Paracrine signalling by cardiac calcitonin controls atrial fibrogenesis and arrhythmia.

Atrial fibrillation, the most common cardiac arrhythmia, is an important contributor to mortality and morbidity, and particularly to the risk of stroke in humans1. Atrial-tissue fibrosis is a central pathophysiological feature of atrial fibrillation that also hampers its treatment; the underlying molecular mechanisms are poorly understood and warrant investigation given the inadequacy of present therapies2. Here we show that calcitonin, a hormone product of the thyroid gland involved in bone metabolism3, is also produced by atrial cardiomyocytes in substantial quantities and acts as a paracrine signal that affects neighbouring collagen-producing fibroblasts to control their proliferation and secretion of extracellular matrix proteins. Global disruption of calcitonin receptor signalling in mice causes atrial fibrosis and increases susceptibility to atrial fibrillation. In mice in which liver kinase B1 is knocked down specifically in the atria, atrial-specific knockdown of calcitonin promotes atrial fibrosis and increases and prolongs spontaneous episodes of atrial fibrillation, whereas atrial-specific overexpression of calcitonin prevents both atrial fibrosis and fibrillation. Human patients with persistent atrial fibrillation show sixfold lower levels of myocardial calcitonin compared to control individuals with normal heart rhythm, with loss of calcitonin receptors in the fibroblast membrane. Although transcriptome analysis of human atrial fibroblasts reveals little change after exposure to calcitonin, proteomic analysis shows extensive alterations in extracellular matrix proteins and pathways related to fibrogenesis, infection and immune responses, and transcriptional regulation. Strategies to restore disrupted myocardial calcitonin signalling thus may offer therapeutic avenues for patients with atrial fibrillation.

Atrial cardiomyocytes contribute to the inflammatory status associated with atrial fibrillation in right heart disease.

AIMS: Right heart disease (RHD), characterized by right ventricular (RV) and atrial (RA) hypertrophy, and cardiomyocytes' (CM) dysfunctions have been described to be associated with the incidence of atrial fibrillation (AF). Right heart disease and AF have in common, an inflammatory status, but the mechanisms relating RHD, inflammation, and AF remain unclear. We hypothesized that right heart disease generates electrophysiological and morphological remodelling affecting the CM, leading to atrial inflammation and increased AF susceptibility. METHODS AND RESULTS: Pulmonary artery banding (PAB) was surgically performed (except for sham) on male Wistar rats (225-275 g) to provoke an RHD. Twenty-one days (D21) post-surgery, all rats underwent echocardiography and electrophysiological studies (EPS). Optical mapping was performed in situ, on Langendorff-perfused hearts. The contractility of freshly isolated CM was evaluated and recorded during 1 Hz pacing in vitro. Histological analyses were performed on formalin-fixed RA to assess myocardial fibrosis, connexin-43 levels, and CM morphology. Right atrial levels of selected genes and proteins were obtained by qPCR and Western blot, respectively. Pulmonary artery banding induced severe RHD identified by RV and RA hypertrophy. Pulmonary artery banding rats were significantly more susceptible to AF than sham. Compared to sham RA CM from PAB rats were significantly elongated and hypercontractile. Right atrial CM from PAB animals showed significant augmentation of mRNA and protein levels of pro-inflammatory interleukin (IL)-6 and IL1ß. Sarcoplasmic-endoplasmic reticulum Ca2+-ATPase-2a (SERCA2a) and junctophilin-2 were decreased in RA CM from PAB compared to sham rats. CONCLUSIONS: Right heart disease-induced arrhythmogenicity may occur due to dysfunctional SERCA2a and inflammatory signalling generated from injured RA CM, which leads to an increased risk of AF.

Distinct protein kinase C isoforms drive the cell cycle re-entry of two separate populations of neonatal rat ventricular cardiomyocytes.

