AMPK and glucose deprivation exert an isoform-specific effect on the expression of Na+,K+-ATPase subunits in cultured myotubes.

In skeletal muscle, Na+,K+-ATPase (NKA), a heterodimeric (α/ß) P-type ATPase, has an essential role in maintenance of Na+ and K+ homeostasis, excitability, and contractility. AMP-activated protein kinase (AMPK), an energy sensor, increases the membrane abundance and activity of NKA in L6 myotubes, but its potential role in regulation of NKA content in skeletal muscle, which determines maximum capacity for Na+ and K+ transport, has not been clearly delineated. We examined whether energy stress and/or AMPK affect expression of NKA subunits in rat L6 and primary human myotubes. Energy stress, induced by glucose deprivation, increased protein content of NKAα1 and NKAα2 in L6 myotubes, while decreasing the content of NKAα1 in human myotubes. Pharmacological AMPK activators (AICAR, A-769662, and diflunisal) modulated expression of NKA subunits, but their effects only partially mimicked those that occurred in response to glucose deprivation, indicating that AMPK does not mediate all effects of energy stress on NKA expression. Gene silencing of AMPKα1/α2 increased protein levels of NKAα1 in L6 myotubes and NKAα1 mRNA levels in human myotubes, while decreasing NKAα2 protein levels in L6 myotubes. Collectively, our results suggest a role for energy stress and AMPK in modulation of NKA expression in skeletal muscle. However, their modulatory effects were not conserved between L6 myotubes and primary human myotubes, which suggests that coupling between energy stress, AMPK, and regulation of NKA expression in vitro depends on skeletal muscle cell model.

Inhibition of the ubiquitin-proteasome system reduces the abundance of pyruvate dehydrogenase kinase 1 in cultured myotubes.

Pyruvate dehydrogenase kinase (PDK), which phosphorylates the pyruvate dehydrogenase complex, regulates glucose metabolism in skeletal muscle. PDK1, an isozyme whose expression is controlled by hypoxia-inducible factor-1α (HIF-1α), is thought to play a role in muscle adaptation to hypoxia. While transcriptional upregulation of PDK1 by HIF-1α is well characterised, mechanisms controlling proteolysis of PDK1 in skeletal muscle have not been thoroughly investigated. Proteasome inhibitor MG132 paradoxically reduced the abundance of PDK1 in human cancer cells and rat L6 myotubes, suggesting that MG132 might direct PDK1 towards autophagic degradation. The objectives of our current study were to determine (1) whether MG132 suppresses PDK1 levels in primary human myotubes, (2) whether chloroquine, an inhibitor of autophagy, prevents MG132-induced suppression of PDK1 in L6 myotubes, and (3) whether PYR-41, an inhibitor of ubiquitination, suppresses PDK1 in L6 myotubes. Using qPCR and/or immunoblotting, we found that despite markedly upregulating HIF-1α protein, MG132 did not alter the PDK1 expression in cultured primary human myotubes, while it suppressed both PDK1 mRNA and protein in L6 myotubes. The PDK1 levels in L6 myotubes were suppressed also during co-treatment with chloroquine and MG132. PYR-41 markedly increased the abundance of HIF-1α in primary human and L6 myotubes, while reducing the abundance of PDK1. In L6 myotubes treated with PYR-41, chloroquine increased the abundance of the epidermal growth factor receptor, but did not prevent the suppression of PDK1. Collectively, our results suggest that cultured myotubes degrade PDK1 via a pathway that cannot be inhibited by MG132, PYR-41, and/or chloroquine.

Importance of the electrophoresis and pulse energy for siRNA-mediated gene silencing by electroporation in differentiated primary human myotubes.

