Volume: 14, Issue: 4

Preface

Computational Cardiology — A New Discipline of Translational Research

Benjamin Meder, Hugo A. Katus, Andreas Keller

过去20年，为了对抗心衰、冠心病、心律不齐等易发生的致命疾病，诊断、药物治疗和介入治疗水平不断提高，这也促使了现代医疗设备的不断完善。目前该领域的革新大多基于假设性的方法，如心脏生物标志物利钠肽和心脏特异性肌钙蛋白等，其中计算心脏病学起着重要作用。计算心脏病学将分子水平信息、临床水平信息和生物信息学分析相结合，探索心脏病的发病机制和治疗的新方法。目前，为了提高和完善个体化治疗方法，对于流行性和偶发性心血管疾病的特征性描述越来越重要。本期文章中，我们列出了一些计算心脏病学的预见性挑战和其扮演的角色，如新型生物标志物的识别、人群大数据整合的实施方法以及新个体化药物的靶标定义等。计算心脏病学将为心血管疾病病人的医疗护理的整合方法开辟一条新道路。

Page 177-178

Research Highlight

Hide and Seek: Protein-coding Sequences Inside “Non-coding” RNAs

Daniel Oehler, Jan Haas

本文点评了Nelson等于2016年1月在Science发表的题为“A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle”的文章。作者发现，Dwarf开放阅读框（Dwarf open reading frame, DWORF）是长链非编码RNA中的隐藏肽，具有34个氨基酸残基，通过间接地激活SERCA蛋白的表达改善人体肌肉的放松状况，从而提高肌肉的收缩性；在患有扩张型心肌病的小鼠和人类的心衰组织中，DWORF基因均明显下调表达，说明DWORF既可作为心衰病人的新型生物标志物，也可作为新的药物靶标。隐藏在基因组“黑暗地带”中的蛋白质为深入解析人类心脏的复杂性打开了一扇新的大门。生物信息学分析是未来新发现的出发点。
Die Erregbarkeit von Zellen ist kritisch mit dem Gleichgewicht der Ionenkonzentrationen verknüpft. In Muskelzellen hängt die Kontraktilität von vor allem der Kalziumhomöostase ab. Hierbei ist die SERCA (Calciumpumpe des sarcoplasmatischen und endoplasmatischen Reticulums) entscheidend beteiligt. In einer kürzlich in „Science“ publizierten Arbeit von Nelson et Al. konnte eine bisher als long non-coding RNA annotierte Sequenz als für das DWORF-Protein kodierend identifiziert werden. DWORF ist dabei ein neuer indirekter Aktivator der SERCA und somit im essentiellen Kalziumhaushalt der Muskelzellen beteiligt. Es führt zu einer gesteigerten Kontraktilität durch Verbesserung der Relaxation der Muskelzellen. Aufgrund dessen ist ein potentieller diagnostischer oder therapeutischer Nutzen dieses neuen Proteins bei Patienten mit einer Herzinsuffizienz, zum Beispiel aufgrund einer Kardiomyopathie, denkbar.

Page 179-180

Resource Review

A Biobank for Long-term and Sustainable Research in the Field of Congenital Heart Disease in Germany

Thomas Pickardt, Eva Niggemeyer, Ulrike M.M. Bauer, Hashim Abdul-Khaliq, Competence Network for Congenital Heart Defects Investigators

Congenital heart disease (CHD) is the most frequent birth defect (0.8%–1% of all live births). Due to the advance in prenatal and postnatal early diagnosis and treatment, more than 90% of these patients survive into adulthood today. However, several mid- and long-term morbidities are dominating the follow-up of these patients. Due to the rarity and heterogeneity of the phenotypes of CHD, multicenter registry-based studies are required. The CHD-Biobank was established in 2009 with the aim to collect DNA from patients and their parents (trios) or from affected families, as well as cardiovascular tissues from patients undergoing corrective heart surgery for cardiovascular malformations. Clinical/phenotype data are matched to the International Paediatric and Congenital Cardiac Code (IPCCC) and the International Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD-10). The DNA collection currently comprises samples from approximately 4200 participants with a wide range of CHD phenotypes. The collection covers about 430 trios and 120 families with more than one affected member. The cardiac tissue collection comprises 1143 tissue samples from 556 patients after open heart surgery. The CHD-Biobank provides a comprehensive basis for research in the field of CHD with high standards of data privacy, IT management, and sample logistics.
先天性心脏病（Congenital heart disease, CHD）是最常见的新生儿器官畸形病，目前亟需建立该领域长期的提供可持续研究的生物样本库（Biobank），为揭示其发病机制提供更多的样本。生物样本库CHD-biobank于2009年在德国建立，旨在收集病人及其家属、受影响家庭以及患有心血管畸形并接受了纠正手术治疗病人的心血管组织DNA。目前已收集到具广泛CHD表型的约4200个个体的DNA，包括430个三口之家和120个家庭（至少包含1个被影响成员）；心血管组织收集则包含曾接受开心手术的556个病人的1143个组织样本。该新型生物样本库的建立为CHD领域的研究提供了高标准的个性化数据，并为信息技术和样品管理奠定了较全面的研究基础。

