Atrial Fibroblast Proliferation Research Articles

SIRT3 sulfhydration using hydrogen sulfide inhibited angiotensin II-induced atrial fibrosis and vulnerability to atrial fibrillation via suppression of the TGF-β1/smad2/3 signalling pathway

Atrial fibrosis is associated with the occurrence of atrial fibrillation (AF) and regulated by the transforming growth factor-β1 (TGF-β1)/Smad2/3 signalling pathway. Unfortunately, the mechanisms of regulation of TGF-β1/Smad2/3-induced atrial fibrosis and vulnerability to AF remain still unknown. Previous studies have shown that sirtuin3 (SIRT3) sulfhydration has strong anti-fibrotic effects. We hypothesised that SIRT3 sulfhydration inhibits angiotensin II (Ang-II)-induced atrial fibrosis via blocking the TGF-β1/Smad2/3 signalling pathway. In this study, we found that SIRT3 expression was decreased in the left atrium of patients with AF compared to that in those with sinus rhythm (SR). In vitro, SIRT3 knockdown by small interfering RNA significantly expanded Ang-II-induced atrial fibrosis and TGF-β1/Smad2/3 signalling pathway activation, whereas supplementation with Sodium Hydrosulfide (NaHS, exogenous hydrogen sulfide donor and sulfhydration agonist) and SIRT3 overexpression using adenovirus ameliorated Ang-II-induced atrial fibrosis. Moreover, we observed suppression of the TGF-β1/Smad2/3 pathway when Ang-II was combined with NaHS treatment, and the effect of this co-treatment was consistent with that of Ang-II combined with LY3200882 (Smad pathway inhibitor) on reducing atrial fibroblast proliferation and cell migration in vitro. Supplementation with dithiothreitol (DTT, a sulfhydration inhibitor) and adenovirus SIRT3 shRNA blocked the ameliorating effect of NaHS and AngII co-treatment on atrial fibrosis in vitro. Finally, continued treatment with NaHS in rats ameliorated atrial fibrosis and remodelling, and further improved AF vulnerability induced by Ang-II, which was reversed by DTT and adenovirus SIRT3 shRNA, suggesting that SIRT3 sulfhydration might be a potential therapeutic target in atrial fibrosis and AF.

Ezh2 Inhibits Replicative Senescence of Atrial Fibroblasts Through Promotion of H3K27me3 in the Promoter Regions of CDKN2a and Timp4 Genes.

BackgroundIn most cell types, replicative senescence (RS) is supposed to be a principle causative factor for aging. Atrial fibrosis, pathologically characterized by proliferation of atrial fibroblasts (AFs) and excessive accumulation of extracellular matrix proteins, is the most common substrate of atrial fibrillation (Afib) in the elderly. However, whether AFs’ RS develops in the aged and fibrotic left atrium (LA) and, if yes, what is the key regulator for the pathogenesis of AFs’ RS remain largely unknown.MethodsWe obtained the left atrial tissues from young (6–8 weeks old) and aged (24 months old) C57BL/6 male mice. Screening and validation of differential genes were performed using comparative analysis of RNA-seq results. Replicative senescence was examined in primary AFs after cell passage. Further gain-of-function and loss-of-function experiments were performed to explore the regulation of the AFs’ RS progression.ResultsIn the present study, we demonstrated that there was a considerable extent of AFs’ RS in the aged and fibrotic LA. Transcriptome screening showed that Ezh2 (Enhancer of zeste homolog 2) was significantly downregulated in the LA tissue of aged mice. Ezh2 is a histone methyltransferase that catalyzes H3K27me3 and mediates transcriptional silencing. We confirmed that Ezh2 was downregulated in the isolated pure senescent AFs. Knockdown of Ezh2 by siRNA or inhibition of Ezh2ʹs methyltransferase activities by GSK-126 and GSK-343 accelerated RS in the early passage of AFs, while its overexpression deaccelerated RS in the late passage of AFs. Mechanistically, Ezh2 suppressed CDKN2a (p16, p19) and Timp4 gene transcription by forming canonical H3K27me3 modifications in their promoter regions. Furthermore, the functional balance between Timp4 and MMP8 in AFs could be collapsed by changes in Ezh2 expression.ConclusionThese results thus indicate that Ezh2 is a key regulator of AFs’ RS and this work may provide a basis for future treatments for atrial fibrosis in the elderly.

