c Ischemia (30 min) Reperfusion (8 w) Supplementary Figure bp 300 bp Ischemia (30 min) Reperfusion (4 h) Dox 20 mg/kg i.p.

Transcription

1 a Marker Ripk3 +/ 5 bp 3 bp b Ischemia (3 min) Reperfusion (4 h) d 2 mg/kg i.p. 1 w 5 w Sacrifice for IF size A subset for echocardiography and morphological analysis c Ischemia (3 min) Reperfusion (8 w) e 5 mg/kg i.p. weekly for 4 weeks 4 w Sacrifice Echocardiography at 4 and 8 w after reperfusion Echocardiography and sacrifice Supplementary Figure 1 Nature Medicine: doi:1.138/nm.417

2 a Collagen I/18S 1 5 b Collagen III/18S 6 3 Sham I/R 8 w Sham I/R 8 w c d e LDH concentration (U/ml) EF (%) FS (%) IVSs (mm) LVPWs (mm) Supplementary Figure 2 Nature Medicine: doi:1.138/nm.417

3 a b RIP3 mrna ( M) ( M).1 1. c d RIP3 mrna H/R Control H/R RIP3 RIP3 GAPDH GAPDH e RIP3 protein ( M) f RIP3 protein Control H/R Normal area Ischemic area Supplementary Figure 3 Nature Medicine: doi:1.138/nm.417

4 a RIP1/GAPDH RIP1 sirna1 RIP1 sirna2 b Cell viability 1..5 Nec1 NS c Cell viability 1..5 Control Nec1 H/R d MLKL/GAPDH MLKL sirna1 MLKL sirna Supplementary Figure 4 Nature Medicine: doi:1.138/nm.417

5 a Ischemia (3 min) Reperfusion (4 h) b 2 mg/kg i.p. Daily KN-93 i.p. Sacrifice KN-93 i.p. 15 min before ischemia 1 w Echocardiography and sacrifice c Cell viability 1..5 Control KN-93 H/R d Cell viability 1..5 KN-93 e Cell viability 1..5 KN-93 Supplementary Figure 5 Nature Medicine: doi:1.138/nm.417

6 a Control H/R DAPI HA DAPI HA Ad-HA-RIP3 Ad-myc-CaMKII myc Merged myc Merged b DAPI RIP3 c DAPI RIP3 Sham CaMKII DAPI Merged RIP3 CaMKII DAPI Merged RIP3 I/R CaMKII Merged CaMKII Merged CaMKII RIP3 Supplementary Figure 6 Nature Medicine: doi:1.138/nm.417

7 a p-camkii t-camkii GAPDH p-camkii/t-camkii * K51A b c Cell viability LDH release K51A Supplementary Figure 7 Nature Medicine: doi:1.138/nm.417

8 a b f CypD -tubulin CypD/ -tubulin c CypD sirna1 CypD sirna1 CypD sirna2 * * CypD sirna2 Cell viability LDH release CypD sirna1 CypD sirna2 CypD sirna1 CypD sirna2 g JC-1 fluorescence (%) JC-1 fluorescence (%) * I/R d TMRM fluorescence (%) 1 5 CypD sirna1 CypD sirna2 e TMRM positive area/ Total cell area (%) CypD sirna1 CypD sirna2 Supplementary Figure 8 Nature Medicine: doi:1.138/nm.417

9 a K51A b TMRM fluorescence (%) 1 5 K51A c TMRM positive area/ Total cell area (%) 6 3 K51A d Ad-CaMKII-DN e f TMRM fluorescence (%) TMRM positive area/ Total cell area (%) Ad-CaMKII-DN Ad-CaMKII-DN Supplementary Figure 9 Nature Medicine: doi:1.138/nm.417

10 a Nox2 GAPDH Nox2/GAPDH 2 1 Nox2 sirna1 * Nox2 sirna2 b Cell viability 1..5 Nox2 sirna1 Nox2 sirna2 c Cell viability 1..5 Tiron d e ox-camkii t-camkii h KN-93 DHE intensity 9 45 ox-camkii/t-camkii (fold of wt vehicle) 2 1 DCF intensity 4 2 KN-93 f PYGL GAPDH GLUD1 GAPDH GLUL GAPDH sirna g sirna PYGL GLUL GLUD1 DCF intensity 2 1 PYGL sirna GLUD1 sirna GLUL sirna Supplementary Figure 1 Nature Medicine: doi:1.138/nm.417

11 DAPI I/R 8 w TUNEL Merged TUNEL positive cells (%) 8 4 Supplementary Figure 11 Nature Medicine: doi:1.138/nm.417

12 a 2 mg/kg DAPI TUNEL Merged TUNEL positive cells (%) 8 4 b 5 mg/kg X 4 DAPI TUNEL Merged TUNEL positive cells (%) 8 4 Supplementary Figure 12 Nature Medicine: doi:1.138/nm.417

