ABSTRACT
Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) provide an in vitro model of the human myocardium. Complex 3D scaffolded culture methods improve the phenotypical maturity of iPSC-CMs, although typically at the expense of throughput. We have developed a novel, scalable approach that enables the use of iPSC-CM 3D spheroid models in a label-free readout system in a standard 96-well plate-based format. Spheroids were accurately positioned onto recording electrodes using a magnetic gold-iron oxide nanoparticle approach. Remarkably, both contractility (impedance) and extracellular field potentials (EFPs) could be detected from the actively beating spheroids over long durations and after automated dosing with pharmacological agents. The effects on these parameters of factors, such as co-culture (including human primary cardiac fibroblasts), extracellular buffer composition, and electrical pacing, were investigated. Beat amplitudes were increased greater than 15-fold by co-culture with fibroblasts. Optimization of extracellular Ca2+ fluxes and electrical pacing promoted the proper physiological response to positive inotropic agonists of increased beat amplitude (force) rather than the increased beat rate often observed in iPSC-CM studies. Mechanistically divergent repolarizations in different spheroid models were indicated by their responses to BaCl2 compared with E-4031. These studies demonstrate a new method that enables the pharmacological responses of 3D iPSC-CM spheroids to be determined in a label-free, standardized, 96-well plate-based system. This approach could have discovery applications across cardiovascular efficacy and safety, where parameters typically sought as readouts of iPSC-CM maturity or physiological relevance have the potential to improve assay predictivity.
Subject(s)
Anti-Arrhythmia Agents/pharmacology , Fibroblasts/drug effects , Myocardial Contraction/drug effects , Myocytes, Cardiac/drug effects , Piperidines/pharmacology , Pyridines/pharmacology , Spheroids, Cellular/drug effects , Barium Compounds/pharmacology , Biological Assay , Calcium/metabolism , Cell Differentiation , Chlorides/pharmacology , Coculture Techniques , Ferric Compounds/chemistry , Fibroblasts/cytology , Fibroblasts/metabolism , Gold/chemistry , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Ion Transport , Metal Nanoparticles/chemistry , Metal Nanoparticles/ultrastructure , Models, Biological , Myocardial Contraction/physiology , Myocardium/cytology , Myocardium/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Spheroids, Cellular/cytology , Spheroids, Cellular/metabolismABSTRACT
BACKGROUND AND PURPOSE: The TRPC1, TRPC4, and TRPC5 proteins form homotetrameric or heterotetrameric, calcium-permeable cation channels that are involved in various disease states. Recent research has yielded specific and potent xanthine-based TRPC1/4/5 inhibitors. Here, we investigated the possibility of xanthine-based activators of these channels. EXPERIMENTAL APPROACH: An analogue of the TRPC1/4/5 inhibitor Pico145, AM237, was synthesized and its activity was investigated using HEK cells overexpressing TRPC4, TRPC5, TRPC4-C1, TRPC5-C1, TRPC1:C4 or TRPC1:C5 channels, and in A498 cells expressing native TRPC1:C4 channels. TRPC1/4/5 channel activities were assayed by measuring intracellular concentration of Ca2+ ([Ca2+ ]i ) and by patch-clamp electrophysiology. Selectivity of AM237 was tested against TRPC3, TRPC6, TRPV4, or TRPM2 channels. KEY RESULTS: AM237 potently activated TRPC5:C5 channels (EC50 15-20 nM in [Ca2+ ]i assay) and potentiated their activation by sphingosine-1-phosphate but suppressed activation evoked by (-)-englerin A (EA). In patch-clamp studies, AM237 activated TRPC5:C5 channels, with greater effect at positive voltages, but with lower efficacy than EA. Pico145 competitively inhibited AM237-induced TRPC5:C5 activation. AM237 did not activate TRPC4:C4, TRPC4-C1, TRPC5-C1, TRPC1:C5, and TRPC1:C4 channels, or native TRPC1:C4 channels in A498 cells, but potently inhibited EA-dependent activation of these channels with IC50 values ranging from 0.9 to 7 nM. AM237 (300 nM) did not activate or inhibit TRPC3, TRPC6, TRPV4, or TRPM2 channels. CONCLUSIONS AND IMPLICATIONS: This study suggests the possibility for selective activation of TRPC5 channels by xanthine derivatives and supports the general principle that xanthine-based compounds can activate, potentiate, or inhibit these channels depending on subunit composition.