BACKGROUND: Electrotransfection is based on application of high-voltage pulses that transiently increase membrane permeability, which enables delivery of DNA and RNA in vitro and in vivo. Its advantage in applications such as gene therapy and vaccination is that it does not use viral vectors. Skeletal muscles are among the most commonly used target tissues. While siRNA delivery into undifferentiated myoblasts is very efficient, electrotransfection of siRNA into differentiated myotubes presents a challenge. Our aim was to develop efficient protocol for electroporation-based siRNA delivery in cultured primary human myotubes and to identify crucial mechanisms and parameters that would enable faster optimization of electrotransfection in various cell lines. RESULTS: We established optimal electroporation parameters for efficient siRNA delivery in cultured myotubes and achieved efficient knock-down of HIF-1α while preserving cells viability. The results show that electropermeabilization is a crucial step for siRNA electrotransfection in myotubes. Decrease in viability was observed for higher electric energy of the pulses, conversely lower pulse energy enabled higher electrotransfection silencing yield. Experimental data together with the theoretical analysis demonstrate that siRNA electrotransfer is a complex process where electropermeabilization, electrophoresis, siRNA translocation, and viability are all functions of pulsing parameters. However, despite this complexity, we demonstrated that pulse parameters for efficient delivery of small molecule such as PI, can be used as a starting point for optimization of electroporation parameters for siRNA delivery into cells in vitro if viability is preserved. CONCLUSIONS: The optimized experimental protocol provides the basis for application of electrotransfer for silencing of various target genes in cultured human myotubes and more broadly for electrotransfection of various primary cell and cell lines. Together with the theoretical analysis our data offer new insights into mechanisms that underlie electroporation-based delivery of short RNA molecules, which can aid to faster optimisation of the pulse parameters in vitro and in vivo.

Gene Immunotherapy of Colon Carcinoma with IL-2 and IL-12 Using Gene Electrotransfer.

Gene immunotherapy has become an important approach in the treatment of cancer. One example is the introduction of genes encoding immunostimulatory cytokines, such as interleukin 2 and interleukin 12, which stimulate immune cells in tumours. The aim of our study was to determine the effects of gene electrotransfer of plasmids encoding interleukin 2 and interleukin 12 individually and in combination in the CT26 murine colon carcinoma cell line in mice. In the in vitro experiment, the pulse protocol that resulted in the highest expression of IL-2 and IL-12 mRNA and proteins was used for the in vivo part. In vivo, tumour growth delay and also complete response were observed in the group treated with the plasmid combination. Compared to the control group, the highest levels of various immunostimulatory cytokines and increased immune infiltration were observed in the combination group. Long-term anti-tumour immunity was observed in the combination group after tumour re-challenge. In conclusion, our combination therapy efficiently eradicated CT26 colon carcinoma in mice and also generated strong anti-tumour immune memory.

Neuronal Agrin Promotes Proliferation of Primary Human Myoblasts in an Age-Dependent Manner.

Neuronal agrin, a heparan sulphate proteoglycan secreted by the α-motor neurons, promotes the formation and maintenance of the neuromuscular junction by binding to Lrp4 and activating muscle-specific kinase (MuSK). Neuronal agrin also promotes myogenesis by enhancing differentiation and maturation of myotubes, but its effect on proliferating human myoblasts, which are often considered to be unresponsive to agrin, remains unclear. Using primary human myoblasts, we determined that neuronal agrin induced transient dephosphorylation of ERK1/2, while c-Abl, STAT3, and focal adhesion kinase were unresponsive. Gene silencing of Lrp4 and MuSK markedly reduced the BrdU incorporation, suggesting the functional importance of the Lrp4/MuSK complex for myoblast proliferation. Acute and chronic treatments with neuronal agrin increased the proliferation of human myoblasts in old donors, but they did not affect the proliferation of myoblasts in young donors. The C-terminal fragment of agrin which lacks the Lrp4-binding site and cannot activate MuSK had a similar age-dependent effect, indicating that the age-dependent signalling pathways activated by neuronal agrin involve the Lrp4/MuSK receptor complex as well as an Lrp4/MuSK-independent pathway which remained unknown. Collectively, our results highlight an age-dependent role for neuronal agrin in promoting the proliferation of human myoblasts.

Phosphorylation of Na+,K+-ATPase at Tyr10 of the α1-Subunit is Suppressed by AMPK and Enhanced by Ouabain in Cultured Kidney Cells.

