Electrophysiologic and molecular mechanisms of a frameshift NPPA mutation linked with familial atrial fibrillation.

A frameshift (fs) mutation in the natriuretic peptide precursor A (NPPA) gene, encoding a mutant atrial natriuretic peptide (Mut-ANP), has been linked with familial atrial fibrillation (AF) but the underlying mechanisms by which the mutation causes AF remain unclear. We engineered 2 transgenic (TG) mouse lines expressing the wild-type (WT)-NPPA gene (H-WT-NPPA) and the human fs-Mut-NPPA gene (H-fsMut-NPPA) to test the hypothesis that mice overexpressing the human NPPA mutation are more susceptible to AF and elucidate the underlying electrophysiologic and molecular mechanisms. Transthoracic echocardiography and surface electrocardiography (ECG) were performed in H-fsMut-NPPA, H-WT-NPPA, and Non-TG mice. Invasive electrophysiology, immunohistochemistry, Western blotting and patch clamping of membrane potentials were performed. To examine the role of the Mut-ANP in ion channel remodeling, we measured plasma cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) activity in the 3 groups of mice. In H-fsMut-NPPA mice mean arterial pressure (MAP) was reduced when compared to H-WT-NPPA and Non-TG mice. Furthermore, injection of synthetic fs-Mut-ANP lowered the MAP in H-WT-NPPA and Non-TG mice while synthetic WT-ANP had no effect on MAP in the 3 groups of mice. ECG characterization revealed significantly prolonged QRS duration in H-fsMut-NPPA mice when compared to the other two groups. Trans-Esophageal (TE) atrial pacing of H-fsMut-NPPA mice showed increased AF burden and AF episodes when compared with H-WT-NPPA or Non-TG mice. The cardiac Na+ (NaV1.5) and Ca2+ (CaV1.2/CaV1.3) channel expression and currents (INa, ICaL) and action potential durations (APD90/APD50/APD20) were significantly reduced in H-fsMut-NPPA mice while the rectifier K+ channel current (IKs) was markedly increased when compared to the other 2 groups of mice. In addition, plasma cGMP levels were only increased in H-fsMut-NPPA mice with a corresponding reduction in plasma cAMP levels and PKA activity. In summary, we showed that mice overexpressing an AF-linked NPPA mutation are more prone to develop AF and this risk is mediated in part by remodeling of the cardiac Na+, Ca2+ and K+ channels creating an electrophysiologic substrate for reentrant AF.

Cardiac Arrhythmias Related to Sodium Channel Dysfunction.

The voltage-gated cardiac sodium channel (Nav1.5) is a mega-complex comprised of a pore-forming α subunit and 4 ancillary ß-subunits together with numerous protein partners. Genetic defects in the form of rare variants in one or more sodium channel-related genes can cause a loss- or gain-of-function of sodium channel current (INa) leading to the manifestation of various disease phenotypes, including Brugada syndrome, long QT syndrome, progressive cardiac conduction disease, sick sinus syndrome, multifocal ectopic Purkinje-related premature contractions, and atrial fibrillation. Some sodium channelopathies have also been shown to be responsible for sudden infant death syndrome (SIDS). Although these genetic defects often present as pure electrical diseases, recent studies point to a contribution of structural abnormalities to the electrocardiographic and arrhythmic manifestation in some cases, such as dilated cardiomyopathy. The same rare variants in SCN5A or related genes may present with different clinical phenotypes in different individuals and sometimes in members of the same family. Genetic background and epigenetic and environmental factors contribute to the expression of these overlap syndromes. Our goal in this chapter is to review and discuss what is known about the clinical phenotype and genotype of each cardiac sodium channelopathy, and to briefly discuss the underlying mechanisms.

Subcellular structures and function of myocytes impaired during heart failure are restored by cardiac resynchronization therapy.

