Membrane Electrical Activity Elicits Inositol 1,4,5-Trisphosphate-dependent Slow Ca2+ Signals through a Gβγ/Phosphatidylinositol 3-Kinase γ Pathway in Skeletal Myotubes

Abstract

Tetanic electrical stimulation of myotubes evokes a ryanodine receptor-related fast calcium signal, during the stimulation, followed by a phospholipase C/inositol 1,4,5-trisphosphate-dependent slow calcium signal few seconds after stimulus end. L-type calcium channels (Cav 1.1, dihydropyridine receptors) acting as voltage sensors activate an unknown signaling pathway involved in phospholipase C activation. We demonstrated that both G protein and phosphatidylinositol 3-kinase were activated by electrical stimulation, and both the inositol 1,4,5-trisphosphate rise and slow calcium signal induced by electrical stimulation were blocked by pertussis toxin, by a G␤␥ scavenger peptide, and by phosphatidylinositol 3-kinase inhibitors. Immunofluorescence using anti-phosphatidylinositol 3-kinase ␥ antibodies showed a clear location in striations within the cytoplasm, consistent with a position near the I band region of the sarcomere. The time course of phosphatidylinositol 3-kinase activation, monitored in single living cells using a pleckstrin homology domain fused to green fluorescent protein, was compatible with sequential phospholipase C␥1 activation as confirmed by phosphorylation assays for the enzyme. Co-transfection of a dominant negative form of phosphatidylinositol 3-kinase ␥ inhibited the phosphatidylinositol 3-kinase activity as well as the slow calcium signal. We conclude that G␤␥/phosphatidylinositol 3-kinase ␥ signaling pathway is involved in phospholipase C activation and the generation of the slow calcium signal induced by tetanic stimulation. We postulate that membrane potential fluctuations in skeletal muscle cells can activate a pertussis toxin-sensitive G protein, phosphatidylinositol 3-kinase, phospholipase C pathway toward modulation of long term, activity-dependent plastic changes.The role of the voltage-dependent calcium channel (Cav 1.1) or dihydropyridine receptor (DHPR) 3 in excitation-contraction (EC) coupling in skeletal muscle is well known (1-4). Recently, another sequence of molecular processes activated by cell membrane depolarization involving the DHPR also acting as membrane voltage sensor has been reported. A slow calcium signal, first described in rat myotubes stimulated with K ϩ depolarization, occurs several seconds after the fast calcium signal that is due to ryanodine receptor opening and mediates EC coupling. The former is refractory to ryanodine treatment and depends on inositol 1,4,5-trisphosphate (IP 3 ) generation (5, 6). This slow calcium signal has a predominant nuclear component, and its propagation between adjacent nuclei in the myotube can be frequently recorded (5). Besides the fact that the three types of IP 3 receptors are located at different sites within rat myotubes, including the nuclear region, a functional calcium store in isolated nuclei has been recently described (7). Treatment of these isolated nuclei with IP 3 induces calcium release to the nucleoplasm, regulating phosphorylation of the transcription factor cAMP response element-binding protein (7). The slo...

Electrical stimulation induces IL-6 in skeletal muscle through extracellular ATP by activating Ca2+signals and an IL-6 autocrine loop

“…Our laboratory has described that electrical stimulation of skeletal muscle cells evokes the release of ATP, subsequent activation of metabotropic purinergic receptors, and the generation of IP 3 -dependent Ca 2ϩ transients (6,11,23,40). In this work, we have determined that this pathway is required for IL-6 expression.…”

“…7B). We also tested the blockade of other critical mediators of the slow calcium signal such as PLC, PI3K, and IP 3 R (11,16,23,24,39). Ten micromolars of U-73122, a PLC inhibitor, abolished STAT3 activation evoked by ES (Fig.…”

“…We have established that extracellular ATP is a relevant mediator between membrane depolarization and signaling pathways leading to gene expression both in rat newborn-derived myotubes and in mouse adult skeletal fibers (11,16,42,43). Extracellular ATP activates metabotropic P2Y receptors; ␤/␥-subunits of the attached heterotrimeric G protein subsequently activate phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC), increasing the intracellular IP 3 levels and cytosolic Ca 2ϩ concentration via IP 3 receptor (IP 3 R) activation in the sarcoplasmic reticulum membrane (11,23,24). This calcium signal is mainly nuclear in distribution (12) and has been related to expression of early genes (Fos/Jun, Egr1), late genes as IL-6, and structural genes as Troponin I via activation of several signal transduction cascades (ERK1/2, CREB, NF-B, AP-1) in skeletal muscle cells (11-13, 16, 45, 62, 85).…”

Cav1.1 controls frequency-dependent events regulating adult skeletal muscle plasticity

“…These slow transients, not related to muscle contraction, regulate several transcription-related events following membrane depolarization (Carrasco et al, 2003;Juretić et al, 2007). The signaling pathway activated by depolarization of muscle cells and triggered by Cav1.1 includes the sequential activation of G protein, phosphatidylinositide 3-kinase (PI3K) and phospholipase C (PLC) to produce inositol (1,4,5)-trisphosphate [Ins(1,4,5)P 3 ] that causes Ca 2+ release via Ins(1,4,5)P 3 R present in the SR membrane as well as in the nucleus (Cárdenas et al, 2005;Eltit et al, 2006). Electrical stimulation of cultured myotubes also promotes ATP release, likely through pannexin-1 (Panx1) channels.…”

Altered Gene Expression Pathways in Duchenne Muscular Dystrophy

“…Slow Ca 2+ transients are involved in the "E-T coupling" mechanism, which relates membrane depolarization with gene expression (Powell et al 2001;Araya et al 2003;Carrasco et al 2003;Juretic et al 2006;Juretic et al 2007). The signaling pathway begins at the DHPR, which by a mechanism involving G protein (Eltit et al 2006), activates PI3 kinase and PLC to produce inositol 1,4,5-trisphosphate (IP 3 ) that diffuses in the cytosol and reaches IP 3 receptors (IP 3 Rs) located both at the SR membrane and at the nuclear envelope, promoting Ca 2+ release (Araya et al 2003). IP 3 mediated Ca 2+ signals induce both a transient activation of ERK½ and transcription factor CREB, and an increase in early genes (c-fos, c-jun and egr-1) and in late genes (troponin I, interleukin-6, hmox and hsp70) mRNA levels after depolarization of normal skeletal muscle cells Juretic et al 2006;Juretic et al 2007;Jorquera et al 2009).…”