Acute myocardial infarction (MI) on initial presentation was diagnosed if there was 20 minutes
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1 SUPPLEMENTAL MATERIAL Supplemental Methods Diagnosis for acute myocardial infarction Acute myocardial infarction (MI) on initial presentation was diagnosed if there was 20 minutes or more of chest pain and (1) a serial rise and fall of creatine phosphokinase (CPK) and CK-MB fraction, or a cardiac troponin T (ctnt) level 0.1 ng/ml; the decision limit in effect during hospitalization; (2) new Q-wave formation during the initial 24 hours after presentation; or (3) coronary arteriogram with documented acute occlusion of a major coronary vessel within 24 hours of admission. ST-segment elevation (STEMI) was determined by ST-segment elevation of >0.2 mv in at least 2 contiguous electrocardiogram (ECG) leads. Unstable angina was diagnosed in patients who were without CPK, CK-MB, and ctnt elevation or ECG criteria for acute MI, and the treating physician diagnosed acute coronary syndrome (ACS)-related chest pain. In situ hybridization of mir-133a A mouse was perfused with 4% paraformaldehyde (PFA)/phosphate buffered saline (PBS) and the heart was removed. The sections were re-fixed in 4% PFA/PBS at 4 C for 10 min. After
2 fixation, the specimens were incubated in 15% sucrose/pbs at 4 C for 1 hour and in 30% sucrose/pbs overnight at 4 C. The next day, the tissue samples were embedded in Tissue-Tek OCT (Sakura, Japan) compound. We used LNA-modified DNA oligonucleotide probes (mmu-mir-133a, product # ; Scramble-miR, negative control, # ; Exiqon, Vedbaek, Denmark). Probes were labeled enzymatically using a 3 -end digoxigenin (DIG) labeling kit (Roche) in accordance with manufacturer s instructions and purified using illustra MicroSpin G-25 Columns (GE Healthcare). mir-133a in situ hybridization was performed on 10 μm frozen sections of mouse hearts as described previously with slight modifications 18. Tissue sections were dried for 20 min, washed, and fixed in 4% PFA/ PBS at 4 C for 10 min. The heart tissue sections were treated in 1 μg/ml proteinase K for 12 min, washed, and re-fixed in 4% PFA/PBS at 4 C for 10 min. Subsequently, the sections were washed and acetylated in 1.15% triethanolamine and 0.25% acetic anhydride. After post-acetylation washing, the sections were prehybridized in hybridization buffer (50% formamide, 0.3 M NaCl, 20 mm ph 8.0 Tris-HCl, 5 mm EDTA, 10 mm ph 8.0 NaPO 4, 10% dextran sulfate, 1 Denhardt s solution, 0.5 mg/ml yeast RNA) for 4 hours. The digoxigenin (DIG)-labeled detection probes (20 nm) were added to the slides, and the heart tissues sections were covered with plastic coverslips (HybriSlip, Grace Bio-Labs) and hybridized with the probes overnight at 55 C for mir-133a. The next day, the slides were soaked in 5 SSC (20 SSC: 3 M NaCl, 0.3 M sodium citrate) and
3 the coverslips were removed. Post-hybridization stringency washing in 50% formamide/1 SSC containing 0.1% Tween-20 was performed twice at 51 C for 1 hour each, followed by washing in 0.2 SSC and PBS. After blocking for 1 hour in blocking solution (0.5% Roche Blocking reagent, 10% goat serum, 0.1% Tween-20, 1 PBS), the sections were incubated in blocking solution containing alkaline phosphatase conjugated anti-dig Fab fragments (1/4000, Roche). The slides were then washed in PBS containing 0.1% Tween-20 and treated in NTM buffer (100 mm Tris-HCl ph 9.5, 5 M NaCl, 1 M MgCl 2 ). Color development was performed in Developer solution (1-Step NBT/BCIP [Pierce] and 2 mm levamisol [Sigma]). Unless otherwise indicated, all washing steps were conducted in PBS and all incubations were performed at room temperature. Images were captured using a microscope (BZ-8100, Keyence). Triphenyltetrazolium chloride (TTC) staining A removed heart was sliced into three separate sections. The middle section was incubated in 1.5% triphenyltetrazolium chloride (TTC) at 37 C for 15 min. After TTC staining, viable myocardium appeared red. Cell culture and evaluation of cell viability A rat cardiomyoblast cell line, H9c2, and 293FT cells were obtained from the American