Abstract 6970: Extracellular Vesicles Prevent Post-Operative Atrial Fibrillation

Half of patients recovering from cardiac surgery experience a malignant form of atrial fibrillation that results from inflammation in the pericardial space surrounding the heart. Given that post-operative atrial fibrillation (POAF) increases mortality, effective measures to prevent or treat POAF are highly desirable. In this study, we built on previous work to see if extracellular vesicles (EVs) isolated from human atrial explant-derived cell conditioned media can prevent POAF. Hypothesis: Human EVs will reduce AF inducibility in a rat model of sterile pericarditis by reducing inflammation and the malignant pro-fibrillatory transformation of atrial fibroblasts. Methods and Results: Middle aged female and male Sprague-Dawley rats (6 months old) were randomized to sham operation (n=24; 12 females, 12 males) or induction of sterile pericarditis followed by intra-atrial injection of 10 8 human EVs (n=34; 17 females, 17 males) or vehicle (n=34; 17 females, 17 males). Three days later, all rats underwent invasive electrophysiological testing prior to sacrifice. As expected, pericarditis increased the probability of inducing AF (Figure Panel A). EV treatment reduced the probability of AF. Recipient sex did not alter this effect. Analysis of atrial tissue histology (H&E staining) and lysate (Luminex assays for IL-2, Il-18, MCP-1 and PDGF-AB) revealed that EV treatment abrogated the pro-inflammatory effects of pericarditis. As shown in Panel B, pericarditis increased both atrial fibrosis and mass while EV treatment limited the effect of pericarditis on atrial fibrosis and mass. These effects were attributable to the anti-fibrotic effect of EV treatment on atrial fibroblast proliferation profiled using in-vitro cultures exposed to pro-fibrillatory stimuli (i.e., IL-6 and TGFβ1). Conclusion: Intramyocardial injection of EVs at the time of open chest surgery may provide a facile acellular strategy to prevent POAF by reducing inflammation and fibrosis.

LncRNA Nuclear-Enriched Abundant Transcript 1 Regulates Atrial Fibrosis via the miR-320/NPAS2 Axis in Atrial Fibrillation.

Atrial fibrosis is a key contributor to atrial fibrillation (AF). Long non-coding ribonucleic acids (lncRNAs) were demonstrated to exhibit a key role in fibrotic remodeling; however, the function of nuclear-enriched abundant transcript 1 (NEAT1) in atrial fibrosis remains unclear. In the present study, we showed that NEAT1 was upregulated in atrial tissues of AF patients and was positively related to collagen I (coll I) and collagen III (coll III) expressions. Furthermore, the deletion of NEAT1 attenuated angiotensin II (Ang II)-caused atrial fibroblast proliferation, migration, and collagen production. We further observed that NEAT1 knockdown improved Ang II caused mouse atrial fibrosis in in vivo experiments. Moreover, we demonstrated that NEAT1 could negatively regulate miR-320 expression by acting as a competitive endogenous RNA (ceRNA). miR-320 directly targeted neuronal per arnt sim domain protein 2 (NPAS2) and suppressed its expression. We observed that NEAT1 exerted its function via the miR-320–NPAS2 axis in cardiac fibroblasts. These findings indicate that NEAT1 exerts a significant effect on atrial fibrosis and that this lncRNA is a new potential molecular target for AF treatment.

Long non-coding RNA LICPAR regulates atrial fibrosis via TGF-β/Smad pathway in atrial fibrillation

Long non-coding RNA predicting cardiac remodeling (lnc LIPCAR) was implicated in several human diseases, while its role in atrial fibrillation (AF) remained poorly understood. Our study aimed to discover the role of LICPAR played in AF.Samples of atrial muscle tissues from patients diagnosed with sinus rhythm (SR) and atrial fibrillation (AF) were collected, and human atrial fibroblasts were isolated and identified under immunofluorescence staining. After Angiotensin II (Ang II, as a activator of TGF-β) stimulation with LICPAR overexpression or knockdown, the viability and proliferation of atrial fibroblasts were respectively determined using cell counting kit-8 (CCK-8) assay and clone formation assay. Relative expressions of LICPAR, fibrosis- and transforming growth factor-β (TGF-β)/Smad2/3-pathway related proteins were measured using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as needed.LICPAR and TGF-β1 were upregulated and were positively correlated in atrial muscle tissues from AF. Atrial fibroblasts were identified as Vimentin positive. Further analysis indicated that Ang II enhanced the levels of LIPCAR, Smad2/3 phosphorylation and α-smooth muscle actin (α-SMA). Also, upregulating LIPCAR further promoted the promotive effects of Ang II on levels of LIPCAR, Collagen I, Collagen II, α-SMA and Smad2/3 phosphorylation, cell viability and proliferation of atrial fibroblasts, whereas silencing LIPCAR resulted in opposite effects.LICPAR regulates atrial fibrosis via modulating TGF-β/Smad pathway, which provided a potential therapeutic method for AF in clinical practice in the future.

