hemodynamic stress. A. Echocardiographic quantification of cardiac dimensions and function in

Transcription

1 SUPPLEMENTAL FIGURE LEGENDS Supplemental Figure 1. Fbn1 C1039G/+ hearts display normal cardiac function in the absence of hemodynamic stress. A. Echocardiographic quantification of cardiac dimensions and function in Fbn1 +/+ and Fbn1 C1039G/+ mice with and without valvular regurgitation at 4 and 12 months of age. EDD, end-diastolic diameter; ESD, end-systolic diameter; FS, fractional shortening; LVM, left ventricular mass. n>=14 per group. *p<5,, p<1, p<01, 2w ANOVA, Tukey s correction. B. Representative PV loop of a 12mo Fbn1 +/+ (blue line, blue fill) and Fbn1 C1039G/+ mouse (red line, red fill) without valvular regurgitation. Summary data obtained from PV loop analysis shown in bar graphs: Ees, end-systolic elastance and PRSW, preload recruitable stroke work, reflect load-independent indices of ventricular contractility. Tau, relaxation time constant reflects a load-independent index of ventricular stiffness; n=3-6 per group. C. dp/dtmax, peak rate of left ventricular pressure rise, an index of contractility; dp/dtmin, peak rate of left ventricular pressure decline, an index of lusitropy. Invasive blood pressure measurements taken from PV loop analysis of Fbn1 +/+ and Fbn1 C1039G/+ mice without valvular regurgitation at 4 and 12 months of age. n=3-6 per group. D. Gross morphometric analysis of post-natal Fbn1 +/+, Fbn1 C1039G/+, Fbn1 C1039G/C1039G mice at 8do, 6mo and 12mo age, in mice without valvular regurgitation. HW/BW, heart weight/body weight ratio; HW/TL, heart weight/tibial length ratio. n>=4 per group. Supplemental Figure 2. Fbn1 C1039G/+ hearts have normal TGFβ signaling in the absence of hemodynamic stress. A-C. Representative Western blot and summary quantification for phosphorylated/total (p/t), p/tsmad2, p/terk1/2, and GAPDH using left ventricular tissue lysates. n=4 per group. D. mrna expression normalized to 18S and then to Fbn1 +/+ data, assessed by real-time RT-PCR from left ventricular tissue of 3mo Fbn1 +/+ and 1

2 Fbn /C1039G/+ hearts without regurgitation. Ctgf, connective tissue growth factor. Serpine1, plasminogen activator inhibitor, type 1. n>=4 per group. Supplemental Figure 3. Fbn1 C1039G/+ hearts develop hypertrophy and fibrosis in response to pressure overload. A. Baseline echocardiographic quantification of cardiac dimensions in 5mo Fbn1 +/+ and Fbn1 C1039G/+ mice without valvular regurgitation. EDD, end-diastolic diameter; ESD, end-systolic diameter; IVST, interventricular septum thickness; PWT, posterior wall thickness. n=4-6 per group. B. Myocyte hypertrophy, as assessed by cross-sectional area (CSA) in hematoxylin-eosin staining. Summary quantification of averaged data, >20 cells per heart. n=4 per group. C. Fibrotic area, as assessed by Masson s Trichrome stain and quantified by total number of blue pixels normalized to whole tissue area and then to Fbn1 +/+ :SHAM data. n=4-6 per group. D. mrna expression of hypertrophy genes, normalized to Gapdh and then to Fbn1 +/+ :SHAM data, assessed by real-time RT-PCR. n=4-6 per group. Myh7, myosin heavy chain 7 (βmhc), Nppa, natriuretic peptide A (ANP), Nppb, natriuretic peptide B (BNP). p<1, p<01, 1w ANOVA, Tukey s correction. Supplemental Figure 4. Fibrillin-1 protein fails to deposit in myocardium in load-induced heart failure in Fbn1 C1039G/+ mice. Additional representative images of immunofluorescent fibrillin-1 staining (red) of heart sections from Fbn1 +/+ and Fbn1 C1039G/+ mice subjected to 4w. Red, fibrillin-1 (interstitial space); blue, DAPI (nuclei); Green, lipofuscin (myocytes). Top 4 panels, Scale bar: 20 µm. Bottom 4 panels, 2x zoom. White arrow head points to an example nonmyocyte cell in the interstitial space. Supplemental Figure 5. Smad2 and ERK1/2 activation are differentially increased in the myocyte and nonmyocyte compartments of failing Fbn1 C1039G/+ hearts. A. Representative serial sections of psmad2 (red, top panels) and perk1/2 (red, bottom panels) immunostaining in Fbn1 C1039G/+ 2

