Search

Atrial fibrillation (AF) is a condition in which the electrical signals in the upper heart chambers (atria) are rapid and disorganized, producing an irregular and chaotical heartbeat. The sinus rhythm should be between 60 to 100 bpm at rest, while the heart rhythm in AF patients may be over 140 bpm. Either structural and electro-mechanical remodeling of the atrial tissue underlies the perpetuation and evolution of AF from the paroxysmal to persistent form. Unravelling the different pathological pathways involved in AF that lead to arrhythmogenesis and atrial remodeling is needed to discovery new and effective therapeutic approaches. A variety of drugs are available to convert and maintain the AF patient in a normal sinus rhythm; however, these strategies have limited chances of success or fail with the progression of AF to more persistent/permanent forms. Consequently, it is necessary to find new therapeutic targets for the relief of persistent or chronic AF forms, as well as the development of new and more effective pharmacological tools. The atrial specific two-pore domain K+ channels (K2P) constitute the background K+ current on atrial cardiomyocytes and modulate cell excitability emerging as novel targets in this disease and avoiding ventricle side effects. Moreover, several antiarrhythmic drugs used in AF treatment exert their mechanism of action in part by modulation of K2P channels. Thus far, TWIK-1, TREK-1, TASK-1, TASK-2 and TASK-3 channel have been identified as responsible for background currents IK2P current in atrial cells; however, it is not excluded that other K2PX subunits or subfamilies have physiological roles in atria. To date, a great diversity openers, activators and blockers of K2P channel have been identified, particularly those targeting TASK and TREK channels. Several studies have demonstrated that the expression of TWIK-1, TREK-1, TASK-1, TASK-2 and TASK-3 are dysregulated in AF and their pharmacology rescue could suppose a novel therapy in AF. The main objective is to examine the regulation of K2P channels and the current K2P channels pharmacological modulators for AF treatment., Competing Interests: The author declares no conflict of interest., (© 2022 The Author(s). Published by IMR Press.)

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety assessment models is a critical step during drug development. Currently, cardiac safety assessment is still highly dependent on animal studies. However, animal models are plagued by poor translational specificity to humans due to species-specific differences, particularly in terms of cardiac electrophysiological characteristics. Thus, there is an urgent need to develop a reliable, efficient, and human-based model for preclinical cardiac safety assessment. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as an invaluable in vitro model for drug-induced cardiotoxicity screening and disease modeling. hiPSC-CMs can be obtained from individuals with diverse genetic backgrounds and various diseased conditions, making them an ideal surrogate to assess drug-induced cardiotoxicity individually. Therefore, methodologies to comprehensively investigate the functional characteristics of hiPSC-CMs need to be established. In this protocol, we detail various functional assays that can be assessed on hiPSC-CMs, including the measurement of contractility, field potential, action potential, and calcium handling. Overall, the incorporation of hiPSC-CMs into preclinical cardiac safety assessment has the potential to revolutionize drug development.

