Atrial Excitation-contraction Coupling Research Articles

The 'Reverse FDUF' Mechanism of Atrial Excitation-Contraction Coupling Sustains Calcium Alternans-A Hypothesis.

Cardiac calcium alternans is defined as beat-to-beat alternations of Ca transient (CaT) amplitude and has been linked to cardiac arrhythmia, including atrial fibrillation. We investigated the mechanism of atrial alternans in isolated rabbit atrial myocytes using high-resolution line scan confocal Ca imaging. Alternans was induced by increasing the pacing frequency until stable alternans was observed (1.6-2.5 Hz at room temperature). In atrial myocytes, action potential-induced Ca release is initiated in the cell periphery and subsequently propagates towards the cell center by Ca-induced Ca release (CICR) in a Ca wave-like fashion, driven by the newly identified 'fire-diffuse-uptake-fire' (FDUF) mechanism. The development of CaT alternans was accompanied by characteristic changes of the spatio-temporal organization of the CaT. During the later phase of the CaT, central [Ca]i exceeded peripheral [Ca]i that was indicative of a reversal of the subcellular [Ca]i gradient from centripetal to centrifugal. This gradient reversal resulted in a reversal of CICR propagation, causing a secondary Ca release during the large-amplitude alternans CaT, thereby prolonging the CaT, enhancing Ca-release refractoriness and reducing Ca release on the subsequent beat, thus enhancing the degree of CaT alternans. Here, we propose the 'reverse FDUF' mechanism as a novel cellular mechanism of atrial CaT alternans, which explains how the uncoupling of central from peripheral Ca release leads to the reversal of propagating CICR and to alternans.

Inositol 1,4,5-trisphosphate receptor - reactive oxygen signaling domain regulates excitation-contraction coupling in atrial myocytes

The inositol 1,4,5-trisphosphate receptor (InsP3R) is up-regulated in patients with atrial fibrillation (AF) and InsP3-induced Ca2+ release (IICR) is linked to pro-arrhythmic spontaneous Ca2+ release events. Nevertheless, knowledge of the physiological relevance and regulation of InsP3Rs in atrial muscle is still limited. We hypothesize that InsP3R and NADPH oxidase 2 (NOX2) form a functional signaling domain where NOX2 derived reactive oxygen species (ROS) regulate InsP3R agonist affinity and thereby Ca2+ release. To quantitate the contribution of IICR to atrial excitation-contraction coupling (ECC) atrial myocytes (AMs) were isolated from wild type and NOX2 deficient (Nox2−/−) mice and changes in the cytoplasmic Ca2+ concentration ([Ca2+]i; fluo-4/AM, indo-1) or ROS (2′,7′-dichlorofluorescein, DCF) were monitored by fluorescence microscopy. Superfusion of AMs with Angiotensin II (AngII: 1 μmol/L) significantly increased diastolic [Ca2+]i (F/F0, Ctrl: 1.00 ± 0.01, AngII: 1.20 ± 0.03; n = 7; p < 0.05), the field stimulation induced Ca2+ transient (CaT) amplitude (ΔF/F0, Ctrl: 2.00 ± 0.17, AngII: 2.39 ± 0.22, n = 7; p < 0.05), and let to the occurrence of spontaneous increases in [Ca2+]i. These changes in [Ca2+]i were suppressed by the InsP3R blocker 2-aminoethoxydiphenyl-borate (2-APB; 1 μmol/L). Concomitantly, AngII induced an increase in ROS production that was sensitive to the NOX2 specific inhibitor gp91ds-tat (1 μmol/L). In NOX2−/− AMs, AngII failed to increase diastolic [Ca2+]i, CaT amplitude, and the frequency of spontaneous Ca2+ increases. Furthermore, the enhancement of CaTs by exposure to membrane permeant InsP3 was abolished by NOX inhibition with apocynin (1 μM). AngII induced IICR in Nox2−/− AMs could be restored by addition of exogenous ROS (tert-butyl hydroperoxide, tBHP: 5 μmol/L). In saponin permeabilized AMs InsP3 (5 μmol/L) induced Ca2+ sparks that increased in frequency in the presence of ROS (InsP3: 9.65 ± 1.44 sparks*s−1*(100μm)−1; InsP3 + tBHP: 10.77 ± 1.5 sparks*s−1*(100μm)−1; n = 5; p < 0.05). The combined effect of InsP3 + tBHP was entirely suppressed by 2-APB and Xestospongine C (XeC). Changes in IICR due to InsP3R glutathionylation induced by diamide could be reversed by the reducing agent dithiothreitol (DTT: 1 mmol/L) and prevented by pretreatment with 2-APB, supporting that the ROS-dependent post-translational modification of the InsP3R plays a role in the regulation of ECC. Our data demonstrate that in AMs the InsP3R is under dual control of agonist induced InsP3 and ROS formation and suggest that InsP3 and NOX2-derived ROS co-regulate atrial IICR and ECC in a defined InsP3R/NOX2 signaling domain.

