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.

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.

Can flow cytometry reinvent the sentinel lymph node concept in gastric cancer patients?

BACKGROUND: The focused sentinel lymph node (SLN) concept we proposed previously relied on real time-quantitative polymerase chain reaction (RT-qPCR) to detect tumor cells, which is too elaborate for intraoperative use. Therefore, we evaluated flow cytometry for intraoperative detection of tumor cells in SLNs. METHODS: Sixty-five consecutive gastric cancer patients were included. SLN analysis was carried out for a single SLN from each patient, using the molecular methods of RT-qPCR (first 30 patients) and flow cytometry (final 35 patients). All LNs underwent hematoxylin and eosin staining and immunohistochemical examination. RESULTS: Extraction of the SLN from a high-risk station was an important determinant for accurate prediction of LN metastases. For RT-qPCR, the sensitivity and specificity of detection were 72.7% and 81.8%, respectively, and for flow cytometry, 36.8% and 100%, respectively. When only high-risk SLNs were analyzed and specimens with <10% viability of leukocytes were excluded, the sensitivity and specificity of flow cytometry were 60% and 100%, respectively. Multivariate analysis identified significant predictors for LN metastases as the molecular method of SLN analysis (P = 0.021; 95% confidence interval [CI]: 1.304-24.284) and lymphovascular invasion (P = 0.002; 95% CI: 2.142-28.555). In subgroup analysis of high-risk SLNs, only RT-qPCR was a significant predictor for LN metastases (P = 0.016; 95% CI: 1.581-91.084). CONCLUSIONS: Flow cytometry of high-risk SLNs, excluding specimens with low cell viability is a rapid, cost-effective, widely obtainable, and highly specific method for SLN metastases detection although it lacks the necessary sensitivity. Therefore, it cannot be recommended as a stand-alone method for the detection of LN metastases during operations.

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.

Dexamethasone does not diminish sugammadex reversal of neuromuscular block - clinical study in surgical patients undergoing general anesthesia.

BACKGROUND: Sugammadex reverses neuromuscular block (NMB) through binding aminosteroid neuromuscular blocking agents. Although sugammadex appears to be highly selective, it can interact with other drugs, like corticosteroids. A prospective single-blinded randomized clinical trial was designed to explore the significance of interactions between dexamethasone and sugammadex. METHODS: Sixty-five patients who were anesthetized for elective abdominal or urological surgery were included. NMB was assessed using train-of-four stimulation (TOF), with rocuronium used to maintain the desired NMB depth. NMB reversal at the end of anaesthesia was achieved using sugammadex. According to their received antiemetics, the patients were randomized to either the granisetron or dexamethasone group. Blood samples were taken before and after NMB reversal, for plasma dexamethasone and rocuronium determination. Primary endpoint was time from sugammadex administration to NMB reversal. Secondary endpoints included the ratios of the dexamethasone and rocuronium concentrations after NMB reversal versus before sugammadex administration. RESULTS: There were no differences for time to NMB reversal between the control (mean 121 ± 61 s) and the dexamethasone group (mean 125 ± 57 s; P = 0.760). Time to NMB reversal to a TOF ratio ≥0.9 was significantly longer in patients with lower TOF prior to sugammadex administration (Beta = -0.268; P = 0.038). The ratio between the rocuronium concentrations after NMB reversal versus before sugammadex administration was significantly affected by sugammadex dose (Beta = -0.375; P = 0.004), as was rocuronium dose per hour of operation (Beta = -0.366; p = 0.007), while it was not affected by NMB depth before administration of sugammadex (Beta = -0.089; p = 0.483) and dexamethasone (Beta = -0.186; p = 0.131). There was significant drop in plasma dexamethasone after sugammadex administration and NMB reversal (p < 0.001). CONCLUSIONS: Administration of dexamethasone to anesthetized patients did not delay NMB reversal by sugammadex. TRIAL REGISTRATION: The trial was retrospectively registered with The Australian New Zealand Clinical Trials Registry (ANZCTR) on February 28th 2012 (enrollment of the first patient on February 2nd 2012) and was given a trial ID number ACTRN12612000245897 and universal trial number U1111-1128-5104.

CA19-9 serum levels predict micrometastases in patients with gastric cancer.

