Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Stichting LSH-TKI Introduction In cardiac gene therapy, two prototypic viral vectors are typically considered for long-term transgene expression, i.e. lentivirus (LV) and adeno-associated virus (AAV) vectors. Compared to AAV vectors, LV vectors have the advantages of a larger insert capacity and the ability to confer permanent transgene expression due to integration of the vector genome in the host cell’s chromosomal DNA . However, standard VSV G protein-pseudotyped LV vectors (VSV G-LV vectors) has shown suboptimal transduction of cardiac myocytes in vivo. Purpose In this study, we aimed to improve LV-mediated transduction of cardiomyocytes in vivo by evaluating LV vectors carrying various different envelopes and compared the most potent variants with AAV1 capsid-pseudotyped AAV2 vectors (AAV2/1 vectors). Methods We made a panel of LV vectors pseudotyped with envelope proteins from 76 different viruses carrying an expression cassette for enhanced green fluorescent protein (eGFP). Transduction efficiencies in neonatal rat ventricular myocytes (NRVMs), human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and cardiomyocyte progenitor cells (CMPCs) were determined by fluorescence-activated cell sorting (FACS) as percentage eGFP-positive cells. Male NOD -SCID mice aged 3-6 months were injected directly into the left ventricular myocardium with eGFP-encoding VSV G-LV and AAV2/1 vectors at doses of 1.4 x 10^7 infectious units and 2.0 x 10^10 genome copies, respectively, or PBS as a control. Mice were sacrificed one and four weeks after surgery. Per heart, left ventricular and eGFP-positive area was determined from 40 – 50 slides and the ratio was used as a measure of transduction efficiency. Results Of the panel of pseudotyped LVs none was able to consistently outperform VSV G-LV vectors, which reached transduction efficiencies in vitro of 58% in NRVMs to 96% in hiPSC-CMs and 100% in CMPCs . In AAV2/1 vector-injected hearts, the eGFP expressing area in the left ventricle increased from 1.07% in week 1 to 5.42% in week 4. In contrast, eGFP expression in LV-injected hearts was 0.31% and 0.16% after 1 week and 4 weeks, respectively (figure 1). Conclusion In vitro screening of a panel of pseudotyped LV vectors showed standard VSV G-LV vectors as top performer. Despite encouraging in vitro results and previously reported findings, VSV G-LV vectors was unable to provide for robust in vivo transduction. The observation that none of the 76 different envelopes yielded a meaningful improvement of lentiviral transduction rates together with the discrepancy between in vitro and in vivo transduction efficiencies, suggest that a post-entry block of transduction likely represents an important component of the poor in vivo performance.Figure 1in vivo transduction

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia, progressive in nature, and known to have a negative impact on mortality, morbidity, and quality of life. Patients requiring acute termination of AF to restore sinus rhythm are subjected to electrical cardioversion, which requires sedation and therefore hospitalization due to pain resulting from the electrical shocks. However, considering the progressive nature of AF and its detrimental effects, there is a clear need for acute out-of-hospital (i.e., ambulatory) cardioversion of AF. In the search for shock-free cardioversion methods to realize such ambulatory therapy, a method referred to as optogenetics has been put forward. Optogenetics enables optical control over the electrical activity of cardiomyocytes by targeted expression of light-activated ion channels or pumps and may therefore serve as a means for cardioversion. First proof-of-principle for such light-induced cardioversion came from in vitro studies, proving optogenetic AF termination to be very effective. Later, these results were confirmed in various rodent models of AF using different transgenes, illumination methods, and protocols, whereas computational studies in the human heart provided additional translational insight. Based on these results and fueled by recent advances in molecular biology, gene therapy, and optoelectronic engineering, a basis is now being formed to explore clinical translations of optoelectronic control of cardiac rhythm. In this review, we discuss the current literature regarding optogenetic cardioversion of AF to restore normal rhythm in a shock-free manner. Moreover, key translational steps will be discussed, both from a biological and technological point of view, to outline a path toward realizing acute shock-free ambulatory termination of AF.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia, progressive in nature, and known to have a negative impact on mortality, morbidity, and quality of life. Patients requiring acute termination of AF to restore sinus rhythm are subjected to electrical cardioversion, which requires sedation and therefore hospitalization due to pain resulting from the electrical shocks. However, considering the progressive nature of AF and its detrimental effects, there is a clear need for acute out-of-hospital (i.e., ambulatory) cardioversion of AF. In the search for shock-free cardioversion methods to realize such ambulatory therapy, a method referred to as optogenetics has been put forward. Optogenetics enables optical control over the electrical activity of cardiomyocytes by targeted expression of light-activated ion channels or pumps and may therefore serve as a means for cardioversion. First proof-of-principle for such light-induced cardioversion came from in vitro studies, proving optogenetic AF termination to be very effective. Later, these results were confirmed in various rodent models of AF using different transgenes, illumination methods, and protocols, whereas computational studies in the human heart provided additional translational insight. Based on these results and fueled by recent advances in molecular biology, gene therapy, and optoelectronic engineering, a basis is now being formed to explore clinical translations of optoelectronic control of cardiac rhythm. In this review, we discuss the current literature regarding optogenetic cardioversion of AF to restore normal rhythm in a shock-free manner. Moreover, key translational steps will be discussed, both from a biological and technological point of view, to outline a path toward realizing acute shock-free ambulatory termination of AF.

