Ange Maguy

Omega-3 Polyunsaturated Fatty Acids Prevent Atrial Fibrillation Associated With Heart Failure but Not Atrial Tachycardia Remodeling

Background— There is epidemiological evidence that omega-3 polyunsaturated fatty acids (PUFAs) reduce the risk of atrial fibrillation (AF), but clinical data are conflicting. The present study assessed the effects of PUFA on AF in experimental models. Methods and Results— We studied the effects of oral PUFA supplements in 2 experimental AF paradigms: electrical remodeling induced by atrial tachypacing (400 bpm for 1 week) and congestive heart failure–associated structural remodeling induced by ventricular tachypacing (240 bpm for 2 weeks). PUFA pretreatment did not directly change atrial effective refractory period (128±6 [mean±SEM] versus 127±2 ms; all effective refractory periods at 300-ms cycle lengths) or burst pacing–induced AF duration (5±4 versus 34±18 seconds). Atrial tachypacing dogs had shorter refractory periods (73±6 ms) and greater AF duration (1185±300 seconds) than shams (119±5 ms and 20±11 seconds; P<0.01 for each). PUFAs did not significantly alter atrial tachypacing effects on refractory periods (77±8 ms) or AF duration (1128±412 seconds). PUFAs suppressed ventricular tachypacing–induced increases in AF duration (952±221 versus 318±249 seconds; P<0.05) and attenuated congestive heart failure–related atrial fibrosis (from 19.2±1.1% to 5.8±1.0%; P<0.001) and conduction abnormalities. PUFAs also attenuated ventricular tachypacing–induced hemodynamic dysfunction (eg, left ventricular end-diastolic and left atrial pressure from 12.2±0.5 and 11.4±0.6 mm Hg, respectively, to 6.4±0.5 and 7.0±0.8 mm Hg; P<0.01) and phosphorylation of mitogen-activated protein kinases (extracellular-signal related and P38 kinase). Conclusions— PUFAs suppress congestive heart failure–induced atrial structural remodeling and AF promotion but do not affect atrial tachycardia–induced electrical remodeling. The beneficial effects of PUFAs on structural remodeling, possibly related to prevention of mitogen-activated protein kinase activation, may contribute to their clinical anti-AF potential.

TRPC3-Dependent Fibroblast Regulation in Atrial Fibrillation

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

A mutation in the drug transporter gene ABCC2 associated with impaired methotrexate elimination.

Human multidrug resistance protein 2 (MRP2, encoded by ABCC2) is involved in active efflux of anionic drugs such as methotrexate. MRP2 is expressed on the luminal side of hepatocytes and renal proximal tubular cells, indicating an important role in drug elimination. We postulated that loss-of-function mutations in ABCC2, which are involved in the Dubin–Johnson syndrome, may be associated with impaired methotrexate elimination and an increased risk of toxicity. We studied the biological phenotype and ABCC2 coding sequence in a patient receiving a high-dose methotrexate infusion for large B-cell lymphoma and who had an unusual pharmacokinetic profile, mainly characterized by a three-fold reduction in the methotrexate elimination rate. This resulted in severe methotrexate over-dosing and reversible nephrotoxicity. An inversion of the urinary coproporphyrin isomer I/III ratio (a specific biological marker of the Dubin–Johnson syndrome) was observed in this patient. Genetic analysis of ABCC2 identified a heterozygous mutation replacing a highly conserved arginine by glycine in the cytoplasmic part of the second membrane-spanning domain (position 412 of MRP2), a region associated with substrate affinity. This genetic variant was not found in a control population. Functional analysis in transiently transfected Chinese hamster ovary cells revealed a loss of transport activity of the G412 MRP2 mutant protein. An ABCC2 mutation altering MRP2-mediated methotrexate transport and resulting in impaired drug elimination and subsequent renal toxicity was identified. Candidates for methotrexate therapy should be considered for MRP2 functional testing.

Membrane cholesterol modulates Kv1.5 potassium channel distribution and function in rat cardiomyocytes.

