Intrafusal-fiber LRP4 for muscle spindle formation and maintenance in adult and aged animals.

Proprioception is sensed by muscle spindles for precise locomotion and body posture. Unlike the neuromuscular junction (NMJ) for muscle contraction which has been well studied, mechanisms of spindle formation are not well understood. Here we show that sensory nerve terminals are disrupted by the mutation of Lrp4, a gene required for NMJ formation; inducible knockout of Lrp4 in adult mice impairs sensory synapses and movement coordination, suggesting that LRP4 is required for spindle formation and maintenance. LRP4 is critical to the expression of Egr3 during development; in adult mice, it interacts in trans with APP and APLP2 on sensory terminals. Finally, spindle sensory endings and function are impaired in aged mice, deficits that could be diminished by LRP4 expression. These observations uncovered LRP4 as an unexpected regulator of muscle spindle formation and maintenance in adult and aged animals and shed light on potential pathological mechanisms of abnormal muscle proprioception.

Muscle spindles of the multifidus muscle undergo structural change after intervertebral disc degeneration.

PURPOSE: Proprioceptive deficits are common in low back pain. The multifidus muscle undergoes substantial structural change after back injury, but whether muscle spindles are affected is unclear. This study investigated whether muscle spindles of the multifidus muscle are changed by intervertebral disc (IVD) degeneration in a large animal model. METHODS: IVD degeneration was induced by partial thickness annulus fibrosus lesion to the L3-4 IVD in nine sheep. Multifidus muscle tissue at L4 was harvested at six months after lesion, and from six age-/sex-matched naïve control animals. Muscle spindles were identified in Van Gieson's-stained sections by morphology. The number, location and cross-sectional area (CSA) of spindles, the number, type and CSA of intrafusal fibers, and thickness of the spindle capsule were measured. Immunofluorescence assays examined Collagen I and III expression. RESULTS: Multifidus muscle spindles were located centrally in the muscle and generally near connective tissue. There were no differences in the number or location of muscle spindles after IVD degeneration and only changes in the CSA of nuclear chain fibers. The thickness of connective tissue surrounding the muscle spindle was increased as was the expression of Collagen I and III. CONCLUSION: Changes to the connective tissue and collagen expression of the muscle spindle capsule are likely to impact their mechanical properties. Changes in capsule stiffness may impact the transmission of length change to muscle spindles and thus transduction of sensory information. This change in muscle spindle structure may explain some of the proprioceptive deficits identified with low back pain.

Consequences of ankle joint immobilisation: insights from a morphometric analysis about fibre typification, intramuscular connective tissue, and muscle spindle in rats.

Orthosis immobilisations are routinely used in orthopaedic procedures. This intervention is applicable in bone fractures, ligament injuries, and tendonitis, among other disorders of the musculoskeletal system. We aimed to evaluate the effects of ankle joint functional immobilisation on muscle fibre morphology, connective tissue, muscle spindle and fibre typification triggered by a novel metallic orthosis. We developed a rodent-proof experimental orthosis able to hold the tibiotalar joint in a functional position for short and long terms. The tibialis anterior muscles of free and immobilised legs were collected and stained by histology and histochemistry techniques to investigate general muscle morphology, connective tissue and muscle fibre typification. Morphometric analysis of muscle cross-section area, fibre type cross-section area, fibre type density, percentage of intramuscular connective tissue, and thickness of the muscle spindle capsule were obtained to gain insights into the experimental protocol. We found that short- and long-term immobilisation decreased the cross-section area of the muscles and induced centralisation of myonuclei. The connective tissue of immobilised muscle increased after 2 and 4 weeks mainly by deposition of type III and type I collagen fibres in the perimysium and endomysium, respectively, in addition to muscle spindle capsule thickening. Type IIB muscle fibre was severely affected in our study; the profile assumed odd shapes, and our data suggest interconversion of these fibre types within long-term immobilisation. In conclusion, our protocol has produced structural and histochemical changes in muscle biology. This method might be applied to various rodent models that enable genetic manipulation for the investigation of muscle degeneration/regeneration processes.

Post Orgasmic Illness Syndrome (POIS) and Delayed Onset Muscle Soreness (DOMS): Do They Have Anything in Common?

