Gerard A. Marchal

Cardiac fibrosis contributes to electrical conduction disturbances, yet its specific impact on conduction remains unclear, hindering predictive insight into cardiac electrophysiology and arrhythmogenesis. Arrhythmogenic cardiomyopathy is associated with fibrotic remodeling, and it accounts for most cases of stress-related arrhythmic sudden death. Here we develop a correlative imaging approach to integrate macroscale cardiac electrophysiology with three-dimensional microscale reconstructions of the ventricles. We apply this tool to a desmoglein-2 mutant mouse model to characterize the dynamics of conduction wavefronts and relate them to the underlying structural substrate. We observed that conduction through fibrotic tissue areas shows a frequency-dependent behavior, where conduction fails at high stimulation frequencies; this promotes reentrant arrhythmias, even in regions that were electrophysiologically inconspicuous at lower stimulation rates. We found that fibrotic areas undergo electrophysiological remodeling that acts as a low-pass filter for conduction, quantitatively explained by computational models informed by structural data. Collectively, our study provides a structure–function mapping pipeline and describes a pro-arrhythmogenic mechanism in arrhythmogenic cardiomyopathy.

Patients with mutations in SCN5A encoding Na_V1.5 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 Scn5a^1798insD/+ mouse model. 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 Scn5a^1798insD/+ (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. Genetic background and age both modulate disease severity in Scn5a^1798insD/+ 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.

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.

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.

The cardiac sodium channel Na_V1.5 is an essential modulator of cardiac excitability, with decreased Na_V1.5 levels at the plasma membrane and consequent reduction in sodium current (I_Na) leading to potentially lethal cardiac arrhythmias. Na_V1.5 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. Na_V1.5 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 Na_V1.5 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 Na_V1.5, we here provide an overview of previously demonstrated interactions between Na_V1.5 interacting proteins and +TIPs, which potentially (in)directly impact on Na_V1.5 trafficking. Strikingly, +TIPs interact extensively with several intercalated disc- and lateral membrane-specific Na_V1.5 interacting proteins. Recent work indicates that this interplay of +TIPs and Na_V1.5 interacting proteins mediates the targeted delivery of Na_V1.5 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 Na_V1.5 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.

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.

In cardiomyocytes, the rapid depolarisation of the membrane potential is mediated by the α‐subunit of the cardiac voltage‐gated Na⁺ channel (Na_V1.5), 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 Na_V1.5 is associated with high risk for arrhythmias and sudden cardiac death. However, development of therapeutic interventions regulating Na_V1.5 has been limited due to the complexity of Na_V1.5 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 Na_V1.5 as well as the proteins which it interacts with in distinct cardiomyocyte microdomains. We here review these insights, detailing the potential role of Na_V1.5 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 Na_V1.5, and their potential as novel targets. Unravelling the complexity of Na_V1.5 (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 Na_V1.5. image

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. 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 Bcat2^{p.Q300*/p.Q300*} in the Bcat2 gene encoding branched chain amino acid transaminase 2. At the age of 4–5 weeks, Bcat2^{p.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 Bcat2^{p.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. 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.

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 GSK3β (glycogen synthase kinase 3β) inhibitor known to modulate CLASP2-EB1 interaction, reduced GSK3β 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.

Aim: The voltage‐gated sodium channel Na_V1.5, 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 (I_Na) becomes functionally relevant in the murine embryonic heart and how Scn5a/Na_V1.5 dysfunction impacts on cardiac development.Methods: Involvement of Na_V1.5‐generated I_Na 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). I_Na 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 I_Na 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 I_Na, in addition to reduced cardiac tissue volume compared to WT littermates.Conclusion: We here demonstrate that Na_V1.5 is involved in cardiac electrical function from E10.5 onwards. Scn5a‐1798insD^−/− embryos displayed cardiac structural abnormalities at E9.5, indicating that Na_V1.5 dysfunction impacts on embryonic cardiac development in a non‐electrogenic manner. These findings are potentially relevant for understanding structural defects observed in relation to Na_V1.5 dysfunction.