Quantitative Evaluation of the Sarcomere Network of Human hiPSC-Derived Cardiomyocytes Using Single-Molecule Localization Microscopy

Abstract

The maturation of iPSC-derived cardiomyocytes is still a critical point for their application in cardiovascular research as well as for their clinical use. Although multiple differentiation protocols have been established, researchers failed to generate fully mature cardiomyocytes in vitro possessing identical phenotype-related and functional properties as their native adult counterparts. Besides electrophysiological and metabolic changes, the establishment of a well structured sarcomere network is important for the development of a mature cardiac phenotype. Here, we present a super resolution-based approach to quantitatively evaluate the structural maturation of iPSC-derived cardiomyocytes. Fluorescence labelling of the α-actinin cytoskeleton and subsequent visualization by photoactivated localization microscopy allows the acquisition of highly resolved images for measuring sarcomere length and z-disc thickness. Our image analysis revealed that iPSC and neonatal cardiomyocyte share high similarity with respect to their sarcomere organization, however, contraction capacity was inferior in iPSC-derived cardiac cells, indicating an early maturation level. Moreover, we demonstrate that this imaging approach can be used as a tool to monitor cardiomyocyte integrity, helping to optimize iPSC differentiation as well as somatic cell direct-reprogramming strategies.

Switching in the expression pattern of actin isoforms marks the onset of contractility and distinct mechanodynamic behavior during cardiomyocyte differentiation

“…The sarcomeric α‐actinin is an integral component of Z‐disks which define the unit length of a sarcomere. By analyzing the distance between bands in images of mCM cells, we determined a length of 1.65 ± 0.03 µm (mean ± SEM), which is smaller than that of typical primary adult cells, but in line with previous reports on iPSC‐derived cardiomyocytes (Figure ; Lemcke et al, 2020). Finally, we wanted to ascertain if the expression of smooth muscle α‐actin (SMA) detected in Western Blots was a feature of the iCM cytoskeleton (Figure 4b, bottom).…”

Monitoring the maturation of the sarcomere network: a super-resolution microscopy-based approach

“…1 ). This morphological and structural phenotype of iPSC CMs is more comparable to immature, neonatal cardiac cells that also exhibit an irregular morphology and a disorganized contraction apparatus [ 8 , 22 , 38 ]. A fact that is verified by our approach, showing that CMs derived from iPSCs share more similarities with neonatal CMs as with native cardiac cells (Figs.…”

“…In adult CMs, the sarcomere length is ~ 2 µm and the respective filaments are aligned perpendicular to the longitudinal axis to generate maximum contraction forces. In contrast, iPSC-CMs often demonstrate a reduced sarcomere length and disorganized filaments, scattered throughout the cytoplasm [ 8 ]. Furthermore, the formation of a proper contraction network encompasses changes in gene expression and isoform switching of sarcomeric proteins.…”

Left ventricular noncompaction associated with titin-truncating variants in the TTN gene

“…Однако, в связи с большим размером гена TTN и технической сложностью его изучения с применением генно-инженерных подходов, возможности изучения патогенной роли вариантов в этом гене в эксперименте ограничены. Активно применяющийся на сегодняшний день подход с применением индуцированных плюрипотентных клеток, полученных от пациентов, также имеет свои ограничения при изучении функциональной значимости вариантов гена TTN в связи с незрелой и зачастую несостоятельной структурой саркомера, получаемой при дифференцировке индуцированных плюрипотентных стволовых клеток [13]. Это повышает важность биоинформатических методов анализа вариантов в гене TTN.…”