Contributions of bone morphogenetic proteins in cardiac repair cells in three-dimensional in vitro models and angiogenesis.

One of the main efforts in myocardial tissue engineering is towards designing cardiac tissues able to rescue the reduction in heart function once implanted at the site of myocardial infarction. To date, the efficiency of this approach in preclinical applications is limited in part by our incomplete understanding of the inflammatory environment known to be present at the site of myocardial infarct and by poor vascularization. It was recently reported that polarized macrophages known to be present at the site of myocardial infarction secrete bone morphogenetic proteins (BMPs)-2 and -4 causing changes in the expression of cardiac proteins in a 2D in vitro model. Here, these findings were extended towards cardiac tissues composed of human embryonic stem cell derived cardiomyocytes embedded in collagen gel. By preconditioning cardiac tissues with BMPs, constructs were obtained with enhanced expression of cardiac markers. Additionally, after BMP preconditioning, the resulting cardiac-tissues were able to sustain diffusion of the BMPs with the added benefit of supporting human umbilical vein endothelial cell tube formation. Here, a model is proposed of cardiac tissues preconditioned with BMPs that results in stimulation of cardiomyocyte function and diffusion of BMPs able to support angiogenesis. This platform represents a step towards the validation of more complex bioengineered constructs for in vivo applications.

The Impact of Heterozygous KCNK3 Mutations Associated With Pulmonary Arterial Hypertension on Channel Function and Pharmacological Recovery.

BACKGROUND: Heterozygous loss of function mutations in the KCNK3 gene cause hereditary pulmonary arterial hypertension (PAH). KCNK3 encodes an acid-sensitive potassium channel, which contributes to the resting potential of human pulmonary artery smooth muscle cells. KCNK3 is widely expressed in the body, and dimerizes with other KCNK3 subunits, or the closely related, acid-sensitive KCNK9 channel. METHODS AND RESULTS: We engineered homomeric and heterodimeric mutant and nonmutant KCNK3 channels associated with PAH. Using whole-cell patch-clamp electrophysiology in human pulmonary artery smooth muscle and COS7 cell lines, we determined that homomeric and heterodimeric mutant channels in heterozygous KCNK3 conditions lead to mutation-specific severity of channel dysfunction. Both wildtype and mutant KCNK3 channels were activated by ONO-RS-082 (10 µmol/L), causing cell hyperpolarization. We observed robust gene expression of KCNK3 in healthy and familial PAH patient lungs, but no quantifiable expression of KCNK9, and demonstrated in functional studies that KCNK9 minimizes the impact of select KCNK3 mutations when the 2 channel subunits co-assemble. CONCLUSIONS: Heterozygous KCNK3 mutations in PAH lead to variable loss of channel function via distinct mechanisms. Homomeric and heterodimeric mutant KCNK3 channels represent novel therapeutic substrates in PAH. Pharmacological and pH-dependent activation of wildtype and mutant KCNK3 channels in pulmonary artery smooth muscle cells leads to membrane hyperpolarization. Co-assembly of KCNK3 with KCNK9 subunits may provide protection against KCNK3 loss of function in tissues where both KCNK9 and KCNK3 are expressed, contributing to the lung-specific phenotype observed clinically in patients with PAH because of KCNK3 mutations.

Directed Differentiation of Human Pluripotent Stem Cells to Microglia.

Microglia, the immune cells of the brain, are crucial to proper development and maintenance of the CNS, and their involvement in numerous neurological disorders is increasingly being recognized. To improve our understanding of human microglial biology, we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs). Further differentiation of the progenitors resulted in ramified microglia with highly motile processes, expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG) and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca2+ transients, whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell lines.

Dynamic subunit stoichiometry confers a progressive continuum of pharmacological sensitivity by KCNQ potassium channels.

