Fig 1.
The effect of E2 on myocardial infarct size, p38β activation and MnSOD expression.
(A) Representative TTC staining of the heart sections in OVX mice with or without E2 supplementation. (B) The phosphorylation of p38β in the left ventricle of OVX mice with or without E2. (C) The protein level of MnSOD in the left ventricle of OVX mice with or without E2 supplementation.*P<0.05 vs. Sham; † P<0.05 vs. I/R; n = 3 in each group. E2, 17β-estradiol; I/R, ischemia/reperfusion; MnSOD, manganese superoxide dismutase.
Fig 2.
The effect of E2 on the mitochondrial p38β activation and MnSOD activity.
(A) Western blots and quantitative analysis of p-p38β and total p38β in mitochondria isolated from the left ventricle of the indicated treatment groups of OVX mice. (B) The protein level of MnSOD in mitochondria isolated from the left ventricle of the indicated treatment groups of OVX mice. (C) The activity of MnSOD in mitochondria isolated from the left ventricle of the indicated treatment groups of OVX mice.*P<0.05 vs. Sham; † P<0.05 vs. I/R; n = 3 in each group. E2, 17β-estradiol; I/R, ischemia/reperfusion; MnSOD, manganese superoxide dismutase, COX IV, cytochrome c oxidase subunit IV.
Fig 3.
The effect of ER subtypes on the infarct size and the activity of mitochondrial p38β and MnSOD.
(A) Representative TTC staining and quantitative analysis of the heart sections in wild type and estrogen receptor knockout mice after myocardial I/R. *P<0.05 vs. WT; n = 3 in each group. (B) Immunoblotting of the active p38β (p-p38β) level in mitochondrial fraction isolated from the left ventricle of WT and DERKO mice after sham or I/R surgery and quantitative analysis of the ratio between p-p38β over total p38β in mitochondria. *P<0.05 vs. WT Sham; † P<0.05 vs. DERKO Sham; n = 3 in each group. (C) The protein level of MnSOD in mitochondrial fraction isolated from the left ventricle of WT and DERKO mice after sham or I/R surgery, shown in immunoblots with quantitative analysis. *P<0.05 vs. WT Sham; # P<0.05 vs. DERKO Sham; n = 3 in each group. (D) The activity of MnSOD in mitochondrial fraction isolated from the left ventricle of WT and DERKO mice after sham or I/R surgery. *P<0.05 vs. WT Sham; † P<0.05 vs. DERKO Sham; n = 3 in each group. I/R, ischemia/reperfusion; MI, myocardial infarction; MnSOD, manganese superoxide dismutase, COX IV, cytochrome c oxidase subunit IV.
Fig 4.
Physical interaction between mitochondrial p38β and MnSOD in vivo.
(A) Cellular localization of p38β in neonatal rat cardiomyocytes. The white scale bar represents 25 μm. (B) Cellular localization of p38β in the left ventricle of the female mouse heart. The white scale bar represents 25 μm. (C) Immunoblotting of a cytosolic marker, PGM1, in the cytosolic fraction and of p38β and MnSOD in the mitochondrial fraction from OVX female heart, with Cox IV as a mitochondrial marker. (D) Immunoblotting of MnSOD in the p38β-immunoprecipitate and immunoblotting of p38β in the MnSOD-immunoprecipitate from the mitochondrial fractions in the OVX hearts of the indicated treatment groups. NS IgG, nonspecific IgG input. Lysate, unfractionated LV homogenate. DAPI, 4’,6-diamidino-2-phenylindole; PGM1, phosphoglucomutase-1; MnSOD, manganese superoxide dismutase; COX IV, cytochrome c oxidase subunit IV; E2, 17β-estradiol; IP, immunoprecipitation.
Fig 5.
The phosphorylation of MnSOD peptides and mutants T79A and S106A by p38β.
(A) Radioactive kinase activity of p38β on wild type MnSOD peptides. MnSOD-1, MnSOD-2, MnSOD-3, MnSOD-4 peptide contains the human WT MnSOD sequence from amino acid position 16–35, 61–80, 91–110, and 181–200, respectively. The dotted box marks the peptides phosphorylated and radiolabeled by p38β. (B) Radioactive kinase activity of p38β on wild type and mutant MnSOD peptides. MnSOD 2–1 had T65 mutated to alanine (T65A). MnSOD-2-2 had T79 changed to alanine (T79A). MnSOD-3-1, MnSOD-3-2 and MnSOD-3-3 contained point mutations S99A, T103A, and S106A, respectively. The dotted box marks the peptides radiolabeled by p38β and those mutants not phosphorylated by the kinase. (C) Kinase activity of p38β on full length wild type and mutant MnSOD, T79A and S106A. *P<0.05 vs. WT MnSOD. MnSOD, manganese superoxide dismutase; ATF2, activating transcription factor 2; SB, SB 203580 (1 μM). 2 μg of purified MnSOD and ATF2 was used, respectively.
Fig 6.
The effect of T79A or S106A mutant MnSOD on ROS generation in cardiomyocytes after H/R.
Intracellular ROS (fluorescent green) was detected in NRCM after full length WT, T79A mutant MnSOD, or S106A mutant MnSOD plasmid was transfected. Mitochondria are co-stained with MitoTracker (red). Representative images of cells are shown with quantitative analysis. *P<0.05 vs. N; † P<0.05 vs. H/R. The white scale bar represents 25 μm. N, normoxia; H/R, hypoxia/reoxygenation; MnSOD, manganese superoxide dismutase; ROS, reactive oxygen species.
Fig 7.
A working mechanism representing the E2/ER-mediated cardioprotection from I/R injury via p38β and MnSOD.
In cardiomyocytes, E2, via ERα and ERβ, increases mitochondrial p38β activation. Phosphorylated p38β (activated p38β kinase), enhances MnSOD activity through phosphorylating the dismutase at T79 and S106 residue, leading to suppression of intracellular ROS production and decreases the myocardial injury and infarct size during I/R.