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Dilated cardiomyopathy (DCM), a myocardial disease, is heterogeneous and often results in heart failure and sudden cardiac death. Unavailability of cardiac tissue has hindered the comprehensive exploration of gene regulatory networks and nodal players in DCM. In this study, we carried out integrated analysis of transcriptome and methylome data using non-negative matrix factorization from a cohort of DCM patients to uncover underlying latent factors and covarying features between whole-transcriptome and epigenome omics datasets from tissue biopsies of living patients. DNA methylation data from Infinium HM450 and mRNA Illumina sequencing of n = 33 DCM and n = 24 control probands were filtered, analyzed and used as input for matrix factorization using R NMF package. Mann-Whitney U test showed 4 out of 5 latent factors are significantly different between DCM and control probands (PP = 3.97E-21), nucleic acid binding (P = 1.42E-18), extracellular matrix (P = 9.23E-14) and myofibrillar structure (P = 8.46E-12). Correlation network analysis revealed interaction of important sarcomeric genes like Nebulin, Tropomyosin alpha-3 and ERC-protein 2 with CpG methylation of ATPase Phospholipid Transporting 11A0, Solute Carrier Family 12 Member 7 and Leucine Rich Repeat Containing 14B, all with significant P values associated with correlation coefficients >0.7. Using matrix factorization, multi-omics data derived from human tissue samples can be integrated and novel interactions can be identified. Hypothesis generating nature of such analysis could help to better understand the pathophysiology of complex traits such as DCM.

Background Non-ischemic dilated cardiomyopathy (DCM) can be complicated by sustained ventricular arrhythmias (SVA) and sudden cardiac death (SCD). By now, left-ventricular ejection fraction (LV-EF) is the main guideline criterion for primary prophylactic ICD implantation, potentially leading either to overtreatment or failed detection of patients at risk without severely impaired LV-EF. The aim of the European multi-center study DETECTIN-HF was to establish a clinical risk calculator for individualized risk stratification of DCM patients. Methods 1393 patients (68% male, mean age 50.7 ± 14.3y) from four European countries were included. The outcome was occurrence of first potentially life-threatening ventricular arrhythmia. The model was developed using Cox proportional hazards, and internally validated using cross validation. The model included seven independent and easily accessible clinical parameters sex, history of non-sustained ventricular tachycardia, history of syncope, family history of cardiomyopathy, QRS duration, LV-EF, and history of atrial fibrillation. The model was also expanded to account for presence of LGE as the eight8h parameter for cases with available cMRI and scar information. Results During a mean follow-up period of 57.0 months, 193 (13.8%) patients experienced an arrhythmic event. The calibration slope of the developed model was 00.97 (95% CI 0.90–1.03) and the C-index was 0.72 (95% CI 0.71–0.73). Compared to current guidelines, the model was able to protect the same number of patients (5-year risk ≥8.5%) with 15% fewer ICD implantations. Conclusions This DCM-SVA risk model could improve decision making in primary prevention of SCD in non-ischemic DCM using easily accessible clinical information and will likely reduce overtreatment.

Dilated cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial origin in 20–40% of cases. Genetic testing by next-generation sequencing (NGS) has yielded a definite diagnosis in many cases; however, some remain elusive. In this study, we used a combination of NGS, human-induced pluripotent-stem-cell-derived cardiomyocytes (iPSC-CMs) and nanopore long-read sequencing to identify the causal variant in a multi-generational pedigree of DCM. A four-generation family with familial DCM was investigated. Next-generation sequencing (NGS) was performed on 22 family members. Skin biopsies from two affected family members were used to generate iPSCs, which were then differentiated into iPSC-CMs. Short-read RNA sequencing was used for the evaluation of the target gene expression, and long-read RNA nanopore sequencing was used to evaluate the relevance of the splice variants. The pedigree suggested a highly penetrant, autosomal dominant mode of inheritance. The phenotype of the family was suggestive of laminopathy, but previous genetic testing using both Sanger and panel sequencing only yielded conflicting evidence for LMNA p.R644C (rs142000963), which was not fully segregated. By re-sequencing four additional affected family members, further non-coding LMNA variants could be detected: rs149339264, rs199686967, rs201379016, and rs794728589. To explore the roles of these variants, iPSC-CMs were generated. RNA sequencing showed the LMNA expression levels to be significantly lower in the iPSC-CMs of the LMNA variant carriers. We demonstrated a dysregulated sarcomeric structure and altered calcium homeostasis in the iPSC-CMs of the LMNA variant carriers. Using targeted nanopore long-read sequencing, we revealed the biological significance of the variant c.356+1G>A, which generates a novel 5′ splice site in exon 1 of the cardiac isomer of LMNA, causing a nonsense mRNA product with almost complete RNA decay and haploinsufficiency. Using novel molecular analysis and nanopore technology, we demonstrated the pathogenesis of the rs794728589 (c.356+1G>A) splice variant in LMNA. This study highlights the importance of precise diagnostics in the clinical management and workup of cardiomyopathies.

