The pharmacology of the TMEM16A channel: therapeutic opportunities.

The TMEM16A Ca2+-gated Cl- channel is involved in a variety of vital physiological functions and may be targeted pharmacologically for therapeutic benefit in diseases such as hypertension, stroke, and cystic fibrosis (CF). The determination of the TMEM16A structure and high-throughput screening efforts, alongside ex vivo and in vivo animal studies and clinical investigations, are hastening our understanding of the physiology and pharmacology of this channel. Here, we offer a critical analysis of recent developments in TMEM16A pharmacology and reflect on the therapeutic opportunities provided by this target.

Atrial AMP-activated protein kinase is critical for prevention of dysregulation of electrical excitability and atrial fibrillation.

Metabolic stress is an important cause of pathological atrial remodeling and atrial fibrillation. AMPK is a ubiquitous master metabolic regulator, yet its biological function in the atria is poorly understood in both health and disease. We investigated the impact of atrium-selective cardiac AMPK deletion on electrophysiological and structural remodeling in mice. Loss of atrial AMPK expression caused atrial changes in electrophysiological properties and atrial ectopic activity prior to the onset of spontaneous atrial fibrillation. Concomitant transcriptional downregulation of connexins and atrial ion channel subunits manifested with delayed left atrial activation and repolarization. The early molecular and electrophysiological abnormalities preceded left atrial structural remodeling and interstitial fibrosis. AMPK inactivation induced downregulation of transcription factors (Mef2c and Pitx2c) linked to connexin and ion channel transcriptional reprogramming. Thus, AMPK plays an essential homeostatic role in atria, protecting against adverse remodeling potentially by regulating key transcription factors that control the expression of atrial ion channels and gap junction proteins.

The Ca2+-gated channel TMEM16A amplifies capillary pericyte contraction and reduces cerebral blood flow after ischemia.

Pericyte-mediated capillary constriction decreases cerebral blood flow in stroke after an occluded artery is unblocked. The determinants of pericyte tone are poorly understood. We show that a small rise in cytoplasmic Ca2+ concentration ([Ca2+]i) in pericytes activated chloride efflux through the Ca2+-gated anion channel TMEM16A, thus depolarizing the cell and opening voltage-gated calcium channels. This mechanism strongly amplified the pericyte [Ca2+]i rise and capillary constriction evoked by contractile agonists and ischemia. In a rodent stroke model, TMEM16A inhibition slowed the ischemia-evoked pericyte [Ca2+]i rise, capillary constriction, and pericyte death; reduced neutrophil stalling; and improved cerebrovascular reperfusion. Genetic analysis implicated altered TMEM16A expression in poor patient recovery from ischemic stroke. Thus, pericyte TMEM16A is a crucial regulator of cerebral capillary function and a potential therapeutic target for stroke and possibly other disorders of impaired microvascular flow, such as Alzheimer's disease and vascular dementia.

Arrhythmia Mechanism and Dynamics in a Humanized Mouse Model of Inherited Cardiomyopathy Caused by Phospholamban R14del Mutation.

BACKGROUND: Arginine (Arg) 14 deletion (R14del) in the calcium regulatory protein phospholamban (hPLNR14del) has been identified as a disease-causing mutation in patients with an inherited cardiomyopathy. Mechanisms underlying the early arrhythmogenic phenotype that predisposes carriers of this mutation to sudden death with no apparent structural remodeling remain unclear. METHODS: To address this, we performed high spatiotemporal resolution optical mapping of intact hearts from adult knock-in mice harboring the human PLNWT (wildtype [WT], n=12) or the heterozygous human PLNR14del mutation (R14del, n=12) before and after ex vivo challenge with isoproterenol and rapid pacing. RESULTS: Adverse electrophysiological remodeling was evident in the absence of significant structural or hemodynamic changes. R14del hearts exhibited increased arrhythmia susceptibility compared with wildtype. Underlying this susceptibility was preferential right ventricular action potential prolongation that was unresponsive to ß-adrenergic stimulation. A steep repolarization gradient at the left ventricular/right ventricular interface provided the substrate for interventricular activation delays and ultimately local conduction block during rapid pacing. This was followed by the initiation of macroreentrant circuits supporting the onset of ventricular tachycardia. Once sustained, these circuits evolved into high-frequency rotors, which in their majority were pinned to the right ventricle. These rotors exhibited unique spatiotemporal dynamics that promoted their increased stability in R14del compared with wildtype hearts. CONCLUSIONS: Our findings highlight the crucial role of primary electric remodeling caused by the hPLNR14del mutation. These inherently arrhythmogenic features form the substrate for adrenergic-mediated VT at early stages of PLNR14del induced cardiomyopathy.