13 a b c d e IL-6/18S 8 4 TNF- /18S 4 2 IL-6/18S 4 2 Sham I/R TNF- /18S 6 3 Sham I/R CD3 + cell number/mm f Sham I/R CD3 + cell number/mm Sham I/R Supplementary Figure 13 Nature Medicine: doi:1.138/nm.417

14 a IL-6/18S Nec1 NS b TNF- /18S Nec1 NS c IL-6/18S RIP1 sirna1 RIP1 sirna2 NS NS d TNF-a/18S RIP1 sirna1 RIP1 sirna2 NS NS e IL-6/18S Ad-CaMKII-DN f TNF- /18S Ad-CaMKII-DN Supplementary Figure 14 Nature Medicine: doi:1.138/nm.417

15 Supplementary Table 1. Heart physiological data HR (bpm) LVIDd (mm) LVPWd (mm) LVIDs (mm) LVPWs (mm) EF (%) FS (%) HW/BW (mg/g) 44.8± ±.18.82±3 2.53± ± ± ± ± ± ±7.85±2 2.57±8 1.18±5 68.4± ± ±.37 p value Values are mean s.e.m.; n = 1, Student s t test. HR, heart rate; LVID, left ventricle internal diameter; LVPW, left ventricle posterior wall thickness; EF, ejection fraction; FS, fraction shortening; HW/BW, heart weight/body weight; d, diastolic. s, systolic.. Nature Medicine: doi:1.138/nm.417

16 Supplemental Figure Legends Supplementary Figure 1. Genotyping of mice and experimental protocols for in vivo studies. (a) PCR genotyping of, Ripk3 +/, and mice. (b,c) Acute (b) and chronic (c) in vivo I/R in mouse hearts (3-min ischemia followed by 4-h or 8-week reperfusion, respectively). (d,e) Acute (2 mg/kg, i.p.) (d) and chronic treatment (5 mg/kg, i.p. weekly for 4 weeks) of mice (e). Supplementary Figure 2. Chronic I/R- and -induced cardiac fibrosis and remodeling are reduced in mice. (a,b) Averaged mrna levels of collagen I (a) and III (b) assessed by real-time PCR in the hearts of and mice with sham surgery or 3-min cardiac ischemia followed by 8-week reperfusion (n = 1). (c) Serum LDH concentrations in and mice 4 weeks after the final or vehicle injection (n = 11). (d) Representative photomicrographs of Masson's trichrome-stained sections from the hearts of and mice with or without treatment (5 mg/kg, i.p. weekly for 4 weeks) (scale bar, 1 µm). (e) Cardiac contractile function (EF, ejection fraction; FS, fractional shortening) and geometry (LVPWs, systolic left ventricular posterior wall thickness; IVSs, systolic interventricular septum thickness) assayed by echocardiography in and mice 4 weeks after the final or vehicle injection (n = 19 for vehicle, n = 11 for, n = 15 for vehicle, n = 11 for ). Data are mean s.e.m., p <1, one-way ANOVA. Nature Medicine: doi:1.138/nm.417

17 Supplementary Figure 3. Upregulation of RIP3 induced by and I/R (or H/R) in cultured cardiomyocytes and in vivo. (a) Averaged RIP3 mrna levels assayed by real-time PCR in cultured cardiomyocytes treated with at different concentrations for 24 h (n = 12). (b) Typical western blots and averaged RIP3 protein levels in cardiomyocytes treated with at different concentrations for 24 h (n = 3). (c) Averaged RIP3 mrna levels assayed by real-time PCR in NRVMs subjected to hypoxia/reoxygenation (H/R) (n = 8 for control, n = 9 for H/R). (d) Typical western blots and averaged RIP3 protein levels in NRVMs with and without H/R (n = 7). (e) Representative immunostaining for RIP3 in mouse hearts 3 days after a single treatment (2 mg/kg, i.p.) or vehicle. (f) Representative photomicrographs of immunostaining for RIP3 in normal and ischemic areas from rat hearts subjected to I/R injury (45-min ischemia followed by 24-h reperfusion). Scale bar, 1 µm. Data are mean s.e.m., p <1 vs control group. In a,b, one-way ANOVA; in c,d, Student s t test. Supplementary Figure 4. RIP3-mediated cardiomyocyte necroptosis is independent of RIP1 or MLKL. (a) Averaged data showing the expression of RIP1 in cells transfected with scrambled or RIP1 sirnas (n = 3). Representative examples are shown in Fig. 3c. (b) Cell viability indexed by cellular ATP content in NRVMs infected with Ad-β-gal or with or without Nec1 treatment (3 μm) (n = 16 for vehicle, n = 19 for Nec1). (c) Cell viability indexed by cellular ATP content in Nature Medicine: doi:1.138/nm.417