BACKGROUND: We explored the prognostic value of the up-regulated carbohydrate antigen (CA19-9) in node-negative patients with gastric cancer as a surrogate marker for micrometastases. PATIENTS AND METHODS: Micrometastases were determined using reverse transcription quantitative polymerase chain reaction (RT-qPCR) for a subgroup of 30 node-negative patients. This group was used to determine the cut-off for preoperative CA19-9 serum levels as a surrogate marker for micrometastases. Then 187 node-negative T1 to T4 patients were selected to validate the predictive value of this CA19-9 threshold. RESULTS: Patients with micrometastases had significantly higher preoperative CA19-9 serum levels compared to patients without micrometastases (p = 0.046). CA19-9 serum levels were significantly correlated with tumour site, tumour diameter, and perineural invasion. Although not reaching significance, subgroup analysis showed better five-year survival rates for patients with CA19-9 serum levels below the threshold, compared to patients with CA19-9 serum levels above the cut-off. The cumulative survival for T2 to T4 node-negative patients was significantly better with CA19-9 serum levels below the cut-off (p = 0.04). CONCLUSIONS: Preoperative CA19-9 serum levels can be used to predict higher risk for haematogenous spread and micrometastases in node-negative patients. However, CA19-9 serum levels lack the necessary sensitivity and specificity to reliably predict micrometastases.

Electrotransfection and lipofection show comparable efficiency for in vitro gene delivery of primary human myoblasts.

Transfection of primary human myoblasts offers the possibility to study mechanisms that are important for muscle regeneration and gene therapy of muscle disease. Cultured human myoblasts were selected here because muscle cells still proliferate at this developmental stage, which might have several advantages in gene therapy. Gene therapy is one of the most sought-after tools in modern medicine. Its progress is, however, limited due to the lack of suitable gene transfer techniques. To obtain better insight into the transfection potential of the presently used techniques, two non-viral transfection methods--lipofection and electroporation--were compared. The parameters that can influence transfection efficiency and cell viability were systematically approached and compared. Cultured myoblasts were transfected with the pEGFP-N1 plasmid either using Lipofectamine 2000 or with electroporation. Various combinations for the preparation of the lipoplexes and the electroporation media, and for the pulsing protocols, were tested and compared. Transfection efficiency and cell viability were inversely proportional for both approaches. The appropriate ratio of Lipofectamine and plasmid DNA provides optimal conditions for lipofection, while for electroporation, RPMI medium and a pulsing protocol using eight pulses of 2 ms at E = 0.8 kV/cm proved to be the optimal combination. The transfection efficiencies for the optimal lipofection and optimal electrotransfection protocols were similar (32 vs. 32.5%, respectively). Both of these methods are effective for transfection of primary human myoblasts; however, electroporation might be advantageous for in vivo application to skeletal muscle.

Dexamethasone produces dose-dependent inhibition of sugammadex reversal in in vitro innervated primary human muscle cells.

BACKGROUND: Corticosteroids are frequently used during anesthesia to provide substitution therapy in patients with adrenal insufficiency, as a first-line treatment of several life-threatening conditions, to prevent postoperative nausea and vomiting, and as a component of multimodal analgesia. For these last 2 indications, dexamethasone is most frequently used. Due to the structural resemblance between aminosteroid muscle relaxants and dexamethasone, concerns have been raised about possible corticosteroid inhibition in the reversal of neuromuscular block by sugammadex. We thus investigated the influence of dexamethasone on sugammadex reversal of rocuronium-induced neuromuscular block, which could be relevant in certain clinical situations. METHODS: The unique co-culture model of human muscle cells innervated in vitro with rat embryonic spinal cord explants to form functional neuromuscular junctions was first used to explore the effects of 4 and 10 µM rocuronium on muscle contractions, as quantitatively evaluated by counting contraction units in contraction-positive explant co-cultures. Next, equimolar and 3-fold equimolar sugammadex was used to investigate the recovery of contractions from 4 and 10 µM rocuronium block. Finally, 1, 100, and 10 µM dexamethasone (normal, elevated, and high clinical levels) were used to evaluate any effects on the reversal of rocuronium-induced neuromuscular block by sugammadex. RESULTS: Seventy-eight explant co-cultures from 3 time-independent experiments were included, where the number of contractions increased to 10 days of co-culturing. Rocuronium showed a time-dependent effect on depth of neuromuscular block (4 µM rocuronium: baseline, 10, 20 minutes administration; P < 0.0001), while the dose-dependent effect was close to nominal statistical significance (4, 10 µM; P = 0.080). This was reversed by equimolar concentrations of sugammadex, with further and virtually complete recovery of contractions with 3-fold equimolar sugammadex (P < 0.0001). Dexamethasone diminished 10 µM sugammadex-induced recovery of contractions from rocuronium-induced neuromuscular block in a dose-dependent manner (P = 0.026) with a higher sugammadex concentration (30 µM) being close to statistically significantly improving recovery (P = 0.065). The highest concentration of dexamethasone decreased the recovery of contractions by equimolar sugammadex by 26%; this effect was more pronounced when 3-fold equimolar (30 µM) sugammadex was used for reversal (48%). CONCLUSIONS: This is the first report in which the effects of rocuronium and sugammadex interactions with dexamethasone have been studied in a highly accessible in vitro experimental model of functionally innervated human muscle cells. Sugammadex reverses rocuronium-induced neuromuscular block; however, concomitant addition of high dexamethasone concentrations diminishes the efficiency of sugammadex. Further studies are required to determine the clinical relevance of these interactions.

