N003 Caractérisation électrophysiologique de progéniteurs cardiaques issus de cellules souches embryonnaires humaines

Les progéniteurs cardiaques issus de cellules souches embryonnaires humaines apparaissent comme de bons candidates dans la prévention et le traitement de la dysfonction cardiaque par thérapie cellulaire. Cependant, la greffe de cellules présentant des propriétés électriques indésirables pourrait prédisposer les patients à des arythmies. Il est donc important de caractériser les propriétés électrophysiologiques des progéniteurs cardiaques avant transplantation. Les cellules progénitrices cardiaques utilisées sont dérivées de la lignée HUES-24 et sélectionnées sur leur capacité à exprimer SSEA-1 après une induction de 4 jours par le BMP2. A l’aide de la technique de patch-clamp en configuration cellule entière, nous avons pu enregistrer des courants Ca2+ et K+, 24 à 48 H après la sélection. Les courants Ca2+ observés (2,22 ± 0.30 Pa/pF) sont de type L, aucun courant de type T n’ayant été détecté dans ces conditions. Ces courants sont insensibles à l’isoprotérénol (1μm) et à la forskoline (30μm) suggérant l’absence de regulation β-adrénergique. Des courants K+ activés par depolarization (6.79 ± 1.10 Pa/pF) sensibles au tétraéthylammonium (10 mM) et à la 4-aminopyridine (5 mM) ont été caractérisés. Ces courants rectifiants sortants ressemblent aux courants rectifiants sortants retardés (IKDR) déjà décrits sur des cellules souches embryonnaires murines. En revanche, aucun courant entrant activé par hyperpolarisation n’a été observé. Finalement, aucune conductance Na+ n’a pu être mise en évidence. Sur les cellules n’exprimant pas SSEA-1, utilisées comme contrôles négatifs, aucun courant Ca2+, K+ ou Na+ n’a été détecté. Le profil moléculaire des canaux ioniques exprimés par les cellules progénitrices est abordé parallèlement par une approche génomique à l’aide de la RT-PCR haut débit.En conclusion, les cellules souches embryonnaires humaines présentent les courants majeurs impliqués dans l’électrogenèse cardiaque dès 24 h après leur orientation cardiaque. Néanmoins, l’absence de régulation β-adrénergique et de courants Na+ souligne l’immaturité de ces cellules comparées aux cardiomyocytes matures. Le suivi de la genèse des propriétés électrophysiologiques de ces progéniteurs cardiaques, dans le contexte d’une thérapie cellulaire cardiovasculaire, devrait nous permettre d’explorer la capacité de ces cellules à exprimer un phénotype électrophysiologique mature et à établir des couplages excitation-contraction avec les cellules hôte

Concise Review: Pluripotent Stem Cell-Derived Cardiac Cells, A Promisingă Cell Source for Therapy of Heart Failure: Where Do We Stand?

International audienceHeart failure is still a major cause of hospitalization and mortality ină developed countries. Many clinical trials have tested the use ofă multipotent stem cells as a cardiac regenerative medicine. The benefită for the patients of this therapeutic intervention has remained limited.ă Herein, we review the pluripotent stem cells as a cell source foră cardiac regeneration. We more specifically address the variousă challenges of this cell therapy approach. We question the cell deliveryă systems, the immune tolerance of allogenic cells, the potentială proarrhythmic effects, various drug mediated interventions to facilitateă cell grafting and, finally, we describe the pathological conditions thată may benefit from such an innovative approach. As members of aă transatlantic consortium of excellence of basic science researchers andă clinicians, we propose some guidelines to be applied to cell types andă modes of delivery in order to translate pluripotent stem cell cardiacă derivatives into safe and effective clinical trials

Functional Interaction of Ca(V) Channel Isoforms with Ryanodine Receptors Studied in Dysgenic Myotubes

The L-type Ca(2+) channels Ca(V)1.1 (α(1S)) and Ca(V)1.2 (α(1C)) share properties of targeting but differ by their mode of coupling to ryanodine receptors in muscle cells. The brain isoform Ca(V)2.1 (α(1A)) lacks ryanodine receptor targeting. We studied these three isoforms in myotubes of the α(1S)-deficient skeletal muscle cell line GLT under voltage-clamp conditions and estimated the flux of Ca(2+) (Ca(2+) input flux) resulting from Ca(2+) entry and release. Surprisingly, amplitude and kinetics of the input flux were similar for α(1C) and α(1A) despite a previously reported strong difference in responsiveness to extracellular stimulation. The kinetic flux characteristics of α(1C) and α(1A) resembled those in α(1S)-expressing cells but the contribution of Ca(2+) entry was much larger. α(1C) but not α(1A)-expressing cells revealed a distinct transient flux component sensitive to sarcoplasmic reticulum depletion by 30 μM cyclopiazonic acid and 10 mM caffeine. This component likely results from synchronized Ca(2+)-induced Ca(2+) release that is absent in α(1A)-expressing myotubes. In cells expressing an α(1A)-derivative (α(1)Aas(1592-clip)) containing the putative targeting sequence of α(1S), a similar transient component was noticeable. Yet, it was considerably smaller than in α(1C), indicating that the local Ca(2+) entry produced by the chimera is less effective in triggering Ca(2+) release despite similar global Ca(2+) inward current density

A possible role of the junctional face protein JP-45 in modulating Ca(2+) release in skeletal muscle

