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.
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
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
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
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
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.
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 β) 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 NKAβ2, NKAβ3, 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.
The cardiotonic steroids (CTS), such as ouabain and marinobufagenin, are thought to be adrenocortical hormones secreted during exercise and the stress response. The catalytic α-subunit of Na,K-ATPase ...(NKA) is a CTS receptor, whose largest pool is located in skeletal muscles, indicating that muscles are a major target for CTS. Skeletal muscles contribute to adaptations to exercise by secreting interleukin-6 (IL-6) and plethora of other cytokines, which exert paracrine and endocrine effects in muscles and non-muscle tissues. Here, we determined whether ouabain, a prototypical CTS, modulates IL-6 signaling and secretion in the cultured human skeletal muscle cells. Ouabain (2.5–50 nM) suppressed the abundance of STAT3, a key transcription factor downstream of the IL-6 receptor, as well as its basal and IL-6-stimulated phosphorylation. Conversely, ouabain (50 nM) increased the phosphorylation of ERK1/2, Akt, p70S6K, and S6 ribosomal protein, indicating activation of the ERK1/2 and the Akt-mTOR pathways. Proteasome inhibitor MG-132 blocked the ouabain-induced suppression of the total STAT3, but did not prevent the dephosphorylation of STAT3. Ouabain (50 nM) suppressed hypoxia-inducible factor-1α (HIF-1α), a modulator of STAT3 signaling, but gene silencing of HIF-1α and/or its partner protein HIF-1β did not mimic effects of ouabain on the phosphorylation of STAT3. Ouabain (50 nM) failed to suppress the phosphorylation of STAT3 and HIF-1α in rat L6 skeletal muscle cells, which express the ouabain-resistant α1-subunit of NKA. We also found that ouabain (100 nM) promoted the secretion of IL-6, IL-8, GM-CSF, and TNF-α from the skeletal muscle cells of healthy subjects, and the secretion of GM-CSF from cells of subjects with the type 2 diabetes. Marinobufagenin (10 nM), another important CTS, did not alter the secretion of these cytokines. In conclusion, our study shows that ouabain suppresses the IL-6 signaling via STAT3, but promotes the secretion of IL-6 and other cytokines, which might represent a negative feedback in the IL-6/STAT3 pathway. Collectively, our results implicate a role for CTS and NKA in regulation of the IL-6 signaling and secretion in skeletal muscle.
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.
Total Plasma Sulfide in Congestive Heart Failure Kovačić, Dragan, MD, PhD; Glavnik, Nina, MD; Marinšek, Matej, MD ...
Journal of cardiac failure,
07/2012, Letnik:
18, Številka:
7
Journal Article
Recenzirano
Abstract Background Hydrogen sulfide (H2 S) has emerged as a third gaseous transmitter in mammals. In animal models of heart failure, treatment with an H2 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 (R2 cubic, 0.692; P = .001) and pulmonary artery systolic pressure (R2 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.
Abstract Sprouting of uninjured nociceptive axons was examined in young adult, middle aged and aged rats. Axon sprouting from the spared sural nerve, both into adjacent denervated skin and into ...end-to-side coapted nerve graft, was significantly higher in young rats than in aged rats. Cross-transplantations of the end-to-side coapted nerve grafts between young and aged rats demonstrated that axon sprouting from young recipient nerves into aged donor nerve grafts was significantly deteriorated, whereas the axon sprouting from aged recipient nerves into young donor nerve grafts was not statistically significantly affected. The levels of laminin polypeptides in peripheral nerves were 50–100% higher in young adult than in aged rats. However, the levels of peripherin, NGF isoforms and TrkA in skin, peripheral nerves and DRG, respectively, were not significantly reduced in aged rats. Therefore, impaired sprouting of nociceptive axons in aged rats is due rather to the alterations in peripheral neural pathways, than to the limited sprouting capacity of aged sensory neurons. Decreased levels of extracellular matrix components might be important in this respect.
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.
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 CO
2
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.
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