Transcriptomic and spatial dissection of human ex vivo right atrial tissue reveals proinflammatory microvascular changes in ischemic heart disease.

Cardiovascular disease plays a central role in the electrical and structural remodeling of the right atrium, predisposing to arrhythmias, heart failure, and sudden death. Here, we dissect with single-nuclei RNA sequencing (snRNA-seq) and spatial transcriptomics the gene expression changes in the human ex vivo right atrial tissue and pericardial fluid in ischemic heart disease, myocardial infarction, and ischemic and non-ischemic heart failure using asymptomatic patients with valvular disease who undergo preventive surgery as the control group. We reveal substantial differences in disease-associated gene expression in all cell types, collectively suggesting inflammatory microvascular dysfunction and changes in the right atrial tissue composition as the valvular and vascular diseases progress into heart failure. The data collectively suggest that investigation of human cardiovascular disease should expand to all functionally important parts of the heart, which may help us to identify mechanisms promoting more severe types of the disease.

Unraveling the evolutionary origin of the complex Nuclear Receptor Element (cNRE), a cis-regulatory module required for preferential expression in the atrial chamber.

Cardiac function requires appropriate proteins in each chamber. Atria requires slow myosin to act as reservoirs, while ventricles demand fast myosin for swift pumping. Myosins are thus under chamber-biased cis-regulation, with myosin gene expression imbalances leading to congenital heart dysfunction. To identify regulatory inputs leading to cardiac chamber-biased expression, we computationally and molecularly dissected the quail Slow Myosin Heavy Chain III (SMyHC III) promoter that drives preferential expression to the atria. We show that SMyHC III gene states are orchestrated by a complex Nuclear Receptor Element (cNRE) of 32 base pairs. Using transgenesis in zebrafish and mice, we demonstrate that preferential atrial expression is achieved by a combinatorial regulatory input composed of atrial activation motifs and ventricular repression motifs. Using comparative genomics, we show that the cNRE might have emerged from an endogenous viral element through infection of an ancestral host germline, revealing an evolutionary pathway to cardiac chamber-specific expression.

Administration of USP7 inhibitor p22077 alleviates Angiotensin II (Ang II)-induced atrial fibrillation in Mice.

Atrial fibrillation (AF), the most common cardiac arrhythmia, is an important contributor to mortality and morbidity. Ubquitin-specific protease 7 (USP7), one of the most abundant ubiquitin-specific proteases (USP), participated in many cellular events, such as cell proliferation, apoptosis, and tumourigenesis. However, its role in AF remains unknown. Here, the mice were treated with Ang II infusion to induce the AF model. Echocardiography was used to measure the atrial diameter. Electrical stimulation was programmed to measure the induction and duration of AF. The changes in atrial remodeling were measured using routine histologic analysis. Here, a significant increase in USP7 expression was observed in Ang II-stimulated atrial cardiomyocytes and atrial tissues, as well as in atrial tissues from patients with AF. The administration of p22077, the inhibitor of USP7, attenuated Ang II-induced inducibility and duration of AF, atrial dilatation, connexin dysfunction, atrial fibrosis, atrial inflammation, and atrial oxidase stress, and then inhibited the progression of AF. Mechanistically, the administration of p22077 alleviated Ang II-induced activation of TGF-ß/Smad2, NF-κB/NLRP3, NADPH oxidases (NOX2 and NOX4) signals, the up-regulation of CX43, ox-CaMKII, CaMKII, Kir2.1, and down-regulation of SERCA2a. Together, this study, for the first time, suggests that USP7 is a critical driver of AF and revealing USP7 may present a new target for atrial fibrillation therapeutic strategies.

HRAS-Mutant Cardiomyocyte Model of Multifocal Atrial Tachycardia.

