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.

Long QT Syndrome KCNH2 Variant Induces hERG1a/1b Subunit Imbalance in Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

[Figure: see text].

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.

Caveolae-Mediated Activation of Mechanosensitive Chloride Channels in Pulmonary Veins Triggers Atrial Arrhythmogenesis.

Background Atrial fibrillation often occurs in the setting of hypertension and associated atrial dilation with pathologically increased cardiomyocyte stretch. In the setting of atrial dilation, mechanoelectric feedback has been linked to the development of ectopic beats that trigger paroxysmal atrial fibrillation mainly originating from pulmonary veins (PVs). However, the precise mechanisms remain poorly understood. Methods and Results We identify mechanosensitive, swelling-activated chloride ion channels (ICl,swell) as a crucial component of the caveolar mechanosensitive complex in rat and human cardiomyocytes. In vitro optical mapping of rat PV, single rat PV, and human cardiomyocyte patch clamp studies showed that stretch-induced activation of ICl,swell leads to membrane depolarization and decreased action potential amplitude, which trigger conduction discontinuities and both ectopic and reentrant activities within the PV. Reverse transcription quantitative polymerase chain reaction, immunofluorescence, and coimmunoprecipitation studies showed that ICl,swell likely consists of at least 2 components produced by mechanosensitive ClC-3 (chloride channel-3) and SWELL1 (also known as LRRC8A [leucine rich repeat containing protein 8A]) chloride channels, which form a macromolecular complex with caveolar scaffolding protein Cav3 (caveolin 3). Downregulation of Cav3 protein expression and disruption of caveolae structures during chronic hypertension in spontaneously hypertensive rats facilitates activation of ICl,swell and increases PV sensitivity to stretch 10- to 50-fold, promoting the development of atrial fibrillation. Conclusions Our findings identify caveolae-mediated activation of mechanosensitive ICl,swell as a critical cause of PV ectopic beats that can initiate atrial arrhythmias including atrial fibrillation. This mechanism is exacerbated in the setting of chronically elevated blood pressures.

Mouse ERG K(+) channel clones reveal differences in protein trafficking and function.

BACKGROUND: The mouse ether-a-go-go-related gene 1a (mERG1a, mKCNH2) encodes mERG K(+) channels in mouse cardiomyocytes. The mERG channels and their human analogue, hERG channels, conduct IKr. Mutations in hERG channels reduce IKr to cause congenital long-QT syndrome type 2, mostly by decreasing surface membrane expression of trafficking-deficient channels. Three cDNA sequences were originally reported for mERG channels that differ by 1 to 4 amino acid residues (mERG-London, mERG-Waterston, and mERG-Nie). We characterized these mERG channels to test the postulation that they would differ in their protein trafficking and biophysical function, based on previous findings in long-QT syndrome type 2. METHODS AND RESULTS: The 3 mERG and hERG channels were expressed in HEK293 cells and neonatal mouse cardiomyocytes and were studied using Western blot and whole-cell patch clamp. We then compared our findings with the recent sequencing results in the Welcome Trust Sanger Institute Mouse Genomes Project (WTSIMGP). CONCLUSIONS: First, the mERG-London channel with amino acid substitutions in regions of highly ordered structure is trafficking deficient and undergoes temperature-dependent and pharmacological correction of its trafficking deficiency. Second, the voltage dependence of channel gating would be different for the 3 mERG channels. Third, compared with the WTSIMGP data set, the mERG-Nie clone is likely to represent the wild-type mouse sequence and physiology. Fourth, the WTSIMGP analysis suggests that substrain-specific sequence differences in mERG are a common finding in mice. These findings with mERG channels support previous findings with hERG channel structure-function analyses in long-QT syndrome type 2, in which sequence changes in regions of highly ordered structure are likely to result in abnormal protein trafficking.

Mechanism of loss of Kv11.1 K+ current in mutant T421M-Kv11.1-expressing rat ventricular myocytes: interaction of trafficking and gating.