We investigated the functional role of JP-45, a recently discovered protein of the junctional face membrane (JFM) of skeletal muscle. For this purpose, we expressed JP-45 C-terminally tagged with the fluorescent protein DsRed2 by nuclear microinjection in myotubes derived from the C2C12 skeletal muscle cell line and performed whole-cell voltage-clamp experiments. We recorded in parallel cell membrane currents and Ca(2+) signals using fura-2 during step depolarization. It was found that properties of the voltage-activated Ca(2+) current were not significantly changed in JP-45–DsRed2-expressing C2C12 myotubes whereas the amplitude of depolarization-induced Ca(2+) transient was decreased compared to control myotubes expressing only DsRed2. Converting Ca(2+) transients to Ca(2+) input flux using a model fit approach to quantify Ca(2+) removal, the change could be attributed to an alteration in voltage-activated Ca(2+) permeability rather than to altered removal properties or a lower Ca(2+) content of the sarcoplasmic reticulum (SR). Determining non-linear capacitive currents revealed a reduction of Ca(2+) permeability per voltage-sensor charge. The results may be explained by a modulatory effect of JP-45 related to its reported in vitro interaction with the dihydropyridine receptor and the SR Ca(2+) binding protein calsequestrin (CSQ)

Sustained calcium signalling and caspase-3 activation involve NMDA receptors in thymocytes in contact with dendritic cells

L-glutamate, the major excitatory neurotransmitter, also has a role in non-neuronal tissues and modulates immune responses. Whether NMDA receptor (NMDAR) signalling is involved in T-cell development is unknown. In this study, we show that mouse thymocytes expressed an array of glutamate receptors, including NMDARs subunits. Sustained calcium (Ca2+) signals and caspase-3 activation in thymocytes were induced by interaction with antigen-pulsed dendritic cells (DCs) and were inhibited by NMDAR antagonists MK801 and memantine. NMDARs were transiently activated, triggered the sustained Ca2+ signal and were corecruited with the PDZ-domain adaptor postsynaptic density (PSD)-95 to thymocyte-DC contact zones. Although T-cell receptor (TCR) activation was sufficient for relocalization of NMDAR and PSD-95 at the contact zone, NMDAR could be activated only in a synaptic context. In these T-DC contacts, thymocyte activation occurred in the absence of exogenous glutamate, indicating that DCs could be a physiological source of glutamate. DCs expressed glutamate, glutamate-specific vesicular glutamate transporters and were capable of fast glutamate release through a Ca2+-dependent mechanism. We suggest that glutamate released by DCs could elicit focal responses through NMDAR-signalling in T cells undergoing apoptosis. Thus, synapses between T and DCs could provide a functional platform for coupling TCR activation and NMDAR signalling, which might reflect on T-cell development and modulation of the immune response

The role of CACNA1S in predisposition to malignant hyperthermia

BACKGROUND Malignant hyperthermia (MH) is an inherited pharmacogenetic disorder of skeletal muscle, characterised by an elevated calcium release from the skeletal muscle sarcoplasmic reticulum. The dihydropyridine receptor (DHPR) plays an essential role in excitation-contraction coupling and calcium homeostasis in skeletal muscle. This study focuses on the gene CACNA1S which encodes the α1 subunit of the DHPR, in order to establish whether CACNA1S plays a major role in MH susceptibility in the UK. METHODS We investigate the CACNA1S locus in detail in 50 independent MH patients, the largest study to date, to identify novel variants that may predispose to disease and also to characterise the haplotype structure across CACNA1S. RESULTS We present CACNA1S cDNA sequencing data from 50 MH patients in whom RYR1 mutations have been excluded, and subsequent mutation screening analysis. Furthermore we present haplotype analysis of unphased CACNA1S SNPs to (1) assess CACNA1S haplotype frequency differences between susceptible MH cases and a European control group and (2) analyse population-based association via clustering of CACNA1S haplotypes based on disease risk. CONCLUSION The study identified a single potentially pathogenic change in CACNA1S (p.Arg174Trp), and highlights that the haplotype structure across CACNA1S is diverse, with a high degree of variability

Inactivation of TRPM7 kinase in mice results in enlarged spleens, reduced T-cell proliferation and diminished store-operated calcium entry

Abstract T lymphocytes enlarge (blast) and proliferate in response to antigens in a multistep program that involves obligatory cytosolic calcium elevations. Store-operated calcium entry (SOCE) pathway is the primary source of Ca2+ in these cells. Here, we describe a novel modulator of blastogenesis, proliferation and SOCE: the TRPM7 channel kinase. TRPM7 kinase-dead (KD) K1646R knock-in mice exhibited splenomegaly and impaired blastogenic responses elicited by PMA/ionomycin or anti-CD3/CD28 antibodies. Splenic T-cell proliferation in vitro was weaker in the mutant compared to wildtype littermates. TRPM7 current magnitudes in WT and KD mouse T cells were, however, similar. We tested the dependence of T-cell proliferation on external Ca2+ and Mg2+ concentrations. At a fixed [Mg2+ o] of ~0.4 mM, Ca2+ o stimulated proliferation with a steep concentration dependence and vice versa, at a fixed [Ca2+ o] of ~0.4 mM, Mg2+ o positively regulated proliferation but with a shallower dependence. Proliferation was significantly lower in KD mouse than in wildtype at all Ca2+ and Mg2+ concentrations. Ca2+ elevations elicited by anti-CD3 antibody were diminished in KD mutant T cells and SOCE measured in activated KD splenocytes was reduced. These results demonstrate that a functional TRPM7 kinase supports robust SOCE, blastogenesis and proliferation, whereas its inactivation suppresses these cellular events