The heart produces bioelectrical signals, which can be measured as an electrocardiogram (ECG) for the detection of rhythm disturbances. Rapid and precise detection of these arrhythmias is crucial for their termination by closed‐looped therapeutic interventions to counteract detrimental effects. However, there is a current lack of such systems tailored for experimental cardiovascular applications. This hampers not only in‐depth mechanistic studies but also translational testing of new therapeutic strategies, especially in an untethered manner in awake animal models. To break new ground, recent advances to develop a non‐invasive AI‐supported heart rhythm monitoring system for untethered automated arrhythmia detection in a continuous manner is combined. This system is housed in a lightweight jacket for mobile use and includes an on‐skin ECG sensor, a low‐power microprocessor unit, a massive data storage unit, and a power‐management system. By implementing a novel hybrid algorithm based on so‐called heart rate (R‐R) variability and a case‐specific AI model, 100% sensitivity and 95% specificity is achieved in detecting atrial arrhythmias within 2 s upon initiation in adult rats. Thereby, the novel system sets the stage for advanced mechanistic studies and therapeutic testing, including closed‐loop applications aiming for the termination of a broad range of atrial arrhythmias.

BackgroundOptogenetics could offer a solution to the current lack of an ambulatory method for the rapid automated cardioversion of atrial fibrillation (AF), but key translational aspects remain to be studied. ObjectiveTo investigate whether optogenetic cardioversion of AF is effective in the aged heart and whether sufficient light penetrates the human atrial wall. MethodsAtria of adult and aged rats were optogenetically modified to express light-gated ion channels (i.e., red-activatable channelrhodopsin), followed by AF induction and atrial illumination to determine the effectivity of optogenetic cardioversion. The irradiance level was determined by light transmittance measurements on human atrial tissue. ResultsAF could be effectively terminated in the remodeled atria of aged rats (97%, n = 6). Subsequently, ex vivo experiments using human atrial auricles demonstrated that 565-nm light pulses at an intensity of 25 mW/mm(2) achieved the complete penetration of the atrial wall. Applying such irradiation onto the chest of adult rats resulted in transthoracic atrial illumination as evidenced by the optogenetic cardioversion of AF (90%, n = 4). ConclusionTransthoracic optogenetic cardioversion of AF is effective in the aged rat heart using irradiation levels compatible with human atrial transmural light penetration.

