Carnegie Institute for Science, Department of Plant Biology, 260 Panama Street, Stanford, CA94305, USA.
Heart Center, Department of Cardiology and Pneumology, University Medical Center, Göttingen University, Robert-Koch-Strasse 40, D-37075, Göttingen, Germany.
Biol Chem. 2020 Oct 27;402(1):113-121. doi: 10.1515/hsz-2020-0212. Print 2020 Nov 18.
We developed a new approach for combined analysis of calcium (Ca) handling and beating forces in contractile cardiomyocytes. We employed human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from dilated cardiomyopathy (DCM) patients carrying an inherited mutation in the sarcomeric protein troponin T (TnT), and isogenic TnT-KO iPSC-CMs generated via CRISPR/Cas9 gene editing. In these cells, Ca handling as well as beating forces and -rates using single-cell atomic force microscopy (AFM) were assessed. We report impaired Ca handling and reduced contractile force in DCM iPSC-CMs compared to healthy WT controls. TnT-KO iPSC-CMs display no contractile force or Ca transients but generate Ca sparks. We apply our analysis strategy to Ca traces and AFM deflection recordings to reveal maximum rising rate, decay time, and duration of contraction with a multi-step background correction. Our method provides adaptive computing of signal peaks for different Ca flux or force levels in iPSC-CMs, as well as analysis of Ca sparks. Moreover, we report long-term measurements of contractile force dynamics on human iPSC-CMs. This approach enables deeper and more accurate profiling of disease-specific differences in cardiomyocyte contraction profiles using patient-derived iPSC-CMs.
我们开发了一种新的方法，用于对收缩型心肌细胞中的钙（Ca）处理和搏动力进行联合分析。我们使用来自扩张型心肌病（DCM）患者的人诱导多能干细胞衍生的心肌细胞（iPSC-CMs），这些患者携带肌钙蛋白 T（TnT）的遗传性突变，以及通过 CRISPR/Cas9 基因编辑产生的同源 TnT-KO iPSC-CMs。在这些细胞中，使用单细胞原子力显微镜（AFM）评估 Ca 处理以及搏动力和速率。我们报告与健康 WT 对照相比，DCM iPSC-CMs 中的 Ca 处理受损，收缩力降低。TnT-KO iPSC-CMs 没有收缩力或 Ca 瞬变，但会产生 Ca 火花。我们将我们的分析策略应用于 Ca 轨迹和 AFM 挠度记录，以揭示收缩的最大上升率、衰减时间和持续时间，具有多步背景校正。我们的方法为不同的 Ca 通量或力水平在 iPSC-CMs 中的信号峰提供自适应计算，并分析 Ca 火花。此外，我们报告了对人类 iPSC-CMs 收缩力动力学的长期测量。这种方法可以使用患者来源的 iPSC-CMs 更深入、更准确地分析心肌病收缩特征的疾病特异性差异。