4 Type Culture Collection (Rockville, MD, USA) and maintained in Dulbecco s modified Eagle s medium (D-MEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 C in 5% CO2 incubator. Oligomyocin and Z-VAD-FMK, a Caspase inhibitor Ⅰ, were purchased from Calbiochem. Deferoxamine was purchased from Sigma. H9c2 cells were stimulated with calcium ionophore A23187 (Sigma) or indicated agent, and cultured in serum-free medium (SFM). In Z-VAD-FMK experiment, H9c2 cells were pre-incubated in Z-VAD-FMK contained medium for 1 hour. Total LDH activity in the culture medium was measured using a LDH Cytotoxicity Assay Kit (Cayman Chemical Company) in accordance with the manufacturer s instructions. Cell viability was evaluated using a MTT (Thiazolyl Blue Tetrazolium Bromide, Sigma) assay. Briefly, H9c2 cells were stimulated with A23187 in SFM for 1 hour and labeled with MTT at a final concentration of 0.5 mg/ml for at least 4 hours at 37 C. Viability was then evaluated by measuring the absorbance at 570 nm using an Elx800 Microplate Reader (BIO-TEK Instruments Inc.). Plasmids mir-133a sensor vector was constructed as described previously 20. Detailed methods are described in the Supplemental Methods. Unmodified pmir-report vector was used as the sensor control vector. prl-tk Renilla reniformis luciferase (RL) plasmid was purchased
5 from Promega. An expression vector for mir-133a was generated using a BLOCK-iT PolⅡ mir RNAi Expression Vector Kit in accordance with the manufacturer s instructions (Invitrogen). Dual-luciferase assay For the sensor vector efficacy study, 0.05 μg of FL reporter gene (mir-133a sensor or sensor control), 0.2 μg of mir-133a expression vector, and 0.02 μg of prl-tk RL plasmid for normalizing transfection efficiency were transfected into 293FT cells. After 2 days incubation, both luciferase activities were measured using a dual-luciferase reporter assay system (Toyo Ink Co.). For the exosome mirna functional study, 0.02 μg of FL reporter gene and 0.02 μg of RL plasmid were tranfected. At 24 hours after transfection, the exosome pellet of H9c2 cells was re-suspended in SFM, and the medium of the 293FT cells was changed to exosome pellet re-suspended media. After 2 days incubation, both luciferase activities were measured as described above.
6 Supplemental Table 1. Clinical characteristics of acute coronary syndrome patients. Blood No. Sex Age [year] sampling time after onset HFABP ctnt [pg/ml] Maximum CPK [IU/L] Maximum CK-MB [IU/L] Diagnosis ST-elevation leads in 12-lead ECG [hour] 1 M Positive > STEMI Ⅱ, Ⅲ, avf 2 M Positive STEMI V2-V5 3 M 67 8 Positive STEMI V1-V5 4 M Positive STEMI V1-V4 5 M 62 2 Positive STEMI Ⅱ, Ⅲ, avf 6 M 92 6 Positive > STEMI Ⅰ, avl, V2-V6 7 M 88 2 Positive Negative UAP N.D. 8 M Positive NSTEMI N.D. 9 M N.A. N.A STEMI Ⅱ, Ⅲ, avf 10 M 69 2 Negative Negative STEMI Ⅱ, Ⅲ, avf 11 M 81 N.D. N.A. N.A UAP N.D. 12 F 88 N.D. Positive <0.1 N.A. N.A. UAP N.D. 13 F Positive NSTEMI N.D. 14 M 79 3 Positive Negative 2361 N.A. STEMI Ⅱ, Ⅲ, avf 15 F 58 N.D. N.A. N.A. 36 N.A. UAP N.D. 16 M 55 1 Positive Negative STEMI Ⅱ, Ⅲ, avf 17 M 62 2 Positive Negative STEMI Ⅱ, Ⅲ, avf 18 M 59 6 Positive Negative STEMI Ⅰ, avl, V2-V6 19 M N.A. N.A STEMI avl, V1-V5 20 M 68 3 Positive STEMI Ⅱ, Ⅲ, avf 21 M 54 9 N.A STEMI Ⅱ, Ⅲ, avf, V1-V6 22 F 60 N.D. Positive Negative UAP N.D. 23 F 49 5 N.A. N.A STEMI Ⅱ, Ⅲ, avf 24 M Positive NSTEMI N.D. 25 M 45 1 Negative Negative STEMI Ⅱ, Ⅲ, avf 26 M 74 N.D. Negative Negative UAP N.D.