Na+,K+-ATPase (NKA) is essential for maintenance of cellular and whole-body water and ion homeostasis. In the kidney, a major site of ion transport, NKA consumes ~ 50% of ATP, indicating a tight coordination of NKA and energy metabolism. AMP-activated protein kinase (AMPK), a cellular energy sensor, regulates NKA by modulating serine phosphorylation of the α1-subunit, but whether it modulates other important regulatory phosphosites, such as Tyr10, is unknown. Using human kidney (HK-2) cells, we determined that the phosphorylation of Tyr10 was stimulated by the epidermal growth factor (EGF), which was opposed by inhibitors of Src kinases (PP2), tyrosine kinases (genistein), and EGF receptor (EGFR, gefitinib). AMPK activators AICAR and A-769662 suppressed the EGF-stimulated phosphorylation of EGFR (Tyr1173) and NKAα1 at Tyr10. The phosphorylation of Src (Tyr416) was unaltered by AICAR and increased by A-769662. Conversely, ouabain (100 nM), a pharmacological NKA inhibitor and a putative adrenocortical hormone, enhanced the EGF-stimulated Tyr10 phosphorylation without altering the phosphorylation of EGFR (Tyr1173) or Src (Tyr416). Ouabain (100-1000 nM) increased the ADP:ATP ratio, while it suppressed the lactate production and the oxygen consumption rate in a dose-dependent manner. Treatment with ouabain or gene silencing of NKAα1 or NKAα3 subunit did not activate AMPK. In summary, AMPK activators and ouabain had antagonistic effects on the phosphorylation of NKAα1 at Tyr10 in cultured HK-2 cells, which implicates a role for Tyr10 in coordinated regulation of NKA-mediated ion transport and energy metabolism.

The role of AMPK in regulation of Na+,K+-ATPase in skeletal muscle: does the gauge always plug the sink?

AMP-activated protein kinase (AMPK) is a cellular energy gauge and a major regulator of cellular energy homeostasis. Once activated, AMPK stimulates nutrient uptake and the ATP-producing catabolic pathways, while it suppresses the ATP-consuming anabolic pathways, thus helping to maintain the cellular energy balance under energy-deprived conditions. As much as ~ 20-25% of the whole-body ATP consumption occurs due to a reaction catalysed by Na+,K+-ATPase (NKA). Being the single most important sink of energy, NKA might seem to be an essential target of the AMPK-mediated energy saving measures, yet NKA is vital for maintenance of transmembrane Na+ and K+ gradients, water homeostasis, cellular excitability, and the Na+-coupled transport of nutrients and ions. Consistent with the model that AMPK regulates ATP consumption by NKA, activation of AMPK in the lung alveolar cells stimulates endocytosis of NKA, thus suppressing the transepithelial ion transport and the absorption of the alveolar fluid. In skeletal muscles, contractions activate NKA, which opposes a rundown of transmembrane ion gradients, as well as AMPK, which plays an important role in adaptations to exercise. Inhibition of NKA in contracting skeletal muscle accentuates perturbations in ion concentrations and accelerates development of fatigue. However, different models suggest that AMPK does not inhibit or even stimulates NKA in skeletal muscle, which appears to contradict the idea that AMPK maintains the cellular energy balance by always suppressing ATP-consuming processes. In this short review, we examine the role of AMPK in regulation of NKA in skeletal muscle and discuss the apparent paradox of AMPK-stimulated ATP consumption.

Activation of (un)regulated cell death as a new perspective for bispyridinium and imidazolium oximes.

Oximes, investigated as antidotes against organophosphates (OP) poisoning, are known to display toxic effects on a cellular level, which could be explained beyond action on acetylcholinesterase as their main target. To investigate this further, we performed an in vitro cell-based evaluation of effects of two structurally diverse oxime groups at concentrations of up to 800 µM, on several cell models: skeletal muscle, kidney, liver, and neural cells. As indicated by our results, compounds with an imidazolium core induced necrosis, unregulated cell death characterized by a cell burst, increased formation of reactive oxygen species, and activation of antioxidant scavenging. On the other hand, oximes with a pyridinium core activated apoptosis through specific caspases 3, 8, and/or 9. Interestingly, some of the compounds exhibited a synergistic effect. Moreover, we generated a pharmacophore model for each oxime series and identified ligands from public databases that map to generated pharmacophores. Several interesting hits were obtained including chemotherapeutics and specific inhibitors. We were able to define the possible structural features of tested oximes triggering toxic effects: chlorine atoms in combination with but-2(E)-en-1,4-diyl linker and adding a second benzene ring with substituents such as chlorine and/or methyl on the imidazolium core. Such oximes could not be used in further OP antidote development research, but could be introduced in other research studies on new specific targets. This could undoubtedly result in an overall improved wider use of unexplored oxime database created so far in OP antidotes field of research in a completely new perspective.

Functional and molecular adaptations of quadriceps and hamstring muscles to blood flow restricted training in patients with ACL rupture.