The large K + channel functional diversity in the pulmonary vasculature results from the multitude of genes expressed encoding K + channels, alternative RNA splicing, the post-transcriptional modifications, the presence of homomeric or heteromeric assemblies of the pore-forming α-subunits and the existence of accessory β-subunits modulating the functional properties of the channel. K + channels can also be regulated at multiple levels by different factors controlling channel activity, trafficking, recycling and degradation. The activity of these channels is the primary determinant of membrane potential (Em) in pulmonary artery smooth muscle cells (PASMC), providing an essential regulatory mechanism to dilate or contract pulmonary arteries (PA). K + channels are also expressed in pulmonary artery endothelial cells (PAEC) where they control resting Em, Ca 2+ entry and the production of different vasoactive factors. The activity of K + channels is also important in regulating the population and phenotype of PASMC in the pulmonary vasculature, since they are involved in cell apoptosis, survival and proliferation. Notably, K + channels play a major role in the development of pulmonary hypertension (PH). Impaired K + channel activity in PH results from: 1) loss of function mutations, 2) downregulation of its expression, which involves transcription factors and microRNAs, or 3) decreased channel current as a result of increased vasoactive factors (e.g., hypoxia, 5-HT, endothelin-1 or thromboxane), exposure to drugs with channel-blocking properties, or by a reduction in factors that positively regulate K + channel activity (e.g., NO and prostacyclin). Restoring K + channel expression, its intracellular trafficking and the channel activity is an attractive therapeutic strategy in PH., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Congenital long QT syndrome type 2 (LQT2) results from KCNH2 mutations that cause loss of Kv11.1 channel function which can lead to arrhythmias, syncope, and sudden death. Here, we generated three human-induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) of two LQT2 patients carrying pathogenic variants (c.1714G > A and c.2960del) and one LQT2 patient carrying a variant of uncertain significance (c.1870A > T) in KCNH2. All lines show typical iPSC morphology, high expression of pluripotent markers, normal karyotype, and differentiate into three germ layers in vitro. These lines are valuable resources for studying the pathological mechanisms of LQTS caused by caused by KCNH2 mutations., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Rationale: Cigarette smoke is considered the chief leading cause of chronic obstructive pulmonary disease (COPD). Its impact on the progressive deterioration of airways has been extensively studied, but its direct effects on the pulmonary vasculature are less known. Objectives: To prove that pulmonary arterial remodeling in patients with COPD is not just a consequence of alveolar hypoxia but also due to the direct effects of cigarette smoke on the pulmonary vascular bed. Methods: We have used different molecular and cell biology approaches, as well as traction force microscopy, wire myography, and patch-clamp techniques in human cells and freshly isolated pulmonary arteries. In addition, we relied on in vivo models and human samples to analyze the effects of cigarette smoke on pulmonary vascular tone alterations. Measurements and Main Results: Cigarette smoke extract exposure directly promoted a hypertrophic, senescent phenotype that in turn contributed, through the secretion of inflammatory molecules, to an increase in the proliferative potential of nonexposed cells. Interestingly, these effects were significantly reversed by antioxidants. Furthermore, cigarette smoke extract affected cell contractility and dysregulated the expression and activity of the voltage-gated K + channel Kv7.4. This contributed to the impairment of vasoconstriction and vasodilation responses. Most importantly, the levels of this channel were diminished in the lungs of smoke-exposed mice, smokers, and patients with COPD. Conclusions: Cigarette smoke directly contributes to pulmonary arterial remodeling through increased cell senescence, as well as vascular tone alterations because of diminished levels and function in the Kv7.4 channel. Strategies targeting these pathways may lead to novel therapies for COPD.

Background and Purpose: K V 1.3 channels are expressed in vascular smooth muscle cells (VSMCs), where they contribute to proliferation rather than contraction and participate in vascular remodelling. K V 1.3 channels are also expressed in macrophages, where they assemble with K V 1.5 channels (K V 1.3/K V 1.5), whose activation generates a K V current. In macrophages, the K V 1.3/K V 1.5 ratio is increased by classical activation (M1). Whether these channels are involved in angiotensin II (AngII)-induced vascular remodelling, and whether they can modulate the macrophage phenotype in hypertension, remains unknown. We characterized the role of K V 1.3 channels in vascular damage in hypertension., Experimental Approach: We used AngII-infused mice treated with two selective K V 1.3 channel inhibitors (HsTX[R14A] and [EWSS]ShK). Vascular function and structure were measured using wire and pressure myography, respectively. VSMC and macrophage electrophysiology were studied using the patch-clamp technique; gene expression was analysed using RT-PCR., Key Results: AngII increased K V 1.3 channel expression in mice aorta and peritoneal macrophages which was abolished by HsTX[R14A] treatment. K V 1.3 inhibition did not prevent hypertension, vascular remodelling, or stiffness but corrected AngII-induced macrophage infiltration and endothelial dysfunction in the small mesenteric arteries and/or aorta, via a mechanism independent of electrophysiological changes in VSMCs. AngII modified the electrophysiological properties of peritoneal macrophages, indicating an M1-like activated state, with enhanced expression of proinflammatory cytokines that induced endothelial dysfunction. These effects were prevented by K V 1.3 blockade., Conclusions and Implications: We unravelled a new role for K V 1.3 channels in the macrophage-dependent endothelial dysfunction induced by AngII in mice which might be due to modulation of macrophage phenotype., (© 2021 The British Pharmacological Society.)