Factor Xa inhibition by rivaroxaban regulates fibrogenesis in human atrial fibroblasts with modulation of nitric oxide synthesis and calcium homeostasis

BackgroundRivaroxaban, a widely used factor Xa inhibitor in reducing stroke in atrial fibrillation (AF) patients has multiple biological effects with activation of protease-activated receptor (PAR) signaling. Atrial fibrosis plays a critical role in the pathophysiology of AF. In this study, we evaluated whether rivaroxaban regulates atrial fibroblast activity and its underlying mechanisms. Methods and resultsMigration, proliferation analyses, nitric oxide (NO) production assay, calcium fluorescence imaging, and western blots were conducted in human atrial fibroblasts with or without rivaroxaban (100 nmol/L or 300 nmol/L) and co-administration of L-NAME (L-NG-nitro arginine methyl ester, 100 μmol/L), EGTA (Ethylene glycol tetra-acetic acid, 1 mmol/L), thrombin (0.5 U/mL), PAR1 agonist peptide (TFLLR-NH2, 100 μmol/L), PAR1 inhibitor (SCH79797, 0.5 μmol/L) and PAR2 inhibitor (GB83, 10 μmol/L). Atrial fibrosis was examined in isoproterenol (100 mg/kg, subcutaneous injection)-treated rats with or without rivaroxaban (10 mg/kg/day orally for 14 consecutive days). Rivaroxaban reduced the migration, pro-collagen type I production, and proliferation of atrial fibroblasts. Rivaroxaban decreased phosphorylated endothelial NO synthase (eNOS) (Thr 495, an inhibitory phosphorylated site of eNOS), and calcium (Ca2+) entry, and increased NO production. Moreover, L-NAME blocked the effects of rivaroxaban on fibroblast collagen and NO production. In the presence of EGTA, the migratory capability was similarly decreased in atrial fibroblasts with and without treatment with rivaroxaban (100 nmol/L), which suggests that rivaroxaban decreases migratory capability of atrial fibroblasts by inhibiting Ca2+ entry. Additionally, rivaroxaban significantly attenuated the effects of thrombin, and TFLLR-NH2 on migratory, proliferative, and pro-collagen type I production capability in atrial fibroblasts. SCH79797 or GB83 decreased pro-collagen type I production, migration, and proliferation capability in fibroblasts, but combined SCH79797 or GB83 with and without rivaroxaban had similar fibroblast activity. Moreover, rivaroxaban significantly decreased atrial fibrosis in isoproterenol-treated rats. ConclusionsRivaroxaban (100–300 nmol/L) regulates atrial fibroblast activity and atrial fibrosis by increasing NO production and decreasing Ca2+ entry through inhibition of PAR signaling.

Reactive oxygen species mediated oxidative stress links diabetes and atrial fibrillation.

Diabetes is an independent risk factor for atrial fibrillation (AF); however, the underlying mechanism linking diabetes and AF remains to be clarified. The present study aimed to explore the molecular mechanism of increased reactive oxygen species (ROS) production in AF and the ROS-mediated downstream events in diabetes. Firstly, the atrial fibroblasts were isolated from the left atrium of rabbits using enzyme digestion and differential adhesion. Then, the isolated cells were identified by morphology analysis under a microscope, collagen distribution using Masson trichrome staining and vimentin by immunofluorescence. Following this, the collected atrial fibroblasts were randomly divided into 7 groups and administered with high glucose (25 mM glucose), H2O2 stimulation (100 nmol/l), glucose + apocynin (100 µg/ml), H2O2 + apocynin, glucose + H2O2, and a combination of glucose, apocynin and H2O2, as well as the negative control (NC). An MTS assay was performed to investigate cell proliferation following the different treatments, and western blotting was conducted to explore the expression of several proteins including NAD(P)H oxidative (NOX) subunits, key factors involved in mitogen-activated protein kinase (MAPK) signaling pathways and matrix metalloproteinases (MMPs). The atrial fibroblasts were spindle-shaped with one or more protuberances. Vimentin was positively expressed in collected cells under confocal laser scanning microscopy. This result indicated that the atrial fibroblasts were successfully prepared. High glucose and H2O2 stimulation significantly increased the proliferation of atrial fibroblasts and apocynin markedly attenuated the promoting effects on cell proliferation induced by high glucose and H2O2 treatment (P<0.05). Additionally, high glucose and H2O2 stimulation increased the expression of Rac1, phospho(p)-c-Jun N-terminal kinase 1, p38, p-p38 and MMP9, which was markedly decreased by the addition of apocynin (P<0.05). The mechanism associated with diabetes and AF may be attributed to oxidative stress (ROS production) derived from NOX activity, and then induced activation of the MAPK signaling pathways and MMP9 expression.

The crucial role of activin A/ALK4 pathway in the pathogenesis of Ang-II-induced atrial fibrosis and vulnerability to atrial fibrillation.

