Search

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.

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.

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.

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

Aims Patients with non‐ischaemic dilated cardiomyopathy (DCM) are at increased risk of sudden cardiac death. Identification of patients that may benefit from implantable cardioverter‐defibrillator implantation remains challenging. In this study, we aimed to determine predictors of sustained ventricular arrhythmias in patients with DCM. Methods and results We searched MEDLINE/Embase for studies describing predictors of sustained ventricular arrhythmias in patients with DCM. Quality and bias were assessed using the Quality in Prognostic Studies tool, articles with high risk of bias in ≥2 areas were excluded. Unadjusted hazard ratios (HRs) of uniformly defined predictors were pooled, while all other predictors were evaluated in a systematic review. We included 55 studies (11 451 patients and 3.7 ± 2.3 years follow‐up). Crude annual event rate was 4.5%. Younger age [HR 0.82; 95% CI (0.74–1.00)], hypertension [HR 1.95; 95% CI (1.26–3.00)], prior sustained ventricular arrhythmia [HR 4.15; 95% CI (1.32–13.02)], left ventricular ejection fraction on ultrasound [HR 1.45; 95% CI (1.19–1.78)], left ventricular dilatation (HR 1.10), and presence of late gadolinium enhancement [HR 5.55; 95% CI (4.02–7.67)] were associated with arrhythmic outcome in pooled analyses. Prior non‐sustained ventricular arrhythmia and several genotypes [mutations in Phospholamban (PLN), Lamin A/C (LMNA), and Filamin‐C (FLNC)] were associated with arrhythmic outcome in non‐pooled analyses. Quality of evidence was moderate, and heterogeneity among studies was moderate to high. Conclusions In patients with DCM, the annual event rate of sustained ventricular arrhythmias is approximately 4.5%. This risk is considerably higher in younger patients with hypertension, prior (non‐)sustained ventricular arrhythmia, decreased left ventricular ejection fraction, left ventricular dilatation, late gadolinium enhancement, and genetic mutations (PLN, LMNA, and FLNC). These results may help determine appropriate candidates for implantable cardioverter‐defibrillator implantation.

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.

We report a rare case of concomitant hypertrophic cardiomyopathy and cardiac myxoma without LEOPARD syndrome. Additionally, 6 similar cases were systemically reviewed, and the characteristics of this first-ever studied patient group were summarized. (Level of Difficulty: Beginner.), Graphical abstract, This article reports a rare case of concomitant hypertrophic cardiomyopathy and cardiac myxoma without LEOPARD syndrome. Six similar cases were reviewed…

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.

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

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 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

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

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.

Background Cardiac troponins are the preferred biomarkers of acute myocardial infarction. Despite superior sensitivity, serial testing of Troponins to identify patients suffering acute coronary syndromes is still required in many cases to overcome limited specificity. Moreover, unstable angina pectoris relies on reported symptoms in the troponin-negative group. In this study, we investigated genome-wide miRNA levels in a prospective cohort of patients with clinically suspected ACS and determined their diagnostic value by applying an in silico neural network. Methods PAXgene blood and serum samples were drawn and hsTnT was measured in patients at initial presentation to our Chest-Pain Unit. After clinical and diagnostic workup, patients were adjudicated by senior cardiologists in duty to their final diagnosis: STEMI, NSTEMI, unstable angina pectoris and non-ACS patients. ACS patients and a cohort of healthy controls underwent deep transcriptome sequencing. Machine learning was implemented to construct diagnostic miRNA classifiers. Results We developed a neural network model which incorporates 34 validated ACS miRNAs, showing excellent classification results. By further developing additional machine learning models and selecting the best miRNAs, we achieved an accuracy of 0.96 (95% CI 0.96–0.97), sensitivity of 0.95, specificity of 0.96 and AUC of 0.99. The one-point hsTnT value reached an accuracy of 0.89, sensitivity of 0.82, specificity of 0.96, and AUC of 0.96. Conclusions Here we show the concept of neural network based biomarkers for ACS. This approach also opens the possibility to include multi-modal data points to further increase precision and perform classification of other ACS differential diagnoses.