Page 181-190

Resource Review

Long non-coding RNA Databases in Cardiovascular Research

Frank Rühle, Monika Stoll

With the rising interest in the regulatory functions of long non-coding RNAs (lncRNAs) in complex human diseases such as cardiovascular diseases, there is an increasing need in public databases offering comprehensive and integrative data for all aspects of these versatile molecules. Recently, a variety of public data repositories that specialized in lncRNAs have been developed, which make use of huge high-throughput data particularly from next-generation sequencing (NGS) approaches. Here, we provide an overview of current lncRNA databases covering basic and functional annotation, lncRNA expression and regulation, interactions with other biomolecules, and genomic variants influencing the structure and function of lncRNAs. The prominent lncRNA antisense noncoding RNA in the INK4 locus (ANRIL), which has been unequivocally associated with coronary artery disease through genome-wide association studies (GWAS), serves as an example to demonstrate the features of each individual database.
在冠心病病人中，仅有约10%的遗传变异变可以用可编码基因中的变异来解释；全基因组关联分析显示，大多数突变发生在非编码基因上。实际上，整个基因组中只有约1%的序列是可编码蛋白的基因，而至少50%的序列是非编码RNA，它们可能是参与表观遗传学调控的重要组分。长非编码RNA（lncRNA）更是在DNA、RNA和蛋白质功能调控方面发挥着广泛作用。同时， lncRNA功能数据库也越来越多。本文综述了现有的28个lncRNA数据库，主要包括lncRNA的功能、表达、调控、分子互作以及影响其结构和功能的突变等信息；列举了这些数据库的链接、数据来源以及包含的信息等情况，并以冠心病相关的lncRNA—ANRIL为例，描述了每个数据库的特征，方便研究者们对比使用。
Das wachsende Interesse an den regulatorischen Funktionen langer nicht-kodierender RNAs (lncRNAs) in komplexen humanen Erkrankungen, wie Herzkreislauferkrankungen, erfordert zunehmend öffentliche Datenbanken mit umfassenden und integrativen Informationen zu allen Aspekten dieser vielseitigen Moleküle. Viele auf lncRNAs spezialisierte Datenbanken wurden jüngst entwickelt, die auf die Hochdurchsatz-Daten der aktuellen Sequenzierverfahren („Next generation sequencing“, NGS) zugreifen. In diesem Review geben wir eine Übersicht der aktuellen lncRNA Datenbanken mit Informationen zu grundlegenden und funktionalen lncRNA-Annotationen, zur lncRNA-Expression und -Regulation, zu Interaktionen mit anderen Biomolekülen sowie zu genomischen Varianten, die die Struktur und Funktion von lncRNAs beeinflussen können. Anhand der gut dokumentierten lncRNA „antisense noncoding RNA in the INK4 locus“ (ANRIL), die in genomweiten Assoziationsstudien (GWAS) bereits eindeutig mit koronarer Herzkrankheit assoziiert wurde, werden die Eigenschaften der einzelnen Datenbanken beispielhaft demonstriert.

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Original Research

The Role of Quality Control in Targeted Next-generation Sequencing Library Preparation

Rouven Nietsch, Jan Haas, Alan Lai, Daniel Oehler, Stefan Mester, Karen S. Frese, Farbod Sedaghat-Hamedani, Elham Kayvanpour, Andreas Keller, Benjamin Meder

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.
二代测序技术正广泛应用于常规的遗传性疾病的检测，如心肌症等。测序数据的可靠性对准确识别疾病相关突变尤为重要。在样品制备过程中，每个文库富集的流程都需要质量评估，但这些流程对数据质量的影响则较少报道。本文研究了不同建库流程阶段对数据质量产生的潜在影响。作者对600个病人样品的数据参数进行了对比分析，包括DNA起始量、剪切的DNA数量、初始DNA的最小和最大长度、初始扩增后的DNA数量、最终DNA的最小和最大长度、最终文库及相应片段的大小以及基因突变的数量等。有趣的是，这些参数均有很好的保守性，说明只要建库流程中的各个参数符合要求，甚至不需要进行测序后的质量评估。