Abstract P498: Polycystin-1 Regulates Excitation-contraction Coupling In Atrial Cardiomyocytes

Patients with Autosomal Dominant Polycystic Kidney disease (ADPKD) have multiple cardiovascular manifestations, including increased susceptibility to arrhythmias. Mutations in polycystin-1 (PC1) encoding gene accounts for 85% cases of ADPKD, whereas mutations in polycystin-2 (PC2) only accounts for 15%. In kidney cells, PC1 interacts with PC2 to form a protein complex at the primary cilia to regulate calcium influx via PC2. However, cardiomyocytes are non-ciliated cells and the role of both PC1 and PC2 in atrial cardiomyocytes remains unknown. We have previously demonstrated that PC1 regulates action potentials and calcium handling to fine-tune ventricular cardiomyocyte contraction. Here, we hypothesize that PC1 regulates action potentials and calcium handling in atrial cardiomyocytes independent of PC2 actions. To test this hypothesis, we differentiated human induced pluripotent stem cells (iPSC) into atrial cardiomyocytes (iPSC-aCM) using previously published protocols. To determine the contribution of PC1/PC2 in atrial excitation-contraction coupling, protein expression was knocked down utilizing specific siRNA constructs, for each protein, or a universal control siRNA transfected using lipofectamine RNAiMAX. We measured action potentials using the potentiometric dye FluoVolt and intracellular calcium with Fura-2 AM or Fluo-4. Changes in fluorescence were monitored using a multiwavelength IonOptix system. iPSC-aCM were paced at 2 Hz to synchronize the beating pattern using field electrical stimulation. Our data shows that PC1 ablation significantly decreased action potential duration at 50% and 80% of repolarization, by 24% and 23%, respectively. Moreover, we observed that PC1 knockdown significantly reduced calcium transient amplitude elicited by field electrical stimulation without changes in calcium transient decay. Interestingly, PC2 knockdown did not modify calcium transients in atrial cardiomyocytes (iPSC-aCM). Our data suggest that PC1 regulates atrial excitation-contraction coupling independent of PC2 actions. This study warrants further investigation into atrial dysfunction in ADPKD patients with PC1 mutations.

Nanoscale imaging of rat atrial myocytes by scanning ion conductance microscopy reveals heterogeneity of T-tubule openings and ultrastructure of the cell membrane.