Background Patients with non-ischemic dilated cardiomyopathy (NIDCM) are at increased risk of ventricular arrhythmias and sudden cardiac death (SCD). However, identifying patients at high risk for life-threatening ventricular arrhythmia (LTVA) who may benefit from an implantable cardioverter defibrillator (ICD) remains challenging. Methods We searched MEDLINE and EMBASE for prognostic studies describing predictors of LTVA (defined as sustained ventricular tachycardia (VT), haemodynamically unstable VT, ventricular fibrillation, (aborted) SCD or appropriate ICD intervention) in patients with NIDCM. We excluded articles with composite heart failure and arrhythmic endpoints but lacking (subgroup) analysis for LTVA. Study quality and risk of bias was assessed using the QUIPS-tool, and articles with high risk of bias in ≥2 areas were excluded from analysis. Univariable hazard ratios of reported predictors were pooled from the remaining studies in a meta-analysis using a random-effects model and presented with 95% confidence interval (CI). Results Out of 1996 unique citations, 51 studies were included comprising 9798 patients with 1493 arrhythmic events. 28 studies were pooled for meta-analysis (mean age 55±4.1 years, 72% male) with a mean follow-up of 3.7±1.9 years. Crude event rate was 4.3% (95% CI 4.02–4.57) per year. From our meta-analysis, hypertension (HR 1.95; CI [1.26–3.00]), history of out of hospital cardiac arrest or sustained VT (HR 4.15; CI [1.32–13.02]), T-wave alternans (HR 6.50; CI [2.46–17.14]), LVEDV per 10ml/m2 increase (HR 1.10; CI [1.10–1.10]), LVESV per 10ml/m2 increase (HR 1.10; CI [1.00–1.22]) and delayed gadolinium enhancement (HR 5.55; CI [4.02–7.67]) were significantly associated with LTVA (figure). The quality of evidence was moderate and there was significant heterogeneity (median i2 57%; IQR 76%) among studies. Additionally from data that could not be pooled, decreased LVEF, history of nsVT and decreased heart rate variability were significantly associated with LTVA. Summary of meta-analysis results Conclusion The risk of LTVA in NIDCM is 4.3% per year and is considerably higher in patients with hypertension, history of LTVA, decreased LVEF, high LVEDV, high LVESV, T-wave alternans, history of nsVT, decreased heart rate variability and delayed gadolinium enhancement. These results may help determine appropriate candidates for ICD implantation. The high heterogeneity in reported results indicate the need for future multicentre studies to further improve risk stratification in NIDCM. Acknowledgement/Funding ERA-CVD JTC2016: DETECTIN-HF, 680969 & Dutch Heart Foundation (2016T096)

Background The diagnosis of cardiac hypertrabecularization has increased considerably in recent years. Whether or not the non-compaction is a pathological condition or a physiological trait is still highly debated. We performed a meta-analysis and systematic review on more than 7,000 adult individuals with left ventricular non-compaction to provide a comprehensive overview on its clinical outcome as well as its genetic background. Methods We first retrieved PubMed/Medline literatures in English language between 2000 to 2018 on clinical outcome and genotype of patients with non-compaction. Altogether, 35 studies with non-compaction cardiomyopathy patients passed the selection criteria and were extensively reviewed and meta-analyzed. Furthermore, we summarized the results of 8 major studies, which investigated the non-compaction in athletes, pregnant women, patients with sickle cell disease, or in individuals from population-based cohorts. Results About 60% of the patients with left-ventricular non-compaction cardiomyopathy were male. The diagnosis was mostly made in the mid of patients' 5th decade. Seven percent of patients had congenital heart diseases (CHD) including atrial/ventricular septum defect or Ebstein anomaly. Around 25% of the patients had positive family history for cardiomyopathy. Frequent phenotypic manifestations were heart rhythm abnormalities including conduction disease (26%), supraventricular tachycardia (17%), and sustained or non-sustained ventricular tachycardia (18%). Neuromuscular disease was a reported comorbidity with a mean frequency of 5%. Three important outcome measures including systemic thromboembolic events (9%), heart transplantation (4%), and adequate ICD therapy (15%) were reported. The genetics of non-compaction cardiomyopathy showed TTN to be the most frequently mutated gene (11%), followed by MYH7 (9%), MYBPC3 (5%), and CASQ2, LDB3 (3% each). TPM1, MIB1, ACTC1, and LMNA mutations had an average frequency of 2% each followed by PLN, HCN4, TAZ, DTNA, TNNT2, and RBM20 (1% each). Eight studies that investigated the occurrence of non-compacted myocardial regions in apparently heart healthy individuals applied different, established imaging-based diagnostic criteria for non-compaction and could confirm its presence in a wide range of 1.3% to 37%. Conclusion This meta-analysis summarizes the clinical presentation of left ventricular non-compaction in a large dataset and indicates that its presence often leads to unfavourable outcome, but can also be observed in heart healthy individuals. Multimodal diagnostic workflows are needed for comprehensive understanding of these individuals and for distinguishing between benign morphological trait and manifest cardiomyopathy.