Post orgasmic illness syndrome is a rare, mysterious condition with an unknown pathomechanism and uncertain treatment. The symptoms of post orgasmic illness syndrome last about 2-7 days after an ejaculation. The current hypothesis proposes that the primary injury in post orgasmic illness syndrome is an acute compression proprioceptive axonopathy in the muscle spindle, as is suspected in delayed onset muscle soreness. The terminal arbor degeneration-like lesion of delayed onset muscle soreness is theorized to be an acute stress response energy-depleted dysfunctional mitochondria-induced impairment of Piezo2 channels and glutamate vesicular release. The recurring symptoms of post orgasmic illness syndrome after each ejaculation are suggested to be analogous to the repeated bout effect of delayed onset muscle soreness. However, there are differences in the pathomechanism, mostly attributed to the extent of secondary tissue damage and to the extent of spermidine depletion. The spermidine depletion-induced differences are as follows: modulation of the acute stress response, flu-like symptoms, opioid-like withdrawal and enhanced deregulation of the autonomic nervous system. The longitudinal dimension of delayed onset muscle soreness, in the form of post orgasmic illness syndrome and the repeated bout effect, have cognitive and memory consequences, since the primary injury is learning and memory-related.

Molecular correlates of muscle spindle and Golgi tendon organ afferents.

Proprioceptive feedback mainly derives from groups Ia and II muscle spindle (MS) afferents and group Ib Golgi tendon organ (GTO) afferents, but the molecular correlates of these three afferent subtypes remain unknown. We performed single cell RNA sequencing of genetically identified adult proprioceptors and uncovered five molecularly distinct neuronal clusters. Validation of cluster-specific transcripts in dorsal root ganglia and skeletal muscle demonstrates that two of these clusters correspond to group Ia MS afferents and group Ib GTO afferent proprioceptors, respectively, and suggest that the remaining clusters could represent group II MS afferents. Lineage analysis between proprioceptor transcriptomes at different developmental stages provides evidence that proprioceptor subtype identities emerge late in development. Together, our data provide comprehensive molecular signatures for groups Ia and II MS afferents and group Ib GTO afferents, enabling genetic interrogation of the role of individual proprioceptor subtypes in regulating motor output.

Differentiating the effects of ß-adrenergic stimulation and stretch on calcium and force dynamics using a novel electromechanical cardiomyocyte model.

Cardiac electrophysiology and mechanics are strongly interconnected. Calcium is crucial in this complex interplay through its role in cellular electrophysiology and sarcomere contraction. We aim to differentiate the effects of acute ß-adrenergic stimulation (ß-ARS) and cardiomyocyte stretch (increased sarcomere length) on calcium-transient dynamics and force generation, using a novel computational model of cardiac electromechanics. We implemented a bidirectional coupling between the O'Hara-Rudy model of human ventricular electrophysiology and the MechChem model of sarcomere mechanics through the buffering of calcium by troponin. The coupled model was validated using experimental data from large mammals or human samples. Calcium transient and force were simulated for various degrees of ß-ARS and initial sarcomere lengths. The model reproduced force-frequency, quick-release, and isotonic contraction experiments, validating the bidirectional electromechanical interactions. An increase in ß-ARS increased the amplitudes of force (augmented inotropy) and calcium transient, and shortened both force and calcium-transient duration (lusitropy). An increase in sarcomere length increased force amplitude even more, but decreased calcium-transient amplitude and increased both force and calcium-transient duration. Finally, a gradient in relaxation along the thin filament may explain the nonmonotonic decay in cytosolic calcium observed with high tension. Using a novel coupled human electromechanical model, we identified differential effects of ß-ARS and stretch on calcium and force. Stretch mostly contributed to increased force amplitude and ß-ARS to the reduction of calcium and force duration. We showed that their combination, rather than individual contributions, is key to ensure force generation, rapid relaxation, and low diastolic calcium levels.NEW & NOTEWORTHY This work identifies the contribution of electrical and mechanical alterations to regulation of calcium and force under exercise-like conditions using a novel human electromechanical model integrating ventricular electrophysiology and sarcomere mechanics. By better understanding their individual and combined effects, this can uncover arrhythmogenic mechanisms in exercise-like situations. This publicly available model is a crucial step toward understanding the complex interplay between cardiac electrophysiology and mechanics to improve arrhythmia risk prediction and treatment.

Systems biology approach to exploring the effect of cyclic stretching on cardiac cell physiology.

