Ayako Takeuchi

Efficient and Scalable Purification of Cardiomyocytes from Human Embryonic and Induced Pluripotent Stem Cells by VCAM1 Surface Expression

Rationale Human embryonic and induced pluripotent stem cells (hESCs/hiPSCs) are promising cell sources for cardiac regenerative medicine. To realize hESC/hiPSC-based cardiac cell therapy, efficient induction, purification, and transplantation methods for cardiomyocytes are required. Though marker gene transduction or fluorescent-based purification methods have been reported, fast, efficient and scalable purification methods with no genetic modification are essential for clinical purpose but have not yet been established. In this study, we attempted to identify cell surface markers for cardiomyocytes derived from hESC/hiPSCs. Method and Result We adopted a previously reported differentiation protocol for hESCs based on high density monolayer culture to hiPSCs with some modification. Cardiac troponin-T (TNNT2)-positive cardiomyocytes appeared robustly with 30–70% efficiency. Using this differentiation method, we screened 242 antibodies for human cell surface molecules to isolate cardiomyocytes derived from hiPSCs and identified anti-VCAM1 (Vascular cell adhesion molecule 1) antibody specifically marked cardiomyocytes. TNNT2-positive cells were detected at day 7–8 after induction and 80% of them became VCAM1-positive by day 11. Approximately 95–98% of VCAM1-positive cells at day 11 were positive for TNNT2. VCAM1 was exclusive with CD144 (endothelium), CD140b (pericytes) and TRA-1-60 (undifferentiated hESCs/hiPSCs). 95% of MACS-purified cells were positive for TNNT2. MACS purification yielded 5−10×105 VCAM1-positive cells from a single well of a six-well culture plate. Purified VCAM1-positive cells displayed molecular and functional features of cardiomyocytes. VCAM1 also specifically marked cardiomyocytes derived from other hESC or hiPSC lines. Conclusion We succeeded in efficiently inducing cardiomyocytes from hESCs/hiPSCs and identifying VCAM1 as a potent cell surface marker for robust, efficient and scalable purification of cardiomyocytes from hESC/hiPSCs. These findings would offer a valuable technological basis for hESC/hiPSC-based cell therapy.

Decreased activity of basolateral organic ion transports in hyperuricemic rat kidney: roles of organic ion transporters, rOAT1, rOAT3 and rOCT2

We investigated organic anion and cation transport activity and the expression of several organic ion transporters in hyperuricemic rat kidney. Feeding oxonic acid, an inhibitor of uric acid metabolism, and uric acid for 10 days significantly increased plasma uric acid level. Plasma creatinine and blood urea nitrogen concentrations also increased in hyperuricemic rats, indicating impaired renal function. The accumulation of organic anions, p-aminohippurate (PAH) and methotrexate, and cations, tetraethylammonium (TEA) and cimetidine, into renal slices was markedly decreased, suggesting decreased transport activity for organic anions and cations at the basolateral membrane in the kidney. The expression levels of basolateral organic anion transporters rOAT1 and rOAT3, and organic cation transporter, rOCT2, significantly decreased in hyperuricemic rat kidney as assessed by mRNA and protein levels. In contrast, the expression of rOCT1 was unaltered by hyperuricemia at both mRNA and protein levels. Moreover, the mRNA expression of kidney-specific organic anion transporters, OAT-K1 and OAT-K2, and organic anion transporting polypeptide (oatp) 1, which localize at the brush-border membrane in the kidney, was unchanged in hyperuricemic rats. In conclusion, we showed decreased basolateral organic anion and cation transport activity, accompanied by a specific decrease in rOAT1, rOAT3 and rOCT2 expression in hyperuricemic rat kidney. These phenomena partly contribute to the changed renal disposition of organic anions and cations in hyperuricemia.

The ion pathway through the opened Na+,K+-ATPase pump

P-type ATPases pump ions across membranes, generating steep electrochemical gradients that are essential for the function of all cells. Access to the ion-binding sites within the pumps alternates between the two sides of the membrane to avoid the dissipation of the gradients that would occur during simultaneous access. In Na+,K+-ATPase pumps treated with the marine agent palytoxin, this strict alternation is disrupted and binding sites are sometimes simultaneously accessible from both sides of the membrane, transforming the pumps into ion channels (see, for example, refs 2, 3). Current recordings in these channels can monitor accessibility of introduced cysteine residues to water-soluble sulphydryl-specific reagents. We found previously that Na+,K+ pump-channels open to the extracellular surface through a deep and wide vestibule that emanates from a narrower pathway between transmembrane helices 4 and 6 (TM4 and TM6). Here we report that cysteine scans from TM1 to TM6 reveal a single unbroken cation pathway that traverses palytoxin-bound Na+,K+ pump-channels from one side of the membrane to the other. This pathway comprises residues from TM1, TM2, TM4 and TM6, passes through ion-binding site II, and is probably conserved in structurally and evolutionarily related P-type pumps, such as sarcoplasmic- and endoplasmic-reticulum Ca2+-ATPases and H+,K+-ATPases.

