Figure 3.
Calcium transients from hESC-CMs replated for 1 to 4 d from day 35 onward without electrical pacing and then paced with 1 Hz. (A) Representative electrical stimulation protocol. Stimulation with 0.5 Hz for 1 min, followed by a stimulation break for registration of basal ratio (basal calcium). The basal ratio was measured at the end of the break where ratio reached a stable phase. Then, the frequency was increased from 0.5 to 1 Hz for ∼1 min. Calcium transients were analyzed during 1 Hz stimulation. (B) Representative averaged calcium transient with analyzed parameters of minimum (diastolic), maximum (systolic, peak ratio) 340/380 nm ratios, calcium amplitude (difference between maximum and minimum 340/380 nm ratios), time to peak, half decay time, and maximum calcium rise velocity (red). Parameters were calculated after fitting the averaged calcium transients using IonWizard software. (C–I) Baseline calcium level (C) without electrical stimulus (baseline ratio 340/380 nm), (D) diastolic (minimum) calcium level (diastolic ratio 340/380 nm), (E) calcium amplitude (ratio 340/380 nm), (F) peak ratio (peak ratio 340/380 nm, maximum calcium level), (G) time to peak (in s), (H) calcium rise velocity (R/s; R: ratio 340/380 nm), and (I) half-decay time of calcium transients (in s). Calcium transients were determined under electrical stimulation with 1 Hz using the ratiometric calcium indicator Fura-2 and compared with non-replated hESC-CMs. Ratio: emission at 510 nm measured after alternating excitation at 340 nm/380 nm. Mean ± SD; n = 28–71 cells from 5–20 individual coverslips derived from two differentiations. Cells are colored according to the hESC-CM differentiation they were derived from (dark grey: differentiation no. 1; light grey: differentiation no. 2). *, P < 0.05. (J) hESC-CMs grown for 35 d on laminin-coated glass coverslips were detached, replated, cultivated for two additional days, and compared with 37-d-old non-replated cells; n = 3. mRNA expression for indicated genes of calcium handling was analyzed by RNA-seq. Data are presented as log2 fold change. The dashed line at ±0.585 indicates a ±1.5-fold change.

Calcium transients from hESC-CMs replated for 1 to 4 d from day 35 onward without electrical pacing and then paced with 1 Hz. (A) Representative electrical stimulation protocol. Stimulation with 0.5 Hz for 1 min, followed by a stimulation break for registration of basal ratio (basal calcium). The basal ratio was measured at the end of the break where ratio reached a stable phase. Then, the frequency was increased from 0.5 to 1 Hz for ∼1 min. Calcium transients were analyzed during 1 Hz stimulation. (B) Representative averaged calcium transient with analyzed parameters of minimum (diastolic), maximum (systolic, peak ratio) 340/380 nm ratios, calcium amplitude (difference between maximum and minimum 340/380 nm ratios), time to peak, half decay time, and maximum calcium rise velocity (red). Parameters were calculated after fitting the averaged calcium transients using IonWizard software. (C–I) Baseline calcium level (C) without electrical stimulus (baseline ratio 340/380 nm), (D) diastolic (minimum) calcium level (diastolic ratio 340/380 nm), (E) calcium amplitude (ratio 340/380 nm), (F) peak ratio (peak ratio 340/380 nm, maximum calcium level), (G) time to peak (in s), (H) calcium rise velocity (R/s; R: ratio 340/380 nm), and (I) half-decay time of calcium transients (in s). Calcium transients were determined under electrical stimulation with 1 Hz using the ratiometric calcium indicator Fura-2 and compared with non-replated hESC-CMs. Ratio: emission at 510 nm measured after alternating excitation at 340 nm/380 nm. Mean ± SD; n = 28–71 cells from 5–20 individual coverslips derived from two differentiations. Cells are colored according to the hESC-CM differentiation they were derived from (dark grey: differentiation no. 1; light grey: differentiation no. 2). *, P < 0.05. (J) hESC-CMs grown for 35 d on laminin-coated glass coverslips were detached, replated, cultivated for two additional days, and compared with 37-d-old non-replated cells; n = 3. mRNA expression for indicated genes of calcium handling was analyzed by RNA-seq. Data are presented as log2 fold change. The dashed line at ±0.585 indicates a ±1.5-fold change.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal