Table 1.
Characteristics of cardiac function in Sham, I/R, and ATRA+I/R groups before and after I/R injury.
Fig 1.
ATRA protects mice from I/R-induced cardiac injury.
Images show transverse, TTC-stained sections of mouse hearts from I/R, ATRA+I/R, RKO-I/R, and ATRA+RKO-I/R groups. After I/R injury, the infarct areas were significantly smaller in ATRA+I/R group than in I/R group (n = 3 in each group). The protective role of ATRA on myocardial I/R injury was not observed in RAGE-KO mice, as the infarct areas did not differ between RKO-I/R and ATRA+RKO-I/R group (n = 3 in each group). Data is expressed as mean ± SD (#P<0.05 vs. I/R group).
Fig 2.
ATRA protects H9c2 cells from H/R-induced cell death.
H9c2 cells subjected to H/R injury were pre-treated with various concentrations of ATRA for 24 h. Cells without H/R injury served as control. The WST-1 method was used to assess cell viability. (n = 5 in each group, #P<0.05 vs. H/R group). Data is expressed as means ± SD.
Fig 3.
ATRA protects H9c2 cells from H/R-induced cell apoptosis.
(A) TUNEL detection of H9c2 cells from H/R (n = 3) and ATRA (n = 3) treatment groups (×200) indicated large numbers of TUNEL-positive cells were visible in the H/R group (arrow), but were lower in the ATRA treatment group. Quantitative analysis of apoptosis revealed significantly fewer apoptotic cells in ATRA treatment groups. (B) After 2 h of treatment with different concentrations of ATRA for 24 h or 1 mM H2O2, H9c2 cells were subjected to H/R injury, and the relative number of annexin-V-positive cells was determined using flow cytometry (n = 3 in every group, #P<0.05 vs. H/R). Data is expressed as means ± SD.
Fig 4.
ATRA protects cardiomyocytes from I/R-induced apoptosis.
(A) TUNEL detection of heart tissues with I/R injury from I/R and ATRA+I/R group (x200), showing that TUNEL-positive cells were prominent in I/R group, but reduced in the ATRA+I/R group. (B) Quantitative analysis of apoptotic cell death showed that there was significantly decreased apoptotic cells in the ATRA+I/R group compared to the I/R group (n = 3 in each group, #P<0.05).
Fig 5.
ATRA decreases cleaved caspase-3 and increases bcl-2 activities after I/R injury.
(A+B) H9c2 cells were subjected to 24 h of pre-treatment with different concentrations of ATRA and then subjected to H/R injury. A DMSO equivalent to ATRA at the highest concentration served as a control (H/R group). Western blotting of cell lysates indicated that there was less cleaved caspase-3 protein and more bcl-2 protein in cells treated with ATRA than in controls (n = 3 in every group, #P<0.05 vs. H/R). (C) The in vitro findings were confirmed in mice cardiomyocyte after I/R injury (n = 3 in every group, #P<0.05 vs. I/R). Data is expressed as means ± SD.
Fig 6.
ATRA decreases ROS production in H9c2 cells after H/R injury.
DCF fluorescence in H/R-induced H9c2 cells pre-treated with different concentrations of ATRA was observed using fluorescence microscopy and measured by FACS analysis (bar graph). Data is expressed as means ± SD of three independent experiments (H/R ratio was set to 1; #P<0.05 vs. H/R).
Fig 7.
ATRA inhibits the MAPK signaling pathway in cardiomyocyte with I/R injury.
(A–C) H9c2 cells were incubated with different concentrations of ATRA for 24 h and then subjected to H/R injury. A DMSO equivalent to ATRA at the highest concentration served as a control (H/R group). Aliquots of cell lysate were analyzed with Western blotting. ATRA inhibited phosphorylation of MAPKs, including p38 MAPK, ERK, and JNK in a concentration-dependent manner. Total MAPKs served as loading controls (n = 3 in each group, #P<0.05 vs. H/R). (D) The in vitro findings were confirmed in mouse cardiomyocytes after I/R injury. Total MAPKs served as loading controls (n = 3 in each group, #P<0.05 vs. I/R). Data is expressed as means ± SD.
Fig 8.
ATRA increases ADAM10 and decreases RAGE expression after I/R injury.
Both in vivo and in vitro studies showed that ATRA treatment increased ADAM10 expression. Levels of RAGE, which is cleaved by ADAM10, were low. GAPDH served as a loading control (control ratio was set to 1; n = 3 in every group, #P < 0.05 vs. control). Data is expressed as means ± SD.
Fig 9.
RAGE, ADAM10, and phospho-specific MAPKs expressions in non-risk (normal) and infarction area of cardiac tissues after I/R injury.
Quantitative measurement of immunohistochemical images as a ratio of positive cells indicated that phospho-specific p38, phospho-specific JNK, phospho-specific ERK, and RAGE expressions were decreased, while ADAM10 expression was increased in the infarction area of the ATRA treatment group. Data is expressed as means ± SD (#P<0.05 vs. I/R).
Fig 10.
One possible mechanism underlying the effect of ATRA on myocardial I/R injury.