Genetic background determines the severity of age-dependent cardiac structural abnormalities and arrhythmia susceptibility in Scn5a-1798insD mice.

AIMS: Patients with mutations in SCN5A encoding NaV1.5 often display variable severity of electrical and structural alterations, but the underlying mechanisms are not fully elucidated. We here investigate the combined modulatory effect of genetic background and age on disease severity in the Scn5a1798insD/+ mouse model. METHODS AND RESULTS: In vivo electrocardiogram and echocardiograms, ex vivo electrical and optical mapping, and histological analyses were performed in adult (2-7 months) and aged (8-28 months) wild-type (WT) and Scn5a1798insD/+ (mutant, MUT) mice from the FVB/N and 129P2 inbred strains. Atrio-ventricular (AV) conduction, ventricular conduction, and ventricular repolarization are modulated by strain, genotype, and age. An aging effect was present in MUT mice, with aged MUT mice of both strains showing prolonged QRS interval and right ventricular (RV) conduction slowing. 129P2-MUT mice were severely affected, with adult and aged 129P2-MUT mice displaying AV and ventricular conduction slowing, prolonged repolarization, and spontaneous arrhythmias. In addition, the 129P2 strain appeared particularly susceptible to age-dependent electrical, functional, and structural alterations including RV conduction slowing, reduced left ventricular (LV) ejection fraction, RV dilatation, and myocardial fibrosis as compared to FVB/N mice. Overall, aged 129P2-MUT mice displayed the most severe conduction defects, RV dilatation, and myocardial fibrosis, in addition to the highest frequency of spontaneous arrhythmia and inducible arrhythmias. CONCLUSION: Genetic background and age both modulate disease severity in Scn5a1798insD/+ mice and hence may explain, at least in part, the variable disease expressivity observed in patients with SCN5A mutations. Age- and genetic background-dependent development of cardiac structural alterations furthermore impacts arrhythmia risk. Our findings therefore emphasize the importance of continued assessment of cardiac structure and function in patients carrying SCN5A mutations.

Decreasing microtubule detyrosination modulates Nav1.5 subcellular distribution and restores sodium current in mdx cardiomyocytes.

AIMS: The microtubule (MT) network plays a major role in the transport of the cardiac sodium channel Nav1.5 to the membrane, where the latter associates with interacting proteins such as dystrophin. Alterations in MT dynamics are known to impact on ion channel trafficking. Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, is associated with an increase in MT detyrosination, decreased sodium current (INa), and arrhythmias. Parthenolide (PTL), a compound that decreases MT detyrosination, has shown beneficial effects on cardiac function in DMD. We here investigated its impact on INa and Nav1.5 subcellular distribution. METHODS AND RESULTS: Ventricular cardiomyocytes (CMs) from wild-type (WT) and mdx (DMD) mice were incubated with either 10 µM PTL, 20 µM EpoY, or dimethylsulfoxide (DMSO) for 3-5 h, followed by patch-clamp analysis to assess INa and action potential (AP) characteristics in addition to immunofluorescence and stochastic optical reconstruction microscopy (STORM) to investigate MT detyrosination and Nav1.5 cluster size and density, respectively. In accordance with previous studies, we observed increased MT detyrosination, decreased INa and reduced AP upstroke velocity (Vmax) in mdx CMs compared to WT. PTL decreased MT detyrosination and significantly increased INa magnitude (without affecting INa gating properties) and AP Vmax in mdx CMs, but had no effect in WT CMs. Moreover, STORM analysis showed that in mdx CMs, Nav1.5 clusters were decreased not only in the grooves of the lateral membrane (LM; where dystrophin is localized) but also at the LM crests. PTL restored Nav1.5 clusters at the LM crests (but not at the grooves), indicating a dystrophin-independent trafficking route to this subcellular domain. Interestingly, Nav1.5 cluster density was also reduced at the intercalated disc (ID) region of mdx CMs, which was restored to WT levels by PTL. Treatment of mdx CMs with EpoY, a specific MT detyrosination inhibitor, also increased INa density, while decreasing the amount of detyrosinated MTs, confirming a direct mechanistic link. CONCLUSION: Attenuating MT detyrosination in mdx CMs restored INa and enhanced Nav1.5 localization at the LM crest and ID. Hence, the reduced whole-cell INa density characteristic of mdx CMs is not only the consequence of the lack of dystrophin within the LM grooves but is also due to reduced Nav1.5 at the LM crest and ID secondary to increased baseline MT detyrosination. Overall, our findings identify MT detyrosination as a potential therapeutic target for modulating INa and subcellular Nav1.5 distribution in pathophysiological conditions.

