Novel integrated workflow allows production and in-depth quality assessment of multifactorial reprogrammed skeletal muscle cells from human stem cells.

Skeletal muscle tissue engineering aims at generating biological substitutes that restore, maintain or improve normal muscle function; however, the quality of cells produced by current protocols remains insufficient. Here, we developed a multifactor-based protocol that combines adenovector (AdV)-mediated MYOD expression, small molecule inhibitor and growth factor treatment, and electrical pulse stimulation (EPS) to efficiently reprogram different types of human-derived multipotent stem cells into physiologically functional skeletal muscle cells (SMCs). The protocol was complemented through a novel in silico workflow that allows for in-depth estimation and potentially optimization of the quality of generated muscle tissue, based on the transcriptomes of transdifferentiated cells. We additionally patch-clamped phenotypic SMCs to associate their bioelectrical characteristics with their transcriptome reprogramming. Overall, we set up a comprehensive and dynamic approach at the nexus of viral vector-based technology, bioinformatics, and electrophysiology that facilitates production of high-quality skeletal muscle cells and can guide iterative cycles to improve myo-differentiation protocols.

Methocarbamol blocks muscular Nav 1.4 channels and decreases isometric force of mouse muscles.

BACKGROUND: The muscle relaxant methocarbamol is widely used for the treatment of muscle spasms and pain syndromes. To elucidate molecular mechanisms of its action, we studied its influence on neuromuscular transmission, on isometric muscle force, and on voltage-gated Na+ channels. METHODS: Neuromuscular transmission was investigated in murine diaphragm-phrenic nerve preparations and muscle force studied on mouse soleus muscles. Nav 1.4 channels and Nav 1.7 channels were functionally expressed in eukaryotic cell lines. RESULTS: Methocarbamol, at 2 mM, decreased the decay of endplate currents, slowed the decay of endplate potentials and reduced tetanic force of soleus muscles. The drug reversibly inhibited current flow through muscular Nav 1.4 channels, while neuronal Nav 1.7 channels were unaffected. CONCLUSIONS: The study provides evidence for peripheral actions of methocarbamol on skeletal muscle. Muscular Na+ channels are a molecular target of methocarbamol. Since Nav 1.7 currents were unaffected, methocarbamol is unlikely to exert its analgesic effect by directly blocking Nav 1.7 channels.

Impaired muscle spindle function in murine models of muscular dystrophy.

KEY POINTS: Muscular dystrophy patients suffer from progressive degeneration of skeletal muscle fibres, sudden spontaneous falls, balance problems, as well as gait and posture abnormalities. Dystrophin- and dysferlin-deficient mice, models for different types of muscular dystrophy with different aetiology and molecular basis, were characterized to investigate if muscle spindle structure and function are impaired. The number and morphology of muscle spindles were unaltered in both dystrophic mouse lines but muscle spindle resting discharge and their responses to stretch were altered. In dystrophin-deficient muscle spindles, the expression of the paralogue utrophin was substantially upregulated, potentially compensating for the dystrophin deficiency. The results suggest that muscle spindles might contribute to the motor problems observed in patients with muscular dystrophy. ABSTRACT: Muscular dystrophies comprise a heterogeneous group of hereditary diseases characterized by progressive degeneration of extrafusal muscle fibres as well as unstable gait and frequent falls. To investigate if muscle spindle function is impaired, we analysed their number, morphology and function in wildtype mice and in murine model systems for two distinct types of muscular dystrophy with very different disease aetiology, i.e. dystrophin- and dysferlin-deficient mice. The total number and the overall structure of muscle spindles in soleus muscles of both dystrophic mouse mutants appeared unchanged. Immunohistochemical analyses of wildtype muscle spindles revealed a concentration of dystrophin and ß-dystroglycan in intrafusal fibres outside the region of contact with the sensory neuron. While utrophin was absent from the central part of intrafusal fibres of wildtype mice, it was substantially upregulated in dystrophin-deficient mice. Single-unit extracellular recordings of sensory afferents from muscle spindles of the extensor digitorum longus muscle revealed that muscle spindles from both dystrophic mouse strains have an increased resting discharge and a higher action potential firing rate during sinusoidal vibrations, particularly at low frequencies. The response to ramp-and-hold stretches appeared unaltered compared to the respective wildtype mice. We observed no exacerbated functional changes in dystrophin and dysferlin double mutant mice compared to the single mutant animals. These results show alterations in muscle spindle afferent responses in both dystrophic mouse lines, which might cause an increased muscle tone, and might contribute to the unstable gait and frequent falls observed in patients with muscular dystrophy.

