Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Supplemental Methods; Determination of early LV infarct size Infarct size was determined as described previously (1,2). In brief, 48 hours post-MI, the heart was quickly excised from anesthetized mice and sliced into five 1.0-mm-thick sections perpendicular to the long axis of the heart. The sections were then incubated with 1% triphenyltetrazolium chloride (TTC) (Sigma T8877) in phosphate buffered saline (pH 7.4) for 15 minutes at room temperature. Pictures were taken under an inverted Nikon microscope using a 4X lens. Infarct size was determined by measuring the TTC negative area (infarcted myocardium) and LV circumference by NIS Elements software. Myocardial infarct size was expressed as a percentage of total LV area. Determination of myocardial apoptosis In situ Cell Death Detection kit, TMR red (Roche No. 12156792910) was used for terminal deoxynucleotidyl-transferase–mediated dUTP nick-end labeling (TUNEL). The 3-week post-MI heart sections were treated according to the manufacturer’s instructions and co-stained for cardiomyocyte specific marker Troponin-I and 4,6-diamidino-2phenylindole (DAPI). Slides were applied with mounting media and covered with a glass cover. A Nikon Eclipse 80i fluorescence microscope was used to visualize the TUNELpositive cardiomyocyte, and NIS Elements software was used to capture the images. In vivo 5-bromo-2'-deoxyuridine (BrdU) labeling BrdU (Sigma B5002) was dissolved in 150 mM NaHCO3 to achieve a final concentration of 5 mg/ml. The solution was passed through a 0.22μm filter and intraperitoneal (i.p.) injected at 50mg/kg body weight twice a day for 5 days during 3rd week of MI. Post 3 week of MI, hearts were harvested from anesthetized mice and fixed in 1 4% PFA followed by paraffin embedding and sectioning. Immunofluorescence staining was performed using BrdU labeling and detection kit (Roche 11296736001) as per manufacturer recommendations. Sample preparation, immunoblotting LV lysates were prepared from 3 week post-MI heart as described previously (3). Protein concentration in the lysate was quantified with the Bicinchoninic acid (BCA) protein assay (#23225 from Pierce). Equal amounts of proteins were subjected to 420% gradient SDS-PAGE and subsequently were transferred to nitrocellulose membranes (GE Healthcare Whatman#10401196). Membranes were blocked for 60 minutes using LI-COR blocking buffer. Primary antibody incubations were performed at different dilutions for different antibodies as described in the antibody list (in Supplementary data). All incubations for primary antibodies were performed overnight at 4°C followed by secondary antibody (Alexa Fluor 680 or Alexa Fluor 800 from LICOR) incubation at 1:4000 dilutions for 1 hour at room temperature. After 3X washing, membranes were scanned with the Odyssey Infrared Imaging System (LI-COR). Neonatal rat ventricular myocyte isolation Primary cultures of neonatal rat ventricular myocytes (NRVMs) were prepared from 12-day-old Sprague-Dawley rats as previously described (4). Cells were plated onto Primaria cell culture dishes (BD) for immunoblotting. Following desired treatments, the cells were lysed for immunoblotting as described previously. Immunohistochemistry Heart sections were deparaffinized with xylene and rehydrated with consecutive incubations in decreasing concentrations of ethanol and finally incubated in distilled 2 water. For antigen retrieval, specimens were boiled for 20 minutes in preheated citrated antigen retrieval system (DAKO S1699) using a 920C hot water incubator. After 3 washes with phosphate buffered saline (PBS), endogenous peroxidase activity was blocked by an additional incubation with 0.15% hydrogen peroxide for 20 minutes at room temperature followed by 45 min incubation with TNB (blocking) buffer (Perkin Elmer, Woodbridge, Ontario). Tissue was incubated with primary antibodies for overnight at 4°C in TNB. After 3 washes in PBS, slides were incubated with secondary antibodies: EnVisionTM+ System-HRP (DAKO K4002). For fluorescence amplification, a TRITC-based Tyramide reagent pack from Perkin Elmer was used for Ki67, phosphohistone H3 (Ser10) and cyclin E1 according to the manufacturer instructions. Slides were then washed 3x with PBS and incubated with cardiomyocyte specific primary antibody (Sarcomeric α-actinin/ Troponin I) for 60 min at 370C followed by 3x wash with PBS. Slides were incubated with fluorescence labeled secondary (Alexa fluor488/ Alexa fluor555) antibodies. After 3X washing with PBS, nuclei were stained by DAPI (1ug/ml) for 15 min. After a quick PBS wash, tissues were mounted