it matches twice the apparent Z-repeat length of 12 nm. We found that the measured periodicities in wide Z-bands in slow and cardiac muscles are all very similar, about 39 nm, just like the nemaline myopathy Z-bands. Hence, the 39 nm periodicity is an important conserved feature of Z-bands and either cannot be explained by titin Z-repeats as previously suggested or may correlate with two Z-repeats.     

Structural changes in muscle crossbridges accompanying force generation

We have investigated the structure of the crossbridges in muscles rapidly frozen while relaxed, in rigor, and at various times after activation from rigor by flash photolysis of caged ATP. We used Fourier analysis of images of cross sections to obtain an average view of the muscle structure, and correspondence analysis to extract information about individual crossbridge shapes. The crossbridge structure changes dramatically between relaxed, rigor, and with time after ATP release. In relaxed muscle, most crossbridges are detached. In rigor, all are attached and have a characteristic asymmetric shape that shows strong left-handed curvature when viewed from the M-line towards the Z-line. Immediately after ATP release, before significant force has developed (20 ms) the homogeneous rigor population is replaced by a much more diverse collection of crossbridge shapes. Over the next few hundred milliseconds, the proportion of attached crossbridges changes little, but the distribution of the crossbridges among different structural classes continues to evolve. Some forms of attached crossbridge (presumably weakly attached) increase at early times when tension is low. The proportion of several other attached non-rigor crossbridge shapes increases in parallel with the development of active tension. The results lend strong support to models of muscle contraction that have attributed force generation to structural changes in attached crossbridges.     

Fatigue-induced modulation of the H reflex of soleus muscle in humans

We studied the effect of fatigue of the mm. gastrocnemius-soleus on the H reflex elicited by transcutaneous stimulation of n. tibialis and recorded from the m. soleus; healthy 18-to 34-year-old volunteers were tested. Fatigue was evoked by long-lasting (6 to 9 min) voluntary tonic static sole flexion of the foot (ankle extension) with a force equal to 75% of the maximum voluntary contraction (MVC). The amplitude of H reflex significantly (P < 0.001) decreased to about 60% of the initial value immediately after the period of fatiguing effort. Within 2 to 3 min, it relatively rapidly recovered and reached about 90% of the control, and this was followed by a period of slow recovery to about 96–97% of the initial value 30 min after conditioning fatigue. We suppose that the initial period of suppression of the H reflex results to a considerable extent in an increase in the intensity of presynaptic inhibition of transmission from Ia afferents due to tonic activation of high-threshold (groups III and IV) afferent fibers induced by intensive fatigue-related metabolic changes in the muscles. More long-lasting (tens of minutes) changes are related to slow reverse development of direct effects of fatigue-induced biochemical shifts in the muscle. Neirofiziologiya/Neurophysiology, Vol. 38, Nos. 5/6, pp. 426–431, September–December, 2006.     

Effects of tendon and muscle belly dissection on muscular force transmission following tendon transfer in the rat

The aim of the present study was to quantify to what extent the scar tissue formation following the transfer of flexor carpi ulnaris (FCU) to the distal tendon of extensor carpi radialis (ECR) affects the force transmission from transferred FCU in the rat. Five weeks after recovery from surgery (tendon transfer group) and in a control group, isometric length-force characteristics of FCU were assessed for progressive stages of dissection: (i) with minimally disrupted connective tissues, (ii) after full dissection of FCU distal tendon exclusively, and (iii) after additional partial dissection of FCU muscle belly. Total and passive length-force characteristics of transferred and control FCU changed significantly by progressive stages of dissection. In both groups, tendon dissection decreased passive FCU force exerted at the distal tendon, as well as the slope of the length-force curve. However, force and slope changes were more pronounced for transferred FCU compared to controls. No additional changes occurred after muscle belly dissection. In contrast, total force increased in transferred FCU following both tendon and muscle belly dissection at all lengths studied, while dissection decreased total force of control FCU. In addition, after tendon and muscle belly dissection, we found decreased muscle belly lengths at equal muscle-tendon complex lengths of transferred FCU. We conclude that scar tissue limits the force transmission from transferred FCU muscle via the tendon of insertion to the skeleton, but that some myofascial connectivity of the muscle should be classified as physiological.     

High fat diet-induced changes in mouse muscle mitochondrial phospholipids do not impair mitochondrial respiration despite insulin resistance

Background

Type 2 diabetes mellitus and muscle insulin resistance have been associated with reduced capacity of skeletal muscle mitochondria, possibly as a result of increased intake of dietary fat. Here, we examined the hypothesis that a prolonged high-fat diet consumption (HFD) increases the saturation of muscle mitochondrial membrane phospholipids causing impaired mitochondrial oxidative capacity and possibly insulin resistance.

Methodology

C57BL/6J mice were fed an 8-week or 20-week low fat diet (10 kcal%; LFD) or HFD (45 kcal%). Skeletal muscle mitochondria were isolated and fatty acid (FA) composition of skeletal muscle mitochondrial phospholipids was analyzed by thin-layer chromatography followed by GC. High-resolution respirometry was used to assess oxidation of pyruvate and fatty acids by mitochondria. Insulin sensitivity was estimated by HOMA-IR.