Membrane lipid composition is a major determinant of cell excitability. In this study, we assessed the role of membrane cholesterol composition in the distribution and function of Kv1.5‐based channels in rat cardiac membranes. In isolated rat atrial myocytes, the application of methyl‐β‐cyclodextrin (MCD), an agent that depletes membrane cholesterol, caused a delayed increase in the Kv1.5‐based sustained component, Ikur, which reached steady state in ∼7 min. This effect was prevented by preloading the MCD with cholesterol. MCD‐increased current was inhibited by low 4‐aminopyridine concentration. Neonatal rat cardiomyocytes transfected with Green Fluorescent Protein (GFP)‐tagged Kv1.5 channels showed a large ultrarapid delayed‐rectifier current (IKur), which was also stimulated by MCD. In atrial cryosections, Kv1.5 channels were mainly located at the intercalated disc, whereas caveolin‐3 predominated at the cell periphery. A small portion of Kv1.5 floated in the low‐density fractions of step sucrose‐gradient preparations. In live neonatal cardiomyocytes, GFP‐tagged Kv1.5 channels were predominantly organized in clusters at the basal plasma membrane. MCD caused reorganization of Kv1.5 subunits into larger clusters that redistributed throughout the plasma membrane. The MCD effect on clusters was sizable 7 min after its application. We conclude that Kv1.5 subunits are concentrated in cholesterol‐enriched membrane microdomains distinct from caveolae, and that redistribution of Kv1.5 subunits by depletion of membrane cholesterol increases their current‐carrying capacity.

Atrial Fibrillation Promotion With Long-Term Repetitive Obstructive Sleep Apnea in a Rat Model

BACKGROUNDnObstructive sleep apnea (OSA) importantly contributes to the occurrence of atrial fibrillation (AF) inxa0humans, but the mechanisms are poorly understood. Experimental research has provided insights into AF promotion byxa0acute OSA episodes. However, patients with OSA usually have frequent nocturnal episodes for some time before manifesting AF.nnnOBJECTIVESnThe goal of this study was to test the hypothesis that repetitive OSA causes cardiac remodeling that predisposes to AF.nnnMETHODSnWe mimicked OSA by using a mechanical ventilator and closing the airway at end-expiration with a 3-way stopcock (OSA rats). Matched control groups included rats with the ventilator stopped but airway left open (open airway rats) and continuously ventilated rats (sham rats). OSA rats were exposed to 20 consecutive 2-min cycles of 40 s of apnea/80 s of ventilation per day, 5 days per week for 4 weeks.nnnRESULTSnOSA significantly increased the duration of AF from (median [interquartile range]) 2.6 s [1.9 s to 8.9 s] (shams) and 16 s [1.8 s to 93 s] (open airway) to 49s [34 s to 444 s]. AF inducibility increased to 56% (9 of 16) of OSA rats; this is up from 15% (2 of 13) and 13% (2 of 15) in open airway and sham rats, respectively (pxa0< 0.05). OSA rats exhibited substantial atrial conduction slowing on optical mapping, along with connexin-43 down-regulation on both quantitative immunofluorescence (expression reduced by 58% vs sham rats) and Western blot (reduced by 38%), as well as increased atrial fibrous tissue content (by 71%). OSA also caused left ventricular hypertrophy, dilation, and diastolic dysfunction and enhanced AF inducibility during superimposed acute OSA episodes to 82.4% of rats.nnnCONCLUSIONSnChronically repeated OSA episodes cause AF-promoting cardiac remodeling, with conduction abnormalities related to connexin dysregulation and fibrosis playing a prominent role. This novel animal model provides mechanistic insights into an important clinical problem and may be useful for further exploration of underlying mechanisms and therapeutic approaches.

Proteomic and metabolomic analysis of atrial profibrillatory remodelling in congestive heart failure

