Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiomyopathy characterized by the replacement of myocardium by fibro-fatty infiltration and cardiomyocyte loss. ACM predisposes to a high risk for ventricular arrhythmias. ACM has initially been defined as a desmosomal disease because most of the known variants causing the disease concern genes encoding desmosomal proteins. Studying this pathology is complex, in particular because human samples are rare and, when available, reflect the most advanced stages of the disease. Usual cellular and animal models cannot reproduce all the hallmarks of human pathology. In the last decade, human-induced pluripotent stem cells (hiPSC) have been proposed as an innovative human cellular model. The differentiation of hiPSCs into cardiomyocytes (hiPSC-CM) is now well-controlled and widely used in many laboratories. This hiPSC-CM model recapitulates critical features of the pathology and enables a cardiomyocyte-centered comprehensive approach to the disease and the screening of anti-arrhythmic drugs (AAD) prescribed sometimes empirically to the patient. In this regard, this model provides unique opportunities to explore and develop new therapeutic approaches. The use of hiPSC-CMs will undoubtedly help the development of precision medicine to better cure patients suffering from ACM. This review aims to summarize the recent advances allowing the use of hiPSCs in the ACM context.

AbstractAimsVentricular fibrillation (VF) occurring in the acute phase of ST-elevation myocardial infarction (STEMI) is the leading cause of sudden cardiac death worldwide. Several studies showed that reduced connexin 43 (Cx43) expression and reduced conduction velocity increase the risk of VF in acute myocardial infarction (MI). Furthermore, genetic background might predispose individuals to primary VF (PVF). The primary objective was to evaluate the presence of GJA1 variants in STEMI patients. The secondary objective was to evaluate the arrhythmogenic impact of GJA1 variants in STEMI patients with VF.Methods and resultsThe MAP-IDM prospective cohort study included 966 STEMI patients and was designed to identify genetic predisposition to VF. A total of 483 (50.0%) STEMI patients with PVF were included. The presence of GJA1 variants increased the risk of VF in STEMI patients [from 49.1 to 70.8%, P = 0.0423; odds ratio (OR): 0.40; 95% confidence interval: 0.16–0.97; P = 0.04]. The risk of PVF decreased with beta-blocker intake (from 53.5 to 44.8%, P = 0.0085), atrial fibrillation (from 50.7 to 26.4%, P = 0.0022), and with left ventricular ejection fraction >50% (from 60.2 to 41.4%, P < 0.0001). Among 16 GJA1 variants, three novel heterozygous missense variants were identified in three patients: V236I, H248R, and I327M. In vitro studies of these variants showed altered Cx43 localization and decreased cellular communication, mainly during acidosis.ConclusionConnexin 43 variants are associated with increased VF susceptibility in STEMI patients. Restoring Cx43 function may be a potential therapeutic target to prevent PVF in patients with acute MI.Clinical trial registrationClinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT00859300

Arrhythmogenic cardiomyopathy (ACM) is a rare genetic disease associated with ventricular arrhythmias in patients. The occurrence of these arrhythmias is due to direct electrophysiological remodeling of the cardiomyocytes, namely a reduction in the action potential duration (APD) and a disturbance of Ca2+ homeostasis. Interestingly, spironolactone (SP), a mineralocorticoid receptor antagonist, is known to block K+ channels and may reduce arrhythmias. Here, we assess the direct effect of SP and its metabolite canrenoic acid (CA) in cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) of a patient bearing a missense mutation (c.394C>T) in the DSC2 gene coding for desmocollin 2 and for the amino acid replacement of arginine by cysteine at position 132 (R132C). SP and CA corrected the APD in the muted cells (vs. the control) in linking to a normalization of the hERG and KCNQ1 K+ channel currents. In addition, SP and CA had a direct cellular effect on Ca2+ homeostasis. They reduced the amplitude and aberrant Ca2+ events. In conclusion, we show the direct beneficial effects of SP on the AP and Ca2+ homeostasis of DSC2-specific hiPSC-CMs. These results provide a rationale for a new therapeutical approach to tackle mechanical and electrical burdens in patients suffering from ACM.

