Balázs Ördög

Transient Receptor Potential Canonical-3 Channel–Dependent Fibroblast Regulation in Atrial Fibrillation

Background —Fibroblast proliferation and differentiation are central in atrial fibrillation (AF)-promoting remodeling. Here, we investigated fibroblast regulation by Ca 2+ -permeable transient receptor potential canonical-3 (TRPC3) channels. Methods and Results —Freshly-isolated rat cardiac-fibroblasts abundantly expressed TRPC3 and had appreciable non-selective cation currents (I NSC ) sensitive to a selective TPRC3-channel blocker, pyrazole-3 (3-μmol/L). Pyrazole-3 suppressed angiotensin-II-induced Ca 2+ -influx, proliferation and α-smooth-muscle actin (αSMA) protein-expression in fibroblasts. Ca 2+ -removal and TRPC3-blockade suppressed extracellular-signal regulated kinase (ERK)-phosphorylation, and ERK-phosphorylation inhibition reduced fibroblast-proliferation. TRPC3-expression was upregulated in atria from AF-patients, goats with electrically-maintained AF and tachypacing-induced heart-failure dogs. TRPC3-knockdown (shRNA-based) decreased canine atrial-fibroblast proliferation. In left-atrial (LA) fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial-tachypacing, TRPC3 protein-expression, currents, ERK-phosphorylation and extracellular-matrix gene-expression were all significantly increased. In cultured LA-fibroblasts from AF-dogs, proliferation-rates, αSMA-expression and ERK-phosphorylation were increased, and suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF-atria; experimental micro-RNA-26 knockdown reproduced AF-induced TRPC3-upregulation and fibroblast-activation. MicroRNA-26 has Nuclear Factor of Activated T-cells (NFAT) binding-sites in the 59-promoter-region. NFAT-activation increased in AF-fibroblasts and NFAT negatively regulated microRNA-26 transcription. In vivo pyrazole-3 administration suppressed AF while decreasing fibroblast proliferation and extracellular-matrix gene-expression. Conclusions —TRPC3-channels regulate cardiac fibroblast proliferation and differentiation, likely by controlling Ca 2+ -influx that activates ERK-signaling. AF increases TRPC3-channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo TRPC3-block prevents AF-substrate development in a dog model of electrically-maintained AF. TRPC3 likely plays an important role in AF-promoting fibroblast pathophysiology and is a novel potential therapeutic target.Background— Fibroblast proliferation and differentiation are central in atrial fibrillation (AF)–promoting remodeling. Here, we investigated fibroblast regulation by Ca2+-permeable transient receptor potential canonical-3 (TRPC3) channels. Methods and Results— Freshly isolated rat cardiac fibroblasts abundantly expressed TRPC3 and had appreciable nonselective cation currents (INSC) sensitive to a selective TPRC3 channel blocker, pyrazole-3 (3 &mgr;mol/L). Pyrazole-3 suppressed angiotensin II–induced Ca2+ influx, proliferation, and &agr;-smooth muscle actin protein expression in fibroblasts. Ca2+ removal and TRPC3 blockade suppressed extracellular signal-regulated kinase phosphorylation, and extracellular signal-regulated kinase phosphorylation inhibition reduced fibroblast proliferation. TRPC3 expression was upregulated in atria from AF patients, goats with electrically maintained AF, and dogs with tachypacing-induced heart failure. TRPC3 knockdown (based on short hairpin RNA [shRNA]) decreased canine atrial fibroblast proliferation. In left atrial fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial tachypacing, TRPC3 protein expression, currents, extracellular signal-regulated kinase phosphorylation, and extracellular matrix gene expression were all significantly increased. In cultured left atrial fibroblasts from AF dogs, proliferation rates, &agr;-smooth muscle actin expression, and extracellular signal-regulated kinase phosphorylation were increased and were suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF atria; experimental microRNA-26 knockdown reproduced AF-induced TRPC3 upregulation and fibroblast activation. MicroRNA-26 has NFAT (nuclear factor of activated T cells) binding sites in the 5′ promoter region. NFAT activation increased in AF fibroblasts, and NFAT negatively regulated microRNA-26 transcription. In vivo pyrazole-3 administration suppressed AF while decreasing fibroblast proliferation and extracellular matrix gene expression. Conclusions— TRPC3 channels regulate cardiac fibroblast proliferation and differentiation, likely by controlling the Ca2+ influx that activates extracellular signal-regulated kinase signaling. AF increases TRPC3 channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo, TRPC3 blockade prevents AF substrate development in a dog model of electrically maintained AF. TRPC3 likely plays an important role in AF by promoting fibroblast pathophysiology and is a novel potential therapeutic target.