3 hearts subjected to 4w. Far left panels, scale bar: 50 µm. Middle and far right panels, zoom 2x. Blue, DAPI (nuclei); Green, lipofuscin (myocytes). White arrows, myocyte nuclei. Yellow arrows, nonmyocyte nuclei. B. Summary quantification of % of total psmad2 and perk1/2 immunofluorescence intensity localized to nonmyocyte (NM) and myocyte (M) cells per unit area. n=10-15 areas quantified. *p<5, p<1, Student s t-test. C. Representative myocyteenriched serial sections. Scale bar: 50 µm. D. Representative co-immunostaining of serial sections with vimentin (yellow) and psmad2 (left panel) and vimentin (yellow) and perk1/2 (right panel). Scale bar: 25 µm. Gray, lipofuscin (myocytes). Supplemental Figure 6. ERK1/2 activation, but not Smad2 activation, is significantly attenuated by MEK inhibition. Immunoblot for psmad2, perk1/2 and GAPDH using cultured neonatal rat cardiac fibroblasts with TGF-ß3 (10 ng/ml) stimulation for 15 minutes with and without treatment with, RDEA119 (100nM) or PD98059 (50μM). n=4 per group. p<1, p<01, 1w ANOVA, Tukey s correction. Supplemental Figure 7. Myocyte hypertrophy in Fbn1 C1039G/+ hearts is prevented with losartan and treatment. A. Representative hematoxylin-eosin staining of formalin-fixed heart sections. White dotted line delineates myocyte diameter. Scale bars: 50µm. B. Myocyte hypertrophy as assessed by wheat germ agglutinin (WGA) staining. Scale bars: 25µm. C. Summary quantification of average cross-sectional area (CSA) data, >1000 cells per heart. n=4-7 per group. D. mrna expression of hypertrophy genes, normalized to Gapdh and then to Fbn1 +/+ :SHAM data, assessed by real-time RT-PCR. Myh7, myosin heavy chain 7 (βmhc), Myh6, myosin heavy chain 6 (αmhc), Nppa, natriuretic peptide A (ANP), Nppb, natriuretic peptide B (BNP). n=4-7 per group. *p<5, p<1, p<01, 1w ANOVA, Tukey s correction. 3

4 Supplemental Figure 8. Smad2 and ERK1/2 activation are differentially increased in response to TGFβ and angiotensin II in cardiac fibroblasts. Immunoblot for psmad2, perk1/2 and GAPDH using cultured neonatal murine cardiac fibroblasts with TGFß1 (5 ng/ml) or AngII (5nM) stimulation for 24 hours. Lanes were run on the same gel but were noncontiguous. n>=3 per group. p<1, 1w ANOVA, Tukey s correction. Supplemental Figure 9. Inhibition of ERK1/2 activation suppresses Smad2 activation in both nonmyocyte and myocyte compartments. A-B. Labeling index (% positive cells) of psmad2 or perk1/2 immunofluorescence in the nonmyocyte and myocyte compartments. n>5 fields per group. C. Total number of nonmyocytes and myocytes normalized to Fbn1 +/+ :SHAM data. n=15-30 fields per group. *p<5, p<1, p<01, 1w ANOVA, Tukey s correction. Supplemental Figure 10. TGFβ3 is predominantly expressed in the nonmyocyte compartment. A- C. Representative in situ hybridization of Tgfb3. A. Fbn1 +/+ : vs Fbn1 C1039G/+ :, Tgfb3, red. Left panel, scale bar: 10µm. Right panel, zoom 2x inset. Dotted line demarcates myocyte compartment. Red arrows point to examples of in situ RNA hybridization (red dots) found throughout the nonmyocyte compartment. B. Myocyte- vs nonmyocyte-enriched areas. Scale bar: 10µm. C. Tgfb3 and Vim. Left panel, Tgfb3, red, left panel and Vim, red, right panel. Dottedline outlines the cardiac myocytes. Scale bar: 10µm. D. mrna expression of Tgfb3 and Vim normalized to Gapdh and then to myocyte data. n=4-6 per group. p<01, Student s t-test. Supplemental Figure 11. AngII-mediated ERK1/2 activation is differentially increased in Fbn1 C1039G/+ vs WT fibroblasts. Immunoblots for psmad2, perk1/2 and GAPDH using cultured neonatal murine cardiac fibroblasts from Fbn1 C1039G/+ and Fbn1 +/+ hearts with AngII (5nM) or TGFß3 (5 ng/ml) stimulation for 12 hours. Lanes were run on the same gel but were noncontiguous. n>=3 per group. p<01, 1w ANOVA, Tukey s correction. 4

