Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency.
Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency.
复制标题
DOI：
10.3389/fphys.2022.1030920
复制
发表时间：
2022
影响因子：
4
通讯作者：
Coppini R
中科院分区：
文献类型：
--
作者：
Pioner JM;Santini L;Palandri C;Langione M;Grandinetti B;Querceto S;Martella D;Mazzantini C;Scellini B;Giammarino L;Lupi F;Mazzarotto F;Gowran A;Rovina D;Santoro R;Pompilio G;Tesi C;Parmeggiani C;Regnier M;Cerbai E;Mack DL;Poggesi C;Ferrantini C;Coppini R
从人诱导多能干细胞（hiPSC- CMs）分化的心肌细胞是建模遗传性心肌病的独特来源。特别是，在一个简单的培养皿中观察成熟过程的可能性，为研究在临床表现出现之前由基因突变引起的早期疾病缺陷开辟了新的视角。例如，钙处理异常被认为是几种遗传性扩张型心肌病（包括罕见类型的杜氏肌营养不良（DMD）相关心肌病）中心肌细胞功能障碍的主要原因。为了更好地定义钙处理的成熟度，我们在特定时间点使用荧光指示器同时测量动作电位和钙瞬态（Ca-Ts）。我们将微图案底物与长期培养相结合，以提高hiPSC-CMs的成熟度（分化后60,75或90天）。对照-(hiPSC)- cms随着时间的推移（90天vs 60天）成熟程度增加，动作电位持续时间（APD）更长，Ca-T振幅增加，Ca-T上升（峰值时间）和Ca-T衰减（RT50）更快。SR对Ca释放的逐渐增加的贡献（通过休息后增强或咖啡因诱导的Ca- ts来估计）似乎是成熟期间Ca- t振幅逐渐上升的主要决定因素。作为早期发病的严重心肌病的一个例子，我们比较了从DMD患者（DMD-ΔExon50）和CRISPR-Cas9基因组编辑细胞系中产生的hiPSC-CMs与DMD基因263位G碱基缺失的健康对照（c.263delG-CMs）。在DMD-hiPSC-CMs中，成熟过程中Ca-Ts的变化不太明显：事实上，与对照hiPSC-CMs相比，DMD细胞在90天时Ca-T振幅降低，Ca-T上升和RT50更快。咖啡因- ca - t的振幅降低，时间过程较慢，表明DMD细胞的SR钙含量和NCX功能低于对照细胞。尽管如此，对福斯克林的肌力和弹性反应仍被保留。crispr诱导的c.263delG-CM细胞系再现了在DMD-CMs中观察到的相同的发育钙处理变化。然后，我们测试了具有更高刚度的微图纹基底的效果。在控制hiPSC-CMs中，更高的刚度导致Ca-T振幅更高，衰变动力学更快。然而，在缺乏全长肌营养不良蛋白的hiPSC-CMs中，更硬的底物不会改变Ca- ts，而只会导致更高的SR Ca含量。这些发现强调了肌营养不良蛋白缺乏的心肌细胞无法调节其钙稳态以响应细胞外基质刚度的增加，这表明在生理和病理发展过程中发生的机制（即纤维化）。
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).
登录
查看更多内容
影响因子：
8.2
作者：
Bremner, Samantha B.;Mandrycky, Christian J.;Leonard, Andrea;Padgett, Ruby M.;Levinson, Alan R.;Rehn, Ethan S.;Pioner, J. Manuel;Sniadecki, Nathan J.;Mack, David L.
通讯作者：
Mack, David L.
影响因子：
5.4
作者：
Ferrantini C;Coppini R;Pioner JM;Gentile F;Tosi B;Mazzoni L;Scellini B;Piroddi N;Laurino A;Santini L;Spinelli V;Sacconi L;De Tombe P;Moore R;Tardiff J;Mugelli A;Olivotto I;Cerbai E;Tesi C;Poggesi C
通讯作者：
Poggesi C
DOI：
10.1073/pnas.0908540107
发表时间：
2010-01-26
影响因子：
11.1
作者：
Fauconnier, Jeremy;Thireau, Jerome;Lacampagne, Alain
通讯作者：
Lacampagne, Alain
影响因子：
5.9
作者：
Duelen R;Costamagna D;Gilbert G;De Waele L;Goemans N;Desloovere K;Verfaillie CM;Sipido KR;Buyse GM;Sampaolesi M
通讯作者：
Sampaolesi M
影响因子：
6
作者：
Jelinkova, Sarka;Fojtik, Petr;Rotrekl, Vladimir
通讯作者：
Rotrekl, Vladimir