The molecular effects of electrical stimulation on the muscle components of the urethra of female rats after trauma by vaginal distention.

AIMS: To evaluate the expression of genes and proteins related to the urethral muscles of female rats after trauma by vaginal distention (VD) and after electrical stimulation therapy (EST). METHODS: We compared the urethras of four groups of 20 animals each: control without trauma (C), 7 (recent-trauma) and 30 days (late-trauma) post-VD, and VD-treated with EST. We evaluated the expression of myogenic regulatory factors MYOD1 and myogenin (MYOG); skeletal muscle myosin heavy chain 1, 2, and 3 (MYH1, MYH2, and MYH3); smooth muscle MYH11; and myosin light chain 9 (MYL9). We used real-time quantitative polymerase chain reaction, Western blot analysis, and immunohistochemistry. RESULTS: MYOD1 and MYOG genes were overexpressed in the recent-trauma group compared with the other groups (P < .05). MYH1 and MYH3 genes were upregulated in the recent-trauma group compared with the control and EST groups (P < .05). The MYH2 gene was overexpressed in the late-trauma group (P < .05), while the MYH2 protein was significantly increased in the EST group compared with control, recent-trauma and late-trauma groups by 5-, 3-, and 2.7-fold change, respectively (P < .05). MYL9 and MYH11 messenger RNA were overexpressed in both trauma groups compared with control and EST groups (P < .05). MYH11 protein was not different among the study groups (P > .05). CONCLUSIONS: EST enhances the recovery of the damaged urethral tissue of rats mainly by acting on the striated-muscle components. The MYH2 pathway underlies the positive effects of EST in the external urethral sphincter.

The effects of microenergy acoustic pulses on an animal model of obesity-associated stress urinary incontinence. Part 1: Functional and histologic studies.

AIM: Obesity is a strong independent risk factor for urinary incontinence. Effective therapeutic approaches for obesity-associated stress urinary incontinence (OA-SUI) are lacking as the mechanisms remain unclear. The aim of our study is to explore the impacts of microenergy acoustic pulse (MAP) therapy on urethral and pelvic floor muscle structure and function in female lean and fatty rats. METHODS: A total 24 Zucker fatty (ZF) and 24 Zucker lean (ZL) female 24-week-old rats were grouped into four groups: ZL control, ZLMAP, ZF control, and ZFMAP. For MAP treatment, 500 pulses were delivered at an energy level of 0.033 mJ/mm 2 and a frequency of 3 Hz and were applied twice a week for 4 weeks. After a 1-week washout, all rats underwent conscious cystometry and leak-point pressure (LPP) measurements followed by ex vivo organ-bath assay and histological study. RESULTS: ZF rats had lower LPP as compared to ZL rats, and MAP treatment significantly improved LPP in ZF rats (P < .05). Impaired muscle contractile activity (MCA) in organ-bath study was noted in ZF rats. MAP treatment significantly increased MCA in ZF rats (P < .05) and also increased the thickness of the striated muscle layer and the number of neuromuscular junctions (NMJs). In situ, MAP activated muscle satellite cells significantly (P < .05). CONCLUSIONS: Obesity impairs the function of both the urethral sphincter and the pelvic floor and leads to atrophy and distortion of the striated muscle in obese female rats. These issues contribute to OA-SUI. MAP improves continence by stimulating muscle regeneration and nerve innervation as well as by activating satellite cells.

Mutations in Caenorhabditis elegans actin, which are equivalent to human cardiomyopathy mutations, cause abnormal actin aggregation in nematode striated muscle.

Actin is a central component of muscle contractile apparatuses, and a number of actin mutations cause diseases in skeletal, cardiac, and smooth muscles. However, many pathogenic actin mutations have not been characterized at cell biological and physiological levels. In this study, we tested whether the nematode Caenorhabditis elegans could be used to characterize properties of actin mutants in muscle cells in vivo. Two representative actin mutations, E99K and P164A, which cause hypertrophic cardiomyopathy in humans, are introduced in a muscle-specific C. elegans actin ACT-4 as E100K and P165A, respectively. When green fluorescent protein-tagged wild-type ACT-4 (GFP-ACT-4), is transgenically expressed in muscle at low levels as compared with endogenous actin, it is incorporated into sarcomeres without disturbing normal structures. GFP-ACT-4 variants with E100K and P165A are incorporated into sarcomeres, but also accumulated in abnormal aggregates, which have not been reported for equivalent actin mutations in previous studies. Muscle contractility, as determined by worm motility, is not apparently affected by expression of ACT-4 mutants. Our results suggest that C. elegans muscle is a useful model system to characterize abnormalities caused by actin mutations.