RATIONALE: Cardiac resynchronization therapy (CRT) is an established treatment for patients with chronic heart failure. However, CRT-associated structural and functional remodeling at cellular and subcellular levels is only partly understood. OBJECTIVE: To investigate the effects of CRT on subcellular structures and protein distributions associated with excitation-contraction coupling of ventricular cardiomyocytes. METHODS AND RESULTS: Our studies revealed remodeling of the transverse tubular system (t-system) and the spatial association of ryanodine receptor (RyR) clusters in a canine model of dyssynchronous heart failure (DHF). We did not find this remodeling in a synchronous heart failure model based on atrial tachypacing. Remodeling in DHF ranged from minor alterations in anterior left ventricular myocytes to nearly complete loss of the t-system and dissociation of RyRs from sarcolemmal structures in lateral cells. After CRT, we found a remarkable and almost complete reverse remodeling of these structures despite persistent left ventricular dysfunction. Studies of whole-cell Ca(2+) transients showed that the structural remodeling and restoration were accompanied with remodeling and restoration of Ca(2+) signaling. CONCLUSIONS: DHF is associated with regional remodeling of the t-system. Myocytes undergo substantial structural and functional restoration after only 3 weeks of CRT. The finding suggests that t-system status can provide an early marker of the success of this therapy. The results could also guide us to an understanding of the loss and remodeling of proteins associated with the t-system. The steep relationship between free Ca(2+) and contraction suggests that some restoration of Ca(2+) release units will have a disproportionately large effect on contractility.

Novel features of the rabbit transverse tubular system revealed by quantitative analysis of three-dimensional reconstructions from confocal images.

With scanning confocal microscopy we obtained three-dimensional (3D) reconstructions of the transverse tubular system (t-system) of rabbit ventricular cells. We accomplished this by labeling the t-system with dextran linked to fluorescein or, alternatively, wheat-germ agglutinin conjugated to an Alexa fluor dye. Image processing and visualization techniques allowed us to reconstruct the t-system in three dimensions. In a myocyte lying flat on a coverslip, t-tubules typically progressed from its upper and lower surfaces. 3D reconstructions of the t-tubules also suggested that some of them progressed from the sides of the cell. The analysis of single t-tubules revealed novel morphological features. The average diameter of single t-tubules from six cells was estimated to 448 +/- 172 nm (mean +/- SD, number of t-tubules 348, number of cross sections 5323). From reconstructions we were able to identify constrictions occurring every 1.87 +/- 1.09 microm along the principal axis of the tubule. The cross-sectional area of these constrictions was reduced to an average of 57.7 +/- 27.5% (number of constrictions 170) of the adjacent local maximal areas. Principal component analysis revealed flattening of t-tubular cross sections, confirming findings that we obtained from electron micrographs. Dextran- and wheat-germ agglutinin-associated signals were correlated in the t-system and are therefore equally good markers. The 3D structure of the t-system in rabbit ventricular myocytes seems to be less complex than that found in rat. Moreover, we found that t-tubules in rabbit have approximately twice the diameter of those in rat. We speculate that the constrictions (or regions between them) are sites of dyadic clefts and therefore can provide geometric markers for colocalizing dyadic proteins. In consideration of the resolution of the imaging system, we suggest that our methods permit us to obtain spatially resolved 3D reconstructions of the t-system in rabbit cells. We also propose that our methods allow us to characterize pathological defects of the t-system, e.g., its remodeling as a result of heart failure.

Electrophysiologic Characterization of Calcium Handling in Human Induced Pluripotent Stem Cell-Derived Atrial Cardiomyocytes.