The present study tested the hypothesis that protein kinase C-α (PKC-α) recruitment in the presence of the p38α/ß MAPK inhibitor SB203580 facilitated the appearance and cell cycle re-entry of nestin(+)-neonatal rat ventricular cardiomyocytes (NNVMs) and induced a transcript profile delineating a proliferative phenotype. Phorbol 12,13-dibutyrate (PDBu) treatment did not induce de novo nestin expression or increase the cell cycle re-entry of 1-day-old NNVMs but significantly increased runt-related transcription factor 1 (Runx1) and p16 cell cycle inhibitor (CDKN2a) mRNA levels and downregulated epithelial cell transforming 2 (ECT2) mRNA expression. SB203580 administration to PDBu-treated NNVMs induced de novo nestin expression, preferentially increased the density (normalized to 500 NNVMs) of nestin(+)-NNVMs that incorporated 5-bromo-2'-deoxyuridine (PDBu, 1.4 ± 3 vs. PDBu/SB203580, 128 ± 34; n = 5 independent litters), significantly inhibited CDKN2a and Runx1 mRNA upregulation and reversed ECT2 mRNA downregulation. PDBu treatment of NNVMs reduced PKC-α, protein kinase-δ (PKC-δ) and protein kinase-ε (PKC-ε) protein levels and GF109203X (conventional PKC isoform inhibitor) selectively attenuated PKC-α protein downregulation. GF109203X administration to PDBu/SB203580-treated NNVMs significantly reduced the density of nestin(+)-NNVMs that incorporated 5-bromo-2'-deoxyuridine (PDBu/SB203580/GF109203X, 40 ± 46; n = 5). Moreover, GF109203X/PDBu/SB203580 treatment unmasked the predominant appearance of a separate NNVM population that incorporated 5-bromo-2'-deoxyuridine (PDBu/SB203580/GF109203X, 192 ± 42; n = 5) delineated by the absence of de novo nestin expression. Sotrastaurin (conventional/novel PKC isoform inhibitor) administration to PDBu/SB203580-treated NNVMs significantly attenuated the density of nestin(+)-NNVMs (PDBu/SB203580/sotrastaurin, 8 ± 10; n = 4) and nestin(-)-NNVMs (PDBu/SB203580/sotrastaurin, 64 ± 30; n = 4) that incorporated 5-bromo-2'-deoxyuridine. These data reveal that the neonatal rat heart contains at least two separate populations of NNVMs that re-enter the cell cycle and the preferential appearance of nestin(+)- or nestin(-)-NNVMs is driven by distinct PKC isoforms in the presence of SB203580.NEW & NOTEWORTHY The appearance of nestin(+)-neonatal rat ventricular cardiomyocytes that re-entered the cell cycle following phorbol ester stimulation in the presence of p38α/ß MAPK inhibitor SB203580 was associated with the inhibition of Runx1 and CDKN2a mRNA upregulation. PKC-α selectively induced the cell cycle re-entry of nestin(+)-neonatal rat ventricular cardiomyocytes. Pharmacological inhibition of PKC-α with concomitant p38α/ß MAPK suppression unmasked the cell cycle re-entry of a second population of neonatal rat ventricular cardiomyocytes in the absence of nestin expression.

Calcium-dependent potassium channels control proliferation of cardiac progenitor cells and bone marrow-derived mesenchymal stem cells.

KEY POINTS: Ex vivo proliferated c-Kit+ endogenous cardiac progenitor cells (eCPCs) obtained from mouse and human cardiac tissues have been reported to express a wide range of functional ion channels. In contrast to previous reports in cultured c-Kit+ eCPCs, we found that ion currents were minimal in freshly isolated cells. However, inclusion of free Ca2+ intracellularly revealed a prominent inwardly rectifying current identified as the intermediate conductance Ca2+ -activated K+ current (KCa3.1) Electrical function of both c-Kit+ eCPCs and bone marrow-derived mesenchymal stem cells is critically governed by KCa3.1 calcium-dependent potassium channels. Ca2+ -induced increases in KCa3.1 conductance are necessary to optimize membrane potential during Ca2+ entry. Membrane hyperpolarization due to KCa3.1 activation maintains the driving force for Ca2+ entry that activates stem cell proliferation. Cardiac disease downregulates KCa3.1 channels in resident cardiac progenitor cells. Alterations in KCa3.1 may have pathophysiological and therapeutic significance in regenerative medicine. ABSTRACT: Endogenous c-Kit+ cardiac progenitor cells (eCPCs) and bone marrow (BM)-derived mesenchymal stem cells (MSCs) are being developed for cardiac regenerative therapy, but a better understanding of their physiology is needed. Here, we addressed the unknown functional role of ion channels in freshly isolated eCPCs and expanded BM-MSCs using patch-clamp, microfluorometry and confocal microscopy. Isolated c-Kit+ eCPCs were purified from dog hearts by immunomagnetic selection. Ion currents were barely detectable in freshly isolated c-Kit+ eCPCs with buffering of intracellular calcium (Ca2+i ). Under conditions allowing free intracellular Ca2+ , freshly isolated c-Kit+ eCPCs and ex vivo proliferated BM-MSCs showed prominent voltage-independent conductances that were sensitive to intermediate-conductance K+ -channel (KCa3.1 current, IKCa3.1 ) blockers and corresponding gene (KCNN4)-expression knockdown. Depletion of Ca2+i induced membrane-potential (Vmem ) depolarization, while store-operated Ca2+ entry (SOCE) hyperpolarized Vmem in both cell types. The hyperpolarizing SOCE effect was substantially reduced by IKCa3.1 or SOCE blockade (TRAM-34, 2-APB), and IKCa3.1 blockade (TRAM-34) or KCNN4-knockdown decreased the Ca2+ entry resulting from SOCE. IKCa3.1 suppression reduced c-Kit+ eCPC and BM-MSC proliferation, while significantly altering the profile of cyclin expression. IKCa3.1 was reduced in c-Kit+ eCPCs isolated from dogs with congestive heart failure (CHF), along with corresponding KCNN4 mRNA. Under perforated-patch conditions to maintain physiological [Ca2+ ]i , c-Kit+ eCPCs from CHF dogs had less negative resting membrane potentials (-58 ± 7 mV) versus c-Kit+ eCPCs from control dogs (-73 ± 3 mV, P < 0.05), along with slower proliferation. Our study suggests that Ca2+ -induced increases in IKCa3.1 are necessary to optimize membrane potential during the Ca2+ entry that activates progenitor cell proliferation, and that alterations in KCa3.1 may have pathophysiological and therapeutic significance in regenerative medicine.