TRPC3-Dependent Fibroblast Regulation in Atrial Fibrillation

Background —Fibroblast proliferation and differentiation are central in atrial fibrillation (AF)-promoting remodeling. Here, we investigated fibroblast regulation by Ca 2+ -permeable transient receptor potential canonical-3 (TRPC3) channels. Methods and Results —Freshly-isolated rat cardiac-fibroblasts abundantly expressed TRPC3 and had appreciable non-selective cation currents (I NSC ) sensitive to a selective TPRC3-channel blocker, pyrazole-3 (3-μmol/L). Pyrazole-3 suppressed angiotensin-II-induced Ca 2+ -influx, proliferation and α-smooth-muscle actin (αSMA) protein-expression in fibroblasts. Ca 2+ -removal and TRPC3-blockade suppressed extracellular-signal regulated kinase (ERK)-phosphorylation, and ERK-phosphorylation inhibition reduced fibroblast-proliferation. TRPC3-expression was upregulated in atria from AF-patients, goats with electrically-maintained AF and tachypacing-induced heart-failure dogs. TRPC3-knockdown (shRNA-based) decreased canine atrial-fibroblast proliferation. In left-atrial (LA) fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial-tachypacing, TRPC3 protein-expression, currents, ERK-phosphorylation and extracellular-matrix gene-expression were all significantly increased. In cultured LA-fibroblasts from AF-dogs, proliferation-rates, αSMA-expression and ERK-phosphorylation were increased, and suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF-atria; experimental micro-RNA-26 knockdown reproduced AF-induced TRPC3-upregulation and fibroblast-activation. MicroRNA-26 has Nuclear Factor of Activated T-cells (NFAT) binding-sites in the 59-promoter-region. NFAT-activation increased in AF-fibroblasts and NFAT negatively regulated microRNA-26 transcription. In vivo pyrazole-3 administration suppressed AF while decreasing fibroblast proliferation and extracellular-matrix gene-expression. Conclusions —TRPC3-channels regulate cardiac fibroblast proliferation and differentiation, likely by controlling Ca 2+ -influx that activates ERK-signaling. AF increases TRPC3-channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo TRPC3-block prevents AF-substrate development in a dog model of electrically-maintained AF. TRPC3 likely plays an important role in AF-promoting fibroblast pathophysiology and is a novel potential therapeutic target.Background— Fibroblast proliferation and differentiation are central in atrial fibrillation (AF)–promoting remodeling. Here, we investigated fibroblast regulation by Ca2+-permeable transient receptor potential canonical-3 (TRPC3) channels. Methods and Results— Freshly isolated rat cardiac fibroblasts abundantly expressed TRPC3 and had appreciable nonselective cation currents (INSC) sensitive to a selective TPRC3 channel blocker, pyrazole-3 (3 &mgr;mol/L). Pyrazole-3 suppressed angiotensin II–induced Ca2+ influx, proliferation, and &agr;-smooth muscle actin protein expression in fibroblasts. Ca2+ removal and TRPC3 blockade suppressed extracellular signal-regulated kinase phosphorylation, and extracellular signal-regulated kinase phosphorylation inhibition reduced fibroblast proliferation. TRPC3 expression was upregulated in atria from AF patients, goats with electrically maintained AF, and dogs with tachypacing-induced heart failure. TRPC3 knockdown (based on short hairpin RNA [shRNA]) decreased canine atrial fibroblast proliferation. In left atrial fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial tachypacing, TRPC3 protein expression, currents, extracellular signal-regulated kinase phosphorylation, and extracellular matrix gene expression were all significantly increased. In cultured left atrial fibroblasts from AF dogs, proliferation rates, &agr;-smooth muscle actin expression, and extracellular signal-regulated kinase phosphorylation were increased and were suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF atria; experimental microRNA-26 knockdown reproduced AF-induced TRPC3 upregulation and fibroblast activation. MicroRNA-26 has NFAT (nuclear factor of activated T cells) binding sites in the 5′ promoter region. NFAT activation increased in AF fibroblasts, and NFAT negatively regulated microRNA-26 transcription. In vivo pyrazole-3 administration suppressed AF while decreasing fibroblast proliferation and extracellular matrix gene expression. Conclusions— TRPC3 channels regulate cardiac fibroblast proliferation and differentiation, likely by controlling the Ca2+ influx that activates extracellular signal-regulated kinase signaling. AF increases TRPC3 channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo, TRPC3 blockade prevents AF substrate development in a dog model of electrically maintained AF. TRPC3 likely plays an important role in AF by promoting fibroblast pathophysiology and is a novel potential therapeutic target.