Mechanical impact of parturition-related strains on rat pelvic striated sphincters.

AIMS: To define the operational resting sarcomere length (Ls ) of the female rat external urethral sphincter (EUS) and external anal sphincter (EAS) and to determine the mechanism of parturition-related injury of EUS and EAS using a simulated birth injury (SBI) vaginal distention model. METHODS: EUS and EAS of 3-month-old Sprague-Dawley control and injured rats were fixed in situ, harvested, and microdissected for Ls measurements and assessment of ultrastructure. EUS and EAS function was determined at baseline, and immediately and 4 weeks after SBI, using leak point pressure (LPP) and anorectal manometry (ARM), respectively. Operational L s was compared to species-specific optimal L s using one sample Student's t test. Data (mean ± SD) were compared between groups and time points using repeated measures one-way analysis of variance, followed by Tukey's post hoc pairwise comparisons, with significance set to 0.05. RESULTS: The operational resting Ls of both sphincters (EUS: 2.09 ± 0.07 µm, EAS: 2.02 ± 0.03 µm) was significantly shorter than optimal rat Ls of 2.4 µm. Strains imposed on EUS and EAS during SBI resulted in significant sarcomere elongation and disruption, compared with the controls (EUS: 3.09 ± 0.11 µm, EAS: 3.37 ± 0.09 µm). Paralleling structural changes, LPP and ARM measures were significantly lower immediately (LPP: 21.5 ± 1.0 cmH2 O, ARM: 5.1 ± 2.31 cmH2 O) and 4 weeks (LPP: 27.7 ± 1.3cmH2 O, ARM: 2.5 ± 1.0 cmH2 O) after SBI relative to the baseline (LPP: 43.4 ± 8.5 cmH2 O, ARM: 8.2 ± 2.0 cmH2 O); P < 0.05. CONCLUSIONS: Analogous to humans, the short resting Ls of rat EUS and EAS favors their sphincteric function. The insult experienced by these muscles during parturition leads to sarcomere hyperelongation, myofibrillar disruption, and dysfunction of the sphincters long-term.

Striated muscle function, regeneration, and repair.

As the only striated muscle tissues in the body, skeletal and cardiac muscle share numerous structural and functional characteristics, while exhibiting vastly different size and regenerative potential. Healthy skeletal muscle harbors a robust regenerative response that becomes inadequate after large muscle loss or in degenerative pathologies and aging. In contrast, the mammalian heart loses its regenerative capacity shortly after birth, leaving it susceptible to permanent damage by acute injury or chronic disease. In this review, we compare and contrast the physiology and regenerative potential of native skeletal and cardiac muscles, mechanisms underlying striated muscle dysfunction, and bioengineering strategies to treat muscle disorders. We focus on different sources for cellular therapy, biomaterials to augment the endogenous regenerative response, and progress in engineering and application of mature striated muscle tissues in vitro and in vivo. Finally, we discuss the challenges and perspectives in translating muscle bioengineering strategies to clinical practice.

A novel mouse model that recapitulates adult-onset glycogenosis type 4.

Glycogen storage disease type IV (GSD IV) is a rare autosomal recessive disorder caused by deficiency of the glycogen-branching enzyme (GBE). The diagnostic hallmark of the disease is the accumulation of a poorly branched form of glycogen known as polyglucosan (PG). The disease is clinically heterogeneous, with variable tissue involvement and age at onset. Complete loss of enzyme activity is lethal in utero or in infancy and affects primarily the muscle and the liver. However, residual enzyme activity as low as 5-20% leads to juvenile or adult onset of a disorder that primarily affects the central and peripheral nervous system and muscles and in the latter is termed adult polyglucosan body disease (APBD). Here, we describe a mouse model of GSD IV that reflects this spectrum of disease. Homologous recombination was used to knock in the most common GBE1 mutation p.Y329S c.986A > C found in APBD patients of Ashkenazi Jewish decent. Mice homozygous for this allele (Gbe1(ys/ys)) exhibit a phenotype similar to APBD, with widespread accumulation of PG. Adult mice exhibit progressive neuromuscular dysfunction and die prematurely. While the onset of symptoms is limited to adult mice, PG accumulates in tissues of newborn mice but is initially absent from the cerebral cortex and heart muscle. Thus, PG is well tolerated in most tissues, but the eventual accumulation in neurons and their axons causes neuropathy that leads to hind limb spasticity and premature death. This mouse model mimics the pathology and pathophysiologic features of human adult-onset branching enzyme deficiency.