Congenital muscle dystrophy and diet consistency affect mouse skull shape differently.

The bones of the mammalian skull respond plastically to changes in masticatory function. However, the extent to which muscle function affects the growth and development of the skull, whose regions have different maturity patterns, remains unclear. Using muscle dissection and 3D landmark-based geometric morphometrics we investigated the effect of changes in muscle function established either before or after weaning, on skull shape and muscle mass in adult mice. We compared temporalis and masseter mass and skull shape in mice with a congenital muscle dystrophy (mdx) and wild type (wt) mice fed on either a hard or a soft diet. We found that dystrophy and diet have distinct effects on the morphology of the skull and the masticatory muscles. Mdx mice show a flattened neurocranium with a more dorsally displaced foramen magnum and an anteriorly placed mandibular condyle compared with wt mice. Compared with hard diet mice, soft diet mice had lower masseter mass and a face with more gracile features as well as labially inclined incisors, suggesting reduced bite strength. Thus, while the early-maturing neurocranium and the posterior portion of the mandible are affected by the congenital dystrophy, the late-maturing face including the anterior part of the mandible responds to dietary differences irrespective of the mdx mutation. Our study confirms a hierarchical, tripartite organisation of the skull (comprising neurocranium, face and mandible) with a modular division based on development and function. Moreover, we provide further experimental evidence that masticatory loading is one of the main environmental stimuli that generate craniofacial variation.

Proteomic profiling of the dystrophin complex and membrane fraction from dystrophic mdx muscle reveals decreases in the cytolinker desmoglein and increases in the extracellular matrix stabilizers biglycan and fibronectin.

The almost complete loss of the membrane cytoskeletal protein dystrophin and concomitant drastic reduction in dystrophin-associated glycoproteins are the underlying mechanisms of the highly progressive neuromuscular disorder Duchenne muscular dystrophy. In order to identify new potential binding partners of dystrophin or proteins in close proximity to the sarcolemmal dystrophin complex, proteomic profiling of the isolated dystrophin-glycoprotein complex was carried out. Subcellular membrane fractionation and detergent solubilisation, in combination with ion exchange, lectin chromatography and density gradient ultracentrifugation, was performed to isolate a dystrophin complex-enriched fraction. Following gradient gel electrophoresis and on-membrane digestion, the protein constituents of the dystrophin fraction were determined by peptide mass spectrometry. This proteomic strategy resulted in the novel identification of desmoglein and desmoplakin, which act as cytolinker proteins and possibly exist in close proximity to the dystrophin complex in the sarcolemma membrane. Interestingly, comparative immunoblotting showed a significant reduction in desmoglein in dystrophin-deficient mdx skeletal muscles, reminiscent of the pathobiochemical fate of the dystrophin-associated core proteins in muscular dystrophy. Comparative membrane proteomics was used to correlate this novel finding to large-scale changes in the dystrophic phenotype. A drastic increase in the extracellular stabilizers biglycan and fibronectin was shown by both mass spectrometric analysis and immunoblotting. The reduced expression of desmoglein in dystrophin-deficient skeletal muscles, and simultaneous increase in components of the extracellular matrix, suggest that muscular dystrophy is associated with plasmalemmal disintegration, loss of cellular linkage and reactive myofibrosis.