Human induced pluripotent stem cell (hiPSC)-derived atrial cardiomyocytes (CMs) hold great promise for elucidating underlying cellular mechanisms that cause atrial fibrillation (AF). In order to use atrial-like hiPSC-CMs for arrhythmia modeling, it is essential to better understand the molecular and electrophysiological phenotype of these cells. We performed comprehensive molecular, transcriptomic, and electrophysiologic analyses of retinoic acid (RA)-guided hiPSC atrial-like CMs and demonstrate that RA results in differential expression of genes involved in calcium ion homeostasis that directly interact with an RA receptor, chicken ovalbumin upstream promoter-transcription factor 2 (COUP-TFII). We report a mechanism by which RA generates an atrial-like electrophysiologic signature through the downstream regulation of calcium channel gene expression by COUP-TFII and modulation of calcium handling. Collectively, our results provide important insights into the underlying molecular mechanisms that regulate atrial-like hiPSC-CM electrophysiology and support the use of atrial-like CMs derived from hiPSCs to model AF.

Channel Activity of Cardiac Ryanodine Receptors (RyR2) Determines Potency and Efficacy of Flecainide and R-Propafenone against Arrhythmogenic Calcium Waves in Ventricular Cardiomyocytes.

Flecainide blocks ryanodine receptor type 2 (RyR2) channels in the open state, suppresses arrhythmogenic Ca2+ waves and prevents catecholaminergic polymorphic ventricular tachycardia (CPVT) in mice and humans. We hypothesized that differences in RyR2 activity induced by CPVT mutations determines the potency of open-state RyR2 blockers like flecainide (FLEC) and R-propafenone (RPROP) against Ca2+ waves in cardiomyocytes. Using confocal microscopy, we studied Ca2+ sparks and waves in isolated saponin-permeabilized ventricular myocytes from two CPVT mouse models (Casq2-/-, RyR2-R4496C+/-), wild-type (c57bl/6, WT) mice, and WT rabbits (New Zealand white rabbits). Consistent with increased RyR2 activity, Ca2+ spark and wave frequencies were significantly higher in CPVT compared to WT mouse myocytes. We next obtained concentration-response curves of Ca2+ wave inhibition for FLEC, RPROP (another open-state RyR2 blocker), and tetracaine (TET) (a state-independent RyR2 blocker). Both FLEC and RPROP inhibited Ca2+ waves with significantly higher potency (lower IC50) and efficacy in CPVT compared to WT. In contrast, TET had similar potency in all groups studied. Increasing RyR2 activity of permeabilized WT myocytes by exposure to caffeine (150 µM) increased the potency of FLEC and RPROP but not of TET. RPROP and FLEC were also significantly more potent in rabbit ventricular myocytes that intrinsically exhibit higher Ca2+ spark rates than WT mouse ventricular myocytes. In conclusion, RyR2 activity determines the potency of open-state blockers FLEC and RPROP for suppressing arrhythmogenic Ca2+ waves in cardiomyocytes, a mechanism likely relevant to antiarrhythmic drug efficacy in CPVT.

Role of common and rare variants in SCN10A: results from the Brugada syndrome QRS locus gene discovery collaborative study.

AIMS: Brugada syndrome (BrS) remains genetically heterogeneous and is associated with slowed cardiac conduction. We aimed to identify genetic variation in BrS cases at loci associated with QRS duration. METHODS AND RESULTS: A multi-centre study sequenced seven candidate genes (SCN10A, HAND1, PLN, CASQ2, TKT, TBX3, and TBX5) in 156 Caucasian SCN5A mutation-negative BrS patients (80% male; mean age 48) with symptoms (64%) and/or a family history of sudden death (47%) or BrS (18%). Forty-nine variants were identified: 18 were rare (MAF <1%) and non-synonymous; and 11/18 (61.1%), mostly in SCN10A, were predicted as pathogenic using multiple bioinformatics tools. Allele frequencies were compared with the Exome Sequencing and UK10K Projects. SKAT methods tested rare variation in SCN10A finding no statistically significant difference between cases and controls. Co-segregation analysis was possible for four of seven probands carrying a novel pathogenic variant. Only one pedigree (I671V/G1299A in SCN10A) showed co-segregation. The SCN10A SNP V1073 was, however, associated strongly with BrS [66.9 vs. 40.1% (UK10K) OR (95% CI) = 3.02 (2.35-3.87), P = 8.07 × 10-19]. Voltage-clamp experiments for NaV1.8 were performed for SCN10A common variants V1073, A1073, and rare variants of interest: A200V and I671V. V1073, A200V and I671V, demonstrated significant reductions in peak INa compared with ancestral allele A1073 (rs6795970). CONCLUSION: Rare variants in the screened QRS-associated genes (including SCN10A) are not responsible for a significant proportion of SCN5A mutation negative BrS. The common SNP SCN10A V1073 was strongly associated with BrS and demonstrated loss of NaV1.8 function, as did rare variants in isolated patients.