Although mechanical forces are involved in pressure-overloaded cardiomyopathy, their effects on gene transcription profiles are not fully understood. Here, we used next-generation sequencing (NGS) to investigate changes in genomic profiles after cyclic mechanical stretching of human cardiomyocytes. We found that 85, 87, 32, 29, and 28 genes were differentially expressed after 1, 4, 12, 24, and 48 hours of stretching. Furthermore, 10 of the 29 genes that were up-regulated and 11 of the 28 that were down-regulated after 24 h showed the same changes after 48 h. We then examined expression of the genes that encode serpin family E member 1 (SERPINE1), DNA-binding protein inhibitor 1 (ID1), DNA-binding protein inhibitor 3 (ID3), and CCL2, a cytokine that acts as chemotactic factor in monocytes, in an RT-PCR experiment. The same changes were observed for all four genes after all cyclic stretching durations, confirming the NGS results. Taken together, these findings suggest that cyclical stretching can alter cardiac cell physiology by activating cardiac cell metabolism and impacting cholesterol biosynthesis signaling.

Physiological cyclic stretch up-regulates angiotensin-converting enzyme 2 expression to reduce proliferation and migration of vascular smooth muscle cells.

Angiotensin-converting enzyme 2 (ACE2) is considered as an endogenous negative regulator of renin-angiotensin system (RAS), exerting multiple cardiovascular protective roles. Whether mechanical stretch modulates ACE2 expression remains unknown. The present study aimed at investigating whether ACE2 is involved in physiological stretch (10% elongation, 1 Hz) mediated cellular functions and the underlying mechanism. Cultured human aortic smooth muscle cells (HASMCs) were exposed to 10% stretch for indicated time, and real-time PCR and Western blot analysis showed 10% stretch increased ACE2 expression and activity significantly compared with static conditions and increased Ang-(1-7) level, but decreased Ang II level; Brdu incorporation assay and Scratch test showed that ACE2 was involved in the inhibition of HASMCs proliferation and migration by 10% stretch; the Dual-Luciferase Reporter Assay demonstrated that 10% increased ACE2 promoter activity, but had no effect on ACE2 mRNA stability; kinase inhibition study and Electrophoretic mobility shift assay (EMSA) showed that JNK1/2 and PKCßII pathway, as well as their downstream transcription factors, AP-1 and NF-κB, were involved in 10% stretch induced ACE2 expression. In conclusion, our study indicates ACE2 is a mechanosensitive gene, and may represent a potential therapeutic target for mechanical forces related vascular diseases.

ASIC1a plays a key role in evoking the metabolic component of the exercise pressor reflex in rats.

The role of the acid-sensing ion channel 1a (ASIC1a) in evoking the exercise pressor reflex is unknown, despite the fact that ASIC1a is opened by decreases in pH in the physiological range. This fact prompted us to test the hypothesis that ASIC1a plays an important role in evoking the exercise pressor reflex in decerebrated rats with freely perfused hindlimb muscles. To test this hypothesis, we measured the effect of injecting two ASIC1a blockers into the arterial supply of the triceps surae muscles on the reflex pressor responses to four maneuvers, namely 1) static contraction of the triceps surae muscles (i.e., the exercise pressor reflex), 2) calcaneal tendon stretch, 3) intra-arterial injection of lactic acid, and 4) intra-arterial injection of diprotonated phosphate. We found that the 2 ASIC1a blockers, psalmotoxin-1 (200 ng/kg) and mambalgin-1 (6.5 µg/kg), decreased the pressor responses to static contraction as well as the peak pressor responses to injection of lactic acid and diprotonated phosphate. In contrast, neither ASIC1a blocker had any effect on the pressor responses to tendon stretch. Importantly, we found that ASIC1a blockade significantly decreased the pressor response to static contraction after a latency of at least 8 s. Our results support the hypothesis that ASIC1a plays a key role in evoking the metabolic component of the exercise pressor reflex.NEW & NOTEWORTHY The role played by acid-sensing ion channel 1a (ASIC1a) in evoking the exercise pressor reflex remains unknown. In decerebrated rats with freely perfused femoral arteries, blocking ASIC1a with psalmotoxin-1 or mambalgin-1 significantly attenuated the pressor response to static contraction, lactic acid, and diprotonated phosphate injection but had no effect on the pressor response to stretch. We conclude that ASIC1a plays a key role in evoking the exercise pressor reflex by responding to contraction-induced metabolites, such as protons.

Muscle spindle reinnervation using transplanted embryonic dorsal root ganglion cells after peripheral nerve transection in rats.