Clinical and Translational MedicineVolume 12, Issue 3 e748 LETTER TO EDITOROpen Access The PPARγ pathway determines electrophysiological remodelling and arrhythmia risks in DSC2 arrhythmogenic cardiomyopathy Jean-Baptiste Reisqs, Jean-Baptiste Reisqs Neuromyogene Institute, Claude Bernard University, Lyon 1, Villeurbanne, France Cardiovascular and Metabolism Research, Sanofi R&D, Chilly Mazarin, FranceSearch for more papers by this authorAdrien Moreau, Adrien Moreau orcid.org/0000-0003-3100-0807 Université de Montpellier, INSERM, CNRS, PhyMedExp, Montpellier, FranceSearch for more papers by this authorAzzouz Charrabi, Azzouz Charrabi Université de Montpellier, INSERM, CNRS, PhyMedExp, Montpellier, FranceSearch for more papers by this authorYvonne Sleiman, Yvonne Sleiman Université de Montpellier, INSERM, CNRS, PhyMedExp, Montpellier, FranceSearch for more papers by this authorAlbano C. Meli, Albano C. Meli Université de Montpellier, INSERM, CNRS, PhyMedExp, Montpellier, FranceSearch for more papers by this authorGilles Millat, Gilles Millat Neuromyogene Institute, Claude Bernard University, Lyon 1, Villeurbanne, France Hospices Civils de Lyon, Lyon, Service de Rythmologie, France Laboratoire de Cardiogénétique Moléculaire, Centre de Biologie et Pathologie Est, Bron, FranceSearch for more papers by this authorVeronique Briand, Veronique Briand Cardiovascular and Metabolism Research, Sanofi R&D, Chilly Mazarin, FranceSearch for more papers by this authorPhilippe Beauverger, Philippe Beauverger Cardiovascular and Metabolism Research, Sanofi R&D, Chilly Mazarin, FranceSearch for more papers by this authorSylvain Richard, Corresponding Author Sylvain Richard sylvain.richard@inserm.fr orcid.org/0000-0001-9460-6705 Université de Montpellier, INSERM, CNRS, PhyMedExp, Montpellier, France Correspondence Sylvain Richard, INSERM U1046, CNRS UMR9214, Université de Montpellier, PhyMedExp, CHU Arnaud de Villeneuve, Bâtiment Crastes de Paulet, 371 Avenue du doyen Gaston Giraud, 34295 Montpellier Cedex 5, France. Email: sylvain.richard@inserm.fr Philippe Chevalier, Service de Rythmologie Cardiaque, CHU de Lyon HCL, GH Est-Hôpital Louis Pradel 59 Boulevard Pinel, 69677 Bron Cedex, France. Email: philippe.chevalier@chu-lyon.frSearch for more papers by this authorPhilippe Chevalier, Corresponding Author Philippe Chevalier philippe.chevalier@chu-lyon.fr Neuromyogene Institute, Claude Bernard University, Lyon 1, Villeurbanne, France Hospices Civils de Lyon, Lyon, Service de Rythmologie, France Correspondence Sylvain Richard, INSERM U1046, CNRS UMR9214, Université de Montpellier, PhyMedExp, CHU Arnaud de Villeneuve, Bâtiment Crastes de Paulet, 371 Avenue du doyen Gaston Giraud, 34295 Montpellier Cedex 5, France. Email: sylvain.richard@inserm.fr Philippe Chevalier, Service de Rythmologie Cardiaque, CHU de Lyon HCL, GH Est-Hôpital Louis Pradel 59 Boulevard Pinel, 69677 Bron Cedex, France. Email: philippe.chevalier@chu-lyon.frSearch for more papers by this author Jean-Baptiste Reisqs, Jean-Baptiste Reisqs Neuromyogene Institute, Claude Bernard University, Lyon 1, Villeurbanne, France Cardiovascular and Metabolism Research, Sanofi R&D, Chilly Mazarin, FranceSearch for more papers by this authorAdrien Moreau, Adrien Moreau orcid.org/0000-0003-3100-0807 Université de Montpellier, INSERM, CNRS, PhyMedExp, Montpellier, FranceSearch for more papers by this authorAzzouz Charrabi, Azzouz Charrabi Université de Montpellier, INSERM, CNRS, PhyMedExp, Montpellier, FranceSearch for more papers by this authorYvonne Sleiman, Yvonne Sleiman Université de Montpellier, INSERM, CNRS, PhyMedExp, Montpellier, FranceSearch for more papers by this authorAlbano C. Meli, Albano C. Meli Université de Montpellier, INSERM, CNRS, PhyMedExp, Montpellier, FranceSearch for more papers by this authorGilles Millat, Gilles Millat Neuromyogene Institute, Claude Bernard University, Lyon 1, Villeurbanne, France Hospices Civils de Lyon, Lyon, Service de Rythmologie, France Laboratoire de Cardiogénétique Moléculaire, Centre de Biologie et Pathologie Est, Bron, FranceSearch for more papers by this authorVeronique Briand, Veronique Briand Cardiovascular and Metabolism Research, Sanofi R&D, Chilly Mazarin, FranceSearch for more papers by this authorPhilippe Beauverger, Philippe Beauverger Cardiovascular and Metabolism Research, Sanofi R&D, Chilly Mazarin, FranceSearch for more papers by this authorSylvain Richard, Corresponding Author Sylvain Richard sylvain.richard@inserm.fr orcid.org/0000-0001-9460-6705 Université de Montpellier, INSERM, CNRS, PhyMedExp, Montpellier, France Correspondence Sylvain Richard, INSERM U1046, CNRS UMR9214, Université de Montpellier, PhyMedExp, CHU Arnaud de Villeneuve, Bâtiment Crastes de Paulet, 371 Avenue du doyen Gaston Giraud, 34295 Montpellier Cedex 5, France. Email: sylvain.richard@inserm.fr Philippe Chevalier, Service de Rythmologie Cardiaque, CHU de Lyon HCL, GH Est-Hôpital Louis Pradel 59 Boulevard Pinel, 69677 Bron Cedex, France. Email: philippe.chevalier@chu-lyon.frSearch for more papers by this authorPhilippe Chevalier, Corresponding Author Philippe Chevalier philippe.chevalier@chu-lyon.fr Neuromyogene Institute, Claude Bernard University, Lyon 1, Villeurbanne, France Hospices Civils de Lyon, Lyon, Service de Rythmologie, France Correspondence Sylvain Richard, INSERM U1046, CNRS UMR9214, Université de Montpellier, PhyMedExp, CHU Arnaud de Villeneuve, Bâtiment Crastes de Paulet, 371 Avenue du doyen Gaston Giraud, 34295 Montpellier Cedex 5, France. Email: sylvain.richard@inserm.fr Philippe Chevalier, Service de Rythmologie Cardiaque, CHU de Lyon HCL, GH Est-Hôpital Louis Pradel 59 Boulevard Pinel, 69677 Bron Cedex, France. Email: philippe.chevalier@chu-lyon.frSearch for more papers by this author First published: 16 March 2022 https://doi.org/10.1002/ctm2.748 Jean-Baptiste Reisqs, Adrien Moreau, Sylvain Richard and Philippe Chevalier contributed equally as junior and senior investigators, respectively. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Dear Editor, Arrhythmogenic cardiomyopathy (ACM) is a rare, life-threatening genetic disease frequently associated with mutations in desmosomal genes.1 Histopathological hallmark includes fibrofatty replacement of myocardial tissue, potentially consisting of cardiomyocytes transdifferentiation into adipocytes.2 The ACM involves electromechanical disorders and risks of developing arrhythmias and sudden cardiac death. We recently evidenced early electrical modifications of human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CM) obtained from an ACM patient with a missense mutation (c.394C>T) in the DSC2 gene encoding desmocollin 2 (DSC2-hiPSC-CMs). These modifications are risk factors for triggering arrhythmias independently of fibrofatty replacement of myocardial tissue.3 We now show that PPARγ, a master regulator of the cardiomyocytes transdifferentiation into adipocytes, is critical early in the pro-arrhythmogenic pathogenesis in ACM-DSC2-hiPSC-CMs. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of heart samples revealed a higher PPARγ gene expression level in the ACM-heart bearing the DSC2 mutation than in the control heart (Figure S1). We derived hiPSCs from the same ACM-DSC2 patient, differentiated them into hiPSC-CMs, and cultured them for 60 days as described.3 We found a similar higher expression of PPARγ and of two pro-adipogenic target genes, perilipin and adiponectin, in DSC2-hiPSC-CMs versus control-hiPSC-CMs (Figure 1A–C). Despite lower expression, the pro-cardiac myosin light chain 2 ventricular (MLC2v) validated the cardiomyocyte phenotype of DSC2-hiPSC-CMs (Figure 1D). To confirm the involvement of PPARγ in the genes switch, we challenged DSC2-hiPSC-CMs with T0070907 (T007), which functions as a transcriptionally corepressor-selective PPARγ inverse agonist.4 Incubation with T007 (1 μM), for 40 days (D20–D60) during the maturation phase, corrected the expression levels of PPARγ, perilipin, adiponectin and MLC2v (Figure 1A–D). These results corroborated the PPARγ-dependent pro-adipogenic switch in the DSC2-hiPSC-CMs, linking with previous studies.2, 5, 6 FIGURE 1Open in figure viewerPowerPoint T0070907 modulates PPARγ pathway and prevents the cardiomyocyte phenotype in DSC2 patient-specific human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CM). Fold change compared to control for (A) PPARγ, (B) perilipin, (C) adiponectin and (D) myosin light chain (MLC) expression level, measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Error bars represent the standard error of the mean (SEM), N = 5/group. *Control versus DSC2, #DSC2 versus T007; *,#p< .05, **,##p< .01, ###p < .001 (Tuckey multiple comparisons test) We next investigated the effects of T007 on the excitation–contraction coupling using a phase-contrast video-based analysis of the hiPSC-CM monolayer's contractile function. We defined several contractile parameters: the beat rate, contraction time, relaxation duration, the resting time between two contractions, asynchronous rate and differentiating factor (Figure 2A–F). For each analysed video, the asynchronous time defines the time-fraction of the area spent in asynchrony. The differentiating factor reflects the linear combination of determinant contractile properties ensuring the best discrimination between the control-hiPSC-CMs and the DSC2-hiPSC-CMs. FIGURE 2Open in figure viewerPowerPoint T0070907 prevents contractile disturbances in DSC2 patient-specific human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs). The contractile activity was studied by video analysis in monolayer condition (control, N = 249 (black); patient, N = 212 (red); T0070907, N = 197 (blue)): beat rate (A), asynchronous rate (B), contraction time (C), relaxation duration (D), resting duration (E) and differentiating factor (F). Results are represented by violin plot with the median, first and third quartile. *Control versus DSC2, #DSC2 versus T007, §control versus T007; **,§§p < .01, ***,###,§§§p < .001 (Kruskal–Wallis test) The mutation increased the beat and asynchronous rates (Figure 2A,B) and reduced the contraction time, relaxation and resting duration in DSC2-hiPSC-CMs versus control-hiPSC-CMs (Figure 2C–E). Incubation with T007 prevented these modifications. The differentiating factor was similar to that of the control-hiPSC-CMs (Figure 2F), showing that PPARγ pathway inhibition counteracts the pathogenesis process. Interestingly, the PPARγ agonist (GW1929) did not mimic the effect of the mutation on the differentiating factor and action potential (AP) duration in control-hiPSC-CMs (Figure S2A) in line with the negligible effect of PPARγ agonists on basal transcription.4 A significant PPARγ expression level may be required for ligand-mediated effects. In cardiomyocytes, the AP initiates the contraction. We studied the effect of T007 on the electrophysiological properties of DSC2-hiPSC-CMs using the patch-clamp technique. Compared to control-hiPSC-CMs, the DSC2-hiPSC-CMs exhibited a twofold shortening of the AP duration at 1.0 Hz and lost action potential duration (APD) modulation according to pacing rate (Figure 3A,B). The voltage-gated Na+ current (INa) induces AP fast depolarisation, playing a master role in cardiac excitability. The DSC2-hiPSC-CMs exhibited a twofold decrease in INa density and a rightward shift in its voltage-dependent activation and steady-state inactivation (Figures 3C,D and S3). Consistently, the expression level of the SCN5A gene coding for the cardiac Nav channels (INa) was decreased (Figure S4). Voltage-gated K+ currents play a crucial role in AP repolarisation. Compared to control-hiPSC-CMs, DSC2-hiPSC-CMs exhibited a 3.5-fold increase in K+ current density, accounting for the AP shortening (Figure 3E,F). RT-qPCR analysis revealed an increase of +227% in the KCNH2 gene coding for IKr and +220% in the KCNQ1 gene coding for IKs in DSC2-hiPSC-CMs compared to control-hiPSC-CMs (Figure S4). Exposure of DSC2-hiPSC-CMs to T007 prevented the differences with control-hiPSC-CMs (Figures 3A–F and S3–S5). FIGURE 3Open in figure viewerPowerPoint Electrical activity of control and patient-specific human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) with or without T0070907. (A) Action potentials (APs) of hiPSC-CM from control (black), DSC2 patient in absence of T0070907 (red) and after 40 days of incubation with 1 μM of T0070907 (blue). (B) AP duration at 90% of repolarisation (APD90) as a function of pacing rate (control, N = 42; DSC2, N = 40; DSC2 + T0070907, N = 40). (C) Whole-cell Na+ current traces of control (top), patient-specific hiPSC-CMs (middle) and after incubation of T007 (bottom). (D) Averaged current density–voltage (I–V) relationship of INa (control, N = 23; DSC2, N = 21; DSC2 + T0070907, N = 23). (E) Whole-cell IK currents raw traces of control (top), patient-specific (middle) and after incubation of T0070907 (bottom) hiPSC-CMs. (F) I–V curves of IK associated currents (control, N = 15; DSC2, N = 18; DSC2 + T007, N = 14). Error bars represent the standard error of the mean (SEM). All differences are statistically different, p < .001, DSC2 versus control and versus DSC2 + T0070907 (Tuckey multiple comparison test) We evaluated the Ca2+ transients of spontaneously beating hiPSC-CMs using the fluorescent non-ratiometric Fluo-4 Ca2+-sensitive dye. The DSC2-hiPSC-CMs (vs. control-hiPSC-CMs) exhibited increased Ca2+ transient frequency but decreased decay time and global Ca2+ mobilised area under the curve (AUC) during the Ca2+ transient (Figure 4A–C) in coherence with impaired contractility. They also exhibited an increase in the number and frequency of pro-arrhythmogenic abnormal spontaneous diastolic microscopic Ca2+ events (Ca2+ sparks), reflecting Ca2+ leak from the sarcoplasmic reticulum through the ryanodine receptor. Treatment of the DSC2-hiPSC-CMs with T007 attenuated all these effects (Figure 4D,E). FIGURE 4Open in figure viewerPowerPoint Spontaneous Ca2+ handlings of control, patient-specific human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) and incubated with T0070907. The Ca2+ transient activity (control, N = 207 (black); patient, N = 222 (red); T0070907, N = 197 (blue)) were studied. Spontaneous frequency (A), decay time (B) and area under the curve (AUC) (C). The Ca2+ sparks were studied by measuring the number of sparks in each cell (D) and the frequency of sparks (E). The violin plot represented the median, first and third quartile, and a Kruskal–Wallis test was performed. *Control versus DSC2, #DSC2 versus T007, §control versus T007; *,§p < .05, ***,###,§§§p < .001 (Tuckey multiple comparisons test were performed) Late T007 incubation (D55–D60) could not reverse the variant-specific modifications of the AP in DSC2-hiPSC-CMs (Figure S5). In addition, when we cultured DSC2-hiPSC-CMs in the presence of T007 between D20 and D60 and then removed the drug at D100, the cells exhibited the typical disease phenotype (Figure S5). Therefore, early and continuous exposure of DSC2-hiPSC-CMs to T007 is required to maintain a wild-type-like cellular electrophysiological signature (Figure S6). In conclusion, the repressing effect of T007 on PPARγ transcriptional activity maintained regular electrical activity and Ca2+ handling in hiPSC-CMs bearing the DSC2 (c.394C>T) mutation. T007 also lowered early pro-arrhythmogenic events intrinsic to cardiomyocytes, likely to contribute to rhythm disturbance independently of fibrofatty replacement of myocardial tissue in ACM patients (Figure S7). The risk of malignant ventricular tachyarrhythmias relies both on (i) shortened AP repolarisation, responsible for short QT (and JTc) intervals in a cohort of patients,3 caused by high KCNH2 (IKr) and KCNQ1 (IKs) expressions and (ii) and abnormal occurrence of Ca2+ sparks known to promote arrhythmias independently of any change in the AP and QT interval.7 Combining a short AP (QT) with disturbed Ca2+ is likely to increase the pro-arrhythmogenic risk. Our results align with different studies in transgenic mice linking PPARγ activation, cardiomyocyte lipid accumulation, dilated cardiomyopathy, changes in electrophysiological profile and intracellular Ca2+ handling, and arrhythmogenic risks, although differences between mice and humans may limit some interpretations.6, 8-10 Overall, our study provides new comprehensive insights directly in human cardiomyocytes regarding the role of PPARγ in genetic ACM. Repression of PPARγ transcription may offer a unique opportunity for ACM treatment. ACKNOWLEDGEMENTS We thank the 'Montpellier Ressources Imagerie' (MRI) platform (https://www.mri.cnrs.fr/fr/). A CIFRE grant from Sanofi R&D supported this work to Jean-Baptiste Reisqs. Fond Marion Elisabeth Brancher supported the post-doctoral fellowship of Adrien Moreau. FUNDING INFORMATION A CIFRE grant from Sanofi R&D supported this work to Jean-Baptiste Reisqs. Fond Marion Elisabeth Brancher supported the post-doctoral fellowship of Adrien Moreau. CONFLICT OF INTERESTS Sanofi, a global biopharmaceutical company focused on human health, employs Jean-Baptiste Reisqs, Veronique Briand and Philippe Beauverger. All other authors have nothing else to disclose. All authors read and approved the final version of the manuscript and ensure it was the case. Supporting Information Filename Description ctm2748-sup-0001-SuppMat.docx2.7 MB Supporting InformationFigure S1: PPARγ expression in patient DSC2 heart sample. Relative level of PPARγ in human heart sample from control and an arrhythmogenic cardiomyopathy (ACM) patient with a missense mutation (c.394C>T) in the DSC2 gene. Data obtained from transcriptome analysis (Affymetrix, HG-U133_plus2 chips)Figure S2: Evaluation of PPARγ agonist in WT human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CM). (A) Differentiating factor of contractile property and (B) action potential (AP) duration at 90% of repolarisation (APD90) in WT hiPSC-CM with or without 40 days of 5 μM GW1929. *Control versus DSC2, §DSC2 versus GW1929; §§p < .01, ***,§§§p < .001 (t-test comparison)Figure S3: Evaluation of Nav biophysical parameters. (A) Voltage-dependence of steady-state activation and inactivation of Nav channelsFigure S4: Evaluation of sodium and potassium channel expression. (A) The expression level of voltage-gated Na+ channel alpha subunit (N = 4/group). Expression level study by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) of potassium voltage-gated channel subfamily H2 (B) and subfamily Q1 (C) (N = 4/group). Error bars represent the standard error of the mean (SEM). *Control versus DSC2, #DSC2 versus T007; *,#p < .05, **,##p< .01, ***p < .001 (Tuckey multiple comparisons test)Figure S5: Electrical activity of control and patient-specific human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) with or without PPARγ for 5 days. (A) raw trace illustrating the AP of control (black), patient-specific hiPSC-CM (red) and after incubation of T0070907 (grey), (B) Action potential (AP) duration at 90% of repolarisation (APD90), (C) adaptation of APD at 0.5, 1 and 2 Hz. Error bars represent the standard error of the mean (SEM). *Control versus DSC2, $control versus T007; *p < .05, ***,$$$p < .001 (Tuckey multiple comparisons test)Figure S6: Inhibition of the PPARγ pathway is reversible. (A) Raw trace illustrating the action potential (AP) of control (black), patient-specific human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) (red) and after temporary incubation with 1 μM T0070907 for 40 days (D20–D60) and then removal of the drug for the next 40 days (D60–D100) (blue). (B) AP duration at 90% of repolarisation (APD90). (C) Adaptation of APD90 at different pacing (0.5, 1.0 and 2.0 Hz). (D) Comparison of APD90 in control, DSC2 and DSC2 + T007 between D60 (blank) and D100 (black square). Error bars represent the mean standard error (SEM) (control, N = 32; DSC2, N = 34; DSC2 + T007, N = 33). *Control versus DSC2, §control versus T007, +DSC2 + T007 D60 versus DSC2 + T007 D100 ; ***,§§§,+++p < .001 (Tuckey multiple comparisons test)Figure S7: A graphical picture summarising the PPARγ mechanism in arrhythmogenic cardiomyopathy (ACM) patient. The DSC2 mutation contributes to destabilising the desmosomal complex in ACM patients. PPARγ pathway contributes to adipogenic transformation and increases the risk of ventricular arrhythmias. Defects in excitation–contraction coupling were associated with cardiomyocytes transdifferentiation into adipocytes in immature human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) from patients with ACM (red panel). The PPARγ inhibitor T0070907 (T007) prevented the molecular genetic expression switch between the cardiac and the pro-adipogenic gene expression profiles. T007 maintained regular electrical activity and calcium handling in hiPSC-CMs bearing the DSC2 (c.394C>T) mutation (green panel) Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. REFERENCES 1Awad MM, Calkins H, Judge DP. 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Arrhythmias can occur in patients with arrhythmogenic cardiomyopathy (ACM, also called arrhythmogenic right ventricular cardiomyopathy), despite the absence of structural abnormalities in the heart.1,2 The mechanism responsible for the onset of ventricular fibrillation (VF) in the early stage of ACM remains poorly understood. Here, we report a clinical case with a 24-year follow-up of a patient that did not have an identified genetic variation, and was resuscitated from VF. A significantly short QTc interval was the only abnormality at the time of the initial event, which argued that it arose from a purely electrical mechanism.