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Original Research

Comparative Gene Expression Analysis of Mouse and Human Cardiac Maturation

Hideki Uosaki, Y-h Taguchi

Understanding how human cardiomyocytes mature is crucial to realizing stem cell-based heart regeneration, modeling adult heart diseases, and facilitating drug discovery. However, it is not feasible to analyze human samples for maturation due to inaccessibility to samples while cardiomyocytes mature during fetal development and childhood, as well as difficulty in avoiding variations among individuals. Using model animals such as mice can be a useful strategy; nonetheless, it is not well-understood whether and to what degree gene expression profiles during maturation are shared between humans and mice. Therefore, we performed a comparative gene expression analysis of mice and human samples. First, we examined two distinct mice microarray platforms for shared gene expression profiles, aiming to increase reliability of the analysis. We identified a set of genes displaying progressive changes during maturation based on principal component analysis. Second, we demonstrated that the genes identified had a differential expression pattern between adult and earlier stages (e.g., fetus) common in mice and humans. Our findings provide a foundation for further genetic studies of cardiomyocyte maturation.
理解人类心肌细胞的成熟对于揭示基于干细胞的心脏再生、心脏病建模以及促进药物研发具有重要意义。然而，由于心肌的成熟发生在胚胎和幼年时期，由此导致的样品采集的局限性不能直接用于人类心肌发育的分析。本文比较分析了人和老鼠心肌细胞的基因表达异同。作者首先对2个具有相似基因表达模式的老鼠基因芯片进行了数据检测，以提升分析结果的可靠性。之后通过主成分分析，鉴定得到500多个在心肌细胞成熟过程中表达量持续变化的基因，并发现这些基因在幼年和成年的老鼠及人类个体中均差异表达，为该领域的研究提供了众多的遗传标记。
多能性幹細胞から分化誘導した心筋細胞(PSC-CM)を再生医療、疾患研究、創薬などへの応用が期待されている。PSC-CMsは幼弱であり、ヒトの心筋細胞がどのように生体内で成熟していくのかを理解することが、PSC-CMsを成熟させ、様々な応用分野へ利用する上で不可欠である。しかしながら、心筋細胞が成熟する胎児期〜乳幼児期の心臓サンプルを得ることは困難であり、同一個体のサンプルを繰り返し得ることも困難であるため、心筋細胞の成熟過程を経時的に観察できない。そこで、マウスなどのモデル動物を用いた研究が行われているが、どの程度ヒトとマウスで遺伝子変化が共通しているかは明らかではない。そこで本研究ではヒトとマウスの遺伝子発現変化を比較した。まず、マウスで実施されたマイクロアレイのデータから主成分分析を用いて胎仔期から成獣に至るまで経時的に変化する遺伝子を同定した。続いてヒト胎児と成人の比較を行い、これらの遺伝子の大半がヒトとマウスで共通して変化していることを明らかにした。

Page 207-215

Original Research

Profiling and Validation of the Circular RNA Repertoire in Adult Murine Hearts

Tobias Jakobi, Lisa F. Czaja-Hasse, Richard Reinhardt, Christoph Dieterich

For several decades, cardiovascular disease has been the leading cause of death throughout all countries. There is a strong genetic component to many disease subtypes (e.g., cardiomyopathy) and we are just beginning to understand the relevant genetic factors. Several studies have related RNA splicing to cardiovascular disease and circular RNAs (circRNAs) are an emerging player. circRNAs, which originate through back-splicing events from primary transcripts, are resistant to exonucleases and typically not polyadenylated. Initial functional studies show clear phenotypic outcomes for selected circRNAs. We provide, for the first time, a comprehensive catalogue of RNase R-resistant circRNA species for the adult murine heart. This work combines state-of-the-art circle sequencing with our novel DCC software to explore the circRNA landscape of heart tissue. Overall, we identified 575 circRNA species that pass a beta-binomial test for enrichment (false discovery rate of 1%) in the exonuclease-treated sequencing sample. Several circRNAs can be directly attributed to host genes that have been previously described as associated with cardiovascular disease. Further studies of these candidate circRNAs may reveal disease-relevant properties or functions of specific circRNAs.
心血管疾病是一种严重威胁人类健康，具有高患病率和高死亡率的疾病。环状RNA（circular RNAs, circRNA）是一类近年受密切关注的非编码RNA，具有难以多聚腺苷酸化的特点，且稳定性较高。本文基于先进的循环测序技术，首次鉴定得到575种成年小鼠心脏组织中具有核糖核酸酶耐受性的circRNA。这些circRNA在核酸外切酶处理的测序样品中，经β-二项式检验其富集程度，错误发现率仅为1%，具有较高的可信度，并且部分circRNA的宿主基因编码目前已发现的与心血管疾病相关的重要因子，具有重要的生物学意义和临床应用前景。这些circRNA经大样本分析和功能验证后，有望进一步揭示circRNA在心血管疾病中的作用，为攻克心血管疾病这一难题提供新途径。