Mutations in mitochondrial aminoacyl-tRNA synthetases (mtARSs) have been reported in patients with mitochondriopathies: most commonly encephalopathy, but also cardiomyopathy. Through a GWAS, we showed possible associations between mitochondrial valyl-tRNA synthetase (VARS2) dysregulations and non-ischemic cardiomyopathy. We aimed to investigate the possible consequences of VARS2 depletion in zebrafish and cultured HEK293A cells. Transient VARS2 loss-of-function was induced in zebrafish embryos using Morpholinos. The enzymatic activity of VARS2 was measured in VARS2-depleted cells via northern blot. Heterozygous VARS2 knockout was established in HEK293A cells using CRISPR/Cas9 technology. BN-PAGE and SDS-PAGE were used to investigate electron transport chain (ETC) complexes, and the oxygen consumption rate and extracellular acidification rate were measured using a Seahorse XFe96 Analyzer. The activation of the integrated stress response (ISR) and possible disruptions in mitochondrial fatty acid oxidation (FAO) were explored using RT-qPCR and western blot. Zebrafish embryos with transient VARS2 loss-of-function showed features of heart failure as well as indications of CNS and skeletal muscle involvements. The enzymatic activity of VARS2 was significantly reduced in VARS2-depleted cells. Heterozygous VARS2-knockout cells showed a rearrangement of ETC complexes in favor of complexes III2, III2 + IV, and supercomplexes without significant respiratory chain deficiencies. These cells also showed the enhanced activation of the ISR, as indicated by increased eIF-2α phosphorylation and a significant increase in the transcript levels of ATF4, ATF5, and DDIT3 (CHOP), as well as disruptions in FAO. The activation of the ISR and disruptions in mitochondrial FAO may underlie the adaptive changes in VARS2-depleted cells.

Background The development of Precision Medicine strategies requires high-dimensional phenotypic and genomic data, both of which are highly privacy-sensitive data types. Conventional data management systems lack the capabilities to sufficiently handle the expected large quantities of such sensitive data in a secure manner. PROMISE is a genetic data management concept that implements a highly secure platform for data exchange while preserving patient interests, privacy, and autonomy. Methods The concept of PROMISE to democratize genetic data was developed by an interdisciplinary team. It integrates a sophisticated cryptographic concept that allows only the patient to grant selective access to defined parts of his genetic information with single DNA base-pair resolution cryptography. The PROMISE system was developed for research purposes to evaluate the concept in a pilot study with nineteen cardiomyopathy patients undergoing genotyping, questionnaires, and longitudinal follow-up. Results The safety of genetic data was very important to 79%, and patients generally regarded the data as highly sensitive. More than half the patients reported that their attitude towards the handling of genetic data has changed after using the PROMISE app for 4 months (median). The patients reported higher confidence in data security and willingness to share their data with commercial third parties, including pharmaceutical companies (increase from 5 to 32%). Conclusion PROMISE democratizes genomic data by a transparent, secure, and patient-centric approach. This clinical pilot study evaluating a genetic data infrastructure is unique and shows that patient’s acceptance of data sharing can be increased by patient-centric decision-making. Graphic abstract

