Experimental muscle pain results in reorganization of coordination among trapezius muscle subdivisions during repetitive shoulder flexion

The aim of the study was to examine the effect of experimental unilateral upper trapezius muscle pain on the relative activation of trapezius muscle subdivisions bilaterally during repetitive movement of the upper limb. Surface EMG signals were detected from nine healthy subjects from the upper, middle and lower divisions of trapezius during a repetitive bilateral shoulder flexion task. Measurements were performed before and after injection of 0.5 ml hypertonic (pain condition) and isotonic (control) saline into the upper division of the right trapezius muscle in two experimental sessions. On the painful side, upper trapezius showed decreased EMG amplitude (average rectified value, ARV) and lower trapezius increased ARV throughout the entire task following the injection of hypertonic saline (40.0 ± 22.2 vs. 26.0 ± 17.4 μV, and 12.5 ± 7.6 vs. 25.6 ± 14.8 μV, respectively, at the beginning of the contraction). On the side contralateral to pain, greater estimates of ARV were identified for the upper division of trapezius as the task progressed (37.4 ± 20.2 vs. 52.7 ± 28.4 μV, at the end of the contraction). Muscle fiber conduction velocity did not change with pain in all three divisions of the right trapezius muscle. The results suggest that local elicitation of nociceptive afferents in the upper division of the trapezius induces reorganization in the coordinated activity of the three subdivisions of the trapezius in repetitive dynamic tasks.     

Partial urethral obstruction of rabbit urinary bladder: stereological evidence that the increase in muscle content is mostly driven by changes in number, rather than size, of smooth muscle cells

The effects of partial urethral obstruction on the detrusor muscle of rabbit urinary bladder were investigated using stereological sampling and estimation tools. Twelve female Norfolk rabbits (2.5-3.0 kg body weight) were divided into four groups: 3, 7 and 12 weeks after surgical intervention to produce a standard partial obstruction and unobstructed controls. Following removal, bladder axes (craniocaudal, dorsoventral and laterolateral) and organ weights were recorded. Bladders were prepared for light microscopy by multistage random sampling procedures. Stereological methods were used to estimate the volume of muscle and the packing density and total number of myocyte nuclei in each bladder. We also estimated mean myocyte volume and the mean cross-sectional area and length of myocytes. Group comparisons were made by one-way analysis of variance. Changes in bladder axes were mainly laterolateral and craniocaudal. Mean bladder weight increased roughly six-fold by 3 weeks and 17-fold by 12 weeks and was accompanied, on average, by 12- and 33-fold increases in total muscle volume. These variables did not differ at 3 and 7 weeks post-obstruction. Increases in muscle content were not accompanied by changes in packing densities but were associated with increases in the total numbers of myocyte nuclei (13-fold by 3 weeks, 28-fold by 12 weeks). Mean myocyte volume did not vary significantly between groups but cells in obstructed groups were shorter and wider. These findings support the notion that partial outflow obstruction leads to an increase in the number, but not mean volume, of myocytes. If due solely to myocyte mitosis, the total of 43 x 10(8) cells found at 12 weeks could be generated by the original complement of 15 x 10(7) cells if an average of only 2.1 x 10(6) new cells was produced every hour. In reality, even this modest proliferation rate is unlikely to be achieved because myocyte proliferation rates are very low and it is possible that new myocytes can arise by differentiation of mesenchymal or other precursor cells.     

Morphological effects of two protocols of passive stretch over the immobilized rat soleus muscle

This study evaluated two different stretching protocols employed during a period of hind-limb immobilization in terms of their effects on muscle morphology. Quantitative data regarding the soleus muscle were obtained based on the clinical hypothesis that a high frequency of this exercise would improve the recovery of muscle structure. Twenty-four male Wistar rats were divided into four groups (n = 6 each): the control group (C); the immobilized group, in which the left hind limb was immobilized in order to maintain the soleus muscle in a fully shortened position for 3 weeks (I); the 'immobilized and stretched every 3 days' group, in which the left hind limb was immobilized as in the immobilized group, but with the soleus muscle stretched every 3 days for 40 min (Ist3); and the 'immobilized (as in the immobilized group) and stretched every 7 days' group (ISt7). All soleus muscles were excised 21 days after the beginning of the experiment, and were processed for (1) haematoxylin and eosin and myosin ATPase to evaluate muscle morphology and cross-sectional area and the proportions of the different fibre types, and (2) ultrastructural analysis. The cross-sectional area was found to have decreased in all fibre types (I, II and C), mainly in ISt7, when compared with the C group and ISt3 group. The proportion of the different fibre types did not show statistical difference between groups. Light and electron microscopy examination revealed signs of cell degeneration that was more intense in the group immobilized and stretched three times a week. In conclusion, sessions of passive stretching applied to the soleus during immobilization induce muscle fibre injury, suggesting that this therapeutic tool should be applied carefully to disused muscles.     