Atrial Fibrillation and SCN5A Variants.

Although atrial fibrillation (AF) is clinically and genetically a highly heterogeneous disease, recent studies suggest that the arrhythmia may arise because of interactions between genetic and acquired risk factors - the so called "double-hit" hypothesis. Genome-wide association studies have identified common AF susceptibility loci, and linkage analysis and candidate gene approaches have identified mutations in genes that encode for cardiac ion channels and signaling proteins; however, most of the heritability of AF still remains unexplained. The voltage-dependent cardiac sodium channel, encoded by SCN5A, conducts the main cardiac inward sodium current (INa) and is responsible for the upstroke of the atrial action potential. Mutations in SCN5A, which encodes the α-subunit of the NaV1.5 channel, have been linked with increased susceptibility to not only AF but also ventricular arrhythmias (long QT syndrome, Brugada syndrome), progressive cardiac conduction disease, and overlap syndromes with mixed arrhythmia phenotypes. Over the last decade, functional characterization of SCN5A mutations by expressing the channel in heterologous expression systems and applying cellular electrophysiological techniques has not only advanced our understanding of molecular mechanisms of AF but also potentially identified a mechanism-based approach to treating this common and morbid condition.

Suppression of spontaneous ca elevations prevents atrial fibrillation in calsequestrin 2-null hearts.

BACKGROUND: Atrial fibrillation (AF) risk has been associated with leaky ryanodine receptor 2 (RyR2) Ca release channels. Patients with mutations in RyR2 or in the sarcoplasmic reticulum Ca-binding protein calsequestrin 2 (Casq2) display an increased risk for AF. Here, we examine the underlying mechanisms of AF associated with loss of Casq2 and test mechanism-based drug therapy. METHODS AND RESULTS: Compared with wild-type Casq2+/+ mice, atrial burst pacing consistently induced atrial flutter or AF in Casq2-/- mice and in isolated Casq2-/- hearts. Atrial optical voltage maps obtained from isolated hearts revealed multiple independent activation sites arising predominantly from the pulmonary vein region. Ca and voltage mapping demonstrated diastolic subthreshold spontaneous Ca elevations (SCaEs) and delayed afterdepolarizations whenever the pacing train failed to induce AF. The dual RyR2 and Na channel inhibitor R-propafenone (3 µmol/L) significantly reduced frequency and amplitude of SCaEs and delayed afterdepolarizations in atrial myocytes and intact atria and prevented induction of AF. In contrast, the S-enantiomer of propafenone, an equipotent Na channel blocker but much weaker RyR2 inhibitor, did not reduce SCaEs and delayed afterdepolarizations and failed to prevent AF. CONCLUSIONS: Loss of Casq2 increases risk of AF by promoting regional SCaEs and delayed afterdepolarizations in atrial tissue, which can be prevented by RyR2 inhibition with R-propafenone. Targeting AF caused by leaky RyR2 Ca channels with R-propafenone may be a more mechanism-based approach to treating this common arrhythmia.

SCN10A/Nav1.8 modulation of peak and late sodium currents in patients with early onset atrial fibrillation.

