BNP mrna expression in DR and DS rat left ventricles (n = 5). (C) Plasma norepinephrine
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1 Kanazawa, et al. Supplementary figure legends Supplementary Figure 1 DS rats had congestive heart failure. (A) DR and DS rat hearts. (B) QRT-PCR analysis of BNP mrna expression in DR and DS rat left ventricles (n = 5). (C) Plasma norepinephrine (NE) concentration in DR and DS rats (n = 5). (D) Tissue NE contents in DR and DS rat left ventricle (n = 5). (E) Triple immunofluorescence staining for α-actinin (purple), TH (red), and CHT (green) in DR and DS left ventricles. DS rat ventricles had fewer TH + nerves but more CHT + nerves than controls (low magnification view, compared with Figure 2). Boxed areas correspond to the high-power photomicrographs in the insets. LA, left atrium; LV, left ventricle. Representative data are shown in each panel. *, P < Scale bars, 25 μm (E, insets); 100 μm (E). Supplementary Figure 2 Cardiac nerve fibers in Dahl salt sensitive rat ventricles have both catecholaminergic and cholinergic activity. (A) Confocal images of double immunofluorescence staining for TH (green) and NF (red) in left ventricles at the epicardial sites in DR and DS rats. (B) Confocal images of double immunofluorescent staining for CHT (green) and NF (red) in left ventricles at the epicardial sites in DR and DS rats. Arrows indicate CHT + nerve fibers in the fasciculated sympathetic nerve bundles. (C) Confocal images of double immunofluorescent staining for calcitonin gene-related peptide (CGRP, green) and NF (red) in left ventricles at the epicardial sites in DR and DS rats. (D) Quantitative analysis of TH +, CHT + and CGRP + nerve areas in left ventricles. *, P < 0.01; ns, not significant. Scale bars, 20 μm. 1
2 Kanazawa, et al. Supplementary Figure 3 DS rats have fewer dopamine beta-hydroxylase (DBH)-immunopositive neurons in the left ventricle and stellate ganglia than do DR rats. (A) Confocal images of double immunofluorescent staining for DBH (green) and NF (red) in the left ventricular epicardium of DR and DS rats. (B) Double immunofluorescent staining with DBH (red) and ChAT (green) of stellate ganglia in DR and DS rats. LV, left ventricle; SG, stellate ganglia. Scale bars, 20 μm (A); 100 μm (B). Supplementary Figure 4 Cholinergic transdifferentiation occurs gradually in accordance with the progression of cardiac hypertrophy to heart failure in DS rats. (A and B) Quantitative analysis of TH + /NF + and CHT + /NF + area ratios in the LV epicardium of 6- (baseline), 11- (hypertrophy phase), 15- (heart failure phase) week-old rats (n = 5). (C) Quantitative analysis of ChAT + /TH + ratio in stellate ganglia (n = 4). *, P < 0.01; **, P < 0.05; ns, not significant. Supplementary Figure 5 Immunofluorescence staining of sympathetic ganglia neurons in DS rats. (A) Immunofluorescence staining for TH (red), ChAT (green), and Toto3 (blue) in DR and DS rat lumbar ganglia. (B) Quantitative analysis of ChAT + /TH + ratio in lumbar ganglia (n = 4). Representative data are shown in each panel. ns: not significant. (C) Immunofluorescence staining for TH (red), ChAT (green) and Toto3 (blue) in DR and DS rat superior cervical ganglia (SCG), upper thoracic ganglia (TG) and lower TG. (D) Quantitative analysis of 2