Treatment with human immunoglobulin G improves the early disease course in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe hereditary myopathy. Standard treatment by glucocorticosteroids is limited because of numerous side effects. The aim of this study was to test immunomodulation by human immunoglobulin G (IgG) as treatment in the experimental mouse model (mdx) of DMD. 2 g/kg human IgG compared to human albumin was injected intraperitoneally in mdx mice at the age of 3 and 7 weeks. Advanced voluntary wheel running parameters were recorded continuously. At the age of 11 weeks, animals were killed so that blood, diaphragm, and lower limb muscles could be removed for quantitative PCR, histological analysis and ex vivo muscle contraction tests. IgG compared to albumin significantly improved the voluntary running performance and reduced muscle fatigability in an ex vivo muscle contraction test. Upon IgG treatment, serum creatine kinase values were diminished and mRNA expression levels of relevant inflammatory markers were reduced in the diaphragm and limb muscles. Macrophage infiltration and myopathic damage were significantly ameliorated in the quadriceps muscle. Collectively, this study demonstrates that, in the early disease course of mdx mice, human IgG improves the running performance and diminishes myopathic damage and inflammation in the muscle. Therefore, IgG may be a promising approach for treatment of DMD. Two monthly intraperitoneal injections of human immunoglobulin G (IgG) improved the early 11-week disease phase of mdx mice. Voluntary running was improved and serum levels of creatine kinase were diminished. In the skeletal muscle, myopathic damage was ameliorated and key inflammatory markers such as mRNA expression of SPP1 and infiltration by macrophages were reduced. The study suggests that IgG could be explored as a potential treatment option for Duchenne muscular dystrophy and that pre-clinical long-term studies should be helpful.

Lack of the serum- and glucocorticoid-inducible kinase SGK1 improves muscle force characteristics and attenuates fibrosis in dystrophic mdx mouse muscle.

Duchenne muscular dystrophy (DMD) is a human genetic disease characterized by fibrosis and severe muscle weakness. Currently, there is no effective treatment available to prevent progressive fibrosis in skeletal muscles. The serum- and glucocorticoid-inducible kinase SGK1 regulates a variety of physiological functions and participates in fibrosis stimulation. Here, we investigated whether SGK1 influences structure, function and/or fibrosis of the muscles from the mdx mouse, an animal model for DMD. As expected, mdx muscles showed the typical pathological features of muscular dystrophy including fiber size variations, central nuclei of muscle fibers, fibrosis in the diaphragm, and force reduction by 30-50 %. Muscles from sgk1 (-/-) mice were histologically overall intact and specific force was only slightly reduced compared to wild-type muscles. Surprisingly, soleus and diaphragm muscles of mdx/sgk1 (-/-) mice displayed forces close to wild-type levels. Most muscle fibers of the double mutants contained central nuclei, but fibrosis was not observed in any of the tested limb and diaphragm muscles. We conclude that the sole lack of SGK1 in mouse muscle does not lead to pronounced changes in muscle structure and function. However, dystrophin-deficient mdx muscle seems to benefit from SGK1 deficiency. SGK1 appears to be an important enzyme in the process of fibrotic remodeling and subsequent weakness of dystrophin-deficient mouse muscle.

Comparative Label-Free Mass Spectrometric Analysis of Mildly versus Severely Affected mdx Mouse Skeletal Muscles Identifies Annexin, Lamin, and Vimentin as Universal Dystrophic Markers.

The primary deficiency in the membrane cytoskeletal protein dystrophin results in complex changes in dystrophic muscles. In order to compare the degree of secondary alterations in differently affected subtypes of skeletal muscles, we have conducted a global analysis of proteome-wide changes in various dystrophin-deficient muscles. In contrast to the highly degenerative mdx diaphragm muscle, which showed considerable alterations in 35 distinct proteins, the spectrum of mildly to moderately dystrophic skeletal muscles, including interosseus, flexor digitorum brevis, soleus, and extensor digitorum longus muscle, exhibited a smaller number of changed proteins. Compensatory mechanisms and/or cellular variances may be responsible for differing secondary changes in individual mdx muscles. Label-free mass spectrometry established altered expression levels for diaphragm proteins associated with contraction, energy metabolism, the cytoskeleton, the extracellular matrix and the cellular stress response. Comparative immunoblotting verified the differences in the degree of secondary changes in dystrophin-deficient muscles and showed that the up-regulation of molecular chaperones, the compensatory increase in proteins of the intermediate filaments, the fibrosis-related increase in collagen levels and the pathophysiological decrease in calcium binding proteins is more pronounced in mdx diaphragm as compared to the less severely affected mdx leg muscles. Annexin, lamin, and vimentin were identified as universal dystrophic markers.

