Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency

Abstract

Cardiomyocytes differentiated from human induced Pluripotent Stem Cells (hiPSC- CMs) are a unique source for modelling inherited cardiomyopathies. In particular, the possibility of observing maturation processes in a simple culture dish opens novel perspectives in the study of early-disease defects caused by genetic mutations before the onset of clinical manifestations. For instance, calcium handling abnormalities are considered as a leading cause of cardiomyocyte dysfunction in several genetic-based dilated cardiomyopathies, including rare types such as Duchenne Muscular Dystrophy (DMD)-associated cardiomyopathy. To better define the maturation of calcium handling we simultaneously measured action potential and calcium transients (Ca-Ts) using fluorescent indicators at specific time points. We combined micropatterned substrates with long-term cultures to improve maturation of hiPSC-CMs (60, 75 or 90 days post-differentiation). Control-(hiPSC)-CMs displayed increased maturation over time (90 vs 60 days), with longer action potential duration (APD), increased Ca-T amplitude, faster Ca-T rise (time to peak) and Ca-T decay (RT50). The progressively increased contribution of the SR to Ca release (estimated by post-rest potentiation or Caffeine-induced Ca-Ts) appeared as the main determinant of the progressive rise of Ca-T amplitude during maturation. As an example of severe cardiomyopathy with early onset, we compared hiPSC-CMs generated from a DMD patient (DMD-ΔExon50) and a CRISPR-Cas9 genome edited cell line isogenic to the healthy control with deletion of a G base at position 263 of the DMD gene (c.263delG-CMs). In DMD-hiPSC-CMs, changes of Ca-Ts during maturation were less pronounced: indeed, DMD cells at 90 days showed reduced Ca-T amplitude and faster Ca-T rise and RT50, as compared with control hiPSC-CMs. Caffeine-Ca-T was reduced in amplitude and had a slower time course, suggesting lower SR calcium content and NCX function in DMD vs control cells. Nonetheless, the inotropic and lusitropic responses to forskolin were preserved. CRISPR-induced c.263delG-CM line recapitulated the same developmental calcium handling alterations observed in DMD-CMs. We then tested the effects of micropatterned substrates with higher stiffness. In control hiPSC-CMs, higher stiffness leads to higher amplitude of Ca-T with faster decay kinetics. In hiPSC-CMs lacking full-length dystrophin, however, stiffer substrates did not modify Ca-Ts but only led to higher SR Ca content. These findings highlighted the inability of dystrophin-deficient cardiomyocytes to adjust their calcium homeostasis in response to increases of extracellular matrix stiffness, which suggests a mechanism occurring during the physiological and pathological development (i.e. fibrosis).

Gene correction and overexpression of TNNI3 improve impaired relaxation in engineered heart tissue model of pediatric restrictive cardiomyopathy

“…Western blot analysis showed that the ratio of TNNI3 to TNNI1 is higher in R170W‐EHT than in Isogenic‐EHT (Figure 4c). Although it is generally accepted that cardiac maturation in iPSC‐CMs contributes to increased contractility and shortening of Ca 2+ tau (Pioner et al, 2022), we observed no difference in the force at the maximum pillar deflection (Figure 4e) and shorter tau in Isogenic‐EHT (Figure 4f). We also observed significantly prolonged Ca 2+ tau in R170W‐iPSC‐CMs (Figure 3d).…”

“…Although it is generally accepted that cardiac maturation in iPSC-CMs contributes to increased contractility and shortening of Ca 2+ tau (Pioner et al, 2022), we observed no difference in the force at the maximum pillar deflection (Figure 4e) and shorter tau in Isogenic-EHT…”

“…Nonetheless, we found significant differences between the disease and gene correction models, which highlight the importance of our findings. In clinical practice, evaluation of diastolic dysfunction in RCM involves determining ventricular pressure through cardiac catheterization (Rapezzi et al, 2022). However, there is currently no methodology available for direct quantification of diastolic dysfunction in vitro.…”

Nongenetic Optical Modulation of Pluripotent Stem Cells Derived Cardiomyocytes Function in the Red Spectral Range

“…Furthermore, hPSC‐CMs plated on polymer show: higher CaT amp (Figure 1D); faster TtP and t 1/2 (Figure 1E,F), and higher spontaneous CaT frequency (Figure 1G), as compared to control samples on GLASS. All these features resemble those of mature CMs, [ 42–44 ] thus supporting a modulation of hPSC‐CMs biological properties and functionality by PCPDTBT thin films, even when used as culturing substrates without optical excitation. Interestingly, these data are in line with those obtained in HL‐1 cells cultured on electrically‐conductive polymeric substrates (polypyrrole‐polycaprolactone, PPy‐PCL).…”

Building blocks of microphysiological system to model physiology and pathophysiology of human heart

“…Substrate stiffness and topography can greatly affect hiPSC-CM maturation. Soft substrates close to the physiological range of myocardium are often reported to promote structural and functional maturation in comparison to rigid surfaces ( Körner et al, 2021 ; Pioner et al, 2022 ). However, the concept of the substrate stiffness is complex and cell response greatly depends on spatial dimension (e.g., 2D vs. 3D) and topography, such as aligned grooves, and the choice of material.…”