Subject(s)
Heterocyclic Compounds, 2-Ring/pharmacology , Purines/pharmacology , TRPC Cation Channels/metabolism , Calcium/analysis , Calcium/metabolism , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , HEK293 Cells , Heterocyclic Compounds, 2-Ring/chemical synthesis , Heterocyclic Compounds, 2-Ring/chemistry , Humans , Molecular Structure , Patch-Clamp Techniques , Purines/chemical synthesis , Purines/chemistry , Structure-Activity Relationship , TRPC Cation Channels/antagonists & inhibitorsABSTRACT
K+/Cl- cotransporter 2 (KCC2) is selectively expressed in the adult nervous system and allows neurons to maintain low intracellular Cl- levels. Thus, KCC2 activity is an essential prerequisite for fast hyperpolarizing synaptic inhibition mediated by type A γ-aminobutyric acid (GABAA) receptors, which are Cl--permeable, ligand-gated ion channels. Consistent with this, deficits in the activity of KCC2 lead to epilepsy and are also implicated in neurodevelopmental disorders, neuropathic pain, and schizophrenia. Accordingly, there is significant interest in developing activators of KCC2 as therapeutic agents. To provide insights into the cellular processes that determine KCC2 activity, we have investigated the mechanism by which N-ethylmaleimide (NEM) enhances transporter activity using a combination of biochemical and electrophysiological approaches. Our results revealed that, within 15 min, NEM increased cell surface levels of KCC2 and modulated the phosphorylation of key regulatory residues within the large cytoplasmic domain of KCC2 in neurons. More specifically, NEM increased the phosphorylation of serine 940 (Ser-940), whereas it decreased phosphorylation of threonine 1007 (Thr-1007). NEM also reduced with no lysine (WNK) kinase phosphorylation of Ste20-related proline/alanine-rich kinase (SPAK), a kinase that directly phosphorylates KCC2 at residue Thr-1007. Mutational analysis revealed that Thr-1007 dephosphorylation mediated the effects of NEM on KCC2 activity. Collectively, our results suggest that compounds that either increase the surface stability of KCC2 or reduce Thr-1007 phosphorylation may be of use as enhancers of KCC2 activity.
Subject(s)
Ethylmaleimide/metabolism , Symporters/metabolism , Animals , Cell Membrane/metabolism , Embryo, Mammalian , Humans , Membrane Transport Modulators/metabolism , Neurons/metabolism , Phosphorylation/physiology , Rats , Rats, Sprague-Dawley , Receptors, GABA/metabolism , Symporters/physiology , K Cl- CotransportersABSTRACT
KCC2 is a neuron specific K+-Cl- co-transporter that controls neuronal chloride homeostasis, and is critically involved in many neurological diseases including brain trauma, epilepsies, autism and schizophrenia. Despite significant accumulating data on the biology and electrophysiological properties of KCC2, structure-function relationships remain poorly understood. Here we used calixarene detergent to solubilize and purify wild-type non-aggregated and homogenous KCC2. Specific binding of inhibitor compound VU0463271 was demonstrated using surface plasmon resonance (SPR). Mass spectrometry revealed glycosylations and phosphorylations as expected from functional KCC2. We show by electron microscopy (EM) that KCC2 exists as monomers and dimers in solution. Monomers are organized into "head" and "core" domains connected by a flexible "linker". Dimers are asymmetrical and display a bent "S-shape" architecture made of four distinct domains and a flexible dimerization interface. Chemical crosslinking in reducing conditions shows that disulfide bridges are involved in KCC2 dimerization. Moreover, we show that adding a tag to the C-terminus is detrimental to KCC2 function. We postulate that the conserved KCC2 C-ter may be at the interface of dimerization. Taken together, our findings highlight the flexible multi-domain structure of KCC2 with variable anchoring points at the dimerization interface and an important C-ter extremity providing the first in-depth functional architecture of KCC2.