Recent advances and current limitations of available technology to optically manipulate and observe cardiac electrophysiology.

Optogenetics, utilising light-reactive proteins to manipulate tissue activity, are a relatively novel approach in the field of cardiac electrophysiology. We here provide an overview of light-activated transmembrane channels (optogenetic actuators) currently applied in strategies to modulate cardiac activity, as well as newly developed variants yet to be implemented in the heart. In addition, we touch upon genetically encoded indicators (optogenetic sensors) and fluorescent dyes to monitor tissue activity, including cardiac transmembrane potential and ion homeostasis. The combination of the two allows for all-optical approaches to monitor and manipulate the heart without any physical contact. However, spectral congestion poses a major obstacle, arising due to the overlap of excitation/activation and emission spectra of various optogenetic proteins and/or fluorescent dyes, resulting in optical crosstalk. Therefore, optogenetic proteins and fluorescent dyes should be carefully selected to avoid optical crosstalk and consequent disruptions in readouts and/or cellular activity. We here present a novel approach to simultaneously monitor transmembrane potential and cytosolic calcium, while also performing optogenetic manipulation. For this, we used the novel voltage-sensitive dye ElectroFluor 730p and the cytosolic calcium indicator X-Rhod-1 in mouse hearts expressing channelrhodopsin-2 (ChR2). By exploiting the isosbestic point of ElectroFluor 730p and avoiding the ChR2 activation spectrum, we here introduce a novel optical imaging and manipulation approach with minimal crosstalk. Future developments in both optogenetic proteins and fluorescent dyes will allow for additional and more optimised strategies, promising a bright future for all-optical approaches in the field of cardiac electrophysiology.

Optogenetic manipulation of cardiac repolarization gradients using sub-threshold illumination.

Introduction: Mechanisms underlying cardiac arrhythmias are typically driven by abnormalities in cardiac conduction and/or heterogeneities in repolarization time (RT) across the heart. While conduction slowing can be caused by either electrophysiological defects or physical blockade in cardiac tissue, RT heterogeneities are mainly related to action potential (AP) prolongation or abbreviation in specific areas of the heart. Importantly, the size of the area with altered RT and the difference between the short RT and long RT (RT gradient) have been identified as critical determinators of arrhythmogenicity. However, current experimental methods for manipulating RT gradient rely on the use of ion channel inhibitors, which lack spatial and temporal specificity and are commonly only partially reversible. Therefore, the conditions facilitating sustained arrhythmia upon the presence of RT heterogeneities and/or defects in cardiac conduction remain to be elucidated. Methods: We here employ an approach based on optogenetic stimulation in a low-intensity fashion (sub-threshold illumination), to selectively manipulate cardiac electrical activity in defined areas of the heart. Results: As previously described, subthreshold illumination is a robust tool able to prolong action potentials (AP), decrease upstroke velocity as well as slow cardiac conduction, in a fully reversible manner. By applying a patterned sub-threshold illumination in intact mouse hearts constitutively expressing the light-gated ion channel channelrhodopsin-2 (ChR2), we optically manipulate RT gradients and cardiac conduction across the heart in a spatially selective manner. Moreover, in a proof-of-concept assessment we found that in the presence of patterned sub-threshold illumination, mouse hearts were more susceptible to arrhythmias. Hence, this optogenetic-based approach may be able to mimic conduction slowing and RT heterogeneities present in pathophysiological conditions.

Microtubule plus-end tracking proteins: novel modulators of cardiac sodium channels and arrhythmogenesis.

