Integrated Ca2+ flux and AFM force analysis in human iPSC-derived cardiomyocytes

Abstract

We developed a new approach for combined analysis of calcium (Ca²⁺) 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, Ca²⁺ handling as well as beating forces and -rates using single-cell atomic force microscopy (AFM) were assessed. We report impaired Ca²⁺ handling and reduced contractile force in DCM iPSC-CMs compared to healthy WT controls. TnT-KO iPSC-CMs display no contractile force or Ca²⁺ transients but generate Ca²⁺ sparks. We apply our analysis strategy to Ca²⁺ 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 Ca²⁺ flux or force levels in iPSC-CMs, as well as analysis of Ca²⁺ 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.

Keywords: atomic force microscopy; cardiovascular disease; contractility; dilated cardiomyopathy; human induced pluripotent stem cell-derived cardiomyocytes; microdomains; signal transduction.