ABSTRACT
OBJECTIVES: The Brown Norway rat is highly susceptible to cerebral haemorrhage when hypertension is induced experimentally, compared with the Long Evans. The aims of the study were to compare the myogenic properties and also the collagen-staining profile of the middle cerebral artery (MCA) and a small systemic artery (cremaster) from Brown Norway and Long Evans rats. METHODS: In-vitro pressure myography was used to compare the myogenic properties and the distensibility of MCA and cremaster arteries from Brown Norway rat, with those of the Long Evans rat. Histologically prepared arterial sections were stained with picrosirius red to compare the collagen-staining profile of MCA and cremaster from these strains of rat. RESULTS: In the presence of myogenic tone, the active pressure-diameter relationship (20-200 âmmHg) was significantly different in MCA from the Brown Norway, but not cremaster arteries, compared with the Long Evans, characterized by in the lack of a myogenic range in the Brown Norway. Midwall collagen staining was significantly increased in MCA from the Brown Norway rat, compared with the Long Evans rat; this difference between rat strains was not observed in the cremaster arteries. However, the stress-strain relationship of MCA and cremaster arteries from the Brown Norway rat was shifted to the right, indicating an increased distensibilty of arteries from both vascular beds, compared with the Long Evans. CONCLUSION: These data demonstrate impaired myogenic properties and differences in the collagen-staining profile of MCA but not cremaster arteries from the Brown Norway rat, compared with the Long Evans. The impaired myogenic properties of MCA from the Brown Norway rat compared with the Long Evans may explain their increased susceptibility to cerebral haemorrhage when hypertension is induced experimentally.
Subject(s)
Cerebral Arteries/pathology , Middle Cerebral Artery/pathology , Muscle Development , Animals , Blood Pressure , Cerebral Arteries/anatomy & histology , Cerebral Hemorrhage/etiology , Cerebral Hemorrhage/physiopathology , Collagen/metabolism , Male , Middle Cerebral Artery/anatomy & histology , Models, Anatomic , Myography/methods , Rats , Rats, Long-Evans , Species SpecificityABSTRACT
The dynamic expression of voltage-gated potassium channels (Kvs) at the cell surface is a fundamental factor controlling membrane excitability. In exploring possible mechanisms controlling Kv surface expression, we identified a region in the extracellular linker between the first and second of the six (S1-S6) transmembrane-spanning domains of the Kv1.4 channel, which we hypothesized to be critical for its biogenesis. Using immunofluorescence microscopy, flow cytometry, patch clamp electrophysiology, and mutagenesis, we identified a single threonine residue at position 330 within the Kv1.4 S1-S2 linker that is absolutely required for cell surface expression. Mutation of Thr-330 to an alanine, aspartate, or lysine prevented surface expression. However, surface expression occurred upon co-expression of mutant and wild type Kv1.4 subunits or mutation of Thr-330 to a serine. Mutation of the corresponding residue (Thr-211) in Kv3.1 to alanine also caused intracellular retention, suggesting that the conserved threonine plays a generalized role in surface expression. In support of this idea, sequence comparisons showed conservation of the critical threonine in all Kv families and in organisms across the evolutionary spectrum. Based upon the Kv1.2 crystal structure, further mutagenesis, and the partial restoration of surface expression in an electrostatic T330K bridging mutant, we suggest that Thr-330 hydrogen bonds to equally conserved outer pore residues, which may include a glutamate at position 502 that is also critical for surface expression. We propose that Thr-330 serves to interlock the voltage-sensing and gating domains of adjacent monomers, thereby yielding a structure competent for the surface expression of functional tetramers.