MicroRNA29 A Mechanistic Contributor and Potential Biomarker in Atrial Fibrillation

Background— Congestive heart failure (CHF) causes atrial fibrotic remodeling, a substrate for atrial fibrillation (AF) maintenance. MicroRNA29 (miR29) targets extracellular matrix proteins. In the present study, we examined miR29b changes in patients with AF and/or CHF and in a CHF-related AF animal model and assessed its potential role in controlling atrial fibrous tissue production. Methods and Results— Control dogs were compared with dogs subjected to ventricular tachypacing for 24 hours, 1 week, or 2 weeks to induce CHF. Atrial miR29b expression decreased within 24 hours in both whole atrial tissue and atrial fibroblasts (−87% and −92% versus control, respectively; p<0.001 for both) and remained decreased throughout the time course. Expression of miR29b extracellular matrix target genes collagen-1A1 (COL1A1), collagen-3A1 (COL3A1), and fibrillin increased significantly in CHF fibroblasts. Lentivirus-mediated miR29b knockdown in canine atrial fibroblasts (−68%; p<0.01) enhanced COL1A1, COL3A1, and fibrillin mRNA expression by 28% (p<0.01), 19% (p<0.05), and 20% (p<0.05), respectively, versus empty virus–infected fibroblasts and increased COL1A1 protein expression by 90% (p<0.05). In contrast, 3-fold overexpression of miR29b decreased COL1A1, COL3A1, and fibrillin mRNA by 65%, 62%, and 61% (all p<0.001), respectively, versus scrambled control and decreased COL1A1 protein by 60% (p<0.05). MiR29b plasma levels were decreased in patients with CHF or AF (by 53% and 54%, respectively; both p<0.001) and were further decreased in patients with both AF and CHF (by 84%; p<0.001). MiR29b expression was also reduced in the atria of chronic AF patients (by 54% versus sinus rhythm; p<0.05). Adenoassociated viral–mediated knockdown of miR29b in mice significantly increased atrial COL1A1 mRNA expression and cardiac tissue collagen content. Conclusions— MiR29 likely plays a role in atrial fibrotic remodeling and may have value as a biomarker and/or therapeutic target.

Ionic mechanisms limiting cardiac repolarization reserve in humans compared to dogs.

•  Cardiac repolarization, through which heart‐cells return to their resting state after having fired, is a delicate process, susceptible to disruption by common drugs and clinical conditions. •  Animal models, particularly the dog, are often used to study repolarization properties and responses to drugs, with the assumption that such findings are relevant to humans. However, little is known about the applicability of findings in animals to man. •  Here, we studied the contribution of various ion‐currents to cardiac repolarization in canine and human ventricle. •  Humans showed much greater repolarization‐impairing effects of drugs blocking the rapid delayed‐rectifier current IKr than dogs, because of lower repolarization‐reserve contributions from two other important repolarizing currents (the inward‐rectifier IK1 and slow delayed‐rectifier IKs). •  Our findings clarify differences in cardiac repolarization‐processes among species, highlighting the importance of caution when extrapolating results from animal models to man.