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.

Nestin is a marker of lung remodeling secondary to myocardial infarction and type I diabetes in the rat.

Upregulation of the intermediate filament protein nestin was identified in a subpopulation of fibroblasts during reactive and reparative fibrosis and directly contributed to the enhanced proliferative phenotype. The present study tested the hypothesis that nestin was expressed in lung fibroblasts and the pattern of expression represented a distinct marker of pulmonary remodeling secondary to myocardial infarction and type I diabetes. Nestin((+)) fibroblasts were detected in rat lungs and a subpopulation exhibited a myofibroblast phenotype delineated by the co-expression of smooth muscle α-actin. In the lungs of myocardial infarcted rats, interstitial collagen content and nestin mRNA/protein levels were significantly increased despite the absence of secondary pulmonary hypertension, whereas smooth muscle α-actin protein expression was unchanged. Exposure of rat pulmonary fibroblasts to pro-fibrotic stimuli angiotensin II and transforming growth factor-ß significantly increased nestin protein levels. In the lungs of type I diabetic rats, the absence of a reactive fibrotic response was associated with a significant downregulation of nestin mRNA/protein expression. Nestin was reported a target of miR-125b, albeit miR-125b levels were unchanged in pulmonary fibroblasts treated with pro-fibrotic stimuli. Nestin((+)) cells lacking smooth muscle α-actin/collagen staining were also identified in rodent lungs and a transgenic approach revealed that expression of the intermediate filament protein was driven by intron 2 of the nestin gene. The disparate regulation of nestin characterized a distinct pattern of pulmonary remodeling secondary to myocardial infarction and type I diabetes and upregulation of the intermediate filament protein in lung fibroblasts may have facilitated in part the reactive fibrotic response.

Role of small-conductance calcium-activated potassium channels in atrial electrophysiology and fibrillation in the dog.

BACKGROUND: Recent evidence points to functional Ca²âº-dependent K⁺ (SK) channels in the heart that may govern atrial fibrillation (AF) risk, but the underlying mechanisms are unclear. This study addressed the role of SK channels in atrial repolarization and AF persistence in a canine AF model. METHODS AND RESULTS: Electrophysiological variables were assessed in dogs subjected to atrial remodeling by 7-day atrial tachypacing (AT-P), as well as controls. Ionic currents and single-channel properties were measured in isolated canine atrial cardiomyocytes by patch clamp. NS8593, a putative selective SK blocker, suppressed SK current with an IC50 of ≈5 µmol/L, without affecting Na⁺, Ca²âº, or other K⁺ currents. Whole-cell SK current sensitive to NS8593 was significantly larger in pulmonary vein (PV) versus left atrial (LA) cells, without a difference in SK single-channel open probability (P(o)), whereas AT-P enhanced both whole-cell SK currents and single-channel P(o). SK-current block increased action potential duration in both PV and LA cells after AT-P; but only in PV cells in absence of AT-P. SK2 expression was more abundant at both mRNA and protein levels for PV versus LA in control dogs, in both control and AT-P; AT-P upregulated only SK1 at the protein level. Intravenous administration of NS8593 (5 mg/kg) significantly prolonged atrial refractoriness and reduced AF duration without affecting the Wenckebach cycle length, left ventricular refractoriness, or blood pressure. CONCLUSIONS: SK currents play a role in canine atrial repolarization, are larger in PVs than LA, are enhanced by atrial-tachycardia remodeling, and appear to participate in promoting AF maintenance. These results are relevant to the potential mechanisms underlying the association between SK single-nucleotide polymorphisms and AF and suggest SK blockers as potentially interesting anti-AF drugs.

Detailed characterization of microRNA changes in a canine heart failure model: Relationship to arrhythmogenic structural remodeling.