18 cardiomyocytes subjected to H/R with or without Nec1 pretreatment (3 μm) (n = 22). (d) Averaged data showing the expression of MLKL in cells infected with scrambled or MLKL sirnas (n = 3). Representative examples are shown in Fig. 3e. Data are mean s.e.m., p <1, one-way ANOVA. NS, not significant. Supplementary Figure 5. Inhibition of CaMKII with KN-93 alleviates I/R- and -induced cardiomyocyte necrosis. (a,b) The experimental protocol for KN-93 treatment of cardiac injury induced by I/R (a) or treatment (b). (c) Viability of cells treated with vehicle or KN-93 (5 μm) in the presence or absence of H/R (n = 12 for vehicle, n = 18 for KN-93). (d) Viability of cells treated with vehicle or KN-93 (5 μm) in response to treatment (1 μm) (n = 9 for vehicle, n = 7 for ). (e) Viability of cells treated with vehicle or KN-93 (5 μm) and infected with Ad-β-gal or (n = 12 for vehicle, n = 15 for KN-93). Data are mean s.e.m., p <1, one-way ANOVA. Supplementary Figure 6. Co-localization of RIP3 and CaMKII in the hearts. (a) Representative immunofluorescent confocal microscopic images of HA-tagged RIP3 and myc-tagged CaMKII in cultured cardiomyocytes co-infected with and Ad-CaMKII with or without H/R challenge. Scale bars, 1 m. Note that H/R markedly enhanced the co-localization of RIP3 and CaMKII. (b) Representative immunofluorescent confocal microscopic images of RIP3 and CaMKII in mouse hearts subjected to sham or I/R surgery (3-min cardiac ischemia followed by 4-h Nature Medicine: doi:1.138/nm.417

19 reperfusion). The heart from a mouse (bottom) served as a negative control. Scale bar is 2 m. (c) Representative immunofluorescent confocal microscopic images of RIP3 and CaMKII in mouse hearts with or without treatment (2 mg/kg, i.p.). Scale bar is 2 m. Note that both I/R and treatment increased the co-localization of RIP3 and CaMKII in myocardium in vivo. Supplementary Figure 7. Kinase activity is required for RIP3-induced CaMKII activation and cardiomyocyte necroptosis. (a) Typical western blots and averaged data showing the phosphorylation of CaMKII (Thr287) in cultured NRVMs infected with Ad-β-gal,, or -K51A (a kinase-defective mutant of RIP3) (n = 4). (b,c) Cell viability indexed by cellular ATP content (b) and LDH concentration in the culture medium (c) of NRVMs infected with Ad-β-gal,, or K51A (MOI 2; n = 2). Data are mean s.e.m., *p <5, p <1, one-way ANOVA. Supplementary Figure 8. mptp plays an essential role in RIP3-mediated cardiomyocyte necroptosis. (a) Typical western blots and averaged data to show the expression of CypD, a key modulator of mptp, in NRVMs transfected with scrambled or CypD sirnas (n = 3). (b) Cell viability indexed by cellular ATP content and LDH concentration in the culture medium of NRVMs infected with or in the presence or absence of CypD sirnas (n = 2). (c e) Representative photomicrographs (c), average fluorescence intensity (d) and percentage of Nature Medicine: doi:1.138/nm.417

20 TMRM-positive area to total cell area (e) of NRVMs subjected to TMRM staining with or infection for 48 h (the data were from 5 independent experiments; for panels d and e, the averaged data were obtained from more than 5 cells for each group). (f,g) Relative fluorescence intensity of JC-1 staining of isolated myocardial mitochondria from perfused and mouse hearts subjected to I/R (3-min cardiac ischemia followed by 1-h reperfusion) (f) or from and mice 1 week after administration of (2 mg/kg, i.p.) (g) (n = 1). Scale bar, 1 m. Data are mean ± s.e.m., * p <5, p <1, in f,g, Student s t test; in the other panels, one-way ANOVA. Supplementary Figure 9. CaMKII is required for RIP3-induced depolarization of the mitochondrial membrane potential ( m ) in cardiomyocytes. (a c) Representative photomicrographs of TMRM staining (a), average fluorescence intensity (b), and percentage of TMRM-positive area to total cell area (c) of NRVMs infected by, or -K51A (n = 4). (d f) Representative micrographs of TMRM staining (d), averaged fluorescence intensity (e), and percentage of TMRM-positive area to total cell area (f) of NRVMs infected with or in the presence or absence of CaMKII inhibition by Ad-CaMKII-DN (n = 5). Scale bar, 1 m. Data are mean ± s.e.m., p <1, one-way ANOVA. Supplementary Figure 1. ROS are required for RIP3-induced cardiomyocyte Nature Medicine: doi:1.138/nm.417