BACKGROUND: Germline HRAS gain-of-function pathogenic variants cause Costello syndrome (CS). During early childhood, 50% of patients develop multifocal atrial tachycardia, a treatment-resistant tachyarrhythmia of unknown pathogenesis. This study investigated how overactive HRAS activity triggers arrhythmogenesis in atrial-like cardiomyocytes (ACMs) derived from human-induced pluripotent stem cells bearing CS-associated HRAS variants. METHODS: HRAS Gly12 mutations were introduced into a human-induced pluripotent stem cells-ACM reporter line. Human-induced pluripotent stem cells were generated from patients with CS exhibiting tachyarrhythmia. Calcium transients and action potentials were assessed in induced pluripotent stem cell-derived ACMs. Automated patch clamping assessed funny currents. HCN inhibitors targeted pacemaker-like activity in mutant ACMs. Transcriptomic data were analyzed via differential gene expression and gene ontology. Immunoblotting evaluated protein expression associated with calcium handling and pacemaker-nodal expression. RESULTS: ACMs harboring HRAS variants displayed higher beating rates compared with healthy controls. The hyperpolarization activated cyclic nucleotide gated potassium channel inhibitor ivabradine and the Nav1.5 blocker flecainide significantly decreased beating rates in mutant ACMs, whereas voltage-gated calcium channel 1.2 blocker verapamil attenuated their irregularity. Electrophysiological assessment revealed an increased number of pacemaker-like cells with elevated funny current densities among mutant ACMs. Mutant ACMs demonstrated elevated gene expression (ie, ISL1, TBX3, TBX18) related to intracellular calcium homeostasis, heart rate, RAS signaling, and induction of pacemaker-nodal-like transcriptional programming. Immunoblotting confirmed increased protein levels for genes of interest and suppressed MAPK (mitogen-activated protein kinase) activity in mutant ACMs. CONCLUSIONS: CS-associated gain-of-function HRASG12 mutations in induced pluripotent stem cells-derived ACMs trigger transcriptional changes associated with enhanced automaticity and arrhythmic activity consistent with multifocal atrial tachycardia. This is the first human-induced pluripotent stem cell model establishing the mechanistic basis for multifocal atrial tachycardia in CS.

Interleukin-33/ST2 axis involvement in atrial remodeling and arrhythmogenesis.

Interleukin (IL)-33, a cytokine involved in immune responses, can activate its receptor, suppression of tumorigenicity 2 (ST2), is elevated during atrial fibrillation (AF). However, the role of IL-33/ST2 signaling in atrial arrhythmia is unclear. This study explored the pathological effects of the IL-33/ST2 axis on atrial remodeling and arrhythmogenesis. Patch clamping, confocal microscopy, and Western blotting were used to analyze the electrical characteristics of and protein activity in atrial myocytes (HL-1) treated with recombinant IL-33 protein and/or ST2-neutralizing antibodies for 48 hrs. Telemetric electrocardiographic recordings, Masson's trichrome staining, and immunohistochemistry staining of the atrium were performed in mice receiving tail vein injections with nonspecific immunoglobulin (control), IL-33, and IL-33 combined with anti-ST2 antibody for 2 weeks. IL-33-treated HL-1 cells had a reduced action potential duration, lower L-type Ca2+ current, greater sarcoplasmic reticulum (SR) Ca2+ content, increased Na+/Ca2+ exchanger (NCX) current, elevation of K+ currents, and increased intracellular calcium transient. IL-33-treated HL-1 myocytes had greater activation of the calcium-calmodulin-dependent protein kinase II (CaMKII)/ryanodine receptor 2 (RyR2) axis and nuclear factor kappa B (NF-κB) / NLR family pyrin domain containing 3 (NLRP3) signaling than did control cells. IL-33 treated cells also had greater expression of Nav1.5, Kv1.5, NCX, and NLRP3 than did control cells. Pretreatment with neutralizing anti-ST2 antibody attenuated IL-33-mediated activation of CaMKII/RyR2 and NF-κB/NLRP3 signaling. IL-33-injected mice had more atrial ectopic beats and increased AF episodes, greater atrial fibrosis, and elevation of NF-κB/NLRP3 signaling than did controls or mice treated with IL-33 combined with anti-ST2 antibody. Thus, IL-33 recombinant protein treatment promotes atrial remodeling through ST2 signaling. Blocking the IL-33/ST2 axis might be an innovative therapeutic approach for patients with atrial arrhythmia and elevated serum IL-33.