3 Kanazawa, et al. ChAT + /TH + ratio in SCG, upper TG and lower TG (n = 4). *, P < 0.01; ns, not significant. Scale bars, 100 μm. Supplementary Figure 6 Anterograde labeling of cardiac sympathetic nerves with BDA. (A) Injected BDA is detected in the cardiac plexus and sympathetic nerve fibers in the left ventricle. (B) Representative transmission electron micrograph of nerve ending in DS rat ventricle that contains both SGV and SAGV. Black arrows indicate SGV (involving catecholamine) and white arrows indicate SAGV (involving acetylcholine). (C) Quantitative analysis of the (SGV)/(SGV+SAGV) ratio (%) in DR and DS rats. (D) Immunoelectron microphotographs of ventricles using gold particles labeled with anti-th antibody and AF64A injection in DS rats. Note there are various patterns of damage in AF64A-treated DS rat sympathetic nerve endings. Red arrows indicate gold particles labeled with anti-th antibody. Arrowhead indicates clumping of organelles. Asterisks indicate vacuolization. Star indicates degeneration of neurofilaments. epi, epicardium. Representative data are shown in each panel. Scale bars, 0.5 μm (B and C); 50 μm (A). Supplementary Figure 7 Evaluation of the mouse model of congestive heart failure. (A-C) Results from the hypoxiainduced pulmonary hypertension (PH) mouse model. (A) Representative transverse sections of the hearts in short axial view for control and PH mice at 8 weeks of age. (B) Right ventricular systolic pressure (RVSP) of the control and PH mice (n = 5). (C) Quantitative 3
4 Kanazawa, et al. RT-PCR analysis of BNP, LIF and CT-1 expression in the RV at 8 weeks of age (n = 5). (D- F) Transaortic constriction (TAC)-induced LV failure model mice. (D) Reduced fractional shortening (FS) in TAC mice at 4 weeks of age. (E) Measurement of the left ventricular end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD), and posterior wall thickness (PW) of TAC mice from echocardiograms. (F) Quantitative RT-PCR analysis of BNP, LIF and CT-1 expression in the LV of TAC mice at 4 weeks (n = 5). (G) Pulse wave Doppler echo of the control and the constricted flow distal to TAC. (H)The bar graph indicated the pressure gradient caused by TAC. *, P < 0.01; **, P < 0.05; ns, not significant. Scale bars, 1 mm (A). Supplementary Figure 8. Measurement of the baseline cardiac hemodynamics and heart weight/body weight ratio in gp130 flox/flox and gp130 DBHCre mice. HW/BW: (heart weight)/(body weight) ratio, ns, not significant. 4
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13 Kanazawa, et al. Supplementary Table 1 Time course of changes in hemodynamics, body and heart weight, and detection of pleural effusion in DR and DS rats 6 Weeks 11 Weeks 15 Weeks DR DS DR DS DR DS SBP (mmhg) 94± ±5.6 ** 114±8.3 * 202±14.8 * 128±6.7 * 204±15.2 * HR (bpm) 400±15 411±25 405±30 428±41 410±35 421±32 BW (g) 155± ±3.5 ** 314±16 311±38 384± ±21 * HW (g) 0.65± ±0.05 ** 1.19± ±0.25 * 1.25± ±0.08 * HW/BW (mg/g) 4.2± ± ± ±0.07 * 3.2± ±0.1 * Pleural effusion Values are means ± SEM. SBP; systolic blood pressure, HR; heart rate, BW; body weight, HW; heart weight. *, P < 0.01 vs. age-matched DR rats. **, P < 0.05 vs. age-matched DR rats., P < 0.01 vs. 6-week-old rats of the same strain., P < 0.05 vs. 6-week-old rats of the same strain., P < 0.01 vs. 11-week-old rats of the same strain., P < 0.05 vs. weekold rats of the same strain. 1
