Identification of novel genetic risk factors of dilated cardiomyopathy: from canine to human.

BACKGROUND: Dilated cardiomyopathy (DCM) is a life-threatening heart disease and a common cause of heart failure due to systolic dysfunction and subsequent left or biventricular dilatation. A significant number of cases have a genetic etiology; however, as a complex disease, the exact genetic risk factors are largely unknown, and many patients remain without a molecular diagnosis. METHODS: We performed GWAS followed by whole-genome, transcriptome, and immunohistochemical analyses in a spontaneously occurring canine model of DCM. Canine gene discovery was followed up in three human DCM cohorts. RESULTS: Our results revealed two independent additive loci associated with the typical DCM phenotype comprising left ventricular systolic dysfunction and dilatation. We highlight two novel candidate genes, RNF207 and PRKAA2, known for their involvement in cardiac action potentials, energy homeostasis, and morphology. We further illustrate the distinct genetic etiologies underlying the typical DCM phenotype and ventricular premature contractions. Finally, we followed up on the canine discoveries in human DCM patients and discovered candidate variants in our two novel genes. CONCLUSIONS: Collectively, our study yields insight into the molecular pathophysiology of DCM and provides a large animal model for preclinical studies.

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Circadian Dependence of the Acute Immune Response to Myocardial Infarction.

Circadian rhythms influence the recruitment of immune cells and the onset of inflammation, which is pivotal in the response to ischemic cardiac injury after a myocardial infarction (MI). The hyperacute immune response that occurs within the first few hours after a MI has not yet been elucidated. Therefore, we characterized the immune response and myocardial damage 3 hours after a MI occurs over a full twenty-four-hour period to investigate the role of the circadian rhythms in this response. MI was induced at Zeitgeber Time (ZT) 2, 8, 14, and 20 by permanent ligation of the left anterior descending coronary artery. Three hours after surgery, animals were terminated and blood and hearts collected to assess the immunological status and cardiac damage. Blood leukocyte numbers varied throughout the day, peaking during the rest-phase (ZT2 and 8). Extravasation of leukocytes was more pronounced during the active-phase (ZT14 and 20) and was associated with greater chemokine release to the blood and expression of adhesion molecules in the heart. Damage to the heart, measured by Troponin-I plasma levels, was elevated during this time frame. Clock gene oscillations remained intact in both MI-induced and sham-operated mice hearts, which could explain the circadian influence of the hyperacute inflammatory response after a MI. These findings are in line with the clinical observation that patients who experience a MI early in the morning (i.e., early active phase) have worse clinical outcomes. This study provides further insight on the immune response occurring shortly after an MI, which may contribute to the development of novel and optimization of current therapeutic approaches.

Massive expansion and cryopreservation of functional human induced pluripotent stem cell-derived cardiomyocytes.

Since the discovery of human induced pluripotent stem cells (hiPSCs), numerous strategies have been established to efficiently derive cardiomyocytes from hiPSCs (hiPSC-CMs). Here, we describe a cost-effective strategy for the subsequent massive expansion (>250-fold) of high-purity hiPSC-CMs relying on two aspects: removal of cell-cell contacts and small-molecule inhibition with CHIR99021. The protocol maintains CM functionality, allows cryopreservation, and the cells can be used in downstream assays such as disease modeling, drug and toxicity screening, and cell therapy. For complete details on the use and execution of this protocol, please refer to Buikema (2020).