Abstract Background To monitor and record the occurrence and duration of cardiac arrhythmias in freely moving animals, ECG telemetry devices are often used. However, traditional telemetry devices require complex equipment unsuitable for on-site application in most animal housing facilities. Additionally, current atrial arrhythmias detection algorithms are mainly based on ECG R-R interval variability for atrial fibrillation (AF) detection. Such algorithms do not allow the detection of stable R-R interval atrial arrhythmias such as flutter. Thus there is currently a lack of non-invasive methods allowing automated detection of all atrial arrhythmias subtypes, thereby precluding in-depth research into general and subtype-specific mechanisms and treatment. Purpose Here we aim to create a non-invasive system allowing fast and accurate on-site detection and characterization of all atrial arrhythmias in rats. Methods Our atrial arrhythmia detector system was made of an on-skin ECG sensor, a low-power microprocessor unit, a large data storage unit and a battery. All components were assembled within a lightweight rat jacket. The filtered ECG signal was sent to the microprocessor allowing algorithm-based, real-time detection of atrial arrhythmias. The device allowed storage of ECG traces as well as arrhythmia characteristics (including type of arrhythmia and its duration). Atrial arrhythmias were induced by transesophageal atrial burst pacing in Wistar rats. These arrhythmia recordings were used to develop an AI detection method. Next, real-time detection algorithms based on R-R variability and AI were both integrated on-chip and compared using the same ECG data. Results ECG traces measured with our new system were similar to classical ECG measurements. Stored ECG traces provided the raw data for the development of an AI-based detection algorithm. The AI training was done using ECG data recorded from five different rats and reached 99.5% self-testing accuracy to detect all sub-types of atrial arrhythmias based on pre-categorized data. Using uncategorized data (n=25 from 5 rats) different from the training dataset, the AI algorithm could detect >80% of all types of atrial arrhythmias within 3s after initiation, while the rest could be detected within 10s. Though R-R interval based algorithm showed a high detection rate of 88% of AF within 10s, this method did not allow the detection of flutter or AF presenting regular R-R intervals within 1 minute after initiation. Conclusion Our atrial arrhythmia detection and analysis system provides a novel method for non-invasive investigation into atrial arrhythmias in rats and could easily be scaled for other species. It allows on-site fast and accurate detection and characterization of atrial arrhythmia subtypes without external equipment, thereby opening new possibilities for mechanistic studies and therapeutic testing, including closed-loop applications aiming for arrhythmia termination.

Optogenetics could offer a solution to the current lack of an ambulatory method for the rapid automated cardioversion of atrial fibrillation (AF), but key translational aspects remain to be studied. To investigate whether optogenetic cardioversion of AF is effective in the aged heart and whether sufficient light penetrates the human atrial wall. Atria of adult and aged rats were optogenetically modified to express light-gated ion channels (i.e., red-activatable channelrhodopsin), followed by AF induction and atrial illumination to determine the effectivity of optogenetic cardioversion. The irradiance level was determined by light transmittance measurements on human atrial tissue. AF could be effectively terminated in the remodeled atria of aged rats (97%, n  = 6). Subsequently, ex vivo experiments using human atrial auricles demonstrated that 565-nm light pulses at an intensity of 25 mW/mm 2 achieved the complete penetration of the atrial wall. Applying such irradiation onto the chest of adult rats resulted in transthoracic atrial illumination as evidenced by the optogenetic cardioversion of AF (90%, n  = 4). Transthoracic optogenetic cardioversion of AF is effective in the aged rat heart using irradiation levels compatible with human atrial transmural light penetration.

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.

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.

AIMS:Ventricular tachyarrhythmias (VTs) are common in the pathologically remodelled heart. These arrhythmias can be lethal, necessitating acute treatment like electrical cardioversion to restore normal rhythm. Recently, it has been proposed that cardioversion may also be realized via optically controlled generation of bioelectricity by the arrhythmic heart itself through optogenetics and therefore without the need of traumatizing high-voltage shocks. However, crucial mechanistic and translational aspects of this strategy have remained largely unaddressed. Therefore, we investigated optogenetic termination of VTs (i) in the pathologically remodelled heart using an (ii) implantable multi-LED device for (iii) in vivo closed-chest, local illumination. METHODS AND RESULTS:In order to mimic a clinically relevant sequence of events, transverse aortic constriction (TAC) was applied to adult male Wistar rats before optogenetic modification. This modification took place 3 weeks later by intravenous delivery of adeno-associated virus vectors encoding red-activatable channelrhodopsin or Citrine for control experiments. At 8-10 weeks after TAC, VTs were induced ex vivo and in vivo, followed by programmed local illumination of the ventricular apex by a custom-made implanted multi-LED device. This resulted in effective and repetitive VT termination in the remodelled adult rat heart after optogenetic modification, leading to sustained restoration of sinus rhythm in the intact animal. Mechanistically, studies on the single cell and tissue level revealed collectively that, despite the cardiac remodelling, there were no significant differences in bioelectricity generation and subsequent transmembrane voltage responses between diseased and control animals, thereby providing insight into the observed robustness of optogenetic VT termination. CONCLUSION:Our results show that implant-based optical cardioversion of VTs is feasible in the pathologically remodelled heart in vivo after local optogenetic targeting because of preserved optical control over bioelectricity generation. These findings add novel mechanistic and translational insight into optical ventricular cardioversion.