AIMS: To test the hypothesis that vulnerability to atrial fibrillation (AF) is associated with rare coding sequence variation in the SCN10A gene, which encodes the voltage-gated sodium channel isoform NaV1.8 found primarily in peripheral nerves and to identify potentially disease-related mechanisms in high-priority rare variants using in-vitro electrophysiology. METHODS AND RESULTS: We re-sequenced SCN10A in 274 patients with early onset AF from the Vanderbilt AF Registry to identify rare coding variants. Engineered variants were transiently expressed in ND7/23 cells and whole-cell voltage clamp experiments were conducted to elucidate their functional properties. Resequencing SCN10A identified 18 heterozygous rare coding variants (minor allele frequency ≤1%) in 18 (6.6%) AF probands. Four probands were carriers of two rare variants each and 14 were carriers of one coding variant. Based on evidence of co-segregation, initial assessment of functional importance, and presence in ≥1 AF proband, three variants (417delK, A1886V, and the compound variant Y158D-R814H) were selected for functional studies. The 417delK variant displayed near absent current while A1886V and Y158D-R814H exhibited enhanced peak and late (INa-L) sodium currents; both Y158D and R818H individually contributed to this phenotype. CONCLUSION: Rare SCN10A variants encoding Nav1.8 were identified in 6.6% of patients with early onset AF. In-vitro electrophysiological studies demonstrated profoundly altered function in 3/3 high-priority variants. Collectively, these data strongly support the hypothesis that rare SCN10A variants may contribute to AF susceptibility.

Prevalence and predictors of atrial fibrillation among patients undergoing bariatric surgery.

BACKGROUND: While AF is a disease of the elderly, it can occur earlier in the presence of risk factors such as obesity. Bariatric surgery patients are significantly younger and more obese than previously described populations with AF. Therefore, it remains to be determined whether current estimates of the prevalence and predictors for AF remain true in the bariatric surgery population. METHODS: We performed a cross-sectional analysis of 1,341 consecutive patients who underwent bariatric surgery from January 2008 to October 2012. Baseline characteristics were compared between patients with and without AF. For additional comparison, 176 patients with AF and body mass index (BMI) >40 kg/m(2) were identified from the Vanderbilt AF Registry. A multivariable logistic regression was performed to identify predictors of AF within the bariatric surgery cohort. RESULTS: The prevalence of AF in the bariatric surgery cohort was 1.9 % (25/1,341). Patients with AF were older (median 56 years (interquartile range [52-64) vs.46 [38-56] years, p < 0.001), were more often male (48 vs. 23 %, p = 0.004), had more comorbidities, but had no difference in BMI (50 kg/m(2) [44-58] vs. 48 [43-54], p = 0.4). In multivariable analysis, the odds of AF increased 2.2-fold by age per decade (95 % CI, 1.4-3.5; p < 0.001) and 2.4-fold by male gender (1.1-5.4, p = 0.03) when adjusted for BMI. BMI was not independently associated with AF (OR 1.15 [95 % CI, 0.98-1.41], p = 0.09). CONCLUSIONS: The prevalence of AF is 1.9 % among patients undergoing bariatric surgery. Risk of AF was found to increase with age and male gender, but not with higher BMI.

Voltage-gated sodium channels: biophysics, pharmacology, and related channelopathies.

Voltage-gated sodium channels (VGSC) are multi-molecular protein complexes expressed in both excitable and non-excitable cells. They are primarily formed by a pore-forming multi-spanning integral membrane glycoprotein (α-subunit) that can be associated with one or more regulatory ß-subunits. The latter are single-span integral membrane proteins that modulate the sodium current (I(Na)) and can also function as cell adhesion molecules. In vitro some of the cell-adhesive functions of the ß-subunits may play important physiological roles independently of the α-subunits. Other endogenous regulatory proteins named "channel partners" or "channel interacting proteins" (ChiPs) like caveolin-3 and calmodulin/calmodulin kinase II (CaMKII) can also interact and modulate the expression and/or function of VGSC. In addition to their physiological roles in cell excitability and cell adhesion, VGSC are the site of action of toxins (like tetrodotoxin and saxitoxin), and pharmacologic agents (like antiarrhythmic drugs, local anesthetics, antiepileptic drugs, and newly developed analgesics). Mutations in genes that encode α- and/or ß-subunits as well as the ChiPs can affect the structure and biophysical properties of VGSC, leading to the development of diseases termed sodium "channelopathies". This review will outline the structure, function, and biophysical properties of VGSC as well as their pharmacology and associated channelopathies and highlight some of the recent advances in this field.