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

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

Inhibition of the acetylcholine-regulated potassium current prevents transient apnea-related atrial arrhythmogenic changes in a porcine model.

BACKGROUND: More than 50% of patients with atrial fibrillation (AF) suffer from sleep disordered breathing (SDB). Obstructive respiratory events contribute to a transient, vagally mediated atrial arrhythmogenic substrate, which is resistant to most available antiarrhythmic drugs. OBJECTIVE: The purpose of this study was to investigate the effect of pharmacologic inhibition of the G-protein-gated acetylcholine-regulated potassium current (IK,ACh) with and without acute autonomic nervous system activation by nicotine in a pig model for obstructive respiratory events. METHODS: In 21 pigs, SDB was simulated by applying an intermittent negative upper airway pressure (INAP). AF inducibility and atrial effective refractory periods (aERPs) were determined before and during INAP by an S1S2 atrial pacing-protocol. Pigs were randomized into 3 groups-group 1: vehicle (n = 4); group 2: XAF-1407 (IK,ACh inhibitor) (n = 7); and group 3: nicotine followed by XAF-1407 (n = 10). RESULTS: In group 1, INAP shortened aERP (ΔaERP -42.6 ms; P = .004) and transiently increased AF inducibility from 0% to 31%. In group 2, XAF-1407 prolonged aERP by 25.2 ms (P = .005) during normal breathing and prevented INAP-induced aERP shortening (ΔaERP -3.6 ms; P = .3) and AF inducibility. In group 3, INAP transiently shortened aERP during nicotine perfusion (ΔaERP -33.6 ms; P = .004) and increased AF inducibility up to 61%, which both were prevented by XAF-1407. CONCLUSION: Simulated obstructive respiratory events transiently shorten aERP and increase AF inducibility, which can be prevented by the IK,ACh-inhibitor XAF-1407. XAF-1407 also prevents these arrhythmogenic changes induced by obstructive respiratory events during nicotine perfusion. Whether IK,ACh channels represent a target for SDB-related AF in humans warrants further study.

Cantharidin and sodium fluoride attenuate the negative inotropic effects of carbachol in the isolated human atrium.

Carbachol, an agonist at muscarinic receptors, exerts a negative inotropic effect in human atrium. Carbachol can activate protein phosphatases (PP1 or PP2A). We hypothesized that cantharidin or sodium fluoride, inhibitors of PP1 and PP2A, may attenuate a negative inotropic effect of carbachol. During bypass-surgery trabeculae carneae of human atrial preparations (HAP) were obtained. These trabeculae were mounted in organ baths and electrically stimulated (1 Hz). Force of contraction was measured under isometric conditions. For comparison, we studied isolated electrically stimulated left atrial preparations (LA) from mice. Cantharidin (100 µM) and sodium fluoride (3 mM) increased force of contraction in LA (n = 5-8, p < 0.05) by 113% ± 24.5% and by 100% ± 38.2% and in HAP (n = 13-15, p < 0.05) by 625% ± 169% and by 196% ± 23.5%, respectively. Carbachol (1 µM) alone exerted a rapid transient maximum negative inotropic effect in LA (n = 6) and HAP (n = 14) to 46.9% ± 3.63% and 19.4% ± 3.74%, respectively (p < 0.05). These negative inotropic effects were smaller in LA (n = 4-6) and HAP (n = 9-12) pretreated with 100 µM cantharidin and amounted to 58.0% ± 2.27% and 59.2% ± 6.19% or 3 mM sodium fluoride to 63.7% ± 9.84% and 46.3% ± 5.69%, (p < 0.05). We suggest that carbachol, at least in part, exerts a negative inotropic effect in the human atrium by stimulating the enzymatic activity of PP1 and/or PP2A.

Chronic kidney disease promotes atrial fibrillation via inflammasome pathway activation.

