Low Density Lipoproteins Promote Unstable Calcium Handling Accompanied by Reduced SERCA2 and Connexin-40 Expression in Cardiomyocytes

The damaging effects of high plasma levels of cholesterol in the cardiovascular system are widely known, but little attention has been paid to direct effects on cardiomyocyte function. We therefore aimed at testing the hypothesis that Low Density Lipoprotein (LDL) cholesterol affects calcium dynamics and signal propagation in cultured atrial myocytes. For this purpose, mRNA and protein expression levels were determined by real time PCR and western blot analysis, respectively, and intracellular calcium was visualized in fluo-4 loaded atrial HL-1 myocyte cultures subjected to field stimulation. At low stimulation frequencies all cultures had uniform calcium transients at all tested LDL concentrations. However, 500 μg LDL/mL maximally reduced the calcium transient amplitude by 43% from 0.30±0.04 to 0.17±0.02 (p<0.05). Moreover, LDL-cholesterol dose-dependently increased the fraction of alternating and irregular beat-to-beat responses observed when the stimulation interval was shortened. This effect was linked to a concurrent reduction in SERCA2, RyR2, IP3RI and IP3RII mRNA levels. SERCA2 protein levels were also reduced by 43% at 200 μg LDL/mL (p<0.05) and SR calcium loading was reduced by 38±6% (p<0.001). By contrast, HDL-cholesterol had no significant effect on SERCA expression or SR calcium loading. LDL-cholesterol also slowed the conduction velocity of the calcium signal from 3.2+0.2 mm/s without LDL to 1.7±0.1 mm/s with 500 μg LDL/mL (p<0.05). This coincided with a reduction in Cx40 expression (by 44±3%; p<0.05 for mRNA and by 79±2%; p<0.05 for Cx40 protein at 200 μg/ml LDL) whereas the Cx-43 expression did not significantly change. In conclusion, LDL-cholesterol destabilizes calcium handling in cultured atrial myocytes subjected to rapid pacing by reducing SERCA2 and Cx40 expression and by slowing the conduction velocity of the calcium signal. © 2013 Barriga et al.This work was supported by grants from the Spanish Ministry of Science and Innovation: SAF2011-30312 and CNIC2009-08 to Leif Hove Madsen, DPI2009-06999 to Rau´ l Benı´tez and from the Spanish Ministry of Health and Consume: FIS PI11/00747 to Vicenta Llorente. Funding from Redes de Investigacio´n Cooperativa, Instituto de Salud Carlos III, red REDINSCOR RD06-0003-0000 and RD06-0003-0015 is also acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer Reviewe

Detection, Properties, and Frequency of Local Calcium Release from the Sarcoplasmic Reticulum in Teleost Cardiomyocytes

Calcium release from the sarcoplasmic reticulum (SR) plays a central role in the regulation of cardiac contraction and rhythm in mammals and humans but its role is controversial in teleosts. Since the zebrafish is an emerging model for studies of cardiovascular function and regeneration we here sought to determine if basic features of SR calcium release are phylogenetically conserved. Confocal calcium imaging was used to detect spontaneous calcium release (calcium sparks and waves) from the SR. Calcium sparks were detected in 16 of 38 trout atrial myocytes and 6 of 15 ventricular cells. The spark amplitude was 1.45±0.03 times the baseline fluorescence and the time to half maximal decay of sparks was 27±3 ms. Spark frequency was 0.88 sparks µm−1 min−1 while calcium waves were 8.5 times less frequent. Inhibition of SR calcium uptake reduced the calcium transient (F/F0) from 1.77±0.17 to 1.12±0.18 (p = 0.002) and abolished calcium sparks and waves. Moreover, elevation of extracellular calcium from 2 to 10 mM promoted early and delayed afterdepolarizations (from 0.6±0.3 min−1 to 8.1±2.0 min−1, p = 0.001), demonstrating the ability of SR calcium release to induce afterdepolarizations in the trout heart. Calcium sparks of similar width and duration were also observed in zebrafish ventricular myocytes. In conclusion, this is the first study to consistently report calcium sparks in teleosts and demonstrate that the basic features of calcium release through the ryanodine receptor are conserved, suggesting that teleost cardiac myocytes is a relevant model to study the functional impact of abnormal SR function