Fibroblast Inward-Rectifier Potassium Current Upregulation in Profibrillatory Atrial Remodeling

RATIONALE Fibroblasts are involved in cardiac arrhythmogenesis and contribute to the atrial fibrillation substrate in congestive heart failure (CHF) by generating tissue fibrosis. Fibroblasts display robust ion currents, but their functional importance is poorly understood. OBJECTIVE To characterize atrial fibroblast inward-rectifier K(+) current (IK1) remodeling in CHF and its effects on fibroblast properties. METHODS AND RESULTS Freshly isolated left atrial fibroblasts were obtained from controls and dogs with CHF (ventricular tachypacing). Patch clamp was used to record resting membrane potential (RMP) and IK1. RMP was significantly increased by CHF (from -43.2±0.8 mV, control, to -55.5±0.9 mV). CHF upregulated IK1 (eg, at -90 mV from -1.1±0.2 to -2.7±0.5 pA/pF) and increased the expression of KCNJ2 mRNA (by 52%) and protein (by 80%). Ba(2+) (300 μmol/L) decreased the RMP and suppressed the RMP difference between controls and dogs with CHF. Store-operated Ca(2+) entry (Fura-2-acetoxymethyl ester) and fibroblast proliferation (flow cytometry) were enhanced by CHF. Lentivirus-mediated overexpression of KCNJ2 enhanced IK1 and hyperpolarized fibroblasts. Functional KCNJ2 suppression by lentivirus-mediated expression of a dominant negative KCNJ2 construct suppressed IK1 and depolarized RMP. Overexpression of KCNJ2 increased Ca(2+) entry and fibroblast proliferation, whereas the dominant negative KCNJ2 construct had opposite effects. Fibroblast hyperpolarization to mimic CHF effects on RMP enhanced the Ca(2+) entry. MicroRNA-26a, which targets KCNJ2, was downregulated in CHF fibroblasts. Knockdown of endogenous microRNA-26 to mimic CHF effects unregulated IK1. CONCLUSIONS CHF upregulates fibroblast KCNJ2 expression and currents, thereby hyperpolarizing RMP, increasing Ca(2+) entry, and enhancing atrial fibroblast proliferation. These effects are likely mediated by microRNA-26a downregulation. Remodeling-induced fibroblast KCNJ2 expression changes may play a role in atrial fibrillation promoting fibroblast remodeling and structural/arrhythmic consequences.

Unique Cardiac Purkinje Fiber Transient-Outward Current Beta-Subunit Composition: A Potential Molecular Link to Idiopathic Ventricular Fibrillation