BACKGROUND: Type 2 long QT syndrome involves mutations in the human ether a-go-go-related gene (hERG or KCNH2). T421M, an S1 domain mutation in the Kv11.1 channel protein, was identified in a resuscitated patient. We assessed its biophysical, protein trafficking, and pharmacological mechanisms in adult rat ventricular myocytes. METHODS AND RESULTS: Isolated adult rat ventricular myocytes were infected with wild-type (WT)-Kv11.1- and T421M-Kv11.1-expressing adenovirus and analyzed with the use of patch clamp, Western blot, and confocal imaging techniques. Expression of WT-Kv11.1 or T421M-Kv11.1 produced peak tail current (I(Kv11.1)) of 8.78±1.18 and 1.91±0.22 pA/pF, respectively. Loss of mutant I(Kv11.1) resulted from (1) a partially trafficking-deficient channel protein with reduced cell surface expression and (2) altered channel gating with a positive shift in the voltage dependence of activation and altered kinetics of activation and deactivation. Coexpression of WT+T421M-Kv11.1 resulted in heterotetrameric channels that remained partially trafficking deficient with only a minimal increase in peak I(Kv11.1) density, whereas the voltage dependence of channel gating became WT-like. In the adult rat ventricular myocyte model, both WT-Kv11.1 and T421M-Kv11.1 channels responded to ß-adrenergic stimulation by increasing I(Kv11.1). CONCLUSIONS: The T421M-Kv11.1 mutation caused a loss of I(Kv11.1) through interactions of abnormal protein trafficking and channel gating. Furthermore, for coexpressed WT+T421M-Kv11.1 channels, different dominant-negative interactions govern protein trafficking and ion channel gating, and these are likely to be reflected in the clinical phenotype. Our results also show that WT and mutant Kv11.1 channels responded to ß-adrenergic stimulation.

Novel chemical suppressors of long QT syndrome identified by an in vivo functional screen.

BACKGROUND: Genetic long QT (LQT) syndrome is a life-threatening disorder caused by mutations that result in prolongation of cardiac repolarization. Recent work has demonstrated that a zebrafish model of LQT syndrome faithfully recapitulates several features of human disease, including prolongation of ventricular action potential duration, spontaneous early afterdepolarizations, and 2:1 atrioventricular block in early stages of development. Because of their transparency, small size, and absorption of small molecules from their environment, zebrafish are amenable to high-throughput chemical screens. We describe a small-molecule screen using the zebrafish KCNH2 mutant breakdance to identify compounds that can rescue the LQT type 2 phenotype. METHODS AND RESULTS: Zebrafish breakdance embryos were exposed to test compounds at 48 hours of development and scored for rescue of 2:1 atrioventricular block at 72 hours in a 96-well format. Only compounds that suppressed the LQT phenotype in 3 of 3 fish were considered hits. Screen compounds were obtained from commercially available small-molecule libraries (Prestwick and Chembridge). Initial hits were confirmed with dose-response testing and time-course studies. Optical mapping with the voltage-sensitive dye di-4 ANEPPS was performed to measure compound effects on cardiac action potential durations. Screening of 1200 small molecules resulted in the identification of flurandrenolide and 2-methoxy-N-(4-methylphenyl) benzamide (2-MMB) as compounds that reproducibly suppressed the LQT phenotype. Optical mapping confirmed that treatment with each compound caused shortening of ventricular action potential durations. Structure activity studies and steroid receptor knockdown suggest that flurandrenolide functions via the glucocorticoid signaling pathway. CONCLUSIONS: Using a zebrafish model of LQT type 2 syndrome in a high-throughput chemical screen, we have identified 2 compounds, flurandrenolide and the novel compound 2-MMB, as small molecules that rescue the zebrafish LQT type 2 syndrome by shortening the ventricular action potential duration. We provide evidence that flurandrenolide functions via the glucocorticoid receptor-mediated pathway. These 2 molecules and future discoveries from this screen should yield novel tools for the study of cardiac electrophysiology and may lead to novel therapeutics for human LQT patients.