| Author/Editor | Jan, Vid; Miš, Katarina; Nikolić, Nataša; Dolinar, Klemen; Petrič, Metka; Bone, Andraž; Thoresen, G. Hege; Rustan, Arild C.; Marš, Tomaž; Chibalin, Alexander V.; Pirkmajer, Sergej | |
| Title | 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 | |
| Type | članek | |
| Vol. and No. | Letnik 16, št. 2 | |
| Publication year | 2021 | |
| Volume | str. 1-24 | |
| ISSN | 1932-6203 - PloS one | |
| Language | eng | |
| Abstract | 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 ([alpha] and [beta]) 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 [alpha]1S, and SERCA2 as well as NKA[alpha]2 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 NKA[alpha]1, NKA[alpha]2, FXYD1, and phospho-FXYD1Ser68 without attendant changes in mRNA levels. Suppression of neuromuscular transmission with [alpha]-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 NKA[beta]2, NKA[beta]3, FXYD1, and FXYD5. In conclusion, low serum concentration promotes NKA[alpha]2 and FXYD1 expression, while de novo innervation is not essential for upregulation of NKA[alpha]2 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. | |
| Keywords | skeletal muscle electrical pulse stimulation diferenciacija mioblastov skeletne mišice stimulacije z električnim pulzom diferenciacija mioblastov |