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1.  Trpm4 Gene Invalidation Leads to Cardiac Hypertrophy and Electrophysiological Alterations 
PLoS ONE  2014;9(12):e115256.
TRPM4 is a non-selective Ca2+-activated cation channel expressed in the heart, particularly in the atria or conduction tissue. Mutations in the Trpm4 gene were recently associated with several human conduction disorders such as Brugada syndrome. TRPM4 channel has also been implicated at the ventricular level, in inotropism or in arrhythmia genesis due to stresses such as ß-adrenergic stimulation, ischemia-reperfusion, and hypoxia re-oxygenation. However, the physiological role of the TRPM4 channel in the healthy heart remains unclear.
We aimed to investigate the role of the TRPM4 channel on whole cardiac function with a Trpm4 gene knock-out mouse (Trpm4-/-) model.
Methods and Results
Morpho-functional analysis revealed left ventricular (LV) eccentric hypertrophy in Trpm4-/- mice, with an increase in both wall thickness and chamber size in the adult mouse (aged 32 weeks) when compared to Trpm4+/+ littermate controls. Immunofluorescence on frozen heart cryosections and qPCR analysis showed no fibrosis or cellular hypertrophy. Instead, cardiomyocytes in Trpm4-/- mice were smaller than Trpm4+/+with a higher density. Immunofluorescent labeling for phospho-histone H3, a mitosis marker, showed that the number of mitotic myocytes was increased 3-fold in the Trpm4-/-neonatal stage, suggesting hyperplasia. Adult Trpm4-/- mice presented multilevel conduction blocks, as attested by PR and QRS lengthening in surface ECGs and confirmed by intracardiac exploration. Trpm4-/-mice also exhibited Luciani-Wenckebach atrioventricular blocks, which were reduced following atropine infusion, suggesting paroxysmal parasympathetic overdrive. In addition, Trpm4-/- mice exhibited shorter action potentials in atrial cells. This shortening was unrelated to modifications of the voltage-gated Ca2+ or K+ currents involved in the repolarizing phase.
TRPM4 has pleiotropic roles in the heart, including the regulation of conduction and cellular electrical activity which impact heart development.
PMCID: PMC4274076  PMID: 25531103
2.  Paradoxical Effect of Increased Diastolic Ca2+ Release and Decreased Sinoatrial Node Activity in a Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia 
Circulation  2012;126(4):392-401.
Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is characterized by stress-triggered syncope and sudden death. CPVT patients manifest sino-atrial node (SAN) dysfunction, the mechanisms of which remain unexplored.
Methods and Results
We investigated SAN [Ca2+]i handling in mice carrying the CPVT-linked mutation of ryanodine receptor (RyR2R4496C) and on their wild-type (WT) littermates. In vivo telemetric recordings showed impaired SAN automaticity in RyR2R4496C mice following Isoproterenol (ISO) injection, analogous to what was observed in CPVT patients after exercise. Pacemaker activity was explored by measuring spontaneous [Ca2+]i transients in SAN cells within the intact SAN by confocal microscopy. RyR2R4496C SAN presented significantly slower pacemaker activity and impaired chronotropic response under β-adrenergic stimulation, accompanied by the appearance of pauses (in spontaneous [Ca2+]i transients and action potentials) in 75% of the cases. Ca2+ spark frequency was increased by 2-fold in RyR2R4496C SAN. Whole-cell patch-clamp experiments performed on isolated RyR2R4496C SAN cells showed that L-type Ca2+ current (ICa,L) density was reduced by ~50%, an effect blunted with internal Ca2+ buffering. ISO dramatically increased the frequency of Ca2+ sparks and waves by ~5 and ~10-fold, respectively. Interestingly, the sarcoplasmic reticulum (SR) Ca2+ content was significantly reduced in RyR2R4496C SAN cells in the presence of ISO, which may contribute to stopping the “Ca2+-clock” rhythm generation, originating SAN pauses.
The increased activity of RyR2R4496C in SAN leads to an unanticipated decrease on SAN automaticity by Ca2+-dependent decrease of ICa,L and SR Ca2+ depletion during diastole, identifying subcellular pathophysiologic alterations contributing to the SAN dysfunction in CPVT patients.
PMCID: PMC3434373  PMID: 22711277
arrhythmia; calcium; catecholaminergic polymorphic ventricular tachycardia; sinoatrial node; L-type calcium current
4.  ACE inhibition prevents diastolic Ca2+ overload and loss of myofilament Ca2+ sensitivity after myocardial infarction 
Current Molecular Medicine  2012;12(2):206-217.