Intraneural and perineural inflammatory changes in piglets after injection of ultrasound gel, endotoxin, 0.9% NaCl, or needle insertion without injection.

BACKGROUND: Ultrasound gel nerve inflammation has been reported. We evaluated the extent and nature of inflammation after gel injection with endotoxin (positive), saline, or dry needle puncture (negative) controls after peripheral blocks in piglets. METHODS: Selected nerves of 12 piglets were localized by landmarks and nerve stimulator. Forty-eight hours after injection, specimens were examined for immunohistochemical cell differentiation/quantification and cytokine expression by using quantitative polymerase chain reaction. RESULTS: Both gel and endotoxin injections resulted in a significantly higher density of inflammatory cells (lymphocytes/granulocytes) as compared with needle insertions and/or saline injections (both P < 0.001). Cytokines were not detected in any of the specimens. CONCLUSIONS: Perineural gel injections cause significant inflammation. The lack of cytokines suggests injectate-related changes rather than mechanical trauma.

Cytokine response of cultured skeletal muscle cells stimulated with proinflammatory factors depends on differentiation stage.

Myoblast proliferation and myotube formation are critical early events in skeletal muscle regeneration. The attending inflammation and cytokine signaling are involved in regulation of skeletal muscle cell proliferation and differentiation. Secretion of muscle-derived cytokines upon exposure to inflammatory factors may depend on the differentiation stage of regenerating muscle cells. Cultured human myoblasts and myotubes were exposed to 24-hour treatment with tumor necrosis factor (TNF)- α or lipopolysaccharide (LPS). Secretion of interleukin 6 (IL-6), a major muscle-derived cytokine, and interleukin 1 (IL-1), an important regulator of inflammatory response, was measured 24 hours after termination of TNF- α or LPS treatment. Myoblasts pretreated with TNF- α or LPS displayed robustly increased IL-6 secretion during the 24-hour period after removal of treatments, while IL-1 secretion remained unaltered. IL-6 secretion was also increased in myotubes, but the response was less pronounced compared with myoblasts. In contrast to myoblasts, IL-1 secretion was markedly stimulated in LPS-pretreated myotubes. We demonstrate that preceding exposure to inflammatory factors stimulates a prolonged upregulation of muscle-derived IL-6 and/or IL-1 in cultured skeletal muscle cells. Our findings also indicate that cytokine response to inflammatory factors in regenerating skeletal muscle partially depends on the differentiation stage of myogenic cells.

Effect of ionizing radiation on human skeletal muscle precursor cells.

BACKGROUND: Long term effects of different doses of ionizing radiation on human skeletal muscle myoblast proliferation, cytokine signalling and stress response capacity were studied in primary cell cultures. MATERIALS AND METHODS: Human skeletal muscle myoblasts obtained from muscle biopsies were cultured and irradiated with a Darpac 2000 X-ray unit at doses of 4, 6 and 8 Gy. Acute effects of radiation were studied by interleukin - 6 (IL-6) release and stress response detected by the heat shock protein (HSP) level, while long term effects were followed by proliferation capacity and cell death. RESULTS: Compared with non-irradiated control and cells treated with inhibitor of cell proliferation Ara C, myoblast proliferation decreased 72 h post-irradiation, this effect was more pronounced with increasing doses. Post-irradiation myoblast survival determined by measurement of released LDH enzyme activity revealed increased activity after exposure to irradiation. The acute response of myoblasts to lower doses of irradiation (4 and 6 Gy) was decreased secretion of constitutive IL-6. Higher doses of irradiation triggered a stress response in myoblasts, determined by increased levels of stress markers (HSPs 27 and 70). CONCLUSIONS: Our results show that myoblasts are sensitive to irradiation in terms of their proliferation capacity and capacity to secret IL-6. Since myoblast proliferation and differentiation are a key stage in muscle regeneration, this effect of irradiation needs to be taken in account, particularly in certain clinical conditions.

