1. Volume 11, Issue 1, Pages 133-140 (January 2014)
Mechanistic basis of excitation-contraction coupling in human pluripotent stem cell- derived ventricular cardiomyocytes revealed by Ca2+ spark characteristics: Direct evidence of functional Ca2+-induced Ca2+ release 
Sen Li, MS, Heping Cheng, PhD, Gordon F. Tomaselli, MD, Ronald A. Li, PhD 
Heart Rhythm 
Volume 11, Issue 1, Pages (January 2014)
DOI: /j.hrthm
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2. Figure 1
Ca2+ sparks in hESC‐VCMs. A: tdTomato-positive hESC-VCMs showed cTnT-, MLC2v-, but not MLC2a-, positive staining. Scale bar = 10 µm. B: hESC-VCMs had ventricular-like action potential. C: Ca2+ sparks were observed in frame-based imaging (upper panel; 3 sequential captures with time points 0, 9.04, and seconds, respectively, were present, and cell nucleus was marked by Hoechst 33342). Scanning line (represent by the red line) was drawn, and Ca2+ sparks were captured in line scanning result (middle panel). The intensity curve of Ca2+ sparks at sites marked by black bar in line scanning figure was generated (lower panel). Scale bar = 10 µm. cTnT = cardiac troponin T; hESC = human embryonic stem cell; MLC2a = myosin light chain 2a; MLC2v = myosin light chain 2v; VCM = ventricular cardiomyocyte.
Heart Rhythm  , DOI: ( /j.hrthm )
Copyright © 2014 Heart Rhythm Society Terms and Conditions

3. Figure 2
Ca2+ sparks pharmacological profiling and parameters. A: In the line‐scanning mode, Ca2+ sparks sensitive to thapsigargin (TG; 1 µM), cyclopiazonic acid (CPA; 50 µM), ryanodine (Ry; 5 µM), or tetracaine (Tetra; 1 mM) pretreatment are shown. Reappearance of Ca2+ sparks was observed after washing out of cyclopiazonic acid. n = 16–17 cells for each treatment; **P < .01. B: Illustration of quantification methods of spark parameters including amplitude, FWHM, FDHM, and TTP. The red curve was averaged fluorescent intensity of the area in the red box in the x direction, while the green curve was averaged fluorescent intensity of the area in the green box in the y direction. Curves were converted by Image J. C: Histograms describing Ca2+ spark parameters in hESC-VCMs (n = 50 sparks from 15 cells). CaSpF = Ca2+ spark frequency; FDHM = full duration at half maximum; FWHM = full width at half maximum; hESC = human embryonic stem cell; TTP = time to peak; VCM = ventricular cardiomyocyte.
Heart Rhythm  , DOI: ( /j.hrthm )
Copyright © 2014 Heart Rhythm Society Terms and Conditions

4. Figure 3
Ca2+ sparks in a high external Ca2+ buffer. A: Under a high external Ca2+ (10 mM) environment, Ca2+ baseline as well as Ca2+ spark frequency were dramatically increased as quantified in (B) and (C), respectively. Also, spark-induced spark activation, as compound sparks, could be observed. n = 16–25 cells; **P < .01. D: Sarcoplasmic reticulum Ca2+ content was not altered significantly by high external Ca2+ buffer examined by caffeine (10 mM) addition (arrow). n = 23–37 cells.
Heart Rhythm  , DOI: ( /j.hrthm )
Copyright © 2014 Heart Rhythm Society Terms and Conditions

5. Figure 4
Ca2+ sparks in permeabilized hESC‐VCMs. A: Cell membrane was permeabilized by saponin (Sap) addition as reflected by the entry of the Fluo-4 salt into the cell (see the inset) and increase in cellular Fluo-4 salt fluorescence (see the curve). Scale bar = 10 µm. B: Sarcoplasmic reticulum remained intact during Sap‐induced permeabilization because the addition of Sap caused a decrease in the preloaded Fluo-4 AM fluorescence without affecting the ER-tracker fluorescence, which was released by the application of Triton X100. A Fluo-4 AM and ER tracker–loaded intact cell was shown (see the inset). Scale bar = 10 µm. C: Gradual increase in the “cytosolic Ca2+” baseline by adjusting the Ca/ethylene glycol tetraacetic acid ratio led to a significant increase in CaSpF. n = 13–18 cells; **P < .01. CaSpF = Ca2+ spark frequency; hESC = human embryonic stem cell; VCM = ventricular cardiomyocyte.
Heart Rhythm  , DOI: ( /j.hrthm )
Copyright © 2014 Heart Rhythm Society Terms and Conditions

6. Figure 5
Contribution of ICa,L channels and the NCX to Ca2+ spark generation in hESC-VCMs. A: NCX inhibition by a Na+-free environment, KB-R7943 (50 µM) or SN-6 (5 µM), had no effect on the Ca2+ spark frequency. n = 11–15 cells. B: Downregulation of the NCX1 was confirmed by Western blotting. C: Ca2+ spark frequency was not altered in NCX1 downregulated hESC-VCMs. n = 26–27 cells. D: NCX1 downregulation prolonged decay phase of caffeine-induced Ca2+ transient without changing SR Ca2+ content. Bar graphs showed the peak amplitude of caffeine-induced Ca2+ transient and decay time to 50% peak amplitude. n = 17–30 cells; **P < .01. E: In hESC-VCMs, ICa,L activation by depolarization instantaneously elicited Ca2+ spark generation. Spark frequency was higher during T1 (1 second long) after electrical stimulation compared with T2 (1 second long). n = 13 cells; **P < .01. F: ICa,L openers (Bay K8644 [5 µM] and FPL [1 µM]) caused a dramatic increase in cytosolic baseline. This effect was abolished by a Ca2+-free environment. n = 14–19 cells; **P < .01. G: After ICa,L activation, SR Ca2+ content was decreased significantly, reflected by Ca2+ release from caffeine-liable store. n = 20 cells; **P < .01. hESC = human embryonic stem cell; ICa,L = L-type Ca2+ current; NCX = Na+-Ca2+ exchanger; NCX1 = sodium/calcium exchanger isoform 1; shRNA = small hairpin RNA; SR = sarcoplasmic reticulum; VCM = ventricular cardiomyocyte.
Heart Rhythm  , DOI: ( /j.hrthm )
Copyright © 2014 Heart Rhythm Society Terms and Conditions

7. Figure 6
A: Ca2+ transient was dose-dependently inhibited by nifedipine. n = 15 cells; **P < .01. (B) Blockage of IP3Rs with xestospongin C (10 µM) or 2-aminoethyl diphenylborinate (10 µM) failed to alter Ca2+ sparks frequency. n = 6–9 cells. C: After carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 10 µM) addition, mitochondrial membrane potential was disrupted as indicated by an increase in rhodamine 123 fluorescence (quench mode). In addition, rhodamine 123 was diffused into the cytosol after mitochondrial depolarization (see the inset). n = 24 cells. Scale bar = 10 µm. D: Ca2+ waves could be observed in hESC-VCMs either in a high concentration of external Ca2+ or by pretreatment of a mitochondrial inhibitor CCCP (10 µM) combined with oligomycin (10 µM), the ATP-synthase inhibitor. n = 11–15 cells. 2-APB = 2-aminoethyl diphenylborinate; hESC = human embryonic stem cell; IP3R = inositol trisphosphate receptor; Oligo = oligomycin; VCM = ventricular cardiomyocyte; XeC = xestospongin C.
Heart Rhythm  , DOI: ( /j.hrthm )
Copyright © 2014 Heart Rhythm Society Terms and Conditions