5 Supplemental Figure 1 A * * * EDD (mm) ESD (mm) FS (%) LVM (mg) Fbn1 +/+ Fbn1 C1039G/+ without regurgitation Fbn1 C1039G/+ with regurgitation 4 mo 12 mo 4 mo 12 mo 4 mo 12 mo 4 mo 12 mo B 160 E es (mmhg/µl/g) PRSW (mmhg) Tau (ms) Pressure (mmhg) C Volume (ml) 4mo 12 mo Systolic Pressure (mmhg) 4mo 12 mo Diastolic Pressure (mmhg) 4mo 12 mo Pulse Pressure (mmhg) dp/dt max (mmhg/s) Thousands 4mo 12 mo 4mo 12 mo 4mo 12 mo 4mo 12 mo dp/dt min (mmhg/s) Thousands D HW/BW (mg/g) Post natal day 8 HW/BW (mg/g) 6mo HW/BW (mg/g) 12mo +/+ C1039G/+ C1039G/ C1039G +/+ C1039G/+ +/+ C1039G/+ Supplemental Figure 1. Fbn1 C1039G/+ hearts display normal cardiac function in the absence of hemodynamic stress. A. Echocardiographic quantification of cardiac dimensions and function in Fbn1 +/+ and Fbn1 C1039G/+ mice with and without valvular regurgitation at 4 and 12 months of age. EDD, end diastolic diameter; ESD, end systolic diameter; FS, fractional shortening; LVM, left ventricular mass. n>=14 per group. *p<5,, p<1, p<01, 2w ANOVA, Tukey s correction. B. Representative PV loop of a 12mo Fbn1 +/+ (blue line, blue fill) and Fbn1 C1039G/+ mouse (red line, red fill) without valvular regurgitation. Summary data obtained from PV loop analysis shown in bar graphs: E es, end systolic elastance and PRSW, preload recruitable stroke work, reflect load independent indices of ventricular contractility. Tau, relaxation time constant reflects a load independent index of ventricular stiffness; n=3 6 per group. C. dp/dt max, peak rate of left ventricular pressure rise, an index of contractility; dp/dt min, peak rate of left ventricular pressure decline, an index of lusitropy. Invasive blood pressure measurements taken from PV loop analysis of Fbn1 +/+ and Fbn1 C1039G/+ mice without valvular regurgitation at 4 and 12 months of age. n=3 6 per group. D. Gross morphometric analysis of post natal Fbn1 +/+, Fbn1 C1039G/+, Fbn1 C1039G/C1039G mice at 8do, 6mo and 12mo age, in mice without valvular regurgitation. HW/BW, heart weight/body weight ratio; HW/TL, heart weight/tibial length ratio. n>=4 per group.

6 Supplemental Figure 2 A Post natal day 8 +/+ C1039G/+ C1039G/ C1039G psmad2 tsmad2 perk1/2 terk1/2 psmad2/tsmad perk1/2/terk1/ B Fbn1 +/+ Fbn1 C1039G/+ GAPDH 1.6 +/+ C1039G/+ C1039G/ C1039G 1.6 +/+ C1039G/+ C1039G/ C1039G 3 mo psmad2 GAPDH perk1/2 psmad2/gapdh perk1/2/gapdh C Fbn1 +/+ Fbn1 C1039G/+ GAPDH 1.6 +/+ C1039G/ /+ C1039G/+ 12 mo psmad2 tsmad2 perk1/2 terk1/2 psmad2/tsmad perk1/2/terk1/ D 2.0 Ctgf 2.0 Serpine1 +/+ C1039G/+ +/+ C1039G/+ Relative expression Relative expression /+ C1039G/+ +/+ C1039G/+ Supplemental Figure 2. Fbn1 C1039G/+ hearts have normal TGFβ signaling in the absence of hemodynamic stress. A C. Representative Western blot and summary quantification for phosphorylated/total (p/t), p/tsmad2, p/terk1/2, and GAPDH using left ventricular tissue lysates. n=4 per group. D. mrna expression normalized to 18S and then to Fbn1 +/+ data, assessed by real time RT PCR from left ventricular tissue of 3mo Fbn1 +/+ and Fbn /C1039G/+ hearts without regurgitation. Ctgf, connective tissue growth factor. Serpine1, plasminogen activator inhibitor, type 1. n>=4 per group.