Heart failure (HF) causes left-atrial (LA) and left-ventricular (LV) remodeling, with particularly-prominent changes in LA that create a substrate for atrial fibrillation (AF). MicroRNAs (miRs) are potential regulators in cardiac remodeling. This study evaluated time-dependent miR expression-changes in LA and LV tissue, fibroblasts and cardiomyocytes in experimental HF. HF was induced in dogs by ventricular tachypacing (varying periods, up to 2weeks). Following screening-microarray, 15 miRs were selected for detailed real-time qPCR assay. Extracellular matrix mRNA-expression was assessed by qPCR. Tachypacing time-dependently reduced LV ejection-fraction, increased LV-volume and AF-duration, and caused tissue-fibrosis with LA changes greater than LV. Tissue miR-expression significantly changed in LA for 10 miRs; in LV for none. Cell-selective analysis showed significant time-dependent changes in LA-fibroblasts for 10/15 miRs, LV-fibroblasts 8/15, LA-cardiomyocytes in 6/15 and LV-cardiomyocytes 3/15. Cell-expression specificity did not predict cell-specificity of VTP-induced expression-changes, e.g. 4/6 cardiomyocyte-selective miRs changed almost exclusively in fibroblasts (miR-1, miR-208b, miR133a/b). Thirteen miRs directly implicated in fibrosis/extracellular-matrix regulation were prominently changed: 9/13 showed fibroblast-selective alterations and 5/13 LA-selective. Multiple miRs changed in relation to associated extracellular-matrix targets. Experimental HF causes tissue and cell-type selective, time-dependent changes in cardiac miR-expression. Expression-changes are greater in LA versus LV, and greater in fibroblasts than cardiomyocytes, even for most cardiomyocyte-enriched miRs. This study, the first to examine time, chamber and cell-type selective changes in an experimental model of HF, suggests that multiple miR-changes underlie the atrial-selective fibrotic response and emphasize the importance of considering cell-specificity of miR expression-changes in cardiac remodeling paradigms.

The role of cellular senescence in profibrillatory atrial remodelling associated with cardiac pathology.

AIMS: Cellular senescence is a stress-related or aging response believed to contribute to many cardiac conditions; however, its role in atrial fibrillation (AF) is unknown. Age is the single most important determinant of the risk of AF. The present study was designed to (i) evaluate AF susceptibility and senescence marker expression in rat models of aging and myocardial infarction (MI), (ii) study the effect of reducing senescent-cell burden with senolytic therapy on the atrial substrate in MI rats, and (iii) assess senescence markers in human atrial tissue as a function of age and the presence of AF. METHODS AND RESULTS: AF susceptibility was studied with programmed electrical stimulation. Gene and protein expression was evaluated by immunoblot or immunofluorescence (protein) and digital polymerase chain reaction (PCR) or reverse transcriptase quantitative PCR (messenger RNA). A previously validated senolytic combination, dasatinib and quercetin, (D+Q; or corresponding vehicle) was administered from the time of sham or MI surgery through 28 days later. Experiments were performed blinded to treatment assignment. Burst pacing-induced AF was seen in 100% of aged (18-month old) rats, 87.5% of young MI rats, and 10% of young control (3-month old) rats (P ≤ 0.001 vs. each). Conduction velocity was slower in aged [both left atrium (LA) and right atrium (RA)] and young MI (LA) rats vs. young control rats (P ≤ 0.001 vs. each). Atrial fibrosis was greater in aged (LA and RA) and young MI (LA) vs. young control rats (P < 0.05 for each). Senolytic therapy reduced AF inducibility in MI rats (from 8/9 rats, 89% in MI vehicle, to 0/9 rats, 0% in MI D + Q, P < 0.001) and attenuated LA fibrosis. Double staining suggested that D + Q acts by clearing senescent myofibroblasts and endothelial cells. In human atria, senescence markers were upregulated in older (≥70 years) and long-standing AF patients vs. individuals ≤60 and sinus rhythm controls, respectively. CONCLUSION: Our results point to a potentially significant role of cellular senescence in AF pathophysiology. Modulating cell senescence might provide a basis for novel therapeutic approaches to AF.

An inflammation resolution-promoting intervention prevents atrial fibrillation caused by left ventricular dysfunction.