The striated muscles in pulmonary arterial hypertension: adaptations beyond the right ventricle.

Pulmonary arterial hypertension (PAH) is a fatal lung disease characterised by progressive remodelling of the small pulmonary vessels. The daily-life activities of patients with PAH are severely limited by exertional fatigue and dyspnoea. Typically, these symptoms have been explained by right heart failure. However, an increasing number of studies reveal that the impact of the PAH reaches further than the pulmonary circulation. Striated muscles other than the right ventricle are affected in PAH, such as the left ventricle, the diaphragm and peripheral skeletal muscles. Alterations in these striated muscles are associated with exercise intolerance and reduced quality of life. In this Back to Basics article on striated muscle function in PAH, we provide insight into the pathophysiological mechanisms causing muscle dysfunction in PAH and discuss potential new therapeutic strategies to restore muscle dysfunction.

[The Linburg-Comstock phenomenon: a review]. / Die Linburg-Comstock-Variante: Eine Übersicht.

Simultaneous flexion of thumb and fingers is described as the Linburg-Comstock phenomenon. Congenital and acquired coupling of the flexor pollicis longus (FPL) and flexor digitorum profundus (FDP) tendons is the reason for this clinical entity. Additionally, coupling of the FPL and the flexor digitorum superficialis II or III has been described. The coupling can be between the muscles, the tendon sheaths or the tendons themselves. Asymptomatic and symptomatic coupling should be differentiated. In general symptomatic congenital or acquired coupling demands surgical intervention. We report about a 35-year-old patient with a congenital asymptomatic coupling of FPL and FDP-II who suffered a distorsion of the thumb. Afterwards she complained of pain and strength loss. An accessory tendon of 3 mm in diameter and 3.5 cm length between FPL and FDP-II was identified during surgery. The patient recovered completely after resection of this coupling.

Effect of myogenic stem cells on the integrity and histomorphology of repaired transected external anal sphincter.

INTRODUCTION AND HYPOTHESIS: The objective was to evaluate the effect of myogenic stem cells on histological properties and the volume of striated muscle of the external anal sphincter after transection and repair. METHODS: Histological analysis was performed on the external anal sphincters of 40 young female rats euthanized at 7 or 90 days after transection and repair and randomization to injection of either phosphate buffered solution (PBS) or myogenic stem cells (SC) at the transection site. Sphincter complexes, previously evaluated for neurophysiological function, were processed for histology and analyzed for possible disruption, amount of inflammation, and volume of striated muscle. The relationship between the muscular disruption and contractile force of sphincters was evaluated. RESULTS: Disruption was seen in 100 % of sphincters 7 days after repair for both SC and control animals. Eighty-nine percent of controls and 78% of SC-administered animals had intact sphincters at 90 days. Significant inflammatory infiltrate was seen in repaired anal sphincters for both the PBS and the SC groups at 7 days, and persisted at 90 days, with no difference between treatment groups. Striated muscle volume increased from 7 to 90 days for both control and SC-administered animals. Although there was no difference in volume between treatments, there was substantial temporal improvement in contractile force generation of the sphincters receiving SC compared with those receiving PBS. CONCLUSION: In this animal model, administration of myogenic stem cells to transected/repaired anal sphincters did not alter the amount of inflammation nor the volume of striated muscle, suggesting that stem cells might improve contractile function through other cellular processes.

Urinary and ejaculatory dysfunction induced by denervation of specific striated muscles anatomically related to the urethra in male rats.

