Electrical Stimulation Induces Calcium-dependent Up-regulation of Neuregulin-1β in Dystrophic Skeletal Muscle Cell Lines

Abstract

Duchenne muscular dystrophy (DMD) is a neuromuscular disease originated by reduced or no expression of dystrophin, a cytoskeletal protein that provides structural integrity to muscle fibres. A promising pharmacological treatment for DMD aims to increase the level of a structural dystrophin homolog called utrophin. Neuregulin-1 (NRG-1), a growth factor that potentiates myogenesis, induces utrophin expression in skeletal muscle cells. Microarray analysis of total gene expression allowed us to determine that neuregulin-1β (NRG-1β) is one of 150 differentially expressed genes in electrically stimulated (400 pulses, 1 ms, 45 Hz) dystrophic human skeletal muscle cells (RCDMD). We investigated the effect of depolarization, and the involvement of intracellular Ca²⁺ and PKC isoforms on NRG-1β expression in dystrophic myotubes. Electrical stimulation of RCDMD increased NRG-1β mRNA and protein levels, and mRNA enhancement was abolished by actinomycin D. NRG-1β transcription was inhibited by BAPTA-AM, an intracellular Ca²⁺ chelator, and by inhibitors of IP₃-dependent slow Ca²⁺ transients, like 2-APB, Ly 294002 and Xestospongin B. Ryanodine, a fast Ca²⁺ signal inhibitor, had no effect on electrical stimulation-induced expression. BIM VI (general inhibitor of PKC isoforms) and Gö 6976 (specific inhibitor of Ca²⁺-dependent PKC isoforms) abolished NRG-1β mRNA induction. Our results suggest that depolarization induced slow Ca²⁺ signals stimulate NRG-1β transcription in RCDMD cells, and that Ca²⁺-dependent PKC isoforms are involved in this process. Based on utrophin´s ability to partially compensate dystrophin disfunction, knowledge on the mechanism involved on NRG-1 up-regulation could be important for new therapeutic strategies design.