Chronic kidney disease (CKD) is associated with a higher risk of atrial fibrillation (AF). The mechanistic link between CKD and AF remains elusive. IL-1ß, a main effector of NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation, is a key modulator of conditions associated with inflammation, such as AF and CKD. Circulating IL-1ß levels were elevated in patients with CKD who had AF (versus patients with CKD in sinus rhythm). Moreover, NLRP3 activity was enhanced in atria of patients with CKD. To elucidate the role of NLRP3/IL-1ß signaling in the pathogenesis of CKD-induced AF, Nlrp3-/- and WT mice were subjected to a 2-stage subtotal nephrectomy protocol to induce CKD. Four weeks after surgery, IL-1ß levels in serum and atrial tissue were increased in WT CKD (WT-CKD) mice versus sham-operated WT (WT-sham) mice. The increased susceptibility to pacing-induced AF and the longer AF duration in WT-CKD mice were associated with an abbreviated atrial effective refractory period, enlarged atria, and atrial fibrosis. Genetic inhibition of NLRP3 in Nlrp3-/- mice or neutralizing anti-IL-1ß antibodies effectively reduced IL-1ß levels, normalized left atrial dimensions, and reduced fibrosis and the incidence of AF. These data suggest that CKD creates a substrate for AF development by activating the NLRP3 inflammasome in atria, which is associated with structural and electrical remodeling. Neutralizing IL-1ß antibodies may be beneficial in preventing CKD-induced AF.

OR-1896 increases force of contraction in the isolated human atrium.

OR-1896 ((R)-N-(4-(4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)acetamide) is the main active metabolite of levosimendan. However, nobody has reported a positive inotropic effect of OR-1896 in isolated human cardiac preparations. The mechanism of action of OR-1896 remains controversial. Hence, we wanted to know whether OR-1896 exerts a positive inotropic effect in humans and what might be the underlying mechanism. Therefore, we measured the contractile effects of OR-1896 (0.01-10 µM cumulatively applied) in isolated electrically stimulated (1 Hz) human right atrial preparations (HAP) obtained during cardiac surgery. OR-1896, given alone, exerted time- and concentration-dependent positive inotropic effects; 1-µM OR-1896 increased force by 72 ± 14.7% (p < 0.05, n = 6) and shortened the time of relaxation by 10.6 ± 3.6% (p < 0.05, n = 11) in HAP started at 0.1 µM, plateaued at 1-µM OR-1896, and was antagonized by 1-µM propranolol. The maximum positive inotropic effect of OR-1896 in human right atrial preparations was less than that of 10-µM isoprenaline. EMD 57033 (10 µM), a calcium sensitizer, enhanced the force of contraction further in the additional presence of 1-µM OR-1896 by 109 ± 19% (p < 0.05, n = 4). Cilostamide (10 µM), an inhibitor of phosphodiesterase III given before OR-1896 (1 µM), blocked the positive inotropic effect of OR-1896 in HAP. Our data suggest that OR-1896 is, indeed, a positive inotropic agent in the human heart. OR-1896 acts as a PDE III inhibitor. OR-1896 is unlikely to act as a calcium sensitizer in the human heart.

KCa3.1 Promotes Proinflammatory Exosome Secretion by Activating AKT/Rab27a in Atrial Myocytes during Rapid Pacing.