AIMS: The purpose of the present study was to determine the contribution of the external urethral sphincter (EUS), the ischiocavernous (IC), or the bulbospongiosus (BS) on the control of micturition, copulatory behavior and semen expulsion in male rats. We hypothesized that the EUS contributes to maintain urinary continence, while all three muscles participate in expulsive urethral functions. METHODS: In Experiment 1, it was analyzed the effects of bilateral denervation of IC, BS or EUS, or sham surgery, on voiding behavior and urinary parameters measured before surgery and 2 and 10 days post-surgery. In Experiment 2, copulatory behavior and the weight of the seminal plug expelled during ejaculation were recorded before and after sham surgery or bilateral denervation of the aforementioned muscles. Immediately after ejaculation, the animals were anesthetized to confirm the denervation and determine whether seminal material had accumulated in the lower urinary tract. RESULTS: In IC-denervated animals, voiding duration and the number of mounts was increased, and intromission or ejaculation patterns were absent. Denervation of BS induced signs of post-micturition dribble, decreased voiding frequency, increased urine volume and reduced the amount of semen ejaculated. Denervation of EUS induced signs of post-micturition dribble and urinary incontinence, as well as retrograde ejaculation. CONCLUSIONS: Striated muscles anatomically related to the urethra contribute differentially to the control of continence and expulsive urethral functions. Damages to the muscles or to their innervation, as may occur during pelvic surgery, would result in sexual and urinary dysfunctions.

Evoked pressure curves from the external anal sphincter following transcranial magnetic stimulation in healthy volunteers and patients with faecal incontinence.

AIM: The hypothesis was tested that evoked pressure curves (EPCs) after transcranial magnetic stimulation (TMS) would provide additional neuropathophysiological information on the descending pathways to the external anal sphincter (EAS) in patients with faecal incontinence (FI). METHOD: Twenty-five healthy subjects and 69 patients with FI were investigated. TMS was applied to the vertex, and EPCs were recorded with a probe placed through the EAS. TMS was performed with the EAS at rest and during contraction (facilitated responses). At least three responses were recorded for each modality. Clinical data and anorectal manometric, electrophysiological perineal and transanal ultrasound recordings were compared with respect to the EPC results. RESULTS: There was no statistically significant difference between the EPCs of healthy subjects and FI patients. Twenty-three per cent of the FI patients had abnormal EPC latencies, with significantly lower voluntary contraction amplitudes (P = 0.03) and significantly higher rectal sensation (P = 0.04) than the other group. We found no significant difference between FI patients with and without abnormal EPC latencies in terms of clinical characteristics and electrophysiological and endoanal ultrasound parameters. There was no difference in the identified causes of the FI between the two groups. CONCLUSION: As abnormal EPC latencies were found in 23% of FI patients with no known central neurological disease, abnormal EPC latencies might reveal undetected lesions of descending pathways in patients with FI.

Sustained improvement in the anal sphincter function following surgical plication of rabbit external anal sphincter muscle.

BACKGROUND: We recently found that the anal canal function and external anal sphincter contraction can be enhanced by surgically adjusting the EAS muscle sarcomere length in rabbits. A 20% length plication of the external anal sphincter muscle results in significant increase in the anal canal pressure and EAS muscle stress without affecting its passive tension. The durability of the beneficial effect of external anal sphincter muscle plication on the anal canal function is not known. OBJECTIVE: We studied the long-term effects of optimal length external anal sphincter plication on the anal canal pressure, external anal sphincter sarcomere length, and anal canal histology. DESIGN: Female rabbits (n = 16) were anesthetized and either sham (n = 4) or external anal sphincter plication (n = 12) surgery was performed. MAIN OUTCOME MEASURES: The effect of external anal sphincter plication on the anal canal pressure was determined every 2 weeks for 6 months in 6 animals. Anal canal was harvested for sarcomere length and histological assessment. RESULTS: External anal sphincter plication resulted in 50% to 60% increase in the anal canal pressure, and 80% to 90% increase in external anal sphincter muscle stress (during maximum electrical stimulus). The effect of plication was durable for the entire study period of 24 weeks. Sarcomere length increased from 2.11 ± 0.08 µm to 2.59 ± 0.03 µm immediately after plication and was 2.35 ± 0.08 µm at the end of 6 months. Histology revealed no significant differences in the muscle (30% vs 29%) or connective tissue components (70% vs 71%) of the anal canal between control and chronically plicated animals. CONCLUSIONS: Normal external anal sphincter muscle plication results in long-term enhancement of the anal canal function without any untoward effects on the tissue architecture in the rabbit. External anal sphincter muscle plication could be an important strategy to improve the anal canal function in patients with anal incontinence.

