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.
Animals
Cell Differentiation
Cell Line
Cells, Cultured
Coculture Techniques
Electric Stimulation
Gene Expression Regulation
Humans
Ion Channels
/ genetics
Membrane Proteins
/ genetics
Microfilament Proteins
/ genetics
Muscle Contraction
Muscle Fibers, Skeletal
/ cytology
Muscle, Skeletal
/ cytology
Phosphoproteins
/ genetics
Rats
Sodium-Potassium-Exchanging ATPase
/ genetics
Journal
PloS one
ISSN: 1932-6203
Titre abrégé: PLoS One
Pays: United States
ID NLM: 101285081
Informations de publication
Date de publication:
2021
2021
Historique:
received:
30
05
2020
accepted:
05
02
2021
entrez:
26
2
2021
pubmed:
27
2
2021
medline:
21
8
2021
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 33635930
doi: 10.1371/journal.pone.0247377
pii: PONE-D-20-16390
pmc: PMC7909653
doi:
Substances chimiques
FXYD5 protein, human
0
Ion Channels
0
Membrane Proteins
0
Microfilament Proteins
0
Phosphoproteins
0
phospholemman
135541-82-1
Sodium-Potassium-Exchanging ATPase
EC 7.2.2.13
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e0247377Déclaration de conflit d'intérêts
The authors have declared that no competing interests exist.
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