Nadjet Belbachir, Vincent Portero, Zeina R Al Sayed, Jean-Baptiste Gourraud, Florian Dilasser, Laurence Jesel, Hongchao Guo, Haodi Wu, Nathalie Gaborit, Christophe Guilluy, Aurore Girardeau, Stephanie Bonnaud, Floriane Simonet, Matilde Karakachoff, Sabine Pattier, Carol Scott, Sophie Burel, Céline Marionneau, Caroline Chariau, Anne Gaignerie, Laurent David, Emmanuelle Genin, Jean-François Deleuze, Christian Dina, Vincent Sauzeau, Gervaise Loirand,

Patients with a deficiency in very long-chain acyl-CoA dehydrogenase (VLCAD), an enzyme that is involved in the mitochondrial beta-oxidation of long-chain fatty acids, are at risk for developing cardiac arrhythmias. In human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), VLCAD deficiency (VLCADD) results in a series of abnormalities, including: 1) accumulation of long-chain acylcarnitines, 2) action potential shortening, 3) higher systolic and diastolic intracellular Ca2+ concentrations, and 4) development of delayed afterdepolarizations. In the fatty acid oxidation process, carnitine is required for bidirectional transport of acyl groups across the mitochondrial membrane. Supplementation has been suggested as potential therapeutic approach in VLCADD, but its benefits are debated. Here, we studied the effects of carnitine supplementation on the long-chain acylcarnitine levels and performed electrophysiological analyses in VLCADD patient-derived hiPSC-CMs with a ACADVL gene mutation (p.Val283Ala/p.Glu381del). Under standard culture conditions, VLCADD hiPSC-CMs showed high concentrations of long-chain acylcarnitines, short action potentials, and high delayed afterdepolarizations occurrence. Incubation of the hiPSC-CMs with 400 µM L-carnitine for 48 h led to increased long-chain acylcarnitine levels both in medium and cells. In addition, carnitine supplementation neither restored abnormal action potential parameters nor the increased occurrence of delayed afterdepolarizations in VLCADD hiPSC-CMs. We conclude that long-chain acylcarnitine accumulation and electrophysiological abnormalities in VLCADD hiPSC-CMs are not normalized by carnitine supplementation, indicating that this treatment is unlikely to be beneficial against cardiac arrhythmias in VLCADD patients.

RATIONALE: Loss-of-function of the cardiac sodium channel Na(V)1.5 causes conduction slowing and arrhythmias. Na(V)1.5 is differentially distributed within subcellular domains of cardiomyocytes, with sodium current (I-Na) being enriched at the intercalated discs (ID). Various pathophysiological conditions associated with lethal arrhythmias display ID-specific I-Na reduction, but the mechanisms underlying microdomain-specific targeting of Na(V)1.5 remain largely unknown. OBJECTIVE: To investigate the role of the microtubule plus-end tracking proteins EB1 (end-binding protein 1) and CLASP2 (cytoplasmic linker associated protein 2) in mediating Na(V)1.5 trafficking and subcellular distribution in cardiomyocytes. METHODS AND RESULTS: EB1 overexpression in human-induced pluripotent stem cell-derived cardiomyocytes resulted in enhanced whole-cell I-Na, increased action potential upstroke velocity (V-max), and enhanced Na(V)1.5 localization at the plasma membrane as detected by multicolor stochastic optical reconstruction microscopy. Fluorescence recovery after photobleaching experiments in HEK293A cells demonstrated that EB1 overexpression promoted Na(V)1.5 forward trafficking. Knockout of MAPRE1 in human induced pluripotent stem cell-derived cardiomyocytes led to reduced whole-cell I-Na, decreased V-max, and action potential duration (APD) prolongation. Similarly, acute knockout of the MAPRE1 homolog in zebrafish (mapre1b) resulted in decreased ventricular conduction velocity and V-max as well as increased APD. Stochastic optical reconstruction microscopy imaging and macropatch I-Na measurements showed that subacute treatment (2-3 hours) with SB216763 (SB2), a GSK3 beta (glycogen synthase kinase 3 beta) inhibitor known to modulate CLASP2-EB1 interaction, reduced GSK3 beta localization and increased Na(V)1.5 and I-Na preferentially at the ID region of wild-type murine ventricular cardiomyocytes. By contrast, SB2 did not affect whole cell I-Na or Na(V)1.5 localization in cardiomyocytes from Clasp2-deficient mice, uncovering the crucial role of CLASP2 in SB2-mediated modulation of Na(V)1.5 at the ID. CONCLUSIONS: Our findings demonstrate the modulatory effect of the microtubule plus-end tracking protein EB1 on Na(V)1.5 trafficking and function, and identify the EB1-CLASP2 complex as a target for preferential modulation of I-Na within the ID region of cardiomyocytes.