Antibodies against potassium channel interacting protein 2 induce necrosis in isolated rat cardiomyocytes.

Auto-antibodies against cardiac proteins have been described in patients with dilated cardiomyopathy. Antibodies against the C-terminal part of KChIP2 (anti-KChIP2 [C-12]) enhance cell death of rat cardiomyocytes. The underlying mechanisms are not fully understood. Therefore, we wanted to explore the mechanisms responsible for anti-KChIP2-mediated cell death. Rat cardiomyocytes were treated with anti-KChIP2 (C-12). KChIP2 RNA and protein expressions, nuclear NF-κB, mitochondrial membrane potential Δψm, caspase-3 and -9 activities, necrotic and apoptotic cells, total Ca(2+) and K(+) concentrations, and the effects on L-type Ca(2+) channels were quantified. Anti-KChIP2 (C-12) induced nuclear translocation of NF-κB. Anti-KChIP2 (C-12)-treatment for 2 h significantly reduced KChIP2 mRNA and protein expression. Anti-KChIP2 (C-12) induced nuclear translocation of NF-κB after 1 h. After 6 h, Δψm and caspase-3 and -9 activities were not significantly changed. After 24 h, anti-KChIP2 (C-12)-treated cells were 75 ± 3% necrotic, 2 ± 1% apoptotic, and 13 ± 2% viable. Eighty-six ± 1% of experimental buffer-treated cells were viable. Anti-KChIP2 (C-12) induced significant increases in total Ca(2+) (plus 11 ± 2%) and K(+) (plus 18 ± 2%) concentrations after 5 min. Anti-KChIP2 (C-12) resulted in an increased Ca(2+) influx through L-type Ca(2+) channels. In conclusion, our results suggest that anti-KChIP2 (C-12) enhances cell death of rat cardiomyocytes probably due to necrosis.

Cardiac and respiratory dysfunction in Duchenne muscular dystrophy and the role of second messengers.

Duchenne muscular dystrophy (DMD) affects young boys and is characterized by the absence of dystrophin, a large cytoskeletal protein present in skeletal and cardiac muscle cells and neurons. The heart and diaphragm become necrotic in DMD patients and animal models of DMD, resulting in cardiorespiratory failure as the leading cause of death. The major consequences of the absence of dystrophin are high levels of intracellular Ca(2+) and the unbalanced production of NO that can finally trigger protein degradation and cell death. Cytoplasmic increase in Ca(2+) concentration directly and indirectly triggers different processes such as necrosis, fibrosis, and activation of macrophages. The absence of the neuronal isoform of nitric oxide synthase (nNOS) and the overproduction of NO by the inducible isoform (iNOS) further increase the intracellular Ca(2+) via a hypernitrosylation of the ryanodine receptor. NO overproduction, which further induces the expression of iNOS but decreases the expression of the endothelial isoform (eNOS), deregulates the muscle tissue blood flow creating an ischemic situation. The high levels of Ca(2+) in dystrophic muscles and the ischemic state of the muscle tissue would culminate in a positive feedback loop. While efforts continue toward optimizing cardiac and respiratory care of DMD patients, both Ca(2+) and NO in cardiac and respiratory muscle pathways have been shown to be important to the etiology of the disease. Understanding the mechanisms behind the fine regulation of Ca(2+) -NO may be important for a noninterventional and noninvasive supportive approach to treat DMD patients, improving the quality of life and natural history of DMD patients.

Immunoblot Analysis of DIGE-Based Proteomics.

Proteins can be separated according to their size by gel electrophoresis and further analyzed by Western blotting. The proteins can be transferred to a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF), which results in a replica of the protein's separation patterns. The proteins on the membrane can be detected by specific antibodies followed by visualization either on the membrane itself, on film, or by CCD cameras. Western blotting is a sensitive technique to verify data obtained from fluorescence two-dimensional difference gel electrophoresis (2D-DIGE)-based proteomics.