Subject(s)
Cell Membrane/metabolism , Kv1.4 Potassium Channel/chemistry , Potassium Channels, Voltage-Gated/metabolism , Threonine/chemistry , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , Electrophysiology , Evolution, Molecular , Humans , Models, Biological , Molecular Sequence Data , Mutation , Sequence Homology, Amino AcidABSTRACT
We have examined the relative contributions of small- and intermediate-conductance Ca(2+)-activated K(+) channels (SK(Ca) and IK(Ca)) to the endothelium-derived hyperpolarizing factor (EDHF) pathway response in small mesenteric arteries of Zucker Diabetic Fatty (ZDF) rats, before and after the development of Type II diabetes, together with Lean controls. Smooth muscle membrane potential was recorded using sharp microelectrodes in the presence of 10 microM indomethacin plus 100 microM N(omega)-nitro-L-arginine. SK(Ca) was selectively inhibited with 100 nM apamin, whereas IK(Ca) was blocked with 10 microM TRAM-39 (2-(2-chlorophenyl)-2,2-diphenylacetonitrile). Resting membrane potentials were similar in arteries from 17- to 20-week-old control and diabetic rats (approximately -54 mV). Responses elicited by 1 and 10 microM acetylcholine (ACh) were significantly smaller in the diabetic group (e.g. hyperpolarizations to -69.5 +/- 0.8 mV (ZDF; n = 12) and -73.2 +/- 0.6 mV (Lean; n = 12; P < 0.05) evoked by 10 microM ACh). The IK(Ca)-mediated components of the ACh responses were comparable between groups (hyperpolarizations to approximately -65 mV on exposure to 10 microM ACh). However, SK(Ca)-mediated responses were significantly reduced in the diabetic group (hyperpolarizations to -63.1 +/- 1.0 mV (ZDF; n = 6) and -71.5 +/- 1.2 mV (Lean; n = 6; P < 0.05) on exposure to 10 microM ACh. Impaired ACh responses were not observed in arteries from 5- to 6-week-old (pre-diabetic) animals. SK(Ca) subunit mRNA expression was increased in the diabetic group. The EDHF pathway, especially the SK(Ca)-mediated response, is impaired in Type II diabetic ZDF rats without a reduction in channel gene expression. These results may be particularly relevant to the microvascular complications of diabetes. The functional separation of SK(Ca) and IK(Ca) pathways is discussed.
Subject(s)
Biological Factors/physiology , Diabetes Mellitus, Type 2/physiopathology , Small-Conductance Calcium-Activated Potassium Channels/physiology , Acetonitriles/pharmacology , Animals , Benzimidazoles/pharmacology , Intermediate-Conductance Calcium-Activated Potassium Channels/physiology , Male , Membrane Potentials/drug effects , Rats , Rats, Zucker , Reverse Transcriptase Polymerase Chain Reaction , Trityl Compounds/pharmacologyABSTRACT
Although it is well established that diabetes impairs endothelium-dependent vasodilation, including those pathways involving vascular myocyte large-conductance Ca(2+)-activated K(+) channels (BK(Ca)), little is known about the effects of diabetes on BK(Ca) activation as an intrinsic response to contractile stimulation. We have investigated this mechanism in a model of Type 2 diabetes, the male Zucker diabetic fatty (ZDF) rat. BK(Ca) function in prediabetic (5-7 wk) and diabetic (17-20 wk) ZDF and lean control animals was assessed in whole arteries using myograph and electrophysiology techniques and in freshly dissociated myocytes by patch clamping. Log EC(25) values for phenylephrine concentration-tension curves were shifted significantly to the left by blockade of BK(Ca) with iberiotoxin (IBTX) in arteries from non- and prediabetic animals but not from diabetic animals. Smooth muscle hyperpolarizations of arteries evoked by the BK(Ca) opener NS-1619 were significantly reduced in the diabetic group. Voltage-clamp recordings indicated that IBTX-sensitive currents were not enhanced to the extent observed in nondiabetic controls by increasing the Ca(2+) concentration in the pipette solution or the application of NS-1619 in myocytes from diabetic animals. An alteration in the expression of BK(Ca) beta(1) subunits was not evident at either the mRNA or protein level in arteries from diabetic animals. Collectively, these results suggest that myocyte BK(Ca) of diabetic animals does not significantly oppose vasoconstriction, unlike that of prediabetic and control animals. This altered function was related to a reduced Ca(2+)-dependent activation of the channel not involving beta(1) subunits.