Effects of low-load blood flow restricted (LL-BFR) training remain unexplored in patients with ACL rupture. Our hypothesis was that LL-BFR training triggers augmented gains in knee muscle strength and size, which are paralleled with transcriptional responses of hypoxia-regulated genes and myokines. Eighteen volunteers (age 37.5 ± 9 years) planned for ACL reconstruction, participated in the study. Twelve were divided between BFR group, performing 9 sessions of LL-BFR exercise, and SHAM-BFR group performing equal training with sham vascular occlusion. Six subjects served as a control for muscle biopsy analysis. Cross-sectional area (CSA) and isokinetic strength of knee muscles were assessed before and after the training. Change in CSAquad was significantly (p < 0.01) larger in BFR (4.9%) compared with SHAM-BFR (1.3%). Similarly, change in peak torque of knee extensors was significantly (p < 0.05) larger in BFR (14%) compared with SHAM-BFR (-1%). The decrease in fatigue index of knee extensors (6%) was larger (p < 0.01) in BFR than in SHAM-BFR (2%). mRNA expression of HIF-1α in the vastus lateralis was reduced (p < 0.05) in SHAM-BFR, while VEGF-A mRNA tended to be higher in BFR. The mRNA expression of myostatin and its receptor were reduced (p < 0.05) in the semitendinosus after both types of training. Expression of IL-6, its receptors IL-6Rα and gp130, as well as musclin were similar in control and training groups. In conclusion, our results show augmented strength and endurance of knee extensors but less of the flexors. LL-BFR training is especially effective for conditioning of knee extensors in this population.

Suppression of Pyruvate Dehydrogenase Kinase by Dichloroacetate in Cancer and Skeletal Muscle Cells Is Isoform Specific and Partially Independent of HIF-1α.

Inhibition of pyruvate dehydrogenase kinase (PDK) emerged as a potential strategy for treatment of cancer and metabolic disorders. Dichloroacetate (DCA), a prototypical PDK inhibitor, reduces the abundance of some PDK isoenzymes. However, the underlying mechanisms are not fully characterized and may differ across cell types. We determined that DCA reduced the abundance of PDK1 in breast (MDA-MB-231) and prostate (PC-3) cancer cells, while it suppressed both PDK1 and PDK2 in skeletal muscle cells (L6 myotubes). The DCA-induced PDK1 suppression was partially dependent on hypoxia-inducible factor-1α (HIF-1α), a transcriptional regulator of PDK1, in cancer cells but not in L6 myotubes. However, the DCA-induced alterations in the mRNA and the protein levels of PDK1 and/or PDK2 did not always occur in parallel, implicating a role for post-transcriptional mechanisms. DCA did not inhibit the mTOR signaling, while inhibitors of the proteasome or gene silencing of mitochondrial proteases CLPP and AFG3L2 did not prevent the DCA-induced reduction of the PDK1 protein levels. Collectively, our results suggest that DCA reduces the abundance of PDK in an isoform-dependent manner via transcriptional and post-transcriptional mechanisms. Differential response of PDK isoenzymes to DCA might be important for its pharmacological effects in different types of cells.

Vitamin B3-Based Biologically Active Compounds as Inhibitors of Human Cholinesterases.

We evaluated the potential of nine vitamin B3 scaffold-based derivatives as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors, as a starting point for the development of novel drugs for treating disorders with cholinergic neurotransmission-linked pathology. As the results indicate, all compounds reversibly inhibited both enzymes in the micromolar range pointing to the preference of AChE over BChE for binding the tested derivatives. Molecular docking studies revealed the importance of interactions with AChE active site residues Tyr337 and Tyr124, which dictated most of the observed differences. The most potent inhibitor of both enzymes with Ki of 4 µM for AChE and 8 µM for BChE was the nicotinamide derivative 1-(4'-phenylphenacyl)-3-carbamoylpyridinium bromide. Such a result places it within the range of several currently studied novel cholinesterase inhibitors. Cytotoxicity profiling did not classify this compound as highly toxic, but the induced effects on cells should not be neglected in any future detailed studies and when considering this scaffold for drug development.

Proposing Urothelial and Muscle In Vitro Cell Models as a Novel Approach for Assessment of Long-Term Toxicity of Nanoparticles.