Voltage-gated KCNQ1 (Kv7.1) potassium channels are expressed abundantly in heart but they are also found in multiple other tissues. Differential coassembly with single transmembrane KCNE beta subunits in different cell types gives rise to a variety of biophysical properties, hence endowing distinct physiological roles for KCNQ1-KCNEx complexes. Mutations in either KCNQ1 or KCNE1 genes result in diseases in brain, heart, and the respiratory system. In addition to complexities arising from existence of five KCNE subunits, KCNE1 to KCNE5, recent studies in heterologous systems suggest unorthodox stoichiometric dynamics in subunit assembly is dependent on KCNE expression levels. The resultant KCNQ1-KCNE channel complexes may have a range of zero to two or even up to four KCNE subunits coassembling per KCNQ1 tetramer. These findings underscore the need to assess the selectivity of small-molecule KCNQ1 modulators on these different assemblies. Here we report a unique small-molecule gating modulator, ML277, that potentiates both homomultimeric KCNQ1 channels and unsaturated heteromultimeric (KCNQ1)4(KCNE1)n (n < 4) channels. Progressive increase of KCNE1 or KCNE3 expression reduces efficacy of ML277 and eventually abolishes ML277-mediated augmentation. In cardiomyocytes, the slowly activating delayed rectifier potassium current, or IKs, is believed to be a heteromultimeric combination of KCNQ1 and KCNE1, but it is not entirely clear whether IKs is mediated by KCNE-saturated KCNQ1 channels or by channels with intermediate stoichiometries. We found ML277 effectively augments IKs current of cultured human cardiomyocytes and shortens action potential duration. These data indicate that unsaturated heteromultimeric (KCNQ1)4(KCNE1)n channels are present as components of IKs and are pharmacologically distinct from KCNE-saturated KCNQ1-KCNE1 channels.

Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics.

Understanding the basis for differential responses to drug therapies remains a challenge despite advances in genetics and genomics. Induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to investigate the pharmacology of disease processes in therapeutically and genetically relevant primary cell types in vitro and to interweave clinical and basic molecular data. We report here the derivation of iPSCs from a long QT syndrome patient with complex genetics. The proband was found to have a de novo SCN5A LQT-3 mutation (F1473C) and a polymorphism (K897T) in KCNH2, the gene for LQT-2. Analysis of the biophysics and molecular pharmacology of ion channels expressed in cardiomyocytes (CMs) differentiated from these iPSCs (iPSC-CMs) demonstrates a primary LQT-3 (Na(+) channel) defect responsible for the arrhythmias not influenced by the KCNH2 polymorphism. The F1473C mutation occurs in the channel inactivation gate and enhances late Na(+) channel current (I(NaL)) that is carried by channels that fail to inactivate completely and conduct increased inward current during prolonged depolarization, resulting in delayed repolarization, a prolonged QT interval, and increased risk of fatal arrhythmia. We find a very pronounced rate dependence of I(NaL) such that increasing the pacing rate markedly reduces I(NaL) and, in addition, increases its inhibition by the Na(+) channel blocker mexiletine. These rate-dependent properties and drug interactions, unique to the proband's iPSC-CMs, correlate with improved management of arrhythmias in the patient and provide support for this approach in developing patient-specific clinical regimens.

Biophysical properties of slow potassium channels in human embryonic stem cell derived cardiomyocytes implicate subunit stoichiometry.

Human embryonic stem cells (hESCs) are an important cellular model for studying ion channel function in the context of a human cardiac cell and will provide a wealth of information about both heritable arrhythmias and acquired electrophysiological disorders. However, detailed electrophysiological characterization of the important cardiac ion channels has been so far overlooked. Because mutations in the gene for the I(Ks) α subunit, KCNQ1, constitute the majority of long QT syndrome (LQT-1) cases, we have carried out a detailed biophysical analysis of this channel expressed in hESCs to establish baseline I(Ks) channel biophysical properties in cardiac myocytes derived from hESCs (hESC-CMs). I(Ks) channels are heteromultimeric proteins consisting of four identical α-subunits (KCNQ1) assembled with auxiliary ß-subunits (KCNE1). We found that the half-maximal I(Ks) activation voltage in hESC-CMs and in myocytes derived from human induced pluripotent stems cells (hiPSC-CMs) falls between that of KCNQ1 channels expressed alone and with full complement of KCNE1, the major KCNE subunit expressed in hESC-CMs as shown by qPCR analysis. Overexpression of KCNE1 by transfection of hESC-CMs markedly shifted and slowed native I(Ks) activation implying assembly of additional KCNE1 subunits with endogenous channels. Our results in hESC-CMs, which indicate an I(Ks) subunit stoichiometry that can be altered by variable KCNE1 expression, suggest the possibility for variable I(Ks) function in the developing heart, in different tissues in the heart, and in disease. This establishes a new baseline for I(Ks) channel properties in myocytes derived from pluripotent stem cells and will guide future studies in patient-specific hiPSCs.