Cardiomyocyte-Specific STIM1 (Stromal Interaction Molecule 1) Depletion in the Adult Heart Promotes the Development of Arrhythmogenic Discordant Alternans.

BACKGROUND: STIM1 (stromal interaction molecule 1) is a calcium (Ca2+) sensor that regulates cardiac hypertrophy by triggering store-operated Ca2+ entry. Because STIM1 binding to phospholamban increases sarcoplasmic reticulum Ca2+ load independent of store-operated Ca2+ entry, we hypothesized that it controls electrophysiological function and arrhythmias in the adult heart. METHODS: Inducible myocyte-restricted STIM1-KD (STIM1 knockdown) was achieved in adult mice using an αMHC (α-myosin heavy chain)-MerCreMer system. Mechanical and electrophysiological properties were examined using echocardiography in vivo and optical action potential (AP) mapping ex vivo in tamoxifen-induced STIM1flox/flox-Cretg/- (STIM1-KD) and littermate controls for STIM1flox/flox (referred to as STIM1-Ctl) and for Cretg/- without STIM deletion (referred to as Cre-Ctl). RESULTS: STIM1-KD mice (N=23) exhibited poor survival compared with STIM1-Ctl (N=22) and Cre-Ctl (N=11) with >50% mortality after only 8-days of cardiomyocyte-restricted STIM1-KD. STIM1-KD but not STIM1-Ctl or Cre-Ctl hearts exhibited a proclivity for arrhythmic behavior, ranging from frequent ectopy to pacing-induced ventricular tachycardia/ventricular fibrillation (VT/VF). Examination of the electrophysiological substrate revealed decreased conduction velocity and increased AP duration (APD) heterogeneity in STIM1-KD. These features, however, were comparable in VT/VF(+) and VT/VF(-) hearts. We also uncovered a marked increase in the magnitude of APD alternans during rapid pacing, and the emergence of a spatially discordant alternans profile in STIM1-KD hearts. Unlike conduction velocity slowing and APD heterogeneity, the magnitude of APD alternans was greater (by 80%, P<0.05) in VT/VF(+) versus VT/VF(-) STIM1-KD hearts. Detailed phase mapping during the initial beats of VT/VF identified one or more rotors that were localized along the nodal line separating out-of-phase alternans regions. CONCLUSIONS: In an adult murine model with inducible and myocyte-specific STIM1 depletion, we demonstrate for the first time the regulation of spatially discordant alternans by STIM1. Early mortality in STIM1-KD mice is likely related to enhanced susceptibility to VT/VF secondary to discordant APD alternans.

