Interleukin-6-Mediated-Ca2+ Handling Abnormalities Contributes to Atrial Fibrillation in Sterile Pericarditis Rats

Pre-existing Ca2+ handling abnormalities constitute the arrhythmogenic substrate in patients developing postoperative atrial fibrillation (POAF), a common complication after cardiac surgery. Postoperative interleukin (IL)-6 levels are associated with atrial fibrosis in several animal models of POAF, contributing to atrial arrhythmias. Here, we hypothesize that IL-6-mediated-Ca2+ handling abnormalities contribute to atrial fibrillation (AF) in sterile pericarditis (SP) rats, an animal model of POAF. SP was induced in rats by dusting atria with sterile talcum powder. Anti-rat-IL-6 antibody (16.7 μg/kg) was administered intraperitoneally at 30 min after the recovery of anesthesia. In vivo electrophysiology, ex vivo optical mapping, western blots, and immunohistochemistry were performed to elucidate mechanisms of AF susceptibility. IL-6 neutralization ameliorated atrial inflammation and fibrosis, as well as AF susceptibility in vivo and the frequency of atrial ectopy and AF with a reentrant pattern in SP rats ex vivo. IL-6 neutralization reversed the prolongation and regional heterogeneity of Ca2+ transient duration, relieved alternans, reduced the incidence of discordant alternans, and prevented the reduction and regional heterogeneity of the recovery ratio of Ca2+ transient. In agreement, western blots showed that IL-6 neutralization reversed the reduction in the expression of ryanodine receptor 2 (RyR2) and phosphorylated phospholamban. Acute IL-6 administration to isolated rat hearts recapitulated partial Ca2+ handling phenotype in SP rats. In addition, intraperitoneal IL-6 administration to rats increased AF susceptibility, independent of fibrosis. Our results reveal that IL-6-mediated-Ca2+ handling abnormalities in SP rats, especially RyR2-dysfunction, independent of IL-6-induced-fibrosis, early contribute to the development of POAF by increasing propensity for arrhythmogenic alternans.

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Abstract P780: Myosin Light Chain Dephosphorylation by Ppp1r12c Promotes Atrial Hypocontractility in Atrial Fibrillation

Stroke ◽

10.1161/str.52.suppl_1.p780 ◽

2021 ◽

Vol 52 (Suppl_1) ◽

Author(s):

Francisco J Gonzalez-Gonzalez ◽

Perike Srikanth ◽

Andrielle E Capote ◽

Alsina Katherina M ◽

Benjamin Levin ◽

...

Keyword(s):

Atrial Fibrillation ◽

Light Chain ◽

Myosin Light Chain ◽

Molecular Mechanisms ◽

Contractile Function ◽

Right Atrial ◽

Western Blots

Atrial fibrillation (AF) is the most common sustained arrhythmia, with an estimated prevalence in the U.S.of 6.1 million. AF increases the risk of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. We have recently identified protein phosphatase 1 subunit 12c (PPP1R12C) as a key molecule targeting myosin light-chain phosphorylation in AF. Objective: We hypothesize that the overexpression of PPP1R12C causes hypophosphorylation of atrial myosin light-chain 2 (MLC2a), thereby decreasing atrial contractility in AF. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluate the role of the PP1c-PPP1R12C interaction in MLC2a de-phosphorylation, we utilized Western blots, co-immunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A), PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, associated with a reduction in atrial contractility and an increase in AF inducibility. All these discoveries suggest that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

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Abstract P458: Myosin Light Chain Dephosphorylation By Ppp1r12c Promotes Atrial Hypocontractility In Atrial Fibrillation

Circulation Research ◽

10.1161/res.129.suppl_1.p458 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Francisco J Gonzalez-Gonzalez ◽

Srikanth Perike ◽

Frederick Damen ◽

Andrielle Capote ◽

Katherina M Alsina ◽

...

