Laurent Bernheim

Cloning of a human ether-a-go-go potassium channel expressed in myoblasts at the onset of fusion

An early sign of human myoblast commitment to fusion is the expression of a non‐inactivating delayed rectifier K+ current, IK(NI), and an associated membrane potential hyperpolarization. We have isolated the full‐length coding region of a human ether‐a‐go‐go K+ channel (h‐eag) from myoblasts undergoing differentiation. The h‐eag gene was localized to chromosome 1q32–41, and is expressed as a ∼9 kb transcript in myogenic cells and in adult brain tissue. Forced expression of h‐eag in undifferentiated myoblasts generates a current with remarkable similarity to IK(NI) indicating that h‐eag constitutes the channel responsible for this current in vivo.

Non-hematopoietic human bone marrow contains long-lasting, pluripotential mesenchymal stem cells.

Mesenchymal stem cells (MSC) are considered as potential agents for reconstructive and gene‐targeting therapies since they differentiate into various cell‐lineages, exhibit an extended survival once injected into a host, and can easily be transfected with engineered DNA. MSC are essentially isolated from hematopoietic bone marrow (BM), a process that is rather invasive and may raise ethical concerns. In an attempt to find an alternative source, we evaluated whether non‐hematopoietic (nh)BM recovered from femoral heads of patients undergoing hip arthroplasty contained MSC. Ex vivo, 99% of nhBM cells were CD45+ leukocytes. After culture, leukocytes were replaced by a homogenous layer of adherent CD45− CD14− CD34− CD11b− CD90+ HLA‐ABC+ cells. Culture doubling time (mean = 4 days, range 1.6–6.7 days) was not correlated with patient age (27–81 years, n = 16). Amplified cultures supported long‐term hematopoiesis, and could be differentiated in vitro into adipocytes and chondrocytes. Moreover, a small fraction of nhBM cells spontaneously expressed MyoD1 and formed myotubes, suggesting that myogenic differentiation also occurred. nhBM contained clonogenic cells whose frequency (1/13,000), doubling time (2.1 days), and maximal amplification (up to 106‐fold) were not age‐related. All 14 clones analyzed (from five patients, ages 27–78 years) differentiated into at least one mesenchymal lineage, and 66% were bipotential (n = 8/12), or tripotential (n = 2/3). In conclusion, nhBM contains pluripotential mesenchymal progenitors which are similar to hematopoietic BM‐derived MSC, and whose biological functions are not altered by aging. Furthermore, if MSC‐based therapies hold their promises, nhBM may become the source of choice for responding to the increasing demand for MSC. J. Cell. Physiol. 198: 110–118, 2004.

STIM1- and Orai1-dependent Store-operated Calcium Entry Regulates Human Myoblast Differentiation

Our previous work on human myoblasts suggested that a hyperpolarization followed by a rise in [Ca2+]in involving store-operated Ca2+ entry (SOCE) channels induced myoblast differentiation. Advances in the understanding of the SOCE pathway led us to examine more precisely its role in post-natal human myoblast differentiation. We found that SOCE orchestrated by STIM1, the endoplasmic reticulum Ca2+ sensor activating Orai Ca2+ channels, is crucial. Silencing STIM1, Orai1, or Orai3 reduced SOCE amplitude and myoblast differentiation, whereas Orai2 knockdown had no effect. Conversely, overexpression of STIM1 with Orai1 increased SOCE and accelerated myoblast differentiation. STIM1 or Orai1 silencing decreased resting [Ca2+]in and intracellular Ca2+ store content, but correction of these parameters did not rescue myoblast differentiation. Remarkably, SOCE amplitude correlated linearly with the expression of two early markers of myoblast differentiation, MEF2 and myogenin, regardless of the STIM or Orai isoform that was silenced. Unexpectedly, we found that the hyperpolarization also depends on SOCE, placing SOCE upstream of K+ channel activation in the signaling cascade that controls myoblast differentiation. These findings indicate that STIM1 and Orai1 are key molecules for the induction of human myoblast differentiation.

STIM1L is a new actin-binding splice variant involved in fast repetitive Ca2+ release

A newly identified splice variant of STIM1 called STIM1L forms constitutive clusters that interact with actin and Orai1 and allows fast repetitive Ca2+ release.