Towards computational modeling of excitation-contraction coupling in cardiac myocytes: reconstruction of structures and proteins from confocal imaging.

Computational models of excitation-contraction (EC) coupling in myocytes are valuable tools for studying the signaling cascade that transduces transmembrane voltage into mechanical responses. A key component of these models is the appropriate description of structures involved in EC coupling, such as the sarcolemma and ion channels. This study aims at developing an approach for spatial reconstruction of these structures. We exemplified our approach by reconstructing clusters of ryanodine receptors (RyRs) together with the sarcolemma of rabbit ventricular myocytes. The reconstructions were based on dual labeling and three-dimensional (3D) confocal imaging of segments of fixed and permeabilized myocytes lying flat or on end. The imaging led to 3D stacks of cross-sections through myocytes. Methods of digital image processing were applied to deconvolve, filter and segment these stacks. Finally, we created point meshes representing RyR distributions together with volume and surface meshes of the sarcolemma. We suggest that these meshes are suitable for computational studies of structure-function relationships in EC coupling. We propose that this approach can be extended to reconstruct other structures and proteins involved in EC coupling.

Sub-micrometer anatomical models of the sarcolemma of cardiac myocytes based on confocal imaging.

We describe an approach to develop anatomical models of cardiac cells. The approach is based on confocal imaging of living ventricular myocytes with submicrometer resolution, digital image processing of three-dimensional stacks with high data volume, and generation of dense triangular surface meshes representing the sarcolemma including the transverse tubular system. The image processing includes methods for deconvolution, filtering and segmentation. We introduce and visualize models of the sarcolemma of whole ventricular myocytes and single transversal tubules. These models can be applied for computational studies of cell and sub-cellular physical behavior and physiology, in particular cell signaling. Furthermore, the approach is applicable for studying effects of cardiac development, aging and diseases, which are associated with changes of cell anatomy and protein distributions.

Mechanical and energetic effects of chronic chagasic patients' antibodies on rat myocardium.

Chagasic (Ch) and nonchagasic (NCh) IgG fraction (20 microg/ml) effects on cardiac performance of adult Wistar rat ventricles were studied with a novel approach applying a microcalorimetric technique. Resting heat (Hr) was significantly decreased by Ch antibodies (DeltaHrCh = 4.8 +/- 0.9 mW/g). Although the Hr decrease can be associated with diminished activity of the Na+/K+ pump, the magnitude of the effect (25% of control Hr) indicates that additional processes may also be affected. Ch antibodies induced an initial increase in developed pressure (P), which was associated with a decreased contractile economy. However, after 30 min of Ch antibody perfusion, P reached a significantly lower level (DeltaPCh = 3.8 +/- 1.2 mN/mm2) without changes in active heat per beat (Ha'). Consequently, Ha'/P ratio increased, indicating that the energetic cost per unit of P was higher. In contrast, P and Ha' were both significantly and reversibly decreased by NCh antibodies (DeltaPNCh = 4.4 +/- 1.2 mN/mm2; DeltaHa'NCh = 9.7 +/- 2.2 mJ/g), but Ha'/P remained unaffected. According to these data, normal hearts exposed to Ch antibodies present a biphasic mechanical response: 1) an initial period of increased contractility (and decreased global muscle economy) consistent with antibodies with beta1-adrenergic activity, such as those used in the present study, and 2) a decrease in P at 30 min of Ch antibody perfusion, which suggests that another Ca(2+)-related mechanism is compromised. These data contribute to redefine the role of antibody-mediated responses in the pathophysiology of chronic chagasic cardiomyopathy as agents of myocardial failure.