Human pluripotent stem cells (hPSCs) offer the potential to generate large numbers of functional cardiomyocytes from clonal and patient-specific cell sources. Here we show that temporal modulation of Wnt signaling is both essential and sufficient for efficient cardiac induction in hPSCs under defined, growth factor-free conditions. shRNA knockdown of β-catenin during the initial stage of hPSC differentiation fully blocked cardiomyocyte specification, whereas glycogen synthase kinase 3 inhibition at this point enhanced cardiomyocyte generation. Furthermore, sequential treatment of hPSCs with glycogen synthase kinase 3 inhibitors followed by inducible expression of β-catenin shRNA or chemical inhibitors of Wnt signaling produced a high yield of virtually (up to 98%) pure functional human cardiomyocytes from multiple hPSC lines. The robust ability to generate functional cardiomyocytes under defined, growth factor-free conditions solely by genetic or chemically mediated manipulation of a single developmental pathway should facilitate scalable production of cardiac cells suitable for research and regenerative applications.

Abstract-Human

Rationale Cardiomyocytes differentiated from human pluripotent stem cells (PSCs) are increasingly being used for cardiovascular research including disease modeling and hold promise for clinical applications. Current cardiac differentiation protocols exhibit variable success across different PSC lines and are primarily based on the application of growth factors. However, extracellular matrix (ECM) is also fundamentally involved in cardiac development from the earliest morphogenetic events such as gastrulation. Objective We sought to develop a more effective protocol for cardiac differentiation of human PSCs by using ECM in combination with growth factors known to promote cardiogenesis. Methods and Results PSCs were cultured as monolayers on Matrigel, an ECM preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin+ mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, BMP4, and bFGF) generated cardiomyocytes with high purity (up to 98%) and yield (up to 11 cardiomyocytes/input PSC) from multiple PSC lines. The resulting cardiomyocytes progressively mature over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial and ventricular cardiomyocytes were observed, and monolayers of electrically coupled cardiomyocytes modeled cardiac tissue and basic arrhythmia mechanisms. Conclusions Dynamic ECM application promoted EMT of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.

Genetic and physiological studies of the Drosophila Hyperkinetic (Hk) The signaling properties of neurons are largely determined by the combination of K+ currents they express. Multiple genes encoding pore-forming K+ channel a subunits (Kva) and the differential splicing of a transcripts contribute to the molecular and functional diversity of K+ channels (1). Additional complexity and regulation are conferred by two recently identified 1 subunits that appear to associate in a 1:1 stoichiometry with the a subunits of mammalian K+ channels and, in one case, modify the inactivation properties of Kva (2, 3). Because outwardly rectifying Kva initially were identified and isolated using Drosophila mutations with distinctive behavioral and electrophysiological defects (4-8), mutations of other loci exhibiting similar defects might identify additional genes key to K+ channel function. One such candidate is the Hyperkinetic (Hk) locus, which was identified by mutations causing an ether-sensitive leg-shaking phenotype (4). Studies of synaptic transmission in Hk larvae revealed a physiological phenotype similar to, but milder than, that of Sh (9). Moreover, Hk has phenotypic interactions in double mutant combinations with eag and inebriated (ine) similar to those observed for Sh (9,10