Principal Findings

At 8 weeks, mono-unsaturated FA (16∶1n7, 18∶1n7 and 18∶1n9) were decreased (−4.0%, p<0.001), whereas saturated FA (16∶0) were increased (+3.2%, p<0.001) in phospholipids of HFD vs. LFD mitochondria. Interestingly, 20 weeks of HFD descreased mono-unsaturated FA while n-6 poly-unsaturated FA (18∶2n6, 20∶4n6, 22∶5n6) showed a pronounced increase (+4.0%, p<0.001). Despite increased saturation of muscle mitochondrial phospholipids after the 8-week HFD, mitochondrial oxidation of both pyruvate and fatty acids were similar between LFD and HFD mice. After 20 weeks of HFD, the increase in n-6 poly-unsaturated FA was accompanied by enhanced maximal capacity of the electron transport chain (+49%, p = 0.002) and a tendency for increased ADP-stimulated respiration, but only when fuelled by a lipid-derived substrate. Insulin sensitivity in HFD mice was reduced at both 8 and 20 weeks.

Conclusions/Interpretation

Our findings do not support the concept that prolonged HF feeding leads to increased saturation of skeletal muscle mitochondrial phospholipids resulting in a decrease in mitochondrial fat oxidative capacity and (muscle) insulin resistance.     

When phenotypes do not match genotypes--unexpected phenotypic diversity and potential environmental constraints in Icelandic stickleback

Divergent lateral plate phenotypes in stickleback represent one of only a few cases known, where a single gene underlies the phenotype under divergent selection between different habitats. However, the selection pressures leading to the repeated loss of lateral plates in freshwater are still not well understood. By genotyping 838 individuals from 9 independently colonized lakes and 1 marine population in Iceland, we found 1) that only in some lakes are phenotypes associated with the expected genotype and 2) that the independent repeated occurrence of a rarely described plate phenotype is expressed in the absence of an allele that is usually associated with this phenotype. This suggests that either other genes such as modifiers might be under divergent selection between lakes or that lateral plate expression in these populations is restricted due to environmental constraints.     

Tandem zyxin LIM sequences do not enhance force sensitive accumulation

The ability to sense mechanical forces is vital to cell physiology. Yet, the molecular basis of mechano-signaling remains unclear. Previous studies have shown that zyxin, a focal adhesion protein, is recruited at force-bearing sites on the actin cytoskeleton and, therefore, identifying zyxin as a mechano-sensing protein candidate. Furthermore, zyxin accumulation at force-bearing sites requires the LIM domain located at the C-terminus of zyxin. The zyxin LIM domain consists of three LIM motifs, each containing two zinc-binding sites. Since individual LIM motifs do not accumulate at focal adhesions or force-bearing sites, we hypothesize that multiple zyxin LIM domains increase force sensitivity. Using a miniature force sensor and GFP-tagged LIM variants, we quantified the relationship between single, tandem dimer and trimer LIM protein localization and traction forces. While the presence of extra LIM domains affected VASP recruitment to focal adhesions, force sensitivity was not enhanced over the single LIM domain. Therefore, zyxin force sensitivity is optimal with a single LIM domain, while additional LIM domains fail to enhance force sensitivity.     

Elevated interstitial adenosine concentrations do not activate the muscle reflex

The purpose of the present study was to examine the effects of adenosine perfusion of the isolated triceps surae muscle group in the decerebrate cat on interstitial adenosine concentrations as well as heart rate and blood pressure responses. In six male cats (6.0 +/- 0.21 kg), the triceps surae muscle group of both legs was perfused with an artificial blood solution containing no additives (control) and then with blood containing 20 mM or 100 microM adenosine for 10 min. An intact muscle reflex was confirmed by bolus injections of 50 mM phosphate and/or saturated KCl administered into the triceps surae muscle via the cannulated popliteal artery before and after adenosine blood perfusion. Microdialysis of the triceps surae muscle group during muscle perfusion revealed that interstitial adenosine was elevated (P < 0.05) from 0.9 +/- 0.3 microM during control blood perfusion to 2,421 +/- 547 microM during 20 mM adenosine perfusion. In addition, interstitial adenosine levels were increased (P < 0.05) from 1.1 +/- 0.3 microM during control blood perfusion to 4.1 +/- 1.2 microM during perfusion with 100 microM adenosine. Despite the large increases in interstitial adenosine levels, perfusion of the triceps surae muscle group with the two blood adenosine solutions resulted in no significant increases in heart rate or blood pressure. These data strongly suggest that elevated interstitial adenosine concentrations do not play a role in activating the muscle reflex and confirm our previous in vivo human findings (J Appl Physiol 83: 1045-1053, 1997).     

Fast coordination changes in cytochrome c do not necessarily imply folding

Folding of globular proteins occurs with rates that range from microseconds to minutes; consequently, it has been necessary to develop new strategies to follow the faster processes that exceed stopped-flow capabilities. Rapid photochemical methods have been employed to study the rate of folding of reduced cytochrome c. In this protein, the iron of the covalently bound heme binds a His and a Met, proximal and distal. Unfolding by guanidine or urea weakens the Fe-Met bond, and the reduced unfolded cytochrome c easily binds CO and other heme ligands, which would react slowly or not at all with the native protein. Therefore in the presence of CO, reduced cytochrome c unfolds at lower denaturant concentrations than in the absence of this ligand, and rapid photochemical removal of CO from unfolded cytochrome c, is expected to trigger at least an incomplete refolding. This approach is complicated by the breakage of the proximal His-Fe bond that may occur as a consequence of CO photodissociation in the unfolded cytochrome c because of the so-called base elimination mechanism. Rebinding of CO to the four-coordinate heme yields kinetic intermediates unrelated to folding. Our hypothesis is supported by parallel observations carried out with protoheme and microperoxidase.     

Skeletal muscle precursors do not arise from bone marrow cells

Cell and Tissue Research - This paper tests the hypothesis that bone marrow stem cells can give rise to circulating muscle precursor cells. Irradiated host mice were reconstituted with bone marrow...