with Vectashield (Vector laboratories, Burlingame). For Massons’ Trichrome, we followed the manufacturers instructions for Trichomes Accustain (Masson), Sigma-Aldrich. Pictures were taken under an inverted Nikon Eclipse 80i fluorescent microscope. Infarct size, fibrosis and other cell quantifications were performed in a blinded manner using NIS Elements software. Cardiomyocyte circumference measurement Cardiomyocyte cross-sectional area was measured from 5-μm-thick 8 week post-MI heart sections that had been stained with Massons’ Trichrome using NIS Element 3 software. Only myocytes that were round (i.e., were cut in cross section) were included in the analysis. An observer blind to the genotypes of the heart performed the measurements. Quantification of viable cardiomyocytes in scar Five images from scar area of each Masson trichrome stained sections of post- 8 week MI WT and KO hearts were captured using 20X lens and quantification was performed using NIS element. Co-immunoprecipitation Cells were harvested in RIPA/lysis buffer. After protein quantification, 500 μg of protein in 500μl lysate was incubated with 2 μg of an antibodies against E2F-1 and cyclin E1 overnight at 4°C using a rotator followed by another 2 hour incubation with Protein G Plus/ Protein A Agarose beads (Calbiochem#IP05). The beads were washed 3X with ice cold PBS. Protein complexes were released by 5 min of boiling in SDS sample buffer, resolved by SDS-PAGE and subjected to immunoblotting. A detailed list of different antibodies used and applications S. Antibody Vendor Catalog No Dilution Application 1 Beta-Catenin Cell Signaling 9587 1;1000 Western Blotting 2 p-Beta Catenin (Thr41/Ser45) Cell Signaling 9565 1;500 Western Blotting 3 GAPDH Fitzgerald 10R-G109a 1;10,000 Western Blotting 4 GSK-3α/β Cell Signaling 5676 1;1000 Western Blotting 5 GSK-3α Cell Signaling 4337 1;1000 Western Blotting No. 4 6 GSK-3β Cell Signaling 9315 1;1000 Western Blotting 7 phospho-GSK-3α(Ser21) Cell Signaling 9316 1;1000 Western Blotting 8 phospho-GSK-3β(Ser9) Cell Signaling 9336 1;1000 Western Blotting 9 Bax Cell Signaling 2772 1;1000 Western Blotting 10 Bcl-2 Cell Signaling 2870 1;1000 Western Blotting 11 Cyclin D1 Cell Signaling 2926 1;500 Western Blotting 12 Cdk4 Santa Cruz Biotech sc-260 1;100 Western Blotting 13 Cdk2 Santa Cruz Biotech sc-163 1;100 Western Blotting 14 p-Cdk2 (Thr 160) Cell Signaling 2561 1;500 Western Blotting 15 Retinoblastoma Cell Signaling 9313 1;500 Western Blotting 16 p-Retinoblastoma (Ser780) Cell Signaling 3590 1;1000 Western Blotting 17 Cyclin E1 Santa Cruz Biotech sc-481 1;100 Western Blotting 18 E2F-1 Santa Cruz Biotech sc-193 1;100 Western Blotting 19 phospho-cyclin E1(Thr395) Santa Cruz Biotech sc-12917-R 1;100 Western Blotting 20 α-Smooth muscle actin Sigma A5228 1;2000 Western Blotting 21 Vimentin Cell Signaling 5741 1;1000 Western Blotting 22 Axin-2 Thermo Scientific PA5-21093 1;500 Western Blotting 23 phospho-NFkB P65 (Ser536) Cell Signaling 3033 1;500 Western Blotting 24 NFkB P65 Santa Cruz Biotech Sc-109 1;100 Western Blotting 25 phospho-4E-BP1 (Thr37/46) Cell Signaling 2855 1;1000 Western Blotting 26 4E-BP1 Cell Signaling 9644 1;1000 Western Blotting 27 p-S6 Ribosomal (Ser235/236) Cell Signaling 4856 1;1000 Western Blotting 28 S6 Ribosomal protein Cell Signaling 2317 1;1000 Western Blotting 29 p-P70S6Kinase (Thr389) Cell Signaling 9205 1;1000 Western Blotting 30 P70S6Kinase Cell Signaling 2708 1;1000 Western Blotting 5 31 Cyclin E1 Santa Cruz Biotech sc-481 2μg/500μg Immunoprecipitation 32 E2F-1 Santa Cruz Biotech sc-193 2μg/500μg Immunoprecipitation 33 Ki67 Vector Lab VP-RM04 1;50 Immunofluorescence 34 Sarcomeric α-actinin Sigma A7811 1;200 Immunofluorescence 35 Troponin I Cell Signaling 4002 1;200 Immunofluorescence 36 phospho-Histone H3 (Ser 10) Upstate 06-570 1;400 Immunofluorescence 37 Cyclin E1 Abcam ab52189 1;300 Immunofluorescence References; 1. Most P, Seifert H, Gao E et al. Cardiac S100A1 protein levels determine contractile performance and propensity toward heart failure after myocardial infarction. Circulation 2006;114:1258-68. 2. Gao E, Lei YH, Shang X, Huang ZM, Zuo L, Boucher M, Fan Q, Chuprun JK, Ma XL, Koch WJ. A novel and efficient model of coronary artery ligation and myocardial infarction in the mouse. Circ Res 2010;107:1445-53. 3. Lal H, Zhou J, Ahmad F et al. Glycogen synthase kinase-3alpha limits ischemic injury, cardiac rupture, post-myocardial infarction remodeling and death. Circulation 2012;125:65-75. 4. Haq S, Choukroun G, Kang ZB et al. Glycogen synthase kinase-3ß is a negative regulator of cardiomyocyte hypertrophy. J Cell Biol 2000;151:117-129. 6 7