Patients with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) can present with life-threatening cardiac arrhythmias. The pathophysiological mechanism is unknown. We reprogrammed fibroblasts from one mildly and one severely affected VLCADD patient, into human induced pluripotent stem cells (hiPSCs) and differentiated these into cardiomyocytes (VLCADD-CMs). VLCADD-CMs displayed shorter action potentials (APs), more delayed afterdepolarizations (DADs) and higher systolic and diastolic intracellular Ca2+ concentration ([Ca2+]i) than control CMs. The mitochondrial booster resveratrol mitigated the biochemical, electrophysiological and [Ca2+]i changes in the mild but not in the severe VLCADD-CMs. Accumulation of potentially toxic intermediates of fatty acid oxidation was blocked by substrate reduction with etomoxir. Incubation with etomoxir led to marked prolongation of AP duration and reduced DADs and [Ca2+]i in both VLCADD-CMs. These results provide compelling evidence that reduced accumulation of fatty acid oxidation intermediates, either by enhanced fatty acid oxidation flux through increased mitochondria biogenesis (resveratrol) or by inhibition of fatty acid transport into the mitochondria (etomoxir), rescues pro-arrhythmia defects in VLCADD-CMs and open doors for new treatments.

AIMS The Brugada syndrome (BrS) is an inherited cardiac disorder predisposing to ventricular arrhythmias. Despite considerable efforts, its genetic basis and cellular mechanisms remain largely unknown. The objective of this study was to identify a new susceptibility gene for BrS through familial investigation. METHODS AND RESULTS Whole-exome sequencing performed in a three-generation pedigree with five affected members allowed the identification of one rare non-synonymous substitution (p.R211H) in RRAD, the gene encoding the RAD GTPase, carried by all affected members of the family. Three additional rare missense variants were found in 3/186 unrelated index cases. We detected higher levels of RRAD transcripts in subepicardium than in subendocardium in human heart, and in the right ventricle outflow tract compared to the other cardiac compartments in mice. The p.R211H variant was then subjected to electrophysiological and structural investigations in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs). Cardiomyocytes derived from induced pluripotent stem cells from two affected family members exhibited reduced action potential upstroke velocity, prolonged action potentials and increased incidence of early afterdepolarizations, with decreased Na+ peak current amplitude and increased Na+ persistent current amplitude, as well as abnormal distribution of actin and less focal adhesions, compared with intra-familial control iPSC-CMs Insertion of p.R211H-RRAD variant in control iPSCs by genome editing confirmed these results. In addition, iPSC-CMs from affected patients exhibited a decreased L-type Ca2+ current amplitude. CONCLUSION This study identified a potential new BrS-susceptibility gene, RRAD. Cardiomyocytes derived from induced pluripotent stem cells expressing RRAD variant recapitulated single-cell electrophysiological features of BrS, including altered Na+ current, as well as cytoskeleton disturbances.

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.

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.

As part of a large-scale phenotype-driven screen we identified a line exhibiting sudden death. Mapping and whole genome sequencing identified a missense mutation in the Bcat2 gene, encoding mitochondrial branched chained aminotransferase, resulting in an early stop (Q300*) and a truncated protein. Homozygous mice exhibited increased plasma and urine levels of branched chain amino acids (BCAAs). Mutations in this pathway have previously been associated with Maple Syrup Urine Disease (MSUD) and can result in neurological symptoms. All homozygous mice died suddenly at 7 weeks of age, without any preceding symptoms. No cardiac abnormalities were observed on histological analysis, nor were there any other significant findings related to MSUD. An accumulation of branched chain amino acids was identified in urine and serum from homozygous mice, but unlike MSUD there was no accumulation of branched chain keto-acids. Homozygous mice showed QTc-prolongation in vivo on surface ECG analysis and prolonged action potential duration (APD) ex vivo (assessed by optical mapping in isolated hearts). Moreover, isolated hearts from mutant animals displayed increased inducibility of atrial and ventricular arrhythmias. In line with this, patch clamp measurements revealed significant APD90 prolongation and increased incidence of pro-arrhythmic events in isolated cardiomyocytes which was prevented by pharmacological inhibition of the late sodium current. Our current data suggests a direct effect of the mutation on cardiac function rather than the observed phenotypes resulting from an accumulation of BCAAs. Thus we have identified a novel model of sudden cardiac death resulting from abnormal BCAA metabolism.