Page 216-223

Original Research

Absent MicroRNAs in Different Tissues of Patients with Acquired Cardiomyopathy

Christine S. Siegismund, Maria Rohde, Uwe Kühl, Felicitas Escher, Heinz Peter Schultheiss, Dirk Lassner

MicroRNAs (miRNAs) can be found in a wide range of tissues and body fluids, and their specific signatures can be used to determine diseases or predict clinical courses. The miRNA profiles in biological samples (tissue, serum, peripheral blood mononuclear cells or other body fluids) differ significantly even in the same patient and therefore have their own specificity for the presented condition. Complex profiles of deregulated miRNAs are of high interest, whereas the importance of non-expressed miRNAs was ignored. Since miRNAs regulate gene expression rather negatively, absent miRNAs could indicate genes with unaltered expression that therefore are normally expressed in specific compartments or under specific disease situations. For the first time, non-detectable miRNAs in different tissues and body fluids from patients with different diseases (cardiomyopathies, Alzheimer’s disease, bladder cancer, and ocular cancer) were analyzed and compared in this study. miRNA expression data were generated by microarray or TaqMan PCR-based platforms. Lists of absent miRNAs of primarily cardiac patients (myocardium, blood cells, and serum) were clustered and analyzed for potentially involved pathways using two prediction platforms, i.e., miRNA enrichment analysis and annotation tool (miEAA) and DIANA miRPath. Extensive search in biomedical publication databases for the relevance of non-expressed miRNAs in predicted pathways revealed no evidence for their involvement in heart-related pathways as indicated by software tools, confirming proposed approach.
研究表明，microRNA（miRNA）广泛参与各种生命活动的调控过程，并与个体的生理病理状态的转换有着密切联系。本文对比分析了不同疾病患者的各种组织及体液中non-detectable miRNA，结合多种生物信息学分析方法预测了心脏病患者（心肌、血细胞、体液）中non-detectable miRNA可能参与的信号通路。之后将这些miRNA在生物医学出版数据库中进行关联性搜索，证实了它们与心脏疾病相关的通路无关。本文同时表明不同的软件会造成分析结果的差异性，因此对于分析结果要小心解释，并应将未表达miRNA纳入考虑范围内。

Page 224-234

Original Research

Comparison of Cox Model Methods in A Low-dimensional Setting with Few Events

Francisco M. Ojeda, Christian Müller, Daniela Börnigen, David-Alexandre Trégouët, Arne Schillert, Matthias Heinig, Tanja Zeller, Renate B. Schnabel