Background and Purpose: Arterial diameter is dictated by the contractile state of the vascular smooth muscle cells (VSMCs), which is modulated by direct and indirect inputs from endothelial cells (ECs). Modulators of KCNQ-encoded k V 7 channels have considerable impact on arterial diameter and these channels are known to be expressed in VSMCs but not yet defined in ECs. However, expression of k V 7 channels in ECs would add an extra level of vascular control. This study aims to characterize the expression and function of K V 7 channels within rat mesenteric artery ECs. Experimental Approach: In rat mesenteric artery, KCNQ transcript and K V 7 channel protein expression were determined via RT-qPCR, immunocytochemistry, immunohistochemistry and immunoelectron microscopy. Wire myography was used to determine vascular reactivity. Key Results: KCNQ transcript was identified in isolated ECs and VSMCs. K V 7.1, K V 7.4 and K V 7.5 protein expression was determined in both isolated EC and VSMC and in whole vessels. Removal of ECs attenuated vasorelaxation to two structurally different K V 7.2-5 activators S-1 and ML213. K IR 2 blockers ML133, and BaCl 2 also attenuated S-1 or ML213-mediated vasorelaxation in an endothelium-dependent process. K V 7 inhibition attenuated receptor-dependent nitric oxide (NO)-mediated vasorelaxation to carbachol, but had no impact on relaxation to the NO donor, SNP. Conclusion and Implications: In rat mesenteric artery ECs, K V 7.4 and K V 7.5 channels are expressed, functionally interact with endothelial K IR 2.x channels and contribute to endogenous eNOS-mediated relaxation. This study identifies K V 7 channels as novel functional channels within rat mesenteric ECs and suggests that these channels are involved in NO release from the endothelium of these vessels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Baldwin, Sandow, Mondéjar-Parreño, Stott and Greenwood.)

K + channels play a fundamental role regulating membrane potential of pulmonary artery (PA) smooth muscle cells and their impairment is a common feature in pulmonary arterial hypertension (PAH). K + voltage-gated channel subfamily Q ( KCNQ1-5 ) or Kv7 channels and their regulatory subunits subfamily E (KCNE) regulatory subunits are known to regulate vascular tone, but whether Kv7 channel function is impaired in PAH and how this can affect the rationale for targeting Kv7 channels in PAH remains unknown. Here, we have studied the role of Kv7/KCNE subunits in rat PA and their possible alteration in PAH. Using the patch-clamp technique, we found that the total K + current is reduced in PA smooth muscle cells from pulmonary hypertension animals (SU5416 plus hypoxia) and Kv7 currents made a higher contribution to the net K + current. Likewise, enhanced vascular responses to Kv7 channel modulators were found in pulmonary hypertension rats. Accordingly, KCNE4 subunit was highly upregulated in lungs from pulmonary hypertension animals and patients. Additionally, Kv7 channel activity was enhanced in the presence of Kv1.5 and TASK-1 channel inhibitors and this was associated with an increased KCNE4 membrane abundance. Compared with systemic arteries, PA showed a poor response to Kv7 channel modulators which was associated with reduced expression and membrane abundance of Kv7.4 and KCNE4. Our data indicate that Kv7 channel function is preserved and KCNE4 is upregulated in PAH. Therefore, compared with other downregulated channels, the contribution of Kv7 channels is increased in PAH resulting in an enhanced sensitivity to Kv7 channel modulators. This study provides insight into the potential usefulness of targeting Kv7 channels in PAH.

Background and Purpose: The NO/cGMP pathway represents a major physiological signalling controlling tone in pulmonary arteries (PA), and drugs activating this pathway are used to treat pulmonary arterial hypertension. K v channels expressed in PA smooth muscle cells (PASMCs) are key determinants of vascular tone. We aimed to analyse the contribution of K v 1.5 and K v 7 channels in the electrophysiological and vasodilating effects evoked by NO donors and the GC stimulator riociguat in PA., Experimental Approach: K v currents were recorded in isolated rat PASMCs using the patch-clamp technique. Vascular reactivity was assessed in a wire myograph., Key Results: The NO donors diethylamine NONOate diethylammonium (DEA-NO) and sodium nitroprusside hyperpolarized the membrane potential and induced a bimodal effect on K v currents (augmenting the current between -40 and -10 mV and decreasing it at more depolarized potentials). The hyperpolarization and the enhancement of the current were suppressed by K v 7 channel inhibitors and by the GC inhibitor ODQ but preserved when K v 1.5 channels were inhibited. Additionally, DEA-NO enhanced K v 7.5 currents in COS7 cells expressing the KCNQ5 gene. Riociguat increased K v currents at all potentials ≥-40 mV and induced membrane hyperpolarization. Both effects were prevented by K v 7 inhibition. Likewise, PA relaxation induced by NO donors and riociguat was attenuated by K v 7 inhibitors., Conclusions and Implications: NO donors and riociguat enhance K v 7 currents, leading to PASMC hyperpolarization. This mechanism contributes to NO/cGMP-induced PA vasodilation. Our study identifies K v 7 channels as a novel mechanism of action of vasodilator drugs used in the treatment of pulmonary arterial hypertension., (© 2019 The British Pharmacological Society.)