OBJECTIVES: Muscle spindles are proprioceptive receptors in the skeletal muscle. Peripheral nerve injury results in a decreased number of muscle spindles and their morphologic deterioration. However, the muscle spindles recover when skeletal muscles are reinnervated with surgical procedures, such as nerve suture or nerve transfer. Morphological changes in muscle spindles by cell transplantation procedure have not been reported so far. Therefore, we hypothesized that transplantation of embryonic sensory neurons may improve sensory neurons in the skeletal muscle and reinnervate the muscle spindles. MATERIALS AND METHODS: We collected sensory neurons from dorsal root ganglions of 14-day-old rat embryos and prepared a rat model of peripheral nerve injury by performing sciatic nerve transection and allowing for a period of one week before which we performed the cell transplantations. Six months later, the morphological changes of muscle spindles in the cell transplantation group were compared with the naïve control and surgical control groups. RESULTS: Our results demonstrated that transplantation of embryonic dorsal root ganglion cells induced regeneration of sensory nerve fibre and reinnervation of muscle spindles in the skeletal muscle. Moreover, calbindin D-28k immunoreactivity in intrafusal muscle fibres was maintained for six months after denervation in the cell transplantation group, whereas it disappeared in the surgical control group. CONCLUSIONS: Cell transplantation therapies could serve as selective targets to modulate mechanosensory function in the skeletal muscle.

The loss of slow skeletal muscle isoform of troponin T in spindle intrafusal fibres explains the pathophysiology of Amish nemaline myopathy.

KEY POINTS: The pathogenic mechanism and the neuromuscular reflex-related phenotype (e.g. tremors accompanied by clonus) of Amish nemaline myopathy, as well as of other recessively inherited TNNT1 myopathies, remain to be clarified. The truncated slow skeletal muscle isoform of troponin T (ssTnT) encoded by the mutant TNNT1 gene is unable to incorporate into myofilaments and is degraded in muscle cells. By contrast to extrafusal muscle fibres, spindle intrafusal fibres of normal mice contain a significant level of cardiac TnT and a low molecular weight splice form of ssTnT. Intrafusal fibres of ssTnT-knockout mice have significantly increased cardiac TnT. Rotarod and balance beam tests have revealed abnormal neuromuscular co-ordination in ssTnT-knockout mice and a blunted response to a spindle sensitizer, succinylcholine. The loss of ssTnT and a compensatory increase of cardiac TnT in intrafusal nuclear bag fibres may increase myofilament Ca2+ -sensitivity and tension, impairing spindle function, thus identifying a novel mechanism for the development of targeted treatment. ABSTRACT: A nonsense mutation at codon Glu180 of TNNT1 gene causes Amish nemaline myopathy (ANM), a recessively inherited disease with infantile lethality. TNNT1 encodes the slow skeletal muscle isoform of troponin T (ssTnT). The truncated ssTnT is unable to incorporate into myofilament and is degraded in muscle cells. The symptoms of ANM include muscle weakness, atrophy, contracture and tremors accompanied by clonus. An ssTnT-knockout (KO) mouse model recapitulates key features of ANM such as atrophy of extrafusal slow muscle fibres and increased fatigability. However, the neuromuscular reflex-related symptoms of ANM have not been explained. By isolating muscle spindles from ssTnT-KO and control mice aiming to examine the composition of myofilament proteins, we found that, in contrast to extrafusal fibres, intrafusal fibres contain a significant level of cardiac TnT and the low molecular weight splice form of ssTnT. Intrafusal fibres from ssTnT-KO mice have significantly increased cardiac TnT. Rotarod and balance beam tests revealed impaired neuromuscular co-ordination in ssTnT-KO mice, indicating abnormality in spindle functions. Unlike the wild-type control, the beam running ability of ssTnT-KO mice had a blunted response to a spindle sensitizer, succinylcholine. Immunohistochemistry detected ssTnT and cardiac TnT in nuclear bag fibres, whereas fast skeletal muscle TnT was detected in nuclear chain fibres, and cardiac α-myosin was present in one of the two nuclear bag fibres. The loss of ssTnT and a compensatory increase of cardiac TnT in nuclear bag fibres would increase myofilament Ca2+ -sensitivity and tension, thus affecting spindle activities. This mechanism provides an explanation for the pathophysiology of ANM, as well as a novel target for treatment.

Effects of myostatin on the mechanical properties of muscles during repeated active lengthening in the mouse.

