Transcriptomic changes in C2C12 myotubes triggered by electrical stimulation: Role of Ca²⁺_i-mediated and Ca²⁺_i-independent signaling and elevated [Na⁺]_i/[K⁺]_i ratio

Elevation of Ca²⁺_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 Ca²⁺_i-mediated and Ca²⁺_i-independent signaling in transcriptomic changes in EPS-treated C2C12 myotubes. Electrical pulse stimulation (40 V, 1 Hz, 10 ms, 2 h) resulted in [Ca²⁺]_i oscillations, gain of Na⁺_i, loss of K⁺_i, and differential expression of 3215 transcripts. Additions of 10 μM nicardipine abolished [Ca²⁺]_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 Ca²⁺_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 Ca²⁺_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.