In contrast to ventricular myocytes, the structural and functional importance of atrial transverse tubules (T-tubules) is not fully understood. Therefore, we investigated the ultrastructure of T-tubules of living rat atrial myocytes in comparison with ventricular myocytes. Nanoscale cell surface imaging by scanning ion conductance microscopy (SICM) was accompanied by confocal imaging of intracellular T-tubule network, and the effect of removal of T-tubules on atrial excitation-contraction coupling (EC-coupling) was observed. By SICM imaging, we classified atrial cell surface into 4 subtypes. About 38% of atrial myocytes had smooth cell surface with no clear T-tubule openings and intracellular T-tubules (smooth-type). In 33% of cells, we found a novel membrane nanostructure running in the direction of cell length and named it 'longitudinal fissures' (LFs-type). Interestingly, T-tubule openings were often found inside the LFs. About 17% of atrial cells resembled ventricular myocytes, but they had smaller T-tubule openings and a lower Z-groove ratio than the ventricle (ventricular-type). The remaining 12% of cells showed a mixed structure of each subtype (mixed-type). The LFs-, ventricular-, and mixed-type had an appreciable amount of reticular form of intracellular T-tubules. Formamide-induced detubulation effectively removed atrial T-tubules, which was confirmed by both confocal images and decreased cell capacitance. However, the LFs remained intact after detubulation. Detubulation reduced action potential duration and L-type Ca2+ channel (LTCC) density, and prolonged relaxation time of the myocytes. Taken together, we observed heterogeneity of rat atrial T-tubules and membranous ultrastructure, and the alteration of atrial EC-coupling by disruption of T-tubules.

Abstract 376: Functional Significance of Anatomically Heterogeneous Distribution of Transversal-axial Tubule System in Mouse and Human Atrial Myocardium

Background: In cardiac myocytes, efficient and synchronous excitation-contraction coupling (ECC) occurs through a specialized network of membrane invaginations called transverse-axial tubule system (TATS). In contrast to an extensive TATS in ventricular myocytes, atrial cells show a significantly heterogeneous organization of TATS, varying from a ventricular-like organized TATS to absent/irregular TATS. However, anatomical distribution of atrial TATS and its functional significance remains unknown. We hypothesized that atrial myocytes with a different TATS organization have a specific anatomical location within the atria that determines their impact on atrial ECC and contraction. Methods and Results: In order to avoid the potential bias associated with cell isolation, we applied a mosaic imaging protocol at the whole-mount isolated mouse atrial preparation stained with a membrane dye RH-237. We developed an algorithm that can automatically remove the outer membrane and analyze TATS organization. This analysis showed that myocytes located within the inter-caval region (ICR, i.e., between the superior and inferior vena cava and between the crista terminalis in atrioventricular junction) have a lower TATS organization than cells located within the right atrial appendage (RAA): 0.21±0.01 A.U. vs. 0.50±0.02 A.U. in ICR vs. RAA, respectively, P&lt;0.01. Fluorescent optical mapping of Ca 2+ transients from isolated mouse atrial preparations revealed that ICR region has a longer Ca 2+ transient rise up time compared to RAA (7.8±0.3 ms vs. 6.3±0.3 ms, P&lt;0.01). Moreover, a similar region-specific TATS organization was found in human non-failing (donor) atrial tissue samples. The analysis of TATS density in specimens selected from ICR and RAA regions of the human hearts (n=4) showed a significantly lower density of TATS in ICR compared to RAA region (5.8±0.2% vs. 10.6±0.4%, P&lt;0.01). Conclusion: Our findings show a region-specific structural and functional differences of myocytes from RAA and ICR regions of mouse and human heart that could represent a general organization of the mammalian atria. It may have important implications into establishment of different functional roles of cells with a specific TATS structure in atrial physiology and pathology.

Effect of carvedilol on atrial excitation-contraction coupling, Ca2+ release, and arrhythmogenicity.