The cardiac sodium channel NaV1.5 is an essential modulator of cardiac excitability, with decreased NaV1.5 levels at the plasma membrane and consequent reduction in sodium current (INa) leading to potentially lethal cardiac arrhythmias. NaV1.5 is distributed in a specific pattern at the plasma membrane of cardiomyocytes, with localization at the crests, grooves, and T-tubules of the lateral membrane and particularly high levels at the intercalated disc region. NaV1.5 forms a large macromolecular complex with and is regulated by interacting proteins, some of which are specifically localized at either the lateral membrane or intercalated disc. One of the NaV1.5 trafficking routes is via microtubules (MTs), which are regulated by MT plus-end tracking proteins (+TIPs). In our search for mechanisms involved in targeted delivery of NaV1.5, we here provide an overview of previously demonstrated interactions between NaV1.5 interacting proteins and +TIPs, which potentially (in)directly impact on NaV1.5 trafficking. Strikingly, +TIPs interact extensively with several intercalated disc- and lateral membrane-specific NaV1.5 interacting proteins. Recent work indicates that this interplay of +TIPs and NaV1.5 interacting proteins mediates the targeted delivery of NaV1.5 at specific cardiomyocyte subcellular domains, while also being potentially relevant for the trafficking of other ion channels. These observations are especially relevant for diseases associated with loss of NaV1.5 specifically at the lateral membrane (such as Duchenne muscular dystrophy), or at the intercalated disc (for example, arrhythmogenic cardiomyopathy), and open up potential avenues for development of new anti-arrhythmic therapies.

Desmosomal protein degradation as an underlying cause of arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is an inherited progressive cardiac disease. Many patients with ACM harbor mutations in desmosomal genes, predominantly in plakophilin-2 (PKP2). Although the genetic basis of ACM is well characterized, the underlying disease-driving mechanisms remain unresolved. Explanted hearts from patients with ACM had less PKP2 compared with healthy hearts, which correlated with reduced expression of desmosomal and adherens junction (AJ) proteins. These proteins were also disorganized in areas of fibrotic remodeling. In vitro data from human-induced pluripotent stem cell-derived cardiomyocytes and microtissues carrying the heterozygous PKP2 c.2013delC pathogenic mutation also displayed impaired contractility. Knockin mice carrying the equivalent heterozygous Pkp2 c.1755delA mutation recapitulated changes in desmosomal and AJ proteins and displayed cardiac dysfunction and fibrosis with age. Global proteomics analysis of 4-month-old heterozygous Pkp2 c.1755delA hearts indicated involvement of the ubiquitin-proteasome system (UPS) in ACM pathogenesis. Inhibition of the UPS in mutant mice increased area composita proteins and improved calcium dynamics in isolated cardiomyocytes. Additional proteomics analyses identified lysine ubiquitination sites on the desmosomal proteins, which were more ubiquitinated in mutant mice. In summary, we show that a plakophilin-2 mutation can lead to decreased desmosomal and AJ protein expression through a UPS-dependent mechanism, which preceded cardiac remodeling. These findings suggest that targeting protein degradation and improving desmosomal protein stability may be a potential therapeutic strategy for the treatment of ACM.

Differential Sodium Current Remodelling Identifies Distinct Cellular Proarrhythmic Mechanisms in Paroxysmal vs Persistent Atrial Fibrillation.

