1 1 SUPPLEMENTAL MATERIAL Supplementary Methods Echocardiography. Cardiac function was assessed by echocardiography using a Vevo 770 High- Resolution Imaging Systems (Visual Sonics, Toronto) with an integrated rail system and a 30MHz transducer at 16 weeks for the fructose/TAC experiment. After performing this experiment, we obtained a Vevo 2100 with a 40MHz transducer, and used this machine to assess cardiac function at 11 weeks for the infarct experiment and at 6 weeks for the first TACexperiment. Measurements were performed on mice anesthetized with isoflurane. The level of isoflurane was 2.5% for the Fructose/TAC study, 1.5% for the infarct study, and 1.0% for the six week severe TAC study. The higher level of isoflurane resulted in lower ejection fraction in (Figure 1G and 5C) compared to the six week TAC study (Figure 3G). Mice were shaved and placed in a supine position on a warming pad. M-mode frames were recorded from the parasternal short axis, and Doppler measurements were recorded from the apex. Absolute wall thickness (AWT) and relative wall thickness (RWT) were calculated as: (PWTd+AWTd) and (PWTd+AWTd)/EDD, where PWTd is diastolic posterior wall thickness, AWTd is diastolic anterior wall thickness, and EDD is end diastolic diameter. Ejection fraction was calculated as: (EDV-ESV)/EDVx100%, where EDV is end diastolic volume and ESV is end systolic volume. From Vevo 2100 measurements, EDV and ESV were calculated as: ((7.0/(2.4+EDD))*EDD 3 and ((7.0/(2.4+ESD))*ESD 3 respectively, where ESD is end systolic diameter. From Vevo 770 measurements, EDV and ESV were calculated as: 1.047xEDD 3 and 1.047xESD 3 . Myocardial performance index (MPI) was calculated as (isovolumetric contraction time+isovolumetric relaxation time)/ejection time.
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SUPPLEMENTAL MATERIAL
Supplementary Methods
Echocardiography. Cardiac function was assessed by echocardiography using a Vevo 770 High-
Resolution Imaging Systems (Visual Sonics, Toronto) with an integrated rail system and a
30MHz transducer at 16 weeks for the fructose/TAC experiment. After performing this
experiment, we obtained a Vevo 2100 with a 40MHz transducer, and used this machine to assess
cardiac function at 11 weeks for the infarct experiment and at 6 weeks for the first
TACexperiment. Measurements were performed on mice anesthetized with isoflurane. The level
of isoflurane was 2.5% for the Fructose/TAC study, 1.5% for the infarct study, and 1.0% for the
six week severe TAC study. The higher level of isoflurane resulted in lower ejection fraction in
(Figure 1G and 5C) compared to the six week TAC study (Figure 3G). Mice were shaved and
placed in a supine position on a warming pad. M-mode frames were recorded from the
parasternal short axis, and Doppler measurements were recorded from the apex. Absolute wall
thickness (AWT) and relative wall thickness (RWT) were calculated as: (PWTd+AWTd) and
(PWTd+AWTd)/EDD, where PWTd is diastolic posterior wall thickness, AWTd is diastolic
anterior wall thickness, and EDD is end diastolic diameter. Ejection fraction was calculated as:
(EDV-ESV)/EDVx100%, where EDV is end diastolic volume and ESV is end systolic volume.
From Vevo 2100 measurements, EDV and ESV were calculated as: ((7.0/(2.4+EDD))*EDD3 and
((7.0/(2.4+ESD))*ESD3 respectively, where ESD is end systolic diameter. From Vevo 770
measurements, EDV and ESV were calculated as: 1.047xEDD3 and 1.047xESD3. Myocardial
performance index (MPI) was calculated as (isovolumetric contraction time+isovolumetric
Heart Mass/Body Mass (mg/g) 3.68±0.38 4.63±0.28* 3.88±0.38 5.37±0.23*#
Data were obtained using mice at 12 weeks after LAD ligation or sham surgery; *P <0.05 vs. Sham; #P <0.05 vs. WT; Sham n=10/group, WT Infarct n=19, G6PDX n=28.