MOTIVATION Cardiomyocytes derived from stem cells are closely followed, notably since the discovery in 2007 of human induced pluripotent stem cells (hiPSC). Cardiomyocytes (hiPSC-CM) derived from hiPSC are indeed more and more used to study specific cardiac diseases as well as for developing novel applications such as drug safety experiments. Robust dedicated tools to characterize hiPSC-CM are now required. The hiPSC-CM morphology constitutes an important parameter since these cells do not demonstrate the expected rod shape, characteristic of native human cardiomyocytes. Similarly, the presence, the density and the organization of contractile structures would be a valuable parameter to study. Precise measurements of such characteristics would be useful in many situations: for describing pathological conditions, for pharmacological screens or even for studies focused on the hiPSC-CM maturation process. RESULTS For this purpose, we developed a MATLAB based image analysis toolbox, which gives accurate values for cellular morphology parameters as well as for the contractile cell organization. IMPLEMENTATION To demonstrate the power of this automated image analysis, we used a commercial maturation medium intended to promote the maturation status of hiPSC-CM, and compare the parameters with the ones obtained with standard culture medium, and with freshly dissociated mouse cardiomyocytes. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Polymorphic ventricular tachycardia (PMVT) can occur in patients with structurally normal hearts and in 8% of cases can lead to sudden cardiac death, typically exercise-induced. The role of the cardiac type 2 ryanodine receptor (RyR2) in pathogenesis of PMVT presenting at rest is unclear. We aimed here at modelling PMVT observed in a patient harboring the RyR2-H29D mutation by comparing the molecular and functional properties of RyR2-H29D hiPSC-derived cardiomyocytes (hiPSC-CMs) with their isogenic control counterparts with a particular focus on the RyR2 properties. We collected blood samples from the patient and generated several clones of RyR2-H29D hiPSC, in addition to generating an isogenic control by reverting the RyR2-H29D mutation using CRIPSR/Cas9 technology. We used fluorescent confocal microscopy, patch-clamp and video-image-based analysis to investigate the molecular and functional consequences of the RyR2-H29D mutation. We first hypothesized that PMVT hiPSC-CMs expressing the RyR2-H29D mutation would exhibit abnormal Ca2+ homeostasis. Thus, we measured and analyzed the intracellular Ca2+ variation. We found that the RyR2-H29D hiPSC-CMs exhibit clone-independent aberrant properties including intracellular sarcoplasmic reticulum (SR) Ca2+ leak through RyR2 under physiological pacing. The contribution of inositol 1,4,5-trisphosphate receptors to excitation-contraction coupling exacerbate the abnormal intracellular Ca2+ release in the RyR2-H29D hiPSC-CMs. Moreover, the RyR2-H29D hiPSC-CMs exhibit RyR2 post-translational remodeling, shorter action potentials, delayed afterdepolarizations, arrhythmias and aberrant contractile properties compared to isogenic controls. These abnormalities are fully reversed with isogenic control. Our results suggest that RyR2-mediated Ca2+ leak induces an impairment of Ca2+ homeostasis and provide support to decipher the molecular mechanisms of short-coupled PMVT at rest.

