Unt inter-line variations, hiPSC lines 1516647 from additional 3 healthy subjects were examined and similar (no statistical difference) Ca2+ properties were observedamong the cardiomyocytes (with same post cardiac differentiation time point) derived from all 4 lines, including the one presented in this study (Table S3). There was no significant difference in Ca2+ spark properties in hiPSC-CMs differentiated from Sudan I different clones. Electrophysiological property of pluripotent stem cell-derived CMs may vary due to culture duration of hiPSC-CMs 
[34]. In our study, cardiomyocytes maintained under culture conditions from 4 to 7 weeks post cardiac differentiation were compared in theirCalcium 370-86-5 sparks in iPSC-Derived CardiomyocytesFigure 7. Effects of ryanodine on spontaneous Ca2+ sparks in hiPSC-CMs. (A) Representative line-scan (X-T) images of spontaneous Ca2+ sparks (top) and the corresponding intensity-time profiles of typical sparks (bottom) before and after the application of ryanodine. (B ) show the mean values for frequency, F/F0, FDHM and FWHM of Ca2+ sparks before (nspark = 163) and after (nspark = 347) application of ryanodine, respectively. ncell = 11. *P,0.05 vs. control. Abbreviations: F/F0, fluorescence (F) normalized to baseline fluorescence (F0); FDHM, full duration at half maximum; FWHM, full width at half maximum. doi:10.1371/journal.pone.0055266.gcharacteristics of Ca2+ sparks and no significant differences were identified (data not shown). Nevertheless, long-term following up studies were not performed due to low yield of cardiac differentiation. In summary, we identified spontaneous Ca2+ sparks and documented their fundamental characteristics in hiPSC-CMs. We found that the Ca2+ sparks in hiPSC-CMs share similar temporal and spatial properties with adult cardiomyocytes. Moreover, RyRs are functioning in hiPSC-CMs and a majority of spontaneous Ca2+ sparks is L-type Ca2+ channel dependent. However, the Ca2+ sparks in hiPSC-CMs appear to be stochastic with a tendency of repetitive occurrence at some sites. Such phenomenon might be attributed to a heterogeneous array ofRyRs due to the lack of T tubules or immature T-tubule system in hiPSC-CMs.Supporting InformationMeasurement of [Ca2+]i by using ionomycin. (A) Representative line scan (X-T) image of Ca2+ transients before and after the application of ionomycin and EGTA. (B) 1662274 The fluorescent intensity profiles of Ca2+ transients in A. (C) The Ca2+ concentrations of spontaneous Ca2+ transients were calculated by using equation: [Ca2+]I = Kd[(F2Fmin)/(Fmax2F)]. Abbreviations: Kd, the dissociation constant value of a fluorescence; F, the measured fluorescence value; Fmax, the fluorescence value withFigure SCalcium Sparks in iPSC-Derived Cardiomyocytes2 mM 
ionomycin; Fmin, the fluorescence value with Ca2+-free bath solution containing 5 mM EGTA. (TIFF)Figure S2 The characteristics of Ca2+ transients in ratnrat = 5, ncell = 13. Abbreviations: F/F0, fluorescence (F) normalized to baseline fluorescence (F0); s, seconds. (TIFF)Table S1 The percentages of hiPSC-CM subtypes and the action potential properties. (DOCX) Table S2 Spatio-temporal properties of Ca2+ sparks in rat cardiomyocytes. (DOCX) Table S3 Characteristics of spontaneous Ca2+ sparks incardiomyocytes. A representative line-scan (X-T) image of Ca2+ transient recorded from field stimulated rat cardiomyocyte (top) and the corresponding intensity profiles (bottom) of Ca2+ transient. nrat = 5, ncell = 12. Abbreviations: F/F0, fluorescence.Unt inter-line variations, hiPSC lines 1516647 from additional 3 healthy subjects were examined and similar (no statistical difference) Ca2+ properties were observedamong the cardiomyocytes (with same post cardiac differentiation time point) derived from all 4 lines, including the one presented in this study (Table S3). There was no significant difference in Ca2+ spark properties in hiPSC-CMs differentiated from different clones. Electrophysiological property of pluripotent stem cell-derived CMs may vary due to culture duration of hiPSC-CMs [34]. In our study, cardiomyocytes maintained under culture conditions from 4 to 7 weeks post cardiac differentiation were compared in theirCalcium Sparks in iPSC-Derived CardiomyocytesFigure 7. Effects of ryanodine on spontaneous Ca2+ sparks in hiPSC-CMs. (A) Representative line-scan (X-T) images of spontaneous Ca2+ sparks (top) and the corresponding intensity-time profiles of typical sparks (bottom) before and after the application of ryanodine. (B ) show the mean values for frequency, F/F0, FDHM and FWHM of Ca2+ sparks before (nspark = 163) and after (nspark = 347) application of ryanodine, respectively. ncell = 11. *P,0.05 vs. control. Abbreviations: F/F0, fluorescence (F) normalized to baseline fluorescence (F0); FDHM, full duration at half maximum; FWHM, full width at half maximum. doi:10.1371/journal.pone.0055266.gcharacteristics of Ca2+ sparks and no significant differences were identified (data not shown). Nevertheless, long-term following up studies were not performed due to low yield of cardiac differentiation. In summary, we identified spontaneous Ca2+ sparks and documented their fundamental characteristics in hiPSC-CMs. We found that the Ca2+ sparks in hiPSC-CMs share similar temporal and spatial properties with adult cardiomyocytes. Moreover, RyRs are functioning in hiPSC-CMs and a majority of spontaneous Ca2+ sparks is L-type Ca2+ channel dependent. However, the Ca2+ sparks in hiPSC-CMs appear to be stochastic with a tendency of repetitive occurrence at some sites. Such phenomenon might be attributed to a heterogeneous array ofRyRs due to the lack of T tubules or immature T-tubule system in hiPSC-CMs.Supporting InformationMeasurement of [Ca2+]i by using ionomycin. (A) Representative line scan (X-T) image of Ca2+ transients before and after the application of ionomycin and EGTA. (B) 1662274 The fluorescent intensity profiles of Ca2+ transients in A. (C) The Ca2+ concentrations of spontaneous Ca2+ transients were calculated by using equation: [Ca2+]I = Kd[(F2Fmin)/(Fmax2F)]. Abbreviations: Kd, the dissociation constant value of a fluorescence; F, the measured fluorescence value; Fmax, the fluorescence value withFigure SCalcium Sparks in iPSC-Derived Cardiomyocytes2 mM ionomycin; Fmin, the fluorescence value with Ca2+-free bath solution containing 5 mM EGTA. (TIFF)Figure S2 The characteristics of Ca2+ transients in ratnrat = 5, ncell = 13. Abbreviations: F/F0, fluorescence (F) normalized to baseline fluorescence (F0); s, seconds. (TIFF)Table S1 The percentages of hiPSC-CM subtypes and the action potential properties. (DOCX) Table S2 Spatio-temporal properties of Ca2+ sparks in rat cardiomyocytes. (DOCX) Table S3 Characteristics of spontaneous Ca2+ sparks incardiomyocytes. A representative line-scan (X-T) image of Ca2+ transient recorded from field stimulated rat cardiomyocyte (top) and the corresponding intensity profiles (bottom) of Ca2+ transient. nrat = 5, ncell = 12. Abbreviations: F/F0, fluorescence.