Background Postcardiac injury syndrome (PCIS) is an inflammatory complication that derives from injury to the epicardium, myocardium, or endocardium. It occurs after trauma, myocardial infarction, percutaneous coronary intervention, cardiac surgery, intracardiac ablation, and implantation of cardiac implantable electronic device (CIED). In this study we assessed the incidence of PCIS after CIED implantation and its possible risk factors. Material and methods All patients who received CIED implantation at Heidelberg University Hospital between 2000 and 2014 were evaluated (n = 4989 patients). Clinical data including age, sex, underlying cardiac disease, type of implanted CIED, location of electrode implantation, clinical symptoms, time of symptom onset of PCIS, therapy, and outcome were extracted and analyzed. Results We identified 19 cases of PCIS in 4989 patients, yielding an incidence of 0.38%. The age of patients with PCIS ranged from 39 to 86 years. Dilated cardiomyopathy (DCM) as underlying cardiac disease and right atrial (RA) lead implantation had a significant association with occurrence of PCIS (p = 0.045 in DCM and p Conclusion To the best of our knowledge, this is the largest cohort evaluating the incidence of PCIS after CIED implantation. The data show that PCIS is a rare complication after CIED implantation and occurs more frequently in patients with DCM and those with RA lead implantation. Although rare and mostly benign, PCIS can lead to potentially lethal complications and physicians must be aware of its symptoms.

Background: The current gold standard biomarker for myocardial infarction (MI), cardiac troponin (cTn), is recognized for its high sensitivity and organ specificity; however, it lacks diagnostic specificity. Numerous studies have introduced circulating microRNAs as potential biomarkers for MI. This study investigates the MI-specificity of these serum microRNAs by investigating myocardial stress/injury due to strenuous exercise. Methods: MicroRNA biomarkers were retrieved by comprehensive review of 109 publications on diagnostic serum microRNAs for MI. MicroRNA levels were first measured by next-generation sequencing in pooled sera from runners (n = 46) before and after conducting a full competitive marathon. Hereafter, reverse transcription quantitative real-time PCR (qPCR) of 10 selected serum microRNAs in 210 marathon runners was performed (>10,000 qPCR measurements). Results: 27 potential diagnostic microRNA for MI were retrieved by the literature review. Eight microRNAs (miR-1-3p, miR-21-5p, miR-26a-5p, miR-122-5p, miR-133a-3p, miR-142-5p, miR-191-5p, miR-486-3p) showed positive correlations with cTnT in marathon runners, whereas two miRNAs (miR-134-5p and miR-499a-5p) showed no correlations. Upregulation of miR-133a-3p (p = 0.03) and miR-142-5p (p = 0.01) went along with elevated cTnT after marathon. Conclusion: Some MI-associated microRNAs (e.g., miR-133a-3p and miR-142-5p) have similar kinetics under strenuous exercise and MI as compared to cTnT, which suggests that their diagnostic specificity could be limited. In contrast, several MI-associated microRNAs (miR-26a-5p, miR-134-5p, miR-191-5p) showed different release behavior; hence, combining cTnT with these microRNAs within a multi-marker strategy may add diagnostic accuracy in MI.

Background Mutations in the splice factor RBM20 account for ∼3% of genetic cardiomyopathies. Mutations at position R634 in the hotspot RS-domain were found to cause dilative cardiomyopathy (DCM) (R634W) or left ventricular non-compaction cardiomyopathy (LVNC) (R634L), but the pathophysiological mechanisms that govern the heterogeneity in phenotype presentation remain unknown. Purpose We aimed here to identify the molecular events caused by the distinct RBM20 mutations from DCM and LVNC patients using a patient-specific induced stem cell model (iPSC) and test if the currently clinically used β-blockers (Metroprolol) are suitable for different RBM20-dependent cardiomyopathies. Methods We generated iPSC-cardiomyocytes of 2 DCM- and 2 LVNC-patients harboring the RBM20-mutations R634W (DCM) or R634L (LVNC). We investigated alternative splicing, sarcomeric regularity, cAMP-level, kinase-specific phosphorylation of Ca2+ players and Ca2+ handling. To investigate the impact of the genetic background, isogenic rescue lines were generated by CRISPR/Cas9. Different clinical drugs as Metoprolol and Verapamil were used to analyze the pharmacological improvement in vitro. Results We investigated the splicing pattern of the 2 RBM20 mutations in DCM and LVNC iPSC-CMs and observed common isoform changes in titin and a 24bp-insertion in the gene RYR2. The Ca2+ handling gene triadin is misspliced in LVNC-CMs, whereas the structural gene LDB3 is misspliced in DCM-CMs. As a possible consequence of splice defects in sarcomeric genes, both DCM and LVNC-CMs exhibited an irregular sarcomeric structure. The Ca2+ handling gene CAMK2δ was predominantly misspliced in LVNC-CMs leading to CAMK2δ-dependent hyperphosphorylation of its target PLN-Thr17 and subsequently to shortened Ca2+ elimination time and weakened response to β-adrenergic stimulation. By contrast, DCM-CMs exhibited increased Ca2+ sparks and decreased systolic and diastolic Ca2+ levels. RBM20 expression itself was decreased in LVNC-CMs, but not in DCM-CMs. This highlights that 2 distinct RBM20 mutations can lead to different pathological Ca2+ phenotypes. Isogenic CRISPR/Cas9 repair of both RBM20 mutations in LVNC and DCM demonstrated a rescue in gene missplicing, sarcomeric regularity and the Ca2+ handling aberrations and underscored the causative nature of the 2 mutations and their diverging effects. Ca2+ channel blockage with Verapamil showed a significant improvement of some of the LVNC disease characteristics compared to commonly clinically used β-blocker Metoprolol and underpins the potential clinical use of this drug in patients with LVNC. Conclusion We show the first iPSC-model of splice-defect associated RBM20-dependent LVNC and DCM. In summary, our results suggest that the molecular aberrations in alternative splicing differ depending on the distinct mutation in RBM20 and lead to shared and differential pathologies. Verapamil could be a good candidate in the treatment of RBM20-dependent LVNC. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Bunderministerium für Bildung und Forschung BMBFGerman Center for Cardiovascular Research DZHK

