Ouabain Suppresses IL-6/STAT3 Signaling and Promotes Cytokine Secretion in Cultured Skeletal Muscle Cells

Pirkmajer, Sergej; Bezjak, Katja; Matkovič, Urška; Dolinar, Klemen; Jiang, Lake Q.; Miš, Katarina; Gros, Katarina; Milovanova, Kseniya; Pirkmajer, Katja Perdan; Marš, Tomaž; Капилевич, Л. В.; Chibalin, Alexander V.

doi:10.3389/fphys.2020.566584

Cited by 20 publications

(30 citation statements)

References 145 publications

(240 reference statements)

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“…As assessed by mRNA measurements, human skeletal muscle expresses primarily NKAα2 (~87% of NKAα transcripts) and NKAα1 (~13% of NKAα transcripts) [23], consistent with data from rat muscles, which indicated that NKAα2 represents~60-90% of NKAα subunits [12,[70][71][72][73]. NKAα2 was robustly expressed in differentiated CD56 + myotubes (~48% of NKAα transcripts), but NKAα1 remained the predominant NKAα isoform (>50% of NKAα transcripts) in cells from most donors (Fig 8), consistent with observations in our recent study [24]. Innervation or EPS did not further increase NKAα2 mRNA, indicating neither formation of functional neuromuscular junctions nor contractions are essential for regulation of NKAα2 expression.…”

Section: Plos Onesupporting

confidence: 81%

“…Primary human skeletal muscle cells for differentiation and co-culture (innervation) experiments were prepared from samples of the semitendinosus muscles, which were obtained during routine orthopaedic surgery as surgical waste, and cultured as described [24,30,32]. All donors of skeletal muscle tissue required surgery for medical reasons and no tissue was removed solely for the purpose of research.…”

Section: Culturing and Differentiation Of Human Skeletal Muscle Cellsmentioning

confidence: 99%

“…Differentiated primary human skeletal muscle cells (myotubes), the most widely used model to study human skeletal muscle in vitro [21,22], are usually cultured without neurons. Unlike in human skeletal muscle tissue, in which NKAα2 is by far the most prominently expressed NKAα isoform [23], NKAα1 predominates in aneurally cultured myotubes [24]. Innervation can be established de novo by co-culturing myotubes with explants of the embryonic rat spinal cord [25], which leads to formation of functional neuromuscular junctions, maturation of contractile apparatus, and spontaneous contractions of myotubes ( [25], reviewed in [26]).…”

Section: Introductionmentioning

confidence: 99%

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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

et al. 2021

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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.

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Section: Plos Onesupporting

confidence: 81%

Section: Culturing and Differentiation Of Human Skeletal Muscle Cellsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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

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“…Notably, although ouabain is proposed to be a specific inhibitor of the Na,K-ATPase, its ability to activate the Na,K-ATPase at concentrations comparable to its endogenous level [ 33 , 34 , 35 ] and thus, to hyperpolarize the sarcolemma [ 36 ] was also shown. While the ability of nanomolar ouabain to activate intracellular signaling in cultured human skeletal muscle cells is well-documented [ 37 , 48 ], little is known about its role in the regulation of skeletal muscle function in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…However, at subnanomolar concentrations that correspond to the level of circulating endogenous analog, ouabain is shown to activate the Na,K-ATPase [ 33 , 34 , 35 ] and to hyperpolarize the sarcolemma [ 36 ]. Moreover, it has been shown that nanomolar concentrations of ouabain modulate interleikin-6 (IL-6) secretion and IL-6/STAT3 signaling pathways [ 37 ].…”

Section: Introductionmentioning

confidence: 99%

Chronic Ouabain Prevents Na,K-ATPase Dysfunction and Targets AMPK and IL-6 in Disused Rat Soleus Muscle

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Vilchinskaya

et al. 2021

IJMS

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Sustained sarcolemma depolarization due to loss of the Na,K-ATPase function is characteristic for skeletal muscle motor dysfunction. Ouabain, a specific ligand of the Na,K-ATPase, has a circulating endogenous analogue. We hypothesized that the Na,K-ATPase targeted by the elevated level of circulating ouabain modulates skeletal muscle electrogenesis and prevents its disuse-induced disturbances. Isolated soleus muscles from rats intraperitoneally injected with ouabain alone or subsequently exposed to muscle disuse by 6-h hindlimb suspension (HS) were studied. Conventional electrophysiology, Western blotting, and confocal microscopy with cytochemistry were used. Acutely applied 10 nM ouabain hyperpolarized the membrane. However, a single injection of ouabain (1 µg/kg) prior HS was unable to prevent the HS-induced membrane depolarization. Chronic administration of ouabain for four days did not change the α1 and α2 Na,K-ATPase protein content, however it partially prevented the HS-induced loss of the Na,K-ATPase electrogenic activity and sarcolemma depolarization. These changes were associated with increased phosphorylation levels of AMP-activated protein kinase (AMPK), its substrate acetyl-CoA carboxylase and p70 protein, accompanied with increased mRNA expression of interleikin-6 (IL-6) and IL-6 receptor. Considering the role of AMPK in regulation of the Na,K-ATPase, we suggest an IL-6/AMPK contribution to prevent the effects of chronic ouabain under skeletal muscle disuse.

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Activation of (un)regulated cell death as a new perspective for bispyridinium and imidazolium oximes

et al. 2021

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Ouabain Suppresses IL-6/STAT3 Signaling and Promotes Cytokine Secretion in Cultured Skeletal Muscle Cells

Abstract: Pirkmajer et al. Ouabain Modulates Myocyte IL-6/STAT3 Signaling 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.

Cited by 20 publications

References 145 publications

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

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

Chronic Ouabain Prevents Na,K-ATPase Dysfunction and Targets AMPK and IL-6 in Disused Rat Soleus Muscle

Activation of (un)regulated cell death as a new perspective for bispyridinium and imidazolium oximes

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