Prevention of adverse cardiac remodeling after myocardial infarction (MI) remains a therapeutic challenge. Angiotensin-converting enzyme inhibitors (ACE-I) are a well-established first-line treatment. ACE-I delay fibrosis, but little is known about their molecular effects on cardiomyocytes. We investigated the effects of the ACE-I delapril on cardiomyocytes in a mouse model of heart failure (HF) after MI. Mice were randomly assigned to three groups: Sham, MI, and MI-D (6 weeks of treatment with a non-hypotensive dose of delapril started 24h after MI). Echocardiography and pressure-volume loops revealed that MI induced hypertrophy and dilatation, and altered both contraction and relaxation of the left ventricle. At the cellular level, MI cardiomyocytes exhibited reduced contraction, slowed relaxation, increased diastolic Ca2+ levels, decreased Ca2+-transient amplitude, and diminished Ca2+ sensitivity of myofilaments. In MI-D mice, however, both mortality and cardiac remodeling were decreased when compared to non-treated MI mice. Delapril maintained cardiomyocyte contraction and relaxation, prevented diastolic Ca2+ overload and retained the normal Ca2+ sensitivity of contractile proteins. Delapril maintained SERCA2a activity through normalization of P-PLB/PLB (for both Ser16-PLB and Thr17-PLB) and PLB/SERCA2a ratios in cardiomyocytes, favoring normal reuptake of Ca2+ in the sarcoplasmic reticulum. In addition, delapril prevented defective cTnI function by normalizing the expression of PKC, enhanced in MI mice. In conclusion, early therapy with delapril after MI preserved the normal contraction/relaxation cycle of surviving cardiomyocytes with multiple direct effects on key intracellular mechanisms contributing to preserve cardiac function.
PMCID: PMC3472404  PMID: 22280358
Angiotensin-Converting Enzyme Inhibitors; pharmacology; therapeutic use; Animals; Calcium; metabolism; Diastole; Disease Models, Animal; Excitation Contraction Coupling; drug effects; Male; Mice; Myocardial Contraction; drug effects; Myocardial Infarction; drug therapy; metabolism; mortality; Myofibrils; metabolism; Ryanodine Receptor Calcium Release Channel; metabolism; Sarcoplasmic Reticulum; metabolism; Ventricular Remodeling; drug effects; Angiotensin-converting enzyme inhibitors; excitation-contraction coupling; heart failure; hypertrophy; myofilaments; sarcoplasmic reticulum Ca2+ ATPase
5.  Carbon Monoxide Induces Cardiac Arrhythmia via Induction of the Late Na+ Current 
Rationale: Clinical reports describe life-threatening cardiac arrhythmias after environmental exposure to carbon monoxide (CO) or accidental CO poisoning. Numerous case studies describe disruption of repolarization and prolongation of the QT interval, yet the mechanisms underlying CO-induced arrhythmias are unknown.
Objectives: To understand the cellular basis of CO-induced arrhythmias and to indentify an effective therapeutic approach.
Methods: Patch-clamp electrophysiology and confocal Ca2+ and nitric oxide (NO) imaging in isolated ventricular myocytes was performed together with protein S-nitrosylation to investigate the effects of CO at the cellular and molecular levels, whereas telemetry was used to investigate effects of CO on electrocardiogram recordings in vivo.
Measurements and Main Results: CO increased the sustained (late) component of the inward Na+ current, resulting in prolongation of the action potential and the associated intracellular Ca2+ transient. In more than 50% of myocytes these changes progressed to early after-depolarization–like arrhythmias. CO elevated NO levels in myocytes and caused S-nitrosylation of the Na+ channel, Nav1.5. All proarrhythmic effects of CO were abolished by the NO synthase inhibitor l-NAME, and reversed by ranolazine, an inhibitor of the late Na+ current. Ranolazine also corrected QT variability and arrhythmias induced by CO in vivo, as monitored by telemetry.
Conclusions: Our data indicate that the proarrhythmic effects of CO arise from activation of NO synthase, leading to NO-mediated nitrosylation of NaV1.5 and to induction of the late Na+ current. We also show that the antianginal drug ranolazine can abolish CO-induced early after-depolarizations, highlighting a novel approach to the treatment of CO-induced arrhythmias.
PMCID: PMC3622900  PMID: 22822026
carbon monoxide; arrhythmia; late Na+ channel; nitric oxide; S-nitrosylation
6.  RyRCa2+ Leak Limits Cardiac Ca2+ Window Current Overcoming the Tonic Effect of Calmodulin in Mice 
PLoS ONE  2011;6(6):e20863.