Ionic Mechanisms of Cardiac Cell Swelling Induced by Blocking Na+/K+ Pump As Revealed by Experiments and Simulation

Although the Na+/K+ pump is one of the key mechanisms responsible for maintaining cell volume, we have observed experimentally that cell volume remained almost constant during 90 min exposure of guinea pig ventricular myocytes to ouabain. Simulation of this finding using a comprehensive cardiac cell model (Kyoto model incorporating Cl− and water fluxes) predicted roles for the plasma membrane Ca2+-ATPase (PMCA) and Na+/Ca2+ exchanger, in addition to low membrane permeabilities for Na+ and Cl−, in maintaining cell volume. PMCA might help maintain the [Ca2+] gradient across the membrane though compromised, and thereby promote reverse Na+/Ca2+ exchange stimulated by the increased [Na+]i as well as the membrane depolarization. Na+ extrusion via Na+/Ca2+ exchange delayed cell swelling during Na+/K+ pump block. Supporting these model predictions, we observed ventricular cell swelling after blocking Na+/Ca2+ exchange with KB-R7943 or SEA0400 in the presence of ouabain. When Cl− conductance via the cystic fibrosis transmembrane conductance regulator (CFTR) was activated with isoproterenol during the ouabain treatment, cells showed an initial shrinkage to 94.2 ± 0.5%, followed by a marked swelling 52.0 ± 4.9 min after drug application. Concomitantly with the onset of swelling, a rapid jump of membrane potential was observed. These experimental observations could be reproduced well by the model simulations. Namely, the Cl− efflux via CFTR accompanied by a concomitant cation efflux caused the initial volume decrease. Then, the gradual membrane depolarization induced by the Na+/K+ pump block activated the window current of the L-type Ca2+ current, which increased [Ca2+]i. Finally, the activation of Ca2+-dependent cation conductance induced the jump of membrane potential, and the rapid accumulation of intracellular Na+ accompanied by the Cl− influx via CFTR, resulting in the cell swelling. The pivotal role of L-type Ca2+ channels predicted in the simulation was demonstrated in experiments, where blocking Ca2+ channels resulted in a much delayed cell swelling.

Isx Participates in the Maintenance of Vitamin A Metabolism by Regulation of β-Carotene 15,15′-Monooxygenase (Bcmo1) Expression

Isx (intestine specific homeobox) is an intestine-specific transcription factor. To elucidate its physiological function, we generated Isx-deficient mice by knocking in the β-galactosidase gene (LacZ) in the Isx locus (IsxLacZ/LacZ mice). LacZ staining of heterozygous (IsxLacZ/+) mice revealed that Isx was expressed abundantly in intestinal epithelial cells from duodenum to proximal colon. Quantitative mRNA expression profiling of duodenum and jejunum showed that β-carotene 15,15′-monooxygenase (EC1.14.99.36 Bcmo1) and the class B type I scavenger receptor, which are involved in vitamin A synthesis and carotenoid uptake, respectively, were drastically increased in IsxLacZ/LacZ mice. Although mild vitamin A deficiency decreased Isx expression in duodenum of wild-type (Isx+/+) mice, severe vitamin A deficiency decreased Isx mRNA expression in both duodenum and jejunum of Isx+/+ mice. On the other hand, vitamin A deficiency increased Bcmo1 expression in both duodenum and jejunum of Isx+/+ mice. However, Bcmo1 expression was not increased in duodenum of IsxLacZ/LacZ mice by mild vitamin A deficiency. These data suggest that Isx participates in the maintenance of vitamin A metabolism by regulating Bcmo1 expression in the intestine.

Functional analysis of rat renal organic anion transporter OAT-K1: bidirectional methotrexate transport in apical membrane

Renal organic anion transporter OAT‐K1 was stably transfected in MDCK cells and examined for its transport characteristics and membrane localization. OAT‐K1 mediated both uptake and efflux of methotrexate in the apical membranes. Immunoblotting showed that the apparent molecular mass of the expressed OAT‐K1 was 50 kDa, which was comparable to that found in the rat renal brush‐border membranes. The OAT‐K1‐mediated methotrexate transport was significantly inhibited in the presence of several organic anions such as folate and sulfobromophthalein. These findings suggest that OAT‐K1 mediates bidirectional methotrexate transport across the apical membranes, and may be involved in the renal handling of methotrexate.

The mitochondrial Na+-Ca2+ exchanger, NCLX, regulates automaticity of HL-1 cardiomyocytes

Mitochondrial Ca2+ is known to change dynamically, regulating mitochondrial as well as cellular functions such as energy metabolism and apoptosis. The NCLX gene encodes the mitochondrial Na+-Ca2+ exchanger (NCXmit), a Ca2+ extrusion system in mitochondria. Here we report that the NCLX regulates automaticity of the HL-1 cardiomyocytes. NCLX knockdown using siRNA resulted in the marked prolongation of the cycle length of spontaneous Ca2+ oscillation and action potential generation. The upstrokes of action potential and Ca2+ transient were markedly slower, and sarcoplasmic reticulum (SR) Ca2+ handling were compromised in the NCLX knockdown cells. Analyses using a mathematical model of HL-1 cardiomyocytes demonstrated that blocking NCXmit reduced the SR Ca2+ content to slow spontaneous SR Ca2+ leak, which is a trigger of automaticity. We propose that NCLX is a novel molecule to regulate automaticity of cardiomyocytes via modulating SR Ca2+ handling.