Carvedilol is an FDA-approved β-blocker commonly used for treatment of high blood pressure, congestive heart failure, and cardiac tachyarrhythmias, including atrial fibrillation. We investigated at the cellular level the mechanisms through which carvedilol interferes with sarcoplasmic reticulum (SR) Ca2+ release during excitation-contraction coupling (ECC) in single rabbit atrial myocytes. Carvedilol caused concentration-dependent (1-10 µM) failure of SR Ca2+ release. Failure of ECC and Ca2+ release was the result of dose-dependent inhibition of voltage-gated Na+ (INa) and L-type Ca2+ (ICa) currents that are responsible for the rapid depolarization phase of the cardiac action potential (AP) and the initiation of Ca2+-induced Ca2+ release from the SR, respectively. Carvedilol (1 µM) led to AP duration shortening, AP failures, and peak INa inhibition by ~80%, whereas ICa was not markedly affected. Carvedilol (10 µM) blocked INa almost completely and reduced ICa by ~40%. No effect on Ca2+-transient amplitude, ICa, and INa was observed in control experiments with the β-blocker metoprolol, suggesting that the carvedilol effect on ECC is unlikely the result of its β-blocking property. The effects of carvedilol (1 µM) on subcellular SR Ca2+ release was spatially inhomogeneous, where a selective inhibition of peripheral subsarcolemmal Ca2+ release from the junctional SR accounted for the cell-averaged reduction in Ca2+-transient amplitude. Furthermore, carvedilol significantly reduced the probability of spontaneous arrhythmogenic Ca2+ waves without changes of SR Ca2+ load. The data suggest a profound antiarrhythmic action of carvedilol in atrial myocytes resulting from an inhibitory effect on the SR Ca2+ release channel.NEW & NOTEWORTHY Here we show that the clinically widely used β-blocker carvedilol has profound effects on Ca2+ signaling and ion currents, but also antiarrhythmic effects in adult atrial myocytes. Carvedilol inhibits sodium and calcium currents and leads to failure of ECC but also prevents spontaneous Ca2+ release from cellular sarcoplasmic reticulum (SR) Ca2+ stores in form of arrhythmogenic Ca2+ waves. The antiarrhythmic effect occurs by carvedilol acting directly on the SR ryanodine receptor Ca2+ release channel.

P1230Impact of regulated junctophilin-2 clustering at axial tubule junctions on atrial excitation-contraction coupling and therapeutic implications

Abstract Objectives Atrial dysfunction is highly prevalent and known to significantly aggravate heart failure. While rapid excitation-contraction (EC) coupling depends on axial tubule junctions in atrial myocytes (AMs), the mechanisms leading to atrial loss-of-function remain unclear. Junctophilin-2 (JP2), a tail-anchored protein of the sarcoplasmic reticulum, stabilizes the integrity of ventricular Ca2+ release units, which is disrupted in ventricular myocytes by reduced JP2 expression or proteolysis. Here we aim to characterize the abundance and subcellular localisation of JP2 in AMs, to assess the impact of decreased JP2 expression on atrial remodelling, and to investigate the potential to correct JP2 expression and atrial dysfunction. Results We identified 5-fold lower JP2 levels in atrial compared to ventricular tissue in mouse and human hearts by SDS-PAGE. Surprisingly, in AMs, this resulted in subcellular expression of large JP2 clusters at axial tubule junctions together with highly phosphorylated ryanodine receptor (RyR2) channels visualized by STED superresolution microscopy. Importantly, left atrial hypertrophy induced by aortic pressure overload led to an additional strong decrease in JP2 expression compared to sham control, disrupted junctional RyR2 clustering and EC-coupling. This loss-of-function mechanism was confirmed by conditional shRNA-mediated JP2 knockdown. Quantitative image analysis after atrial JP2 knockdown showed a 50% decrease in area overlap between RyR2 and JP2 in AMs (JP2 knockdown 0.03±0.003 μm2 vs. control 0.06±0.004 μm2, p&lt;0.001), and a ∼2-fold increased Ca2+ spark frequency, consistent with decreased left atrial fractional shortening (JP2 knockdown 12.9±0.8% vs. control 16.5±0.9%, p&lt;0.01). Whereas atrial-ventricular dysfunction due to aortic pressure overload resulted in 40% mortality, additional JP2 knockdown exacerbated mortality to 100% (n: 10 control vs. 9 JP2 knockdown mice). In contrast, transgenic JP2 overexpressor mice showed greatly improved atrial contractility without mortality after induced aortic pressure overload (n: 21 control vs. 16 JP2 overexpressor mice). JP2-OE not only augmented atrial RyR2-clustering, but induced the de-novo biogenesis of large poly-adic junctional membrane complexes, which were resolved by STED microscopy via high-resolution cholesterol-based membrane staining in live AMs and electron tomography. Conclusions Nanoscale imaging identifies a new subcellular mechanism of significantly limited atrial JP2 protein expression in large clusters at axial tubule junctions. In atrial hypertrophy, JP2 is further decreased with junctional RyR2 cluster disruption leading to impaired Ca2+ release and decreased contractility. Importantly, JP2 overexpression effectively protected from atrial dysfunction, providing a novel therapeutic rationale for atrial cardiomyopathies.