Keyword(s):

Atrial Fibrillation ◽

Light Chain ◽

Myosin Light Chain ◽

Molecular Mechanisms ◽

Contractile Function ◽

Right Atrial ◽

Western Blots

Introduction: Atrial fibrillation (AF), is the most common sustained arrhythmia, with an estimated prevalence in the U.S. of 2.7 million to 6.1 million and is predictive to increase to 12.1 million in 2030. AF increases the chances of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. Objective: The overexpression of PPP1R12C, causes hypophosphorylation of atrial myosin light chain 2 (MLC2a), decreasing atrial contractility. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluated the role of PP1c-PPP1R12C interaction in MLC2a de-phosphorylation we used Western blots, coimmunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A) PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The Overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, that cause a reduction in atrial contractility and increases AF inducibility. All these discoveries advocate that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

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Abstract 607: Antiarrhythmic Effects Of Omega-3 Fatty Acids And Cyp-eicosanoids In A Murine Atrial Fibrillation Model

Hypertension ◽

10.1161/hyp.60.suppl_1.a607 ◽

2012 ◽

Vol 60 (suppl_1) ◽

Author(s):

Bastian Spallek ◽

Christina Westphal ◽

Anne Konkel ◽

Gerd Wallukat ◽

Darryl C Zeldin ◽

...

Keyword(s):

Atrial Fibrillation ◽

Fatty Acids ◽

Vehicle Group ◽

Synthetic Analog ◽

Omega 3 ◽

Atrial Fibrosis ◽

Adrenergic Stimulation ◽

Connexin 40 ◽

Normal Chow

Atrial fibrillation (AF) is the most common arrhythmia in man. Small animal models of AF are rare, limiting the opportunities of mechanistic studies and of evaluating novel treatment strategies. We found that mice can be rendered susceptible to AF by chronic ß-adrenergic stimulation and used this model to test the hypothesis that dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) and endogenously produced cytochrome P450 (CYP)-dependent epoxyeicosanoids protect against AF. Our study included male wild type C57BL/6 mice given either normal chow (WT-n6-group) or a diet supplemented with n-3 PUFAs (2.5% EPA/DHA added to normal chow; WT-n3-group), as well as transgenic littermates with cardiomyocyte-specific overexpression of the human epoxygenase CYP2J2 (CYP-n6 group). All animal groups received 40mg/kg/d of isoproterenol (ISO) or vehicle over 14 days via osmotic mini-pumps. AF induction was tested in vivo by programmed electrical stimulation (PES) and reached 48% (13 of 27 protocols) in ISO-treated WT-n6, compared to 9% (2 of 22) in the vehicle group. ISO-treated WT-n6 mice also featured increased atrial fibrosis, decreased atrial connexin-40 expression, and significantly reduced atrial refractoriness (AERP: 13.1±0.5 vs. 21.7±0.9 ms in vehicle-infused controls). Ventricular refractoriness remained unchanged and ventricular arrhythmias were not inducible. EPA/DHA-supplementation as well as CYP2J2 overexpression significantly reduced ISO-stimulated AF inducibility to 17% (5 of 30 in WT-n3, and 4 of 24 in CYP-n6), decreased atrial fibrosis, and partially prevented connexin-40 down-regulation and AERP reduction (16,1±0,9 in WT-n3 and 17.5±0.5 ms in CYP-n6). Finally, we tested the antiarrhythmic potential of a synthetic analog of 17,18-epoxyeicosatetraenoic acid (17,18-EEQ), an EPA metabolite predominantly produced by CYP2J2 and other epoxygenases. Acute i.v. injection of the 17,18-EEQ compound decreased AF inducibility in ISO-stimulated WT-n6 mice from 67% (14 of 21 in vehicle control) to 33% (10 of 30) and also the mean duration of AF episodes. These results show that ISO-infusion allows establishing a suitable mouse model of AF and indicate an important role of CYP-dependent n-3 PUFA metabolites in preventing AF.

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Abstract 13383: The Vascular Endothelial Barrier: A Novel Therapeutic Target for Preventing Atrial Fibrillation

Circulation ◽

10.1161/circ.142.suppl_3.13383 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Louisa Mezache ◽

Heather Struckman ◽

Anna Phillips ◽

Stephen Baine ◽

Amara Greer-short ◽

...

