305 Modeling and Rescue of PLN-R14del Cardiomyopathy Phenotype in Human iPSC-Derived Cardiac Spheroids 12 RESULTS PLN-R14del hCSs show reduced Ca2+ amplitude and impaired Ca2+ handling To evaluate the PLN-R14del phenotype, we used patient-specific hiPSC lines (D4iR14del, 6BiR14del, and 10BiR14del) and compared them to control hiPSC lines derived from 1) a healthy proband of the D4iR14del line (1CiCTR), 2) an individual with no history of heart disease (273iCTR), and 3) an isogenic control of the D4iR14del line corrected by CRISPR-Cas9 (C31iCTR) (Supplementary Figure 1). As three-dimensional (3D) hCSs more closely recapitulate intercellular biomechanics, we generated 3D spheroids after step-wise differentiation, in which guided mesodermal induction of human induced pluripotent stem cells (hiPSC) was followed by cardiomyocyte (CM) differentiation.21 After 11 days, the differentiated CMs were re-plated to T75 culture flasks to induce controlled CM expansion, as previously described.35,36 After differentiation and subsequent expansion of the CMs, 10.000 CMs were placed in one well to induce spheroid formation as previously described23 (Figure 1A). PLN-R14del is generally associated with susceptibility to Ca2+ handling, and multiple electrophysiological abnormalities, resulting in heart failure.1,16,37 Therefore, we assessed Ca2+ handling in hCSs at 1,2, 3, and 6 weeks after their generation in 96-well plates (Supplementary Figure 2). The previously reported consistent difference in all Ca2+ handling parameters between C31iCTR and D4iR14del PLN-R14del hCSs was present at weeks 2 and 3 (Supplementary Figure 2C). We extended these analyses on 2 control lines; 1CiCTR, 2731CTR, and PLN-R14del; 6BiR14del and 10BiR14del, and recorded spontaneous Ca2+ sparks and observed reduced Ca2+ intensity (Figure 1B) and a very ‘spiky and narrow’ peak width in the PLN-R14del hCSs (Figure 1C). Next, we performed automated high-throughput screening of the spontaneous Ca2+ transients’ parameters (Figure 1D). We observed significantly decreased decay and rise times, peak amplitude, and Ca2+ transient durations of 10% (CTD10) and 90% (CTD90) (Figure 1E-I), which hint towards a decreased Ca2+ handling function. Hence, we observed an increase in the beating rate in all hCSs from the PLN-R14del lines (Figure 1J).
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