Purpose: The aim of this study was to investigate the role of the medium-conductance calcium-activated potassium channel (KCNN4, KCa3.1) in the secretion of proinflammatory exosomes by atrial myocytes. Methods: Eighteen beagles were randomly divided into the sham group (n = 6), pacing group (n = 6), and pacing+TRAM-34 group (n = 6). Electrophysiological data, such as the effective refractory period, atrial fibrillation (AF) induction, and AF duration, were collected by programmed stimulation. Atrial tissues were subjected to hematoxylin and eosin, Masson's trichrome, and immunofluorescence staining. The expression of KCa3.1 and Rab27a was assessed by immunohistochemistry and western blotting. The downstream signaling pathways involved in KCa3.1 were examined by rapid pacing or overexpressing KCNN4 in HL-1 cells. Results: Atrial rapid pacing significantly induced electrical remodeling, inflammation, fibrosis, and exosome secretion in the canine atrium, while TRAM-34 (KCa3.1 blocker) inhibited these changes. Compared with those in control HL-1 cells, the levels of exosome markers and inflammatory factors were increased in pacing HL-1 cells. Furthermore, the levels of CD68 and iNOS in macrophages incubated with exosomes derived from HL-1 cells were higher in the pacing-exo group than in the control group. More importantly, KCa3.1 regulated exosome secretion through the AKT/Rab27a signaling pathway. Similarly, inhibiting the downstream signaling pathway of KCa3.1 significantly inhibited exosome secretion. Conclusions: KCa3.1 promotes proinflammatory exosome secretion through the AKT/Rab27a signaling pathway. Inhibiting the KCa3.1/AKT/Rab27a signaling pathway reduces myocardial tissue structural remodeling in AF.

Serum Proteomic Profiles Reflect the Stages of Myxomatous Mitral Valve Disease in Dogs.

Canine myxomatous mitral valve disease (MMVD) is similar to Barlow's form of MMVD in humans. These valvulopathies are complex, with varying speeds of progression. We hypothesized that the relative abundances of serum proteins would help identify the consecutive MMVD stages and discover new disease pathways on a systemic level. To identify distinction-contributing protein panels for disease onset and progression, we compared the proteomic profiles of serum from healthy dogs and dogs with different stages of naturally occurring MMVD. Dogs were divided into experimental groups on the basis of the left-atrium-to-aorta ratio and normalized left ventricular internal dimension in diastole values. Serum was collected from healthy (N = 12) dogs, dogs diagnosed with MMVD in stages B1 (N = 13) and B2 (N = 12) (asymptomatic), and dogs diagnosed with MMVD in chronic stage C (N = 13) (symptomatic). Serum biochemistry and selected ELISAs (galectin-3, suppression of tumorigenicity, and asymmetric dimethylarginine) were performed. Liquid chromatography-mass spectrometry (LC-MS), tandem mass tag (TMT) quantitative proteomics, and statistical and bioinformatics analysis were employed. Most of the 21 serum proteins with significantly different abundances between experimental groups (p < 0.05, FDR ˂ 0.05) were classified as matrix metalloproteinases, protease inhibitors, scaffold/adaptor proteins, complement components, anticoagulants, cytokine, and chaperone. LC-MS TMT proteomics results obtained for haptoglobin, clusterin, and peptidase D were further validated analytically. Canine MMVD stages, including, for the first time, asymptomatic B1 and B2 stages, were successfully distinguished in dogs with the disease and healthy dogs on the basis of the relative abundances of a panel of specific serum proteins. Most proteins with significantly different abundances were involved in immune and inflammatory pathways. Their role in structural remodeling and progression of canine MMVD must be further investigated. Further research is needed to confirm the resemblance/difference with human MMVD. Proteomics data are available via ProteomeXchange with the unique dataset identifier PXD038475.

Cardiac Hamp mRNA Is Predominantly Expressed in the Right Atrium and Does Not Respond to Iron.

Hepcidin is a liver-derived hormone that controls systemic iron traffic. It is also expressed in the heart, where it acts locally. We utilized cell and mouse models to study the regulation, expression, and function of cardiac hepcidin. Hepcidin-encoding Hamp mRNA was induced upon differentiation of C2C12 cells to a cardiomyocyte-like phenotype and was not further stimulated by BMP6, BMP2, or IL-6, the major inducers of hepatic hepcidin. The mRNAs encoding hepcidin and its upstream regulator hemojuvelin (Hjv) are primarily expressed in the atria of the heart, with ~20-fold higher Hamp mRNA levels in the right vs. left atrium and negligible expression in the ventricles and apex. Hjv-/- mice, a model of hemochromatosis due to suppression of liver hepcidin, exhibit only modest cardiac Hamp deficiency and minor cardiac dysfunction. Dietary iron manipulations did not significantly affect cardiac Hamp mRNA in the atria of wild-type or Hjv-/- mice. Two weeks following myocardial infarction, Hamp was robustly induced in the liver and heart apex but not atria, possibly in response to inflammation. We conclude that cardiac Hamp is predominantly expressed in the right atrium and is partially regulated by Hjv; however, it does not respond to iron and other inducers of hepatic hepcidin.