The aim of the present study was to investigate how myostatin dysfunction affects fast and slow muscle stiffness and viscosity during severe repeated loading. Isolated extensor digitorum longus (EDL) and soleus muscles of young adult female mice of the BEH (dysfunctional myostatin) and BEH+/+ (functional myostatin) strains were subjected to 100 contraction-stretching loading cycles during which contractile and mechanical properties were assessed. BEH mice exhibited greater exercise-induced muscle damage, although the effect was muscle- and age-dependent and limited to the early phases of simulated exercise. The relative reduction of the EDL muscle isometric force recorded during the initial 10-30 loading cycles was greater in BEH mice than in BEH+/+ mice and exceeded that of the soleus muscle of either strain. The induced damage was associated with lower muscle stiffness. The effects of myostatin on the mechanical properties of muscles depend on muscle type and maturity.

GABAergic contribution to the muscle mechanoreflex-mediated heart rate responses at the onset of exercise in humans.

Previous studies have indicated that central GABAergic mechanisms are involved in the heart rate (HR) responses at the onset of exercise. On the basis of previous research that showed similar increases in HR during passive and active cycling, we reasoned that the GABAergic mechanisms involved in the HR responses at the exercise onset are primarily mediated by muscle mechanoreceptor afferents. Therefore, in this study, we sought to determine whether central GABA mechanisms are involved in the muscle mechanoreflex-mediated HR responses at the onset of exercise in humans. Twenty-eight healthy subjects (14 men and 14 women) aged between 18 and 35 yr randomly performed three bouts of 5-s passive and active cycling under placebo and after oral administration of diazepam (10 mg), a benzodiazepine that produces an enhancement in GABAA activity. Beat-to-beat HR (electrocardiography) and arterial blood pressure (finger photopletysmography) were continuously measured. Electromyography of the vastus lateralis was obtained to confirm no electrical activity during passive trials. HR increased from rest under placebo and further increased after administration of diazepam in both passive (change: 12 ± 1 vs. 17 ± 1 beats/min, P < 0.01) and active (change: 14 ± 1 vs. 18 ± 1 beats/min, P < 0.01) cycling. Arterial blood pressure increased from rest similarly during all conditions ( P > 0.05). Importantly, no sex-related differences were found in any variables during experiments. These findings demonstrate, for the first time, that the GABAergic mechanisms significantly contribute to the muscle mechanoreflex-mediated HR responses at the onset of exercise in humans. NEW & NOTEWORTHY We found that passive and voluntary cycling evokes similar increases in heart rate and that these responses were enhanced after diazepam administration, a benzodiazepine that enhances GABAA activity. These findings suggest that the GABAergic system may contribute to the muscle mechanoreflex-mediated vagal withdrawal at the onset of exercise in humans.

Chronic femoral artery ligation exaggerates the pressor and sympathetic nerve responses during dynamic skeletal muscle stretch in decerebrate rats.

Mechanical and metabolic signals arising during skeletal muscle contraction reflexly increase sympathetic nerve activity and blood pressure (i.e., the exercise pressor reflex). In a rat model of simulated peripheral artery disease in which a femoral artery is chronically (~72 h) ligated, the mechanically sensitive component of the exercise pressor reflex during 1-Hz dynamic contraction is exaggerated compared with that found in normal rats. Whether this is due to an enhanced acute sensitization of mechanoreceptors by metabolites produced during contraction or involves a chronic sensitization of mechanoreceptors is unknown. To investigate this issue, in decerebrate, unanesthetized rats, we tested the hypothesis that the increases in mean arterial blood pressure and renal sympathetic nerve activity during 1-Hz dynamic stretch are larger when evoked from a previously "ligated" hindlimb compared with those evoked from the contralateral "freely perfused" hindlimb. Dynamic stretch provided a mechanical stimulus in the absence of contraction-induced metabolite production that closely replicated the pattern of the mechanical stimulus present during dynamic contraction. We found that the increases in mean arterial blood pressure (freely perfused: 14 ± 1 and ligated: 23 ± 3 mmHg, P = 0.02) and renal sympathetic nerve activity were significantly greater during dynamic stretch of the ligated hindlimb compared with the increases during dynamic stretch of the freely perfused hindlimb. These findings suggest that the exaggerated mechanically sensitive component of the exercise pressor reflex found during dynamic muscle contraction in this rat model of simulated peripheral artery disease involves a chronic sensitizing effect of ligation on muscle mechanoreceptors and cannot be attributed solely to acute contraction-induced metabolite sensitization. NEW & NOTEWORTHY We found that the pressor and sympathetic nerve responses during dynamic stretch were exaggerated in rats with a ligated femoral artery (a model of peripheral artery disease). Our findings provide mechanistic insights into the exaggerated exercise pressor reflex in this model and may have important implications for peripheral artery disease patients.