Hypertrophic obstructive cardiomyopathy (HOCM) increases the risk for mother and fetus during pregnancy. Alcohol septal ablation (ASA) is an established procedure in nonpregnant patients with HOCM. In this report, we present a case of a 29-year-old woman in her 29th gestational week with decompensated HOCM undergoing a successful ASA. (Level of Difficulty: Advanced.), Graphical abstract, Hypertrophic obstructive cardiomyopathy (HOCM) increases the risk for mother and fetus during pregnancy…

Cardiac sarcoidosis is a chronic inflammatory disease with a large spectrum of symptoms that can mimic diseases such as dilated, hypertrophic, or arrhythmogenic cardiomyopathies. It can be asymptomatic but can also present with ventricular arrhythmias, conduction disease, and heart failure (HF) or even sudden cardiac death (SCD). We present here the case of a patient transplanted due to end‐stage arrhythmogenic right ventricular cardiomyopathy (ARVC), fulfilling the task force criteria. A few years after successful heart transplantation (HTX), the patient developed similar symptoms and morphofunctional changes of the heart, which led to critical re‐evaluation of his primary diagnosis.

Importance The overall low survival rate of patients with lung cancer calls for improved detection tools to enable better treatment options and improved patient outcomes. Multivariable molecular signatures, such as blood-borne microRNA (miRNA) signatures, may have high rates of sensitivity and specificity but require additional studies with large cohorts and standardized measurements to confirm the generalizability of miRNA signatures. Objective To investigate the use of blood-borne miRNAs as potential circulating markers for detecting lung cancer in an extended cohort of symptomatic patients and control participants. Design, Setting, and Participants This multicenter, cohort study included patients from case-control and cohort studies (TREND and COSYCONET) with 3102 patients being enrolled by convenience sampling between March 3, 2009, and March 19, 2018. For the cohort study TREND, population sampling was performed. Clinical diagnoses were obtained for 3046 patients (606 patients with non–small cell and small cell lung cancer, 593 patients with nontumor lung diseases, 883 patients with diseases not affecting the lung, and 964 unaffected control participants). No samples were removed because of experimental issues. The collected data were analyzed between April 2018 and November 2019. Main Outcomes and Measures Sensitivity and specificity of liquid biopsy using miRNA signatures for detection of lung cancer. Results A total of 3102 patients with a mean (SD) age of 61.1 (16.2) years were enrolled. Data on the sex of the participants were available for 2856 participants; 1727 (60.5%) were men. Genome-wide miRNA profiles of blood samples from 3046 individuals were evaluated by machine-learning methods. Three classification scenarios were investigated by splitting the samples equally into training and validation sets. First, a 15-miRNA signature from the training set was used to distinguish patients diagnosed with lung cancer from all other individuals in the validation set with an accuracy of 91.4% (95% CI, 91.0%-91.9%), a sensitivity of 82.8% (95% CI, 81.5%-84.1%), and a specificity of 93.5% (95% CI, 93.2%-93.8%). Second, a 14-miRNA signature from the training set was used to distinguish patients with lung cancer from patients with nontumor lung diseases in the validation set with an accuracy of 92.5% (95% CI, 92.1%-92.9%), sensitivity of 96.4% (95% CI, 95.9%-96.9%), and specificity of 88.6% (95% CI, 88.1%-89.2%). Third, a 14-miRNA signature from the training set was used to distinguish patients with early-stage lung cancer from all individuals without lung cancer in the validation set with an accuracy of 95.9% (95% CI, 95.7%-96.2%), sensitivity of 76.3% (95% CI, 74.5%-78.0%), and specificity of 97.5% (95% CI, 97.2%-97.7%). Conclusions and Relevance The findings of the study suggest that the identified patterns of miRNAs may be used as a component of a minimally invasive lung cancer test, complementing imaging, sputum cytology, and biopsy tests.