Keyword(s):

Atrial Fibrillation ◽

Ex Vivo ◽

Vascular Dysfunction ◽

Super Resolution ◽

Vascular Endothelial ◽

Vascular Leak ◽

Barrier Breakdown ◽

Endothelial Growth Factor ◽

Intercalated Disk

Atrial fibrillation (AF), the most common arrhythmia, is associated with inflammation and vascular dysfunction. AF patients have elevated levels of vascular endothelial growth factor (VEGF; 90-580 pg/ml), which promotes vascular leak and edema. We have previously identified edema-induced disruption of sodium channel (Na V 1.5) -rich intercalated disk (ID) nanodomains as a novel arrhythmia mechanism. We hypothesized that (i) elevated VEGF levels promote AF by disrupting ID nanodomains, and slowing atrial conduction, and (ii) protection of the vascular barrier can prevent these arrhythmias. Clinically-relevant VEGF levels (500 pg/ml, 60 minutes) increased FITC-dextran extravasation (99.3% vs. 24.3% in vehicle controls) in WT mouse hearts, consistent with increased vascular leak. Electron microscopy revealed ID nanodomain swelling, near both gap junctions (perinexi; 64±9nm vs 17±1nm) and mechanical junctions (63±4nm vs 27±2nm) in VEGF-treated hearts relative to controls. Super-resolution STORM microscopy revealed Na V 1.5 enrichment at perinexi (9±2 fold) and N-cadherin-rich sites (7±1 fold) relative to non-junctional ID sites in control hearts. VEGF reduced Na V 1.5 enrichment at both sites (6±1 and 4±1 fold, respectively), consistent with Na V 1.5 translocation from ID nanodomains. Atrial conduction, assessed by optical mapping, was slowed by VEGF (10±0.4 cm/s vs 21.3±1.3 cm/s at baseline). VEGF increased atrial arrhythmia burden both ex vivo (80% vs 0% in vehicle controls) and in vivo (70% vs 20% in vehicle controls). Next, we tested two strategies shown to prevent vascular barrier breakdown. Blocking connexin43 hemichannels (αCT11 peptide) decreased both incidence (40%) and duration (1.45±3.42s) of VEGF-induced arrhythmias. Likewise, blocking pannexin1 channels (Panx1-IL2 peptide) shortened VEGF-induced arrhythmias (2.48±0.83s). Mefloquine and spironolactone, which are small molecules that respectively inhibit Cx43 hemichannels and pannexin channels, were also found to effectively prevent VEGF-induced atrial arrhythmias. These results highlight VEGF-induced vascular leak as a novel mechanism for AF, and suggest vascular barrier protection as an anti-arrhythmic strategy.

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A new implantable tool for repeated assessment of supraventricular electrophysiology and atrial fibrillation susceptibility in freely moving rats

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00676.2020 ◽

2020 ◽

Author(s):

Michael Murninkas ◽

Roni Gillis ◽

Danielle I Lee ◽

Sigal Elyagon ◽

Nikhil Suresh Bhandarkar ◽

...

Keyword(s):