Rationale: A chromosomal haplotype producing cardiac overexpression of dipeptidyl peptidase-like protein-6 (DPP6) causes familial idiopathic ventricular fibrillation. The molecular basis of transient outward current (Ito) in Purkinje fibers (PFs) is poorly understood. We hypothesized that DPP6 contributes to PF Ito and that its overexpression might specifically alter PF Ito properties and repolarization. Objective: To assess the potential role of DPP6 in PF Ito. Methods and Results: Clinical data in 5 idiopathic ventricular fibrillation patients suggested arrhythmia origin in the PF-conducting system. PF and ventricular muscle Ito had similar density, but PF Ito differed from ventricular muscle in having tetraethylammonium sensitivity and slower recovery. DPP6 overexpression significantly increased, whereas DPP6 knockdown reduced, Ito density and tetraethylammonium sensitivity in canine PF but not in ventricular muscle cells. The K+-channel interacting &bgr;-subunit K+-channel interacting protein type-2, essential for normal expression of Ito in ventricular muscle, was weakly expressed in human PFs, whereas DPP6 and frequenin (neuronal calcium sensor-1) were enriched. Heterologous expression of Kv4.3 in Chinese hamster ovary cells produced small Ito; Ito amplitude was greatly enhanced by coexpression with K+-channel interacting protein type-2 or DPP6. Coexpression of DPP6 with Kv4.3 and K+-channel interacting protein type-2 failed to alter Ito compared with Kv4.3/K+-channel interacting protein type-2 alone, but DPP6 expression with Kv4.3 and neuronal calcium sensor-1 (to mimic PF Ito composition) greatly enhanced Ito compared with Kv4.3/neuronal calcium sensor-1 and recapitulated characteristic PF kinetic/pharmacological properties. A mathematical model of cardiac PF action potentials showed that Ito enhancement can greatly accelerate PF repolarization. Conclusions: These results point to a previously unknown central role of DPP6 in PF Ito, with DPP6 gain of function selectively enhancing PF current, and suggest that a DPP6-mediated PF early-repolarization syndrome might be a novel molecular paradigm for some forms of idiopathic ventricular fibrillation.Rationale: A chromosomal haplotype producing cardiac overexpression of dipeptidyl peptidase-like protein-6 (DPP6) causes familial idiopathic ventricular fibrillation. The molecular basis of transient outward current ( I to) in Purkinje fibers (PFs) is poorly understood. We hypothesized that DPP6 contributes to PF I to and that its overexpression might specifically alter PF I to properties and repolarization. Objective: To assess the potential role of DPP6 in PF I to. Methods and Results: Clinical data in 5 idiopathic ventricular fibrillation patients suggested arrhythmia origin in the PF-conducting system. PF and ventricular muscle I to had similar density, but PF I to differed from ventricular muscle in having tetraethylammonium sensitivity and slower recovery. DPP6 overexpression significantly increased, whereas DPP6 knockdown reduced, I to density and tetraethylammonium sensitivity in canine PF but not in ventricular muscle cells. The K+-channel interacting β-subunit K+-channel interacting protein type-2, essential for normal expression of I to in ventricular muscle, was weakly expressed in human PFs, whereas DPP6 and frequenin (neuronal calcium sensor-1) were enriched. Heterologous expression of Kv4.3 in Chinese hamster ovary cells produced small I to; I to amplitude was greatly enhanced by coexpression with K+-channel interacting protein type-2 or DPP6. Coexpression of DPP6 with Kv4.3 and K+-channel interacting protein type-2 failed to alter I to compared with Kv4.3/K+-channel interacting protein type-2 alone, but DPP6 expression with Kv4.3 and neuronal calcium sensor-1 (to mimic PF I to composition) greatly enhanced I to compared with Kv4.3/neuronal calcium sensor-1 and recapitulated characteristic PF kinetic/pharmacological properties. A mathematical model of cardiac PF action potentials showed that I to enhancement can greatly accelerate PF repolarization. Conclusions: These results point to a previously unknown central role of DPP6 in PF I to, with DPP6 gain of function selectively enhancing PF current, and suggest that a DPP6-mediated PF early-repolarization syndrome might be a novel molecular paradigm for some forms of idiopathic ventricular fibrillation. # Novelty and Significance {#article-title-55}