With more than 25 million people affected, heart failure (HF) is a global threat. As energy production pathways are known to play a pivotal role in HF, we sought here to identify key metabolic changes in ischemic- and non-ischemic HF by using a multi-OMICS approach. Serum metabolites and mRNAseq and epigenetic DNA methylation profiles were analyzed from blood and left ventricular heart biopsy specimens of the same individuals. In total we collected serum from n = 82 patients with Dilated Cardiomyopathy (DCM) and n = 51 controls in the screening stage. We identified several metabolites involved in glycolysis and citric acid cycle to be elevated up to 5.7-fold in DCM (p = 1.7 × 10−6). Interestingly, cardiac mRNA and epigenetic changes of genes encoding rate-limiting enzymes of these pathways could also be found and validated in our second stage of metabolite assessment in n = 52 DCM, n = 39 ischemic HF and n = 57 controls. In conclusion, we identified a new set of metabolomic biomarkers for HF. We were able to identify underlying biological cascades that potentially represent suitable intervention targets.

The heart muscle diseases hypertrophic (HCM) and dilated (DCM) cardiomyopathies are leading causes of sudden death and heart failure in young, otherwise healthy, individuals. We conducted genome-wide association studies and multi-trait analyses in HCM (1,733 cases), DCM (5,521 cases) and nine left ventricular (LV) traits (19,260 UK Biobank participants with structurally normal hearts). We identified 16 loci associated with HCM, 13 with DCM and 23 with LV traits. We show strong genetic correlations between LV traits and cardiomyopathies, with opposing effects in HCM and DCM. Two-sample Mendelian randomization supports a causal association linking increased LV contractility with HCM risk. A polygenic risk score explains a significant portion of phenotypic variability in carriers of HCM-causing rare variants. Our findings thus provide evidence that polygenic risk score may account for variability in Mendelian diseases. More broadly, we provide insights into how genetic pathways may lead to distinct disorders through opposing genetic effects.

Introduction Myocardial fibrosis occurs during pathological remodeling of the heart and can be associated with worse outcome in affected patients. Genetic background is also known to affect patients' survival. In this study we sought to estimate patients' overall fibrosis burden by combining independent modalities including left ventricular endomyocardial biopsy (LV-EMB), circulating biomarkers, and cMRI. We also aimed to use patients' genetics information to predict outcome. Furthermore, we investigated the correlation between cardiac fibrosis and genetic variations to detect novel susceptibility loci for fibrosis in DCM. Methods A total number of 542 DCM patients were included. Collagen volume fraction (CVF) was automatically estimated from biopsies. 13 circulating fibrosis biomarkers were measured using Human Magnetic Luminex Screening Assays, and the cMRIs were screened for presence of LGE. Whole exome sequencing (WES) was performed in 410 patients of the cohort using illumina HiSeq 2000. Common (MAF ≥0.05 in the study population OR gnomAD NFE AF ≥0.01) and non-common missense variants (MAF Results The median follow-up time was 43.2 months (2084 patient-years). Sixty-two patients reached the composite end point and 55 died. LV-EMB proved to be a safe procedure with a total complication rate of 2.3%. Machine learning based characterization of biopsies was highly accurate. Although the 3 different modalities did not significantly correlate with one another, the extent of CVF, levels of MMP-2, TIMP-1, OPN, and GDF-15, and presence of LGE were each significantly associated with worse outcome. Four possible susceptibility loci for cardiac fibrosis in DCM were introduced and underwent eQTL analyses. Rare missense variants in a list of 11 DCM-related genes showed to be associated with the 2 outcome measures or fibrosis burden. Conclusions LV-EMB, fibrosis biomarkers, and cMRI likely capture different aspects of a detrimental fibrosis process and may be combined to estimate patients' prognosis and monitor therapeutic success. Phenotype-genotype association studies help elucidate novel disease pathomechanisms and individualize patients' treatment. Funding Acknowledgement Type of funding source: Other. Main funding source(s): German Centre for Cardiovascular Research: DZHK

