NFAT activation by membrane potential follows a calcium pathway distinct from other activity-related transcription factors in skeletal muscle cells

Depolarization of skeletal muscle cells triggers intracellular Ca ²⁺ signals mediated by ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors. Previously, we have reported that K⁺-induced depolarization activates transcriptional regulators ERK, cAMP response element-binding protein, c-fos, c-jun, and egr-1 through IP₃- dependent Ca²⁺ release, whereas NF-κB activation is elicited by both ryanodine and IP₃ receptor-mediated Ca²⁺ signals. We have further shown that field stimulation with electrical pulses results in an NF-κB activation increase dependent of the amount of pulses and independent of their frequency. In this work, we report the results obtained for nuclear factor of activated T cells (NFAT)-mediated transcription and translocation generated by both K⁺ and electrical stimulation protocols in primary skeletal muscle cells and C2C12 cells. The Ca²⁺ source for NFAT activation is through release by ryanodine receptors and extracellular Ca²⁺ entry. We found this activation to be independent of the number of pulses within a physiological range of stimulus frequency and enhanced by long-lasting low-frequency stimulation. Therefore, activation of the NFAT signaling pathway differs from that of NF-κB and other transcription factors. Calcineurin enzyme activity correlated well with the relative activation of NFAT translocation and transcription using different stimulation protocols. Furthermore, both K⁺-induced depolarization and electrical stimulation increased mRNA levels of the type 1 IP₃ receptor mediated by calcineurin activity, which suggests that depolarization may regulate IP₃ receptor transcription. These results confirm the presence of at least two independent pathways for excitation-transcription coupling in skeletal muscle cells, both dependent on Ca²⁺ release and triggered by the same voltage sensor but activating different intracellular release channels.