Abstract Background Severe ventricular rhythm disturbances are the hallmark of arrhythmogenic cardiomyopathy (ACM), and are often explained by structural conduction abnormalities. However, comprehensive investigations of ACM cell electrical instability are lacking. This study aimed to elucidate early electrical myogenic signature of ACM. Methods We investigated a 41‐year‐old ACM patient with a missense mutation (c.394C>T) in the DSC2 gene, which encodes desmocollin 2. Pathogenicity of this variant was confirmed using a zebrafish DSC2 model system. Control and DSC2 patient‐derived pluripotent stem cells were reprogrammed and differentiated into cardiomyocytes (hiPSC‐CM) to examine the specific electromechanical phenotype and its modulation by antiarrhythmic drugs (AADs). Samples of the patient's heart and hiPSC‐CM were examined to identify molecular and cellular alterations. Results A shortened action potential duration was associated with reduced Ca2+ current density and increased K+ current density. This finding led to the elucidation of previously unknown abnormal repolarization dynamics in ACM patients. Moreover, the Ca2+ mobilised during transients was decreased, and the Ca2+ sparks frequency was increased. AAD testing revealed the following: (1) flecainide normalised Ca2+ transients and significantly decreased Ca2+ spark occurrence and (2) sotalol significantly lengthened the action potential and normalised the cells’ contractile properties. Conclusions Thorough analysis of hiPSC‐CM derived from the DSC2 patient revealed abnormal repolarization dynamics, prompting the discovery of a short QT interval in some ACM patients. Overall, these results confirm a myogenic origin of ACM electrical instability and provide a rationale for prescribing class 1 and 3 AADs in ACM patients with increased ventricular repolarization reserve.