Atrial fibrosis, the hallmark of structural remodeling associated with atrial fibrillation (AF), is characterized by abnormal proliferation of atrial fibroblasts and excessive deposition of extracellular matrix. Transforming growth factor-β1 (TGF-β1)/activin receptor-like kinase 5 (ALK5)/Smad2/3/4 pathway has been reported to be involved in the process. Recent studies have implicated both activin A and its specific downstream component activin receptor-like kinase 4 (ALK4) in stimulating fibrosis in non-cardiac organs. We recently reported that ALK4 haplodeficiency attenuated the pressure overload- and myocardial infarction-induced ventricular fibrosis. However, the role of activin A/ALK4 in the pathogenesis of atrial fibrosis and vulnerability to AF remains unknown. Our study provided experimental and clinical evidence for the involvement of activin A and ALK4 in the pathophysiology of atrial fibrosis and AF. Patients with AF had higher activin A and ALK4 expression in atriums as compared to individuals devoid of AF. After angiotensin-II (Ang-II) stimulation which mimicked atrial fibrosis progression, ALK4-deficient mice showed lower expression of ALK4 in atriums, reduced activation of atrial fibroblasts, blunted atrial enlargement and atrial fibrosis, and further reduced AF vulnerability upon right atrial electrophysiological studies as compared to wild-type littermates. Moreover, we found that apart from the well-known TGF-β1/ALK5 pathway, the activation of activin A/ALK4/smad2/3 pathway played an important role in the pathogenesis of Ang-II-mediated atrial fibrosis and inducibility of AF, suggesting that targeting ALK4 might be a potential therapy for atrial fibrosis and AF.

The Role of the Rho/ROCK Pathway in Ang II and TGF-β1-Induced Atrial Remodeling.

ObjectivesTo study the role of the Rho/ROCK pathway in Ang II and TGF-β1-induced atrial remodeling.Methods and ResultsA canine atrial fibrillation (AF) model was established by rapid atrial pacing (RAP) of the left atrium. The roles of TGF-β1, the RhoA/ROCK signaling pathway and connective tissue growth factor (CTGF) in atrial remodeling were studied via both in vitro and in vivo experiments. Each of the dogs that received RAP developed persistent AF within 4 weeks. The mRNA expression levels of TGF-β1 (1.32±0.38), Collagen-I(1.33±0.91), CTGF(5.83±3.71), RhoA(1.23±0.57) and ROCK-1 (1.02±0.27) in the left atrium were significantly increased following 4 weeks of RAP. Angiotensin II (Ang II) induced the proliferation of atrial fibroblasts and up-regulated the expression of both CTGF and ROCK-1 in a dose-dependent manner. Simvastatin and Y27632 reversed Ang II-induced CFs proliferation, as well as ROCK-1(0.89±0.05 and 1.27±0.03, respectively) and CTGF (0.87±0.04 and 0.91±0.02, respectively) expression. The expression mRNA of ROCK-1(1.74±0.13) and CTGF (2.28±0.11) can upregulated by TGF-β1, and down-regulated by Simvastatin (1.22±0.03 vs 2.27±0.11), Y27632 (1.01±0.04 vs 1.64±0.03), Los (1.04±0.11 vs 1.26±0.05), respectively. Losartan and Simvastatin attenuated the effects of TGF-β1, inhibited RhoA activity as opposed to RhoA protein expression. Y27632 had no effect on either the expression or the activity of RhoA.ConclusionsThe increased expression of profibrotic factors (CTGF, ROCK1 and Smad2/3) played an important role in our RAP-induced AF model. Increased atrial profibrotic factors involve the activation of either the TGF-β1/RhoA/ROCK-1 or the TGF-β1/Smad2/3 signaling pathway.

EHRA/HRS/APHRS/SOLAECE expert consensus on atrial cardiomyopathies: definition, characterization, and clinical implication.

The atria provide an important contribution to cardiac function.1,2 Besides their impact on ventricular filling, they serve as a volume reservoir, host pacemaker cells and important parts of the cardiac conduction system (e.g. sinus node, AV node), and secrete natriuretic peptides like atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) that regulate fluid homeostasis. Atrial myocardium is affected by many cardiac and non-cardiac conditions3 and is, in some respects, more sensitive than ventricular.4 The atria are activated, besides the three specialized intermodal tracts,5,6 through working cardiomyocytes, so that any architectural or structural change in the atrial myocardium may cause significant electrophysiological disturbances. In addition, atrial cells (both cardiomyocytes and non-cardiomyocyte elements like fibroblasts, endothelial cells, and neurons) react briskly and extensively to pathological stimuli3 and are susceptible to a range of genetic influences.7 Responses include atrial cardiomyocyte hypertrophy and contractile dysfunction, arrhythmogenic changes in cardiomyocyte ion-channel and transporter function, atrial fibroblast proliferation, hyperinnervation, and thrombogenic changes.2 Thus, atrial pathologies have a substantial impact on cardiac performance, arrhythmia occurrence, and stroke risk.1,8 Ventricular cardiomyopathies have been well classified; however, a …

Chamber-specific differences in human cardiac fibroblast proliferation and responsiveness toward simvastatin.

