atttgaaatgaaggcagaaaccaggcttaaacaaagactgaaactcattctcttttcaaa tctcctgccgataaacatatgtgcccagtcttttgtttcccagacatcaggtttccatta tttaaacagagcttctacctggatctgtcaagagcatgaggcagacatatttaagatttt tacaaaccggtgtatgagttaggtgagttttgcaaatgttcaaattcatttaatcaaaag aagcgctacaatgg tgtcct taatccttttatgattactgcaattttcagacaatatgaa caacacttattgcttctatatgctcttgtcttgtagcatttactttaatatcagtggaca gacagatatcactagtcatctgctgtttcttgagacatttgtaattttgtactctggtac acttctctctcct tgttct actgaaccccatacttacctacgaaaatactaagaacaaac atatgtattcctctgcttgcagcttgggaggcattatttattcaaatgacccaaagcttt taaagtcaatcttcaagttaaaaataaaaaaaaatggtactaccaa tgtacc acgccctg cttccttgaagaataggattcaggggaatcattgtaattgcacatctctttgttgtgttgt gggttgcttggttatcagcactaactgcagaaaatttcacgattctatatttctcttccc tgctttctgttttgctaatacattaaaaaaagaacacttttgcttgcatagcaaactttc ttcttctttgactcttttgcaatagcttgttaactgtccataacgaagactgtctgtaaa tgctgtatgtatcctctttgctgaacaaaaggtgaaacaaaaggatcagcaagcagggta gagattaaaacatatgagtcctggcaagagtttacttttgttgaaattcaca tgtcct ca taacagctatgggccccttgggaatttatttaaaatctgtcaccctgtgtttcctgaatc gtagattggcaataatattaatactttttagggcatgcgctgtgtgttaggcatcatttt cagtactctaaatataataattcatttgcttttagtaacttcatgatatgagtaactaat atccccatttctcagatgagaaagca gagaca agaagaaggcaagtcatgtgactccatt catagaactgagatgtggcttgtgaagccaagtgtttggccttcagaggccattgcttac cacttgctatggtgctctctgttgatctactgagcacacagtacttagtacaccattagg cacggagcacctgctgtcagctggtactgtccagtaccatcacctcgtatggactcatc catttttattttattttattttattttttttgctgctgctggctggtatgaagggg attgt tct tccacagaaaaggaaaaacagtcataacaatagcagcataatcatcctttaccttgt ctggtgtatatttgagctacagttgtgacagtcacatgaaaaagggcactacatggaact ttatttttgtcgtgacaaaattttatccttcttatcctcaaccttctagcagcagggcaa ctgaccaaaatggttcaaagtaaggtaaaaacttaaaagtcacagacgtcaagtagaatt gctaccattctttaataaacaatgttaaaaaatttaaaatgtactgaaagctgtaaaaga tgcttgtgaaccaactttgtactctttctagtccttaactgattttagaaagccaggatc agaaaaatagaaaaaaattctgctaaaaattatattcttctcttaaagaaagccacggag tttaaaactctctaacacatttgtggaaactttattttttttttgtttgagatttatttg aatgagctgttatgattggagacatgagaatttcagattaatgttttgcagccagaaaaa aaagccctctggaaagctggcaag tgttca taagtcagctccagaattatgtaggttgaa Ggctccccagtggacagagcgaata tataa gaaggaaaccagaggtctggtgc agttaca Tcccagagtct ggggatggagcg agcaca gaatt GRE1 GRE2 GRE3 GRE4 GRE5 GRE6 GRE7 GRE8 +1 Figure S1. Rat AT 2 R gene promoter sequence. The TATAA element is labeled in red. GREs are labeled in blue. Supplemental Results