O-glycosylation of the cardiac I(Ks) complex.

Post-translational modifications of the KCNQ1­KCNE1 (Kv7) K+ channel complex are vital for regulation of the cardiac IKs current and action potential duration. Here, we show the KCNE1 regulatory subunit is O-glycosylated with mucin-type glycans in vivo. As O-linked glycosylation sites are not recognizable by sequence gazing, we designed a novel set of glycosylation mutants and KCNE chimeras and analysed their glycan content using deglycosylation enzymes. Our results show that KCNE1 is exclusively O-glycosylated at Thr-7, which is also required for N-glycosylation at Asn-5. For wild type KCNE1, the overlapping N- and O-glycosylation sites are innocuous for subunit biogenesis; however, mutation of Thr-7 to a non-hydroxylated residue yielded mostly unglycosylated protein and a small fraction of mono-N-glycosylated protein. The compounded hypoglycosylation was equally deleterious for KCNQ1­KCNE1 cell surface expression, demonstrating that KCNE1 O-glycosylation is a post-translational modification that is integral for the proper biogenesis and anterograde trafficking of the cardiac IKs complex. The enzymatic assays and panel of glycosylation mutants used here will be valuable for identifying the different KCNE1 glycoforms in native cells and determining the roles N- and O-glycosylation play in KCNQ1­KCNE1 function and localization in cardiomyocytes,

The cardiac IKs potassium channel macromolecular complex includes the phosphodiesterase PDE4D3.

The cardiac I(Ks) potassium channel is a macromolecular complex consisting of alpha-(KCNQ1) and beta-subunits (KCNE1) and the A kinase-anchoring protein (AKAP) Yotiao (AKAP-9), which recruits protein kinase A) and protein phosphatase 1 to the channel. Here, we have tested the hypothesis that specific cAMP phosphodiesterase (PDE) isoforms of the PDE4D family that are expressed in the heart are also part of the I(Ks) signaling complex and contribute to its regulation by cAMP. PDE4D isoforms co-immunoprecipitated with I(Ks) channels in hearts of mice expressing the I(Ks) channel. In myocytes isolated from these mice, I(Ks) was increased by pharmacological PDE inhibition. PDE4D3, but not PDE4D5, co-immunoprecipitated with the I(Ks) channel only in Chinese hamster ovary cells co-expressing AKAP-9, and PDE4D3, but not PDE4D5, co-immunoprecipitated with AKAP-9. Functional experiments in Chinese hamster ovary cells expressing AKAP-9 and either PDE4D3 or PDE4D5 isoforms revealed modulation of the I(Ks) response to cAMP by PDE4D3 but not PDE4D5. We conclude that PDE4D3, like protein kinase A and protein phosphatase 1, is recruited to the I(Ks) channel via AKAP-9 and contributes to its critical regulation by cAMP.

Adrenergic regulation of a key cardiac potassium channel can contribute to atrial fibrillation: evidence from an I Ks transgenic mouse.

Inherited gain-of-function mutations of genes coding for subunits of the heart slow potassium (I Ks) channel can cause familial atrial fibrillation (AF). Here we consider a potentially more prevalent mechanism and hypothesize that beta-adrenergic receptor (beta-AR)-mediated regulation of the I Ks channel, a natural gain-of-function pathway, can also lead to AF. Using a transgenic I Ks channel mouse model, we studied the role of the channel and its regulation by beta-AR stimulation on atrial arrhythmias. In vivo administration of isoprenaline (isoproterenol) predisposes I Ks channel transgenic mice but not wild-type (WT) littermates that lack I Ks to prolonged atrial arrhythmias. Patch-clamp analysis demonstrated expression and isoprenaline-mediated regulation of I Ks in atrial myocytes from transgenic but not WT littermates. Furthermore, computational modelling revealed that beta-AR stimulation-dependent accumulation of open I Ks channels accounts for the pro-arrhythmic substrate. Our results provide evidence that beta-AR-regulated I Ks channels can play a role in AF and imply that specific I Ks deregulation, perhaps through disruption of the I Ks macromolecular complex necessary for beta-AR-mediated I Ks channel regulation, may be a novel therapeutic strategy for treating this most common arrhythmia.