Background Post cardiac injury syndrome (PCIS) is an inflammatory process, which occurs in the setting of injury to the pericardium, epicardium or myocardium. It may follow cardiac surgery, myocardial infarction, trauma, intracardiac ablation, percutaneous coronary intervention, and implantation of heart rhythm devices (HRD). In this study we aimed to assess the incidence of PCIS after HRD implantation and its possible risk factors. Material and methods All patients who received HRD implantation at our University Hospital between the years 2000 to 2014 were evaluated (n=4,989 patients) for developing PCIS. Clinical data including age, sex, underlying cardiac disease, type of implanted HRD, location of electrode implantation, clinical symptoms, time of symptom onset of PCIS, therapy, and outcome were extracted and analysed. Results 19 cases of PCIS were identified in altogether 4,989 patients resulting in an incidence of 0.38%. The age in patients with PCIS ranged from 39 to 86 years. Dilated cardiomyopathy (DCM) as underlying cardiac disease and right atrial (RA) lead implantation showed significant association to occurrence of PCIS (p=0.045 in DCM and p Conclusion To the best of our knowledge, this is the thus far largest cohort evaluating the incidence of PCIS after HRD implantation. PCIS remains a rare, but potentially lethal complication of heart rhythm device implantation. It should be considered particularly in patients with right atrial lead active fixation and DCM as underlying cardiac disease.

Background Arrhythmogenic cardiomyopathy (AC) is an important cause of ventricular arrhythmias in children and young adults. AC is associated with mutation of desmosomal proteins, however, cardiac disease penetrance is incomplete and the clinical course varies widely without recognizable exogenous or epi/genetic co-factors. Importantly, DSP mutation carriers may also display entirely non-cardiac e.g. dermatological phenotypes. Methods and results In two brothers with recurrent fulminant myocarditis, mutation screening of 218 cardiomyopathy-related genes identified a truncating mutation Arg1458* of desmoplakin (DSP). DSP immunhistology unexpectedly revealed complete loss (“knockout”) of DSP protein in endomyocardial biopsies (EMBs), but none of the histological anomalies of AC. Criteria for histological diagnosis of myocarditis were not either fulfilled, and cardiac MRI revealed no features associated with AC. Screening for infections was negative, there was no substance abuse, medication or vaccination. Possible disease triggers were competitive sport events. Myosin and troponin I autoantibodies were detected at titers up to 1:320. We used allele-specific RT-PCR to distinguish if the patients' allele classified as “normal” was actually defective due to promotor mutation or epigenetic silencing. RT-PCRs were done on EMBs and peripheral blood mononuclear cells (PBMCs). In a cohort of dilated cardiomyopathy (DCM) patients we were able to detect DSP transcripts in both, PBMC and left-ventricular heart tissue. RNA sequencing of human PBMC subpopulations suggested that DSP transcription may be restricted to certain immune cell subtypes. RT-PCRs revealed that both Arg1458* carriers have a functional second DSP allele, indicating that their “DSP knockout” occurs at the protein level and may be due to protein instability and degradation within desmosomes. We screened additional existing cohorts for such variants and identified stopgain variant Gln307Ter in a 37-yrs-old woman with ARVC. This patient's sister died from heart failure at the age of 39. In a 59-yrs-old female LVNC patient, stopgain variant Y1391X was identified. Here, family history was unclear, her brother probably died from coronary artery disease. In a 71-yrs-old female DCM patient with no family history, stopgain variant Tyr1512Ter was identified. Conclusions The described patients with DSP truncations strongly suggest the existence of additional genetic or exogenous modifiers driving pathogenesis either way. DSP defects may cause recurrent myocarditis, and mutation screening is advisable to enable early detection of high-risk patients with similar phenotypes. Our finding of complete myocardial DSP protein loss emphasizes that DNA sequencing may miss critical molecular disturbances. It is indispensable to also analyze transcriptome and protein level in the tissue actually affected in a patient in order to recognize his/her individual pathogenesis.

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.