Fibroblasts, the most abundant cells in the heart, contribute to cardiac fibrosis, the substrate for the development of arrythmogenesis, and therefore are potential targets for preventing arrhythmic cardiac remodeling. A chamber-specific difference in the responsiveness of fibroblasts from the atria and ventricles toward cytokine and growth factors has been described in animal models, but it is unclear whether similar differences exist in human cardiac fibroblasts (HCFs) and whether drugs affect their proliferation differentially. Using cardiac fibroblasts from humans, differences between atrial and ventricular fibroblasts in serum-induced proliferation, DNA synthesis, cell cycle progression, cyclin gene expression, and their inhibition by simvastatin were determined. The serum-induced proliferation rate of human atrial fibroblasts was more than threefold greater than ventricular fibroblasts with faster DNA synthesis and higher mRNA levels of cyclin genes. Simvastatin predominantly decreased the rate of proliferation of atrial fibroblasts, with inhibition of cell cycle progression and an increase in the G0/G1 phase in atrial fibroblasts with a higher sensitivity toward inhibition compared with ventricular fibroblasts. The DNA synthesis and mRNA levels of cyclin A, D, and E were significantly reduced by simvastatin in atrial but not in ventricular fibroblasts. The inhibitory effect of simvastatin on atrial fibroblasts was abrogated by mevalonic acid (500 μM) that bypasses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition. Chamber-specific differences exist in the human heart because atrial fibroblasts have a higher proliferative capacity and are more sensitive to simvastatin-mediated inhibition through HMG-CoA reductase pathway. This mechanism may be useful in selectively preventing excessive atrial fibrosis without inhibiting adaptive ventricular remodeling during cardiac injury.

Fibroblast inward-rectifier potassium current upregulation in profibrillatory atrial remodeling.

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. To characterize atrial fibroblast inward-rectifier K(+) current (IK1) remodeling in CHF and its effects on fibroblast properties. 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. 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.

Hydrogen sulphide suppresses human atrial fibroblast proliferation and transformation to myofibroblasts

Cardiac fibroblasts are crucial in pathophysiology of the myocardium whereby their aberrant proliferation has significant impact on cardiac function. Hydrogen sulphide (H2S) is a gaseous modulator of potassium channels on cardiomyocytes and has been reported to attenuate cardiac fibrosis. Yet, the mechanism of H2S in modulating proliferation of cardiac fibroblasts remains poorly understood. We hypothesized that H2S inhibits proliferative response of atrial fibroblasts through modulation of potassium channels. Biophysical property of potassium channels in human atrial fibroblasts was examined by whole-cell patch clamp technique and their cellular proliferation in response to H2S was assessed by BrdU assay. Large conductance Ca2+-activated K+ current (BKCa), transient outward K+ current (Ito) and inwardly rectifying K+ current (IKir) were found in human atrial fibroblasts. Current density of BKCa (IC50 = 69.4 μM; n = 6), Ito (IC50 = 55.1 μM; n = 6) and IKir (IC50 = 78.9 μM; n = 6) was significantly decreased (P < 0.05) by acute exposure to NaHS (a H2S donor) in atrial fibroblasts. Furthermore, NaHS (100–500 μM) inhibited fibroblast proliferation induced by transforming growth factor-β1 (TGF-β1; 1 ng/ml), Ang II (100 nM) or 20% FBS. Pre-conditioning of fibroblasts with NaHS decreased basal expression of Kv4.3 (encode Ito), but not KCa1.1 (encode BKCa) and Kir2.1 (encode IKir). Furthermore, H2S significantly attenuated TGF-β1–stimulated Kv4.3 and α-smooth muscle actin expression, which coincided with its inhibition of TGF-β–induced myofibroblast transformation. Our results show that H2S attenuates atrial fibroblast proliferation via suppression of K+ channel activity and moderates their differentiation towards myofibroblasts.

Ca 2+ Entry Through TRP-C Channels Regulates Fibroblast Biology in Chronic Atrial Fibrillation

In this issue of Circulation , Harada et al1 provide fundamental new insights into the cellular mechanism(s) for initiation and maintenance of chronic atrial fibrillation in the human heart. The authors, taking what most might still consider to be an unconventional approach to understanding this proarrhythmic substrate,2,3,4 have identified the atrial fibroblast as an important player. More specifically, this international group of investigators concludes that a particular member of the transient receptor potential or TRP family5 of ion channels, TRPC3, when expressed/upregulated in human atrial fibroblasts, can contribute to chronic atrial fibrillation. Activation or enhanced expression of TRPC3 provides a means for increased transmembrane calcium entry into the fibroblast. This trigger calcium can then result in a marked increase in proliferation, followed by transformation to the myofibroblast phenotype.6,7 A previous study had drawn attention to the possibility that a different TRP channel subtype, TRPM7, could play a somewhat similar proarrhythmic role in the atrium.7,8 Article see p 2051 Atrial fibrillation is the most common form of cardiac arrhythmia in adult humans.9 Importantly, its incidence is projected to increase substantially as a consequence of the association of atrial fibrillation with healthy aging,10 diabetes mellitus, and hypothyroidism. Harada et al1 provide the first evidence for the presence of TRPC3 current during the proliferative phase in cultured human atrial fibroblasts. Knockdown of TRPC3 is able to suppress atrial fibroblast proliferation, and similar results were obtained with the pharmacological inhibitor PYR3, a relatively new pyrazole-based compound. This particular TRP channel exhibits significant calcium permeability. This calcium influx contributes to ERK phosphorylation, which is involved in mediating atrial fibroblast proliferation. A very interesting observation …

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

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. 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. 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.