Congestive heart failure (CHF) leads to atrial structural remodelling and increased susceptibility to atrial fibrillation. The underlying molecular mechanisms are poorly understood. We applied high-throughput proteomic and metabolomic analysis to left-atrial cardiomyocytes and tissues obtained from sham and ventricular-tachypaced (VTP, 240 bpm × 24 h and × 2 weeks) CHF dogs. Protein-extracts were subjected to two-dimensional gel electrophoresis using differential in-gel electrophoresis technology. Differentially expressed (P<0.05) proteins were identified by tandem mass-spectrometry. Cardiac metabolites were assayed with high-resolution NMR spectroscopy. Extensive changes occurred in structural proteins, particularly at 2-week VTP, with desmin and filamin fragmentation suggesting structural damage, which was confirmed by electron-microscopy. Oxidant stress was evidenced by decreased antioxidant proteins (superoxide dismutase and peroxiredoxin) at 2-week VTP. Extensive changes in cardioprotective heat shock proteins (HSPs) occurred, with several proteins increasing rapidly (HSP27, HSP60 and HSP70) and others showing a delayed rise (GRP78, α-B-crystallin, and HSP90). An evolving adaptive response to metabolic stress was suggested by early upregulation of malate dehydrogenase (DH), α-/β-enolase and pyruvate dehydrogenase (α-subunit of E1 component) and delayed downregulation of a host of enzymes, along with extensive metabolomic changes. Early changes in metabolite expression that persisted as CHF developed included increased concentrations of glucose and alanine. ADP/ATP accumulation and alpha-ketoisovalerate depletion at 2-week VTP suggested a combination of metabolic stress and less effective energy utilization, as well as a shift from glycolysis to alpha-ketoacid metabolism. We conclude that VTP-induced CHF causes time-dependent changes in the atrial proteome and metabolome, providing insights into molecular mechanisms contributing to arrhythmogenic atrial remodelling.

The role of pulmonary veins vs. autonomic ganglia in different experimental substrates of canine atrial fibrillation

AIMSnPulmonary vein (PV)-encircling ablation, which is effective in suppressing atrial fibrillation (AF), damages autonomic ganglia near the PV ostia. This study examined the effects of PV isolation (PVI) vs. peri-PV ganglionic plexus ablation (GPA) in two discrete canine AF models: ventricular tachypacing (240 bpm, 2 weeks)-induced congestive heart failure (CHF), and atrial tachypacing (400 bpm, 1 week)-induced atrial tachycardia remodeling (ATR).nnnMETHODS AND RESULTSnAll PVs were isolated with an epicardial radiofrequency clamp in nine CHF and eight ATR dogs. Peri-PV ganglionic plexi (identified by bradycardic responses to high-frequency stimulation) were ablated in six CHF and five ATR dogs with an epicardial radiofrequency-ablation pen. Electrophysiologic measurements, including 240-electrode AF mapping, were obtained and dominant frequencies (DFs) determined. Atrial growth associated protein-43 (GAP-43) and neurofilament-M (NF-M) expression were determined immunohistologically. In CHF, neither PVI nor GPA affected AF duration, DF or the already low AF vulnerability. In ATR, PVI reduced AF vulnerability (75 ± 6% to 55 ± 11%, P< 0.05) but did not alter AF duration or DF. In contrast, GPA prolonged atrial refractory period and decreased AF vulnerability (75 ± 8 to 30 ± 10%, P< 0.05), AF duration (617 ± 246 to 39 ± 23 s, **P< 0.01), and DF (11.4 ± 0.6 to 8.6 ± 0.3** Hz, left atrium) in ATR dogs. Both GAP-43 and NF-M expression were decreased in CHF (by 63.1** and 60.0%**) and increased in ATR (by 65.5** and 92.1%, P< 0.001) compared with control.nnnCONCLUSIONSnPVs play a minor role in experimental AF due to CHF or ATR, but autonomic ganglia are important in AF related to ATR. Differential neural remodelling may contribute to varying effects of GPA in discrete AF substrates.

Differences in atrial fibrillation properties under vagal nerve stimulation versus atrial tachycardia remodeling

BACKGROUNDnThere are many similarities between atrial effects of atrial tachycardia remodeling (ATR) and vagal nerve stimulation (VS): both promote atrial fibrillation (AF), reduce atrial effective refractory period (AERP) and AERP rate accommodation, enhance AERP heterogeneity, and increase inward-rectifier K+ current.nnnOBJECTIVEnThis study sought to compare the consequences of ATR and VS at similar levels of AERP abbreviation in dogs.nnnMETHODSnATR dogs (n = 6) were subjected to 7-day atrial tachypacing at 400 beats/min, with radiofrequency-induced atrioventricular block and ventricular demand pacing (80 beats/min) to control ventricular response. VS was applied in 6 matched dogs with stimulation parameters selected to produce similar mean AERP values to ATR dogs.nnnRESULTSnATR and VS produced similarly short AERPs (79 +/- 12 and 80 +/- 12 ms, respectively), AERP rate-adaptation loss, and AERP heterogeneity increases. Although both ATR and VS increased AF duration, VS was significantly more effective in AF promotion, with mean AF duration of 992 +/- 134 seconds, versus 440 +/- 240 seconds (P <.05) under ATR. The greater AF-promoting effect of VS was associated with greater mean dominant frequency values during AF (11.7 +/- 1.8 versus 10.0 +/- 1.3 Hz ATR, P <.05). VS greatly enhanced the spatial dominant frequency variability, increasing the coefficient of variation to 15.2 +/- 1.9 Hz, versus 8.9 +/- 1.5 Hz for ATR (P <.05), primarily by increasing the per-dog maximum dominant frequency (15.4 +/- 0.6 Hz versus 12.5 +/- 0.6 for ATR, P <.01).nnnCONCLUSIONnFor matched AERP values, VS promotes AF more strongly than ATR. Despite similar AERP changes, VS produces considerably greater increases in dominant frequencies, particularly maximum values, consistent with previous suggestions that inward-rectifier K+ current enhancement is particularly effective at accelerating and stabilizing spiral wave rotors that maintain AF.