The transcriptome needs to be tightly regulated by mechanisms that include transcription factors, enhancers, and repressors as well as non‐coding RNAs. Besides this dynamic regulation, a large part of phenotypic variability of eukaryotes is expressed through changes in gene transcription caused by genetic variation. In this study, we evaluate genome‐wide structural genomic variants (SVs) and their association with gene expression in the human heart. We detected 3,898 individual SVs affecting all classes of gene transcripts (e.g., mRNA, miRNA, lncRNA) and regulatory genomic regions (e.g., enhancer or TFBS). In a cohort of patients (n = 50) with dilated cardiomyopathy (DCM), 80,635 non‐protein‐coding elements of the genome are deleted or duplicated by SVs, containing 3,758 long non‐coding RNAs and 1,756 protein‐coding transcripts. 65.3% of the SV‐eQTLs do not harbor a significant SNV‐eQTL, and for the regions with both classes of association, we find similar effect sizes. In case of deleted protein‐coding exons, we find downregulation of the associated transcripts, duplication events, however, do not show significant changes over all events. In summary, we are first to describe the genomic variability associated with SVs in heart failure due to DCM and dissect their impact on the transcriptome. Overall, SVs explain up to 7.5% of the variation of cardiac gene expression, underlining the importance to study human myocardial gene expression in the context of the individual genome. This has immediate implications for studies on basic mechanisms of cardiac maladaptation, biomarkers, and (gene) therapeutic studies alike.

Background: Biochemical DNA modification resembles a crucial regulatory layer among genetic information, environmental factors, and the transcriptome. To identify epigenetic susceptibility regions and novel biomarkers linked to myocardial dysfunction and heart failure, we performed the first multi-omics study in myocardial tissue and blood of patients with dilated cardiomyopathy and controls. Methods: Infinium human methylation 450 was used for high-density epigenome-wide mapping of DNA methylation in left-ventricular biopsies and whole peripheral blood of living probands. RNA deep sequencing was performed on the same samples in parallel. Whole-genome sequencing of all patients allowed exclusion of promiscuous genotype-induced methylation calls. Results: In the screening stage, we detected 59 epigenetic loci that are significantly associated with dilated cardiomyopathy (false discovery corrected P ≤0.05), with 3 of them reaching epigenome-wide significance at P ≤5×10 −8 . Twenty-seven (46%) of these loci could be replicated in independent cohorts, underlining the role of epigenetic regulation of key cardiac transcription regulators. Using a staged multi-omics study design, we link a subset of 517 epigenetic loci with dilated cardiomyopathy and cardiac gene expression. Furthermore, we identified distinct epigenetic methylation patterns that are conserved across tissues, rendering these CpGs novel epigenetic biomarkers for heart failure. Conclusions: The present study provides to our knowledge the first epigenome-wide association study in living patients with heart failure using a multi-omics approach.

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiovascular disease, which goes along with increased risk for sudden cardiac death (SCD). Despite the knowledge about the different causal genes, the relationship between individual genotypes and phenotypes is incomplete. We retrieved PubMed/Medline literatures on genotype–phenotype associations in patients with HCM and mutations in MYBPC3, MYH7, TNNT2, and TNNI3. Altogether, 51 studies with 7675 HCM patients were included in our meta-analysis. The average frequency of mutations in MYBPC3 (20%) and MYH7 (14%) was higher than TNNT2 and TNNI3 (2% each). The mean age of HCM onset for MYH7 mutation positive patients was the beginning of the fourth decade, significantly earlier than patients without sarcomeric mutations. A high male proportion was observed in TNNT2 (69%), MYBPC3 (62%) and mutation negative group (64%). Cardiac conduction disease, ventricular arrhythmia and heart transplantation (HTx) rate were higher in HCM patients with MYH7 mutations in comparison to MYBPC3 (p

To adapt to changing hemodynamic demands, regulatory mechanisms modulate actin-myosin-kinetics by calcium-dependent and -independent mechanisms. We investigate the posttranslational modification of human essential myosin light chain (ELC) and identify NIMA-related kinase 9 (NEK9) to interact with ELC. NEK9 is highly expressed in the heart and the interaction with ELC is calcium-dependent. Silencing of NEK9 results in blunting of calcium-dependent ELC-phosphorylation. CRISPR/Cas9-mediated disruption of NEK9 leads to cardiomyopathy in zebrafish. Binding to ELC is mediated via the protein kinase domain of NEK9. A causal relationship between NEK9 activity and ELC-phosphorylation is demonstrated by genetic sensitizing in-vivo. Finally, we observe significantly upregulated ELC-phosphorylation in dilated cardiomyopathy patients and provide a unique map of human ELC-phosphorylation-sites. In summary, NEK9-mediated ELC-phosphorylation is a calcium-dependent regulatory system mediating cardiac contraction and inotropy.