8+ NE 1+ NE+C-CGRE 2+ NE+C-CGRE 1+ NE 2+ NE 6+ NE  3+ NE 3+ NE+C-3 3+ NE+C-CGRE 3+ NE+C-AGRE  5+ NE+C-CGRE 5+ NE+C-AGRE 6+ NE 5+ NE 7+ NE 7+ NE+C-CGRE 7+ NE+C-AGRE 8+ NE+C-CGRE 8+ NE+C-AGRE  4 4+ NE+C-4 4+ NE 6 6+ NE 6+ NE+C-6 4+ NE+C-AGRE 4+ NE 4+ NE+C-CGRE 6+ NE 6+ NE+C-CGRE 6+ NE+C-AGRECGRE+NE CGRE+NE+C-CGREAGRE+NE+C-AGRE AGRE+NE    Figure S2. Characterization of GREs in the AT 2 R promoter. EMSA was performed with nuclear extracts (NE) and biotin labeled ds-oligo probes containing GREs (1 through 8), and consensus GRE (CGRE), AT1aR-GRE (AGRE). C-3, C-4, C-6, C- CGRE, C-AGRE etc are cold ds-oligos used for competition experiments. Each EMSA generated band of same electrophoretic mobility as those of authentic GREs CGRE and AGRE, and competed out by homologous, heterlogous, and consensus oligos. GRE oligos used in EMSA GRE 1 (-1853)5’- CTACAATGGTGTCCTTAATCCTTTTA GRE 2 (-1674)5’-TCTCTCTCCTTGTTCTACTGAACCCC GRE 3 (-1526)5’-GTACTACCAATGTACCACGCCCTGCT GRE 4 (-1159)5’-GAAATTCACATGTCCTCATAACAGCT GRE 5 (-947)5’-TGAGAAAGCAGAGACAAGAAGAAGGC GRE 6 (-676)5’-ATGAAGGGGATTGTTCTTCCACAGAA GRE 7 (-107)5’-AGCTGGCAAGTGTTCATAAGTCAGCT GRE 8 (+13)5’-GGATGGAGCGAGCACAGAATTGAAAG Consensus GRE oligos used for heterologous competition in EMSA CGRE-S 5’-TATGGTTACAAACTGTTCTAAAAC CGRE-AS 5’-GTTTTAGAACAGTTTGTAACCATA AT1a-GRE-S 5’-AAGCTTGTACACTATTGTCTGAGTT AT1a-GRE-AS 5’-AACTCAGACAATAGTGTACAAGCTT

Figure S3. Effect of dexamethasone (Dex) on AT 2 R protein and mRNA abundance. Intact 17-day fetal hearts were treated with Dex for 48 hours in the absence or presence of RU 486. Data are mean  SEM. * P < 0.05 (t-test), treatment vs. control. n = 6.

MaleFemale Figure S4. Effect of AT 1 R and AT 2 R inhibitors on cardiac ischemia and reperfusion injury. Hearts were isolated from 3 month old male and female rats and were pretreated in the absence or presence of losartan (1 μM), or PD123,319 (PD, 0.3 μM), or losartan + PD for 5 min before subjecting to 20 min of ischemia and 30 min of reperfusion in a Langendorff preparation. Post- ischemic recoveries of left ventricle dP/dt max, dP/dt min, and end diastolic pressure (LVEDP) were determined. Data are mean  SEM. * P < 0.05 (2-way ANOVA), treatment vs. control. n = 5.

Figure S5. Rescue effect of PD123,319 on hypoxia-mediated ischemic vulnerability. Hearts were isolated from 3 month old male offspring that had been exposed to normoxia (control) or hypoxia before birth, and were treated in the absence or presence of PD123,319 (0.3 μM) for 5 min before subjecting to 20 min of ischemia and 30 min of reperfusion in a Langendorff preparation. Post-ischemic recovery of dP/dt max and dP/dt min were determined. Data are mean  SEM. Data were analyzed by two-way ANOVA. * P<0.05, hypoxia vs. control; † P<0.05, +PD123,319 vs. -PD123,319. n = 5