Unique Cardiac Purkinje Fiber Transient Outward Current β-Subunit Composition

Rationale: A chromosomal haplotype producing cardiac overexpression of dipeptidyl peptidase-like protein-6 (DPP6) causes familial idiopathic ventricular fibrillation. The molecular basis of transient outward current (Ito) in Purkinje fibers (PFs) is poorly understood. We hypothesized that DPP6 contributes to PF Ito and that its overexpression might specifically alter PF Ito properties and repolarization. Objective: To assess the potential role of DPP6 in PF Ito. Methods and Results: Clinical data in 5 idiopathic ventricular fibrillation patients suggested arrhythmia origin in the PF-conducting system. PF and ventricular muscle Ito had similar density, but PF Ito differed from ventricular muscle in having tetraethylammonium sensitivity and slower recovery. DPP6 overexpression significantly increased, whereas DPP6 knockdown reduced, Ito density and tetraethylammonium sensitivity in canine PF but not in ventricular muscle cells. The K+-channel interacting &bgr;-subunit K+-channel interacting protein type-2, essential for normal expression of Ito in ventricular muscle, was weakly expressed in human PFs, whereas DPP6 and frequenin (neuronal calcium sensor-1) were enriched. Heterologous expression of Kv4.3 in Chinese hamster ovary cells produced small Ito; Ito amplitude was greatly enhanced by coexpression with K+-channel interacting protein type-2 or DPP6. Coexpression of DPP6 with Kv4.3 and K+-channel interacting protein type-2 failed to alter Ito compared with Kv4.3/K+-channel interacting protein type-2 alone, but DPP6 expression with Kv4.3 and neuronal calcium sensor-1 (to mimic PF Ito composition) greatly enhanced Ito compared with Kv4.3/neuronal calcium sensor-1 and recapitulated characteristic PF kinetic/pharmacological properties. A mathematical model of cardiac PF action potentials showed that Ito enhancement can greatly accelerate PF repolarization. Conclusions: These results point to a previously unknown central role of DPP6 in PF Ito, with DPP6 gain of function selectively enhancing PF current, and suggest that a DPP6-mediated PF early-repolarization syndrome might be a novel molecular paradigm for some forms of idiopathic ventricular fibrillation.Rationale: A chromosomal haplotype producing cardiac overexpression of dipeptidyl peptidase-like protein-6 (DPP6) causes familial idiopathic ventricular fibrillation. The molecular basis of transient outward current ( I to) in Purkinje fibers (PFs) is poorly understood. We hypothesized that DPP6 contributes to PF I to and that its overexpression might specifically alter PF I to properties and repolarization. Objective: To assess the potential role of DPP6 in PF I to. Methods and Results: Clinical data in 5 idiopathic ventricular fibrillation patients suggested arrhythmia origin in the PF-conducting system. PF and ventricular muscle I to had similar density, but PF I to differed from ventricular muscle in having tetraethylammonium sensitivity and slower recovery. DPP6 overexpression significantly increased, whereas DPP6 knockdown reduced, I to density and tetraethylammonium sensitivity in canine PF but not in ventricular muscle cells. The K+-channel interacting β-subunit K+-channel interacting protein type-2, essential for normal expression of I to in ventricular muscle, was weakly expressed in human PFs, whereas DPP6 and frequenin (neuronal calcium sensor-1) were enriched. Heterologous expression of Kv4.3 in Chinese hamster ovary cells produced small I to; I to amplitude was greatly enhanced by coexpression with K+-channel interacting protein type-2 or DPP6. Coexpression of DPP6 with Kv4.3 and K+-channel interacting protein type-2 failed to alter I to compared with Kv4.3/K+-channel interacting protein type-2 alone, but DPP6 expression with Kv4.3 and neuronal calcium sensor-1 (to mimic PF I to composition) greatly enhanced I to compared with Kv4.3/neuronal calcium sensor-1 and recapitulated characteristic PF kinetic/pharmacological properties. A mathematical model of cardiac PF action potentials showed that I to enhancement can greatly accelerate PF repolarization. Conclusions: These results point to a previously unknown central role of DPP6 in PF I to, with DPP6 gain of function selectively enhancing PF current, and suggest that a DPP6-mediated PF early-repolarization syndrome might be a novel molecular paradigm for some forms of idiopathic ventricular fibrillation. # Novelty and Significance {#article-title-55}

Exchange Protein Directly Activated by cAMP Mediates Slow Delayed-Rectifier Current Remodeling by Sustained β-Adrenergic Activation in Guinea Pig Hearts