Many studies evaluated the short-term in vitro toxicity of nanoparticles (NPs); however, long-term effects are still not adequately understood. Here, we investigated the potential toxic effects of biomedical (polyacrylic acid and polyethylenimine coated magnetic NPs) and two industrial (SiO2 and TiO2) NPs following different short-term and long-term exposure protocols on two physiologically different in vitro models that are able to differentiate: L6 rat skeletal muscle cell line and biomimetic normal porcine urothelial (NPU) cells. We show that L6 cells are more sensitive to NP exposure then NPU cells. Transmission electron microscopy revealed an uptake of NPs into L6 cells but not NPU cells. In L6 cells, we obtained a dose-dependent reduction in cell viability and increased reactive oxygen species (ROS) formation after 24 h. Following continuous exposure, more stable TiO2 and polyacrylic acid (PAA) NPs increased levels of nuclear factor Nrf2 mRNA, suggesting an oxidative damage-associated response. Furthermore, internalized magnetic PAA and TiO2 NPs hindered the differentiation of L6 cells. We propose the use of L6 skeletal muscle cells and NPU cells as a novel approach for assessment of the potential long-term toxicity of relevant NPs that are found in the blood and/or can be secreted into the urine.

Comparison of the effects of metformin on MDA-MB-231 breast cancer cells in a monolayer culture and in tumor spheroids as a function of nutrient concentrations.

Metabolic pathways of cancer cells depend on the concentrations of nutrients in their micro-environment as well as on the cell-to-cell interactions. Here we examined the effects of glucose, pyruvate and glutamine on the sensitivity of MDA-MB-231 cells to metabolic drug metformin using standard 2D culture, in which cells are grown in a monolayer, and 3D tumor spheroids, in which three-dimensional growth of cells better mimics a tumor. To examine effects of nutrients on metformin action, MDA-MB-231 cells were grown in commonly used media (DMEM, MEM and RPMI-1640) that differ mainly in the concentrations of amino acids. We used MTS assay and Hoechst and propidium iodide staining to determine cell number, viability and survival, respectively. We also determined the size of tumor spheroids and assessed effects of nutrients on metformin-stimulated AMP-activated protein kinase activation. Non-essential amino acids suppressed the effects of metformin on MDA-MB-231 cells in a 2D culture and in 3D tumor spheroids. Glutamine and pyruvate weakly diminished the effects of metformin in 2D culture. Furthermore, glucose protected tumor spheroids against metformin-induced disintegration. Our results show that nutrient availability must be considered when we evaluate the effects of metformin in 2D culture and in biologically more relevant 3D tumor spheroids.

Hormonal regulation of Na+-K+-ATPase from the evolutionary perspective.

Na+-K+-ATPase, an α/ß heterodimer, is an ancient enzyme that maintains Na+ and K+ gradients, thus preserving cellular ion homeostasis. In multicellular organisms, this basic housekeeping function is integrated to fulfill the needs of specialized organs and preserve whole-body homeostasis. In vertebrates, Na+-K+-ATPase is essential for many fundamental physiological processes, such as nerve conduction, muscle contraction, nutrient absorption, and urine excretion. During vertebrate evolution, three key developments contributed to diversification and integration of Na+-K+-ATPase functions. Generation of novel α- and ß-subunits led to formation of multiple Na+-K+-ATPase isoenyzmes with distinct functional characteristics. Development of a complex endocrine system enabled efficient coordination of diverse Na+-K+-ATPase functions. Emergence of FXYDs, small transmembrane proteins that regulate Na+-K+-ATPase, opened new ways to modulate its function. FXYDs are a vertebrate innovation and an important site of hormonal action, suggesting they played an especially prominent role in evolving interaction between Na+-K+-ATPase and the endocrine system in vertebrates.

Nucleosides block AICAR-stimulated activation of AMPK in skeletal muscle and cancer cells.

AMP-activated kinase (AMPK) is a major regulator of energy metabolism and a promising target for development of new treatments for type 2 diabetes and cancer. 5-Aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), an adenosine analog, is a standard positive control for AMPK activation in cell-based assays. Some broadly used cell culture media, such as minimal essential medium α (MEMα), contain high concentrations of adenosine and other nucleosides. We determined whether such media alter AICAR action in skeletal muscle and cancer cells. In nucleoside-free media, AICAR stimulated AMPK activation, increased glucose uptake, and suppressed cell proliferation. Conversely, these effects were blunted or completely blocked in MEMα that contains nucleosides. Addition of adenosine or 2'-deoxyadenosine to nucleoside-free media also suppressed AICAR action. MEMα with nucleosides blocked AICAR-stimulated AMPK activation even in the presence of methotrexate, which normally markedly enhances AICAR action by reducing its intracellular clearance. Other common media components, such as vitamin B-12, vitamin C, and α-lipoic acid, had a minor modulatory effect on AICAR action. Our findings show that nucleoside-containing media, commonly used in AMPK research, block action of the most widely used pharmacological AMPK activator AICAR. Results of cell-based assays in which AICAR is used for AMPK activation therefore critically depend on media formulation. Furthermore, our findings highlight a role for extracellular nucleosides and nucleoside transporters in regulation of AMPK activation.