Subject(s)
Calcium/metabolism , Diabetes Mellitus, Type 2/metabolism , Mesenteric Arteries/metabolism , Myocytes, Cardiac/metabolism , Obesity/metabolism , Potassium Channels, Calcium-Activated/metabolism , Vasodilation , Animals , Diabetes Mellitus, Type 2/complications , Ion Channel Gating , Male , Obesity/complications , Rats , Rats, ZuckerABSTRACT
The aims of the study were to compare the myogenic and structural properties of middle cerebral arteries (MCAs) from the stroke-prone spontaneously hypertensive rat (SHRSP) with MCAs from the spontaneously hypertensive rat (SHR) before stroke development in SHRSP. Rats were fed a "Japanese" diet (low-protein rat chow and 1% NaCl in drinking water) for 8 wk, and cerebral arteries were studied in vitro at 12 wk using a pressure arteriograph. Systolic pressure was significantly increased in SHRSP compared with SHR at 12 wk. Between 60 and 180 mmHg, MCAs from SHR maintained an essentially constant diameter, i.e., displayed a "myogenic range," whereas the diameter of MCAs from SHRSP progressively increased as a function of pressure. Passive lumen diameter of MCAs from SHRSP was reduced at high pressure, and wall thickness and wall/lumen were increased, compared with SHR. Wall cross-sectional area was also increased in MCAs from SHRSP compared with the SHR, indicating growth. The stress-strain relationship was shifted to the left in MCAs from SHRSP, indicating decreased MCA distensibility compared with SHR. However, collagen staining with picrosirius red revealed a redistribution of collagen to the outer half of the MCA wall in SHRSP compared with SHR. These data demonstrate impaired myogenic properties in prestroke SHRSP compared with SHR, which may explain stroke development. The structural differences in MCAs from SHRSP compared with SHR were a consequence of both growth and a reduced distensibility.
Subject(s)
Cerebral Arteries/physiopathology , Genetic Predisposition to Disease , Hypertension/genetics , Hypertension/physiopathology , Muscle, Smooth, Vascular/physiopathology , Stroke/genetics , Vasodilation , Animals , Blood Pressure , Cerebral Angiography , Cerebral Arteries/diagnostic imaging , Cerebral Arteries/pathology , Collagen/metabolism , Dietary Proteins/administration & dosage , Hypertension/diagnostic imaging , Hypertension/pathology , Male , Muscle, Smooth, Vascular/diagnostic imaging , Muscle, Smooth, Vascular/pathology , Rats , Rats, Inbred SHR/genetics , Sodium Chloride, Dietary/administration & dosage , Tissue DistributionABSTRACT
1. This study characterizes the K(+) channel(s) underlying charybdotoxin-sensitive hyperpolarization of porcine coronary artery endothelium. 2. Two forms of current-voltage (I/V) relationship were evident in whole-cell patch-clamp recordings of freshly-isolated endothelial cells. In both cell types, iberiotoxin (100 nM) inhibited a current active only at potentials over +50 mV. In the presence of iberiotoxin, charybdotoxin (100 nM) produced a large inhibition in 38% of cells and altered the form of the I/V relationship. In the remaining cells, charybdotoxin also inhibited a current but did not alter the form. 3. Single-channel, outside-out patch recordings revealed a 17.1+/-0.4 pS conductance. Pipette solutions containing 100, 250 and 500 nM free Ca(2+) demonstrated that the open probability was increased by Ca(2+). This channel was blocked by charybdotoxin but not by iberiotoxin or apamin. 4. Hyperpolarizations of intact endothelium elicited by substance P (100 nM; 26.1+/-0.7 mV) were reduced by apamin (100 nM; 17.0+/-1.8 mV) whereas those to 1-ethyl-2-benzimidazolinone (1-EBIO, 600 microM, 21.0+/-0.3 mV) were unaffected (21.7+/-0.8 mV). Substance P, bradykinin (100 nM) and 1-EBIO evoked charybdotoxin-sensitive, iberiotoxin-insensitive whole-cell perforated-patch currents. 5 A porcine homologue of the intermediate-conductance Ca(2+)-activated K(+) channel (IK1) was identified in endothelial cells. 6. In conclusion, porcine coronary artery endothelial cells express an intermediate-conductance Ca(2+)-activated K(+) channel and the IK1 gene product. This channel is opened by activation of the EDHF pathway and likely mediates the charybdotoxin-sensitive component of the EDHF response.