Contribution of two-pore K+ channels to cardiac ventricular action potential revealed using human iPSC-derived cardiomyocytes.

Two-pore K+ (K2p) channels have been described in modulating background conductance as leak channels in different physiological systems. In the heart, the expression of K2p channels is heterogeneous with equivocation regarding their functional role. Our objective was to determine the K2p expression profile and their physiological and pathophysiological contribution to cardiac electrophysiology. Induced pluripotent stem cells (iPSCs) generated from humans were differentiated into cardiomyocytes (iPSC-CMs). mRNA was isolated from these cells, commercial iPSC-CM (iCells), control human heart ventricular tissue (cHVT), and ischemic (iHF) and nonischemic heart failure tissues (niHF). We detected 10 K2p channels in the heart. Comparing quantitative PCR expression of K2p channels between human heart tissue and iPSC-CMs revealed K2p1.1, K2p2.1, K2p5.1, and K2p17.1 to be higher expressed in cHVT, whereas K2p3.1 and K2p13.1 were higher in iPSC-CMs. Notably, K2p17.1 was significantly lower in niHF tissues compared with cHVT. Action potential recordings in iCells after K2p small interfering RNA knockdown revealed prolongations in action potential depolarization at 90% repolarization for K2p2.1, K2p3.1, K2p6.1, and K2p17.1. Here, we report the expression level of 10 human K2p channels in iPSC-CMs and how they compared with cHVT. Importantly, our functional electrophysiological data in human iPSC-CMs revealed a prominent role in cardiac ventricular repolarization for four of these channels. Finally, we also identified K2p17.1 as significantly reduced in niHF tissues and K2p4.1 as reduced in niHF compared with iHF. Thus, we advance the notion that K2p channels are emerging as novel players in cardiac ventricular electrophysiology that could also be remodeled in cardiac pathology and therefore contribute to arrhythmias.NEW & NOTEWORTHY Two-pore K+ (K2p) channels are traditionally regarded as merely background leak channels in myriad physiological systems. Here, we describe the expression profile of K2p channels in human-induced pluripotent stem cell-derived cardiomyocytes and outline a salient role in cardiac repolarization and pathology for multiple K2p channels.

Effects of hydrogen sulfide synthesis inhibitors on posthypoxic ventilatory behavior in the C57BL/6J mouse.

BACKGROUND: H(2)S synthesis inhibitors (HSSI) have been shown to impact respiratory control. For instance, the HSSI hydroxylamine (HA) decreases the respiratory discharge rate from isolated medullary sections, and HA in addition to other HSSIs propargylglycine and amino-oxyacetic acid (AOAA) have been found to reduce hypoxic responsiveness. OBJECTIVES: The aim of this study was to determine if administration of HSSIs could improve respiratory stability in an intact organism prone to recurrent central apneas. METHODS: Saline and HSSI compounds were administered to C57BL/6J mice (n = 24), a strain predisposed to recurrent central apneas, prior to measurement of hypoxic and posthypoxic ventilatory behavior. RESULTS: Administration of HA and AOAA resulted in a significantly smaller percentage of animals expressing one or more apneas during reoxygenation compared to saline control, and animals given AOAA demonstrated a smaller coefficient of variation for frequency during reoxygenation, a marker suggesting greater respiratory stability. This occurred despite varying effects of the three HSSI compounds on hypoxic ventilatory response. CONCLUSIONS: Instability and pause expression are improved by targeting H(2)S synthesis, an effect not predicted by effects on hypoxic responsiveness.

Cardiovascular pharmacology of K2P17.1 (TASK-4, TALK-2) two-pore-domain K+ channels.