Insulin, dibutyryl-cAMP, and glucose modulate expression of patatin-like domain containing protein 7 in cultured human myotubes.

Expression of patatin-like phospholipase domain containing protein 7 (PNPLA7), also known as neuropathy target esterase-related esterase (NRE), a lysophospholipase, increases with fasting and decreases with feeding in mouse skeletal muscle, indicating it is regulated by insulin, counterregulatory hormones, such as glucocorticoids and catecholamines, and/or nutrients. In cultured mouse adipocytes insulin reduces Pnpla7 expression, underscoring the possibility that insulin regulates PNPLA7 in skeletal muscle. The first aim of this study was to establish whether PNPLA7 is functionally expressed in cultured human skeletal muscle cells. The second aim was to determine whether PNPLA7 is regulated by insulin, glucocorticoids, cAMP/protein kinase A pathway, and/or glucose. Cultured human skeletal muscle cells expressed PNPLA7 mRNA and protein. Gene silencing of PNPLA7 in myoblasts reduced the phosphorylation of 70 kDa ribosomal protein S6 kinase and ribosomal protein S6 as well as the abundance of α1-subunit of Na+,K+-ATPase and acetyl-CoA carboxylase, indirectly suggesting that PNPLA7 is functionally important. In myotubes, insulin suppressed PNPLA7 mRNA at 1 g/L glucose, but not at low (0.5 g/L) or high (4.5 g/L) concentrations. Treatment with synthetic glucocorticoid dexamethasone and activator of adenylyl cyclase forskolin had no effect on PNPLA7 regardless of glucose concentration, while dibutyryl-cAMP, a cell-permeable cAMP analogue, suppressed PNPLA7 mRNA at 4.5 g/L glucose. The abundance of PNPLA7 protein correlated inversely with the glucose concentrations. Collectively, our results highlight that PNPLA7 in human myotubes is regulated by metabolic signals, implicating a role for PNPLA7 in skeletal muscle energy metabolism.

Total plasma sulfide in congestive heart failure.

BACKGROUND: Hydrogen sulfide (H(2)S) has emerged as a third gaseous transmitter in mammals. In animal models of heart failure, treatment with an H(2)S donor can protect the heart against adverse remodeling and attenuate cardiac dysfunction. The aim was to determine total plasma sulfide in patients with congestive heart failure. METHODS AND RESULTS: Total plasma sulfide was determined in 57 patients on admission to an outpatient clinic or cardiology department. Total plasma sulfide concentrations in these patients was lower compared with a control group (5.32 [2.22, 8.00] µM vs. 8.5 [6.00, 14.00] µM; P = .05). Total plasma sulfide decreased significantly across the New York Heart Association (NYHA) functional classes (II, 5.84 [4.33, 8.00] µM vs. III, 4.67 [4.00, 7.17] µM vs. IV, 2.67 [2.22, 4.31] µM; P = .001). The total plasma sulfide negatively correlated with pro-BNP (R(2) cubic, 0.692; P = .001) and pulmonary artery systolic pressure (R(2) cubic, 0.569; P = .001). The receiver operating characteristic analysis of the area under the curve for total plasma sulfide as a predictor of mortality was 0.904 (95% CI, 0.822-0.987; P = .001), and of rehospitalization was 0.779 (95% CI, 0.650-0.908; P = .001). Total plasma sulfide was a univariate predictor of mortality (odds ratio, 0.245; 95% CI, 0.108-0.555; P = .001). CONCLUSION: Total plasma sulfide is negatively related to severity of congestive heart failure: it is lowest in NYHA Class IV and in patients with high pro-BNP and high pulmonary artery pressure. Low total plasma sulfide predicts a higher mortality rate.

The Influence of a Single Intra-Articular Lidocaine Injection on the Viability of Articular Cartilage in the Knee.

