TY - JOUR
T1 - Transcriptomic changes in C2C12 myotubes triggered by electrical stimulation
T2 - Role of Ca 2+ i -mediated and Ca 2+ i -independent signaling and elevated [Na + ] i /[K + ] i ratio
AU - Sidorenko, Svetlana
AU - Klimanova, Elizaveta
AU - Milovanova, Kseniya
AU - Lopina, Olga D.
AU - Kapilevich, Leonid V.
AU - Chibalin, Alexander V.
AU - Orlov, Sergei N.
N1 - Funding Information:
Calculating femoral arterial-venous Na + and K + concentration differences, it was found that in humans intense knee extensor exercise rapidly increased intracellular Na + concentration ([Na + ] i ) in skeletal muscle from 13 to 23 mM and reduced [K + ] i from 162 to 129 mM [ 39 ]. Using flame photometry, Murphy and coworkers found that in rat soleus muscle 60 min of intermittent running resulted in ∼2-fold elevation in [Na + ] i and attenuation of [K + ] i by 10–20% [ 40 ]. Recently, we reported that 4-h EPS of C2C12 cells resulted in ∼5-fold elevation in [Na + ] i /[K + ] i ratio [ 16 ]. Considering this, it might be proposed that along with the above-listed signaling pathways, elevation of [Na + ] i /[K + ] i ratio detected in exercising skeletal muscle and EPS-treated myotubes contributes to myokine production [ 3 ]. This hypothesis is supported by several observations. First , sustained inhibition of Na + ,K + -ATPase affects expression of hundreds of genes in the presence of extracellular and intracellular Ca 2+ chelators [ 41 ] and inhibitors of Ca 2+ i -mediated signaling pathways [ 42 ]. Second, in cultured vascular smooth muscle cells subjected to hypoxia and glucose-deprivation, transcriptomic changes are partially caused by activation of [Na + ] i /[K + ] i -mediated, HIF-1α-independent signaling [ 43 ]. The present study examines the role of dissipation of transmembrane gradient of monovalent cations in transcriptomic changes triggered by EPS in C2C12 myotubes.
Funding Information:
This work was supported by a grant from the Russian Scientific Foundation RNF #16-15-10026 (for SNO) and by the Swedish Research Council , Novo Nordisk Foundation , and Strategic Research Program in Diabetes at Karolinska Institutet (for AVC).
Publisher Copyright:
© 2018 Elsevier Ltd
Copyright:
Copyright 2019 Elsevier B.V., All rights reserved.
PY - 2018/12
Y1 - 2018/12
N2 - Elevation of Ca 2+ i and AMP-activated protein kinase (AMPK) are considered as major signals triggering transcriptomic changes in exercising skeletal muscle. Electrical pulse stimulation (EPS) of cultured myotubes is widely employed as an in vitro model of muscle contraction. This study examines the impact of Ca 2+ i -mediated and Ca 2+ i -independent signaling in transcriptomic changes in EPS-treated C2C12 myotubes. Electrical pulse stimulation (40 V, 1 Hz, 10 ms, 2 h) resulted in [Ca 2+ ] i oscillations, gain of Na + i , loss of K + i , and differential expression of 3215 transcripts. Additions of 10 μM nicardipine abolished [Ca 2+ ] i oscillations but did not affect elevation of the [Na + ] i /[K + ] i ratio seen in EPS-treated myotubes. Differential expression of 1018 transcripts was preserved in the presence of nicardipine, indicating a Ca 2+ i -independent mechanism of excitation–transcription coupling. Among nicardipine-resistant transcripts, we noted 113 transcripts whose expression was also affected by partial Na + ,K + -ATPase inhibition with 30 μM ouabain providing the same elevation of the [Na + ] i /[K + ] i ratio as in EPS-treated cells. Electrical pulse stimulation increased phosphorylation of CREB, ATF-1, Akt, ERK, and p38 MAPK without any impact on phosphorylation of acetyl-CoA carboxylase and Unc-51 like autophagy activating kinase-1, i.e. downstream markers of AMPK activation. Unlike CREB, ATF-1, and MAPKs, an increment in Akt phosphorylation was abolished by nicardipine. Thus, our results show that Ca 2+ i -independent signaling plays a key role in altered expression of 30% of studied genes in EPS-treated myotubes. This signaling pathway is at least partially triggered by dissipation of transmembrane gradients of monovalent cations.
AB - Elevation of Ca 2+ i and AMP-activated protein kinase (AMPK) are considered as major signals triggering transcriptomic changes in exercising skeletal muscle. Electrical pulse stimulation (EPS) of cultured myotubes is widely employed as an in vitro model of muscle contraction. This study examines the impact of Ca 2+ i -mediated and Ca 2+ i -independent signaling in transcriptomic changes in EPS-treated C2C12 myotubes. Electrical pulse stimulation (40 V, 1 Hz, 10 ms, 2 h) resulted in [Ca 2+ ] i oscillations, gain of Na + i , loss of K + i , and differential expression of 3215 transcripts. Additions of 10 μM nicardipine abolished [Ca 2+ ] i oscillations but did not affect elevation of the [Na + ] i /[K + ] i ratio seen in EPS-treated myotubes. Differential expression of 1018 transcripts was preserved in the presence of nicardipine, indicating a Ca 2+ i -independent mechanism of excitation–transcription coupling. Among nicardipine-resistant transcripts, we noted 113 transcripts whose expression was also affected by partial Na + ,K + -ATPase inhibition with 30 μM ouabain providing the same elevation of the [Na + ] i /[K + ] i ratio as in EPS-treated cells. Electrical pulse stimulation increased phosphorylation of CREB, ATF-1, Akt, ERK, and p38 MAPK without any impact on phosphorylation of acetyl-CoA carboxylase and Unc-51 like autophagy activating kinase-1, i.e. downstream markers of AMPK activation. Unlike CREB, ATF-1, and MAPKs, an increment in Akt phosphorylation was abolished by nicardipine. Thus, our results show that Ca 2+ i -independent signaling plays a key role in altered expression of 30% of studied genes in EPS-treated myotubes. This signaling pathway is at least partially triggered by dissipation of transmembrane gradients of monovalent cations.
KW - C2C12 myoblasts
KW - Electrical pulse stimulation
KW - excitation–transcription coupling
KW - Intracellular Na , K and Ca
KW - Ouabain
KW - Transcriptomics
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U2 - 10.1016/j.ceca.2018.09.007
DO - 10.1016/j.ceca.2018.09.007
M3 - Article
C2 - 30300758
AN - SCOPUS:85054321430
VL - 76
SP - 72
EP - 86
JO - Cell Calcium
JF - Cell Calcium
SN - 0143-4160
ER -