Extracellular matrix promotes highly efficient cardiac differentiation of human pluripotent stem cells: the matrix sandwich method.

RATIONALE: Cardiomyocytes (CMs) 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 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 extracellular matrix in combination with growth factors known to promote cardiogenesis. METHODS AND RESULTS: PSCs were cultured as monolayers on Matrigel, an extracellular matrix preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin-positive mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, bone morphogenetic protein 4, and basic fibroblast growth factor) generated CMs with high purity (up to 98%) and yield (up to 11 CMs/input PSC) from multiple PSC lines. The resulting CMs progressively matured over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial, and ventricular CMs were observed, and monolayers of electrically coupled CMs modeled cardiac tissue and basic arrhythmia mechanisms. CONCLUSIONS: Dynamic extracellular matrix application promoted epithelial-mesenchymal transition of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.

Functional cardiomyocytes derived from human induced pluripotent stem cells.

Human induced pluripotent stem (iPS) cells hold great promise for cardiovascular research and therapeutic applications, but the ability of human iPS cells to differentiate into functional cardiomyocytes has not yet been demonstrated. The aim of this study was to characterize the cardiac differentiation potential of human iPS cells generated using OCT4, SOX2, NANOG, and LIN28 transgenes compared to human embryonic stem (ES) cells. The iPS and ES cells were differentiated using the embryoid body (EB) method. The time course of developing contracting EBs was comparable for the iPS and ES cell lines, although the absolute percentages of contracting EBs differed. RT-PCR analyses of iPS and ES cell-derived cardiomyocytes demonstrated similar cardiac gene expression patterns. The pluripotency genes OCT4 and NANOG were downregulated with cardiac differentiation, but the downregulation was blunted in the iPS cell lines because of residual transgene expression. Proliferation of iPS and ES cell-derived cardiomyocytes based on 5-bromodeoxyuridine labeling was similar, and immunocytochemistry of isolated cardiomyocytes revealed indistinguishable sarcomeric organizations. Electrophysiology studies indicated that iPS cells have a capacity like ES cells for differentiation into nodal-, atrial-, and ventricular-like phenotypes based on action potential characteristics. Both iPS and ES cell-derived cardiomyocytes exhibited responsiveness to beta-adrenergic stimulation manifest by an increase in spontaneous rate and a decrease in action potential duration. We conclude that human iPS cells can differentiate into functional cardiomyocytes, and thus iPS cells are a viable option as an autologous cell source for cardiac repair and a powerful tool for cardiovascular research.

Camguk/CASK enhances Ether-á-go-go potassium current by a phosphorylation-dependent mechanism.

Signaling complexes are essential for the modulation of excitability within restricted neuronal compartments. Adaptor proteins are the scaffold around which signaling complexes are organized. Here, we demonstrate that the Camguk (CMG)/CASK adaptor protein functionally modulates Drosophila Ether-á-go-go (EAG) potassium channels. Coexpression of CMG with EAG in Xenopus oocytes results in a more than twofold average increase in EAG whole-cell conductance. This effect depends on EAG-T787, the residue phosphorylated by calcium- and calmodulin-dependent protein kinase II (Wang et al., 2002). CMG coimmunoprecipitates with wild-type and EAG-T787A channels, indicating that T787, although necessary for the effect of CMG on EAG current, is not required for the formation of the EAG-CMG complex. Both CMG and phosphorylation of T787 increase the surface expression of EAG channels, and in COS-7 cells, EAG recruits CMG to the plasma membrane. The interaction of EAG with CMG requires a noncanonical Src homology 3-binding site beginning at position R1037 of the EAG sequence. Mutation of basic residues, but not neighboring prolines, prevents binding and prevents the increase in EAG conductance. Our findings demonstrate that membrane-associated guanylate kinase adaptor proteins can modulate ion channel function; in the case of CMG, this occurs via an increase in the surface expression and phosphorylation of the EAG channel.

Protein kinase modulation of a neuronal cation channel requires protein-protein interactions mediated by an Src homology 3 domain.

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.

A voltage-driven switch for ion-independent signaling by ether-à-go-go K+ channels.

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 p44/42 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.

Calcium/calmodulin-dependent protein kinase II phosphorylates and regulates the Drosophila eag potassium channel.

Modulation of neuronal excitability is believed to be an important mechanism of plasticity in the nervous system. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been postulated to regulate the ether à go-go (eag) potassium channel in Drosophila. Inhibition of CaMKII and mutation of the eag gene both cause hyperexcitability at the larval neuromuscular junction (NMJ) and memory formation defects in the adult. In this study, we identify a single site, threonine 787, as the major CaMKII phosphorylation site in Eag. This site can be phosphorylated by CaMKII both in a heterologous cell system and in vivo at the larval NMJ. Expression of Eag in Xenopus oocytes was used to assess the function of phosphorylation. Injection of either a specific CaMKII inhibitor peptide or lavendustin C, another CaMKII inhibitor, reduced Eag current amplitude acutely. Mutation of threonine 787 to alanine also reduced amplitude. Moreover, both CaMKII inhibition and the alanine mutation accelerated inactivation. The reduction in current amplitudes and the accelerated inactivation of dephosphorylated Eag channels would result in decreased outward potassium currents and hyperexcitability at presynaptic terminals and, thus, are consistent with the NMJ phenotype observed when CaMKII is inhibited. These results show that Eag is a substrate of CaMKII and suggest that direct modulation of potassium channels may be an important function of this kinase.