How can we use stem cell-derived cardiomyocytes to understand the involvement of energetic metabolism in alterations of cardiac function?

Mutations in the mitochondrial-DNA or mitochondria related nuclear-encoded-DNA lead to various multisystemic disorders collectively termed mitochondrial diseases. One in three cases of mitochondrial disease affects the heart muscle, which is called mitochondrial cardiomyopathy (MCM) and is associated with hypertrophic, dilated, and noncompact cardiomyopathy. The heart is an organ with high energy demand, and mitochondria occupy 30%–40% of its cardiomyocyte-cell volume. Mitochondrial dysfunction leads to energy depletion and has detrimental effects on cardiac performance. However, disease development and progression in the context of mitochondrial and nuclear DNA mutations, remains incompletely understood. The system of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) is an excellent platform to study MCM since the unique genetic identity to their donors enables a robust recapitulation of the predicted phenotypes in a dish on a patient-specific level. Here, we focus on recent insights into MCM studied by patient-specific iPSC-CM and further discuss research gaps and advances in metabolic maturation of iPSC-CM, which is crucial for the study of mitochondrial dysfunction and to develop novel therapeutic strategies

Generation of iPSC-lines from two independent Takotsubo syndrome patients with recurrent Takotsubo events

The Takotsubo syndrome (TTS) is characterized by acute transient left ventricular dysfunction in the absence of obstructive coronary lesions. An enhanced β-adrenergic signaling and higher sensitivity to catecholamine-induced-toxicity were identified as mechanisms associated with TTS. It is still elusive, whether TTS patients with recurrent events show similar underlying signaling pathomechanism. Induced pluripotent stem cell (iPSC)-lines were generated from skin fibroblasts of two independent female Takotsubo syndrome patients with a severe phenotype characterized by recurrent TTS events. For reprogramming, a non-integrative plasmid technique was used. All generated iPSCs maintained full pluripotency, genomic integrity, and spontaneous in vitro and in vivo differentiation capacity

Generation of a pluripotent stem cell line (UMGi270-A) and a corresponding CRISPR/Cas9 modified isogenic control (UMGi270-A-1) from a patient with sudden onset dilated cardiomyopathy harboring a FLNC p.R2187P mutation

Filamin C (FLNC) is a highly important actin crosslinker and multi-adaptor protein in striated skeletal and cardiac muscle. Mutations have been linked to a range of cardiomyopathy types. Here, we generated induced pluripotent stem cells (iPSC) from a patient with dilated cardiomyopathy (DCM) harboring a new, unique heterozygous FLNC mutation p.R2187P. From this patient-specific iPSC line, a corresponding isogenic control line was created by CRISPR/Cas9 genome editing. Both, the patient-specific and isogenic-control iPSC maintained full pluripotency, genomic integrity, and in vitro differentiation capacity. All iPSC lines differentiate into iPSC-cardiomyocytes, hence providing the possibility to study the pathogenesis of FLNC-mediated DCM further

Generation of an RBM20-mutation-associated left-ventricular non-compaction cardiomyopathy iPSC line (UMGi255-A) into a DCM genetic background to investigate monogenetic cardiomyopathies

RBM20 mutations account for 3 % of genetic cardiomypathies and manifest with high penetrance and arrhythmogenic effects. Numerous mutations in the conserved RS domain have been described as causing dilated cardiomyopathy (DCM), whereas a particular mutation (p.R634L) drives development of a different cardiac phenotype: left-ventricular non-compaction cardiomyopathy. We generated a mutation-induced pluripotent stem cell (iPSC) line in which the RBM20-LVNC mutation p.R634L was introduced into a DCM patient line with rescued RBM20-p.R634W mutation. These DCM-634L-iPSC can be differentiated into functional cardiomyocytes to test whether this RBM20 mutation induces development of the LVNC phenotype within the genetic context of a DCM patient

Generation and cardiac differentiation of an induced pluripotent stem cell line from a patient with arrhythmia-induced cardiomyopathy