Ejaculation induced by i.c.v. injection of the preferential dopamine D(3) receptor agonist 7-hydroxy-2-(di-N-propylamino)tetralin in anesthetized rats

In addition to serotonin, dopamine within the CNS is known to play a primary role in the control of ejaculation. However, whether D(2) and/or D(3) dopamine receptor subtypes mediate this effect is still unclear. In order to clarify this issue, a pharmacological competitive study using the preferential D(3) agonist 7-hydroxy-2-(di-N-propylamino)tetralin (7-OH-DPAT) alone or in combination with competitive nonpreferential or preferential D(2) and D(3) antagonists delivered intracerebroventricularly (i.c.v.) was undertaken in anesthetized rats. Urethane-anesthetized male rats were implanted into the cerebral ventricle with a cannula for i.c.v. injections, and recording electrodes were placed within the bulbospongiosus (BS) muscle to monitor BS muscle contractions, which were used as a marker for the expulsion phase of ejaculation. Following i.c.v. injection, 7-OH-DPAT induced ejaculation and rhythmic BS muscle contractions. Co-injected i.c.v. with 7-OH-DPAT, the nonselective D(2)/D(3) antagonist (raclopride), and the preferential D(3) antagonist (S(-)-N[n-butyl-2-pyrrolidinyl)methyl]-1-methoxy-4-cyanonaphtalene-2-carboxamide; nafadotride) but not the preferential D(2) antagonist ((+/-)-3-[4-(4-chlorophenyl)-4-hydroxypiperidinyl]methylindole; L 741,626) inhibited the occurrence of ejaculation and BS muscle contractions. These results suggest that i.c.v. delivery of 7-OH-DPAT does represent a pertinent model to investigate the physio-pharmacology of ejaculation. It is inferred that targeting brain D(3) receptors may provide a therapeutic approach for treating ejaculatory disorders in humans.     

Uterine smooth muscle cells increase invasive ability of endometrial carcinoma cells through tumor–stromal interaction

The incidence of lymph node metastasis by endometrial carcinoma (EMCA) increases with the depth of myometrial invasion, and this depth of invasion has been found to have a major impact on the outcome. In the present study, we assessed the effect of tumor–stromal interactions on the invasive behavior of EMCA cells and examined the involvement of SDF-1alpha/CXCL12-CXCR4 in the interaction of EMCA cells and uterine smooth muscle cells (UtSMCs). We investigated whether SDF-1alpha/CXCL12 produced and secreted from UtSMCs induces EMCA cell migration by using 5 human EMCA cell lines such as AMEC and RL95 cells. The SDF-1alpha/CXCL12 concentration in conditioned medium (CM) of UtSMCs(was 4,120 ± 530 pg/ml. Treatments with CM of UtSMCs and plated UtSMCs significantly induced both AMEC and RL95 cell migration. The induced cell migrations were significantly inhibited by CXCR4 mAb (12G5) and CXCR4 antagonist (AMD3100) pre-treatments. Treatments with UtSMCs CM to AMEC and RL95 cells stimulated Akt phosphorylation in a time-dependent manner. Pre-treatment of AMEC and RL95 cells with wortmannin as a PI3K inhibitor significantly inhibited UtSMCs CM-induced cell migration. The SDF-1alpha/CXCL12-CXCR4 chemokine axis between UtSMCs and EMCA played an important role in the muscular infiltration of endometrial cancer through activation of PI3K-Akt signaling pathway. Suppression of this pathway could be an effective target for the treatment of early uterine body cancer in particular.     