An ether ‐à‐go‐go K+ current, Ih‐eag, contributes to the hyperpolarization of human fusion‐competent myoblasts

1 Two early signs of human myoblast commitment to fusion are membrane potential hyperpolarization and concomitant expression of a non‐inactivating delayed rectifier K+ current, IK(NI). This current closely resembles the outward K+ current elicited by rat ether‐à‐go‐go (r‐eag) channels in its range of potential for activation and unitary conductance. 2 It is shown that activation kinetics of IK(NI), like those of r‐eag, depend on holding potential and on [Mg2+]o, and that IK(NI), like r‐eag, is reversibly inhibited by a rise in [Ca2+]. 3 Forced expression of an isolated human ether‐à‐go‐go K+ channel (h‐eag) cDNA in undifferentiated myoblasts generates single‐channel and whole‐cell currents with remarkable similarity to IK(NI). 4 h‐eag current (Ih‐eag) is reversibly inhibited by a rise in [Ca2+]i, and the activation kinetics depend on holding potential and [Mg2+]o. 5 Forced expression of h‐eag hyperpolarizes undifferentiated myoblasts from −9 to −50 mV, the threshold for the activation of both Ih‐eag and IK(NI). Similarly, the higher the density of IK(NI), the more hyperpolarized the resting potential of fusion‐competent myoblasts. 6 It is concluded that h‐eag constitutes the channel underlying IK(NI) and that it contributes to the hyperpolarization of fusion‐competent myoblasts. To our knowledge, this is the first demonstration of a physiological role for a mammalian eag K+ channel.

Lentivector-mediated transfer of Bmi-1 and telomerase in muscle satellite cells yields a Duchenne myoblast cell line with long-term genotypic and phenotypic stability

Conditionally immortalized human cells are valuable substrates for basic biologic studies, as well as for the production of specific proteins and for the creation of bioartificial organs. We previously demonstrated that the lentivector-mediated transduction of immortalizing genes into human primary cells is an efficient method for obtaining such cell lines. Here, we used human muscle satellite cells as model targets to examine the impact of the transduced genes on the genotypic and phenotypic characteristics of the immortalized cells. The most commonly used immortalizing gene, the SV40 large T antigen (T-Ag), was extremely efficient at inducing the continuous growth of primary myoblasts, but the resulting cells rapidly accumulated major chromosomal aberrations and exhibited profound phenotypic changes. In contrast, the constitutive expression of telomerase and Bmi-1 in satellite cells from a control individual and from a patient suffering from Duchennes muscular dystrophy yielded cell lines that remained diploid and conserved their growth factor dependence for proliferation. However, despite the absence of detectable cytogenetic abnormalities, clones derived from satellite cells of a control individual exhibited a differentiation block in vitro. In contrast, a Duchenne-derived cell line exhibited all the phenotypic characteristics of its primary parent, including an ability to differentiate fully into myotubes when placed in proper culture conditions. This cell line should constitute a useful reagent for a wide range of studies aimed at this disease.

Role of an inward rectifier K+ current and of hyperpolarization in human myoblast fusion.

1 The role of K+ channels and membrane potential in myoblast fusion was evaluated by examining resting membrane potential and timing of expression of K+ currents at three stages of differentiation of human myogenic cells: undifferentiated myoblasts, fusion‐competent myoblasts (FCMBs), and freshly formed myotubes. 2 Two K+ currents contribute to a hyperpolarization of myoblasts prior to fusion: IK(NI), a non‐inactivating delayed rectifier, and IK(IR), an inward rectifier. 3 I K(NI) density is low in undifferentiated myoblasts, increases in FCMBs and declines in myotubes. On the other hand, IK(IR) is expressed in 28 % of the FCMBs and in all myotubes. 4 I K(IR) is reversibly blocked by Ba2+ or Cs+. 5 Cells expressing IK(IR) have resting membrane potentials of −65 mV. A block by Ba2+ or Cs+ induces a depolarization to a voltage determined by IK(NI) (‐32 mV). 6 Cs+ and Ba2+ ions reduce myoblast fusion. 7 It is hypothesized that the IK(IR)‐mediated hyperpolarization allows FCMBs to recruit Na+, K+ and T‐type Ca2+ channels which are present in these cells and would otherwise be inactivated. FCMBs, rendered thereby capable of firing action potentials, could amplify depolarizing signals and may accelerate fusion.