Intra-tracheal gene delivery of aerosolized SERCA2a to the lung suppresses ventricular arrhythmias in a model of pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) results in right ventricular (RV) failure, electro-mechanical dysfunction and heightened risk of sudden cardiac death (SCD), although exact mechanisms and predisposing factors remain unclear. Because impaired chronotropic response to exercise is a strong predictor of early mortality in patients with PAH, we hypothesized that progressive elevation in heart rate can unmask ventricular tachyarrhythmias (VT) in a rodent model of monocrotaline (MCT)-induced PAH. We further hypothesized that intra-tracheal gene delivery of aerosolized AAV1.SERCA2a (AAV1.S2a), an approach which improves pulmonary vascular remodeling in PAH, can suppress VT in this model. OBJECTIVE: To determine the efficacy of pulmonary AAV1.S2a in reversing electrophysiological (EP) remodeling and suppressing VT in PAH. METHODS: Male rats received subcutaneous injection of MCT (60 mg/kg) leading to advanced PAH. Three weeks following MCT, rats underwent intra-tracheal delivery of aerosolized AAV1.S2a (MCT + S2a, N = 8) or saline (MCT, N = 9). Age-matched rats served as controls (CTRL, N = 7). The EP substrate and risk of VT were determined using high-resolution optical action potential (AP) mapping ex vivo. The expression levels of key ion channel subunits, fibrosis markers and hypertrophy indices were measured by RT-PCR and histochemical analyses. RESULTS: Over 80% of MCT but none of the CTRL hearts were prone to sustained VT by rapid pacing (P < .01). Aerosolized gene delivery of AAV1.S2a to the lung suppressed the incidence of VT to <15% (P < .05). Investigation of the EP substrate revealed marked prolongation of AP duration (APD), increased APD heterogeneity, a reversal in the trans-epicardial APD gradient, and marked conduction slowing in untreated MCT compared to CTRL hearts. These myocardial EP changes coincided with major remodeling in the expression of K and Ca channel subunits, decreased expression of Cx43 and increased expression of pro-fibrotic and pro-hypertrophic markers. Intra-tracheal gene delivery of aerosolized AAV1 carrying S2a but not luciferase resulted in selective upregulation of the human isoform of SERCA2a in the lung but not the heart. This pulmonary intervention, in turn, ameliorated MCT-induced APD prolongation, reversed spatial APD heterogeneity, normalized myocardial conduction, and suppressed the incidence of pacing-induced VT. Comparison of the minimal conduction velocity (CV) generated at the fastest pacing rate before onset of VT or at the end of the protocol revealed significantly lower values in untreated compared to AAV1.S2a treated PAH and CTRL hearts. Reversal of EP remodeling by pulmonary AAV1.S2a gene delivery was accompanied by restored expression of key ion channel transcripts. Restored expression of Cx43 and collagen but not the pore-forming Na channel subunit Nav1.5 likely ameliorated VT by improving CV at rapid rates in PAH. CONCLUSION: Aerosolized AAV1.S2a gene delivery selectively to the lungs ameliorates myocardial EP remodeling and VT susceptibility at rapid heart rates. Our findings highlight for the first time the utility of a non-cardiac gene therapy approach for arrhythmia suppression.

The Mitochondrial Translocator Protein and the Emerging Link Between Oxidative Stress and Arrhythmias in the Diabetic Heart.

The mitochondrial translocator protein (TSPO) is a key outer mitochondrial membrane protein that regulates the activity of energy-dissipating mitochondrial channels in response to oxidative stress. In this article, we provide an overview of the role of TSPO in the systematic amplification of reactive oxygen species (ROS) through an autocatalytic process known as ROS-induced ROS-release (RIRR). We describe how this TSPO-driven process destabilizes the mitochondrial membrane potential leading to electrical instability at the cellular and whole heart levels. Finally, we provide our perspective on the role of TSPO in the pathophysiology of diabetes, in general and diabetes-related arrhythmias, in particular.

Immobilization of Nonactivated Unfixed Platelets for Real-Time Single-Cell Analysis.