OBJECTIVE: To evaluate the in vivo effect of a single intra-articular injection of local anesthetic (LA) lidocaine on the viability of articular cartilage in the intact or osteoarthritic (OA) human knees, and to measure the synovial postinjection concentration of lidocaine in the knee. DESIGN: This study includes 3 interconnected experiments: (A) Synovial LA concentration measurement after a 2% lidocaine injection before knee arthroscopy in 10 patients by liquid chromatography-tandem mass spectrometry (LC-MS/MS). (B) Human osteochondral explants (N = 27) from intact knees procured at autopsies were incubated for different time intervals (30 minutes, 2 hours, 24 hours) with 2% lidocaine, 0.04% lidocaine (measured), or culture medium (control), and later evaluated for cell viability by LIVE/DEAD staining. (C) Ten out of 19 matched patients scheduled for knee replacement received a single intra-articular injection of 2% lidocaine approximately 30 minutes prior to the procedure; 9 patients served as control. Osteochondral samples with OA changes were harvested during surgery and analyzed for chondrocyte viability by LIVE/DEAD staining. RESULTS: (A) The synovial LA concentration was significantly lower than the primary concentration injected: average 0.23 mg/mL (0.02%), highest measured 0.37 mg/mL (0.04%). (B) In vitro exposure to a reduced LA concentration had no significant influence on chondrocyte viability in intact cartilage explants (24-hour averages: control, 93%; 0.04% lidocaine, 92%; 2% lidocaine, 79%). (C) Viability of chondrocytes in OA knees was similar between 2% lidocaine injection (85%) and control (80%). CONCLUSIONS: A single intra-articular knee injection of 2% lidocaine did not influence the chondrocyte viability neither in healthy nor in OA cartilage. A fast postinjection reduction of synovial LA concentration (more than 40 times) is the most likely protective mechanism.

Acute Cartilage Injury Induced by Trans-Articular Sutures.

OBJECTIVE: To determine the extent of acute cartilage injury by using trans-articular sutures. METHODS: Five different absorbable sutures, monofilament polydioxanone (PDS) and braided polyglactin (Vicryl), were compared on viable human osteochondral explants. An atraumatic needle with 30 cm of thread was advanced through the cartilage with the final thread left in the tissue. A representative 300 µm transversal slice from the cartilage midportion was stained with Live/Dead probes, scanned under the confocal laser microscope, and analyzed for the diameters of (a) central "Black zone" without any cells, representing in situ thread thickness and (b) "Green zone," including the closest Live cells, representing the maximum injury to the tissue. The exact diameters of suture needles and threads were separately measured under an optical microscope. RESULTS: The diameters of the Black (from 144 to 219 µm) and the Green zones (from 282 to 487 µm) varied between the different sutures (P < 0.001). The Green/Black zone ratio remained relatively constant (from 1.9 to 2.2; P = 0.767). A positive correlation between thread diameters and PDS suturing material, toward the Black and Green zone, was established, but needle diameters did not reveal any influence on the zones. CONCLUSIONS: The width of acute cartilage injury induced by the trans-articular sutures is about twice the thread thickness inside of the tissue. Less compressible monofilament PDS induced wider tissue injury in comparison to a softer braided Vicryl. Needle diameter did not correlate to the extent of acute cartilage injury.

Effect of differentiation, de novo innervation, and electrical pulse stimulation on mRNA and protein expression of Na+,K+-ATPase, FXYD1, and FXYD5 in cultured human skeletal muscle cells.

Denervation reduces the abundance of Na+,K+-ATPase (NKA) in skeletal muscle, while reinnervation increases it. Primary human skeletal muscle cells, the most widely used model to study human skeletal muscle in vitro, are usually cultured as myoblasts or myotubes without neurons and typically do not contract spontaneously, which might affect their ability to express and regulate NKA. We determined how differentiation, de novo innervation, and electrical pulse stimulation affect expression of NKA (α and ß) subunits and NKA regulators FXYD1 (phospholemman) and FXYD5 (dysadherin). Differentiation of myoblasts into myotubes under low serum conditions increased expression of myogenic markers CD56 (NCAM1), desmin, myosin heavy chains, dihydropyridine receptor subunit α1S, and SERCA2 as well as NKAα2 and FXYD1, while it decreased expression of FXYD5 mRNA. Myotubes, which were innervated de novo by motor neurons in co-culture with the embryonic rat spinal cord explants, started to contract spontaneously within 7-10 days. A short-term co-culture (10-11 days) promoted mRNA expression of myokines, such as IL-6, IL-7, IL-8, and IL-15, but did not affect mRNA expression of NKA, FXYDs, or myokines, such as musclin, cathepsin B, meteorin-like protein, or SPARC. A long-term co-culture (21 days) increased the protein abundance of NKAα1, NKAα2, FXYD1, and phospho-FXYD1Ser68 without attendant changes in mRNA levels. Suppression of neuromuscular transmission with α-bungarotoxin or tubocurarine for 24 h did not alter NKA or FXYD mRNA expression. Electrical pulse stimulation (48 h) of non-innervated myotubes promoted mRNA expression of NKAß2, NKAß3, FXYD1, and FXYD5. In conclusion, low serum concentration promotes NKAα2 and FXYD1 expression, while de novo innervation is not essential for upregulation of NKAα2 and FXYD1 mRNA in cultured myotubes. Finally, although innervation and EPS both stimulate contractions of myotubes, they exert distinct effects on the expression of NKA and FXYDs.