Atrial Fibrillation ◽

Ex Vivo ◽

Adult Rats ◽

Electrophysiological Properties ◽

Multiple Risk Factors ◽

Electrophysiological Studies ◽

Cycle Lengths ◽

Major Factors

The complex pathophysiology of atrial fibrillation (AF) is governed by multiple risk factors in ways that are still elusive. Basic electrophysiological properties including atrial effective refractory period (AERP) and conduction velocity are major factors determining the susceptibility of the atrial myocardium to AF. Although there is a great need for affordable animal models in this field of research, in-vivo rodent studies are limited by technical challenges. Recently, we introduced an implantable system for long-term assessment of AF susceptibility in ambulatory rats. However, technical considerations did not allow us to perform concomitant supraventricular electrophysiology measurements. Here, we designed a novel quadripolar-electrode specifically adapted for comprehensive atrial studies in ambulatory rats. Electrodes were fabricated from medical-grade silicone, four platinum-iridium poles and stainless steel fixating pins. Initial quality validation was performed ex-vivo, followed by implantation in adult rats and repeated electrophysiological studies 1, 4 and 8 weeks post implantation. Capture threshold was stable. Baseline AERP values (38.1±2.3 and 39.5±2.0 using 70ms and 120ms S1-S1 cycle lengths, respectively) confirmed the expected absence of rate-adaptation in the unanesthetized state and validated our prediction that markedly higher values reported under anesthesia are non-physiological. Evaluation of AF substrate in parallel with electrophysiological parameters validated our recent finding of a gradual increase in AF susceptibility over-time and demonstrated that this phenomenon is associated with an electrical remodeling process characterized by AERP shortening. Our findings indicate that the miniature quadripolar-electrode is a potent new tool, which opens a window of opportunities for better utilization of rats in AF research.

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P936Atrial remodeling in rat hearts after exposure to high-intensity infrasound

EP Europace ◽

10.1093/europace/euaa162.014 ◽

2020 ◽

Vol 22 (Supplement_1) ◽

Author(s):

A Lousinha ◽

G Pereira ◽

G Borrecho ◽

J Brito ◽

A Oliveira De Carvalho ◽

...

Keyword(s):

High Intensity ◽

Connexin 43 ◽

Noise Exposure ◽

Experimental Studies ◽

Blue Staining ◽

P Value ◽

Atrial Remodeling ◽

Atrial Fibrosis ◽

Rat Hearts ◽

Arrhythmogenic Substrate

Abstract Background Noise is an important environmental risk factor. Industrial environments are rich in high-intensity infrasound (hi-IFS), which we have found to induce myocardial and coronary perivascular fibrosis in rats. Recently, a significant association between noise exposure and the incidence of atrial fibrillation (AF) was found in large cohort studies but the pathophysiology is unclear. Atrial fibrosis remains the cornerstone of atrial pathology in AF. Purpose: We hypothesized that rats exposed to hi-IFS develop atrial remodeling involving fibrosis and connexin 43, which we sought to evaluate. Material and Methods: Seventy-two Wistar rats, half exposed to hi-IFS (120dB, <20Hz) during a maximum period of 12 weeks and half age-matched controls, were studied. Atrial fibrosis was analyzed by Chromotrope-aniline blue staining. The immunohistochemical evaluation of Cx43 was performed using the polyclonal antibody connexin-43m diluted 1:1000 at 4ºC overnight. Digitized images were obtained with an optical microscope using 400× magnifications. The measurements were performed using image J software. A two-way ANOVA model was used to compare the groups. Results: The mean values of the ratio "atrial fibrosis / cardiomyocytes" increased to a maximum of 0,1095 ± 0,04 and 0,5408 ± 0,01, and of the ratio "CX43 / cardiomyocytes" decreased to 0,0834 ± 0,03 and 0,0966 ± 0,03, respectively in IFS-exposed rats and controls. IFS-exposed rats exhibited a significantly higher ratio of fibrosis (p < 0,001) and lower ratio of Cx43 (p = 0,009). Conclusion: High-intensity infrasound exposure triggers atrial remodeling in rat hearts. Whether this finding correlates to arrhythmogenic substrate in noise induced-AF is not known and reinforces the need for further experimental studies. Table 1 Time of exposure (weeks) Group IFS (n = 36) Group CTL (n = 36) P value Ratio of atrial fibrosis / cardiomyocytesMean ± SD 1 0,0896 ± 0,04 0,0460 ± 0,03 0,007 6 0,0936 ± 0,03 0,0491 ± 0,01 0,001 12 0,1095 ± 0,04 0,0541 ± 0,01 0,001 Ratio of atrial CX43 / cardiomyocytesMean ± SD 1 0,1100 ± 0,03 0,1371 ± 0,03 0,047 6 0,0829 ± 0,04 0,1036 ± 0,03 0,170 12 0,0834 ± 0,03 0,0966 ± 0,03 0,259 Mean ± SD of the two measured outcomes in the two groups. IFS – Infrasound; CTL – Control; SD – standard deviation; Cx43 – Connexin 43 Abstract Figure. Atrial fibrosis and Cx43 /cardiomyocytes