Background: Nav1.5, which is encoded by the SCN5A gene, is the predominant voltage-gated Na+ channel in the heart. Several muta-tions of this gene have been identified and reported to be involved in several cardiac rhythm disorders, including type 3 long QT interval syndrome, that can cause sudden cardiac death. We analyzed the biophysical properties of 2 novel variants of the Nav1.5 channel (Q1491H and G1481V) detected in 5-and 12-week-old infants diag-nosed with a prolonged QT interval. Methods: The Nav1.5 wild-type and the Q1491H and G1481V mutant channels were reproduced in vitro. Wild-type or mutant channels were cotransfected in human embryonic kidney (HEK) 293 cells with the beta 1 regulatory subunit. Na+ currents were recorded using the whole -cell configuration of the patch-clamp technique. Results: The Q1491H mutant channel exhibited a lower current den-sity, a persistent Na+ current, an enhanced window current due to a +20-mV shift of steady-state inactivation, a +10-mV shift of steady-state activation, a faster onset of slow inactivation, and a recovery from fast inactivation with fast and slow time constants of recovery. The G1481V mutant channel exhibited an increase in current density and a +7-mV shift of steady-state inactivation. The observed defects are characteristic of gain-of-function mutations typical of type 3 long QT interval syndrome. Conclusions: The 5-and 12-week-old infants displayed prolonged QT intervals. Our analyses of the Q1491H and G1481V mutations corre-lated with the clinical diagnosis. The observed biophysical dysfunctions associated with both mutations were most likely responsible for the sudden deaths of the 2 infants.

