1、 The manuscript number: CVR-2012-1184R11Supplementary materials1. Methods1.1. Immunohistofluorescence analysisIt was performed using the methods described in our previous studies 1. Briefly, Cardiac tissues at papillary muscles were collected from mice and fixed in 4% paraformaldehyde for 2 hrs, and
2、 incubated with 30% sucrose overnight. The tissues were frozen in OCT and sectioned at 5 m. After blocking with 3.5% normal goat serum for 30 min, the cryosections were incubated with rabbit anti-mouse/human HSPA12B antibody (1:100) and rat anti-mouse PCAM-1 antibody (1:250) overnight. Subsequently,
3、 the sections were washed and incubated with FITC-labeled goat anti rabbit IgG (1:100) and Cy3-labelled goat anti-rat IgG (1:100) for 60min. The sections were observed under a fluorescent microscope (Axiovert 200, Zeiss Ltd., Germany) at a magnification of 200 x. 1.2. Myocardial infiltration of infl
4、ammatory cellsHeart tissues at the papillary muscle level were collected at 24 hrs after MI or sham operation. Paraffin section was prepared and subjected to Hematoxylin-Eosin staining (H89:109-118.2. Lancel S, Tissier S, Mordon S, Marechal X, Depontieu F, Scherpereel A et al. Peroxynitrite decompos
5、ition catalysts prevent myocardial dysfunction and inflammation in endotoxemic rats. J Am Coll Cardiol 2004; 43: 2348-2358.3. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin In
6、vest 1973; 52:2745-2756.4. Tang D, Kang R, Xiao W, Jiang L, Liu M, Shi Y, et al. Nuclear heat shock protein 72 as a negative regulator of oxidative stress (hydrogen peroxide)-induced HMGB1 cytoplasmic translocation and release. J Immuno 2007; 178: 7376-7384.The manuscript number: CVR-2012-1184R18Fig
7、ure LegendsFig.S1. Transgene of hspa12b increases the expression of HSPA12B without changing the expression of other major heat shock proteins in heart. The hearts were collected from WT and HSPA12B Tg mice (8-10 weeks old). The cellular proteins were prepared for immunoblot analysis. The same membr
8、ane was also blotted with -Tubulin as a loading control. Each lane represents an individual animal. n=4/group, * P0.01.Fig.S2. HSPA12B is selectively expressed in endothelial cells in cardiac tissues in Tg mice both at basal level and post-MI.Cardiac tissues at papillary muscles were collected from
9、Tg mice at basal level (A) or at 24 hrs post-MI (B). The tissues were then prepared for cryosectioning. The sections were subsequently subjected to immunofluorescence staining against HSPA12B (green) and PCAM-1 (red), a selective marker of endothelial cells. The staining was observed using a fluores
10、cence microscope at a magnification of 200 x. n=3/group.Fig.S3. Sham operation did not significantly change LV morphometric features and cardiac function.LV morphometric features and cardiac function were evaluated at 1 and 4 week(s) The manuscript number: CVR-2012-1184R19after sham operation by ech
11、ocardiography. The representative M-mode images of echocardiography are shown. The mice without any interventions served as basal controls (baseline). n=6-17/group.Fig.S4. Overexpression of HSPA12B reduces infarct size. Cardiac infarct size was evaluated at 24 hrs after MI. AAR, area at risk; LV, le
12、ft ventricular volume. n=5/group, *P0.01.Fig.S5. Inflammatory cells infiltration in ischemic hearts. WT and Tg mice were subjected to sham or MI operation. Twenty-four hours later, cardiac tissues at papillary muscles level were collected and prepared for paraffin-section. HE counterstaining and leu
13、kocyte infiltration examination were subsequently performed. Original magnification: 200 . *P0.01, n=3-4/group.Fig.S6. Overexpression of HSPA12B increased NOS activity after MI. Hearts were collected at 24 hrs after MI. NOS activity was then analyzed. *P0.01 and #P0.05, n=6/group.Fig.S7. Effects of