168-03: Axial membrane tubules in atrial myocytes initiate rapid intracellular calcium signals through a new “super-hub” mechanism

Invaginated sarcolemmal membrane structures in cardiac myocytes form transverse tubules (TTs). The TTs form electrical and chemical signaling conduits from the myocyte exterior to regions deep in the cell such as the junctional sarcoplasmic reticulum (jSR) critical in excitation-contraction (EC) coupling. Local control of jSR Ca2+ release in ventricular myocytes (VM) depends critically on this organization. In contrast, atrial myocytes (AM) normally have few if any TTs (although the exact abundance has been controversial). In atrial myocytes, the normally sparse distribution of TTs suggests that a critical role in EC coupling remains unlikely. Here, we identify a structurally distinct and mechanistically novel role for even sparsely set TTs in atrial EC coupling. Using novel membrane-preserving live cell techniques, we identify relatively abundant axial membrane tubules (ATs). These ATs are deep intracellular branches of the relatively rare TTs in 100% of AM investigated (n = 29). Using live cell STimulated Emission Depletion (STED) superresolution microscopy, we show that atrial AT structures are characteristically different from TTs. ATs represent both the major component of the cell-wide tubule network and the largest tubules: AT circumference in AM 921 ± 24 nm vs. VM 630 ± 26 nm; p < 0.001 (physiological live cell conditions). These dimensions indicate unprecedentedly large AT surfaces with a positive membrane curvature at the AM center suggesting that ATs could function as a central-axial Ca2+ signaling hub during excitation-contraction (EC)-coupling. To test this hypothesis, we combined AT and intracellular Ca2+ transient (CaT) imaging. High-amplitude CaTs originated directly from ATs creating highly localized central-axial Ca2+ signals. Importantly, local CaT onset measurements showed that atrial CaTs occurred significantly earlier at AT compared to surface membrane locations (p < 0.05). To explain the observed CaT behaviors, we imaged in situ the Ser-2808 phospho-epitope of ryanodine receptor (RyR2) Ca2+ release channels necessary for PKA phosphorylation. RyR2 channel clusters at AT-hubs showed increased PKA phosphorylation in contrast to the low phosphorylation state of non-junctional RyR2 clusters in unstimulated AM. Sarcomere shortening experiments confirmed significantly larger and faster AM compared with VM axial contractions (p < 0.05); this effect was significantly augmented by isoproterenol (1 µm) through non-junctional cluster recruitment. Consistent with the proposed AT super-hub model, genetic ablation of RyR2 PKA phosphorylation in Ser-2808-Ala knockin mice significantly attenuated atrial sarcomere shortening and in vivo atrial but not ventricular contractile function. Our data suggest a fundamentally new atrial EC-coupling model where AT structures in atrial myocytes function as large signaling super-hubs that couple central-axial Ca2+ signaling to rapid contractile activation. This supports a critical role of the transverse-axial tubular system in cell-specific atrial EC-coupling mechanisms (i.e. faster contractions than in ventricles) and questions previous models of atrial EC-coupling based on slow intracellular Ca2+ diffusion. Genetic ablation of RyR2 cluster phosphorylation unmasks a previously unknown role of atrial PKA regulation, which may explain atrial function in health versus decreased contractility occuring in atrial fibrillation and heart failure.