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Structural, Pro-Inflammatory and Calcium Handling Remodeling Underlies Spontaneous Onset of Paroxysmal Atrial Fibrillation in JDP2-Overexpressing Mice

International Journal of Molecular Sciences ◽

10.3390/ijms21239095 ◽

2020 ◽

Vol 21 (23) ◽

pp. 9095

Author(s):

Mariana S. Parahuleva ◽

Jens Kockskämper ◽

Jacqueline Heger ◽

Wolfram Grimm ◽

Anna Scherer ◽

...

Keyword(s):

Atrial Fibrillation ◽

Immune Cell ◽

Marker Gene ◽

Fold Increase ◽

Calcium Handling ◽

In Vivo Studies ◽

Western Blots ◽

The Impact ◽

Spontaneous Onset

Background: Cardiac-specific JDP2 overexpression provokes ventricular dysfunction and atrial dilatation in mice. We performed in vivo studies on JDP2-overexpressing mice to investigate the impact of JDP2 on the predisposition to spontaneous atrial fibrillation (AF). Methods: JDP2-overexpression was started by withdrawal of a doxycycline diet in 4-week-old mice. The spontaneous onset of AF was documented by ECG within 4 to 5 weeks of JDP2 overexpression. Gene expression was analyzed by real-time RT-PCR and Western blots. Results: In atrial tissue of JDP2 mice, besides the 3.6-fold increase of JDP2 mRNA, no changes could be detected within one week of JDP2 overexpression. Atrial dilatation and hypertrophy, combined with elongated cardiomyocytes and fibrosis, became evident after 5 weeks of JDP2 overexpression. Electrocardiogram (ECG) recordings revealed prolonged PQ-intervals and broadened P-waves and QRS-complexes, as well as AV-blocks and paroxysmal AF. Furthermore, reductions were found in the atrial mRNA and protein level of the calcium-handling proteins NCX, Cav1.2 and RyR2, as well as of connexin40 mRNA. mRNA of the hypertrophic marker gene ANP, pro-inflammatory MCP1, as well as markers of immune cell infiltration (CD68, CD20) were increased in JDP2 mice. Conclusion: JDP2 is an important regulator of atrial calcium and immune homeostasis and is involved in the development of atrial conduction defects and arrhythmogenic substrates preceding paroxysmal AF.

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Arrhythmia susceptibility in senescent rat hearts

Journal of Biological Research - Bollettino della Società Italiana di Biologia Sperimentale ◽

10.4081/jbr.2014.2143 ◽

2014 ◽

Vol 87 (1) ◽

Author(s):

Stefano Rossi ◽

Silvana Baruffi ◽

Domenico Corradi ◽

Sergio Callegari ◽

Ezio Musso ◽

...

Keyword(s):

Cardiac Electrophysiology ◽

The Elderly ◽

Detailed Knowledge ◽

Activation Patterns ◽

Electrophysiological Properties ◽

Age Related ◽

Rat Hearts ◽

Arrhythmogenic Substrate ◽

Ventricular Activation

Cardiovascular disease increases with age as well as alterations of cardiac electrophysiological properties, but a detailed knowledge about changes in cardiac electrophysiology relevant to arrhythmogenesis in the elderly is relatively lacking. The aim of this study was to determine specific age-related changes in electrophysiological properties of the ventricles which can be related to a structural-functional arrhythmogenic substrate. Multiple epicardial electrograms were recorded on the ventricular surface of in vivo control and aged rats, while arrhythmia vulnerability was investigated by premature stimulation protocols. Single or multiple ectopic beats and sustained ventricular arrhythmias were frequently induced in aged but not in control hearts. Abnormal ventricular activation patterns during sinus rhythm and unchanged conduction velocity during point stimulation in aged hearts suggest the occurrence of impaired impulse conduction through the distal Purkinje system that might create a potential reentry substrate.

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