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

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 activates chloride efflux through the Ca2+-gated anion channel TMEM16A, thus depolarizing the cell and opening voltage-gated calcium channels. This mechanism strongly amplifies the pericyte [Ca2+]i rise and capillary constriction evoked by contractile agonists and ischemia. In a rodent stroke model, TMEM16A inhibition slows the ischemia-evoked pericyte [Ca2+]i rise, capillary constriction and pericyte death, reduces neutrophil stalling and improves cerebrovascular reperfusion. Genetic analysis implicates 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

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. We 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 microscope slides within flow chambers that allowed application of physiological and pathological shear stress. Arterial shear (1002.6s-1) induced a sustained increase in intracellular calcium ([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 GsMTx-4 or by chelation of extracellular Ca2+. Thrombus formation was studied on collagen-coated surfaces using 3,3'-dihexyloxacarbocyanine iodide (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 and 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 in both platelets and Meg-01 cells using qRT-PCR and Western blotting. 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 stress

Mechanosensitive and FcγRIIa-mediated Platelet Calcium Entry Mechanisms

Elevation of intracellular Ca2+ ([Ca2+]i) is essential for platelet function. Despite the established role of shear stress in haemostasis and thrombosis, the possible contribution of mechanosensitive (MS) Ca2+-permeable ion channels to platelet activation remains unknown. One well-established Ca2+-permeable ion channel which enhances platelet responses at high shear is the ATP-gated P2X1 channel. The relative contribution of this channel to platelet function was studied following the activation of the FcγRIIa immune receptor. qRT-PCR and Western blotting revealed that human platelets and a megakaryocytic cell line, Meg-01, express the MS cation channel Piezo1. To investigate Ca2+-permeable MS channel activity, single platelets and Meg-01 cells were loaded with the Ca2+ indicator Fluo-3, and exposed to arterial shear in flow chambers which induced increases in [Ca2+]i in both cell types in physiological salines. The MS channel blocker GsMTx-4 inhibited these responses and reduced thrombus formation over collagen, whereas Piezo1 channel agonist Yoda1 potentiated platelet shear-induced Ca2+ transients. In Fura-2-loaded platelet suspensions, the GsMTx-4-sensitive shear-evoked responses were shown to be independent of P2X1, Orai1 and TRPC6. FcγRIIa-mediated [Ca2+]i elevations and aggregation were monitored using ratiometric [Ca2+]i measurements and light transmission, respectively. The contribution of P2X1 channels was assessed following inhibition by NF449, and inactivation by pre-addition of α,β-meATP or apyrase exclusion. These treatments significantly reduced antibody- or bacteria-induced FcγRIIa-mediated responses, indicating a significant P2X1 channel contribution. Phosphorylation assays indicated that P2X1 amplifies FcγRIIa-mediated responses via direct Ca2+ influx, rather than via a feedforward effect on early tyrosine phosphorylation. In conclusion, this thesis provides evidence that platelets express functional MS Piezo1 channels which can provide a direct route for Ca2+ entry under normal and pathological arterial shear, and contribute to thrombus formation. Additionally, P2X1 channels were shown to amplify FcγRIIa-mediated platelet Ca2+ signalling and aggregation, which can contribute to platelet activation under shear in infective endocarditis

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

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)

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

International audienceBackground: STIM1 (stromal interaction molecule 1) is a calcium (Ca 2+ ) sensor that regulates cardiac hypertrophy by triggering store-operated Ca 2+ entry. Because STIM1 binding to phospholamban increases sarcoplasmic reticulum Ca 2+ load independent of store-operated Ca 2+ 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 STIM1 flox/flox -Cre tg /− (STIM1-KD) and littermate controls for STIM1 flox/flox (referred to as STIM1-Ctl) and for Cre tg/− 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

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

International audienceMetabolic 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