Ergotamine Stimulates Human 5-HT4-Serotonin Receptors and Human H2-Histamine Receptors in the Heart.

Ergotamine (2'-methyl-5'α-benzyl-12'-hydroxy-3',6',18-trioxoergotaman) is a tryptamine-related alkaloid from the fungus Claviceps purpurea. Ergotamine is used to treat migraine. Ergotamine can bind to and activate several types of 5-HT1-serotonin receptors. Based on the structural formula of ergotamine, we hypothesized that ergotamine might stimulate 5-HT4-serotonin receptors or H2-histamine receptors in the human heart. We observed that ergotamine exerted concentration- and time-dependent positive inotropic effects in isolated left atrial preparations in H2-TG (mouse which exhibits cardiac-specific overexpression of the human H2-histamine receptor). Similarly, ergotamine increased force of contraction in left atrial preparations from 5-HT4-TG (mouse which exhibits cardiac-specific overexpression of the human 5-HT4-serotonin receptor). An amount of 10 µM ergotamine increased the left ventricular force of contraction in isolated retrogradely perfused spontaneously beating heart preparations of both 5-HT4-TG and H2-TG. In the presence of the phosphodiesterase inhibitor cilostamide (1 µM), ergotamine 10 µM exerted positive inotropic effects in isolated electrically stimulated human right atrial preparations, obtained during cardiac surgery, that were attenuated by 10 µM of the H2-histamine receptor antagonist cimetidine, but not by 10 µM of the 5-HT4-serotonin receptor antagonist tropisetron. These data suggest that ergotamine is in principle an agonist at human 5-HT4-serotonin receptors as well at human H2-histamine receptors. Ergotamine acts as an agonist on H2-histamine receptors in the human atrium.

Impaired calcium handling mechanisms in atrial trabeculae of diabetic patients.

The aim of this study was to investigate cardiomyocyte Ca2+ handling and contractile function in freshly excised human atrial tissue from diabetic and non-diabetic patients undergoing routine surgery. Multicellular trabeculae (283 ± 20 µm in diameter) were dissected from the endocardial surface of freshly obtained right atrial appendage samples from consenting surgical patients. Trabeculae were mounted in a force transducer at optimal length, electrically stimulated to contract, and loaded with fura-2/AM for intracellular Ca2+ measurements. The response to stimulation frequencies encompassing the physiological range was recorded at 37°C. Myofilament Ca2+ sensitivity was assessed from phase plots and high potassium contractures of force against [Ca2+ ]i . Trabeculae from diabetic patients (n = 12) had increased diastolic (resting) [Ca2+ ]i (p = 0.03) and reduced Ca2+ transient amplitude (p = 0.04) when compared to non-diabetic patients (n = 11), with no difference in the Ca2+ transient time course. Diastolic stress was increased (p = 0.008) in trabeculae from diabetic patients, and peak developed stress decreased (p ≤ 0.001), which were not accounted for by reduction in the cardiomyocyte, or contractile protein, content of trabeculae. Trabeculae from diabetic patients also displayed diminished myofilament Ca2+ sensitivity (p = 0.018) compared to non-diabetic patients. Our data provides evidence of impaired calcium handling during excitation-contraction coupling with resulting contractile dysfunction in atrial tissue from patients with type 2 diabetes in comparison to the non-diabetic. This highlights the importance of targeting cardiomyocyte Ca2+ homeostasis in developing more effective treatment options for diabetic heart disease in the future.

Increased Density of Endogenous Adenosine A2A Receptors in Atrial Fibrillation: From Cellular and Porcine Models to Human Patients.