Long-term blocking of calcium channels in mdx mice results in differential effects on heart and skeletal muscle.

The disease mechanisms underlying dystrophin-deficient muscular dystrophy are complex, involving not only muscle membrane fragility, but also dysregulated calcium homeostasis. Specifically, it has been proposed that calcium channels directly initiate a cascade of pathological events by allowing calcium ions to enter the cell. The objective of this study was to investigate the effect of chronically blocking calcium channels with the aminoglycoside antibiotic streptomycin from onset of disease in the mdx mouse model of Duchenne muscular dystrophy (DMD). Treatment in utero onwards delayed onset of dystrophic symptoms in the limb muscle of young mdx mice, but did not prevent degeneration and regeneration events occurring later in the disease course. Long-term treatment had a positive effect on limb muscle pathology, reduced fibrosis, increased sarcolemmal stability, and promoted muscle regeneration in older mice. However, streptomycin treatment did not show positive effects in diaphragm or heart muscle, and heart pathology was worsened. Thus, blocking calcium channels even before disease onset does not prevent dystrophy, making this an unlikely treatment for DMD. These findings highlight the importance of analyzing several time points throughout the life of the treated mice, as well as analyzing many tissues, to get a complete picture of treatment efficacy.

Functional TRPV4 channels are expressed in mouse skeletal muscle and can modulate resting Ca2+ influx and muscle fatigue.

Skeletal muscle contraction is basically controlled by Ca(2+) release and its reuptake into the sarcoplasmic reticulum. However, the long-term maintenance of muscle function requires an additional Ca(2+) influx from extracellular. Several mechanisms seem to contribute to the latter process, such as store-operated Ca(2+) entry, stretch-activated Ca(2+) influx and resting Ca(2+) influx. Candidate channels that may control Ca(2+) influx into muscle fibers are the STIM proteins, Orai, and the members of the transient receptor potential (TRP) family of cation channels. Here we show that TRPV4, an osmo-sensitive cation channel of the vanilloid subfamily of TRP channels is functionally expressed in mouse skeletal muscle. Western blot analysis showed the presence of TRPV4-specific bands at about 85 and 100 kDa in all tested muscles. The bands were absent when muscle proteins from TRPV4 deficient mice were analyzed. Using the manganese quench technique, we studied the resting influx of divalent cations into isolated wild-type muscle fibers. The specific TRPV4-channel activator 4α-phorbol-12,13-didecanoate (4α-PDD) stimulated resting influx by about 60% only in wild-type fibers. Electrical stimulation of soleus muscles did not reveal changes in isometric twitch contractions upon application of 4α-PDD, but tetanic contractions (at 120 Hz) were slightly increased by about 15%. When soleus muscles were stimulated with a fatigue protocol, muscle fatigue was significantly attenuated in the presence of 4α-PDD. The latter effect was not observed with muscles from TRPV4(-/-) mice. We conclude that TRPV4 is functionally expressed in mouse skeletal muscle and that TRPV4 activation modulates resting Ca(2+) influx and muscle fatigue.

TRP channels in skeletal muscle: gene expression, function and implications for disease.

Besides the well known voltage-gated Ca(2+) channels skeletal muscle fibres contain several non-voltage gated Ca(2+) conducting cation channels. They have been physiologically characterized as stretch activated, store operated and Ca(2+) leak channels. TRP channels are good candidates to account for these sarcolemmal channels and Ca(2+) influx pathways or at least contribute to the responsible macromolecular complexes. Several members of the TRPC, TRPV and TRPM subfamilies of TRP channels are expressed in skeletal muscle as shown by RT-PCR, Western blot and immunohistochemistry. The most prominent and consistently found are TRPC1, C3, C4 and C6, TRPV2 and V4 as well as TRPM4 and M7. However, the precise function of individual channels is largely unknown. Linking physiologically characterized channels of the muscle fibre membrane to TRP channel proteins has been a major challenge during the last years. It has been successful only in a few cases and is complicated by the fact that some channels have dual functions in cultured, immature muscle cells and adult fibres. The best characterized TRP channel in skeletal muscle is TRPC1, a small-conductance channel of the sarcolemma. It is needed for Ca(2+) homeostasis during sustained contractile muscle activity. In addition to certain physiological functions TRP channels seem to be involved in the pathomechanisms of muscle disorders. There is a broad body of evidence that dysregulation of Ca(2+) conducting channels plays a key role in the pathomechanism of Duchenne muscular dystrophy. Lack of the cytoskeletal protein dystrophin or δ-sarcoglycan, seems to disturb the function of one or several Ca(2+) channels of the muscle fibre membrane, leading to pathological dystrophic changes. Almost 10 different TRP channels have been detected in skeletal muscle. They seem to be involved in muscle development, Ca(2+) homeostasis, Ca(2+) signalling and in disease progression of certain muscle disorders. However, we are still at the beginning of understanding the impact of TRP channel functions in skeletal muscle.