14 Kanazawa, et al. Supplementary Table 2 Time course of the echocardiographic findings in DR and DS rats 6 Weeks 11 Weeks 15 Weeks DR DS DR DS DR DS LVEDD (mm) 4.9± ± ± ± ± ±0.2 * LVESD (mm) 2.6± ± ± ±0.1 * 4.4± ±0.2 * FS (%) 46±2.6 49±3.0 42±1.2 54±0.7 * 39±1.8 32±1.0 * PWT (mm) 1.5± ±0.04 * 1.4± ±0.1 * 1.6± ±0.08 ** Values are means ± SEM. LVEDD; left ventricular (LV) end-diastolic diameter, LVESD; end-systolic diameter, FS; fractional shortening, PWT; posterior wall thickness. *, P < 0.01 vs. age-matched DR rats. **, P < 0.05 vs. age-matched DR rats., P < 0.01 vs. 6-week-old rats of the same strain., P < 0.05 vs. 6-week-old rats of the same strain., P < 0.01 vs. 11-week-old rats of the same strain., P < 0.05 vs. week-old rats of the same strain. 2
15 Kanazawa, et al. Supplementary Table 3 Measurements of hemodynamics, and body and heart weight, in WT, αmhc-cre, LIF-loxP, and LIF transgenic mice (Line 3 and Line 10) WT αmhc-cre LIF-loxP Line 3 Line 10 SBP (mmhg) 109± ± ± ± ±5.2 HR (bpm) 420±12 416±15 425±10 430±15 423±13 BW (g) 12.9± ± ± ± ±1.1 * HW (g) 0.059± ± ± ±0.01 * 0.078±0.003 * HW/BW (mg/g) 4.55± ± ± ±1.47 ** 8.52±0.71 * Values are means ± SEM. SBP; systolic blood pressure, HR; heart rate, BW; body weight, HW; heart weight. *, P < 0.01 vs. age-matched αmhc-cre mice. **, P < 0.05 vs. agematched αmhc-cre mice., P < 0.05 vs. age-matched Line 3 mice., P < 0.01 vs. agematched LIF-loxP mice., P < 0.05 vs. age-matched LIF-loxP mice., P < 0.01 vs. agematched WT mice., P < 0.05 vs. age-matched WT mice. 3
hemodynamic stress. A. Echocardiographic quantification of cardiac dimensions and function in
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Postn MCM Smad2 fl/fl Postn MCM Smad3 fl/fl Postn MCM Smad2/3 fl/fl. Postn MCM. Tgfbr1/2 fl/fl TAC
A Smad2 fl/fl Smad3 fl/fl Smad2/3 fl/fl Tgfbr1/2 fl/fl 1. mm B Tcf21 MCM Tcf21 MCM Smad3 fl/fl Tcf21 MCM Smad2/3 fl/fl Tcf21 MCM Tgfbr1/2 fl/fl αmhc MCM C 1. mm 1. mm D Smad2 fl/fl Smad3 fl/fl Smad2/3
c Ischemia (30 min) Reperfusion (8 w) Supplementary Figure bp 300 bp Ischemia (30 min) Reperfusion (4 h) Dox 20 mg/kg i.p.
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
E10.5 E18.5 P2 10w 83w NF1 HF1. Sham ISO. Bmi1. H3K9me3. Lung weight (g)
Myociyte cross-sectional Relative mrna levels Relative levels Relative mrna levels Supplementary Figures and Legends a 8 6 4 2 Ezh2 E1.5 E18.5 P2 1w 83w b Ezh2 p16 amhc b-actin P2 43w kd 37 86 16 wt mouse
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a c e doi:10.1038/nature10407 b d f Supplementary Figure 1. SERCA2a complex analysis. (a) Two-dimensional SDS-PAGE gels of SERCA2a complexes. A silver-stained SDSPAGE gel is shown, which reveals a 12 kda
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Tcf21 MCM ; R26 mtmg Sham GFP Col 1/3 TAC 8W TAC 2W. Postn MCM ; R26 mtmg Sham GFP Col 1/3 TAC 8W TAC 2W
A Tcf21 MCM ; R26 mtmg Sham GFP Col 1/3 Tcf21 MCM ; R26 mtmg TAC 2W Tcf21 MCM ; R26 mtmg TAC 8W B Postn MCM ; R26 mtmg Sham GFP Col 1/3 Postn MCM ; R26 mtmg TAC 2W Postn MCM ; R26 mtmg TAC 8W Supplementary
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marker. DAPI labels nuclei. Flies were 20 days old. Scale bar is 5 µm. Ctrl is
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Supplemental data Supplemental Figure Legends Supplemental Figure 1. Supplemental Figure 2.
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