Adenosine, an endogenous nucleoside, plays a critical role in maintaining homeostasis during stressful situations, such as energy deprivation or cellular damage. Therefore, extracellular adenosine is generated locally in tissues under conditions such as hypoxia, ischemia, or inflammation. In fact, plasma levels of adenosine in patients with atrial fibrillation (AF) are elevated, which also correlates with an increased density of adenosine A2A receptors (A2ARs) both in the right atrium and in peripheral blood mononuclear cells (PBMCs). The complexity of adenosine-mediated effects in health and disease requires simple and reproducible experimental models of AF. Here, we generate two AF models, namely the cardiomyocyte cell line HL-1 submitted to Anemonia toxin II (ATX-II) and a large animal model of AF, the right atrium tachypaced pig (A-TP). We evaluated the density of endogenous A2AR in those AF models. Treatment of HL-1 cells with ATX-II reduced cell viability, while the density of A2AR increased significantly, as previously observed in cardiomyocytes with AF. Next, we generated the animal model of AF based on tachypacing pigs. In particular, the density of the key calcium regulatory protein calsequestrin-2 was reduced in A-TP animals, which is consistent with the atrial remodelling shown in humans suffering from AF. Likewise, the density of A2AR in the atrium of the AF pig model increased significantly, as also shown in the biopsies of the right atrium of subjects with AF. Overall, our findings revealed that these two experimental models of AF mimicked the alterations in A2AR density observed in patients with AF, making them attractive models for studying the adenosinergic system in AF.

Beta-blocker treatment of patients with atrial fibrillation attenuates spontaneous calcium release-induced electrical activity.

AIMS: Atrial fibrillation (AF) has been associated with excessive spontaneous calcium release, linked to cyclic AMP (cAMP)-dependent phosphorylation of calcium regulatory proteins. Because ß-blockers are expected to attenuate cAMP-dependent signaling, we aimed to examine whether the treatment of patients with ß-blockers affected the incidence of spontaneous calcium release events or transient inward currents (ITI). METHODS: The impact of treatment with commonly used ß-blockers was analyzed in human atrial myocytes from 371 patients using patch-clamp technique, confocal calcium imaging or immunofluorescent labeling. Data were analyzed using multivariate regression analysis taking into account potentially confounding effects of relevant clinical factors RESULTS: The L-type calcium current (ICa) density was diminished significantly in patients with chronic but not paroxysmal AF and the treatment of patients with ß-blockers did not affect ICa density in any group. By contrast, the ITI frequency was elevated in patients with either paroxysmal or chronic AF that did not receive treatment, and ß-blocker treatment reduced the frequency to levels observed in patients without AF. Confocal calcium imaging showed that ß-blocker treatment also reduced the calcium spark frequency in patients with AF to levels observed in those without AF. Furthermore, phosphorylation of the ryanodine receptor (RyR2) at Ser-2808 and phospholamban at Ser-16 was significantly lower in patients with AF that received ß-blockers. CONCLUSION: Together, our findings demonstrate that ß-blocker treatment may be of therapeutic utility to prevent spontaneous calcium release-induced atrial electrical activity; especially in patients with a history of paroxysmal AF displaying preserved ICa density.

Investigation on the positive chronotropic action of 6-nitrodopamine in the rat isolated atria.

6-Nitrodopamine (6-ND) is released from rat isolated atria being 100 times more potent than noradrenaline and adrenaline, and 10,000 times more potent than dopamine as a positive chronotropic agent. The present study aimed to investigate the interactions of 6-ND with the classical catecholamines, phosphodiesterase (PDE)-3 and PDE4, and the protein kinase A in rat isolated atria. Atrial incubation with 1 pM of dopamine, noradrenaline, or adrenaline had no effect on atrial frequency. Similar results were observed when the atria were incubated with 0.01 pM of 6-ND. However, co-incubation of 6-ND (0.01 pM) with dopamine, noradrenaline, or adrenaline (1 pM each) resulted in significant increases in atrial rate, which persisted over 30 min after washout of the agonists. The increased atrial frequency induced by co-incubation of 6-ND with the catecholamines was significantly reduced by the voltage-gated sodium channel blocker tetrodotoxin (1 µM, 30 min), indicating that the positive chronotropic effect of 6-ND is due in part to activation of nerve terminals. Pre-treatment of the animals with reserpine had no effect on the positive chronotropic effect induced by dopamine, noradrenaline, or adrenaline; however, reserpine markedly reduced the 6-ND (1 pM)-induced positive chronotropic effect. Incubation of the rat isolated atria with the protein kinase A inhibitor H-89 (1 µM, 30 min) abolished the increased atrial frequency induced by dopamine, noradrenaline, and adrenaline, but only attenuated the increases induced by 6-ND. 6-ND induces catecholamine release from adrenergic terminals and increases atrial frequency independently of PKA activation.