Existing methods for measuring the response of individual platelets to stimulation are limited. They either measure each platelet at one discrete time-point (flow cytometry) or rely on adhesive ligands to immobilize platelets that concomitantly generate activation signals (microscopy). Such methods of immobilization make it impossible to assess resting platelets, the changes that occur as platelets transition from resting to active states, or the signals generated by soluble agonists, such as ADP and thrombin, or by mechanical stimulus, independently from those generated by the adhesive ligand. Here we describe a microscopy method that allows the immobilization of platelets to a glass cover slip without triggering platelet activation. This method makes use of specific antibodies that bind platelet PECAM-1 without activating it. Platelets can therefore be immobilized to PECAM-1 antibody coated biochips without causing activation and perfused with agonists or inhibitors. Using this method, platelets can be stimulated by an array of soluble agonists at any concentration or combination, in the presence or absence of inhibitors or shear forces. This chapter describes in detail this PECAM-1 mediated immobilized platelet method and its use for measuring changes in Ca2+ signaling in individual platelets under a number of different conditions. While we focus on the measurement of Ca2+ dynamics in this chapter, it is important to consider that the basic method we describe will easily lend its self to other measures of platelet activation (integrin activation, shape change, actin dynamics, degranulation), and may, therefore, be used to measure almost any facet of platelet activation.

Optical Action Potential Mapping in Acute Models of Ischemia-Reperfusion Injury: Probing the Arrhythmogenic Role of the Mitochondrial Translocator Protein.

Ischemia-reperfusion (I/R) injury causes dynamic changes in electrophysiological properties that promote the incidence of post-ischemic arrhythmias. High-resolution optical action potential mapping allows for a quantitative assessment of the electrophysiological substrate at a cellular resolution within the intact heart, which is critical for elucidation of arrhythmia mechanisms. We and others have found that pharmacological inhibition of the translocator protein (TSPO) is highly effective against postischemic arrhythmias. A major hurdle that has limited the translation of this approach to patients is the fact that available TSPO ligands have several confounding effects, including a potent negative ionotropic property. To circumvent such limitations we developed an in vivo cardiac specific TSPO gene silencing approach as an alternative. Here, we provide the methodological details of our optical action potential mapping studies that were designed to probe the effects of TSPO silencing in hearts from spontaneously hypertensive rats (SHR) that are prone to I/R injury.

P2X1 Receptors Amplify FcγRIIa-Induced Ca2+ Increases and Functional Responses in Human Platelets.

Platelets express key receptors of the innate immune system such as FcγRIIa and Toll-like receptors (TLR). P2X1 cation channels amplify the platelet responses to several major platelet stimuli, particularly glycoprotein (GP)VI and TLR2/1, whereas their contribution to Src tyrosine kinase-dependent FcγRIIa receptors remains unknown. We investigated the role of P2X1 receptors during activation of FcγRIIa in human platelets, following stimulation by cross-linking of an anti-FcγRIIa monoclonal antibody (mAb) IV.3, or bacterial stimulation with Streptococcus sanguinis. Activation was assessed in washed platelet suspensions via measurement of intracellular Ca2+ ([Ca2+]i) increases, ATP release and aggregation. P2X1 activity was abolished by pre-addition of α,ß-meATP, exclusion of apyrase or the antagonist NF449. FcγRIIa activation evoked a robust increase in [Ca2+]i (441 ± 33 nM at 30 µg/mL mAb), which was reduced to a similar extent (to 66-70% of control) by NF449, pre-exposure to α,ß-meATP or apyrase omission, demonstrating a significant P2X1 receptor contribution. FcγRIIa activation-dependent P2X1 responses were partially resistant to nitric oxide (NO), but abrogated by 500 nM prostacyclin (PGI2). Aggregation responses to bacteria and FcγRIIa activation were also inhibited by P2X1 receptor desensitization (to 66 and 42% of control, respectively). However, FcγRIIa-mediated tyrosine phosphorylation and ATP release were not significantly altered by the loss of P2X1 activity. In conclusion, we show that P2X1 receptors enhance platelet FcγRIIa receptor-evoked aggregation through an increase in [Ca2+]i downstream of the initial tyrosine phosphorylation events and early dense granule release. This represents a further route whereby ATP-gated cation channels can contribute to platelet-dependent immune responses in vivo.