Nonlinearities due to refractoriness in SR Ca release

Calcium alternans is a pro-arrhytmic cardiac dysfunction related to beat-to-beat changes in the amplitude of intracellular calcium transient, that typically occurs at rapid pacing rates. Although oscillations in sarcoplasmic reticulum (SR) content have been related with calcium alternans, the experimental appearance of alternans without these oscillations suggests that another mechanism related with refractoriness of SR calcium release might be key, at least, under certain conditions. We investigate how RyR2 refractoriness modulates calcium handling on a beat-to-beat basis using a numerical rabbit cardiomyocyte model. We find that a slow recovery from inactivation of the RyR2 might be crucial. On one hand, a steep relation between sarcoplasmic reticulum (SR) load and calcium release makes regular calcium cycling unstable at high SR calcium load and/or fast pacing rates, in agreement with previous explanation when RyR2 inactivation is not important. On the other hand, we show that calcium release can also depend strongly on the number of RyR2 ready to open if an important number of RyR2s inactivate after the release. This gives rise to a steep nonlinear relation between the calcium release and the level of recovered RyR2, so that a small change in the later produces big changes in calcium release. A conclusion of this result is that RyR2 refractoriness can be the main nonlinearity behind alternans even when alternation in SR-Ca load is presentPostprint (published version

Impact of R-carvedilol on ß2-adrenergic receptor-mediated spontaneous calcium release in human atrial myocytes

A hallmark of atrial fibrillation is an excess of spontaneous calcium release events, which can be mimicked by ß1- or ß2-adrenergic stimulation. Because ß1-adrenergic receptor blockers (ß1-blockers) are primarily used in clinical practice, we here examined the impact of ß2-adrenergic stimulation on spontaneous calcium release and assessed whether the R- and S-enantiomers of the non-selective ß- blocker carvedilol could reverse these effects. For this purpose, human atrial myocytes were isolated from patients undergoing cardiovascular surgery and subjected to confocal calcium imaging or immunofluorescent labeling of the ryanodine receptor (RyR2). Interestingly, the ß2-adrenergic agonist fenoterol increased the incidence of calcium sparks and waves to levels observed with the non-specific ß-adrenergic agonist isoproterenol. Moreover, fenoterol increased both the amplitude and duration of the sparks, facilitating their fusion into calcium waves. Subsequent application of the non ß-blocking R-Carvedilol enantiomer reversed these effects of fenoterol in a dose-dependent manner. R-Carvedilol also reversed the fenoterol-induced phosphorylation of the RyR2 at Ser-2808 dose-dependently, and 1 µM of either R- or S-Carvedilol fully reversed the effect of fenoterol. Together, these findings demonstrate that ß2-adrenergic stimulation alone stimulates RyR2 phosphorylation at Ser-2808 and spontaneous calcium release maximally, and points to carvedilol as a tool to attenuate the pathological activation of ß2-receptors.Peer ReviewedPostprint (published version

Modeling Calcium Dynamics in Human Atria

Abstract Mathematical models of cardiac electrophysiology are an important tool to investigate the underlying mechanisms responsible of arrhythmias. In particular, an important question is the origin of atrial fibrillation (AF). Often, AF initiation is preceded by action potential duration (APD) alternans, i.e., beat to beat oscillations in the APD, that arise at slower rates in patients with persistent AF than in those without AF or with paroxymal AF. Most of these arrhythmias appear as a consequence of malfunctions in calcium dynamics that produce oscillations in intracellular calcium, inducing subsequent APD alternans through electromechanical coupling. The aim of this work is to present a human atrial mathematical model that gives insight into the presence of calcium alternans. For that the model by Nygren et al was modified in order to reproduce calcium alternans at high pacing rhythms, as has been observed in experiments. The model reproduces the nonlinear dependence of gain and fractional SR Ca release upon SR Ca load. At fast pacing rates it presents alternans, due to slow recovery from inactivation of the RyR. Finally, we compare the results from this new model with other human atrial models well established in the literature

Expression and impact of Adenosine A3 receptors on calcium homeostasis in human right atrium