Left ventricular non-compaction has been increasingly diagnosed in recent years. However, it is still debated whether non-compaction is a pathological condition or a physiological trait. In this meta-analysis and systematic review, we compare studies, which investigated these two different perspectives. Furthermore, we provide a comprehensive overview on the clinical outcome as well as genetic background of left ventricular non-compaction cardiomyopathy in adult patients. We retrieved PubMed/Medline literatures in English language from 2000 to 19/09/2018 on clinical outcome and genotype of patients with non-compaction. We summarized and extensively reviewed all studies that passed selection criteria and performed a meta-analysis on key phenotypic parameters. Altogether, 35 studies with 2271 non-compaction patients were included in our meta-analysis. The mean age at diagnosis was the mid of their fifth decade. Two-thirds of patients were male. Congenital heart diseases including atrial or ventricular septum defect or Ebstein anomaly were reported in 7% of patients. Twenty-four percent presented with family history of cardiomyopathy. The mean frequency of neuromuscular diseases was 5%. Heart rhythm abnormalities were reported frequently: conduction disease in 26%, supraventricular tachycardia in 17%, and sustained or non-sustained ventricular tachycardia in 18% of patients. Three important outcome measures were reported including systemic thromboembolic events with a mean frequency of 9%, heart transplantation with 4%, and adequate ICD therapy with 15%. Nine studies investigated the genetics of non-compaction cardiomyopathy. The most frequently mutated gene was TTN with a pooled frequency of 11%. The average frequency of MYH7 mutations was 9%, for MYBPC3 mutations 5%, and for CASQ2 and LDB3 3% each. TPM1, MIB1, ACTC1, and LMNA mutations had an average frequency of 2% each. Mutations in PLN, HCN4, TAZ, DTNA, TNNT2, and RBM20 were reported with a frequency of 1% each. We also summarized the results of eight studies investigating the non-compaction in altogether 5327 athletes, pregnant women, patients with sickle cell disease, as well as individuals from population-based cohorts, in which the presence of left ventricular hypertrabeculation ranged from 1.3 to 37%. The summarized data indicate that non-compaction may lead to unfavorable outcome in different cardiomyopathy entities. The presence of key features in a multimodal diagnostic approach could distinguish between benign morphological trait and manifest cardiomyopathy.

Routine genetic testing in Dilated Cardiomyopathy (DCM) has recently become reality using Next-Generation Sequencing. Several studies have explored the relationship between genotypes and clinical phenotypes to support risk estimation and therapeutic decisions, however, most studies are small or restricted to a few genes. This study provides to our knowledge the first systematic meta-analysis on genotype-phenotype associations in DCM. We retrieved PubMed/Medline literature on genotype–phenotype associations in patients with DCM and mutations in LMNA, PLN, RBM20, MYBPC3, MYH7, TNNT2 and TNNI3. We summarized and extensively reviewed all studies that passed selection criteria and performed a meta-analysis on key phenotypic parameters. Together, 48 studies with 8097 patients were included. Furthermore, we reviewed recent studies investigating genotype-phenotype associations in DCM patients with TTN mutations. The average frequency of mutations in the investigated genes was between 1 and 5 %. The mean age of DCM onset was the beginning of the fifth decade for all genes. Heart transplantation (HTx) rate was highest in LMNA mutation carriers (27 %), while RBM20 mutation carriers were transplanted at a markedly younger age (mean 28.5 years). While 73 % of DCM patients with LMNA mutations showed cardiac conduction diseases, low voltage was the reported ECG hallmark in PLN mutation carriers. The frequency of ventricular arrhythmia in DCM patients with LMNA (50 %) and PLN (43 %) mutations was significantly higher. The penetrance of DCM phenotype in subjects with TTN truncating variants increased with age and reached 100 % by age of 70. A pooled analysis of available genotype-phenotype data shows a higher prevalence of sudden cardiac death (SCD), cardiac transplantation, or ventricular arrhythmias in LMNA and PLN mutation carriers compared to sarcomeric gene mutations. This study will further support the clinical interpretation of genetic findings.