Prognostic models based on survival data frequently make use of the Cox proportional hazards model. Developing reliable Cox models with few events relative to the number of predictors can be challenging, even in low-dimensional datasets, with a much larger number of observations than variables. In such a setting we examined the performance of methods used to estimate a Cox model, including (i) full model using all available predictors and estimated by standard techniques, (ii) backward elimination (BE), (iii) ridge regression, (iv) least absolute shrinkage and selection operator (lasso), and (v) elastic net. Based on a prospective cohort of patients with manifest coronary artery disease (CAD), we performed a simulation study to compare the predictive accuracy, calibration, and discrimination of these approaches. Candidate predictors for incident cardiovascular events we used included clinical variables, biomarkers, and a selection of genetic variants associated with CAD. The penalized methods, i.e., ridge, lasso, and elastic net, showed a comparable performance, in terms of predictive accuracy, calibration, and discrimination, and outperformed BE and the full model. Excessive shrinkage was observed in some cases for the penalized methods, mostly on the simulation scenarios having the lowest ratio of a number of events to the number of variables. We conclude that in similar settings, these three penalized methods can be used interchangeably. The full model and backward elimination are not recommended in rare event scenarios.
预后模型可用于预测病人的病程、评估患某种疾病或疾病复发的风险以及指导选择治疗方案等。预后模型的构建基于临床随访数据，这类数据常因随访对象的失访而产生截尾数据。本研究中，作者基于平均随访时间为5.7年的包括1731个样本和209个事件的冠心病真实数据集的模拟，对比分析了full model（考虑所有因素）、后向消除法（BE）、岭回归法（ridge regression）、lasso回归法以及弹性神经网络（elastic net）等5种方法对计算Cox比例风险模型的影响，包括该模型的预测准确性和判断力等。结果表明，后3种基于罚分的方法具有相近的评估能力，而前2种方法的评估能力较差。因此在事件删失较多的情况下，作者推荐使用岭回归法、lasso回归法或弹性神经网络建立预测模型。
Los módelos pronósticos están basados frecuentemente en el modelo de riesgos proporcionales de Cox. El desarrollo de modelos de Cox fiables con pocos eventos respecto al número de predictores puede ser díficil, aún con datos de baja dimension y con muchas más observaciones que variables. En este escenario examinamos el desempeño de métodos utilizados para estimar un módelo de Cox, incluyendo (i) modelo completo usando todo los predictores disponibles y estimado usando técnicas estándar, (ii) eliminación retrógrada (BE, por sus siglas en inglés), (iii) regresión contraída (ridge), (iv) lazo (lasso, least absolute shrinkage and selection operator), y (v) la red elástica. Basado en estudio de cohorte prospectivo de pacientes con enfermedad de las arterias coronarias (EAC), realizamos un estudio de simulación para comparar la exactitud predictiva, la calibración, y la discriminación de los distintos métodos. Como predictores usamos variables clínicas, biomarcadores, y una selección de variantes genéticas asociadas con EAC. Los métodos penalizados, es decir, la regresión contraída, el lazo y la red elástica mostraron un rendimiento comparable, en términos de exactitud predictiva, calibración y discriminación, y su desempeño fue superior al de BE y el modelo completo. Reducción excesiva fue observada en algunos casos para los métodos penalizados, en su mayoría, en simulaciones que tenían el menor proporción de número de eventos a número de variables. Concluímos que en escenarios similares, los tres métodos penalizados pueden ser usados de manera indistinta. El modelo completo y la eliminación retrógrada no son recomendados en escenarios con pocos eventos.

Page 235-243

Original Research

Personalized Computer Simulation of Diastolic Function in Heart Failure

Ali Amr, Elham Kayvanpour, Farbod Sedaghat-Hamedani, Tiziano Passerini, Viorel Mihalef, Alan Lai, Dominik Neumann, Bogdan Georgescu, Sebastian Buss, Derliz Mereles, Edgar Zitron, Andreas E. Posch, Maximilian Würstle, Tommaso Mansi, Hugo A. Katus, Benjamin Meder

The search for a parameter representing left ventricular relaxation from non-invasive and invasive diagnostic tools has been extensive, since heart failure (HF) with preserved ejection fraction (HF-pEF) is a global health problem. We explore here the feasibility using patient-specific cardiac computer modeling to capture diastolic parameters in patients suffering from different degrees of systolic HF. Fifty eight patients with idiopathic dilated cardiomyopathy have undergone thorough clinical evaluation, including cardiac magnetic resonance imaging (MRI), heart catheterization, echocardiography, and cardiac biomarker assessment. A previously-introduced framework for creating multi-scale patient-specific cardiac models has been applied on all these patients. Novel parameters, such as global stiffness factor and maximum left ventricular active stress, representing cardiac active and passive tissue properties have been computed for all patients. Invasive pressure measurements from heart catheterization were then used to evaluate ventricular relaxation using the time constant of isovolumic relaxation Tau (τ). Parameters from heart catheterization and the multi-scale model have been evaluated and compared to patient clinical presentation. The model parameter global stiffness factor, representing diastolic passive tissue properties, is correlated significantly across the patient population with τ. This study shows that multi-modal cardiac models can successfully capture diastolic (dys) function, a prerequisite for future clinical trials on HF-pEF.
正常射血分数心力衰竭（HF-pEF）是全球性的健康问题，许多研究在侵入性和非侵入性诊断方法中致力于寻找代表左心室松弛的参数。本文用具有不同程度的收缩性心力衰竭病人特异的心脏计算机建模来捕获舒张压参数，对58个扩张型心肌病病人进行了系统的临床评价，包括心脏核磁共振成像、心脏导管插入术、超声心动图和心脏生物标记物等评估。作者首先利用一个多尺度病人特异的心脏模型，计算了所有病人的代表心脏主动和被动的组织特点的新参数，如全局劲度因子（global stiffness factor）和最大左心室主动力（maximum left ventricular active stress）；之后用心脏导管术的等容舒张时间常数Tau来评估心室舒张功能，发现该模型的全局劲度因子可代表舒张压被动组织特性，与病人的Tau显著相关，表明多尺度的心脏模型可以成功地捕获舒张压功能，在未来HF-pEF临床试验中有很好的应用前景。

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