Lazzarino Marco, Zanetti Michele, Chen Suet Nee, Gao Shanshan, Peña Brisa, Lam Chi Keung, Wu Joseph C, Taylor Matthew R G, Mestroni Luisa, Sbaizero Orfeo
CNR-IOM, Area Science Park, 34149 Trieste, Italy.
Cardiovascular Institute, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
Int J Mol Sci. 2024 Mar 3;25(5):2942. doi: 10.3390/ijms25052942.
Actin-binding filamin C (FLNC) is expressed in cardiomyocytes, where it localizes to Z-discs, sarcolemma, and intercalated discs. Although FLNC truncation variants () are an established cause of arrhythmias and heart failure, changes in biomechanical properties of cardiomyocytes are mostly unknown. Thus, we investigated the mechanical properties of human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) carrying . CRISPR/Cas9 genome-edited homozygous FLNC hiPSC-CMs and heterozygous knock-out FLNC hiPSC-CMs were analyzed and compared to wild-type FLNC (FLNC) hiPSC-CMs. Atomic force microscopy (AFM) was used to perform micro-indentation to evaluate passive and dynamic mechanical properties. A qualitative analysis of the beating traces showed gene dosage-dependent-manner "irregular" peak profiles in FLNC and FLNC hiPSC-CMs. Two Young's moduli were calculated: E1, reflecting the compression of the plasma membrane and actin cortex, and E2, including the whole cell with a cytoskeleton and nucleus. Both E1 and E2 showed decreased stiffness in mutant FLNC and FLNC iPSC-CMs compared to that in FLNC. The cell adhesion force and work of adhesion were assessed using the retraction curve of the SCFS. Mutant FLNC iPSC-CMs showed gene dosage-dependent decreases in the work of adhesion and adhesion forces from the heterozygous FLNC to the FLNC model compared to FLNC, suggesting damaged cytoskeleton and membrane structures. Finally, we investigated the effect of crenolanib on the mechanical properties of hiPSC-CMs. Crenolanib is an inhibitor of the Platelet-Derived Growth Factor Receptor α (PDGFRA) pathway which is upregulated in hiPSC-CMs. Crenolanib was able to partially rescue the stiffness of FLNC hiPSC-CMs compared to control, supporting its potential therapeutic role.
肌动蛋白结合细丝蛋白C（FLNC）在心肌细胞中表达，定位于Z线、肌膜和闰盘。尽管FLNC截短变体是心律失常和心力衰竭的既定病因，但心肌细胞生物力学特性的变化大多未知。因此，我们研究了携带FLNC截短变体的人诱导多能干细胞衍生心肌细胞（hiPSC-CMs）的力学特性。对经CRISPR/Cas9基因组编辑的纯合FLNC hiPSC-CMs和杂合敲除FLNC hiPSC-CMs进行分析，并与野生型FLNC（FLNC）hiPSC-CMs进行比较。使用原子力显微镜（AFM）进行微压痕以评估被动和动态力学特性。对搏动轨迹的定性分析显示，FLNC和FLNC hiPSC-CMs中存在基因剂量依赖性的“不规则”峰值轮廓。计算了两个杨氏模量：E1反映质膜和肌动蛋白皮质的压缩，E2包括带有细胞骨架和细胞核的整个细胞。与FLNC相比，突变型FLNC和FLNC iPSC-CMs中的E1和E2刚度均降低。使用SCFS的回缩曲线评估细胞粘附力和粘附功。与FLNC相比，突变型FLNC iPSC-CMs从杂合FLNC到FLNC模型的粘附功和粘附力呈基因剂量依赖性降低，表明细胞骨架和膜结构受损。最后，我们研究了克伦洛尼对hiPSC-CMs力学特性的影响。克伦洛尼是血小板衍生生长因子受体α（PDGFRA）途径的抑制剂，该途径在hiPSC-CMs中上调。与对照组相比，克伦洛尼能够部分恢复FLNC hiPSC-CMs的刚度，支持其潜在的治疗作用。
