Sequeira Vasco, Wang Lili, Wijnker Paul J M, Kim Kyungsoo, Pinto Jose R, Dos Remedios Cris, Redwood Charles, Knollmann Bjorn C, van der Velden Jolanda
Amsterdam UMC, Vrije Universiteit Amsterdam, Physiology, Amsterdam Cardiovascular Sciences, Amsterdam, the Netherlands.
Netherlands Heart Institute, Utrecht, the Netherlands.
J Mol Cell Cardiol Plus. 2022 Sep;1:100007. doi: 10.1016/j.jmccpl.2022.100007.
Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder with patients typically showing heterozygous inheritance of a pathogenic variant in a gene encoding a contractile protein. Here, we study the contractile effects of a rare homozygous mutation using explanted tissue and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to gain insight into how the balance between mutant and WT protein expression affects cardiomyocyte function.
Force measurements were performed in cardiomyocytes isolated from a HCM patient carrying a homozygous troponin T mutation (cTnT-K280N) and healthy donors. To discriminate between mutation-mediated and phosphorylation-related effects on Ca-sensitivity, cardiomyocytes were treated with alkaline phosphatase (AP) or protein kinase A (PKA). Troponin exchange experiments characterized the relation between mutant levels and myofilament function. To define mutation-mediated effects on Ca-dynamics we used CRISPR/Cas9 to generate hiPSC-CMs harbouring heterozygous and homozygous TnT-K280N mutations. Ca-transient and cell shortening experiments compared these lines against isogenic controls.
Myofilament Ca-sensitivity was higher in homozygous cTnT-K280N cardiomyocytes and was not corrected by AP- and PKA-treatment. In cTnT-K280N cells exchanged with cTnT-WT, a low level (14%) of cTnT-K280N mutation elevated Ca-sensitivity. Similarly, exchange of donor cells with 45 ± 2% cTnT-K280N increased Ca-sensitivity and was not corrected by PKA. cTnT-K280N hiPSC-CMs show elevated diastolic Ca and increases in cell shortening. Impaired cardiomyocyte relaxation was only evident in homozygous cTnT-K280N hiPSC-CMs.
The cTnT-K280N mutation increases myofilament Ca-sensitivity, elevates diastolic Ca, enhances contractility and impairs cellular relaxation. A low level (14%) of the cTnT-K280N sensitizes myofilaments to Ca, a universal finding of human HCM.
肥厚型心肌病（HCM）是一种常染色体显性遗传病，患者通常表现为编码收缩蛋白的基因中致病性变异的杂合子遗传。在此，我们使用移植组织和人诱导多能干细胞衍生的心肌细胞（hiPSC-CMs）研究一种罕见纯合突变的收缩效应，以深入了解突变型和野生型蛋白表达之间的平衡如何影响心肌细胞功能。
对一名携带肌钙蛋白T纯合突变（cTnT-K280N）的HCM患者和健康供体分离的心肌细胞进行力测量。为了区分突变介导的和磷酸化相关的对钙敏感性的影响，心肌细胞用碱性磷酸酶（AP）或蛋白激酶A（PKA）处理。肌钙蛋白交换实验表征了突变水平与肌丝功能之间的关系。为了确定突变介导的对钙动力学的影响，我们使用CRISPR/Cas9生成携带杂合和纯合TnT-K280N突变的hiPSC-CMs。钙瞬变和细胞缩短实验将这些细胞系与同基因对照进行比较。
纯合cTnT-K280N心肌细胞的肌丝钙敏感性更高，且未通过AP和PKA处理得到校正。在与cTnT-WT交换的cTnT-K280N细胞中，低水平（14%）的cTnT-K280N突变提高了钙敏感性。同样，用45±2%的cTnT-K280N交换供体细胞增加了钙敏感性，且未被PKA校正。cTnT-K280N hiPSC-CMs显示舒张期钙升高和细胞缩短增加。心肌细胞舒张受损仅在纯合cTnT-K280N hiPSC-CMs中明显。
cTnT-K280N突变增加了肌丝钙敏感性，升高了舒张期钙，增强了收缩力并损害了细胞舒张。低水平（14%）的cTnT-K280N使肌丝对钙敏感，这是人类HCM的普遍发现。