Next-generation sequencing (NGS) is getting routinely used in the diagnosis of hereditary diseases, such as human cardiomyopathies. Hence, it is of utter importance to secure high quality sequencing data, enabling the identification of disease-relevant mutations or the conclusion of negative test results. During the process of sample preparation, each protocol for target enrichment library preparation has its own requirements for quality control (QC); however, there is little evidence on the actual impact of these guidelines on resulting data quality. In this study, we analyzed the impact of QC during the diverse library preparation steps of Agilent SureSelect XT target enrichment and Illumina sequencing. We quantified the parameters for a cohort of around 600 samples, which include starting amount of DNA, amount of sheared DNA, smallest and largest fragment size of the starting DNA; amount of DNA after the pre-PCR, and smallest and largest fragment size of the resulting DNA; as well as the amount of the final library, the corresponding smallest and largest fragment size, and the number of detected variants. Intriguingly, there is a high tolerance for variations in all QC steps, meaning that within the boundaries proposed in the current study, a considerable variance at each step of QC can be well tolerated without compromising NGS quality.

Background and aim: This systematic review with meta-analysis sought to determine the strength of evidence for effects of antioxidants (AO) such as N-acetyl cysteine (NAC), vitamin C, vitamin E, and alpha-lipoic acid on the incidence of contrast-in­duced nephropathy (CIN), requirement for haemodialysis, level of serum creatinine, and mortality after coronary angiography. Methods and results: After Medline, Embase, Elsevier, Sciences online database, and Google Scholar literature searches, studies with randomised controlled design were selected for the meta-analysis. The effect sizes measured were odds ratio (OR) for categorical variables and standard mean difference (SMD) with 95% confidence interval (CI) for calculating differences between mean changes of serum creatinine in intervention and control groups. A value of p 50% indicated significant heterogeneity between the studies. Literature search of all major databases retrieved 2350 studies. After screening, a total of 49 trials were identified that reported outcomes. Pooled treatment effect analysis revealed that NAC (OR of 0.79; 95% CI 0.69–0.9; p = 0.000), vitamin C (0.63; 95% CI 0.45–0.89; p = 0.000), and vitamin E (OR of 0.5; 95% CI 0.27–0.92; p = 0.026) could significantly reduce the incidence of CIN. NAC (SMD of –0.119; 95% CI –0.191 – 0.046; p = 0.000), but not vitamin C (SMD of –0.08; 95% CI –0.22–0.04; p = 0.1) and vitamin E (–0.25; 95% CI –0.46–0.05; p = 0.1), could significantly reduce mean levels of serum creatinine. Nevertheless, AO could not reduce the incidence of mortality, with an OR of 0.94 (95% CI 0.69–1.28; p = 0.7). Conclusions: Overall, antioxidants such as NAC, vitamin C, and vitamin E can reduce the incidence of CIN, while only NAC might be able to significantly lower serum creatinine levels. There is no impact of AO supplementation on mortality.

BACKGROUND Biomarkers are well established for diagnosis of myocardial infarction [cardiac troponins, high-sensitivity cardiac troponins (hs-cTn)], exclusion of acute and chronic heart failure [B-type natriuretic peptide (BNP), N-terminal proBNP (NT-proBNP)] and venous thromboembolism (d-dimers). Several studies have demonstrated acute increases in cardiac biomarkers and altered cardiac function after strenuous sports that can pretend a cardiovascular emergency and interfere with state-of-the-art clinical assessment. METHODS We performed a systematic review and metaanalysis of biomarker and cardiovascular imaging changes after endurance exercise. We searched for observational studies published in the English language from 1997 to 2014 that assessed these biomarkers or cardiac function and morphology directly after endurance exercise. Of 1787 identified abstracts, 45 studies were included. RESULTS Across all studies cardiac troponin T (cTnT) exceeded the cutoff value (0.01 ng/mL) in 51% (95% CI, 37%–64%) of participants. The measured pooled changes from baseline for high-sensitivity cTnT (hs-cTnT) were +26 ng/L (95% CI, 5.2–46.0), for cTnI +40 ng/L (95% CI, 21.4; 58.0), for BNP +10 ng/L (95% CI, 4.3; 16.6), for NT-proBNP +67 ng/L (95% CI, 49.9; 84.7), and for d-dimer +262 ng/mL (95% CI, 165.9; 358.7). Right ventricular end diastolic diameter increased and right ventricular ejection fraction as well as the ratio of the early to late transmitral flow velocities decreased after exercise, while no significant changes were observed in left ventricular ejection fraction. CONCLUSIONS Current cardiovascular biomarkers (cTnT, hs-cTnT, BNP, NT-proBNP, and d-dimer) that are used in clinical diagnosis of pulmonary embolism, acute coronary syndrome, and heart failure are prone to alterations due to strenuous exercise. Hence, it is necessary to take previous physical exercise into account when a cardiac emergency is suspected.