K2P17.1 (TASK-4, TALK-2) potassium channels are expressed in the heart and represent potential targets for pharmacological management of atrial and ventricular arrhythmias. Reduced K2P17.1 expression was found in atria and ventricles of heart failure (HF) patients. Modulation of K2P17.1 currents by antiarrhythmic compounds has not been comprehensively studied to date. The objective of this study was to investigate acute effects of clinically relevant antiarrhythmic drugs on human K2P17.1 channels to provide a more complete picture of K2P17.1 electropharmacology. Whole-cell patch clamp and two-electrode voltage clamp electrophysiology was employed to study human K2P17.1 channel pharmacology. K2P17.1 channels expressed in Xenopus laevis oocytes were screened for sensitivity to antiarrhythmic drugs, revealing significant activation by propafenone (+ 296%; 100 µM), quinidine (+ 58%; 100 µM), mexiletine (+ 21%; 100 µM), propranolol (+ 139%; 100 µM), and metoprolol (+ 17%; 100 µM) within 60 min. In addition, the currents were inhibited by amiodarone (- 13%; 100 µM), sotalol (- 10%; 100 µM), verapamil (- 21%; 100 µM), and ranolazine (- 8%; 100 µM). K2P17.1 channels were not significantly affected by ajmaline and carvedilol. Concentration-dependent K2P17.1 activation by propafenone was characterized in more detail. The onset of activation was fast, and current-voltage relationships were not modulated by propafenone. K2P17.1 activation was confirmed in mammalian Chinese hamster ovary cells, revealing 7.8-fold current increase by 100 µM propafenone. Human K2P17.1 channels were sensitive to multiple antiarrhythmic drugs. Differential pharmacological regulation of repolarizing K2P17.1 background K+ channels may be employed for personalized antiarrhythmic therapy.

Physiological genomics identifies genetic modifiers of long QT syndrome type 2 severity.

Congenital long QT syndrome (LQTS) is an inherited channelopathy associated with life-threatening arrhythmias. LQTS type 2 (LQT2) is caused by mutations in KCNH2, which encodes the potassium channel hERG. We hypothesized that modifier genes are partly responsible for the variable phenotype severity observed in some LQT2 families. Here, we identified contributors to variable expressivity in an LQT2 family by using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and whole exome sequencing in a synergistic manner. We found that iPSC-CMs recapitulated the clinical genotype-phenotype discordance in vitro. Importantly, iPSC-CMs derived from the severely affected LQT2 patients displayed prolonged action potentials compared with cells from mildly affected first-degree relatives. The iPSC-CMs derived from all patients with hERG R752W mutation displayed lower IKr amplitude. Interestingly, iPSC-CMs from severely affected mutation-positive individuals exhibited greater L-type Ca2+ current. Whole exome sequencing identified variants of KCNK17 and the GTP-binding protein REM2, providing biologically plausible explanations for this variable expressivity. Genome editing to correct a REM2 variant reversed the enhanced L-type Ca2+ current and prolonged action potential observed in iPSC-CMs from severely affected individuals. Thus, our findings showcase the power of combining complementary physiological and genomic analyses to identify genetic modifiers and potential therapeutic targets of a monogenic disorder. Furthermore, we propose that this strategy can be deployed to unravel myriad confounding pathologies displaying variable expressivity.

Brugada syndrome disease phenotype explained in apparently benign sodium channel mutations.

BACKGROUND: Brugada syndrome (BrS) is an arrhythmogenic disorder that has been linked to mutations in SCN5A, the gene encoding for the pore-forming α-subunit of the cardiac sodium channel. Typically, BrS mutations in SCN5A result in a reduction of sodium current with some mutations even exhibiting a dominant-negative effect on wild-type (WT) channels, thus leading to an even more prominent decrease in current amplitudes. However, there is also a category of apparently benign (atypical) BrS SCN5A mutations that in vitro demonstrates only minor biophysical defects. It is therefore not clear how these mutations produce a BrS phenotype. We hypothesized that similar to dominant-negative mutations, atypical mutations could lead to a reduction in sodium currents when coexpressed with WT to mimic the heterozygous patient genotype. METHODS AND RESULTS: WT and atypical BrS mutations were coexpressed in Human Embryonic Kidney-293 cells, showing a reduction in sodium current densities similar to typical BrS mutations. Importantly, this reduction in sodium current was also seen when the atypical mutations were expressed in rat or human cardiomyocytes. This decrease in current density was the result of reduced surface expression of both mutant and WT channels. CONCLUSIONS: Taken together, we have shown how apparently benign SCN5A BrS mutations can lead to the ECG abnormalities seen in patients with BrS through an induced defect that is only present when the mutations are coexpressed with WT channels. Our work has implications for risk management and stratification for some SCN5A-implicated BrS patients.

