Figure 1.
Effect of EpiC treatment on CStC growth, viability and senescence.
(A) Growth curve of CStC cultured in GM or EpiC for 5, 7, and 14 days (n = 7). (B) Quantification of free nucleosomes, used as markers of apoptosis, in CStC exposed to GM or EpiC for7 days. Ctrl+ = positive control supplied by the manufacturer. (C) Staining for senescence-related acidic β-galactosidase (β-gal ) performed on CStC grown either in GM or in EpiC medium. Positivity for β-gal is indicated by the presence of a dark grey stain within the cytoplasm. Ctrl+ = primary bone marrow mesenchymal stromal cells at passage 10 (replicative senescence). Bar Graph shows average percentage of β-gal positive cells (n = 4). Original magnification: 20×.
Figure 2.
Effect of EpiC treatment on stem cell marker expression.
Western blotting analysis of (A) adult cardiac stem cell marker c-Kit and MDR-1 (n = 4) and (B) Notch, Jagged-1, Numb and nucleostemin (NS) in CStC, grown either in GM or in EpiC (n = 6). Densitometry is shown in the bar graphs at the bottom of each panel (**P≤0.01, *P≤0.05 vs GM). FL = full length protein; TM = Transmembrane domain.
Figure 3.
Effect of EpiC treatment of cardiovascular marker expression.
Immunoblots showing expression analyses of (A) vascular markers: VEGFR-2, GATA6, and α-smooth muscle actin (α-SMA); (B) early cardiomyogenic markers: Mef2c, GATA4, and Nkx2.5; (C) late cardiomyogenic markers α-MHC, α-Sarc, and Tn-TC in EpiC-treated CStC compared to cells in GM. Bar graphs represent average results, normalized to β-actin, of western blot densitometric analyses (*P≤0.05, **P<0.01, and ***P≤0.005, vs GM). The data represent the mean ± SD of 4 independent experiments performed in duplicate.
Figure 4.
Hierarchical clustering of differentially expressed microRNAs in GM and EpiC-treated CStC.
Unsupervised cluster analysis was performed using the 31 microRNAs that showed a significant modulation induced by the EpiC treatment. The dendrogram above shows that the differential expression profile completely discriminates the two treatment groups. The dendrogram on the left shows two distinct clusters of microRNAs up- and down-regulated by EpiC treatment in CStC (respectively at the top and the bottom of the heatmap). The mean centered value of normalized log2 transformed relative expression level of each microRNA is represented with a green, black, and red color scale (green indicates below mean; black, equal to mean; and red, above mean).
Table 1.
Differentially expressed microRNAs.
Figure 5.
EpiC-treated CStCs express functionally competent ion channels.
In panel (A) a family sodium current recorded from a representative EpiC-treated TTX (10 µM) low -sensitive CStC, following the application of a standard depolarizing protocol (see inset), is shown. (B) Mean current-voltage relation of normalized TTX-sensitive currents showing a threshold of activation around −40 mV and a peak around 0 mV. (C) SCN5A and SCN2A genes were up-regulated (n = 3, *P≤0.05) by real-time PCR analysis. (D) Western Blot evidences increase of the type V (NaV1.5) and type II (NaV1.2) voltage-gated sodium channel protein level in CStC cells exposed to EpiC (n = 5, *P≤0.05). FL = full length protein. (E) In few cells, membrane hyperpolarization in the range −35 to −125 mV (see inset) revealed an inward current with the kinetic features of the native pacemaker If current. (F) Plot of the mean activation curve obtained from the analysis of the If currents recorded in a subset of EpiC-treated CStC.
Figure 6.
EpiC treatment modifies CStC epigenetic landscape.
Western Blot analysis showing histone modification changes in EpiC-treated CStC compared to GM. (A) H3 modifications: H3K9Ac, H3K4Me3, H3K27Me3, H3K9Me3, and H3S10P. (B) H4 modifications: H4K16Ac, H4K20Me, and H4K20Me3. The same filter was probed with anti-total histone H3 or H4, respectively, to control for equal nuclear protein loading. Band densitometric analyses are reported in the bar graphs on the right (n = 3, *P≤0.05).
Figure 7.
Effects of EpiC treatment on specific-gene promoters.
ChIP experiments, performed on CStC isolated from 2 different patients, show that H3K4Me3 association to (A) c-Kit and (B) MDR-1 promoter is enriched after EpiC treatment, while H3S10P is decreased. Data are expressed as relative enrichment of specific histone modifications i in EpiC-treated cells compared to GM measured by real-time PCR amplification. (C) Real-time RT-PCR analysis of c-Kit and MDR-1 mRNA in EpiC-treated CStC (n = 3, *P≤0.05 and ****P≤0.005).