Modelling Cl− homeostasis and volume regulation of the cardiac cell

We aim at introducing a Cl− homeostasis to the cardiac ventricular cell model (Kyoto model), which includes the sarcomere shortening and the mitochondria oxidative phosphorylation. First, we examined mechanisms underlying the cell volume regulation in a simple model consisting of Na+/K+ pump, Na+–K+–2Cl− cotransporter 1 (NKCC1), cystic fibrosis transmembrane conductance regulator, volume-regulated Cl− channel and background Na+, K+ and Cl− currents. The high intracellular Cl− concentration of approximately 30 mM was achieved by the balance between the secondary active transport via NKCC1 and passive currents. Simulating responses to Na+/K+ pump inhibition revealed the essential role of Na+/K+ pump in maintaining the cellular osmolarity through creating the negative membrane potential, which extrudes Cl− from a cell, confirming the previous model study in the skeletal muscle. In addition, this model well reproduced the experimental data such as the responses to hypotonic shock in the presence or absence of β-adrenergic stimulation. Finally, the volume regulation via Cl− homeostasis was successfully incorporated to the Kyoto model. The steady state was well established in the comprehensive cell model in respect to both the intracellular ion concentrations and the shape of the action potential, which are all in the physiological range. The source code of the model, which can reproduce every result, is available from http://www.sim-bio.org/.

Peering into an ATPase ion pump with single-channel recordings

In principle, an ion channel needs no more than a single gate, but a pump requires at least two gates that open and close alternately to allow ion access from only one side of the membrane at a time. In the Na+,K+-ATPase pump, this alternating gating effects outward transport of three Na+ ions and inward transport of two K+ ions, for each ATP hydrolysed, up to a hundred times per second, generating a measurable current if assayed in millions of pumps. Under these assay conditions, voltage jumps elicit brief charge movements, consistent with displacement of ions along the ion pathway while one gate is open but the other closed. Binding of the marine toxin, palytoxin, to the Na+,K+-ATPase uncouples the two gates, so that although each gate still responds to its physiological ligand they are no longer constrained to open and close alternately, and the Na+,K+-ATPase is transformed into a gated cation channel. Millions of Na+ or K+ ions per second flow through such an open pump–channel, permitting assay of single molecules and allowing unprecedented access to the ion transport pathway through the Na+,K+-ATPase. Use of variously charged small hydrophilic thiol-specific reagents to probe cysteine targets introduced throughout the pumps transmembrane segments allows mapping and characterization of the route traversed by transported ions.

Impairment of Ubiquitin–Proteasome System by E334K cMyBPC Modifies Channel Proteins, Leading to Electrophysiological Dysfunction

Cardiac arrhythmogenesis is regulated by channel proteins whose protein levels are in turn regulated by the ubiquitin-proteasome system (UPS). We have previously reported on UPS impairment induced by E334K cardiac myosin-binding protein C (cMyBPC), which causes hypertrophic cardiomyopathy (HCM) accompanied by arrhythmia. We hypothesized that UPS impairment induced by E334K cMyBPC causes accumulation of cardiac channel proteins, leading to electrophysiological dysfunction. Wild-type or E334K cMyBPC was overexpressed in HL-1 cells and primary cultured neonatal rat cardiac myocytes. The expression of E334K cMyBPC suppressed cellular proteasome activities. The protein levels of K(v)1.5, Na(v)1.5, Hcn4, Ca(v)3.2, Ca(v)1.2, Serca, RyR2, and Ncx1 were significantly higher in cells expressing E334K cMyBPC than in wild type. They further increased in cells pretreated with MG132 and had longer protein decays. The channel proteins retained the correct localization. Cells expressing E334K cMyBPC exhibited higher Ca(2+) transients and longer action potential durations (APDs), accompanied by afterdepolarizations and higher apoptosis. Those augments of APD and Ca(2+) transients were recapitulated by a simulation model. Although a Ca(2+) antagonist, azelnidipine, neither protected E334K cMyBPC from degradation nor affected E334K cMyBPC incorporation into the sarcomere, it normalized the APD and Ca(2+) transients and partially reversed the levels of those proteins regulating apoptosis, thereby attenuating apoptosis. In conclusion, UPS impairment caused by E334K cMyBPC may modify the levels of channel proteins, leading to electrophysiological dysfunction. Therefore, UPS impairment due to a mutant cMyBPC may partly contribute to the observed clinical arrhythmias in HCM patients.