BACKGROUND: The cellular mechanisms underlying progression from paroxysmal to persistent atrial fibrillation (AF) are not fully understood, but alterations in (late) sodium current (INa) have been proposed. Human studies investigating electrophysiological changes at the paroxysmal stage of AF are sparse, with the majority employing right atrial appendage cardiomyocytes (CMs). We here investigated action potential (AP) characteristics and (late) INa remodelling in left atrial appendage CMs (LAA-CMs) from patients with paroxysmal and persistent AF and patients in sinus rhythm (SR), as well as the potential contribution of the "neuronal" sodium channel SCN10A/NaV1.8. METHODS: Peak INa, late INa and AP properties were investigated through patch-clamp analysis on single LAA-CMs, whereas quantitative polymerase chain reaction was used to assess SCN5A/SCN10A expression levels in LAA tissue. RESULTS: In paroxysmal and persistent AF LAA-CMs, AP duration was shorter than in SR LAA-CMs. Compared with SR, peak INa and SCN5A expression were significantly decreased in paroxysmal AF, whereas they were restored to SR levels in persistent AF. Conversely, although late INa was unchanged in paroxysmal AF compared with SR, it was significantly increased in persistent AF. Peak or late Nav1.8-based INa was not detected in persistent AF LAA-CMs. Similarly, expression of SCN10A was not observed in LAAs at any stage. CONCLUSIONS: Our findings demonstrate differences in (late) INa remodeling in LAA-CMs from patients with paroxysmal vs persistent AF, indicating distinct cellular proarrhythmic mechanisms in different AF forms. These observations are of particular relevance when considering potential pharmacologic approaches targeting (late) INa in AF.

Subcellular diversity of Nav1.5 in cardiomyocytes: distinct functions, mechanisms and targets.

In cardiomyocytes, the rapid depolarisation of the membrane potential is mediated by the α-subunit of the cardiac voltage-gated Na+ channel (NaV 1.5), encoded by the gene SCN5A. This ion channel allows positively charged Na+ ions to enter the cardiomyocyte, resulting in the fast upstroke of the action potential and is therefore crucial for cardiac excitability and electrical propagation. This essential role is underscored by the fact that dysfunctional NaV 1.5 is associated with high risk for arrhythmias and sudden cardiac death. However, development of therapeutic interventions regulating NaV 1.5 has been limited due to the complexity of NaV 1.5 structure and function and its diverse roles within the cardiomyocyte. In particular, research from the last decade has provided us with increased knowledge on the subcellular distribution of NaV 1.5 as well as the proteins which it interacts with in distinct cardiomyocyte microdomains. We here review these insights, detailing the potential role of NaV 1.5 within subcellular domains as well as its dysfunction in the setting of arrhythmia disorders. We furthermore provide an overview of current knowledge on the pathways involved in (microdomain-specific) trafficking of NaV 1.5, and their potential as novel targets. Unravelling the complexity of NaV 1.5 (dys)function may ultimately facilitate the development of therapeutic strategies aimed at preventing lethal arrhythmias. This is not only of importance for pathophysiological conditions where sodium current is specifically decreased within certain subcellular regions, such as in arrhythmogenic cardiomyopathy and Duchenne muscular dystrophy, but also for other acquired and inherited disorders associated with NaV 1.5.

Chronically elevated branched chain amino acid levels are pro-arrhythmic.

AIMS: Cardiac arrhythmias comprise a major health and economic burden and are associated with significant morbidity and mortality, including cardiac failure, stroke, and sudden cardiac death (SCD). Development of efficient preventive and therapeutic strategies is hampered by incomplete knowledge of disease mechanisms and pathways. Our aim is to identify novel mechanisms underlying cardiac arrhythmia and SCD using an unbiased approach. METHODS AND RESULTS: We employed a phenotype-driven N-ethyl-N-nitrosourea mutagenesis screen and identified a mouse line with a high incidence of sudden death at young age (6-9 weeks) in the absence of prior symptoms. Affected mice were found to be homozygous for the nonsense mutation Bcat2p.Q300*/p.Q300* in the Bcat2 gene encoding branched chain amino acid transaminase 2. At the age of 4-5 weeks, Bcat2p.Q300*/p.Q300* mice displayed drastic increase of plasma levels of branch chain amino acids (BCAAs-leucine, isoleucine, valine) due to the incomplete catabolism of BCAAs, in addition to inducible arrhythmias ex vivo as well as cardiac conduction and repolarization disturbances. In line with these findings, plasma BCAA levels were positively correlated to electrocardiogram indices of conduction and repolarization in the German community-based KORA F4 Study. Isolated cardiomyocytes from Bcat2p.Q300*/p.Q300* mice revealed action potential (AP) prolongation, pro-arrhythmic events (early and late afterdepolarizations, triggered APs), and dysregulated calcium homeostasis. Incubation of human pluripotent stem cell-derived cardiomyocytes with elevated concentration of BCAAs induced similar calcium dysregulation and pro-arrhythmic events which were prevented by rapamycin, demonstrating the crucial involvement of mTOR pathway activation. CONCLUSIONS: Our findings identify for the first time a causative link between elevated BCAAs and arrhythmia, which has implications for arrhythmogenesis in conditions associated with BCAA metabolism dysregulation such as diabetes, metabolic syndrome, and heart failure.