Voltage-gated channels maintain cellular resting potentials and generate neuronal action potentials by regulating ion flux. Here, we show that Ether-à -go-go (EAG) K ؉ channels also regulate intracellular signaling pathways by a mechanism that is independent of ion flux and depends on the position of the voltage sensor. Regulation of intracellular signaling was initially inferred from changes in proliferation. Specifically, transfection of NIH 3T3 fibroblasts or C2C12 myoblasts with either wild-type or nonconducting (F456A) eag resulted in dramatic increases in cell density and BrdUrd incorporation over vector-and Shaker-transfected controls. The effect of EAG was independent of serum and unaffected by changes in extracellular calcium. Inhibitors of p38 mitogen-activated protein (MAP) kinases, but not p44͞42 MAP kinases (extracellular signal-regulated kinases), blocked the proliferation induced by nonconducting EAG in serum-free media, and EAG increased p38 MAP kinase activity. Importantly, mutations that increased the proportion of channels in the open state inhibited EAG-induced proliferation, and this effect could not be explained by changes in the surface expression of EAG. These results indicate that channel conformation is a switch for the signaling activity of EAG and suggest an alternative mechanism for linking channel activity to the activity of intracellular messengers, a role that previously has been ascribed only to channels that regulate calcium influx.intracellular messenger ͉ mitogen-activated protein kinase ͉ neuromodulation ͉ proliferation ͉ gating V oltage-gated ion channels generate neuronal action potentials, the primary units of information transfer in the brain, by regulating ion f lux (1). Effects of ion channels on synaptic connectivity, transmitter release, plasticity, and other cellular processes are generally assumed to be a secondary consequence of ion f lux. Specifically, changes in membrane potential and action potentials alter Ca 2ϩ inf lux, and Ca 2ϩ regulates multiple intracellular signaling pathways (2-7). Several recent studies, however, have indicated that some voltagegated ion channels are bifunctional proteins (5, 8 -11). These studies show that voltage-gated channels can contribute to transcriptional regulation, protein scaffolding, cell adhesion, and intracellular signaling, and the effects appear largely independent of ion conduction. Recent studies of Ether-á-go-go [EAG (KCNH1)] voltagedependent K ϩ channels suggest that EAG may also be bifunctional. First, a region of Drosophila EAG with similarity to the autoinhibitory domain of Ca 2ϩ ͞calmodulin-dependent protein kinase II can associate with activated, Ca 2ϩ ͞calmodulin-bound Ca 2ϩ ͞calmodulin-dependent protein kinase II. In vitro assays indicate that, once Ca 2ϩ levels decline, EAG-bound kinase retains 5-10% of its maximum Ca 2ϩ -stimulated activity (12). Second, human EAG has been implicated in cell-cycle regulation and cancer: transfection can induce oncogenic transformation, EAG is present in some cancer cell line...

Tyrosine kinases and tyrosine phosphatases are abundant in central nervous system tissue, yet the role of these enzymes in the modulation of neuronal excitability is unknown. Patch-clamp studies of an Aplysia voltage-gated cation channel now demonstrate that a tyrosine phosphatase endogenous to excised patches determines both the gating mode of the channel and the response of the channel to protein kinase A. Moreover, a switch in gating modes similar to that triggered by the phosphatase occurs at the onset of a prolonged change in the excitability of Aplysia bag cell neurons.

SUMMARY1. Patch clamp studies of whole-cell ionic currents and biochemical studies of proliferation were carried out on Schwann cells of myelinated axons in explant segments of sciatic nerves of adult rabbit maintained in culture for 0-10 days.2. Schwann cell proliferation, as assayed by [3H]thymidine incorporation and by electron microscopic autoradiography, showed an increase following nerve explant. Proliferation proceeded in parallel with a gradual hyperpolarization of the resting potential and an increase in K+ currents in Schwann cells of myelinated axons.3. The relation between K+ channels and proliferation was studied by incubating explant nerves in the presence of various K+ channel blockers. Quinine, TEA and 4-aminopyridine (4-AP), which blocked K+ currents in Schwann cells, were found also to block Schwann cell proliferation in a dose-dependent fashion and over similar concentrations. Electron microscopy showed that TEA did not retard myelin and axonal break-down which is thought to be the source of mitogens for Schwann cell proliferation.4. The relation between resting potential and proliferation was studied by incubating explant nerves in depolarizing culture media. Depolarizing monovalent cations (K+ and Rb+) led to a marked inhibition of Schwann cell proliferation. However, Cs+ and NH4+, which did not depolarize Schwann cells in patch clamp studies, also inhibited proliferation. Gramicidin and veratridine also inhibited proliferation.5. The results suggest that the expression of K+ channels is functionally important for Schwann cell proliferation in Wallerian degeneration. A possible link between K+ channel and proliferation might be via a hyperpolarization of the resting membrane potential which occurs when Schwann cells proliferate.

Accumulating evidence suggests that many ion channels reside within a multiprotein complex that contains kinases and other signaling molecules. The role of the adaptor proteins that physically link these complexes together for the purposes of ion channel modulation, however, has been little explored. Here, we examine the protein-protein interactions required for regulation of an Aplysia bag cell neuron cation channel by a closely associated protein kinase C (PKC). In inside-out patches, the PKC-dependent enhancement of cation channel open probability could be prevented by the src homology 3 (SH3) domain, presumably by disrupting a link between the channel and the kinase. SH3 and PDZ domains from other proteins were ineffective. Modulation was also prevented by an SH3 motif peptide that preferentially binds the SH3 domain of src. Furthermore, whole-cell depolarizations elicited by cation channel activation were decreased by the src SH3 domain. These data suggest that the cation channel-PKC association may require SH3 domain-mediated interactions to bring about modulation, promote membrane depolarization, and initiate prolonged changes in bag cell neuron excitability. In general, protein-protein interactions between ion channels and protein kinases may be a prominent mechanism underlying neuromodulation.