14、L-NAME on eNOS expression after MI. Hearts were collected at 24 hrs after MI in the presence or absence of L-NAME. The protein levels of eNOS were examined by western blot. *P0.01, n=3/group.The manuscript number: CVR-2012-1184R110Fig.S8. L-NAME did not significantly change cardiac function and stru
15、cture in sham mice. WT mice were administrated with L-NAME in drinking water immediately after sham surgery. Cardiac function and morphometric features were evaluated by echocardiography 1 week later. WT mice that did not receive any interventions served as a basal control. The representative M-mode
16、 images of echocardiography are shown. n=7-17/group. Fig.S9. Fibrosis in ischemic hearts following L-NAME treatment. WT and Tg mice were subjected to MI for 1 week in the presence of L-NAME. Cardiac tissues at papillary muscle were collected and prepared for paraffin-sectioning. Massons trichrome st
17、aining was used to analyze myocardial interstitial fibrosis. n=3/group. Fig.S10. The expression of HSPA12B was upregulated by ischemic stress in myocardium. Hearts were harvested at 24 hrs after MI. The cellular protein extracts were then prepared for western blotting. n=4/group, * P0.01.Fig.S11. Th
18、e expression of HSPA12B was upregulated by heat shock in HUVEC. The manuscript number: CVR-2012-1184R111HUVECs were subjected to heat shock (42C) for 45 min and were then recovered (HSR) for 0, 0.5, 1, 2, 4 or 24 hrs. Untreated HUVECs served as controls (Con). n=3/group, *P0.01 vs. Con.The manuscrip
19、t number: CVR-2012-1184R112Tab.S1. Sham operation did not significantly change cardiac performanceBaseline 1 week post-sham 4 week post-shamWT Tg WT Tg WT Tgn=17 n=16 n=6 n=6 n=6 n=6EF (%) 65.96 6.50 65.67 8.34 65.986.17 63.915.98 65.501.39 63.883.11FS (%) 35.80 4.79 35.73 6.53 35.614.70 34.064.44 3
20、5.101.22 34.022.04SV (l) 33.96 5.51 32.47 5.66 32.345.85 32.007.08 32.635.56 31.337.44LVIDd (mm) 3.51 0.28 3.46 0.33 3.360.36 3.380.67 3.300.30 3.570.48LVIDs (mm) 2.26 0.30 2.24 0.39 2.170.33 2.360.73 2.130.17 2.360.36LVVd (l) 51.91 9.95 50.25 11.86 50.8710.35 48.4118.04 47.908.07 51.4413.17LVVs (l)
21、 17.95 6.11 17.79 7.77 16.426.08 16.0715.55 15.283.10 20.117.52IVSd (mm) 0.72 0.10 0.71 0.11 0.730.09 0.720.07 0.660.08 0.680.07IVSs (mm) 1.09 0.16 1.04 0.12 1.020.16 0.950.07 0.951.10 1.010.15LVPWd (mm) 0.70 0.11 0.66 0.14 0.710.12 0.690.1 0.690.10 0.710.17LVPWs (mm) 1.15 0.16 1.08 0.23 1.170.4 1.1
22、10.41 1.130.14 1.130.08%EF: Ejection fraction; %FS: Fractional Shortening; SV, stroke volume; LVIDd: left ventricular internal diameter at diastolic phase; LVIDs: left ventricular internal diameter at systolic phase; LVVd: left ventricle end-diastolic volume; LVVs: left ventricle end-systolic volume
23、; IVSd: interventricular septal thickness at diastolic phase; IVSs: interventricular septal thickness at systolic phase; LVPWd: left ventricular posterior wall thickness at diastolic phase; LVPWs: left ventricular posterior wall thickness at systolic phase. The manuscript number: CVR-2012-1184R113Ta
24、b.S2. Treatment with L-NAME did not significantly change cardiac performance in sham mice%EF: Ejection fraction; %FS: Fractional Shortening; SV, stroke volume; LVIDd: left ventricular internal diameter at diastolic phase; LVIDs: left ventricular internal diameter at systolic phase; LVVd: left ventri
25、cle end-diastolic volume; LVVs: left ventricle end-systolic volume; IVSd: interventricular septal thickness at diastolic phase; IVSs: interventricular septal thickness at systolic phase; LVPWd: left ventricular posterior wall thickness at diastolic phase; LVPWs: left ventricular posterior wall thick
26、ness at systolic phase. Baseline Sham/L-NAMEWT WTn=17 n=7EF (%) 65.96 6.50 64.475.02FS (%) 35.80 4.79 34.343.59SV (l) 33.96 5.51 27.136.39LVIDd (mm) 3.51 0.28 3.220.35LVIDs (mm) 2.26 0.30 2.120.29LVVd (l) 51.91 9.95 42.3710.42LVVs (l) 17.95 6.11 15.244.53IVSd (mm) 0.72 0.10 0.700.11IVSs (mm) 1.09 0.16 1.080.15LVPWd (mm) 0.70 0.11 0.750.10LVPWs (mm) 1.15 0.16 1.070.10