Voltage-gated Na⁺ channels: novel players in fibroblast-to-myofibroblast transition with a potential role in atrial arrhythmogenesis?

Fibrosis entails the accumulation of excess extracellular matrix (ECM) components such as collagen. Excessive ECM deposition distorts normal tissue architecture, compromises cardiac pump function and disrupts cytoarchitecture of electrically coupled cardiomyocytes, predisposing to arrhythmias. Various triggers can promote cardiac fibrosis. Regardless of the initiating event, cardiac fibroblasts generally first proliferate and then differentiate into ECM-secreting myofibroblasts, the key mediators of fibrotic-tissue remodelling. Atria are more susceptible to fibrotic stimuli than ventricles, and fibrosis is important in atrial fibrillation (AF; Yue et al. 2011). Atrial-fibrotic remodelling causes conduction abnormalities and may promote electrical fibroblast–cardiomyocyte interactions that favour AF. While it is known that ECM-secreting myofibroblasts are central to atrial fibrosis formation, the precise regulatory mechanisms of fibroblast-to-myofibroblast transitions remain unclear (Wakili et al. 2011). A better understanding of the molecular biology underlying AF-promoting fibrosis may help to identify novel drugs for effective AF treatment (Dobrev et al. 2012). Cardiac fibroblasts express ion channels (Du et al. 2010; Li et al. 2009) but their functional role is poorly understood. Recent studies have provided evidence that Ca2+-permeable melastatin type-7 transient-receptor potential (TRPM7) channels regulate human atrial fibroblast/myofibroblast function, potentially contri-buting to AF pathophysiology (Du et al. 2010). Ca2+-permeable TRP canonical-3 (TRPC3) channels control atrial fibroblast proliferation and differentiation by regulating the Ca2+ influx that activates ERK signalling. TRPC3-channel expression is increased in human AF, probably contributing to fibroblast-to-myofibroblast transition (Harada et al. 2011). Thus, TRPM7 and TRPC3 channels and the related Ca2+ influx may play important roles in AF-promoting fibrotic remodelling. However, whether voltage-gated ion channels also contribute to the activation of atrial myofibroblasts is unknown. In a recent issue of The Journal of Physiology, Chatelier et al. (2012) demonstrate a unique de novo functional expression of Na+ currents in human atrial myofibroblasts. Human atrial fibroblasts did not show specific labelling for α-smooth muscle actin (α-SMA), an established marker of myofibroblasts, before 7 days in culture, but a strong staining after 12 days, indicating that the differentiation of fibroblasts into myofibroblasts occurred between 7 and 12 days of culture. The same time course was observed for expression of the Na+-channel subunit Nav1.5, with INa being recordable in every cell after 12 days in culture. RT-qPCR, immunofluorescence and pharmacological experiments showed that INa is predominantly conducted by Nav1.5. The most common method to identify myofibroblasts is immunostaining for α-SMC. One important merit of the work of Chatelier et al. (2012), which is the first to show INa in human atrial myofibroblasts, is the identification of INa as a potential marker of human atrial myofibroblasts, which should foster the electrophysiological separation of fibroblasts and myofibroblasts in diseased human atria. They also add INa to the growing list of ion channels potentially controlling the transition of fibroblasts to ECM-secreting myofibroblasts. Ca2+-handling abnormalities are critical determinants of atrial cardiomyocyte dysfunction in AF (Voigt et al. 2012), and abnormal Ca2+ entry is suggested to be essential for fibroblast proliferation, migration and differentiation into myofibroblasts in diseased hearts (Yue et al. 2011). Fibroblasts lack voltage-gated Ca2+ channels and Ca2+ influx is likely to occur predominantly via TRPM7 and TRPC3 channels. In addition, reverse-mode (Ca2+ influx) Na+–Ca2+ exchanger (NCX) may also participate in Ca2+ signalling in myofibroblasts. Myofibroblast INa had a greater window current than cardiomyocyte INa, which should produce a larger persistent Na+ entry, enhancing Ca2+ influx through NCX that may contribute to cell proliferation. However, whether INa participates in the regulation of Ca2+ fluxes in myofibroblasts needs exploration. Taken together, the work of Chatelier et al. (2012) opens new avenues in the understanding of voltage-gated ion channels in the molecular biology of myofibroblasts in the human atrium. Their study raises several important questions that need addressing in subsequent work. What is the source (endothelial cells?) of myofibroblasts de novo expressing Na+ channels? Do Na+ channels promote fibroblasts differentiation into myofibroblasts? Is ion-channel composition of fibroblasts and myofibroblasts different? Do Na+ channels accelerate myofibroblast proliferation, migration and secretion properties? Is the inhibition of Na+ channels in myofibroblasts an exploitable therapeutic strategy for AF? Answers to these questions should provide important insights into the multifunctional role of ion channels in cardiac myofibroblasts and their precise role in structural remodelling in AF. The authors’ work is supported by the European Network for Translational Research in Atrial Fibrillation, the Atrial Fibrillation Competence Network and German Center for Cardiovascular Research, the Deutsche Forschungsgemeinschaft, and by Fondation Leducq (European–North American Atrial Fibrillation Research Alliance).