Targeting the Microtubule EB1-CLASP2 Complex Modulates NaV1.5 at Intercalated Discs.

[Figure: see text].

Low human dystrophin levels prevent cardiac electrophysiological and structural remodelling in a Duchenne mouse model.

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder caused by loss of dystrophin. This lack also affects cardiac structure and function, and cardiovascular complications are a major cause of death in DMD. Newly developed therapies partially restore dystrophin expression. It is unclear whether this will be sufficient to prevent or ameliorate cardiac involvement in DMD. We here establish the cardiac electrophysiological and structural phenotype in young (2-3 months) and aged (6-13 months) dystrophin-deficient mdx mice expressing 100% human dystrophin (hDMD), 0% human dystrophin (hDMDdel52-null) or low levels (~ 5%) of human dystrophin (hDMDdel52-low). Compared to hDMD, young and aged hDMDdel52-null mice displayed conduction slowing and repolarisation abnormalities, while only aged hDMDdel52-null mice displayed increased myocardial fibrosis. Moreover, ventricular cardiomyocytes from young hDMDdel52-null animals displayed decreased sodium current and action potential (AP) upstroke velocity, and prolonged AP duration at 20% and 50% of repolarisation. Hence, cardiac electrical remodelling in hDMDdel52-null mice preceded development of structural alterations. In contrast to hDMDdel52-null, hDMDdel52-low mice showed similar electrophysiological and structural characteristics as hDMD, indicating prevention of the cardiac DMD phenotype by low levels of human dystrophin. Our findings are potentially relevant for the development of therapeutic strategies aimed at restoring dystrophin expression in DMD.

Patch-Clamp Recordings of Action Potentials From Human Atrial Myocytes: Optimization Through Dynamic Clamp.

Introduction: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Consequently, novel therapies are being developed. Ultimately, the impact of compounds on the action potential (AP) needs to be tested in freshly isolated human atrial myocytes. However, the frequent depolarized state of these cells upon isolation seriously hampers reliable AP recordings. Purpose: We assessed whether AP recordings from single human atrial myocytes could be improved by providing these cells with a proper inward rectifier K+ current (IK1), and consequently with a regular, non-depolarized resting membrane potential (RMP), through "dynamic clamp". Methods: Single myocytes were enzymatically isolated from left atrial appendage tissue obtained from patients with paroxysmal AF undergoing minimally invasive surgical ablation. APs were elicited at 1 Hz and measured using perforated patch-clamp methodology, injecting a synthetic IK1 to generate a regular RMP. The injected IK1 had strong or moderate rectification. For comparison, a regular RMP was forced through injection of a constant outward current. A wide variety of ion channel blockers was tested to assess their modulatory effects on AP characteristics. Results: Without any current injection, RMPs ranged from -9.6 to -86.2 mV in 58 cells. In depolarized cells (RMP positive to -60 mV), RMP could be set at -80 mV using IK1 or constant current injection and APs could be evoked upon stimulation. AP duration differed significantly between current injection methods (p < 0.05) and was shortest with constant current injection and longest with injection of IK1 with strong rectification. With moderate rectification, AP duration at 90% repolarization (APD90) was similar to myocytes with regular non-depolarized RMP, suggesting that a synthetic IK1 with moderate rectification is the most appropriate for human atrial myocytes. Importantly, APs evoked using each injection method were still sensitive to all drugs tested (lidocaine, nifedipine, E-4031, low dose 4-aminopyridine, barium, and apamin), suggesting that the major ionic currents of the atrial cells remained functional. However, certain drug effects were quantitatively dependent on the current injection approach used. Conclusion: Injection of a synthetic IK1 with moderate rectification facilitates detailed AP measurements in human atrial myocytes. Therefore, dynamic clamp represents a promising tool for testing novel antiarrhythmic drugs.