Directed evolution of adeno-associated virus (AAV) as vector for muscle gene therapy.

Adeno-associated virus (AAV) is emerging as a vector of choice for muscle gene therapy because of its effective and stable transduction in striated muscles. AAV naturally evolve into multiple serotypes with diverse capsid gene sequences that are apparently the determinants of their tissue tropism and infectivity. Certain AAV serotypes show robust gene transfer upon direct intramuscular injection, while others are effective in crossing the endothelial barrier to reach muscle when delivered intravenously. Muscular dystrophy gene therapy requires efficient body-wide muscle gene transfer. However, preferential liver transduction by nearly all natural AAV serotypes could be an undesirable feature for muscle-directed applications, especially by means of systemic gene delivery. Here we describe a method of in vitro evolution and in vivo selection of AAV capsids that target striated muscles and detarget the liver. Using DNA shuffling technology, we have generated a capsid gene library by in vitro scrambling and shuffling the capsid genes of natural AAV1 to AAV9. To minimize the bias and limitation of in vitro screening on culture cells, we performed direct in vivo panning in adult mice after intravenous injection of the shuffled capsid library that packaged their own coding sequences. The AAV variants enriched in the heart and muscle are retrieved by capsid gene PCR and subsequently characterized for their tissue tropisms. This directed evolution and in vivo selection method should be useful in generating novel gene therapy vectors for muscle and heart and other tissues.

Improved motor function in dko mice by intravenous transplantation of bone marrow-derived mesenchymal stromal cells.

BACKGROUND AIMS: We explored the potential therapeutic value of transplanting bone marrow (BM)-derived mesenchymal stromal cells (MSC) into utrophin/dystrophin-deficient double knock-out (dko) mice, a murine model of Duchenne muscular dystrophy. METHODS: MSC from male rats were isolated and transplanted into female dko mice via the caudal vein. Behavior and locomotor function were later evaluated, along with the expression of dystrophin and utrophin in the sarcolemma of myofiber tissues. The presence of grafted cells was confirmed via polymerase chain reaction for the sex-determining region of the Y-chromosome. RESULTS: Locomotor activity improved significantly (P < 0.05) from 5 to 15 weeks after cell transplantation, as measured by traction, rotating rod and running wheel tests. We also found that the expression of dystrophin and utrophin increased significantly (P < 0.05) and progressively in the sarcolemma from 5 to 15 weeks after transplantation. The median lifespan of mice in the normal group (74.1 weeks) was significantly (P < 0.001) higher than those in the control (22.0 weeks) and transplantation (35.0 weeks) groups, and the median lifespan of mice in the transplantation group was significantly (P < 0.001) higher than that in the control group. CONCLUSIONS: Results of this study demonstrate that BM MSC have potential value in xenogeneic transplantation therapy for muscular dystrophy.

[Functional anatomy of the male continence mechanism]. / Funktionelle Anatomie des männlichen Kontinenzmechanismus.

The basic structures and organs contributing to continence in men are far less well investigated than in women. This concerns anatomical and functional aspects as well. Especially the cooperation of single components and the dynamic anchoring in the pelvic floor require further investigation. An improved anatomical-functional interpretation is needed to generate therapeutic concepts orientated at the physiology of the bladder neck.Therefore, the focus of anatomical investigations should be on the external sphincter which is the main muscle responsible for urethral closure as well as on the connective tissue, smooth muscular and neuronal structures in the pelvis. The smooth muscular structures involved are the internal sphincter, the inner parts of the external sphincter, the urethral longitudinal musculature, and parts of the centrum perinei and of the ventral suspension apparatus which fixes the position of the bladder neck and seems to be vital for continence and initiation of micturition. These new findings imply an integral concept for men as was developed for women. A first step in this regard would be a consistent and updated anatomical nomenclature.