AIMS: Recent studies suggest that bioactive mediators called resolvins promote an active resolution of inflammation. Inflammatory signalling is involved in the development of the substrate for atrial fibrillation (AF). The aim of this study is to evaluate the effects of resolvin-D1 on atrial arrhythmogenic remodelling resulting from left ventricular (LV) dysfunction induced by myocardial infarction (MI) in rats. METHODS AND RESULTS: MI was produced by left anterior descending coronary artery ligation. Intervention groups received daily intraperitoneal resolvin-D1, beginning before MI surgery (early-RvD1) or Day 7 post-MI (late-RvD1) and continued until Day 21 post-MI. AF vulnerability was evaluated by performing an electrophysiological study. Atrial conduction was analysed by using optical mapping. Fibrosis was quantified by Masson's trichrome staining and gene expression by quantitative polymerase chain reaction and RNA sequencing. Investigators were blinded to group identity. Early-RvD1 significantly reduced MI size (17 ± 6%, vs. 39 ± 6% in vehicle-MI) and preserved LV ejection fraction; these were unaffected by late-RvD1. Transoesophageal pacing induced atrial tachyarrhythmia in 2/18 (11%) sham-operated rats, vs. 18/18 (100%) MI-only rats, in 5/18 (28%, P < 0.001 vs. MI) early-RvD1 MI rats, and in 7/12 (58%, P < 0.01) late-RvD1 MI rats. Atrial conduction velocity significantly decreased post-MI, an effect suppressed by RvD1 treatment. Both early-RvD1 and late-RvD1 limited MI-induced atrial fibrosis and prevented MI-induced increases in the atrial expression of inflammation-related and fibrosis-related biomarkers and pathways. CONCLUSIONS: RvD1 suppressed MI-related atrial arrhythmogenic remodelling. Early-RvD1 had MI sparing and atrial remodelling suppressant effects, whereas late-RvD1 attenuated atrial remodelling and AF promotion without ventricular protection, revealing atrial-protective actions unrelated to ventricular function changes. These results point to inflammation resolution-promoting compounds as novel cardio-protective interventions with a particular interest in attenuating AF substrate development.

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 nonselective 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 protein expression in fibroblasts. Ca(2+) 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, α-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 Ca(2+) 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.

Early ion-channel remodeling and arrhythmias precede hypertrophy in a mouse model of complete atrioventricular block.

Complete atrioventricular block (CAVB) and related ventricular bradycardia are known to induce ventricular hypertrophy and arrhythmias. Different animal models of CAVB have been established with the most common being the dog model. Related studies were mainly focused on the consequences on the main repolarizing currents in these species, i.e. IKr and IKs, with a limited time point kinetics post-AVB. In order to explore at a genomic scale the electrical remodeling induced by AVB and its chronology, we have developed a novel model of CAVB in the mouse using a radiofrequency-mediated ablation procedure. We investigated transcriptional changes in ion channels and contractile proteins in the left ventricles as a function of time (12h, 1, 2 and 5 days after CAVB), using high-throughput real-time RT-PCR. ECG in conscious and anesthetized mice, left ventricular pressure recordings and patch-clamp were used for characterization of this new mouse model. As expected, CAVB was associated with a lengthening of the QT interval. Moreover, polymorphic ventricular tachycardia was recorded in 6/9 freely-moving mice during the first 24h post-ablation. Remarkably, myocardial hypertrophy was only evident 48 h post-ablation and was associated with increased heart weight and altered expression of contractile proteins. During the first 24 hours post-CAVB, genes encoding ion channel subunits were either up-regulated (such as Nav1.5, +74%) or down-regulated (Kv4.2, -43%; KChIP2, -47%; Navß1, -31%; Cx43, -29%). Consistent with the transient alteration of Kv4.2 expression, I(to) was reduced at day 1, but restored at day 5. In conclusion, CAVB induces two waves of molecular remodeling: an early one (≤24 h) leading to arrhythmias, a later one related to hypertrophy. These results provide new molecular basis for ventricular tachycardia induced by AV block.

The inflammation-resolution promoting molecule resolvin-D1 prevents atrial proarrhythmic remodelling in experimental right heart disease.