Differential Behaviors of Atrial Versus Ventricular Fibroblasts

In various heart disease paradigms, atria show stronger fibrotic responses than ventricles. The possibility that atrial and ventricular fibroblasts respond differentially to pathological stimuli has not been examined. We compared various morphological, secretory, and proliferative response indexes of canine atrial versus ventricular fibroblasts. Cultured atrial fibroblasts showed faster cell surface area increases, distinct morphology at confluence, and greater alpha-smooth muscle actin expression than ventricular fibroblasts. Atrial fibroblast proliferation ([(3)H]thymidine incorporation) responses were consistently greater for a range of growth factors, including fetal bovine serum, platelet-derived growth factor (PDGF), basic fibroblast growth factor, angiotensin II, endothelin-1, and transforming growth factor-beta(1). Normal atrial tissue showed larger myofibroblast density compared with ventricular tissue, and the difference was exaggerated by congestive heart failure. Congestive heart failure atria showed larger fractions of fibroblasts in mitotic phases compared with ventricles and displayed enhanced gene expression of fibroblast-selective markers (collagen-1, collagen-3, fibronectin-1). Gene microarrays revealed 225 differentially expressed transcript probe sets between paired atrial and ventricular fibroblast samples, including extracellular matrix (eg, fibronectin, laminin, fibulin), cell signaling (PDGF, PDGF receptor, angiopoietin, vascular endothelial growth factor), structure (keratin), and metabolism (xanthine dehydrogenase) genes, identifying PDGF as a candidate contributor to atrial-ventricular fibroblast differences. PDGF receptor gene expression was greater in normal atrium compared with ventricle, and congestive heart failure differentially enhanced atrial versus ventricular PDGF and PDGF receptor gene expression. PDGF receptor protein expression and alpha-smooth muscle actin protein expression were enhanced in isolated congestive heart failure fibroblasts. The PDGF receptor tyrosine kinase inhibitor AG1295 eliminated fetal bovine serum- and transforming growth factor-beta(1)-stimulated atrial-ventricular fibroblast proliferative response differences. Atrial fibroblasts behave differently than ventricular fibroblasts over a range of in vitro and in vivo paradigms, with atrial fibroblasts showing enhanced reactivity that may explain greater atrial fibrotic responses. PDGF signaling is particularly important for atrium-selective fibroblast responses and may represent a novel target for arrhythmogenic atrial structural remodeling prevention.

Rapid electrical pacing of cardiomyocytes alters the behavior of cardiac fibroblasts: Implications for atrial fibrillations

Background: Atrial fibrillation (AF) produces atrial tachycardia-related changes in atrial electrophysiology, providing a possible explanation for the progressive nature of the arrhythmia. Structural remodeling results from ventricular dysfunction due to a rapid response to AF, but whether AF itself can cause atrial fibrosis remains unclear. This study investigated the hypothesis that rapid atrial cardiomyocyte activation produces factors that influence atrial fibroblast proliferation and secretory functions. &#x0D; Methods: Cultured canine atrial fibroblasts were treated with medium from rapidly-paced atrial cardiomyocytes (group 5Hz), non-paced cardiomyocytes (group NP) and cardiomyocyte pacing medium alone (group Ø), and analyzed by [3H]thymidine incorporation, Western-blot and real-time RT-PCR. &#x0D; Results: Rapidly-paced cardiomyocyte-conditioned medium had a potent anti-proliferative effect, reducing [3H]thymidine uptake compared to non-paced cardiomyocyte-conditioned medium and medium alone (2,143±1,490** vs. 15,870±3,343 and 16,440±4,193 respectively, **P &lt; 0.01 vs. both NP and Ø). Conditioned medium from rapidly-paced cardiomyocytes increased αSMA protein reflecting an activated myofibroblast phenotype (1.62±0.20** vs. 1.08±0.26 and 1.01±0.24), collagen-1 (2.41±0.22* vs. 1.49±0.33 and 1.10±0.19, *P &lt; 0.05 both NP and Ø) and fibronectin-1 (3.05±0.28* vs. 1.80±0.45 and 1.20±0.19) mRNA expression compared to control cells. Treatment of the conditioned medium with the AT1 receptor blocker valsartan, partially attenuated the pacing-induced αSMA increase but had no effect on fibroblast proliferation. Atrial tissue from 1-week atrial-tachypaced dogs with AV block/ventricular pacing to control ventricular rate similarly showed upregulation of collagen-1 (2.6±0.5 vs. 0.7±0.3, P &lt; 0.01) and fibronectin-1 (4.9±0.6 vs. 1.1±0.4, P &lt; 0.001) mRNA versus unpaced shams. Conclusion: Rapidly-paced cardiomyocytes release substances that profoundly alter cardiac fibroblast function and induce an activated myofibroblast phenotype that is reflected by altered ECM-gene expression in vivo. These findings are consistent with recent observations that AF per se causes ECM remodeling and may have important consequences for understanding and preventing the mechanisms underlying AF progression.

