Mechanomics study of cardiomyocytes with filamin c truncations

Abstract Background Filamin C (FLNC) is expressed in cardiomyocytes, where it binds to actin and localizes to Z-discs, sarcolemma, and intercalated discs. Although FLNC truncation variants (FLNCtv) are an established cause of arrhythmias and heart failure (arrhythmogenic cardiomyopathy), the underlying mechanisms, in particular changes in mechanical properties of cardiomyocytes, are mostly unknown. Purpose In this study, we applied a biophysical approach to investigate the interaction between altered mechanical properties and biological response in human induced pluripotent stem cells–derived cardiomyocytes (hiPSC-CMs) carrying FLNCtv. Methods CRISPR/Cas9 genome-edited FLNC-/- hiPSC-CMs, a homozygous condition that is lethal in animal models, and FLNC+/- hiPSC-CMs, recapitulating the heterozygous human disease, were analyzed. Wild-type FLNC hiPSC-CMs were used as controls. Atomic Force microscopy (AFM) was used to perform single-cell force spectroscopy (SCFS) microindentation, to evaluate passive and dynamic mechanical properties. From the loading curves, two cell Young’s Moduli were calculated: E1, related to the compression of the plasma membrane and actin cortex, and E2, including the rest of the inner cell layers (cytoskeleton and nucleus). Cell adhesion was assessed using the retraction curve of the SCFS. Data were processed in terms of adhesion force (the highest value of the force exerted by the cell-surface contact to the cantilever during retraction) and work of adhesion (integrating the area under the retraction curve). Finally, FLNC-/- hiPSC-CMs were incubated with 1 µM Crenolanib (a PDGFRA signaling inhibitor) for 3 days, and the microindentation was performed again using DMSO-only-treated FLNC-/- cells as controls. Results The AFM indentation curve for the cells analyzed showed significant differences. Both elastic contributions (E1 and E2) show that wild-type FLNC iPSC-CMs are stiffer than both mutant FLNC+/- and FLNC-/- iPSC-CMs (see Figure upper panel). The adhesion showed the same trend as the elasticity: mutant FLNC iPSC-CMs showed a progressive decrease of work of adhesion as well as max adhesion force from the FLNC+/- to the FLNC-/- models. Rescue of the latter with Crenolanib showed to moderately increase their stiffness. Moreover, a qualitative analysis of the beating traces showed that FLNC+/- but mostly FLNC-/- display "irregular" small peaks, resembling those of delayed after depolarizations (see Figure lower panel). Conclusions We found that FLNCtv causes a decrease in cell stiffness which declines progressively from the heterozygous status, which is associated with FLNC haploinsufficiency, to the homozygous status, which lacks FLNC. This indicates a progressive softening of the cardiomyocytes, suggesting a damaged cytoskeleton. Crenolanib rescue suggests that the inhibition of PDGFRA signaling could partially restore such damage (see Figure upper panel - right corner).