AIMS: Inflammation plays a role in atrial fibrillation (AF), but classical anti-inflammatory molecules are ineffective. Recent evidence suggests that failure of inflammation-resolution causes persistent inflammatory signalling and that a novel drug-family called resolvins promotes inflammation-resolution. Right heart disease (RHD) is associated with AF; experimental RHD shows signs of atrial inflammatory-pathway activation. Here, we evaluated resolvin-therapy effects on atrial arrhythmogenic remodelling in experimental RHD. METHODS AND RESULTS: Pulmonary hypertension and RHD were induced in rats with an intraperitoneal injection of 60 mg/kg monocrotaline (MCT). An intervention group received daily resolvin-D1 (RvD1), starting 1 day before MCT administration. Right atrial (RA) conduction and gene-expression were analysed respectively by optical mapping and qPCR/gene-microarray. RvD1 had no or minimal effects on MCT-induced pulmonary artery or right ventricular remodelling. Nevertheless, in vivo transoesophageal pacing induced atrial tachyarrhythmias in no CTRL rats vs. 100% MCT-only rats, and only 33% RvD1-treated MCT rats (P < 0.001 vs. MCT-only). Conduction velocity was significantly decreased by MCT, an effect prevented by RvD1. RHD caused RA dilation and fibrosis. RvD1 strongly attenuated RA fibrosis but had no effect on RA dilation. MCT increased RA expression of inflammation- and fibrosis-related gene-expression pathways on gene-microarray transcriptomic analysis, effects significantly attenuated by RvD1 (334 pathways enriched in MCT-rats vs. control; only 177 dysregulated by MCT with RvD1 treatment). MCT significantly increased RA content of type 1 (proinflammatory) CD68-positive M1 macrophages without affecting type 2 (anti-inflammatory) M2 macrophages. RvD1-treated MCT-rat RA showed significant reductions in proinflammatory M1 macrophages and increases in anti-inflammatory M2 macrophages vs. MCT-only. MCT caused statistically significant increases in protein-expression (western blot) of COL3A1, ASC, CASP1, CASP8, IL1ß, TGFß3, CXCL1, and CXCL2, and decreases in MMP2, vs. control. RvD1-treatment suppressed all these MCT-induced protein-expression changes. CONCLUSION: The inflammation-resolution enhancing molecule RvD1 prevents AF-promoting RA remodelling, while suppressing inflammatory changes and fibrotic/electrical remodelling, in RHD. Resolvins show potential promise in combating atrial arrhythmogenic remodelling by suppressing ongoing inflammatory signalling.

Transcriptomic Profiling of Canine Atrial Fibrillation Models After One Week of Sustained Arrhythmia.

[Figure: see text].

Role of atrial arrhythmia and ventricular response in atrial fibrillation induced atrial remodelling.

AIMS: No studies have assessed the specific contributions of atrial fibrillation (AF)-related atrial vs. associated ventricular arrhythmia to remodelling. This study assessed the roles of atrial arrhythmia vs. high ventricular rate in AF-associated remodelling. METHODS AND RESULTS: Four primary dog-groups (12/group) were subjected to 3-week pacing: 600-b.p.m. atrial tachypacing maintaining AF [AF w/o- atrioventricular block (AVB)]; atrial tachypacing with atrioventricular-node ablation (AF+AVB) and ventricular-demand pacing (80 b.p.m.); 160-b.p.m. ventricular-tachypacing (V160) reproducing the response rate during AF; and sinus rhythm with AVB/ventricular-pacing at 80-b.p.m. (control group). At terminal study, left-atrial (LA) effective refractory period (ERP) was reduced equally in both AF groups (w/o-AVB and AF+AVB). AF-inducibility was increased strongly in AF groups (w/o-AVB and AF+AVB) and modestly in V160. AF duration was significantly increased in AF w/o-AVB but not in AF+AVB or V160. Conduction velocity was decreased in AF w/o-AVB, to a greater extent than in AF+AVB and V160. Atrial fibrous-tissue content was increased in AF w/o-AVB, AF+AVB and V160, with collagen-gene up-regulation only in AF w/o-AVB. Connexin43 gene expression was reduced only in AF w/o-AVB. An additional group of 240-b.p.m. ventricular tachypacing dogs (VTP240; to induce heart failure) was studied: vs. other tachypaced groups, VTP240 caused greater fibrosis, but no change in LA-ERP or AF-inducibility. VTP240 also increased AF duration, strongly decreased left ventricular ejection fraction, and was the only group with LA natriuretic-peptide activation. CONCLUSION: The atrial tachyarrhythmia and rapid ventricular response during AF produce distinct atrial remodelling; both contribute to the arrhythmogenic substrate, providing new insights into AF-related remodelling and novel considerations for ventricular rate-control.

Comparison of Atrial Remodeling Caused by Sustained Atrial Flutter Versus Atrial Fibrillation.