RNA binding motif protein 20 (RBM20) is an alternative splicing factor and highly expressed in cardiac tissue. Mutations in the RS domain of RBM20 have been shown to cause different cardiomyopathies. Here, we generated induced pluripotent stem cells (iPSCs) from a dilated cardiomyopathy patient harboring the heterozygous RBM20 mutation p.R634W and consecutively produced isogenic control line using CRISPR/Cas9 genome editing. Patient-specific RBM20 iPSCs and isogenic control line maintained full pluripotency, genomic integrity, and in vitro differentiation capacity. All iPSC-lines were able to differentiate into pure cardiomyocytes, thus providing a valuable tool for studying the pathogenesis of human RBM20-mediated cardiac disease.

About 30% of HF patients suitable for CRT do not respond to therapy. Estimating CRT response preoperatively would allow improved patient selection. The ability of a patient-specific computer heart model to introduce new preoperative biomarkers of CRT response was studied. cMRI and 12-lead ECGs were

Aims Circulating immune cells have a significant impact on progression and outcome of heart failure. Long non-coding RNAs (lncRNAs) comprise novel epigenetic regulators which control cardiovascular diseases and inflammatory disorders. We aimed to identify lncRNAs regulated in circulating immune cells of the blood of heart failure patients. Methods and results Next-generation sequencing revealed 110 potentially non-coding RNA transcripts differentially expressed in peripheral blood mononuclear cells of heart failure patients with reduced ejection fraction. The up-regulated lncRNA Heat2 was further functionally characterized. Heat2 expression was detected in whole blood, PBMNCs, eosinophil and basophil granulocytes. Heat2 regulates cell division, invasion, transmigration and immune cell adhesion on endothelial cells. Conclusion Heat2 is an immune cell enriched lncRNA that is elevated in the blood of heart failure patients and controls cellular functions.

This systematic review with meta-analysis sought to determine comparison of efficacy and safety of hydration with sodium bicarbonate versus sodium chloride on contrast induced nephropathy and clinical outcomes. We searched major electronic databases for studies in randomized controlled trials. A value of P0.1 for Q test or I (2)50% indicated significant heterogeneity between the studies. Literature search of all databases retrieved 650 studies. 29 studies enrolled in meta-analysis. Pooled analysis indicated about the incidence of CIN (OR of 0.718; 95% CI: 0.60 to 0.85; P = 0.000), requirement of hemodialysis (OR of 1.00; 95% CI: 0.49 to 2.01; P = 0.9), mean changes of serum creatinine (WMD of 2.321; 95% CI: 1.995 to 2.648; P = 0.000), length of hospital stays (WMD of -0.774; 95% CI: -1.65 to 0.10; P = 0.08), major adverse cardiovascular events (OR = 1.075, 95% CI: 0.59 to 1.95; P = 0.8), and mortality (OR of 0.73; 95% CI: 0.42 to 1.26; P = 0.2). Overall, hydration with sodium bicarbonate could significantly reduce CIN and the length of hospital stay compared to sodium chloride. In addition NAC added as a supplement to sodium bicarbonate could increase prophylactic effects against nephropathy.

Genetic variants contribute to several steps during heart failure pathophysiology. The mechanisms include frequent polymorphisms that increase the susceptibility to heart failure in the general population and rare variants as causes of an underlying cardiomyopathy. In this review, we highlight recent discoveries made by genetic approaches and provide an outlook onto the role of epigenetic modifiers of heart failure.

This systematic review with meta-analysis sought to determine protective effects of erythropoietin on clinical outcomes following percutaneous coronary intervention (PCI). Medline, Embase, Elsevier and Sciences online database as well as Google scholar literature were used for selecting appropriate studies with randomized controlled design. The effect sizes measured were odds ratio (OR) for categorical variables and weighted mean difference (WMD) with 95% confidence interval for calculating differences between mean values of duration of hospitalization in intervention and control groups. Values of P 2 >50% indicated significant heterogeneity between the studies. The literature searches of all major databases retrieved 973 studies. After screening, a total of 15 trials that reported outcomes were identified. Pooled analysis was performed on left ventricular ejection fraction (WMD of −0.047; 95% CI: −0.912 to 0.819; P =0.9), left ventricular end diastolic volume (WMD of −0.363; 95% CI: −3.902 to 3.175; P =0.8), left ventricular end systolic volume (WMD of 0.346; 95% CI: −2.533 to 3.226; P =0.8), infarct size (WMD of −0.446; 95% CI: −2.352 to −1.460; P =0.6), stroke (OR of 2.1; 95% CI: 0.58 to 7.54; P =0.2), re-myocardial infarction (OR of 1.06; 95% CI: 0.52 to 2.185; P =0.8), heart failure (OR of 0.53; 95% CI: 0.259 to 1.105; P =0.09), mortality (OR of 0.56; 95% CI: 0.27 to 1.19; P =0.13), thrombosis (OR of 0.774; 95% CI: 0.41 to 1.45; P =0.4), major adverse cardiovascular events (OR of 0.926; 95% CI: 0.63 to 1.35; P =0.6). Short-term administration of EPO in patients with myocardial infarction (MI) undergoing PCI does not result in improvement in cardiac function, reduction of infarct size and all-cause mortality. Low dose EPO therapy may not be the choice of treatment for the patients with MI, while higher doses might be more effective.