Alterations in triad ultrastructure following repetitive stimulation and intracellular changes associated with exercise in amphibian skeletal muscle

This study used Rana temporaria sartorius muscles to examine the effect of fatiguing electrical stimulation on the gap between the T-tubular and sarcoplasmic reticular membranes (T-SR distance) and the T-tubule diameter and compared this with corresponding effects on resting fibres exposed to a range of extracellular conditions that each replicate one of the major changes associated with muscular activity: membrane depolarisation, isotonic volume increase, acidification and intracellular lactate accumulation. Following each treatment, muscles were immersed in isotonic fixative solution and processed for transmission electron microscopy (TEM). Mean T-SR distances were estimated from orthogonal intercepts to provide estimates of diffusion distances between T and SR membranes and T-tubule diameter was estimated by measuring its shortest axis in the sampled J-SR complexes. Measurements from muscles fatigued by low frequency intermittent stimulation showed significant (P << 0.05) reversible increases in both T-SR distance and T-tubule diameter from 15.97 ± 0.37 nm to 20.15 ± 0.56 nm and from 15.44 ± 0.60 nm to 22.26 ± 0.84 nm (n = 40, 30) respectively. Exposure to increasing concentrations of extracellular [K⁺] in the absence of Cl⁻ to produce membrane depolarisation without accompanying cell swelling reduced T-SR distance and increased T-tubule diameter, whilst comparable increases in [K⁺]_e in the presence of Cl⁻ suggested that isotonic cell swelling has the opposite effect. Acidification alone, produced by NH₄Cl addition and withdrawal, also decreased T-SR distance and T-tubule diameter. A similar reduction in T-SR distance occurred following exposure to extracellular Na-lactate where such acidification was accompanied by elevations of intracellular lactate, but these conditions produced a significant swelling of T-tubules attributable to movement of lactate from the cell into the T-tubules. This study thus confirms previous reports of significant increases in T-SR distance and T-tubule diameter following stimulation. However, of membrane depolarisation, isotonic cell swelling, intracellular acidification and lactate accumulation, only isotonic cell swelling increases T-SR distance whilst membrane depolarisation and intracellular lactate likely contribute to the observed increases in T-tubule diameter.     

Site directed mutagenesis of Drosophila flightin disrupts phosphorylation and impairs flight muscle structure and mechanics

Flightin is a myosin rod binding protein that in Drosophila melanogaster is expressed exclusively in the asynchronous indirect flight muscles (IFM). Hyperphosphorylation of flightin coincides with the completion of myofibril assembly and precedes the emergence of flight competency in young adults. To investigate the role of flightin phosphorylation in vivo we generated three flightin null (fln ⁰ ) Drosophila strains that express a mutant flightin transgene with two (Thr158, Ser 162), three (Ser139, Ser141, Ser145) or all five potential phosphorylation sites mutated to alanines. These amino acid substitutions result in lower than normal levels of flightin accumulation and transgenic strains that are unable to beat their wings. On two dimensional gels of IFM proteins, the transgenic strain with five mutant sites (fln ^5STA ) is devoid of all phosphovariants, the transgenic strain with two mutant sites (fln ^2TSA ) expresses only the two least acidic of the nine phosphovariants, and the transgenic strain with three mutant sites (fln ^3SA ) expresses all nine phosphovariants, as the wild-type strain. These results suggest that phosphorylation of Thr158 and/or Ser162 is necessary for subsequent phosphorylation of other sites. All three transgenic strains show normal, albeit long, IFM sarcomeres in newly eclosed adults. In contrast, sarcomeres in fully mature fln ^5STA and fln ^2TSA adults show extensive breakdown while those in fln ^3SA are not as disordered. The fiber hypercontraction phenotype that characterizes fln ⁰ is fully evident in fln ^5STA and fln ^2TSA but partially rescued in fln ^3SA . Mechanics on skinned fibers from newly eclosed flies show alterations in viscous modulus for fln ^5STA and fln ^2TSA that result in a significant reduction in oscillatory power output. Expression of fln ^5STA and fln ^2TSA , but not fln ^3SA , in a wild-type (fln ⁺ /fln ⁺ ) background resulted in a dominant negative effect manifested as flight impairments and hypercontracted IFM fibers. Our studies indicate that Thr158 and/or Ser162 are (is) indispensable for flightin function and suggest that phosphorylation of one or both residues fulfills an essential role in IFM structural stability and mechanics.