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.

Vid Jan,Natasa Nikolic,Arild C Rustan,Alexander V Chibalin,Metka Petrič,Sergej Pirkmajer,G Hege Thoresen,Katarina Miš,Andraž Bone,Klemen Dolinar,Tomaž Marš,Luis Eduardo M Quintas

doi:10.1371/journal.pone.0247377

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 (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.

Highlights

Skeletal muscle fibers are excited to contract by the α-motor neurons via the neuromuscular junctions
We show that innervation or electrical pulse stimulation (EPS) of cultured myotubes did not lead to major changes in expression pattern of NKA subunits, FXYD1, and FXYD5
We show that low serum promoted upregulation of FXYD1 mRNA and downregulation of FXYD5 mRNA changes in gene expression were not always paralleled with significant changes in the protein abundance

Summary

Introduction

Skeletal muscle fibers are excited to contract by the α-motor neurons via the neuromuscular junctions. Capacity of skeletal muscle to perform ion transport depends on the total NKA content (reviewed in [5, 6]), which is reduced by denervation [7]. Reinnervation increases NKA content [8], but whether innervation exerts differential effects on NKA isozymes and the regulatory FXYD proteins is not known. NKAα1 is the most abundant α-isoform in immature skeletal muscle, but NKAα2 is upregulated during postnatal myogenesis [12] and represents 60–90% of α-subunits (reviewed in [5]). NKAα2 is thought to be important during contractions, while NKAα1 is supposed to primarily maintain ion transport at rest [13], it may participate in the process of muscle hypertrophy [14]. Whether innervation differentially regulates expression of the NKAα subunits has not been established

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