The Effect of Functional Mandibular Shift on the Muscle Spindle Systems in Head-Neck Muscles and the Related Neurotransmitter Histamine.

The aim of this study is to explore the effects of abnormal occlusion and functional recovery caused by functional mandible deviation on the head and neck muscles and muscle spindle sensory-motor system by electrophysiological response and endogenous monoamine neurotransmitters' distribution in the nucleus of the spinal tract. Seven-week-old male Wistar rats were randomly divided into 7 groups: normal control group, 2W experimental control group, 2W functional mandible deviation group, 2W functional mandible deviation recovery group, 4W experimental control group, 4W functional mandible deviation group, 4W functional mandible deviation recovery group. Chewing muscles, digastric muscle, splenius, and trapezius muscle spindles electrophysiological response activities at the opening and closing state were recorded. And then the chewing muscles, digastric, splenius, trapezius, and neck trigeminal nucleus were taken for histidine decarboxylase (HDC) detection by high performance liquid chromatography (HPLC), immunofluorescence, and reverse-transcription polymerase chain reaction (RT-PCR). Histamine receptor proteins in the neck nucleus of the spinal tract were also examined by immunofluorescence and RT-PCR. Electromyography activity of chewing muscles, digastric, and splenius muscle was significantly asymmetric; the abnormal muscle electromyography activity was mainly detected at the ipsilateral side. After functional mandibular deviation, muscle sensitivity on the ipsilateral sides of the chewing muscle and splenius decreased, muscle excitement weakened, modulation depth decreased, and the muscle spindle afferent impulses of excitation transmission speed slowed down. Changes for digastric muscle electrical activity were contrary. The functions recovered at different extents after removing the deflector. However, trapezius in all the experimental groups and recovery groups exhibited bilateral symmetry electrophysiological responses, and no significant difference compared with the control group. After functional mandibular deviation, HDC protein and messenger ribonucleic acid (mRNA) levels on the ipsilateral sides of the chewing muscle and splenius increased significantly. HDC level changes for digastric muscle were contrary. After the removal of the mandibular position deflector, HDC protein and mRNA levels decreased on the ipsilateral sides of the chewing muscle and splenius while they increased in the digastric muscle. The difference of histamine decarboxylase content in the bilateral trapezius in each experimental group was small. After functional mandibular deviation, the temporomandibular joint mechanical receptors not only caused the fusimotor fiber hypoallergenic fatigue slow response on the ipsilateral sides of splenius, but also increased the injury neurotransmitter histamine release. The authors' results further support the opinion that the temporomandibular joint receptors may be involved in the mechanical theory of the head and neck muscles nervous system regulation.

Grb2-associated binder-1 is required for extrafusal and intrafusal muscle fiber development.

The neuregulin-1 (NRG1) signaling pathway plays an important role in the development of the peripheral neuromuscular system, including in muscle spindle and postnatal myelination. We previously showed that NRG1 on the axonal membrane regulates peripheral nerve myelination through Grb2-associated binder 1 (Gab1), a scaffolding mediator of receptor tyrosine kinase signaling. Here, we determined the role of Gab1 in the development of muscles and the muscle spindle using muscle-specific conditional Gab1 knockout mice. The mutant mice showed general retardation in muscular growth and hypotrophy of extrafusal muscle fibers. In addition, the muscle-specific Gab1 knockout mutant exhibited significant underdevelopment of muscle spindles, which are normally regulated by NRG1, and abnormal proprioceptive behavior. Furthermore, the selective knockdown of Gab1 in C2C12 muscle cells reduced NRG1-induced expression of Egr3, a critical transcription factor for muscle spindle development. However, Gab2 knockout mice did not show any defects in the development of muscles or muscle spindles. Our findings suggest that Gab1 is an essential signaling molecule in mediating axonal NRG1 signaling for the development of both extrafusal and intrafusal muscle fibers.

Nek11 regulates asymmetric cell division during mouse oocyte meiotic maturation.