BACKGROUND: Atrial flutter (AFL) and atrial fibrillation (AF) are associated with AF-promoting atrial remodeling, but no experimental studies have addressed remodeling with sustained AFL. OBJECTIVES: This study aimed to define the atrial remodeling caused by sustained atrial flutter (AFL) and/or atrial fibrillation (AF). METHODS: Intercaval radiofrequency lesions created a substrate for sustained isthmus-dependent AFL, confirmed by endocavity mapping. Four groups (6 dogs per group) were followed for 3 weeks: sustained AFL; sustained AF (600 beats/min atrial tachypacing); AF superimposed on an AFL substrate (AF+AFLs); sinus rhythm (SR) with an AFL substrate (SR+AFLs; control group). All dogs had atrioventricular-node ablation and ventricular pacemakers at 80 beats/min to control ventricular rate. RESULTS: Monitoring confirmed spontaneous AFL maintenance >99% of the time in dogs with AFL. At terminal open-chest study, left-atrial (LA) effective refractory period was reduced similarly with AFL, AF+AFLs and AF, while AF vulnerability to extrastimuli increased in parallel. Induced AF duration increased significantly in AF+AFLs and AF, but not AFL. Dogs with AF+AFLs had shorter cycle lengths and substantial irregularity versus dogs with AFL. LA volume increased in AF+AFLs and AF, but not dogs with AFL, versus SR+AFLs. Optical mapping showed significant conduction slowing in AF+AFLs and AF but not AFL, paralleling atrial fibrosis and collagen-gene upregulation. Left-ventricular function did not change in any group. Transcriptomic analysis revealed substantial dysregulation of inflammatory and extracellular matrix-signaling pathways with AF and AF+ALs but not AFL. CONCLUSIONS: Sustained AFL causes atrial repolarization changes like those in AF but, unlike AF or AF+AFLs, does not induce structural remodeling. These results provide novel insights into AFL-induced remodeling and suggest that early intervention may be important to prevent irreversible fibrosis when AF intervenes in a patient with AFL.

Right Atrial Mechanisms of Atrial Fibrillation in a Rat Model of Right Heart Disease.

BACKGROUND: Conditions affecting the right heart, including diseases of the lungs and pulmonary circulation, promote atrial fibrillation (AF), but the mechanisms are poorly understood. OBJECTIVES: This study sought to determine whether right heart disease promotes atrial arrhythmogenesis in a rat model of pulmonary hypertension (PH) and, if so, to define the underlying mechanisms. METHODS: PH was induced in male Wistar rats with a single intraperitoneal injection of 60 mg/kg of monocrotaline, and rats were studied 21 days later when right heart disease was well developed. AF vulnerability was assessed in vivo and in situ, and mechanisms were defined by optical mapping, histochemistry, and biochemistry. RESULTS: Monocrotaline-treated rats developed increased right ventricular pressure and mass, along with right atrial (RA) enlargement. AF/flutter was inducible in 32 of 32 PH rats (100%) in vivo and 11 of 12 (92%) in situ, versus 2 of 32 (6%) and 2 of 12 (17%), respectively, in control rats (p < 0.001 vs. PH for each). PH rats had significant RA (16.1 ± 0.5% of cross-sectional area, vs. 3.0 ± 0.6% in control) and left atrial (LA: 11.8 ± 0.5% vs. 5.4 ± 0.8% control) fibrosis. Multiple extracellular matrix proteins, including collagen 1 and 3, fibronectin, and matrix metalloproteinases 2 and 9, were up-regulated in PH rat RA. Optical mapping revealed significant rate-dependent RA conduction slowing and rotor activity, including stable rotors in 4 of 11 PH rats, whereas no significant conduction slowing or rotor activity occurred in the LA of monocrotaline-treated rats. Transcriptomic analysis revealed differentially enriched genes related to hypertrophy, inflammation, and fibrosis in RA of monocrotaline-treated rats versus control. Biochemical results in PH rats were compared with those of AF-prone rats with atrial remodeling in the context of left ventricular dysfunction due to myocardial infarction: myocardial infarction rat LA shared molecular motifs with PH rat RA. CONCLUSIONS: Right heart disease produces a substrate for AF maintenance due to RA re-entrant activity, with an underlying substrate prominently involving RA fibrosis and conduction abnormalities.

An N-/L-type calcium channel blocker, cilnidipine, suppresses autonomic, electrical, and structural remodelling associated with atrial fibrillation.

AIMS: Autonomic dysfunction can promote atrial fibrillation (AF) and results from AF-related remodelling. N-type Ca2+-channels (NTCCs) at sympathetic nerve terminals mediate Ca2+-entry that triggers neurotransmitter release. AF-associated remodelling plays an important role in AF pathophysiology but the effects of NTCC inhibition on such remodelling is unknown. Here, we investigated the ability of a clinically available Ca2+-channel blocker (CCB) with NTCC-blocking activity to suppress the arrhythmogenic effects of AF-promoting remodelling in dogs. METHODS AND RESULTS: Mongrel dogs were kept in AF by right atrial tachypacing at 600 bpm. Four groups were studied under short-term AF (7 days): (i) Shams, instrumented but without tachypacing (n = 5); (ii) a placebo group, tachypaced while receiving placebo (n = 6); (iii) a control tachypacing group receiving nifedipine (10 mg orally twice-daily; n = 5), an L-type CCB; and (iv) a cilnidipine group, subjected to tachypacing and treatment with cilnidipine (10 mg orally twice-daily; n = 7), an N-/L-type CCB. With cilnidipine therapy, dogs with 1-week AF showed significantly reduced autonomic changes reflected by heart rate variability (decreases in RMSSD and pNN50) and plasma norepinephrine concentrations. In addition, cilnidipine-treated dogs had decreased extracellular matrix gene expression vs. nifedipine-dogs. As in previous work, atrial fibrosis had not yet developed after 1-week AF, so three additional groups were studied under longer-term AF (21 days): (i) Shams, instrumented without tachypacing or drug therapy (n = 8); (ii) a placebo group, tachypaced while receiving placebo (n = 8); (iii) a cilnidipine group, subjected to tachypacing during treatment with cilnidipine (10 mg twice-daily; n = 8). Cilnidipine attenuated 3-week AF effects on AF duration and atrial conduction, and suppressed AF-induced increases in fibrous-tissue content, decreases in connexin-43 expression and reductions in sodium-channel expression. CONCLUSIONS: Cilnidipine, a commercially available NTCC-blocking drug, prevents AF-induced autonomic, electrical and structural remodelling, along with associated AF promotion.