Innervation and electrical pulse stimulation - in vitro effects on human skeletal muscle cells.

Contraction-induced adaptations in skeletal muscles are well characterized in vivo, but the underlying cellular mechanisms are still not completely understood. Cultured human myotubes represent an essential model system for human skeletal muscle that can be modulated ex vivo, but they are quiescent and do not contract unless being stimulated. Stimulation can be achieved by innervation of human myotubes in vitro by co-culturing with embryonic rat spinal cord, or by replacing motor neuron activation by electrical pulse stimulation (EPS). Effects of these two in vitro approaches, innervation and EPS, were characterized with respects to the expression of myosin heavy chains (MyHCs) and metabolism of glucose and oleic acid in cultured human myotubes. Adherent human myotubes were either innervated with rat spinal cord segments or exposed to EPS. The expression pattern of MyHCs was assessed by quantitative polymerase chain reaction, immunoblotting, and immunofluorescence, while the metabolism of glucose and oleic acid were studied using radiolabelled substrates. Innervation and EPS promoted differentiation towards different fiber types in human myotubes. Expression of the slow MyHC-1 isoform was reduced in innervated myotubes, whereas it remained unaltered in EPS-treated cells. Expression of both fast isoforms (MyHC-2A and MyHC-2X) tended to decrease in EPS-treated cells. Both approaches induced a more oxidative phenotype, reflected in increased CO2 production from both glucose and oleic acid. Novelty: Innervation and EPS favour differentiation into different fiber types in human myotubes. Both innervation and EPS promote a metabolically more oxidative phenotype in human myotubes.

HIF-1alpha response to hypoxia is functionally separated from the glucocorticoid stress response in the in vitro regenerating human skeletal muscle.

Injury of skeletal muscle is followed by muscle regeneration in which new muscle tissue is formed from the proliferating mononuclear myoblasts, and by systemic response to stress that exposes proliferating myoblasts to increased glucocorticoid (GC) concentration. Because of its various causes, hypoxia is a frequent condition affecting skeletal muscle, and therefore both processes, which importantly determine the outcome of the injury, often proceed under hypoxic conditions. It is therefore important to identify and characterize in proliferating human myoblasts: 1) response to hypoxia which is generally organized by hypoxia-inducible factor-1α (HIF-1α); 2) response to GCs which is mediated through the isoforms of glucocorticoid receptors (GRs) and 11ß-hydroxysteroid dehydrogenases (11ß-HSDs), and 3) the response to GCs under the hypoxic conditions and the influence of this combination on the factors controlling myoblast proliferation. Using real-time PCR, Western blotting, and HIF-1α small-interfering RNA silencing, we demonstrated that cultured human myoblasts possess both, the HIF-1α-based response to hypoxia, and the GC response system composed of GRα and types 1 and 2 11ß-HSDs. However, using combined dexamethasone and hypoxia treatments, we demonstrated that these two systems operate practically without mutual interactions. A seemingly surprising separation of the two systems that both organize response to hypoxic stress can be explained on the evolutionary basis: the phylogenetically older HIF-1α response is a protection at the cellular level, whereas the GC stress response protects the organism as a whole. This necessitates actions, like downregulation of IL-6 secretion and vascular endothelial growth factor, that might not be of direct benefit for the affected myoblasts.

Echocardiography of isolated subacute left heart tamponade in a patient with cor pulmonale and circumferential pericardial effusion.

Patients with advanced idiopathic pulmonary artery hypertension have often a chronic pericardial effusion. It is the result of increased transudation and impaired re-absorption due to elevated venous pressure. These patients have pre-existent symptoms and signs of chronic right heart failure. High degree of suspicion is required to detect of development of an atypical form of tamponade with isolated compression of left heart chambers as shown in present case report. Transthoracic echocardiography provides a rapid access to the correct diagnosis, a prompt relief of symptoms following the ultrasound guided pericardiocentesis and important diagnostic tool for regular follow up of patients thereafter as shown in our case report.