Introduction Cardiomyopathies (CMPs) are leading causes of heart failure (HF) and sudden cardiac death (SCD). Comparative data of the multiple cardiomyopathy forms are largely missing. The TranslatiOnal Registry for CardiomyopatHies (TORCH) is the largest prospective multicentre CMP registry world-wide. Enrolled patients are comprehensively phenotyped by clinical examinations, state-of-the-art imaging, and molecular investigations. In this study, we present the baseline and 1-year follow-up data. Methods TORCH is a national, prospective, multicentre registry within the German Centre for Cardiovascular Research (DZHK) and includes 2300 patients with non-ischemic (primary and secondary) CMP from 20 centres. The minimum follow up was one year. The DZHK-wide harmonization of datasets and SOPs ensure a high level of data quality and comparability across different CMP forms. Results Dilated cardiomyopathy (DCM) has the highest prevalence with 64% of all enrolled patients, followed by hypertrophic cardiomyopathy (HCM) with 16%. At baseline, patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) were treated more often with ICD implantation and showed high rates of adequate ICD therapies (65.8%, p15% and 12% receiving heart transplantation. In DCM, reverse remodelling with improvement of functional parameters and biomarkers was more often observed in idiopathic and inflammatory cases compared to familial ones. HCM patients had the most favourable outcome. Conclusion and outlook TORCH is the largest prospective study focusing on CMPs. We provided for the first time prospectively the clinical data of patients with diverse cardiomyopathies with outcome. Furthermore, comparing the different CMP forms on the clinical and molecular level will be an important step to enable translational research projects. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): German Centre for Cardiovascular Research (DZHK)

Background and aim Mutations in the splice factor RBM20 have been identified to account for ∼3% of cardiomyopathies. In particular, the highly conserved RS-domain is a hotspot for disease-associated mutations. Distinct mutations at position 634 in the RS-domain were already described to be associated to dilative cardiomyopathy (DCM) (R634W) or to left ventricular non-compaction cardiomyopathy (LVNC) (R634L), but the molecular mechanisms that govern the heterogenic entity of DCM and LVNC remain largely unknown. We aimed to analyze the molecular driver behind the RBM20 mutation-based DCM and LVNC in a patient-specific stem cell model. Methods Human somatic cells from 2 DCM- and 2 LVNC-patients harboring the RBM20-mutations R634W (DCM) or R634L (LVNC) were reprogrammed into induced pluripotent stem cells (iPSC) and differentiated into functional cardiomyocytes (CM). Gene expression, alternative splicing activity, sarcomeric regularity, cAMP level, kinase-specific phosphorylation of important Ca2+ players, and physiological cardiac functions as Ca2+ homeostasis were analyzed (Fluo3 and Fura4). Isogenic rescue lines were generated by CRISPR/Cas9 technology to analyze the direct impact of the RBM20 mutations to the cardiac phenotype. Results We investigated the role of RBM20 mutations in DCM and LVNC-iPSC-CMs RBM20-splicing and observed common splice defects in titin-isoform-switch or a 24bp insertion in the gene ryanodine receptor 2 (RYR2).. In contrast, the calcium-handling gene Camk2δ was predominantly mis-spliced in LVNC-CMs, whereas the structural gene LDB3 was mis-spliced in DCM-CMs. As a possible consequence of splice defects in sarcomeric genes both DCM and LVNC-CMs exhibited an irregular sarcomeric structure at the Z-disk and M-line. Interestingly, the LVNC-CMs showed faster Ca2+ transient decay time and weakened response to β-adrenergic stimulation. In contrast, the DCM-CMs did exhibit increased Ca2+-sparks and decreased systolic and diastolic Ca2+ highlighting that two distinct missense mutations can lead to different pathological Ca2+ phenotypes. Ca2+ kinetic defects in LVNC-iPSC-CMs were independent of cAMP, but in line with Camk2δ-dependent hyperphosphorylation of the specific target PLN. Isogenic WT-iPSC lines were generated using CRISPR/Cas9 technology and underscored the role of RBM20-mutations in cardiomyopathies as the sarcomeric defects, Ca2+ cycling and leakage were rescued for both LVNC-CMs and DCM-CMs. Conclusion We show the first iPSC-model of splice-defect-associated RBM20-dependent LVNC and DCM. Our data demonstrate that RBM20-R634L induce mis-splicing of Camk2δ leading to hyperphosphorylation of PLN-Thr17 along with increased Ca2+ kinetics in LVNC, whereas RBM20-R634W induced RYR2-dependent Ca2+ leak with disturbed systolic and diastolic Ca2+in DCM. Taken together these results suggest that the molecular aberrations in alternative splicing differ depending on the distinct missense mutation in RBM20. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): BMBF, DZHK German Center for Cardiovascular research