Identification of novel mouse genes conferring posthypoxic pauses.

Although central to the susceptibility of adult diseases characterized by abnormal rhythmogenesis, characterizing the genes involved is a challenge. We took advantage of the C57BL/6J (B6) trait of hypoxia-induced periodic breathing and its absence in the C57BL/6J-Chr 1(A/J)/NaJ chromosome substitution strain to test the feasibility of gene discovery for this abnormality. Beginning with a genetic and phenotypic analysis of an intercross study between these strains, we discovered three quantitative trait loci (QTLs) on mouse chromosome 1, with phenotypic effects. Fine-mapping reduced the genomic intervals and gene content, and the introgression of one QTL region back onto the C57BL/6J-Chr 1(A/J)/NaJ restored the trait. mRNA expression of non-synonymous genes in the introgressed region in the medulla and pons found evidence for differential expression of three genes, the highest of which was apolipoprotein A2, a lipase regulator; the apo a2 peptide fragment (THEQLTPLVR), highly expressed in the liver, was expressed in low amounts in the medulla but did not correlate with trait expression. This work directly demonstrates the impact of elements on mouse chromosome 1 in respiratory rhythmogenesis.

Morphological differences of the carotid body among C57/BL6 (B6), A/J, and CSS B6A1 mouse strains.

The C57/BL6 (B6) mouse strain exhibits post-hypoxic frequency decline and periodic breathing, as well as greater amount of irregular breathing during rest in comparison to the A/J and to the B6a1, a chromosomal substitution strain whereby the A/J chromosome 1 is bred onto the B6 background (Han et al., 2002; Yamauchi et al., 2008a,b). The hypothesis was that morphological differences in the carotid body would associate with such trait variations. After confirming strain differences in post-hypoxic ventilatory behavior, histological examination (n=8 in each group) using hematoxylin and eosin (H&E) staining revealed equivalent, well-defined tissue structure at the bifurcation of the carotid arteries, an active secretory parenchyma (type I cells) from the supportive stromal tissue, and clustering of type I cells in all three strains. Tyrosine hydroxylase (TH) immunohistochemical staining revealed a typical organization of type I cells and neurovascular components into glomeruli in all three strains. Image analysis from 5 µm sections from each strain generated a series of cytological metrics. The percent carotid body composition of TH+ type I cells in the A/J, B6 and B6a1 was 20±4%, 39±3%, and 44±3%, respectively (p=0.00004). However, cellular organization in terms of density and ultrastructure in the B6a1 is more similar to the B6 than to the A/J. These findings indicate that genetic mechanisms that produce strain differences in ventilatory function do not associate with carotid body structure or tyrosine hydroxylase morphology, and that A/J chromosome 1 does not contribute much to B6 carotid body morphology.

Ventilatory behavior and carotid body morphology of Brown Norway and Sprague Dawley rats.

Differences in acute ventilatory behavior are associated with carotid body (CB) structural and immunohistologic profiles in some, but not all, reports. Brown Norway (BN) rats exhibit lower acute ventilatory responses to hypoxia and hypercapnia compared to Sprague Dawley (SD) rats. We hypothesized that BN rats possess CB with fewer glomus cells. Ventilation was recorded in 6-month-old BN and SD rats exposed to hypoxia-reoxygenation and hypercapnia. Extracted CBs were examined using H&E staining, and immunohistochemistry with antibodies specific for tyrosine hydroxylase (TH), neural nitric oxide synthase (nNOS), and pyruvate dehydrogenase (PD). Sections were analyzed for cell and immunostaining density. SD displayed greater hypoxic and hypercapnic responses, and post-hypoxic short term potentiation, whereas BN exhibited post-hypoxic frequency decline. Contrary to our hypothesis, BN demonstrated a denser arrangement of glomus cells with a larger TH stained area (31.7% BN, 22.6% SD; p<0.0001), and nNOS stained area (37.3% BN, 32.1%; SD; p=0.01). Hence, respiratory phenotype does not correlate intuitively with these anatomic features.