Fig 1.
Expression of TRPV1 in mESCs and mESC-CMs.
(A) Representative immunostaining images of undifferentiated mESCs (day 0). Oct4 (green) was a marker of undifferentiated MSCs. (B) Representative immunostaining images of differentiating mESCs (day 4). (C) Representative images from differentiated mESC-CMs (day 9). c-TnT was a cardiomyocyte marker. (D) Negative control in the absence of primary antibody for day 9 cells. (E) Negative control after TRPV1-peptide preabsorption. A through E, from left to right, were images of TRPV1 (red), Oct4/c-TnT (green), DAPI (blue) and overlay. Scale bars, 10μm. Experiments were performed 3–5 times.
Fig 2.
Expression pattern of TRPV1 mRNA during the differentiation of mESCs to cardiomyocytes.
Shown was the relative expression of TRPV1 mRNA at different differentiation days. Values were Mean ± SEM. n = 3.
Fig 3.
Capsaicin and camphor-induced rise in basal [Ca2+]i level in mESC-CMs.
(A and B) Representative traces showing the [Ca2+]i responses to 1 μM capsaicin (A) and 2.5 mM camphor (B). (C and D) summary of data comparing the maximal [Ca2+]i, while rise in response to 1 μM capsaicin (C) and 2.5 mM camphor (D). Shown were effects of TRPV1-shRNA and SB366791 (10 μM). (E) Effectiveness of TRPV1-shRNA in suppressing TRPV1 mRNA by qPCR. ctl-shRNA stands for control scrambled shRNA. Values were Mean ± SEM. n = 3–5 experiments as labeled. * P < 0.05, ** P < 0.01, ***P < 0.001.
Fig 4.
Effect of TRPV1 agonist or antagonists on EB sizes and EB beating curves.
(A) (A) Summary of data illustrating EB size distribution on the differentiation day 7 after treatment with 0.1% DMSO (vehicle control), 1 μM capsaicin, 10 μM capsazepine, or 10 μM SB366791. Each group contained more than 200 EBs from 3 independent experiments, with red bar representing the average size. (B) Data summary of EB sizes under different treatments. (C) The percentage of beating EBs after different treatments. The beating was recorded from differentiation day 8 to day 17. Results were Mean ± SEM. n = 3 experiments. * P < 0.05, ** P < 0.01 compared to DMSO control.
Fig 5.
Effect of TRPV1 agonist and antagonists on mRNA expression of cardiac-specific marker genes in mESC-CMs.
Shown were the expressional levels of cardiac actin (car. actin), cardiac troponin T (c-TnT), cardiac troponin I (c-TnI) and alpha-myosin heavy chain (α-MHC) after treatment with 0.1% DMSO (vehicle control), 1 μM capsaicin, 10 μM capsazepine, or 10 μM SB366791. (A) qPCR data on the differentiation day 12. (B) qPCR data on the differentiation day 17. Values were Mean ± SEM. n = 4–5 experiments. **P < 0.01, ***P < 0.001 compared to DMSO control.
Fig 6.
Effect of TRPV1-shRNA on mESC differentiation to cardiomyocytes.
(A) Summary of FACS data showing that TRPV1-shRNA reduced the c-TnT-positive cardiomyocytes on the differentiation day 12. (B) Data summary showing the effect of TRPV1-shRNA on EB sizes on the differentiation day 7. Each group contained more than 80 EBs from 3 independent experiments. (C) The effect of TRPV1-shRNA on the percentage of beating EBs. The beating was recorded from differentiation day 8 to day 17. (D) qPCR data on the differentiation day 12, showing the expressional levels of cardiac actin (car. actin), cardiac troponin T (c-TnT), cardiac troponin I (c-TnI) and alpha-myosin heavy chain (α-MHC). ctl-shRNA stands for control scrambled shRNA. Values were Mean ± SEM. n = 3 experiments. *P < 0.05, **P < 0.01, ***P < 0.001.