Induced KCNQ1 autoimmunity accelerates cardiac repolarization in rabbits: Potential significance in arrhythmogenesis and antiarrhythmic therapy

BACKGROUNDnAutoantibodies directed against various cardiac receptors have been implicated in cardiomyopathy and heart rhythm disturbances. In a previous study among patients with dilated cardiomyopathy, autoantibodies targeting the cardiac voltage-gated KCNQ1 K(+) channel were associated with shortened corrected QT intervals (QTc). However, the electrophysiologic actions of KCNQ1 autoimmunity have not been assessed experimentally in a direct fashion.nnnOBJECTIVEnThe purpose of this study was to investigate the cardiac electrophysiologic effects of KCNQ1 autoantibody production induced by vaccination in a rabbit model.nnnMETHODSnRabbits were immunized with KCNQ1 channel peptide. ECG recordings were obtained during a 1-month follow-up period. Rabbits then underwent in vivo electrophysiologic study, after which cardiomyocytes were isolated for analysis of slow delayed rectifier current (IKs) and action potential properties via patch-clamp.nnnRESULTSnKCNQ1-immunized rabbits exhibited shortening of QTc compared to sham-immunized controls. Reduced ventricular effective refractory periods and increased susceptibility to ventricular tachyarrhythmia induction were noted in KCNQ1-immunized rabbits upon programmed ventricular stimulation. Action potential durations were shortened in cardiomyocytes isolated from KCNQ1-immunized rabbits compared to the sham group. IKs step and tail current densities were enhanced after KCNQ1 immunization. Functional and structural changes of the heart were not observed. The potential therapeutic significance of KCNQ1 immunization was then explored in a dofetilide-induced long QT rabbit model. KCNQ1 immunization prevented dofetilide-induced QTc prolongation and attenuated long QT-related arrhythmias.nnnCONCLUSIONnInduction of KCNQ1 autoimmunity accelerates cardiac repolarization and increases susceptibility to ventricular tachyarrhythmia induction through IKs enhancement. On the other hand, vaccination against KCNQ1 ameliorates drug-induced QTc prolongation and might be useful therapeutically to enhance repolarization reserve in long QT syndrome.

Spatiotemporal Stability of Neonatal Rat Cardiomyocyte Monolayers Spontaneous Activity Is Dependent on the Culture Substrate

In native conditions, cardiac cells must continuously comply with diverse stimuli necessitating a perpetual adaptation. Polydimethylsiloxane (PDMS) is commonly used in cell culture to study cellular response to changes in the mechanical environment. The aim of this study was to evaluate the impact of using PDMS substrates on the properties of spontaneous activity of cardiomyocyte monolayer cultures. We compared PDMS to the gold standard normally used in culture: a glass substrate. Although mean frequency of spontaneous activity remained unaltered, incidence of reentrant activity was significantly higher in samples cultured on glass compared to PDMS substrates. Higher spatial and temporal instability of the spontaneous rate activation was found when cardiomyocytes were cultured on PDMS, and correlated with decreased connexin-43 and increased CaV3.1 and HCN2 mRNA levels. Compared to cultures on glass, cultures on PDMS were associated with the strongest response to isoproterenol and acetylcholine. These results reveal the importance of carefully selecting the culture substrate for studies involving mechanical stimulation, especially for tissue engineering or pharmacological high-throughput screening of cardiac tissue analog.