The N-terminal juxtamembranous domain of KCNQ1 is critical for channel surface expression: implications in the Romano-Ward LQT1 syndrome.

N-terminal mutations in the KCNQ1 channel are frequently linked to fatal arrhythmias in newborn children and adolescents but the cellular mechanisms involved in this dramatic issue remain, however, to be discovered. Here, we analyzed the trafficking of a series of N-terminal truncation mutants and identified a critical trafficking motif of KCNQ1. This determinant is located in the juxtamembranous region preceding the first transmembrane domain of the protein. Three mutations (Y111C, L114P and P117L) implicated in inherited Romano-Ward LQT1 syndrome, are embedded within this domain. Reexpression studies in both COS-7 cells and cardiomyocytes showed that the mutant proteins fail to exit the endoplasmic reticulum. KCNQ1 subunits harboring Y111C or L114P exert a dominant negative effect on the wild-type KCNQ1 subunit by preventing plasma membrane trafficking of heteromultimeric channels. The P117L mutation had a less pronounced effect on the trafficking of heteromultimeric channels but altered the kinetics of the current. Furthermore, we showed that the trafficking determinant in KCNQ1 is structurally and functionally conserved in other KCNQ channels and constitutes a critical trafficking determinant of the KCNQ channel family. Computed structural predictions correlated the potential structural changes introduced by the mutations with impaired protein trafficking. In conclusion, our studies unveiled a new role of the N-terminus of KCNQ channels in their trafficking and its implication in severe forms of LQT1 syndrome.

JAK-STAT signalling and the atrial fibrillation promoting fibrotic substrate.

AIMS: Left-atrial (LA) fibrosis is an important feature of many atrial fibrillation (AF) substrates. The JAK-STAT system contributes to cardiac remodelling, but its role in AF is unknown. Here we investigated JAK-STAT changes in an AF-model and their potential contributions to LA-fibrosis. METHODS AND RESULTS: LA-remodelling was studied in dogs with heart failure (HF) induced by ventricular tachypacing (VTP, 240 bpm), and in mice with left-ventricular (LV) dysfunction due to myocardial infarction (MI). The selective STAT-3 inhibitor S3I-201 was administered to fibroblasts in vitro or mice in vivo (10 mg/kg/d, osmotic mini-pump). HF-dogs developed LA-selective fibrosis and AF-susceptibility at 1-week VTP. The mRNA-expression of platelet-derived growth factor (PDGF, a JAK-STAT activator) isoforms A, C and D, as well as JAK2, increased in LA fibroblasts from 1-week VTP. HF upregulated protein-expression of PDGF-receptor-ß and phosphorylated (activated) signal transducer and activator of transcription 3 (STAT3) in LA. PDGF-AB stimulation of LA fibroblasts increased PDGFR-α, STAT3 and phosphorylated-STAT3 expression, as well as collagen-1 and fibronectin-1 protein secretion (by 1.6- to 20-fold), with smaller changes in LV fibroblasts. Phosphorylated-STAT3 and collagen upregulation were suppressed by the JAK2 inhibitor AG-490, PDGF receptor inhibitor AG1296 and STAT3-inhibitor SI3-201. In vivo S3I-201 treatment of MI-mice attenuated LA-fibrosis, LA-dilation and P-wave duration changes versus vehicle-control. CONCLUSIONS: HF activates the LA JAK-STAT system and enhances PDGF-signalling. JAK-STAT inhibition reduces the profibrotic effects of PDGF stimulation on canine fibroblasts in vitro while attenuating in vivo LA-fibrosis and remodelling in post-MI mice, suggesting that the JAK/STAT pathway contributes to LA-fibrogenesis and might be a potential target for LA-fibrosis prevention.