Role of sodium channels in propagation in heart muscle: how subtle genetic alterations result in major arrhythmic disorders.

Sodium channels play a crucial role in initiation, propagation, and maintenance of cardiac excitation throughout the heart. Indeed, dysfunctional sodium channels have been shown to be responsible for several inherited cardiac electrical disorders, such as Long QT and Brugada syndromes (BrS), potentially leading to fatal arrhythmic events. Genetic approaches and functional experiments using heterologous systems have enabled the characterization of the molecular determinants involved in these disorders and their consequences on ion channel function. The improved understanding of the mechanisms leading to these cardiac arrhythmic events represents a first step in the development of therapeutic treatments.

Stabilization of cardiac ryanodine receptor prevents intracellular calcium leak and arrhythmias.

Catecholaminergic polymorphic ventricular tachycardia is a form of exercise-induced sudden cardiac death that has been linked to mutations in the cardiac Ca2+ release channel/ryanodine receptor (RyR2) located on the sarcoplasmic reticulum (SR). We have shown that catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutations significantly decrease the binding affinity for calstabin-2 (FKBP12.6), a subunit that stabilizes the closed state of the channel. We have proposed that RyR2-mediated diastolic SR Ca2+ leak triggers ventricular tachycardia (VT) and sudden cardiac death. In calstabin-2-deficient mice, we have now documented diastolic SR Ca2+ leak, monophasic action potential alternans, and bidirectional VT. Calstabin-deficient cardiomyocytes exhibited SR Ca2+ leak-induced aberrant transient inward currents in diastole consistent with delayed after-depolarizations. The 1,4-benzothiazepine JTV519, which increases the binding affinity of calstabin-2 for RyR2, inhibited the diastolic SR Ca2+ leak, monophasic action potential alternans and triggered arrhythmias. Our data suggest that calstabin-2 deficiency is as a critical mediator of triggers that initiate cardiac arrhythmias.

Autonomic control of cardiac action potentials: role of potassium channel kinetics in response to sympathetic stimulation.

I(Ks), the slowly activating component of the delayed rectifier current, plays a major role in repolarization of the cardiac action potential (AP). Genetic mutations in the alpha- (KCNQ1) and beta- (KCNE1) subunits of I(Ks) underlie Long QT Syndrome type 1 and 5 (LQT-1 and LQT-5), respectively, and predispose carriers to the development of polymorphic ventricular arrhythmias and sudden cardiac death. beta-adrenergic stimulation increases I(Ks) and results in rate dependent AP shortening, a control system that can be disrupted by some mutations linked to LQT-1 and LQT-5. The mechanisms by which I(Ks) regulates action potential duration (APD) during beta-adrenergic stimulation at different heart rates are not known, nor are the consequences of mutation induced disruption of this regulation. Here we develop a complementary experimental and theoretical approach to address these questions. We reconstituted I(Ks) in CHO cells (ie, KCNQ1 coexpressed with KCNE1 and the adaptator protein Yotiao) and quantitatively examined the effects of beta-adrenergic stimulation on channel kinetics. We then developed theoretical models of I(Ks) in the absence and presence of beta-adrenergic stimulation. We simulated the effects of sympathetic stimulation on channel activation (speeding) and deactivation (slowing) kinetics on the whole cell action potential under different pacing conditions. The model suggests these kinetic effects are critically important in rate-dependent control of action potential duration. We also investigate the effects of two LQT-5 mutations that alter kinetics and impair sympathetic stimulation of I(Ks) and show the likely mechanism by which they lead to tachyarrhythmias and indicate a distinct role of I(KS) kinetics in this electrical dysfunction. The full text of this article is available online at http://circres.ahajournals.org.