Early vertebrate origin and diversification of small transmembrane regulators of cellular ion transport.

KEY POINTS: Small transmembrane proteins such as FXYDs, which interact with Na+ ,K+ -ATPase, and the micropeptides that interact with sarco/endoplasmic reticulum Ca2+ -ATPase play fundamental roles in regulation of ion transport in vertebrates. Uncertain evolutionary origins and phylogenetic relationships among these regulators of ion transport have led to inconsistencies in their classification across vertebrate species, thus hampering comparative studies of their functions. We discovered the first FXYD homologue in sea lamprey, a basal jawless vertebrate, which suggests small transmembrane regulators of ion transport emerged early in the vertebrate lineage. We also identified 13 gene subfamilies of FXYDs and propose a revised, phylogeny-based FXYD classification that is consistent across vertebrate species. These findings provide an improved framework for investigating physiological and pathophysiological functions of small transmembrane regulators of ion transport. ABSTRACT: Small transmembrane proteins are important for regulation of cellular ion transport. The most prominent among these are members of the FXYD family (FXYD1-12), which regulate Na+ ,K+ -ATPase, and phospholamban, sarcolipin, myoregulin and DWORF, which regulate the sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA). FXYDs and regulators of SERCA are present in fishes, as well as terrestrial vertebrates; however, their evolutionary origins and phylogenetic relationships are obscure, thus hampering comparative physiological studies. Here we discovered that sea lamprey (Petromyzon marinus), a representative of extant jawless vertebrates (Cyclostomata), expresses an FXYD homologue, which strongly suggests that FXYDs predate the emergence of fishes and other jawed vertebrates (Gnathostomata). Using a combination of sequence-based phylogenetic analysis and conservation of local chromosome context, we determined that FXYDs markedly diversified in the lineages leading to cartilaginous fishes (Chondrichthyes) and bony vertebrates (Euteleostomi). Diversification of SERCA regulators was much less extensive, indicating they operate under different evolutionary constraints. Finally, we found that FXYDs in extant vertebrates can be classified into 13 gene subfamilies, which do not always correspond to the established FXYD classification. We therefore propose a revised classification that is based on evolutionary history of FXYDs and that is consistent across vertebrate species. Collectively, our findings provide an improved framework for investigating the function of ion transport in health and disease.

In Vitro Innervation as an Experimental Model to Study the Expression and Functions of Acetylcholinesterase and Agrin in Human Skeletal Muscle.

Acetylcholinesterase (AChE) and agrin, a heparan-sulfate proteoglycan, reside in the basal lamina of the neuromuscular junction (NMJ) and play key roles in cholinergic transmission and synaptogenesis. Unlike most NMJ components, AChE and agrin are expressed in skeletal muscle and α-motor neurons. AChE and agrin are also expressed in various other types of cells, where they have important alternative functions that are not related to their classical roles in NMJ. In this review, we first focus on co-cultures of embryonic rat spinal cord explants with human skeletal muscle cells as an experimental model to study functional innervation in vitro. We describe how this heterologous rat-human model, which enables experimentation on highly developed contracting human myotubes, offers unique opportunities for AChE and agrin research. We then highlight innovative approaches that were used to address salient questions regarding expression and alternative functions of AChE and agrin in developing human skeletal muscle. Results obtained in co-cultures are compared with those obtained in other models in the context of general advances in the field of AChE and agrin neurobiology.

Na,K-ATPase regulation in skeletal muscle.

