Mechanisms of stretch-induced electro-anatomical remodeling and atrial arrhythmogenesis.

Atrial fibrillation (AF) is the most common cardiac rhythm disorder, often occurring in the setting of atrial distension and elevated myocardialstretch. While various mechano-electrochemical signal transduction pathways have been linked to AF development and progression, the underlying molecular mechanisms remain poorly understood, hampering AF therapies. In this review, we describe different aspects of stretch-induced electro-anatomical remodeling as seen in animal models and in patients with AF. Specifically, we focus on cellular and molecular mechanisms that are responsible for mechano-electrochemical signal transduction and the development of ectopic beats triggering AF from pulmonary veins, the most common source of paroxysmal AF. Furthermore, we describe structural changes caused by stretch occurring before and shortly after the onset of AF as well as during AF progression, contributing to longstanding forms of AF. We also propose mechanical stretch as a new dimension to the concept "AF begets AF", in addition to underlying diseases. Finally, we discuss the mechanisms of these electro-anatomical alterations in a search for potential therapeutic strategies and the development of novel antiarrhythmic drugs targeted at the components of mechano-electrochemical signal transduction not only in cardiac myocytes, but also in cardiac non-myocyte cells.

Caveolae-associated cAMP/Ca2+-mediated mechano-chemical signal transduction in mouse atrial myocytes.

Caveolae are tiny invaginations in the sarcolemma that buffer extra membrane and contribute to mechanical regulation of cellular function. While the role of caveolae in membrane mechanosensation has been studied predominantly in non-cardiomyocyte cells, caveolae contribution to cardiac mechanotransduction remains elusive. Here, we studied the role of caveolae in the regulation of Ca2+ signaling in atrial cardiomyocytes. In Langendorff-perfused mouse hearts, atrial pressure/volume overload stretched atrial myocytes and decreased caveolae density. In isolated cells, caveolae were disrupted through hypotonic challenge that induced a temporal (<10 min) augmentation of Ca2+ transients and caused a rise in Ca2+ spark activity. Similar changes in Ca2+ signaling were observed after chemical (methyl-ß-cyclodextrin) and genetic ablation of caveolae in cardiac-specific conditional caveolin-3 knock-out mice. Acute disruption of caveolae, both mechanical and chemical, led to the elevation of cAMP level in the cell interior, and cAMP-mediated augmentation of protein kinase A (PKA)-phosphorylated ryanodine receptors (at Ser2030 and Ser2808). Caveolae-mediated stimulatory effects on Ca2+ signaling were abolished via inhibition of cAMP production by adenyl cyclase antagonists MDL12330 and SQ22536, or reduction of PKA activity by H-89. A compartmentalized mathematical model of mouse atrial myocytes linked the observed changes to a microdomain-specific decrease in phosphodiesterase activity, which disrupted cAMP signaling and augmented PKA activity. Our findings add a new dimension to cardiac mechanobiology and highlight caveolae-associated cAMP/PKA-mediated phosphorylation of Ca2+ handling proteins as a novel component of mechano-chemical feedback in atrial myocytes.

Caveolin-3 prevents swelling-induced membrane damage via regulation of ICl,swell activity.

Caveola membrane structures harbor mechanosensitive chloride channels (MCCs; including chloride channel 2, chloride channel 3, and SWELL1, also known as LRRC8A) that form a swelling-activated chloride current (ICl,swell) and play an important role in cell volume regulation and mechanoelectrical signal transduction. However, the role of the muscle-specific caveolar scaffolding protein caveolin-3 (Cav3) in regulation of MCC expression, activity, and contribution to membrane integrity in response to mechanical stress remains unclear. Here we showed that Cav3-transfected (Cav3-positive) HEK293 cells were significantly resistant to extreme (<20 milliosmole) hypotonic swelling compared with native (Cav3-negative) HEK293 cells; the percentage of cells with membrane damage decreased from 45% in Cav3-negative cells to 17% in Cav3-positive cells (p < 0.05). This mechanoprotection was significantly reduced (p < 0.05) when cells were exposed to the ICl,swell-selective inhibitor 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (10 µM). These results were recapitulated in isolated mouse ventricular myocytes, where the percentage of cardiomyocytes with membrane damage increased from 47% in control cells to 78% in 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid-treated cells (p < 0.05). A higher resistance to hypotonic swelling in Cav3-positive HEK293 cells was accompanied by a significant twofold increase of ICl,swell current density and SWELL1 protein expression, whereas ClC-2/3 protein levels remained unchanged. Förster resonance energy transfer analysis showed a less than 10-nm membrane and intracellular association between Cav3 and SWELL1. Cav3/SWELL1 membrane Förster resonance energy transfer efficiency was halved in mild (220 milliosmole) hypotonic solution as well as after disruption of caveola structures via cholesterol depletion by 1-h treatment with 10 mM methyl-ß-cyclodextrin. A close association between Cav3 and SWELL1 was confirmed by co-immunoprecipitation analysis. Our findings indicate that, in the MCCs tested, SWELL1 abundance and activity are regulated by Cav3 and that their association relies on membrane tension and caveola integrity. This study highlights the mechanoprotective role of Cav3, which is facilitated by complimentary SWELL1 expression and activity.

Region-specific distribution of transversal-axial tubule system organization underlies heterogeneity of calcium dynamics in the right atrium.

