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 μmol/L). Pyrazole-3 suppressed angiotensin II-induced Ca2+ influx, proliferation, and α-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, 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 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.

Background— Atrial fibrillation (AF) is characterized by structural remodeling, contractile dysfunction, and AF progression. Histone deacetylases (HDACs) influence acetylation of both histones and cytosolic proteins, thereby mediating epigenetic regulation and influencing cell proteostasis. Because the exact function of HDACs in AF is unknown, we investigated their role in experimental and clinical AF models. Methods and Results— Tachypacing of HL-1 atrial cardiomyocytes and Drosophila pupae hearts significantly impaired contractile function (amplitude of Ca 2+ transients and heart wall contractions). This dysfunction was prevented by inhibition of HDAC6 (tubacin) and sirtuins (nicotinamide). Tachypacing induced specific activation of HDAC6, resulting in α-tubulin deacetylation, depolymerization, and degradation by calpain. Tachypacing-induced contractile dysfunction was completely rescued by dominant-negative HDAC6 mutants with loss of deacetylase activity in the second catalytic domain, which bears α-tubulin deacetylase activity. Furthermore, in vivo treatment with the HDAC6 inhibitor tubastatin A protected atrial tachypaced dogs from electric remodeling (action potential duration shortening, L-type Ca 2+ current reduction, AF promotion) and cellular Ca 2+ -handling/contractile dysfunction (loss of Ca 2+ transient amplitude, sarcomere contractility). Finally, atrial tissue from patients with AF also showed a significant increase in HDAC6 activity and reduction in the expression of both acetylated and total α-tubulin. Conclusions— AF induces remodeling and loss of contractile function, at least in part through HDAC6 activation and subsequent derailment of α-tubulin proteostasis and disruption of the cardiomyocyte microtubule structure. In vivo inhibition of HDAC6 protects against AF-related atrial remodeling, disclosing the potential of HDAC6 as a therapeutic target in clinical AF.

Angiotensin-II (Ang-II) from extracardiac sources and intracardiac synthesis regulates cardiac homeostasis, with mitogenic and growth-promoting effects largely due to altered gene expression. Here, we assessed the possibility that angiotensin-1 (AT1R) or angiotensin-2 (AT2R) receptors on the nuclear envelope mediate effects on cardiomyocyte gene expression. Immunoblots of nucleus-enriched fractions from isolated cardiomyocytes indicated the presence of AT1R and AT2R proteins that copurified with the nuclear membrane marker nucleoporin-62 and histone-3, but not markers of plasma (calpactin-I), Golgi (GRP-78), or endoplasmic reticulum (GM130) membranes. Confocal microscopy revealed AT1R and AT2R proteins on nuclear membranes. Microinjected Ang-II preferentially bound to nuclear sites of isolated cardiomyocytes. AT1R and AT2R ligands enhanced de novo RNA synthesis in isolated cardiomyocyte nuclei incubated with [␣-

Chronic endurance exercise increased AF susceptibility in rats, with autonomic changes, atrial dilation, and fibrosis identified as potential mechanistic contributors. Vagal promotion is particularly important and occurs via augmented baroreflex responsiveness and increased cardiomyocyte sensitivity to cholinergic stimulation, possibly due to RGS protein downregulation.

G protein-coupled receptors (GPCRs) play key physiological roles in numerous tissues, including the heart, and their dysfunction influences a wide range of cardiovascular diseases. Recently, the notion of nuclear localization and action of GPCRs has become more widely accepted. Nuclear-localized receptors may regulate distinct signalling pathways, suggesting that the biological responses mediated by GPCRs are not solely initiated at the cell surface but may result from the integration of extracellular and intracellular signalling pathways. Many of the observed nuclear effects are not prevented by classical inhibitors that exclusively target cell surface receptors, presumably because of their structures, lipophilic properties, or affinity for nuclear receptors. In this topical review, we discuss specifically how angiotensin-II, endothelin, β-adrenergic and opioid receptors located on the nuclear envelope activate signalling pathways, which convert intracrine stimuli into acute responses such as generation of second messengers and direct genomic effects, and thereby participate in the development of cardiovascular disorders.