Arrhythmia-induced cardiomyopathy (AIC) is characterized by left-ventricular systolic dysfunction caused by persistent arrhythmia. To date, genetic or pathological drivers causing AIC remain unknown. Here, we generated induced pluripotent stem cells (iPSCs) from an AIC patient. The AIC-iPSCs exhibited full pluripotency and differentiation characteristics and maintained a normal karyotype after reprogramming. The AIC-iPSCs differentiated into functional beating AIC-iPSCcardiomyocytes (CMs), which represents the cell-type of interest to study molecular, genetic and functional aspects of AIC

A roadmap for the characterization of energy metabolism in human cardiomyocytes derived from induced pluripotent stem cells

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are an increasingly employed model in cardiac research and drug discovery. As cellular metabolism plays an integral role in determining phenotype, the characterization of the metabolic profile of hiPSC-CM during maturation is crucial for their translational application. In this study we employ a combination of methods including extracellular flux, 13C-glucose enrichment and targeted metabolomics to characterize the metabolic profile of hiPSC-CM during their maturation in culture from 6 weeks, up to 12 weeks. Results show a progressive remodeling of pathways involved in energy metabolism and substrate utilization along with an increase in sarcomere regularity. The oxidative capacity of hiPSC-CM and particularly their ability to utilize fatty acids increased with time. In parallel, relative glucose oxidation was reduced while glutamine oxidation was maintained at similar levels. There was also evidence of increased coupling of glycolysis to mitochondrial respiration, and away from glycolytic branch pathways at later stages of maturation. The rate of glycolysis as assessed by lactate production was maintained at both stages but with significant alterations in proximal glycolytic enzymes such as hexokinase and phosphofructokinase. We observed a progressive maturation of mitochondrial oxidative capacity at comparable levels of mitochondrial content between these time-points with enhancement of mitochondrial network structure. These results show that the metabolic profile of hiPSC-CM is progressively restructured, recapitulating aspects of early post-natal heart development. This would be particularly important to consider when employing these cell model in studies where metabolism plays an important role

Atrial Fibrillation Burden Specifically Determines Human Ventricular Cellular Remodeling

BACKGROUND Atrial fibrillation (AF) can either be a consequence or an underlying mechanism of left ventricular systolic dysfunction. Patients included in the CASTLE-AF (Catheter Ablation vs. Standard Conventional Treatment in Patients With LV Dysfunction and AF) trial who suffered from AF and left ventricular systolic dysfunction benefited from an AF burden 50%.OBJECTIVES This analysis tried to explain the clinical findings of the CASTLE-AF trial regarding AF burden in a "back-to-bench" approach. METHODS To study the ventricular effects of different AF burdens, experiments were performed using human ventricular induced pluripotent stem cell-derived cardiomyocytes undergoing in vitro AF simulation. Epifluorescence microscopy, action potential measurements, and measurements of sarcomere regularity were conducted.RESULTS Induced pluripotent stem cell-derived cardiomyocytes stimulated with AF burden of 60% or higher displayed typical hallmarks of heart failure. Ca2 thorn transient amplitude was significantly reduced indicating negative inotropic effects. Action potential duration was significantly prolonged, which represents a potential trigger for arrhythmias. A significant decrease of sarcomere regularity could explain impaired cardiac contractility in patients with high AF burden. These effects were more pronounced after 7 days of AF simulation compared with 48 hours.CONCLUSIONS Significant functional and structural alterations occurred at the cellular level at a threshold of w50% AF burden as it was observed to be harmful in the CASTLE-AF trial. Therefore, these translational results may help to understand the findings of the CASTLE-AF trial. (J Am Coll Cardiol EP 2022;8:1357-1366) (c) 2022 by the American College of Cardiology Foundation

Genotype Complements the Phenotype: Identification of the Pathogenicity of an LMNA Splice Variant by Nanopore Long-Read Sequencing in a Large DCM Family

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