Diagnosis and treatment of dilated cardiomyopathy (DCM) is challenging due to a large variety of causes and disease stages. Computational models of cardiac electrophysiology (EP) can be used to improve the assessment and prognosis of DCM, plan therapies and predict their outcome, but require personalization. In this work, we present a data-driven approach to estimate the electrical diffusivity parameter of an EP model from standard 12-lead electrocardiograms (ECG). An efficient forward model based on a mono-domain, phenomenological Lattice-Boltzmann model of cardiac EP, and a boundary element-based mapping of potentials to the body surface is employed. The electrical diffusivity of myocardium, left ventricle and right ventricle endocardium is then estimated using polynomial regression which takes as input the QRS duration and electrical axis. After validating the forward model, we computed 9500 EP simulations on 19 different DCM patients in just under three seconds each to learn the regression model. Using this database, we quantify the intrinsic uncertainty of electrical diffusion for given ECG features and show in a leave-one-patient-out cross-validation that the regression method is able to predict myocardium diffusion within the uncertainty range. Finally, our approach is tested on the 19 cases using their clinical ECG. 84% of them could be personalized using our method, yielding mean prediction errors of 18.7 ms for the QRS duration and 6.5 ° for the electrical axis, both values being within clinical acceptability. By providing an estimate of diffusion parameters from readily available clinical data, our data-driven approach could therefore constitute a first calibration step toward a more complete personalization of cardiac EP.

This systematic review with meta-analysis sought to determine the impact of posterior pericardiotomy on incidences of atrial fibrillation and supraventricular arrhythmias, pericardial effusion, pleural effusion, tamponade, and the length of hospital stay after cardiac surgery. We searched for randomized controlled trials, using Medline, Embase, Elsevier and Sciences online databases as well as Google Scholar literature. The effect sizes measured were odds ratio for categorical variables and standard mean difference with 95% confidence interval for calculating differences between mean values of hospital stay in intervention and control groups. A value of p 2 > 50% indicated significant heterogeneity between the studies. The literature search of all major databases retrieved 20 studies. After screening, 12 suitable trials were identified, which reported outcomes of 2052 patients undergoing cardiac surgery. Posterior pericardiotomy had an odds ratio of 0.33 [95% confidence interval: 0.18–0.61] p

Post cardiac injury syndrome (PCIS) is an inflammatory process that occurs in the setting of injury to the pericardium, epicardium or myocardium. It may follow cardiac surgery, myocardial infarction, trauma, intracardiac ablation, percutaneous coronary intervention or implantation of a pacemaker or cardioverter–defibrillator. In this paper, we report the first case of PCIS after implantation of a “Cardiac Resynchronization Therapy Defibrillator” (CRT-D) device and review systematically the available literature. To obtain information on PCIS after implantation of heart rhythm devices (HRD), we performed a systematic review of Medline, EMBASE and the Cochrane Central Register of Controlled Trials (CENTRAL). The collected data included age, gender, initial diagnosis, procedure type, time to PCIS, symptoms, clinical manifestations, therapy and outcome. Included were reports in English, French and Spanish. In our systematic review, we found PCIS after HRD implantation in 17 additional cases. The age ranged from 23 to 84 years. Symptoms developed within 1 day–4 months after implantation. The use of active-fixation atrial leads was reported in nine cases. Fever, dyspnoea, chest pain, tachypnoea, tachycardia, palpitation, malaise, ankle edema, dry cough, night sweats, nausea and vomiting were reported as associated symptoms. PCIS should be considered as a rare, but serious complication of HRD implantation, may cause recurrent hospitalization and can be life threatening. The incidence and possible causes of PCIS after HRD, such as active fixation leads and specific lead positions as well as its treatment deserve further investigation in prospective studies.