Rationale: &bgr;-Adrenoceptor activation contributes to sudden death risk in heart failure. Chronic &bgr;-adrenergic stimulation, as occurs in patients with heart failure, causes potentially arrhythmogenic reductions in slow delayed-rectifier K+ current (IKs). Objective: To assess the molecular mechanisms of IKs downregulation caused by chronic &bgr;-adrenergic activation, particularly the role of exchange protein directly activated by cAMP (Epac). Methods and Results: Isolated guinea pig left ventricular cardiomyocytes were incubated in primary culture and exposed to isoproterenol (1 &mgr;mol/L) or vehicle for 30 hours. Sustained isoproterenol exposure decreased IKs density (whole cell patch clamp) by 58% (P<0.0001), with corresponding decreases in potassium voltage-gated channel subfamily E member 1 (KCNE1) mRNA and membrane protein expression (by 45% and 51%, respectively). Potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1) mRNA expression was unchanged. The &bgr;1-adrenoceptor antagonist 1-[2-((3-Carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol dihydrochloride (CGP-20712A) prevented isoproterenol-induced IKs downregulation, whereas the &bgr;2-antagonist ICI-118551 had no effect. The selective Epac activator 8-pCPT-2′-O-Me-cAMP decreased IKs density to an extent similar to isoproterenol exposure, and adenoviral-mediated knockdown of Epac1 prevented isoproterenol-induced IKs/KCNE1 downregulation. In contrast, protein kinase A inhibition with a cell-permeable highly selective peptide blocker did not affect IKs downregulation. 1,2-Bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetate-AM acetoxymethyl ester (BAPTA-AM), cyclosporine, and inhibitor of nuclear factor of activated T cell (NFAT)-calcineurin association-6 (INCA6) prevented IKs reduction by isoproterenol and INCA6 suppressed isoproterenol-induced KCNE1 downregulation, consistent with signal-transduction via the Ca2+/calcineurin/NFAT pathway. Isoproterenol induced nuclear NFATc3/c4 translocation (immunofluorescence), which was suppressed by Epac1 knockdown. Chronic in vivo administration of isoproterenol to guinea pigs reduced IKs density and KCNE1 mRNA and protein expression while inducing cardiac dysfunction and action potential prolongation. Selective in vivo activation of Epac via sp-8-pCPT-2′-O-Me-cAMP infusion decreased IKs density and KCNE1 mRNA/protein expression. Conclusions: Prolonged &bgr;1-adrenoceptor stimulation suppresses IKs by downregulating KCNE1 mRNA and protein via Epac-mediated Ca2+/calcineurin/NFAT signaling. These results provide new insights into the molecular basis of K+ channel remodeling under sustained adrenergic stimulation.

A novel transgenic rabbit model with reduced repolarization reserve: long QT syndrome caused by a dominant-negative mutation of the KCNE1 gene

The reliable assessment of proarrhythmic risk of compounds under development remains an elusive goal. Current safety guidelines focus on the effects of blocking the KCNH2/HERG ion channel‐in tissues and animals with intact repolarization. Novel models with better predictive value are needed that more closely reflect the conditions in patients with cardiac remodelling and reduced repolarization reserve.

Anthrax Lethal Factor Activates K+ Channels To Induce IL-1β Secretion in Macrophages

Anthrax lethal toxin (LeTx) is a virulence factor of Bacilillus anthracis that is a bivalent toxin, containing lethal factor (LF) and protective Ag proteins, which causes cytotoxicity and altered macrophage function. LeTx exposure results in early K+ efflux from macrophages associated with caspase-1 activation and increased IL-1β release. The mechanism of this toxin-induced K+ efflux is unknown. The goals of the current study were to determine whether LeTx-induced K+ efflux from macrophages is mediated by toxin effects on specific K+ channels and whether altered K+-channel activity is involved in LeTx-induced IL-1β release. Exposure of macrophages to LeTx induced a significant increase in the activities of two types of K+ channels that have been identified in mouse macrophages: Ba2+-sensitive inwardly rectifying K+ (Kir) channels and 4-aminopyridine–sensitive outwardly rectifying voltage-gated K+ (Kv) channels. LeTx enhancement of both Kir and Kv required the proteolytic activity of LF, because exposure of macrophages to a mutant LF-protein (LFE687C) combined with protective Ag protein had no effect on the currents. Furthermore, blocking Kir and Kv channels significantly decreased LeTx-induced release of IL-1β. In addition, retroviral transduction of macrophages with wild-type Kir enhanced LeTx-induced release of IL-1β, whereas transduction of dominant-negative Kir blocked LeTx-induced release of IL-1β. Activation of caspase-1 was not required for LeTx-induced activation of either of the K+ channels. These data indicate that a major mechanism through which LeTx stimulates macrophages to release IL-1β involves an LF-protease effect that enhances Kir and Kv channel function during toxin stimulation.