Ca2+ mediates the functional coupling between L-type Ca2+ channel (LTCC) and sarcoplasmic reticulum (SR) Ca2+ release channel (ryanodine receptor, RyR), participating in key pathophysiological processes. This crosstalk manifests as the orthograde Ca2+-induced Ca2+-release (CICR) mechanism triggered by Ca2+ influx, but also as the retrograde Ca2+-dependent inactivation (CDI) of LTCC, which depends on both Ca2+ permeating through the LTCC itself and on SR Ca2+ release through the RyR. This latter effect has been suggested to rely on local rather than global Ca2+ signaling, which might parallel the nanodomain control of CDI carried out through calmodulin (CaM). Analyzing the CICR in catecholaminergic polymorphic ventricular tachycardia (CPVT) mice as a model of RyR-generated Ca2+ leak, we evidence here that increased occurrence of the discrete local SR Ca2+ releases through the RyRs (Ca2+ sparks) causea depolarizing shift in activation and a hyperpolarizing shift inisochronic inactivation of cardiac LTCC current resulting in the reduction of window current. Both increasing fast [Ca2+]i buffer capacity or depleting SR Ca2+ store blunted these changes, which could be reproduced in WT cells by RyRCa2+ leak induced with Ryanodol and CaM inhibition.Our results unveiled a new paradigm for CaM-dependent effect on LTCC gating and further the nanodomain Ca2+ control of LTCC, emphasizing the importance of spatio-temporal relationships between Ca2+ signals and CaM function.
PMCID: PMC3108979  PMID: 21673970
7.  Endothelin-Dependent Vasoconstriction in Human Uterine Artery: Application to Preeclampsia 
PLoS ONE  2011;6(1):e16540.
Reduced uteroplacental perfusion, the initiating event in preeclampsia, is associated with enhanced endothelin-1 (ET-1) production which feeds the vasoconstriction of uterine artery. Whether the treatments of preeclampsia were effective on ET-1 induced contraction and could reverse placental ischemia is the question addressed in this study. We investigated the effect of antihypertensive drugs used in preeclampsia and of ET receptor antagonists on the contractile response to ET-1 on human uterine arteries.
Methodology/Principal Findings
Experiments were performed, ex vivo, on human uterine artery samples obtained after hysterectomy. We studied variations in isometric tension of arterial rings in response to the vasoconstrictor ET-1 and evaluated the effects of various vasodilators and ET-receptor antagonists on this response. Among antihypertensive drugs, only dihydropyridines were effective in blocking and reversing the ET-1 contractile response. Their efficiency, independent of the concentration of ET-1, was only partial. Hydralazine, alpha-methyldopa and labetalol had no effect on ET-1 induced contraction which is mediated by both ETA and ETB receptors in uterine artery. ET receptors antagonists, BQ-123 and BQ-788, slightly reduced the amplitude of the response to ET-1. Combination of both antagonists was more efficient, but it was not possible to reverse the maximal ET-1-induced contraction with antagonists used alone or in combination.
Pharmacological drugs currently used in the context of preeclampsia, do not reverse ET-1 induced contraction. Only dihydropyridines, which partially relax uterine artery previously contracted with ET-1, might offer interesting perspectives to improve placental perfusion.
PMCID: PMC3027698  PMID: 21298073
8.  Increased Ca2+ sensitivity of the ryanodine receptor mutant RyR2R4496C underlies catecholaminergic polymorphic ventricular tachycardia 
Circulation Research  2008;104(2):201-9, 12p following 209.
Cardiac ryanodine receptor (RyR2) mutations are associated with autosomal dominant catecholaminergic polymorphic ventricular tachycardia (CPVT), suggesting that alterations in Ca2+ handling underlie this disease. Here we analyze the underlying Ca2+ release defect that leads to arrhythmia in cardiomyocytes isolated from heterozygous knock-in mice carrying the RyR2R4496C mutation. RyR2R4496C−/− littermates (wild type, WT) were used as controls. [Ca2+]i transients were obtained by field stimulation in fluo-3 loaded cardiomyocytes and viewed using confocal microscopy. In our basal recording conditions (2 Hz stimulation rate), [Ca2+]i transients and sarcoplasmic reticulum (SR) Ca2+ load were similar in WT and RyR2R4496C cells. However, paced RyR2R4496C ventricular myocytes presented abnormal Ca2+ release during the diastolic period, viewed as Ca2+-waves, consistent with the occurrence of delayed after-depolarizations. The occurrence of this abnormal Ca2+ release was enhanced at faster stimulation rates and by β-adrenergic stimulation, which also induced triggered activity. Spontaneous Ca2+-sparks were more frequent in RyR2R4496C myocytes, indicating increased RyR2R4496C activity. When permeabilized cells were exposed to different cytosolic [Ca2+]i, RyR2R4496C showed a dramatic increase in Ca2+ sensitivity. Isoproterenol increased [Ca2+]i transient amplitude and Ca2+ spark frequency to the same extent in WT and RyR2R4496C cells, indicating that the β-adrenergic sensitivity of RyR2R4496C cells remained unaltered. This effect was independent of protein expression variations since no difference was found in the total or phosphorylated RyR2 expression levels. In conclusion, the arrhythmogenic potential of the RyR2R4496C mutation is due to the increased Ca2+ sensitivity of RyR2R4496C, which induces diastolic Ca2+ release and lowers the threshold for triggered activity.
