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

Jan, Vid; Miš, Katarina; Nikolić, Nataša; Dolinar, Klemen; Petrič, Miha; Bone, Andraž; Thoresen, G. Hege; Rustan, Arild C.; Marš, Tomaž; Chibalin, Alexander V.; Pirkmajer, Sergej

doi:10.1371/journal.pone.0247377

Cited by 10 publications

(10 citation statements)

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“…Muscle samples were collected from donors 5–77 years of age. Human skeletal muscle cell cultures were prepared as described [ 29 , 102 , 103 , 104 ]. Briefly, skeletal muscle tissue was cleaned of visible connective and adipose tissue and then cut into small (0.5–1 mm) pieces under the microscope.…”

Section: Methodsmentioning

confidence: 99%

“…Advanced MEM, supplemented with 10% ( v / v ) FBS, 0.3% ( v / v ) Fungizone and 0.15% ( v / v ) gentamicin was used as the growth medium (GM). Before reaching confluence, primary cultures were purified using MACS CD56 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) in order to separate the myoblasts from other cell types contaminating the primary culture [ 103 ]. Only cell cultures with more than 75% of desmin-positive cells (see below) were used for the experiments described here.…”

Section: Methodsmentioning

confidence: 99%

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

Gros

Matkovič

Parato

et al. 2022

IJMS

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Gros

Matkovič

Parato

et al. 2022

IJMS

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“…This implies that muscle activity and innervation regulate FXYD5 expression in human muscle fibres. However, electrical pulse stimulation of human cultured myotubes for 48 h, as a model of chronic muscle activity, upregulated FXYD5 abundance, while innervation did not affect its abundance [ 17 ]. Discrepancies between the in situ and in vitro findings may reflect that other factors also regulate FXYD5 in muscle fibres, including the metabolic and hormonal milieu.…”

Section: Discussionmentioning

confidence: 99%

“…FXYD5 is a dysadherin-related single-pass type I membrane glycoprotein that regulates NKA. While mainly expressed in epithelial tissues and markedly upregulated in carcinomas [ 15 , 16 ], FXYD5 is also expressed in skeletal muscle [ 15 , 16 , 17 , 18 ]. However, not much is known about FXYD5 in human skeletal muscle.…”

Section: Introductionmentioning

confidence: 99%

High-Intensity Training Represses FXYD5 and Glycosylates Na,K-ATPase in Type II Muscle Fibres, Which Are Linked with Improved Muscle K+ Handling and Performance

Hostrup

Lemminger

Thomsen

et al. 2023

IJMS

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Na+/K+ ATPase (NKA) comprises several subunits to provide isozyme heterogeneity in a tissue-specific manner. An abundance of NKA α, β, and FXYD1 subunits is well-described in human skeletal muscle, but not much is known about FXYD5 (dysadherin), a regulator of NKA and β1 subunit glycosylation, especially with regard to fibre-type specificity and influence of sex and exercise training. Here, we investigated muscle fibre-type specific adaptations in FXYD5 and glycosylated NKAβ1 to high-intensity interval training (HIIT), as well as sex differences in FXYD5 abundance. In nine young males (23.8 ± 2.5 years of age) (mean ± SD), 3 weekly sessions of HIIT for 6 weeks enhanced muscle endurance (220 ± 102 vs. 119 ± 99 s, p < 0.01) and lowered leg K+ release during intense knee-extensor exercise (0.5 ± 0.8 vs. 1.0 ± 0.8 mmol·min–1, p < 0.01) while also increasing cumulated leg K+ reuptake 0–3 min into recovery (2.1 ± 1.5 vs. 0.3 ± 0.9 mmol, p < 0.01). In type IIa muscle fibres, HIIT lowered FXYD5 abundance (p < 0.01) and increased the relative distribution of glycosylated NKAβ1 (p < 0.05). FXYD5 abundance in type IIa muscle fibres correlated inversely with the maximal oxygen consumption (r = –0.53, p < 0.05). NKAα2 and β1 subunit abundances did not change with HIIT. In muscle fibres from 30 trained males and females, we observed no sex (p = 0.87) or fibre type differences (p = 0.44) in FXYD5 abundance. Thus, HIIT downregulates FXYD5 and increases the distribution of glycosylated NKAβ1 in type IIa muscle fibres, which is likely independent of a change in the number of NKA complexes. These adaptations may contribute to counter exercise-related K+ shifts and enhance muscle performance during intense exercise.

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“…Furthermore, SERCA1 and SERCA2 isoforms are expressed in fast-twitch and slow-twitch skeletal muscle respectively, whereas SERCA3 isoforms are mainly expressed in non-muscle tissues such as hematopoietic cell lines and epithelial cells ( Periasamy and Kalyanasundaram, 2007 ). In cultured human myotubes, we have recently reported the expression of SERCA1 and SERCA2 ( Jan et al., 2021 ).…”

Section: Introductionmentioning

confidence: 99%

The small molecule SERCA activator CDN1163 increases energy metabolism in human skeletal muscle cells

Mengeste

Lund

Katare

et al. 2021

Current Research in Pharmacology and Drug Discovery

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Background and objective A number of studies have highlighted muscle-specific mechanisms of thermogenesis involving futile cycling of Ca 2+ driven by sarco (endo)plasmic reticulum Ca 2+ -ATPase (SERCA) and generating heat from ATP hydrolysis to be a promising strategy to counteract obesity and metabolic dysfunction. However, to the best of our knowledge, no experimental studies concerning the metabolic effects of pharmacologically targeting SERCA in human skeletal muscle cells have been reported. Thus, in the present study, we aimed to explore the effects of SERCA-activating compound, CDN1163, on energy metabolism in differentiated human skeletal muscle cells (myotubes). Methods In this study, we used primary myotube cultures derived from muscle biopsies of the musculus vastus lateralis and musculi interspinales from lean, healthy male donors. Energy metabolism in myotubes was studied using radioactive substrates. Oxygen consumption rate was assessed with the Seahorse XF24 bioanalyzer, whereas metabolic genes and protein expressions were determined by qPCR and immunoblotting, respectively. Results Both acute (4 h) and chronic (5 days) treatment of myotubes with CDN1163 showed increased uptake and oxidation of glucose, as well as complete fatty acid oxidation in the presence of carbonyl cyanide 4-(trifluromethoxy)phenylhydrazone (FCCP). These effects were supported by measurement of oxygen consumption rate, in which the oxidative spare capacity and maximal respiration were enhanced after CDN1163-treatment. In addition, chronic treatment with CDN1163 improved cellular uptake of oleic acid (OA) and fatty acid β-oxidation. The increased OA metabolism was accompanied by enhanced mRNA-expression of carnitine palmitoyl transferase ( CPT ) 1B , pyruvate dehydrogenase kinase ( PDK ) 4, as well as increased AMP-activated protein kinase (AMPK) Thr172 phosphorylation. Moreover, following chronic CDN1163 treatment, the expression levels of stearoyl-CoA desaturase ( SCD ) 1 was decreased together with de novo lipogenesis from acetic acid and formation of diacylglycerol (DAG) from OA. Conclusion Altogether, these results suggest that SERCA activation by CDN1163 enhances energy metabolism in human myotubes, which might be favourable in relation to disorders that are related to metabolic dysfunction such as obesity and type 2 diabetes mellitus.

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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

Cited by 10 publications

References 86 publications

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

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

High-Intensity Training Represses FXYD5 and Glycosylates Na,K-ATPase in Type II Muscle Fibres, Which Are Linked with Improved Muscle K+ Handling and Performance

The small molecule SERCA activator CDN1163 increases energy metabolism in human skeletal muscle cells

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