Oxidation of Protein Kinase A Regulatory Subunit PKARIα Protects Against Myocardial Ischemia-Reperfusion Injury by Inhibiting Lysosomal-Triggered Calcium Release.

BACKGROUND: Kinase oxidation is a critical signaling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, PKARIα (type-1 protein kinase A) can be reversibly oxidized, forming interprotein disulfide bonds in the holoenzyme complex. However, the effect of PKARIα disulfide formation on downstream signaling in the heart, particularly under states of oxidative stress such as ischemia and reperfusion (I/R), remains unexplored. METHODS: Atrial tissue obtained from patients before and after cardiopulmonary bypass and reperfusion and left ventricular (LV) tissue from mice subjected to I/R or sham surgery were used to assess PKARIα disulfide formation by immunoblot. To determine the effect of disulfide formation on PKARIα catalytic activity and subcellular localization, live-cell fluorescence imaging and stimulated emission depletion super-resolution microscopy were performed in prkar1 knock-out mouse embryonic fibroblasts, neonatal myocytes, or adult LV myocytes isolated from "redox dead" (Cys17Ser) PKARIα knock-in mice and their wild-type littermates. Comparison of intracellular calcium dynamics between genotypes was assessed in fura2-loaded LV myocytes, whereas I/R-injury was assessed ex vivo. RESULTS: In both humans and mice, myocardial PKARIα disulfide formation was found to be significantly increased (2-fold in humans, P=0.023; 2.4-fold in mice, P<0.001) in response to I/R in vivo. In mouse LV cardiomyocytes, disulfide-containing PKARIα was not found to impact catalytic activity, but instead led to enhanced AKAP (A-kinase anchoring protein) binding with preferential localization of the holoenzyme to the lysosome. Redox-dependent regulation of lysosomal two-pore channels by PKARIα was sufficient to prevent global calcium release from the sarcoplasmic reticulum in LV myocytes, without affecting intrinsic ryanodine receptor leak or phosphorylation. Absence of I/R-induced PKARIα disulfide formation in "redox dead" knock-in mouse hearts resulted in larger infarcts (2-fold, P<0.001) and a concomitant reduction in LV contractile recovery (1.6-fold, P<0.001), which was prevented by administering the lysosomal two-pore channel inhibitor Ned-19 at the time of reperfusion. CONCLUSIONS: Disulfide modification targets PKARIα to the lysosome, where it acts as a gatekeeper for two-pore channel-mediated triggering of global calcium release. In the postischemic heart, this regulatory mechanism is critical for protection from extensive injury and offers a novel target for the design of cardioprotective therapeutics.

Functional modulation of atrio-ventricular conduction by enhanced late sodium current and calcium-dependent mechanisms in Scn5a1798insD/+ mice.

AIMS: SCN5A mutations are associated with arrhythmia syndromes, including Brugada syndrome, long QT syndrome type 3 (LQT3), and cardiac conduction disease. Long QT syndrome type 3 patients display atrio-ventricular (AV) conduction slowing which may contribute to arrhythmogenesis. We here investigated the as yet unknown underlying mechanisms. METHODS AND RESULTS: We assessed electrophysiological and molecular alterations underlying AV-conduction abnormalities in mice carrying the Scn5a1798insD/+ mutation. Langendorff-perfused Scn5a1798insD/+ hearts showed prolonged AV-conduction compared to wild type (WT) without changes in atrial and His-ventricular (HV) conduction. The late sodium current (INa,L) inhibitor ranolazine (RAN) normalized AV-conduction in Scn5a1798insD/+ mice, likely by preventing the mutation-induced increase in intracellular sodium ([Na+]i) and calcium ([Ca2+]i) concentrations. Indeed, further enhancement of [Na+]i and [Ca2+]i by the Na+/K+-ATPase inhibitor ouabain caused excessive increase in AV-conduction time in Scn5a1798insD/+ hearts. Scn5a1798insD/+ mice from the 129P2 strain displayed more severe AV-conduction abnormalities than FVB/N-Scn5a1798insD/+ mice, in line with their larger mutation-induced INa,L. Transverse aortic constriction (TAC) caused excessive prolongation of AV-conduction in FVB/N-Scn5a1798insD/+ mice (while HV-intervals remained unchanged), which was prevented by chronic RAN treatment. Scn5a1798insD/+-TAC hearts showed decreased mRNA levels of conduction genes in the AV-nodal region, but no structural changes in the AV-node or His bundle. In Scn5a1798insD/+-TAC mice deficient for the transcription factor Nfatc2 (effector of the calcium-calcineurin pathway), AV-conduction and conduction gene expression were restored to WT levels. CONCLUSIONS: Our findings indicate a detrimental role for enhanced INa,L and consequent calcium dysregulation on AV-conduction in Scn5a1798insD/+ mice, providing evidence for a functional mechanism underlying AV-conduction disturbances secondary to gain-of-function SCN5A mutations.

