Severe DCM phenotype of patient harboring RBM20 mutation S635A can be modeled by patient-specific induced pluripotent stem cell-derived cardiomyocytes

Abstract

The ability to generate patient-specific induced pluripotent stem cells (iPSCs) provides a unique opportunity for modeling heart disease in vitro. In this study, we generated iPSCs from a patient with dilated cardiomyopathy (DCM) caused by a missense mutation S635A in RNA-binding motif protein 20 (RBM20) and investigated the functionality and cell biology of cardiomyocytes (CMs) derived from patient-specific iPSCs (RBM20-iPSCs). The RBM20-iPSC-CMs showed abnormal distribution of sarcomeric α-actinin and defective calcium handling compared to control-iPSC-CMs, suggesting disorganized myofilament structure and altered calcium machinery in CMs of the RBM20 patient. Engineered heart muscles (EHMs) from RBM20-iPSC-CMs showed that not only active force generation was impaired in RBM20-EHMs but also passive stress of the tissue was decreased, suggesting a higher visco-elasticity of RBM20-EHMs. Furthermore, we observed a reduced titin (TTN) N2B-isoform expression in RBM20-iPSC-CMs by demonstrating a reduction of exon skipping in the PEVK region of TTN and an inhibition of TTN isoform switch. In contrast, in control-iPSC-CMs both TTN isoforms N2B and N2BA were expressed, indicating that the TTN isoform switch occurs already during early cardiogenesis. Using next generation RNA sequencing, we mapped transcriptome and splicing target profiles of RBM20-iPSC-CMs and identified different cardiac gene networks in response to the analyzed RBM20 mutation in cardiac-specific processes. These findings shed the first light on molecular mechanisms of RBM20-dependent pathological cardiac remodeling leading to DCM. Our data demonstrate that iPSC-CMs coupled with EHMs provide a powerful tool for evaluating disease-relevant functional defects and for a deeper mechanistic understanding of alternative splicing-related cardiac diseases.

Modelling of Genetic Cardiac Diseases

“…In vitro model of RBM20 hiPSC-CMs successfully mirrored the altered titin Modelling of Genetic Cardiac Diseases DOI: http://dx.doi.org/10.5772/intechopen.84965 isoform expression (titin isoform switch) [89,90]. Furthermore, RBM20 hiPSC-CMs showed delayed Ca 2+ extrusion and reuptake and more Ca 2+ being released during each ECC, which resulted into deficient muscle contraction, the hallmark of cardiac dysfunction of DCM patients [89,90]. In addition, a three-dimensional engineered heart muscle generated from RBM20 hiPSC-CMs showed an impaired force of contraction, and passive stress was decreased in response to stepwise increase in strain, suggesting higher viscoelasticity caused by mutation in RBM20 [89].…”

“…RBM20 is the main regulator of the heart-specific titin splicing, and N2BA isoform is predominantly expressed in CMs from DCM patient carrying mutation in the RBM20 gene [91]. In vitro model of RBM20 hiPSC-CMs successfully mirrored the altered titin Modelling of Genetic Cardiac Diseases DOI: http://dx.doi.org/10.5772/intechopen.84965 isoform expression (titin isoform switch) [89,90]. Furthermore, RBM20 hiPSC-CMs showed delayed Ca 2+ extrusion and reuptake and more Ca 2+ being released during each ECC, which resulted into deficient muscle contraction, the hallmark of cardiac dysfunction of DCM patients [89,90].…”

“…DCM patients with mutations in RBM20, encoding RNA binding motif protein 20 (RBM20), have an early onset of disease phenotype [88]. Isolated CMs from DCM patients carrying mutation in RBM20 displayed elongated and thinner sarcomere structure [88], and such disorganized sarcomeric structure phenotypes were recapitulated in DCM hiPSC-CMs carrying mutation in RBM20 [89,90]. RBM20 is the main regulator of the heart-specific titin splicing, and N2BA isoform is predominantly expressed in CMs from DCM patient carrying mutation in the RBM20 gene [91].…”

“…Furthermore, RBM20 hiPSC-CMs showed delayed Ca 2+ extrusion and reuptake and more Ca 2+ being released during each ECC, which resulted into deficient muscle contraction, the hallmark of cardiac dysfunction of DCM patients [89,90]. In addition, a three-dimensional engineered heart muscle generated from RBM20 hiPSC-CMs showed an impaired force of contraction, and passive stress was decreased in response to stepwise increase in strain, suggesting higher viscoelasticity caused by mutation in RBM20 [89]. Besides HCM, mutation in cTnT also caused DCM and resulted in shifts in Ca 2+ sensitivity and force of contraction [92].…”

“…One of the most questionable issues of hiPSC-CMs is their maturity. Despite expressing relevant ion channels [107] and structural genes [25,26,75,76,89,108], hiPSC-CMs lack t-tubules and exhibit lower expression of Kir2.1 and weaker contractility; thus they do not fully resemble adult CMs. In order to improve the maturity of hiPSC-CMs and consequently upgrade the functionality of hiPSC-CMs, various techniques have been investigated in different groups.…”