21 necroptosis. (a) Typical western blots and averaged data showing the expression of Nox2 in cultured cardiomyocytes transfected with scrambled or Nox2 sirnas (n = 3). (b) Cell viability indexed by cellular ATP content in cells infected with Ad-β-gal or with or without Nox2 knockdown (n = 15). (c) Cell viability indexed by cellular ATP content in cells infected with Ad-β-gal or in the presence or absence of Tiron (1 mm) (n = 24 for, n = 16 for ). (d) Representative photomicrographs and averaged data of ROS production indexed by DHE (dihydroethidium) staining in heart sections from and mice treated once with (2 mg/kg i.p.; scale bar,.3 mm) (n = 4). (e) Typical western blots and averaged data showing the oxidation of CaMKII in and mouse hearts treated once with (2 mg/kg i.p.) (n = 4). (f) Typical western blots showing the expression of PYGL, GLUD1 and GLUL in cultured cardiomyocytes transfected with scrambled, PYGL, GLUD1 or GLUL sirnas (n = 3). (g) Representative photomicrographs and averaged data of ROS production assayed by DCF (CM-H2DCFDA) fluorescence intensity in cultured NRVMs infected with or (MOI 2) for 48 h in the presence or absence of gene silencing by PYGL, GLUD1 or GLUL sirnas (n = 12). (h) Representative photomicrographs and averaged data of ROS production assayed by DCF fluorescence intensity in cultured NRVMs infected with or (MOI 2) for 48 h with or without CaMKII inhibition by Ad-CaMKII-DN (n = 15). Scale bar, 1 m. Data are mean s.e.m., *p <5, p <1 vs other groups (d,e), vs scrambled group (g), or as indicated (other panels), one-way ANOVA. Nature Medicine: doi:1.138/nm.417

22 Supplementary Figure 11. RIP3 deficiency abolishes chronic I/R-induced cardiomyocyte apoptosis. Representative photomicrographs and averaged data of TUNEL staining in cardiac sections from and mice subjected to long-term I/R injury (3-min cardiac ischemia followed by 8 weeks of reperfusion) (n = 1). Data are mean s.e.m.. p <1, Student s t test. Scale bar, 2 m. Supplementary Figure 12. RIP3 deficiency protects cardiomyocytes against -induced apoptosis. (a,b) Representative photomicrographs and averaged data of TUNEL staining in cardiac sections from and mice subjected to acute treatment (2 mg/kg, i.p.) (a) or chronic treatment (5 mg/kg, i.p. weekly for 4 weeks) (b) (n = 1 for each group). Data are mean s.e.m.. p <1, Student s t test. Scale bar, 2 m. Supplementary Figure 13. RIP3 is required for - and I/R-induced myocardial inflammation. (a d) Averaged interleukin-6 (IL-6) and TNF- mrna levels assayed by real-time PCR in the hearts of and mice subjected to treatment (2 mg/kg for 1 week) (a,b) or I/R injury (3-min cardiac ischemia followed by 24 h of reperfusion) (c,d) (n = 1 for sham and vehicle groups, n = 12 for I/R and groups). (e,f) Representative photomicrographs and averaged numbers of the CD3-positive cells in the hearts of and mice subjected to treatment (2 mg/kg for 1 week) (e) or I/R (3-min cardiac ischemia followed by 24 h of reperfusion) (f) (n = 1 for sham and vehicle groups, n = 12 for I/R and groups). Nature Medicine: doi:1.138/nm.417

23 The arrows indicated CD-3 positive cells. Data are mean ± s.e.m., p <1, one-way ANOVA. Supplementary Figure 14. RIP3-induced expression of inflammatory factors in the cardiomyocytes is mediated by activation of CaMKII independently of RIP1. (a,b) Averaged IL-6 (a) and TNF- (b) mrna levels assayed by real-time PCR in cardiomyocytes infected with or (MOI 2) for 48 h with or without Nec1 treatment (3 μm) (n = 1). (c,d) Averaged IL-6 (c) and TNF- (d) mrna levels in cardiomyocytes infected with or (MOI 2) for 48 h in the presence or absence of RIP1 sirnas (n = 8). (e,f) Averaged IL-6 (e) and TNF- (f) mrna levels assayed by real-time PCR in the cardiomyocytes infected with or (MOI 2) for 48 h with or without CaMKII inhibition by Ad-CaMKII-DN (n = 15). Data are mean s.e.m.. p <1, one-way ANOVA. NS, not significant. Nature Medicine: doi:1.138/nm.417