In recent years, the genetic architecture of dilated cardiomyopathy (DCM) has been more thoroughly elucidated. However, there is still insufficient knowledge on the modifiers and regulatory principles that lead to the failure of myocardial function. The current study investigates the association of epigenome-wide DNA methylation and alternative splicing, both of which are important regulatory principles in DCM. We analyzed screening and replication cohorts of cases and controls and identified distinct transcriptomic patterns in the myocardium that differ significantly, and we identified a strong association of intronic DNA methylation and flanking exons usage (p &lt, 2 &times, 10&minus, 16). By combining differential exon usage (DEU) and differential methylation regions (DMR), we found a significant change of regulation in important sarcomeric and other DCM-associated pathways. Interestingly, inverse regulation of Titin antisense non-coding RNA transcript splicing and DNA methylation of a locus reciprocal to TTN substantiate these findings and indicate an additional role for non-protein-coding transcripts. In summary, this study highlights for the first time the close interrelationship between genetic imprinting by DNA methylation and the transport of this epigenetic information towards the dynamic mRNA splicing landscape. This expands our knowledge of the genome&ndash, environment interaction in DCM besides simple gene expression regulation.

Introduction: Familial dilated cardiomyopathy (DCM) is clinically variable and has been associated with mutations in more than 50 genes. Rapid improvements in DNA sequencing have led to the identification of diverse rare variants with unknown significance (VUS), which underlines the importance of functional analyses. In this study, by investigating human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we evaluated the pathogenicity of the p.C335R sodium voltage-gated channel alpha subunit 5 (SCN5a) variant in a large family with familial DCM and conduction disease. Methods: A four-generation family with autosomal dominant familial DCM was investigated. Next-generation sequencing (NGS) was performed in all 16 family members. Clinical deep phenotyping, including endomyocardial biopsy, was performed. Skin biopsies from two patients and one healthy family member were used to generate human-induced pluripotent stem cells (iPSCs), which were then differentiated into cardiomyocytes. Patch-clamp analysis with Xenopus oocytes and iPSC-CMs were performed. Results: A SCN5a variant (c.1003T>C; p.C335R) could be detected in all family members with DCM or conduction disease. A novel truncating TTN variant (p.Ser24998LysfsTer28) could also be identified in two family members with DCM. Family members with the SCN5a variant (p.C335R) showed significantly longer PQ and QRS intervals and lower left ventricular ejection fractions (LV-EF). All four patients who received CRT-D were non-responders. Electrophysiological analysis with Xenopus oocytes showed a loss of function in SCN5a p.C335R. Na+ channel currents were also reduced in iPSC-CMs from DCM patients. Furthermore, iPSC-CM with compound heterozygosity (SCN5a p.C335R and TTNtv) showed significant dysregulation of sarcomere structures, which may be contributed to the severity of the disease and earlier onset of DCM. Conclusion: The SCN5a p.C335R variant is causing a loss of function of peak INa in patients with DCM and cardiac conduction disease. The co-existence of genetic variants in channels and structural genes (e.g., SCN5a p.C335R and TTNtv) increases the severity of the DCM phenotype.