7 Supplemental Table 1. continued. Blood No. Sex Age [year] sampling time after onset HFABP ctnt [pg/ml] Maximum CPK [IU/L] Maximum CK-MB [IU/L] Diagnosis ST-elevation leads in 12-lead ECG [hour] 27 F 69 N.D. N.A. N.A. 68 N.A. UAP N.D. 28 M 66 7 Positive > STEMI Ⅰ, avl, Ⅱ, Ⅲ, avf 29 M 84 N.D. Positive Negative UAP N.D. Mean ± S.E. N.D ± ± 1.6 N.D. N.D ± ± 49.8 N.D. N.D. HFABP, heart-type fatty acid binding protein; ctnt, cardiac troponin T; CPK, creatine phosphokinase; CK-MB, creatine phosphokinase-mb fraction; M, male; F, female; MI, myocardial infarction; STEMI, ST-elevation MI; NSTEMI, non-st-elevation MI; UAP, unstable angina pectoris; N.A., not applicable; N.D., not determined.
8 Supplemental Figure Legends Supplemental Figure 1. mir-17-5p (A), mir-454 (B), and mir-1249 (C) are not suitable as normalizers for evaluating mirna expression levels in serum. Comparisons of RNA levels in serum of patients with non-acute coronary syndrome (non-acs) and ACS using Taqman microrna RT-PCR. mir-17-5p: non-acs patients, n = 7, ACS patients, n = 5; mir-451: non-acs patients, n = 16, ACS patients, n = 13; mir-1249: non-acs patients, n = 7, ACS patients, n = 5. *, p<0.05. Supplemental Figure 2. mir-133a levels were up-regulated in the serum of patients with unstable angina pectoris (UAP) and Takotsubo cardiomyopathy (Takotsubo CM). A. In UAP patients, mir-133a levels were increased significantly in the serum compared with non-acs patients and non-takotsubo CM patients. B. In Takotsubo CM patients, mir-133a levels were also increased in the serum compared with non-acs patients. *, p<0.05; **, p< Supplemental Figure 3. Serum levels of mir-1 (A) and mir-133a (B) in mouse serum after myocardial infarction. Significant elevation was observed at 1 hour after coronary ligation. n = 3-5. *, p<0.05.
9 Supplemental Figure 4. In situ hybridization of mir-133a and triphenyltetrazolium chloride (TTC) staining of a mouse myocardial infarction model. A. Short axis view of mir-133a in situ expression. B. Transverse section of an infarcted heart after TTC staining. Supplemental Figure 5. Mature mir-133a levels decreased after stimulation of oligomycin or deferoxamine (DFO). When H9c2 cells were stimulated with oligomyocin of 20 μmol/l for 24 hours (A) or DFO of 200 μmol/l for 60 hours (B), cell viability significantly decreased. Stimulating of oligomyocin (C) or DFO (D), mature mir-133a levels were down-regulated in H9c2 cells evaluating Taqman micrornas RT-PCR. n = 3. *, p<0.05; **, p<
10 A mir-17-5p * B mir-454 C mir-1249 Supplemental Fig. 1.
11 A Unstable angina B Takotsubo cardiomyopathy * ** non-acs non-takotsubo CM UAP non-acs non-takotsubo CM Takotsubo CM Supplemental Fig. 2.
12 A mir-1 B mir-133a * n.s. n.s. n.s. n.s. * 1 hr 3 hr 6 hr 1 hr 3 hr 6 hr Supplemental Fig. 3.
13 A B Supplemental Fig. 4.
14 A ** ** Oligomycin [μm] DFO [μm] B ** * Oligomycin [μm] DFO [μm] Supplemental Fig. 5.
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