Long-term human IgG treatment improves heart and muscle function in a mouse model of Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by mutations in the dystrophin gene, which leads to structural instability of the dystrophin-glycoprotein-complex with subsequent muscle degeneration. In addition, muscle inflammation has been implicated in disease progression and therapeutically addressed with glucocorticosteroids. These have numerous adverse effects. Treatment with human immunoglobulin G (IgG) improved clinical and para-clinical parameters in the early disease phase in the well-established mdx mouse model. The aim of the present study was to confirm the efficacy of IgG in a long-term pre-clinical study in mdx mice. METHODS: IgG (2 g/kg body weight) or NaCl solution as control was administered monthly over 18 months by intraperitoneal injection in mdx mice beginning at 3 weeks of age. Several clinical outcome measures including endurance, muscle strength, and echocardiography were assessed. After 18 months, the animals were sacrificed, blood was collected for analysis, and muscle samples were obtained for ex vivo muscle contraction tests, quantitative PCR, and histology. RESULTS: IgG significantly improved the daily voluntary running performance (1.9 m more total daily running distance, P < 0.0001) and slowed the decrease in grip strength by 0.1 mN, (P = 0.018). IgG reduced fatigability of the diaphragm (improved ratio to maximum force by 0.09 ± 0.04, P = 0.044), but specific tetanic force remained unchanged in the ex vivo muscle contraction test. Cardiac function was significantly better after IgG, especially fractional area shortening (P = 0.012). These results were accompanied by a reduction in cardiac fibrosis and the infiltration of T cells (P = 0.0002) and macrophages (P = 0.0027). In addition, treatment with IgG resulted in a significant reduction of the infiltration of T cells (P ≤ 0.036) in the diaphragm, gastrocnemius, quadriceps, and a similar trend in tibialis anterior and macrophages (P ≤ 0.045) in gastrocnemius, quadriceps, tibialis anterior, and a similar trend in the diaphragm, as well as a decrease in myopathic changes as reflected by a reduced central nuclear index in the diaphragm, tibialis anterior, and quadriceps (P ≤ 0.002 in all). CONCLUSIONS: The present study underscores the importance of an inflammatory contribution to the disease progression of DMD. The data demonstrate the long-term efficacy of IgG in the mdx mouse. IgG is well tolerated by humans and could preferentially complement gene therapy in DMD. The data call for a clinical trial with IgG in DMD.

Diacylglycerol analogues activate second messenger-operated calcium channels exhibiting TRPC-like properties in cortical neurons.