Skeletal muscle contains one of the largest and the most dynamic pools of Na,K-ATPase (NKA) in the body. Under resting conditions, NKA in skeletal muscle operates at only a fraction of maximal pumping capacity, but it can be markedly activated when demands for ion transport increase, such as during exercise or following food intake. Given the size, capacity, and dynamic range of the NKA pool in skeletal muscle, its tight regulation is essential to maintain whole body homeostasis as well as muscle function. To reconcile functional needs of systemic homeostasis with those of skeletal muscle, NKA is regulated in a coordinated manner by extrinsic stimuli, such as hormones and nerve-derived factors, as well as by local stimuli arising in skeletal muscle fibers, such as contractions and muscle energy status. These stimuli regulate NKA acutely by controlling its enzymatic activity and/or its distribution between the plasma membrane and the intracellular storage compartment. They also regulate NKA chronically by controlling NKA gene expression, thus determining total NKA content in skeletal muscle and its maximal pumping capacity. This review focuses on molecular mechanisms that underlie regulation of NKA in skeletal muscle by major extrinsic and local stimuli. Special emphasis is given to stimuli and mechanisms linking regulation of NKA and energy metabolism in skeletal muscle, such as insulin and the energy-sensing AMP-activated protein kinase. Finally, the recently uncovered roles for glutathionylation, nitric oxide, and extracellular K(+) in the regulation of NKA in skeletal muscle are highlighted.

siRNA delivery into cultured primary human myoblasts--optimization of electroporation parameters and theoretical analysis.

Introduction of genetic material into muscle tissue has been extensively researched, including isolation and in vitro expansion of primary myoblasts as a potential source of cells for skeletal and heart muscle tissue engineering applications. In this study, we optimized the electroporation protocol for introduction of short interfering ribonucleic acid (siRNA) against messenger RNA for Hypoxia Inducible Factor 1α (HIF-1α) into cultured primary human myoblasts. We established optimal pulsing protocol for siRNA electro transfection, and theoretically analyzed the effect of electric field and pulse duration on silencing efficiency and electrophoretic displacement of siRNA. Silencing of HIF-1α was determined with quantitative polymerase chain reaction and Western Blot. The most efficient silencing (71% knockdown) was achieved with 8 × 2 ms pulses, E = 0.6 kV/cm. Viability was determined immediately, 1 h and 48 h after electroporation. In general, there was a trade-off between efficient silencing and preserved viability. Electric field and pulse duration are crucial parameters for silencing, since both increase membrane permeabilization and electrophoretic transfer of siRNA. Short-term viability showed immediate toxicity of pulses due to membrane damage, while indirect effects on cell proliferation were observed after 48 h. Presented results are important for faster optimization of electroporation parameters for ex vivo electrotransfer of short RNA molecules into primary human myoblasts.

Activation of AMP-activated protein kinase stimulates Na+,K+-ATPase activity in skeletal muscle cells.

Contraction stimulates Na(+),K(+)-ATPase and AMP-activated protein kinase (AMPK) activity in skeletal muscle. Whether AMPK activation affects Na(+),K(+)-ATPase activity in skeletal muscle remains to be determined. Short term stimulation of rat L6 myotubes with the AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), activates AMPK and promotes translocation of the Na(+),K(+)-ATPase α(1)-subunit to the plasma membrane and increases Na(+),K(+)-ATPase activity as assessed by ouabain-sensitive (86)Rb(+) uptake. Cyanide-induced artificial anoxia, as well as a direct AMPK activator (A-769662) also increase AMPK phosphorylation and Na(+),K(+)-ATPase activity. Thus, different stimuli that target AMPK concomitantly increase Na(+),K(+)-ATPase activity. The effect of AICAR on Na(+),K(+)-ATPase in L6 myotubes was attenuated by Compound C, an AMPK inhibitor, as well as siRNA-mediated AMPK silencing. The effects of AICAR on Na(+),K(+)-ATPase were completely abolished in cultured primary mouse muscle cells lacking AMPK α-subunits. AMPK stimulation leads to Na(+),K(+)-ATPase α(1)-subunit dephosphorylation at Ser(18), which may prevent endocytosis of the sodium pump. AICAR stimulation leads to methylation and dephosphorylation of the catalytic subunit of the protein phosphatase (PP) 2A in L6 myotubes. Moreover, AICAR-triggered dephosphorylation of the Na(+),K(+)-ATPase was prevented in L6 myotubes deficient in PP2A-specific protein phosphatase methylesterase-1 (PME-1), indicating a role for the PP2A·PME-1 complex in AMPK-mediated regulation of Na(+),K(+)-ATPase. Thus contrary to the common paradigm, we report AMPK-dependent activation of an energy-consuming ion pumping process. This activation may be a potential mechanism by which exercise and metabolic stress activate the sodium pump in skeletal muscle.