7 Supplemental Figure 3 A mm SHAM: Fbn1 +/+ : Fbn1 +/+ SHAM: Fbn1 C1039G/+ : Fbn1 C1039G/+ 1 B Normalized fibrotic area Fbn1 +/+ Fbn1 C1039G/+ EDD ESD IVST PWT C Normalized myocyte area SHAM SHAM 0 SHAM SHAM D Myh7/GAPDH * Nppa/GAPDH Nppb/GAPDH SHAM SHAM SHAM SHAM SHAM SHAM Supplemental Figure 3. Fbn1 C1039G/+ hearts develop hypertrophy and fibrosis in response to pressure overload. A. Baseline echocardiographic quantification of cardiac dimensions in 5mo Fbn1 +/+ and Fbn1 C1039G/+ mice without valvular regurgitation. EDD, end diastolic diameter; ESD, end systolic diameter; IVST, interventricular septum thickness; PWT, posterior wall thickness. n=4 6 per group. B. Myocyte hypertrophy, as assessed by cross sectional area (CSA) in hematoxylin eosin staining. Summary quantification of averaged data, >20 cells per heart. n=4 per group. C. Fibrotic area, as assessed by Masson s Trichrome stain and quantified by total number of blue pixels normalized to whole tissue area and then to Fbn1 +/+ :SHAM data. n=4 6 per group. D. mrna expression of hypertrophy genes, normalized to Gapdh and then to Fbn1 +/+ :SHAM data, assessed by real time RT PCR. n=4 6 per group. Myh7, myosin heavy chain 7 (βmhc), Nppa, natriuretic peptide A (ANP), Nppb, natriuretic peptide B (BNP). p<1, p<01, 1w ANOVA, Tukey s correction.

8 Supplemental Figure 4 Supplemental Figure 4. Fibrillin 1 protein fails to deposit in myocardium in load induced heart failure in Fbn1C1039G/+ mice. Additional representative images of immunofluorescent fibrillin 1 staining (red) of heart sections from Fbn1+/+ and Fbn1C1039G/+ mice subjected to 4w. Red, fibrillin 1 (interstitial space); blue, DAPI (nuclei); Green, lipofuscin (myocytes). Top 4 panels, Scale bar: 20 µm. Bottom 4 panels, 2x zoom. White arrow head points to an example nonmyocyte cell in the interstitial space.

9 Fbn1C1039G/+: Supplemental Figure 5 A B % of total intensity psmad2/3 Myocyte Nuclei D Myocyte Nuclei psmad2 perk1/2 * NM M perk1/2 % of total intensity C NM M Supplemental Figure 5. Smad2 and ERK1/2 activation are differentially increased in the myocyte and nonmyocyte compartments of failing Fbn1C1039G/+ hearts. A. Representative serial sections of psmad2 (red, top panels) and perk1/2 (red, bottom panels) immunostaining in Fbn1C1039G/+ hearts subjected to 4w. Far left panels, scale bar: 50 µm. Middle and far right panels, zoom 2x. Blue, DAPI (nuclei); Green, lipofuscin (myocytes). White arrows, myocyte nuclei. Yellow arrows, nonmyocyte nuclei. B. Summary quantification of % of total psmad2 and perk1/2 immunofluorescence intensity localized to nonmyocyte (NM) and myocyte (M) cells per unit area. n=10 15 areas quantified. *p<5, p<1, Student s t test. C. Representative myocyte enriched serial sections. Scale bar: 50 µm. D. Representative co immunostaining of serial sections with vimentin (yellow) and psmad2 (left panel) and vimentin (yellow) and perk1/2 (right panel). Scale bar: 25 µm. Gray, lipofuscin (myocytes).

10 Supplemental Figure 6 No stim TGFβ3 TGFβ3 + psmad2/gapdh perk1/2/gapdh TGFβ3 + _ + + TGFβ3 + _ + + Supplemental Figure 6. ERK1/2 activation, but not Smad2 activation, is significantly attenuated by MEK inhibition. Immunoblot for psmad2, perk1/2 and GAPDH using cultured neonatal rat cardiac fibroblasts with TGF ß3 (10 ng/ml) stimulation for 15 minutes with and without treatment with, RDEA119 (100nM) or PD98059 (50μM). n=4 per group. p<1, p<01, 1w ANOVA, Tukey s correction.