The atrial myocardium demonstrates the highly heterogeneous organization of the transversal-axial tubule system (TATS), although its anatomical distribution and region-specific impact on Ca2+ dynamics remain unknown. Here, we developed a novel method for high-resolution confocal imaging of TATS in intact live mouse atrial myocardium and applied a custom-developed MATLAB-based computational algorithm for the automated analysis of TATS integrity. We observed a twofold higher (P < 0.01) TATS density in the right atrial appendage (RAA) than in the intercaval regions (ICR, the anatomical region between the superior vena cava and atrioventricular junction and between the crista terminalis and interatrial septum). Whereas RAA predominantly consisted of well-tubulated myocytes, ICR showed partially tubulated/untubulated cells. Similar TATS distribution was also observed in healthy human atrial myocardium sections. In both mouse atrial preparations and isolated mouse atrial myocytes, we observed a strong anatomical correlation between TATS distribution and Ca2+ transient synchronization and rise-up time. This region-specific difference in Ca2+ transient morphology disappeared after formamide-induced detubulation. ICR myocytes showed a prolonged action potential duration at 80% of repolarization as well as a significantly lower expression of RyR2 and Cav1.2 proteins but similar levels of NCX1 and Cav1.3 compared with RAA tissue. Our findings provide a detailed characterization of the region-specific distribution of TATS in mouse and human atrial myocardium, highlighting the structural foundation for anatomical heterogeneity of Ca2+ dynamics and contractility in the atria. These results could indicate different roles of TATS in Ca2+ signaling at distinct anatomical regions of the atria and provide mechanistic insight into pathological atrial remodeling.NEW & NOTEWORTHY Mouse and human atrial myocardium demonstrate high variability in the organization of the transversal-axial tubule system (TATS), with more organized TATS expressed in the right atrial appendage. TATS distribution governs anatomical heterogeneity of Ca2+ dynamics and thus could contribute to integral atrial contractility, mechanics, and arrhythmogenicity.

Caveolin-3 is required for regulation of transient outward potassium current by angiotensin II in mouse atrial myocytes.

Angiotensin II (AngII) is a key mediator of the renin-angiotensin system and plays an important role in the regulation of cardiac electrophysiology by affecting various cardiac ion currents, including transient outward potassium current, Ito. AngII receptors and molecular components of Ito, Kv4.2 and Kv4.3 channels, have been linked to caveolae structures. However, their functional interaction and the importance of such proximity within 50- to 100-nm caveolar nanodomains remain unknown. To address this, we studied the mechanisms of Ito regulation by AngII in atrial myocytes of wild-type (WT) and cardiac-specific caveolin-3 (Cav3) conditional knockout (Cav3KO) mice. We showed that in WT atrial myocytes, a short-term (2 h) treatment with AngII (5 µM) significantly reduced Ito density. This effect was prevented 1) by a 30-min pretreatment with a selective antagonist of AngII receptor 1 (Ang1R) losartan (2 µM) or 2) by a selective inhibition of protein kinase C (PKC) by BIM1 (10 µM). The effect of AngII on Ito was completely abolished in Cav3-KO mice, with no change in a baseline Ito current density. In WT atria, Ang1Rs co-localized with Cav3, and the expression of Ang1Rs was significantly decreased in Cav3KO in comparison with WT mice, whereas no change in Kv4.2 and Kv4.3 protein expression was observed. Overall, our findings demonstrate that Cav3 is involved in the regulation of Ang1R expression and is required for the modulation of Ito by AngII in mouse atrial myocytes.NEW & NOTEWORTHY Angiotensin II receptor 1 is associated with caveolae and caveolar scaffolding protein caveolin-3 in mouse atrial myocytes that is required for the regulation of Ito by angiotensin II. Downregulation of caveolae/caveolin-3 disrupts this regulation and may be implicated in pathophysiological atrial remodeling.

WITHDRAWN: Caveolar Compartmentalization is Required for Stable Rhythmicity of Sinus Nodal Cells and is Disrupted in Heart Failure.

The authors have withdrawn their manuscript owing to technical concerns merged during peer review. Therefore, the authors do not wish this work to be cited as a reference. If you have any questions, please contact the corresponding author.

Local hyperactivation of L-type Ca2+ channels increases spontaneous Ca2+ release activity and cellular hypertrophy in right ventricular myocytes from heart failure rats.

Right ventricle (RV) dysfunction is an independent predictor of patient survival in heart failure (HF). However, the mechanisms of RV progression towards failing are not well understood. We studied cellular mechanisms of RV remodelling in a rat model of left ventricle myocardial infarction (MI)-caused HF. RV myocytes from HF rats show significant cellular hypertrophy accompanied with a disruption of transverse-axial tubular network and surface flattening. Functionally these cells exhibit higher contractility with lower Ca2+ transients. The structural changes in HF RV myocytes correlate with more frequent spontaneous Ca2+ release activity than in control RV myocytes. This is accompanied by hyperactivated L-type Ca2+ channels (LTCCs) located specifically in the T-tubules of HF RV myocytes. The increased open probability of tubular LTCCs and Ca2+ sparks activation is linked to protein kinase A-mediated channel phosphorylation that occurs locally in T-tubules. Thus, our approach revealed that alterations in RV myocytes in heart failure are specifically localized in microdomains. Our findings may indicate the development of compensatory, though potentially arrhythmogenic, RV remodelling in the setting of LV failure. These data will foster better understanding of mechanisms of heart failure and it could promote an optimized treatment of patients.