AMPK is activated by metabolic stress and AF, and helps maintain the intactness of atrial ICa,L, Ca(2+) handling, and cell contractility. AMPK contributes to the atrial compensatory response to AF-related metabolic stress; AF-related metabolic responses may be an interesting new therapeutic target.

Abstract-Transient outward Kϩ current (I to ) downregulation following sustained tachycardia in vivo is usually attributed to tachycardiomyopathy. This study assessed potential direct rate regulation of cardiac I to and underlying mechanisms. Cultured adult canine left ventricular cardiomyocytes (37°C) were paced continuously at 1 or 3 Hz for 24 hours. I to was recorded with whole-cell patch clamp. The 3-Hz pacing reduced I to by 44% (PϽ0.01). Kv4.3 mRNA and protein expression were significantly reduced (by Ϸ30% and Ϸ40%, respectively) in 3-Hz paced cells relative to 1-Hz cells, but KChIP2 expression was unchanged. Prevention of Ca 2ϩ loading with nimodipine or calmodulin inhibition with W-7, A-7, or W-13 eliminated 3-Hz pacing-induced I to downregulation, whereas downregulation was preserved in the presence of valsartan. Inhibition of Ca 2ϩ /calmodulin-dependent protein kinase (CaMK)II with KN93, or calcineurin with cyclosporin A, also prevented I to downregulation. CaMKII-mediated phospholamban phosphorylation at threonine 17 was increased in 3-Hz paced cells, compatible with enhanced CaMKII activity, with functional significance suggested by acceleration of the Ca 2ϩ i transient decay time constant (Indo 1-acetoxymethyl ester microfluorescence). Total phospholamban expression was unchanged, as was expression of Na ϩ /Ca 2ϩ exchange and sarcoplasmic reticulum Ca 2ϩ -ATPase proteins. Nuclear localization of the calcineurin-regulated nuclear factor of activated T cells (NFAT)c3 was increased in 3-Hz paced cells compared to 1-Hz (immunohistochemistry, immunoblot). INCA-6 inhibition of NFAT prevented I to reduction in 3-Hz paced cells. Calcineurin activity increased after 6 hours of 3-Hz pacing. CaMKII inhibition prevented calcineurin activation and NFATc3 nuclear translocation with 3-Hz pacing. We conclude that tachycardia downregulates I to expression, with the Ca 2ϩ /calmodulin-dependent CaMKII and calcineurin/NFAT systems playing key Ca 2ϩ -sensing and signal-transducing roles in rate-dependent I to control. Key Words: potassium channels Ⅲ calcium Ⅲ calmodulin Ⅲ remodeling Ⅲ arrhythmias S udden cardiac death caused by ventricular tachycardia or fibrillation is an important contributor to mortality in congestive heart failure (CHF) patients. 1 Rapid heart-rhythms can impair cardiac function and patients with "tachycardiomyopathy" are at risk of sudden cardiac death. 2 Chronic ventricular tachypacing in experimental animals produces a dilated cardiomyopathy that mimics clinical tachycardiomyopathy and is often used as an experimental model to study CHF-related cardiac remodeling. 3 Changes in cardiac ion channel transport are important components of this remodeling, and extensive evidence suggests that these ion transport changes are crucial contributors to the pathogenesis of CHFrelated ventricular tachyarrhythmias and sudden death. 3,4 Among the most ubiquitous changes are alterations in the transient outward K ϩ current (I to ), 3 which play potentially important roles in repolarization abnormalities, 5,6 c...

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 ( I K1 ) 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 I K1 . RMP was significantly increased by CHF (from −43.2±0.8 mV, control, to −55.5±0.9 mV). CHF upregulated I K1 (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 I K1 and hyperpolarized fibroblasts. Functional KCNJ2 suppression by lentivirus-mediated expression of a dominant negative KCNJ2 construct suppressed I K1 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 I K1 . 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.