Electrical Stimulation Induces Calcium-dependent Up-regulation of Neuregulin-1� 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.

CaMKII-Mediated CREB Phosphorylation Is Involved in Ca2+-Induced BDNF mRNA Transcription and Neurite Outgrowth Promoted by Electrical Stimulation

“…ES increases Ca 2+ levels and activates nerve growth factor in ES-responsive Schwann cells in vitro [28, 46]. Ca 2+ , a critical second messenger, is involved in many intracellular molecular signaling cascades [47, 48]. Previous studies have shown that Ca 2+ plays important roles in regulating neurite extension triggered by extracellular factors, such an chemicals, neurotrophin or physical stimulation [5, 49, 50].…”

A Novel Functional In Vitro Model that Recapitulates Human Muscle Disorders

“…Previous attempts in this line have been focused on promoting contractility of human myotubes, in order to increase maturation and mimic exercise and adaptive responses characteristic of skeletal muscle. Different strategies include exogenous application of electrical pulse stimulation to cultured myotubes [11][12][13][14][15], magnetic fields to induce myoblast differentiation, and co-culturing with spinal cord explants [16][17][18][19] or human motoneurons [20,21] in order to achieve myotube innervation and subsequent maturation. More recently, spontaneous contractile activity has been observed in human myotubes by using a defined differentiation medium containing several trophic factors [22].…”

Computer task performance by subjects with Duchenne muscular dystrophy

“…Several studies have recently described DMD phenotype and genotype, 5 , 6 heart problems, 7 postural adjustment, 8 physical training, 9 multidisciplinary clinical evaluations, 10 , 11 therapeutic treatments, 12 and drug treatment. 13 – 15 However, there is growing interest in the use of computers by subjects with DMD.…”