Atrial Excitation-Contraction Coupling and Ca Wave Propagation in Normal and Failing Hearts

We compared excitation-contraction coupling and Ca wave propagation in normal and heart failure (HF) rabbit atrial myocytes. Cytosolic Ca transients (Fluo-4 or Rhod-2), sarcoplasmic reticulum (SR) [Ca] (Fluo-5N) and mitochondrial localization (Mitotracker Red) were recorded by confocal microscopy. SEA and Ru360 were used to inhibit sarcolemmal Na/Ca exchange (NCX) or mitochondrial Ca uptake, respectively. In normal and HF rabbit atrial myocytes transverse tubules are missing, and the peripheral junctional SR (j-SR) is separated from the central non-junctional SR (nj-SR) by a 1-2 µm wide gap that is largely devoid of mitochondria and SR structures. During action potential (AP) stimulation SR Ca release initiated from the j-SR and propagated in centripetal direction via Ca-induced Ca release (CICR) from nj-SR. The centripetal Ca propagation velocity was highest across the gap between j-SR and nj-SR, slowed during propagation across the nj-SR and increased after Ru360 application. In HF, the centripetal propagation velocity was higher and mitochondrial density was decreased. Spontaneous Ca waves propagated with a leading wave front located to the cell periphery, i.e. arising from j-SR Ca release. Nearly half of all Ca waves in normal cells, but <10% in normal cells pretreated with SEA and none in ventricular cells triggered an AP, followed by a whole-cell Ca transient (termed here 'arrhythmogenic Ca waves'). The incidence of spontaneous Ca waves, the fraction of arrhythmogenic Ca waves and NCX activity were significantly increased in HF. In summary, the lack of mitochondria in the gap between j-SR and nj-SR enables rapid propagation of CICR from the j-SR to the nj-SR. Decreased mitochondrial Ca uptake in HF contributes to an increased centripetal Ca propagation velocity. Atrial myocytes are prone to develop NCX-dependent arrhythmogenic Ca waves, which is further accentuated in HF.

Type 1 Inositol (1,4,5)-Trisphosphate Receptor Activates Ryanodine Receptor 1 to Mediate Calcium Spark Signaling in Adult Mammalian Skeletal Muscle

Functional coupling between inositol (1,4,5)-trisphosphate receptor (IP(3)R) and ryanodine receptor (RyR) represents a critical component of intracellular Ca(2+) signaling in many excitable cells; however, the role of this mechanism in skeletal muscle remains elusive. In skeletal muscle, RyR-mediated Ca(2+) sparks are suppressed in resting conditions, whereas application of transient osmotic stress can trigger activation of Ca(2+) sparks that are restricted to the periphery of the fiber. Here we show that onset of these spatially confined Ca(2+) sparks involves interaction between activation of IP(3)R and RyR near the sarcolemmal membrane. Pharmacological prevention of IP(3) production or inhibition of IP(3)R channel activity abolishes stress-induced Ca(2+) sparks in skeletal muscle. Although genetic ablation of the type 2 IP(3)R does not appear to affect Ca(2+) sparks in skeletal muscle, specific silencing of the type 1 IP(3)R leads to ablation of stress-induced Ca(2+) sparks. Our data indicate that membrane-delimited signaling involving cross-talk between IP(3)R1 and RyR1 contributes to Ca(2+) spark activation in skeletal muscle.

Altered Excitation-Contraction Coupling in Human Chronic Atrial Fibrillation.

This review focuses on the (mal)adaptive processes in atrial excitation-contraction coupling occurring in patients with chronic atrial fibrillation. Cellular remodeling includes shortening of the atrial action potential duration and effective refractory period, depressed intracellular Ca2+ transient, and reduced myocyte contractility. Here we summarize the current knowledge of the ionic bases underlying these changes. Understanding the molecular mechanisms of excitation-contraction-coupling remodeling in the fibrillating human atria is important to identify new potential targets for AF therapy.