Expression of human ERG K+ channels in the mouse heart exerts anti-arrhythmic activity.

OBJECTIVE: The K(+) channel encoded by the human ether-a-go-go-related gene (HERG) is crucial for repolarization in the human heart. In order to investigate the impact of HERG current (I(Kr)) on the incidence of cardiac arrhythmias, we generated a transgenic mouse expressing HERG specifically in the heart. METHODS AND RESULTS: ECG recordings at baseline showed no obvious difference between transgenic and wild-type (WT) mice with the exception of the T wave, which was more negative in transgenic mice than in WT mice. E4031 (20 mg/kg) prolonged the QTc interval and flattened the T wave in transgenic mice, but not in WT mice. Injection of BaCl(2) (25 mg/kg) induced short runs of ventricular tachycardia in 9/10 WT mice, but not in transgenic animals. Atrial pacing reproducibly induced atrial tachyarrhythmias in 11/15 WT mice. In contrast, atrial arrhythmia was inducible in only 2/11 transgenic mice. When pretreated with dofetilide (10 mg/kg), transgenic mice were as sensitive to experimental arrhythmias as WT mice. Microelectrode studies showed that atrial action potentials have a steeper slope of duration-rate adaptation in WT than in transgenic mice. Transgenic mice were also characterized by a post-repolarization refractoriness, which could result from the substantial amount of I(Kr) subsisting after repolarization as assessed with action potential-clamp experiments and simulations with a model of the transgenic mouse action potential. CONCLUSION: HERG expression in the mouse heart can protect against experimental induction of arrhythmias. This is the first report of such a protective effect of HERG in vivo.

Overexpression of beta2-adrenergic receptors cAMP-dependent protein kinase phosphorylates and modulates slow delayed rectifier potassium channels expressed in murine heart: evidence for receptor/channel co-localization.

The cardiac slow delayed rectifier potassium channel (IKs), comprised of (KCNQ1) and beta (KCNE1) subunits, is regulated by sympathetic nervous stimulation, with activation of beta-adrenergic receptors PKA phosphorylating IKs channels. We examined the effects of 2-adrenergic receptors (beta2-AR) on IKs in cardiac ventricular myocytes from transgenic mice expressing fusion proteins of IKs subunits and hbeta2-ARs. KCNQ1 and beta2-ARs were localized to the same subcellular regions, sharing intimate localization within nanometers of each other. In IKs/B2-AR myocytes, IKs density was increased, and activation shifted in the hyperpolarizing direction; IKs was not further modulated by exposure to isoproterenol, and KCNQ1 was found to be PKA-phosphorylated. Conversely, beta2-AR overexpression did not affect L-type calcium channel current (ICaL) under basal conditions with ICaL remaining responsive to cAMP. These data indicate intimate association of KCNQ1 and beta2-ARs and that beta2-AR signaling can modulate the function of IKs channels under conditions of increased beta2-AR expression, even in the absence of exogenous beta-AR agonist.

p11, an annexin II subunit, an auxiliary protein associated with the background K+ channel, TASK-1.

TASK-1 belongs to the 2P domain K+ channel family and is the prototype of background K+ channels that set the resting membrane potential and tune action potential duration. Its activity is highly regulated by hormones and neurotransmitters. Although numerous auxiliary proteins have been described to modify biophysical, pharmacological and expression properties of different voltage- and Ca2+-sensitive K+ channels, none of them is known to modulate 2P domain K+ channel activity. We show here that p11 interacts specifically with the TASK-1 K+ channel. p11 is a subunit of annexin II, a cytoplasmic protein thought to bind and organize specialized membrane cytoskeleton compartments. This association with p11 requires the integrity of the last three C-terminal amino acids, Ser-Ser-Val, in TASK-1. Using series of C-terminal TASK-1 deletion mutants and several TASK-1-GFP chimeras, we demonstrate that association with p11 is essential for trafficking of TASK-1 to the plasma membrane. p11 association with the TASK-1 channel masks an endoplasmic reticulum retention signal identified as Lys-Arg-Arg that precedes the Ser-Ser-Val sequence.