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Calcium homeostasis is critical for several vital functions in excitable and nonexcitable cells and has been shown to be impaired in many pathologies including Duchenne muscular dystrophy (DMD). Various studies using murine models showed the implication of calcium entry in the dystrophic phenotype. However, alteration of store-operated calcium entry (SOCE) and transient receptor potential vanilloid 2 (TRPV2)-dependant cation entry has not been investigated yet in human skeletal muscle cells. We pharmacologically characterized basal and storeoperated cation entries in primary cultures of myotubes prepared from muscle of normal and DMD patients and found, for the first time, an increased SOCE in DMD myotubes. Moreover, this increase cannot be explained by an over expression of the well-known SOCE actors: TRPC1/4, Orai1, and stromal interaction molecule 1 (STIM1) mRNA and proteins. Thus we investigated the modes of regulation of this cation entry. We firstly demonstrated the important role of the scaffolding protein α1-syntrophin, which regulates SOCE in primary human myotubes through its PDZ domain. We also studied the implication of phospholipase C (PLC) and protein kinase C (PKC) in SOCE and showed that their inhibition restores normal levels of SOCE in DMD human myotubes. In addition, the involvement of TRPV2 in calcium deregulation in DMD human myotubes was explored. We showed an abnormal elevation of TRPV2-dependant cation entry in dystrophic primary human myotubes compared with normal ones. These findings show that calcium homeostasis mishandling in DMD myotubes depends on SOCE under the influence of Ca2+/PLC/PKC pathway and α1-syntrophin regulation as well as on TRPV2-dependant cation influx. [ABSTRACT FROM AUTHOR]

Introduction: It has been speculated that pacemaker current (I f) in human atria could play a role in causing ectopic atrial automaticity. Ivabradine is a novel selective and specific I f inhibitor in the sinus node that reduces heart rate without any negative inotropic effect. The aim of the study was to explore possible effects of ivabradine on I f in atrial myocytes. Methods and Results: Using patch-clamp technique, we studied effects of ivabradine on I f present in atrial myocytes isolated from human right appendages of patients undergoing cardiac surgery. The identification of HCN isoforms was obtained by means of multiplex single-cell RT-PCR. Ivabradine induced a marked concentration and use-dependent I f inhibition with an IC50 at steady state of 2.9 μM. Time constant of block development (Tauon) decreases with the increase in the ivabradine concentration. Use-dependent inhibition induced by ivabradine (3 μM) was not modified in the presence of cAMP (10 μM) in the pipette solution. Multiplex single-cell RT-PCR indicates that the major HCN gene subtype detected in atria was HCN2. HCN4 is detected weakly and HCN1 is not significantly detected. Conclusions: Ivabradine inhibits I f current in the nonpacemaker cell with characteristics similar to those described previously in rabbit sinus node cells, but revealed a lesser sensitivity for I f recorded in human atrial cell than hHCN4 subunits considered as the major contributors to native f-channels in human sinoatrial node. A potential protection of atrial arrhythmias by ivabradine is discussed. [ABSTRACT FROM AUTHOR]

Background: Human cardiac stem cells expressing the W8B2 marker (W8B2+ CSCs) were recently identified and proposed as a new model of multipotent CSCs capable of differentiating into smooth muscle cells, endothelial cells and immature myocytes. Nevertheless, no characterization of ion channel or calcium activity during the differentiation of these stem cells has been reported. Methods: The objectives of this study were thus to analyze (using the TaqMan Low-Density Array technique) the gene profile of W8B2+ CSCs pertaining to the regulation of ion channels, transporters and other players involved in the calcium homeostasis of these cells. We also analyzed spontaneous calcium activity (via the GCaMP calcium probe) during the in vitro differentiation of W8B2+ CSCs into cardiac myocytes. Results: Our results show an entirely different electrophysiological genomic profile between W8B2+ CSCs before and after differentiation. Some specific nodal genes, such as Tbx3, HCN, ICaT, L, KV, and NCX, are overexpressed after this differentiation. In addition, we reveal spontaneous calcium activity or a calcium clock whose kinetics change during the differentiation process. A pharmacological study carried out on differentiated W8B2+ CSCs showed that the NCX exchanger and IP3 stores play a fundamental role in the generation of these calcium oscillations. Conclusions: Taken together, the present results provide important information on ion channel expression and intrinsic calcium dynamics during the differentiation process of stem cells expressing the W8B2 marker. [ABSTRACT FROM AUTHOR]

It is generally accepted that voltage-gated Ca 2+ channels, CaV, regulate Ca 2+ homeostasis in excitable cells following plasma membrane depolarization. Here, we show that the Ca 2+ protein α1D of CaV1.3 channel is overexpressed in colorectal cancer biopsies compared to normal tissues. Gene silencing experiments targeting α1D reduced the migration and the basal cytosolic Ca 2+ concentration of HCT116 colon cancer cell line and modified the cytosolic Ca 2+ oscillations induced by the sodium/calcium exchanger NCX1/3 working in its reverse mode. Interestingly, NCX1/3 regulated membrane potential of HCT116 cells only when α1D was silenced, and blocking NCX1/3 increased cytosolic Ca 2+ concentration and cell migration. However, membrane depolarization did not induce an increase in intracellular Ca 2+ . Patch-clamp experiments clearly showed that the inward Ca 2+ current was absent. Finally, flow cytometry and immunofluorescence studies showed that α1D protein was localized at the plasma membrane, in cytosol and cell nuclei. Altogether, we uncover a novel signaling pathway showing that α1D is involved in the regulation of Ca 2+ homeostasis and cell migration by a mechanism independent of its plasma membrane canonical function but that involved plasma membrane Na + /Ca 2+ exchanger.