Stiff substrates inhibit collagen accumulation via integrin α2ß1, FAK and Src kinases in human atrial fibroblast and myofibroblast cultures derived from patients with aortal stenosis.

The aim of the study was to confirm whether cell substrate stiffness may participate in the regulation of fibrosis. The involvement of integrin α2ß1, focal adhesion kinase (FAK) and Src kinase in signal transmission was investigated. Human atrial fibroblasts and myofibroblasts were cultured in both soft (2.23 ± 0.8 kPa) and stiff (8.28 ± 1.06 kPa) polyacrylamide gels. The cells were derived from the right atrium of patients with aortal stenosis undergoing surgery. The isolated cells, identified as fibroblasts or myofibroblasts, were stained positively with α smooth muscle actin, vimentin and desmin. The cultures settled on stiff gel demonstrated lower intracellular collagen and collagen type I telopeptide (PICP) levels; however, no changes in α1 chain of procollagen type I and III expression were noted. Inhibition of α2ß1 integrin by TC-I 15 (10-7 and 10-8 M) or α2 integrin subunit silencing augmented intracellular collagen level. Moreover, FAK or Src kinase inhibitors increased collagen content within the culture. Lower TIMP4 secretion was reported within the stiff gel cultures but neither MMP 2 nor TIMP-1, 2 or 3 release was altered. The stiff substrate cultures also demonstrated lower interleukin-6 release. Substrate stiffness modified collagen deposition within the atrial fibroblast and myofibroblast cultures. The elasticity of the cellular environment exerts a regulatory influence on both synthesis and breakdown of collagen. Integrin α2ß1, FAK and Src kinase activity participates in signal transmission, which may influence fibrosis in the atria of the human heart.

Advanced glycation end products induce senescence of atrial myocytes and increase susceptibility of atrial fibrillation in diabetic mice.

Diabetes mellitus (DM) is a common chronic metabolic disease caused by significant accumulation of advanced glycation end products (AGEs). Atrial fibrillation (AF) is a common cardiovascular complication of DM. Here, we aim to clarify the role and mechanism of atrial myocyte senescence in the susceptibility of AF in diabetes. Rapid transesophageal atrial pacing was used to monitor the susceptibility of mice to AF. Whole-cell patch-clamp was employed to record the action potential (AP) and ion channels in single HL-1 cell and mouse atrial myocytes. More importantly, anti-RAGE antibody and RAGE-siRNA AAV9 were used to investigate the relationship among diabetes, aging, and AF. The results showed that elevated levels of p16 and retinoblastoma (Rb) protein in the atrium were associated with increased susceptibility to AF in diabetic mice. Mechanistically, AGEs increased p16/Rb protein expression and the number of SA-ß-gal-positive cells, prolonged the action potential duration (APD), reduced protein levels of Cav1.2, Kv1.5, and current density of ICa,L , IKur in HL-1 cells. Anti-RAGE antibody or RAGE-siRNA AAV9 reversed these effects in vitro and in vivo, respectively. Furthermore, downregulating p16 or Rb by siRNA prevented AGEs-mediated reduction of Cav1.2 and Kv1.5 proteins expression. In conclusion, AGEs accelerated atrial electrical remodeling and cellular senescence, contributing to increased AF susceptibility by activating the p16/Rb pathway. Inhibition of RAGE or the p16/Rb pathway may be a potential therapeutic target for AF in diabetes.