Computation of a tetrahedral mesh for striated muscle deformation simulation.

Competing concepts exist regarding surgery for instance of the cleft lip and palate to date. Morphology-based simulations at histological scale may one day be used to help the surgeon predict the possible outcome of a variety of approaches. It however can be a challenge to generate volume meshes that are applicable to the mathematical modelling of three-dimensional spatial modifications. Computation of surface meshes may be considered less delicate. The aim of this study is to design and evaluate a novel algorithm that supports finite element methods. Images of histological serial sections of a striated muscle were segmented. Results of the three-dimensional reconstruction of multiple layers of the polygonal segmentation data characterized the hull of the muscle. The corresponding surface mesh was then converted into a tetrahedral mesh to generate volume. This was achieved by mapping multiple template types onto neighbouring intersection polygons. Muscle contraction was subsequently simulated by mesh deformation. The technique successfully generated volumes and was able to provide data on contraction directions. The mesh supported a novel approach to simulate representations of contraction. However, several drawbacks were evident. Mathematical modelling of scenarios with more than one striated muscle will require considerable modification of the currently presented approach. Future studies need to then evaluate the applicability of volume meshes to represent arrays of three-dimensional biological objects. Surface mesh based mathematical modelling of cleft lip and palate surgery and its results are therefore not yet in reach.

Effects of sphincterotomy and pudendal nerve transection on the anal sphincter in a rat model.

PURPOSE: Our objective was to define anal resting pressure and electromyography of the normal rat anal sphincter and investigate the short-term effects of both mechanical trauma to the anal sphincter muscles and pudendal nerve transection. METHODS: Forty-five virgin female Sprague-Dawley rats were randomly allotted to three groups: controls (n = 21), sphincterotomy (n = 12), and pudendal nerve transection (n = 12). Anal pressure was monitored using a saline-filled balloon connected to a pressure transducer. Anal pressure and electromyography of the anal sphincter with use of a needle electrode were recorded both before and after injury or succinylcholine administration. RESULTS: Anal pressure data were consistent with rhythmic pressure contractions. Succinylcholine significantly reduced both pressure and electromyography signals. Electromyography amplitude and frequency decreased after nerve transection but not after sphincterotomy. The histology showed that the rat anal anatomy has muscular components that compare with human anatomy. The sphincterotomy group showed injury to the anal sphincters and the sphincter anatomy of the nerve transection group appeared similar to the control group. The anal pressure wave appears to be created by synergistic activity of both striated and smooth muscle of the anal sphincter. CONCLUSION: The female rat is a suitable and reliable model for studying effect of direct and indirect injury to the anal sphincters.

Congenital myopathies--a comprehensive update of recent advancements.

The congenital myopathies are relatively newly discovered compared with other categories of muscle diseases. Current research continues to clarify and classify the congenital myopathies. These pose a diagnostic problem and cannot be diagnosed by routine hematoxylin and eosin stain. A lot of special techniques are required to diagnose them correctly and it's various subtypes. The disease specific structural changes seen in the muscle are detected by enzyme histochemistry, immunohistochemistry and electron microscopy. Through this review we provide an up-to-date analysis of congenital myopathies including clinical and pathologic aspects.

Diagnosis of glycogenosis type II.

The diagnosis of glycogenosis type II is often complicated by the rarity of the condition and the heterogeneity of the clinical manifestations of the disease. It is a progressive, debilitating, and often fatal neuromuscular disorder that manifests as a continuum of clinical phenotypes, which vary with respect to organ involvement, age at onset, and severity. Early diagnosis requires both increased awareness among physicians regarding the clinical characteristics of the disease and fast and reliable acid alpha-glucosidase (GAA) enzyme activity assays to confirm the GAA deficiency. The clinical diagnosis of glycogenosis type II is confirmed by virtual absence (found in infants) and marked reduced activity (found in juveniles and adults) of GAA enzyme in blood samples, cultured fibroblasts, and muscle biopsies. This article specifically highlights the need for early recognition of the clinical manifestation of the disease in infants, juveniles, and adults. Descriptions of the main clinical features of the condition, as well as differential diagnosis are included. In addition, the tests required for a confirmed diagnosis are described, and use of muscle imaging to evaluate muscle pathology is reviewed.