BACKGROUND:While mutations in the cardiac type 2 ryanodine receptor (RyR2) have been linked to exercise-induced or catecholaminergic polymorphic ventricular tachycardia (CPVT), its association with polymorphic ventricular tachycardia (PMVT) occurring at rest is unclear. We aimed at constructing a patient-specific human-induced pluripotent stem cell (hiPSC) model of PMVT occurring at rest linked to a single point mutation in RyR2.METHODS:Blood samples were obtained from a patient with PMVT at rest due to a heterozygous RyR2-H29D mutation. Patient-specific hiPSCs were generated from the blood samples, and the hiPSC-derived cardiomyocytes (CMs) were generated via directed differentiation. Using CRIPSR/Cas9 technology, isogenic controls were generated by correcting the RyR2-H29D mutation. Using patch-clamp, fluorescent confocal microscopy and video-image-based analysis, the molecular and functional properties of RyR2-H29D hiPSCCMs and control hiPSCCMs were compared.FINDINGS:RyR2-H29D hiPSCCMs exhibit intracellular sarcoplasmic reticulum (SR) Ca2+ leak through RyR2 under physiological pacing. RyR2-H29D enhances the contribution of inositol 1,4,5-trisphosphate receptors to excitation-contraction coupling (ECC) that exacerbates abnormal Ca2+ release in RyR2-H29D hiPSCCMs. RyR2-H29D hiPSCCMs exhibit shorter action potentials, delayed afterdepolarizations, arrhythmias and aberrant contractile properties compared to isogenic controls. The RyR2-H29D mutation causes post-translational remodeling that is fully reversed with isogenic controls.INTERPRETATION:To conclude, in a model based on a RyR2 point mutation that is associated with short-coupled PMVT at rest, RyR2-H29D hiPSCCMs exhibited aberrant intracellular Ca2+ homeostasis, shortened action potentials, arrhythmias and abnormal contractile properties.FUNDING:French Muscular Dystrophy Association (AFM; project 16,073, MNM2 2012 and 20,225), "Fondation de la Recherche Médicale" (FRM; SPF20130526710), "Institut National pour la Santé et la Recherche Médicale" (INSERM), National Institutes of Health (ARM; R01 HL145473) and New York State Department of Health (NYSTEM C029156).

Le traumatisme crânien léger (TCL) est une pathologie fréquente à risque évolutif vers le syndrome post-commotionnel (SPC) comprenant des plaintes somatiques, cognitives, émotionnelles et comportementales. Ce syndrome est souvent difficile à objectiver mais il existe des outils innovants pour quantifier l’examen neurologique. L’objectif de l’étude est de quantifier la cognition, l’équilibre statique et la marche des TCL afin de rechercher des facteurs prédictifs du SPC. Nous avons recueilli de manière prospective, pendant 9 mois, les données concernant 37 patients pris en charge pour un TCL dans un centre de premier recours, l’Hôpital Kremlin-Bicêtre, et/ou de second recours l’Hôpital d’Instruction des Armées de Percy. Le SPC était défini à 3 mois selon la classification DSM-IV-TR. L’évaluation de la cognition se faisait par l’inventaire du syndrome dysexécutif comportemental. L’évaluation quantitative de l’équilibre statique et de la marche, se faisait respectivement par une plateforme de force Wii-balance board et 4 capteurs d’inertie, fixés sur le dos des pieds, le pelvis et le front. Le tout était connecté à une tablette pour collecter les résultats. Le taux de SPC à 3 mois était de 59,5 %. Pour la cognition, il existait un lien significatif entre SPC et syndrome dysexécutif (p = 0,04). Pour l’équilibre statique, il existait un lien significatif entre SPC et vitesse moyenne (de déplacement du centre de pression, CDP) (p = 0,011), sway density (densité de temps de balancement du CDP) (p = 0,002), surface (balayée par le CDP) (p = 0,036) et l’indice de stabilité (p = 0,013). Pour la marche, il existait un lien significatif entre SPC et vitesse moyenne (p = 0,007). Notre étude retrouve un fort taux de SPC chez les TCL. Nos résultats placent la quantification de l’équilibre statique et de la marche comme un nouveau mode de dépistage du SPC et participe à la collecte de données précieuses dans l’ère de la modélisation.