Nek11, a member of the never in mitosis gene A (NIMA) family, is activated in somatic cells associated with G1/S or G2/M arrest. However, its function in meiosis is unknown. In this research, the expression, localization and functions of NEK11 in the mouse oocyte meiotic maturation were examined. Western blotting indicated that NEK11S was the major NEK11 protein in mouse oocyte. MYC-tagged Nek11 mRNA microinjection and immunofluorescent staining showed that NEK11 was localized to the meiotic spindles at MI and MII stage. Knockdown of Nek11 by microinjection of siRNA did not affect germinal vesicle breakdown (GVBD) and the first polar body extrusion, but caused formation of 2-cell-like eggs. These results demonstrate that Nek11 regulates asymmetric cell division during oocyte meiotic maturation.

Muscle spindles exhibit core lesions and extensive degeneration of intrafusal fibers in the Ryr1(I4895T/wt) mouse model of core myopathy.

Muscle spindles from the hind limb muscles of adult Ryr1(I4895T/wt) (IT/+) mice exhibit severe structural abnormalities. Up to 85% of the spindles are separated from skeletal muscle fascicles by a thick layer of connective tissue. Many intrafusal fibers exhibit degeneration, with Z-line streaming, compaction and collapse of myofibrillar bundles, mitochondrial clumping, nuclear shrinkage and pyknosis. The lesions resemble cores observed in the extrafusal myofibers of this animal model and of core myopathy patients. Spindle abnormalities precede those in extrafusal fibers, indicating that they are a primary pathological feature in this murine Ryr1-related core myopathy. Muscle spindle involvement, if confirmed for human core myopathy patients, would provide an explanation for an array of devastating clinical features characteristic of these diseases and provide novel insights into the pathology of RYR1-related myopathies.

Muscle spindles in the human bulbospongiosus and ischiocavernosus muscles.

INTRODUCTION: Muscle spindles are crucial for neuronal regulation of striated muscles, but their presence and involvement in the superficial perineal muscles is not known. METHODS: Bulbospongiosus and ischiocavernosus muscle specimens were obtained from 31 human cadavers. Serial sections were stained with hematoxylin and eosin, Sirius red, antibodies against Podocalyxin, myosin heavy chain isoforms (MyHC-slow tonic, S46; MyHC-2a/2x, A4.74), and neurofilament for the purpose of muscle spindle screening, counting, and characterization. RESULTS: A low but consistent number of spindles were detected in both muscles. The muscles contained few intrafusal fibers, but otherwise showed normal spindle morphology. The extrafusal fibers of both muscles were small in diameter. CONCLUSIONS: The presence of muscle spindles in bulbospongiosus and ischiocavernosus muscles supports physiological models of pelvic floor regulation and may provide a basis for further clinical observations regarding sexual function and micturition. The small number of muscle spindles points to a minor level of proprioceptive regulation.

Differential regulation of AChR clustering in the polar and equatorial region of murine muscle spindles.

Intrafusal fibers of muscle spindles are innervated in the central region by afferent sensory axons and at both polar regions by efferent γ-motoneurons. We previously demonstrated that both neuron-muscle contact sites contain cholinergic synapse-like specialisation, including aggregates of the nicotinic acetylcholine receptor (AChR). In this study we tested the hypothesis that agrin and its receptor complex (consisting of LRP4 and the tyrosine kinase MuSK) are involved in the aggregation of AChRs in muscle spindles, similar to their role at the neuromuscular junction. We show that agrin, MuSK and LRP4 are concentrated at the contact site between the intrafusal fibers and the sensory- and γ-motoneuron, respectively, and that they are expressed in the cell bodies of proprioceptive neurons in dorsal root ganglia. Moreover, agrin and LRP4, but not MuSK, are expressed in γ-motoneuron cell bodies in the ventral horn of the spinal cord. In agrin- and in MuSK-deficient mice, AChR aggregates are absent from the polar regions. In contrast, the subcellular concentration of AChRs in the central region where the sensory neuron contacts the intrafusal muscle fiber is apparently unaffected. Skeletal muscle-specific expression of miniagrin in agrin(-/-) mice in vivo is sufficient to restore the formation of γ-motoneuron endplates. These results show that agrin and MuSK are major determinants during the formation of γ-motoneuron endplates but appear dispensable for the aggregation of AChRs at the central region. Our results therefore suggest different molecular mechanisms for AChR clustering within two domains of intrafusal fibers.