Evidence for shear-mediated Ca2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line.

The role of mechanosensitive (MS) Ca2+-permeable ion channels in platelets is unclear, despite the importance of shear stress in platelet function and life-threatening thrombus formation. We therefore sought to investigate the expression and functional relevance of MS channels in human platelets. The effect of shear stress on Ca2+ entry in human platelets and Meg-01 megakaryocytic cells loaded with Fluo-3 was examined by confocal microscopy. Cells were attached to glass coverslips within flow chambers that allowed applications of physiological and pathological shear stress. Arterial shear (1002.6 s-1) induced a sustained increase in [Ca2+] i in Meg-01 cells and enhanced the frequency of repetitive Ca2+ transients by 80% in platelets. These Ca2+ increases were abrogated by the MS channel inhibitor Grammostola spatulata mechanotoxin 4 (GsMTx-4) or by chelation of extracellular Ca2+ Thrombus formation was studied on collagen-coated surfaces using DiOC6-stained platelets. In addition, [Ca2+] i and functional responses of washed platelet suspensions were studied with Fura-2 and light transmission aggregometry, respectively. Thrombus size was reduced 50% by GsMTx-4, independently of P2X1 receptors. In contrast, GsMTx-4 had no effect on collagen-induced aggregation or on Ca2+ influx via TRPC6 or Orai1 channels and caused only a minor inhibition of P2X1-dependent Ca2+ entry. The Piezo1 agonist, Yoda1, potentiated shear-dependent platelet Ca2+ transients by 170%. Piezo1 mRNA transcripts and protein were detected with quantitative RT-PCR and Western blotting, respectively, in both platelets and Meg-01 cells. We conclude that platelets and Meg-01 cells express the MS cation channel Piezo1, which may contribute to Ca2+ entry and thrombus formation under arterial shear.

Direct Evidence for Microdomain-Specific Localization and Remodeling of Functional L-Type Calcium Channels in Rat and Human Atrial Myocytes.

BACKGROUND: Distinct subpopulations of L-type calcium channels (LTCCs) with different functional properties exist in cardiomyocytes. Disruption of cellular structure may affect LTCC in a microdomain-specific manner and contribute to the pathophysiology of cardiac diseases, especially in cells lacking organized transverse tubules (T-tubules) such as atrial myocytes (AMs). METHODS AND RESULTS: Isolated rat and human AMs were characterized by scanning ion conductance, confocal, and electron microscopy. Half of AMs possessed T-tubules and structured topography, proportional to cell width. A bigger proportion of myocytes in the left atrium had organized T-tubules and topography than in the right atrium. Super-resolution scanning patch clamp showed that LTCCs distribute equally in T-tubules and crest areas of the sarcolemma, whereas, in ventricular myocytes, LTCCs primarily cluster in T-tubules. Rat, but not human, T-tubule LTCCs had open probability similar to crest LTCCs, but exhibited ≈ 40% greater current. Optical mapping of Ca(2+) transients revealed that rat AMs presented ≈ 3-fold as many spontaneous Ca(2+) release events as ventricular myocytes. Occurrence of crest LTCCs and spontaneous Ca(2+) transients were eliminated by either a caveolae-targeted LTCC antagonist or disrupting caveolae with methyl-ß-cyclodextrin, with an associated ≈ 30% whole-cell ICa,L reduction. Heart failure (16 weeks post-myocardial infarction) in rats resulted in a T-tubule degradation (by ≈ 40%) and significant elevation of spontaneous Ca(2+) release events. Although heart failure did not affect LTCC occurrence, it led to ≈ 25% decrease in T-tubule LTCC amplitude. CONCLUSIONS: We provide the first direct evidence for the existence of 2 distinct subpopulations of functional LTCCs in rat and human AMs, with their biophysical properties modulated in heart failure in a microdomain-specific manner.