Increased adenosine A2A receptor (A2AR) expression and activation underlies a higher incidence of spontaneous calcium release in atrial fibrillation (AF). Adenosine A3 receptors (A3R) could counteract excessive A2AR activation, but their functional role in the atrium remains elusive, and we therefore aimed to address the impact of A3Rs on intracellular calcium homeostasis. For this purpose, we analyzed right atrial samples or myocytes from 53 patients without AF, using quantitative PCR, patch-clamp technique, immunofluorescent labeling or confocal calcium imaging. A3R mRNA accounted for 9% and A2AR mRNA for 32%. At baseline, A3R inhibition increased the transient inward current (ITI) frequency from 0.28 to 0.81 events/min (p < 0.05). Simultaneous stimulation of A2ARs and A3Rs increased the calcium spark frequency seven-fold (p < 0.001) and the ITI frequency from 0.14 to 0.64 events/min (p < 0.05). Subsequent A3R inhibition caused a strong additional increase in the ITI frequency (to 2.04 events/min; p < 0.01) and increased phosphorylation at s2808 1.7-fold (p < 0.001). These pharmacological treatments had no significant effects on L-type calcium current density or sarcoplasmic reticulum calcium load. In conclusion, A3Rs are expressed and blunt spontaneous calcium release at baseline and upon A2AR-stimulation in human atrial myocytes, pointing to A3R activation as a means to attenuate physiological and pathological elevations of spontaneous calcium release events

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

Pitx2c deficiency confers cellular electrophysiological hallmarks of atrial fibrillation to isolated atrial myocytes

Aims Atrial fibrillation (AF) has been associated with altered expression of the transcription factor Pitx2c and a high incidence of calcium release-induced afterdepolarizations. However, the relationship between Pitx2c expression and defective calcium homeostasis remains unclear and we here aimed to determine how Pitx2c expression affects calcium release from the sarcoplasmic reticulum (SR) and its impact on electrical activity in isolated atrial myocytes. Methods To address this issue, we applied confocal calcium imaging and patch-clamp techniques to atrial myocytes isolated from a mouse model with conditional atrial-specific deletion of Pitx2c. Results Our findings demonstrate that heterozygous deletion of Pitx2c doubles the calcium spark frequency, increases the frequency of sparks/site 1.5-fold, the calcium spark decay constant from 36 to 42 ms and the wave frequency from none to 3.2 min-1. Additionally, the cell capacitance increased by 30% and both the SR calcium load and the transient inward current (ITI) frequency were doubled. Furthermore, the fraction of cells with spontaneous action potentials increased from none to 44%. These effects of Pitx2c deficiency were comparable in right and left atrial myocytes, and homozygous deletion of Pitx2c did not induce any further effects on sparks, SR calcium load, ITI frequency or spontaneous action potentials. Conclusion Our findings demonstrate that heterozygous Pitx2c deletion induces defects in calcium homeostasis and electrical activity that mimic derangements observed in right atrial myocytes from patients with AF and suggest that Pitx2c deficiency confers cellular electrophysiological hallmarks of AF to isolated atrial myocytes.Peer ReviewedPostprint (published version

Adenosine A2A receptors are upregulated in peripheral blood mononuclear cells from atrial fibrillation patients

Atrial fibrillation (AF) is the most common form of cardiac arrhythmia seen in clinical practice. While some clinical parameters may predict the transition from paroxysmal to persistent AF, the molecular mechanisms behind the AF perpetuation are poorly understood. Thus, oxidative stress, calcium overload and inflammation, among others, are believed to be involved in AF-induced atrial remodelling. Interestingly, adenosine and its receptors have also been related to AF development and perpetuation. Here, we investigated the expression of adenosine A2A receptor (A2AR) both in right atrium biopsies and peripheral blood mononuclear cells (PBMCs) from non-dilated sinus rhythm (ndSR), dilated sinus rhythm (dSR) and AF patients. In addition, plasma adenosine content and adenosine deaminase (ADA) activity in these subjects were also determined. Our results revealed increased A2AR expression in the right atrium from AF patients, as previously described. Interestingly, increased levels of adenosine content and reduced ADA activity in plasma from AF patients were detected. An increase was observed when A2AR expression was assessed in PBMCs from AF subjects. Importantly, a positive correlation (P=0.001) between A2AR expression in the right atrium and PBMCs was observed. Overall, these results highlight the importance of the A2AR in AF and suggest that the evaluation of this receptor in PBMCs may be potentially be useful in monitoring disease severity and the efficacy of pharmacological treatments in AF patients