Arrhythmogenic cardiomyopathy (ACM) primarily involves right ventricle impairment and lethal arrhythmias. Hereditary forms of ACM are mostly caused by mutations in genes coding for desmosomal proteins. Histopathological hallmark includes fibrofatty replacement of myocardial tissue, which may involve transdifferentiation of cardiomyocytes into adipocyte cells through the PPAR-γ pathway. Comprehensive investigation of cardiomyocytes electro-mechanical properties during this process has never been undertaken. We aimed to assess the implication of the PPAR-γ pathway inhibition using T0070907 inhibitor. We used cardiomyocytes (hiPSC-CM) differentiated from an ACM patient-derived pluripotent stem cells (hiPSC) harbouring a mutation (R132 C) in the DSC2 gene. The control and DSC2 hiPSC were reprogrammed and differentiated into hiPSC-CM to examine gene expression, electro-mechanical properties (patch-clamp and video analysis) and calcium handling (fluorescence imaging) profiles. The DSC2 hiPSC-CM were cultured for 40 days in presence or absence of 1 μM T0070907. The RT-qPCR of both DSC2 patient's heart samples and hiPSC-CM showed an increase of PPAR-γ expression when compared to control cells. When compared to control hiPSC-CM, the DSC2 hiPSC-CM exhibited a shorter action potential duration, due to an increase of K+ current density. Culture of DSC2 hiPSC-CM with the PPAR-γ inhibitor normalized both the action potential duration and K+ current density. Differences were also found in both the calcium transient and mechanical properties between control, DSC2 and DSC2 with PPAR-γ hiPSC-CM. PPAR-γ pathway inhibition by T0070907 normalized the disrupted electro-mechanical properties in DSC2 hiPSC-CM. This result opens interesting perspectives for further studies of the adipogenesis pathway to prevent appearance of fibro-fatty adipose tissue in the ACM patient's heart.

This article thoroughly describes a data set of 1020 multivariate gait signals collected with two inertial measurement units, from 230 subjects undergoing a fixed protocol: standing still, walking 10 m, turning around, walking back and stopping. In total, 8.5 h of gait time series are distributed. The measured population was composed of healthy subjects as well as patients with neurological or orthopedic disorders. An outstanding feature of this data set is the amount of signal metadata that are provided. In particular, the start and end time stamps of more than 40,000 footsteps are available, as well as a number of contextual information about each trial. This exact data set was used in [Oudre et al., Template-based step detection with inertial measurement units, Sensors 18, 2018] to design and evaluate a step detection procedure.

Abstract Introduction Duchenne Muscular Dystrophy (DMD) is a X-linked degenerative pathology with a prevalence of 1/3500 boys due to absence of functional dystrophin in muscles. In a late stage of DMD, patients developed a dilated cardiomyopathy (DCM) which can lead to heart failure and premature death. In the past, we showed that DMD (mdx) mice exhibit a perturbation of the intracellular calcium homeostasis correlated to a pathological remodelling of the calcium ryanodine receptor channel (RyR2) leading to DCM with aging. However, mouse model does not represent a pertinent prototype to study DMD. Human pluripotent stem-cell derived-cardiomyocytes (hiPSC-CMs) are a pertinent tool to model patient-specific inherited cardiac diseases and screen pharmacological drugs in a Petri dish. Objective Based on the clinical history of DMD patients in the local Hospital, our main objective is to model DMD-induced DCM using hiPSC-CMs and compare the functional and molecular features with the clinical echocardiography. To that, we hypothesize that hiPSC-CMs are a powerful technology to model in vitro DCM and to better understand the pathophysiological mechanisms underlying DCM. Methods 3 blood samples from DMD patients with different DCM degrees of severity and 3 from healthy control (HC) were collected, reprogrammed in hiPSC and differentiated into cardiomyocytes. Results Our preliminary data indicate that DMD hiPSC-CMs present an abnormal intracellular calcium homeostasis characterized by the presence of leaky diastolic calcium events compared to HC hiPSC-CMs suggesting a RyR2 dysfunction. In DMD hiPSC-CMs, we also observe alterations in the contractile properties and a perturbation of the mitochondrial respiration. Conclusion Our results support the fact that DMD-inducing DCM can be modelled in the dish using patient-specific hiPSC-CMs. Such modelling may provide a better understanding of the pathophysiological mechanisms and the pharmacological treatment of the DMD-induced DCM.

Voltage gated sodium channels (Na-V) are broadly expressed in the human body. They are responsible for the initiation of action potentials in excitable cells. They also underlie several physiological processes such as cognitive, sensitive, motor, and cardiac functions. The Na(V)1.5 channel is the main NaV expressed in the heart. A dysfunction of this channel is usually associated with the development of pure electrical disorders such as long QT syndrome, Brugada syndrome, sinus node dysfunction, atrial fibrillation, and cardiac conduction disorders. However, mutations of Na(v)1.5 have recently been linked to the development of an atypical clinical entity combining complex arrhythmias and dilated cardiomyopathy. Although several Na(v)1.5 mutations have been linked to dilated cardiomyopathy phenotypes, their pathogenic mechanisms remain to be elucidated. The gating pore may constitute a common biophysical defect for all Na(V)1.5 mutations located in the channel's VSDs. The creation of such a gating pore may disrupt the ionic homeostasis of cardiomyocytes, affecting electrical signals, cell morphology, and cardiac myocyte function. The main objective of this article is to review the concept of gating pores and their role in structural heart diseases and to discuss potential pharmacological treatments.