PMCID: PMC2796688  PMID: 19096022
Adrenergic beta-Agonists; pharmacology; Animals; Caffeine; pharmacology; Calcium Signaling; drug effects; Cardiac Pacing, Artificial; Catecholamines; metabolism; Female; Isoproterenol; pharmacology; Male; Membrane Potentials; Mice; Mice, Transgenic; Microscopy, Confocal; Mutation; Myocardial Contraction; drug effects; Myocytes, Cardiac; drug effects; metabolism; Phosphorylation; Ryanodine Receptor Calcium Release Channel; genetics; metabolism; Sarcoplasmic Reticulum; metabolism; Tachycardia, Ventricular; genetics; metabolism; physiopathology; Time Factors; Ca2+ 2+-sparks; [Ca2+ 2+]i transients; ryanodine receptor; excitation-contraction coupling; heart; CPVT
9.  New Insights in the Contribution of Voltage-Gated Nav Channels to Rat Aorta Contraction 
PLoS ONE  2009;4(10):e7360.
Despite increasing evidence for the presence of voltage-gated Na+ channels (Nav) isoforms and measurements of Nav channel currents with the patch-clamp technique in arterial myocytes, no information is available to date as to whether or not Nav channels play a functional role in arteries. The aim of the present work was to look for a physiological role of Nav channels in the control of rat aortic contraction.
Methodology/Principal Findings
Nav channels were detected in the aortic media by Western blot analysis and double immunofluorescence labeling for Nav channels and smooth muscle α-actin using specific antibodies. In parallel, using real time RT-PCR, we identified three Nav transcripts: Nav1.2, Nav1.3, and Nav1.5. Only the Nav1.2 isoform was found in the intact media and in freshly isolated myocytes excluding contamination by other cell types. Using the specific Nav channel agonist veratridine and antagonist tetrodotoxin (TTX), we unmasked a contribution of these channels in the response to the depolarizing agent KCl on rat aortic isometric tension recorded from endothelium-denuded aortic rings. Experimental conditions excluded a contribution of Nav channels from the perivascular sympathetic nerve terminals. Addition of low concentrations of KCl (2–10 mM), which induced moderate membrane depolarization (e.g., from −55.9±1.4 mV to −45.9±1.2 mV at 10 mmol/L as measured with microelectrodes), triggered a contraction potentiated by veratridine (100 µM) and blocked by TTX (1 µM). KB-R7943, an inhibitor of the reverse mode of the Na+/Ca2+ exchanger, mimicked the effect of TTX and had no additive effect in presence of TTX.
These results define a new role for Nav channels in arterial physiology, and suggest that the TTX-sensitive Nav1.2 isoform, together with the Na+/Ca2+ exchanger, contributes to the contractile response of aortic myocytes at physiological range of membrane depolarization.
PMCID: PMC2752992  PMID: 19809503
10.  Akt regulates L-type Ca2+ channel activity by modulating Cavα1 protein stability 
The Journal of Cell Biology  2009;184(6):923-933.
The insulin IGF-1–PI3K–Akt signaling pathway has been suggested to improve cardiac inotropism and increase Ca2+ handling through the effects of the protein kinase Akt. However, the underlying molecular mechanisms remain largely unknown. In this study, we provide evidence for an unanticipated regulatory function of Akt controlling L-type Ca2+ channel (LTCC) protein density. The pore-forming channel subunit Cavα1 contains highly conserved PEST sequences (signals for rapid protein degradation), and in-frame deletion of these PEST sequences results in increased Cavα1 protein levels. Our findings show that Akt-dependent phosphorylation of Cavβ2, the LTCC chaperone for Cavα1, antagonizes Cavα1 protein degradation by preventing Cavα1 PEST sequence recognition, leading to increased LTCC density and the consequent modulation of Ca2+ channel function. This novel mechanism by which Akt modulates LTCC stability could profoundly influence cardiac myocyte Ca2+ entry, Ca2+ handling, and contractility.
PMCID: PMC2699149  PMID: 19307602

Results 1-10 (10)