11 Supplemental Figure 7 A B C Nppb/GAPDH Myocyte Area (μm 3 ) Fbn1 +/+ Fbn1 C1039G/+ D Myh7/GAPDH p=0.10 SHAM SHAM Supplemental Figure 7. Myocyte hypertrophy in Fbn1 C1039G/+ hearts is prevented with losartan and treatment. A. Representative hematoxylin eosin staining of formalin fixed heart sections. White dotted line delineates myocyte diameter. Scale bars: 50µm. B. Myocyte hypertrophy as assessed by wheat germ agglutinin (WGA) staining. Scale bars: 25µm. C. Summary quantification of average cross sectional area (CSA) data, >1000 cells per heart. n=4 7 per group. D. mrna expression of hypertrophy genes, normalized to Gapdh and then to Fbn1 +/+ :SHAM data, assessed by real time RT PCR. Myh7, myosin heavy chain 7 (βmhc), Myh6, myosin heavy chain 6 (αmhc), Nppa, natriuretic peptide A (ANP), Nppb, natriuretic peptide B (BNP). n=4 7 per group. *p<5, p<1, p<01, 1w ANOVA, Tukey s correction. Nppa/GAPDH SHAM SHAM * SHAM SHAM * SHAM SHAM

12 Supplemental Figure 8 psmad2/gapdh Ctrl TGFβ1 AngII perk1/2/gapdh Ctrl TGFβ1 AngII Supplemental Figure 8. Smad2 and ERK1/2 activation are differentially increased in response to TGFβ and angiotensin II in cardiac fibroblasts. Immunoblot for psmad2, perk1/2 and GAPDH using cultured neonatal murine cardiac fibroblasts with TGFß1 (5 ng/ml) or AngII (5nM) stimulation for 24 hours. Lanes were run on the same gel but were noncontiguous. n>=3 per group. p<1, 1w ANOVA, Tukey s correction.

13 Supplemental Figure 9 A psmad2 (% positive) Fbn1 +/+ Fbn1 C1039G/+ Nonmyocytes psmad2 (% positive) Myocytes B perk1/2 (% positive) SHAM SHAM Nonmyocytes perk1/2 (% positive) SHAM SHAM Myocytes C SHAM SHAM Nonmyocytes: Fbn1 +/+ Nonmyocytes: Fbn1 C1039G/+ Myocytes: Fbn1 +/+ Myocytes: Fbn1 C1039G/+ Fold change # of nonmyocytes/mm 2 * * SHAM SHAM SHAM SHAM TGFβNAbLosartan Supplemental Figure 9. Inhibition of ERK1/2 activation suppresses Smad2 activation in both nonmyocyte and myocyte compartments. A B. Labeling index (% positive cells) of psmad2 or perk1/2 immunofluorescence in the nonmyocyte and myocyte compartments. n>5 fields per group. C. Total number of nonmyocytes and myocytes normalized to Fbn1 +/+ :SHAM data. n=15 30 fields per group. *p<5, p<1, p<01, 1w ANOVA, Tukey s correction.

14 Supplemental Figure 10 B Myocyte enriched DAPI D Myocyte Fold increase Vim Tgfb3/GAPDH Nonmyocyte enriched Tgfb3 Myocyte Fibroblast Fold increase Fbn1C1039G/+: C Vim/GAPDH A Myocyte Fibroblast Supplemental Figure 10. TGFβ3 is predominantly expressed in the nonmyocyte compartment. A C. Representative in situ hybridization of Tgfb3. A. Fbn1+/+: vs Fbn1C1039G/+:, Tgfb3, red. Left panel, scale bar: 10µm. Right panel, zoom 2x inset. Dotted line demarcates myocyte compartment. Red arrows point to examples of in situ RNA hybridization (red dots) found throughout the nonmyocyte compartment. B. Myocyte vs nonmyocyte enriched areas. Scale bar: 10µm. C. Tgfb3 and Vim. Left panel, Tgfb3, red, left panel and Vim, red, right panel. Dotted line outlines the cardiac myocytes. Scale bar: 10µm. D. mrna expression of Tgfb3 and Vim normalized to Gapdh and then to myocyte data. n=4 6 per group. p<01, Student s t test.

15 Supplemental Figure 11 psmad2/gapdh Fbn1 +/+ Fbn1 C1039G/+ psmad2/gapdh Ctrl AngII Ctrl AngII 0 Ctrl TGFβ3 Ctrl TGFβ3 3 2 perk1/2/gapdh 2 1 perk1/2/gapdh 1 0 Ctrl AngII Ctrl AngII 0 Ctrl TGFβ3 Ctrl TGFβ3 Supplemental Figure 11. AngII mediated ERK1/2 activation is differentially increased in Fbn1 C1039G/+ vs WT fibroblasts. Immunoblots for psmad2, perk1/2 and GAPDH using cultured neonatal murine cardiac fibroblasts from Fbn1 C1039G/+ and Fbn1 +/+ hearts with AngII (5nM) or TGFß3 (5 ng/ml) stimulation for 12 hours. Lanes were run on the same gel but were noncontiguous. n>=3 per group. p<01, 1w ANOVA, Tukey s correction.