Background: Sarcoplasmic reticulum Ca2+ leak and post-translational modifications under stress have been implicated in catecholaminergic polymorphic ventricular tachycardia (CPVT), a highly lethal inherited arrhythmogenic disorder. Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling. Objective: The aims were to obtain functional hiPSC-derived cardiomyocytes from a CPVT patient harboring a novel ryanodine receptor (RyR2) mutation and model the syndrome, drug responses and investigate the molecular mechanisms associated to the CPVT syndrome. Methods: Patient-specific cardiomyocytes were generated from a young athletic female diagnosed with CPVT. The contractile, intracellular Ca2+ handling and electrophysiological properties as well as the RyR2 macromolecular remodeling were studied. Results: Exercise stress electrocardiography revealed polymorphic ventricular tachycardia when treated with metoprolol and marked improvement with flecainide alone. We found abnormal stress-induced contractile and electrophysiological properties associated with sarcoplasmic reticulum Ca2+ leak in CPVT hiPSC-derived cardiomyocytes. We found inadequate response to metoprolol and a potent response of flecainide. Stabilizing RyR2 with a Rycal compound prevents those abnormalities specifically in CPVT hiPSC-derived cardiomyocytes. The RyR2-D3638A mutation is located in the conformational change inducing-central core domain and leads to RyR2 macromolecular remodeling including depletion of PP2A and Calstabin2. Conclusion: We identified a novel RyR2-D3638A mutation causing 3D conformational defects and aberrant biophysical properties associated to RyR2 macromolecular complex post-translational remodeling. The molecular remodeling is for the first time revealed using patient-specific hiPSC-derived cardiomyocytes which may explain the CPVT proband’s resistance. Our study promotes hiPSC-derived cardiomyocytes as a suitable model for disease modeling, testing new therapeutic compounds, personalized medicine and deciphering underlying molecular mechanisms.

Arrhythmogenic right ventricular dysplasia (ARVD) is a rare cardiomyopathy characterized by the progressive replacement of cardiomyocytes by fatty and fibrous tissue in the right ventricle (RV). These infiltrations lead to cardiac electrical instability and ventricular arrhythmia. Current treatment for ARVD is empirical and essentially based on treatment of arrhythmia. Thus, there is no validated treatment that will prevent the deterioration of RV function in patients with ARVD. The aim of the BRAVE study is to evaluate the effect of ramipril, an angiotensin‐converting enzyme inhibitor, on ventricular myocardial remodeling and arrhythmia burden in patients with ARVD. Despite the fact that myocardial fibrosis is one of the structural hallmarks of ARVD, no study has tested an antifibrotic drug in ARVD patients. The trial is a double‐blind, parallel, multicenter, prospective, randomized, phase 4 drug study. Patients will be randomized into 2 groups, ramipril or placebo. The 120 patients (60 per group) will be enrolled by 26 centers in France. Patients will be followed up every 6 months for 3 years. The 2 co–primary endpoints are defined as the difference of telediastolic RV volume measured by magnetic resonance imaging between baseline and 3 years of follow‐up, and the change in arrhythmia burden during the 3 years of follow‐up. A decrease in RV and/or left ventricular deterioration and in arrhythmia burden are expected in ARVD patients treated with ramipril. This reduction will improve quality of life of patients and will reduce the number of hospitalizations and the risk of terminal heart failure.

AimsCardiac atrial arrhythmias are the most common type of heart rhythm disorders. Its genetic elucidation remains challenging with poor understanding of cellular and molecular processes. These arrhythmias usually affect elderly population but in rare cases, young children may also suffer from such electrical diseases. Severe complications, including stroke, are commonly age related. This study aims to identify a genetic link between electro-mechanic atrial dysfunction and stroke in children.Methods and resultsIn two unrelated boys of 11 and 14 years with both stroke and atrial arrhythmias, the clinical phenotype was determined through a complete physical examination, electrocardiogram (ECG), Holter ECG, and computed tomography. The genetic testing was performed on a large 95 genes panel implicated in myocardial electrical imbalance, using the next generation sequencing method. The panel also includes the genes usually associated with the development of cardiomyopathies. In one child, a left atrial dilation was observed. The 2nd boy suffered from atrial standstill. Both suffered from atrial bradycardia, flutter, and fibrillation. The complete genetic testing revealed the SCN5A c.3823G>A (p.D1275N) mutation in the first family, c.1141-2A>G and c.3157G>A (p.E1053K) mutations in the second family.ConclusionOur results strengthen the association between Nav1.5 mutations and the occurrence of stroke in young patients. It emphasizes the need to look for atrial myopathy in the decision process for anticoagulation in young patients with atrial arrhythmic events.

Dilated cardiomyopathy (DCM) is a structural heart disease that causes dilatation of cardiac chambers and impairs cardiac contractility. The SCN5A gene encodes Nav1.5, the predominant cardiac sodium channel alpha subunit. SCN5A mutations have been identified in patients with arrhythmic disorders associated with DCM. The characterization of Nav1.5 mutations located in the voltage sensor domain (VSD) and associated with DCM revealed divergent biophysical defects that do not fully explain the pathologies observed in these patients. The purpose of this study was to characterize the pathological consequences of a gating pore in the heart arising from the Nav1.5/R219H mutation in a patient with complex cardiac arrhythmias and DCM. We report its properties using cardiomyocytes derived from patient-specific human induced pluripotent stem cells. We showed that this mutation generates a proton leak (called gating pore current). We also described disrupted ionic homeostasis, altered cellular morphology, electrical properties, and contractile function, most probably linked to the proton leak. We thus propose a novel link between SCN5A mutation and the complex pathogenesis of cardiac arrhythmias and DCM. Furthermore, we suggest that leaky channels would constitute a common pathological mechanism underlying several neuronal, neuromuscular, and cardiac pathologies.