The lipid diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) was used to verify the existence of DAG-sensitive channels in cortical neurons dissociated from E13 mouse embryos. Calcium imaging experiments showed that OAG increased the cytosolic concentration of Ca(2+) ([Ca(2+)]i) in nearly 35% of the KCl-responsive cells. These Ca(2+) responses disappeared in a Ca(2+)-free medium supplemented with EGTA. Mn(2+) quench experiments showed that OAG activated Ca(2+)-conducting channels that were also permeant to Ba(2+). The OAG-induced Ca(2+) responses were unaffected by nifedipine or omega-conotoxin GVIA (Sigma-Aldrich, Saint-Quentin Fallavier, France) but blocked by 1-[beta-(3-(4-Methoxyphenyl)propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF)-96365 and Gd(3+). Replacing Na(+) ions with N-methyl-D-glucamine diminished the amplitude of the OAG-induced Ca(2+) responses showing that the Ca(2+) entry was mediated via Na(+)-dependent and Na(+)-independent mechanisms. Experiments carried out with the fluorescent Na(+) indicator CoroNa Green showed that OAG elevated [Na(+)]i. Like OAG, the DAG lipase inhibitor RHC80267 increased [Ca(2+)]i but not the protein kinase C activator phorbol 12-myristate 13-acetate. Moreover, the OAG-induced Ca(2+) responses were not regulated by protein kinase C activation or inhibition but they were augmented by flufenamic acid which increases currents through C-type transient receptor potential protein family (TRPC) 6 channels. In addition, application of hyperforin, a specific activator of TRPC6 channels, elevated [Ca(2+)]i. Whole-cell patch-clamp recordings showed that hyperforin activated non-selective cation channels. They were blocked by SKF-96365 but potentiated by flufenamic acid. Altogether, our data show the presence of hyperforin- and OAG-sensitive Ca(2+)-permeable channels displaying TRPC6-like properties. This is the first report revealing the existence of second messenger-operated channels in cortical neurons.

Transient receptor potential cation channels in normal and dystrophic mdx muscle.

To investigate the defective calcium regulation of dystrophin-deficient muscle fibres we studied gene expression and localization of non-voltage gated cation channels in normal and mdx mouse skeletal muscle. We found TRPC3, TRPC6, TRPV4, TRPM4 and TRPM7 to be the most abundant isoforms. Immunofluorescent staining of muscle cross-sections with antibodies against TRP proteins showed sarcolemmal localization of TRPC6 and TRPM7, both, for mdx and control. TRPV4 was found only in a fraction of fibres at the sarcolemma and around myonuclei, while TRPC3 staining revealed intracellular patches, preferentially in mdx muscle. Transcripts of low abundance coding for TRPC5, TRPA1 and TRPM1 channels were increased in mdx skeletal muscle at certain stages. The increased Ca(2+)-influx into dystrophin-deficient mdx fibres cannot be explained by increased gene expression of major TRP channels. However, a constant TRP channel expression in combination with the well described weaker Ca(2+)-handling system of mdx fibres may indicate an imbalance between Ca(2+)-influx and cellular Ca(2+)-control.

Glutaminyl cyclase activity is a characteristic feature of human cerebrospinal fluid.

BACKGROUND: Proteins and peptides occurring in human body fluids can be useful biological markers for neurological diseases and can even contribute to the pathogenesis of such diseases. However, proteins and peptides are potential substrates of proteases and other enzymes. Proteolysis and enzymatic modification may lead to their degradation and modification. METHODS: Using mass spectrometry we investigated the degradation and modification of indicator peptides in the presence of cerebrospinal fluid (CSF). We further applied a fluorometric assay to study the activity of the presumed enzyme glutaminyl cyclase. RESULTS: In CSF we observed an aminopeptidase activity that could partially be inhibited by protease inhibitors and EDTA. In addition, the formation of pyroglutamate (pGlu) from N-terminal glutamine (Gln) was regularly observed. The reaction to pGlu was rapid and protected the indicator peptides from further N-terminal degradation. The conversion of Gln to pGlu could be attributed to the activity of the enzyme glutaminyl cyclase (QC). The QC activity was a characteristic feature of all 45 CSF samples collected from multiple sclerosis patients and controls. CONCLUSION: Glutaminyl cyclase activity is a characteristic feature of human cerebrospinal fluid. The presence of QC in CSF can stabilize peptides from degradation by aminopeptidases. This may have impact for neurological disorders that are characterized by both, the presence of QC and the occurrence of appropriate peptide substrates.

Immunoblot Analysis of DIGE-Based Proteomics.

Proteins can be separated according to their size by gel electrophoresis and further analyzed by Western blotting. The proteins can be transferred to a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF), which results in a replica of the proteins' separation patterns. The proteins on the membrane can be detected by specific antibodies followed by visualization either on the membrane itself, on film or by CCD cameras. Western blotting is a sensitive technique to verify data obtained from difference gel electrophoresis (DIGE)-based proteomics.