The sodium channel NaV 1.5 impacts on early murine embryonic cardiac development, structure and function in a non-electrogenic manner.

AIM: The voltage-gated sodium channel NaV 1.5, encoded by SCN5A, is essential for cardiac excitability and ensures proper electrical conduction. Early embryonic death has been observed in several murine models carrying homozygous Scn5amutations. We investigated when sodium current (INa ) becomes functionally relevant in the murine embryonic heart and how Scn5a/NaV 1.5 dysfunction impacts on cardiac development. METHODS: Involvement of NaV 1.5-generated INa in murine cardiac electrical function was assessed by optical mapping in wild type (WT) embryos (embryonic day (E)9.5 and E10.5) in the absence and presence of the sodium channel blocker tetrodotoxin (30 µmol/L). INa was assessed by patch-clamp analysis in cardiomyocytes isolated from WT embryos (E9.5-17.5). In addition, cardiac morphology and electrical function was assessed in Scn5a-1798insD-/- embryos (E9.5-10.5) and their WT littermates. RESULTS: In WT embryos, tetrodotoxin did not affect cardiac activation at E9.5, but slowed activation at E10.5. Accordingly, patch-clamp measurements revealed that INa was virtually absent at E9.5 but robustly present at E10.5. Scn5a-1798insD-/- embryos died in utero around E10.5, displaying severely affected cardiac activation and morphology. Strikingly, altered ventricular activation was observed in Scn5a-1798insD-/- E9.5 embryos before the onset of INa , in addition to reduced cardiac tissue volume compared to WT littermates. CONCLUSION: We here demonstrate that NaV 1.5 is involved in cardiac electrical function from E10.5 onwards. Scn5a-1798insD-/- embryos displayed cardiac structural abnormalities at E9.5, indicating that NaV 1.5 dysfunction impacts on embryonic cardiac development in a non-electrogenic manner. These findings are potentially relevant for understanding structural defects observed in relation to NaV 1.5 dysfunction.

Functional Consequences of the SCN5A-p.Y1977N Mutation within the PY Ubiquitylation Motif: Discrepancy between HEK293 Cells and Transgenic Mice.

Dysfunction of the cardiac sodium channel Nav1.5 (encoded by the SCN5A gene) is associated with arrhythmias and sudden cardiac death. SCN5A mutations associated with long QT syndrome type 3 (LQT3) lead to enhanced late sodium current and consequent action potential (AP) prolongation. Internalization and degradation of Nav1.5 is regulated by ubiquitylation, a post-translational mechanism that involves binding of the ubiquitin ligase Nedd4-2 to a proline-proline-serine-tyrosine sequence of Nav1.5, designated the PY-motif. We investigated the biophysical properties of the LQT3-associated SCN5A-p.Y1977N mutation located in the Nav1.5 PY-motif, both in HEK293 cells as well as in newly generated mice harboring the mouse homolog mutation Scn5a-p.Y1981N. We found that in HEK293 cells, the SCN5A-p.Y1977N mutation abolished the interaction between Nav1.5 and Nedd4-2, suppressed PY-motif-dependent ubiquitylation of Nav1.5, and consequently abrogated Nedd4-2 induced sodium current (INa) decrease. Nevertheless, homozygous mice harboring the Scn5a-p.Y1981N mutation showed no electrophysiological alterations nor changes in AP or (late) INa properties, questioning the in vivo relevance of the PY-motif. Our findings suggest the presence of compensatory mechanisms, with additional, as yet unknown, factors likely required to reduce the "ubiquitylation reserve" of Nav1.5. Future identification of such modulatory factors may identify potential triggers for arrhythmias and sudden cardiac death in the setting of LQT3 mutations.