Membrane Hyperpolarization Triggers Myogenin and Myocyte Enhancer Factor-2 Expression during Human Myoblast Differentiation

It is widely thought that myogenin is one of the earliest detectable markers of skeletal muscle differentiation. Here we show that, during human myoblast differentiation, an inward rectifier K+ channel (Kir2.1) and its associated hyperpolarization trigger expression and activity of the myogenic transcription factors, myogenin and myocyte enhancer factor-2 (MEF2). Furthermore, Kir2.1 current precedes and is required for the developmental increase in expression/activity of myogenin and MEF2. Drugs or antisense reducing Kir2.1 current diminished or suppressed fusion as well as expression/activity of myogenin and MEF2. In contrast, LY294002, an inhibitor of phosphatidylinositol 3-kinase (a pathway controlling initiation of the myogenic program) that inhibited both myogenin/MEF2 expression and fusion, did not affect Kir2.1 current. This non-blockade by LY294002 indicates that Kir2.1 acts upstream of myogenin and MEF2. We propose that Kir2.1 channel activation is a required key early event that initiates myogenesis by turning on myogenin and MEF2 transcription factors via a hyperpolarization-activated Ca2+-dependent pathway.

Distinct contributions of high- and low-voltage-activated calcium currents to afterhyperpolarizations in cholinergic nucleus basalis neurons of the guinea pig.

The contributions made by low- (LVA) and high-voltage-activated (HVA) calcium currents to afterhyperpolarizations (AHPs) of nucleus basalis (NB) cholinergic neurons were investigated in dissociated cells. Neurons with somata >25 μm were studied because 80% of them stained positively for choline acetyltransferase and had electrophysiological characteristics identical to those of cholinergic NB neurons previously recorded in basal forebrain slices. Calcium currents of cholinergic NB neurons first were dissected pharmacologically into an amiloride-sensitive LVA and at least five subtypes of HVA currents. Approximately 17% of the total HVA current was sensitive to nifedipine (3 μm), 35% to ω-conotoxin-GVIA (200–400 nm), 10% to ω-Agatoxin-IVA (100 nm), and 20% to ω-Agatoxin-IVA (300–500 nm), suggesting the presence of L-, N-, P-, and Q-type channels, respectively. A remaining current (R-type) resistant to these antagonists was blocked by cadmium (100–200 μm). We then assessed pharmacologically the role that LVA and HVA currents had in activating the apamin-insensitive AHP elicited by a long train of action potentials (sAHP) and the AHP evoked either by a short burst of action potentials or by a single action potential (mAHP) that is known to be apamin-sensitive. During sAHPs, ∼60% of the hyperpolarization was activated by calcium flowing through N-type channels and ∼20% through P-type channels, whereas T-, L-, and Q-type channels were not involved significantly. In contrast, during mAHPs, N- and T-type channels played key roles (∼60 and 30%, respectively), whereas L-, P-, and Q-type channels were not implicated significantly. It is concluded that in cholinergic NB neurons various subtypes of calcium channels can differentially activate the apamin-sensitive mAHP and the apamin-insensitive sAHP.

Mibefradil (Ro 40-5967) inhibits several Ca2+ and K+ currents in human fusion-competent myoblasts

The effect of mibefradil (Ro 40–5967), an inhibitor of T‐type Ca2+ current (ICa(T)), on myoblast fusion and on several voltage‐gated currents expressed by fusion‐competent myoblasts was examined. At a concentration of 5 μM, mibefradil decreases myoblast fusion by 57%. At this concentration, the peak amplitudes of ICa(T) and L‐type Ca2+ current (ICa(L)) measured in fusion‐competent myoblasts are reduced by 95 and 80%, respectively. The IC50 of mibefradil for ICa(T) and ICa(L) are 0.7 and 2 μM, respectively. At low concentrations, mibefradil increased the amplitude of ICa(L) with respect to control. Mibefradil blocked three voltage‐gated K+ currents expressed by human fusion‐competent myoblasts: a delayed rectifier K+ current, an ether‐à‐go‐go K+ current, and an inward rectifier K+ current, with a respective IC50 of 0.3, 0.7 and 5.6 μM. It is concluded that mibefradil can interfere with myoblast fusion, a mechanism fundamental to muscle growth and repair, and that the interpretation of the effect of mibefradil in a given system should take into account the action of this drug on ionic currents other than Ca2+ currents.