Atrial cardiomyocyte tachycardia alters cardiac fibroblast function: A novel consideration in atrial remodeling☆

Atrial fibrillation (AF) causes tachycardia-induced atrial electrical remodeling, contributing to the progressive nature of the arrhythmia. Ventricular dysfunction due to a rapid response to AF can cause structural remodeling, but whether AF itself directly promotes atrial fibrosis is controversial. This study investigated the hypothesis that rapid atrial cardiomyocyte activation produces factors that influence atrial fibroblast proliferation and secretory functions. Cultured canine atrial fibroblasts were treated with medium from rapidly-paced atrial cardiomyocytes, non-paced cardiomyocytes and cardiomyocyte-pacing medium only, and analyzed by [(3)H]thymidine incorporation, Western blot and real-time RT-PCR. Rapidly-paced cardiomyocyte-conditioned medium reduced [(3)H]thymidine uptake compared to non-paced cardiomyocyte-conditioned medium and medium alone (approximately 85%, P<0.01). Rapidly-paced cardiomyocyte medium increased alpha SMA protein (approximately 55%, p<0.001), collagen-1 (approximately 85%, P<0.05) and fibronectin-1 (approximately 205%, P<0.05) mRNA expression vs. controls. The angiotensin-1 receptor blocker valsartan attenuated pacing-induced alpha SMA changes but did not affect fibroblast proliferation. Suppression of contraction with blebbistatin did not prevent tachypacing-induced changes in [(3)H]thymidine uptake or alpha SMA upregulation, pointing to a primary role of electrical over mechanical cardiomyocyte activity. Atrial tissue from 1-week atrial-tachypaced dogs with ventricular rate control similarly showed upregulation of alpha SMA protein (approximately 40%, P<0.05), collagen-1 (approximately 380%, P<0.01) and fibronectin-1 (approximately 430%, P<0.001) mRNA versus shams. Rapidly-paced cardiomyocytes release substances that profoundly alter cardiac fibroblast function, inducing an activated myofibroblast phenotype that is reflected by increased ECM-gene expression in vivo. These findings are consistent with recent observations that AF per se may cause ECM remodeling, and have potentially important consequences for understanding and preventing the mechanisms underlying AF progression.

Effects of simvastatin on the development of the atrial fibrillation substrate in dogs with congestive heart failure☆

Congestive heart failure (CHF) is a common cause of atrial fibrillation (AF). Oxidative stress and inflammation (profibrotic) and peroxisome proliferator-activated receptor-alpha (PPAR-alpha, antifibrotic) factors may be involved in CHF-related remodeling. We evaluated the effects of simvastatin (antioxidant, anti-inflammatory) and fenofibrate (PPAR-alpha activator) on CHF-related atrial remodeling. Dogs were subjected to 2-week ventricular tachypacing (VTP) in the absence and presence of simvastatin (20 or 80 mg/day) or fenofibrate. Induced AF duration (DAF) was increased by VTP from 36+/-14 (non-paced controls) to 1005+/-257 s (p<0.01). Simvastatin prevented VTP-induced DAF increases (147+/-37 and 84+/-37 s at 20 and 80 mg/day, respectively), but fenofibrate did not (1018+/-352 s). Simvastatin also attenuated CHF-induced conduction abnormalities (heterogeneity-index reduced from 1.5+/-0.1 to 1.1+/-0.1 and 1.0+/-0.1 at 20 and 80 mg/day, p<0.01) and atrial fibrosis (from 19.4+/-1.3% to 10.8+/-0.8% and 9.9+/-0.8% at 20 and 80 mg/day, p<0.01), while fenofibrate did not. Simvastatin (but not fenofibrate) also attenuated VTP-induced left-ventricular nitric-oxide synthase and nitrotyrosine increases, along with hemodynamic dysfunction. Atrial fibroblast proliferation increased with 24-h fetal bovine serum (FBS) stimulation from 654+/-153 to 7264+/-1636 DPM (p<0.001). Simvastatin, but not fenofibrate, suppressed fibroblast proliferation (664+/-192 DPM, p<0.001). Simvastatin also significantly attenuated transforming growth factor-beta1-stimulated alpha-smooth muscle actin (alpha-SMA) expression (indicating myofibroblast differentiation) from 1.3+/-0.1 to 1.0+/-0.1 times baseline (p<0.05). CHF-induced atrial structural remodeling and AF promotion are attenuated by simvastatin, but not fenofibrate. Statin-induced inhibition of profibrotic atrial fibroblast responses and attenuation of left-ventricular dysfunction may contribute to preventing the CHF-induced fibrotic AF substrate.