Absence of Functional Nav1.8 Channels in Non-diseased Atrial and Ventricular Cardiomyocytes.

PURPOSE: Several studies have indicated a potential role for SCN10A/NaV1.8 in modulating cardiac electrophysiology and arrhythmia susceptibility. However, by which mechanism SCN10A/NaV1.8 impacts on cardiac electrical function is still a matter of debate. To address this, we here investigated the functional relevance of NaV1.8 in atrial and ventricular cardiomyocytes (CMs), focusing on the contribution of NaV1.8 to the peak and late sodium current (INa) under normal conditions in different species. METHODS: The effects of the NaV1.8 blocker A-803467 were investigated through patch-clamp analysis in freshly isolated rabbit left ventricular CMs, human left atrial CMs and human-induced pluripotent stem cell-derived CMs (hiPSC-CMs). RESULTS: A-803467 treatment caused a slight shortening of the action potential duration (APD) in rabbit CMs and hiPSC-CMs, while it had no effect on APD in human atrial cells. Resting membrane potential, action potential (AP) amplitude, and AP upstroke velocity were unaffected by A-803467 application. Similarly, INa density was unchanged after exposure to A-803467 and NaV1.8-based late INa was undetectable in all cell types analysed. Finally, low to absent expression levels of SCN10A were observed in human atrial tissue, rabbit ventricular tissue and hiPSC-CMs. CONCLUSION: We here demonstrate the absence of functional NaV1.8 channels in non-diseased atrial and ventricular CMs. Hence, the association of SCN10A variants with cardiac electrophysiology observed in, e.g. genome wide association studies, is likely the result of indirect effects on SCN5A expression and/or NaV1.8 activity in cell types other than CMs.

A common co-morbidity modulates disease expression and treatment efficacy in inherited cardiac sodium channelopathy.

Aims: Management of patients with inherited cardiac ion channelopathy is hindered by variability in disease severity and sudden cardiac death (SCD) risk. Here, we investigated the modulatory role of hypertrophy on arrhythmia and SCD risk in sodium channelopathy. Methods and results: Follow-up data was collected from 164 individuals positive for the SCN5A-1795insD founder mutation and 247 mutation-negative relatives. A total of 38 (obligate) mutation-positive patients died suddenly or suffered life-threatening ventricular arrhythmia. Of these, 18 were aged >40 years, a high proportion of which had a clinical diagnosis of hypertension and/or cardiac hypertrophy. While pacemaker implantation was highly protective in preventing bradycardia-related SCD in young mutation-positive patients, seven of them aged >40 experienced life-threatening arrhythmic events despite pacemaker treatment. Of these, six had a diagnosis of hypertension/hypertrophy, pointing to a modulatory role of this co-morbidity. Induction of hypertrophy in adult mice carrying the homologous mutation (Scn5a1798insD/+) caused SCD and excessive conduction disturbances, confirming a modulatory effect of hypertrophy in the setting of the mutation. The deleterious effects of the interaction between hypertrophy and the mutation were prevented by genetically impairing the pro-hypertrophic response and by pharmacological inhibition of the enhanced late sodium current associated with the mutation. Conclusion: This study provides the first evidence for a modulatory effect of co-existing cardiac hypertrophy on arrhythmia risk and treatment efficacy in inherited sodium channelopathy. Our findings emphasize the need for continued assessment and rigorous treatment of this co-morbidity in SCN5A mutation-positive individuals.