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Table S2. Echocardiography Data - 11 Weeks Post-Infarct
Parameter Wild Type G6PD Deficient
Sham Infarct Sham Infarct
Fractional Shortening % 27.0±2.3 16.0±1.7* 31.7±2.3 14.6±1.4* Stroke Volume (µL) 34.0±2.4 27.9±1.7* 35.5±2.4 30.0±1.4* AWT;d (mm) 0.97±0.08 0.92±0.06 1.03±0.08 0.75±0.05*# AWT;s (mm) 1.37±0.10 1.14±0.07 1.40±0.10 0.93±0.06*# End Diastolic Diameter (mm) 3.85±0.20 4.46±0.15* 3.73±0.20 4.90±0.12*# End Systolic Diameter (mm) 2.81±0.26 3.78±0.19* 2.56±0.26 4.24±0.15* PWT;d (mm) 0.97±0.06 1.02±0.04 0.96±0.06 0.98±0.04 PWT;s (mm) 1.26±0.07 1.24±0.05 1.37±0.07 1.19±0.04 Absolute Wall Thickness (mm) 1.94±0.09 1.94±0.07 1.99±0.09 1.73±0.06 End Diastolic Area (mm2) 11.2±1.4 16.3±1.0* 10.7±1.4 17.6±0.9* End Systolic Area (mm2) 7.2±1.5 12.8±1.1* 6.3±1.5 14.5±0.9* Area Fractional Shortening % 35.7±3.0 23.9±2.2* 41.1±3.0 19.3±1.8* Isovolumetric Contraction Time (ms) 16.7±4.0 26.1±3.0 11.1±4.0 26.3±2.5* Isovolumetric Relaxation Time (ms) 26.3±3.3 34.4±2.4 17.2±3.2 37.0±2.0* Ejection Time (ms) 40.7±3.2 21.9±2.3* 44.4±3.2 22.2±1.9* Myocardial Performance Index 1.11±0.74 4.12±0.55* 0.67±0.74 3.75±0.45* Heart Rate (bpm) 445±16 458±12 489±16 471±10 Data were obtained using mice anesthetized with 1.5% isoflurane at 11 weeks after LAD ligation or sham surgery; *P <0.05 vs. Sham; #P <0.05 vs. WT; Sham n=10/group, WT Infarct n=19, G6PDX n=28. AWT;d, anterior wall thickness at diastole; AWT;s, anterior wall thickness at systole; PWT;d, posterior wall thickness at diastole; PWT;s, anterior wall thickness at systole; bpm, beats per minute.
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Table S3. Myocardial PCR Data - 12 Weeks Post-Infarct
Parameter Wild Type G6PD Deficient
Sham Infarct Sham Infarct
ANP (Ct) 18.0±0.3 17.2±0.2* 17.9±0.3 17.0±0.2* MHCβ (Ct) 22.7±0.3 21.2±0.2* 22.3±0.3 20.9±0.2* MHCα (Ct) 14.3±0.2 14.7±0.1 14.3±0.2 14.9±0.1* G6PD (Ct; Exons 1-2) 25.9±0.1 25.6±0.1 28.7±0.1# 28.4±0.1# G6PD (Fold Change; Exons 1-2) 1.00±0.07 1.27±0.05* 0.15±0.07# 0.18±0.04# G6PD (Ct; Exons 12-13) 25.3±0.1 25.0±0.1* 27.5±0.1# 27.1±0.1# 18s (Ct) 6.03±0.23 6.15±0.17 6.11±0.24 6.15±0.14 Data were obtained using myocardium from mice at 12 weeks after LAD ligation or sham surgery; *P <0.05 vs. Sham; #P <0.05 vs. WT; Sham n=9-10/group, WT Infarct n=19, G6PDX n=27. 18s, ribosomal RNA; ANP, atrial natriuretic peptide; Ct, cycle at which threshold is crossed; MHC, myosin heavy chain; G6PD, glucose 6-phosphate dehydrogenase.
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Table S4. Morphometric Data - 6 Weeks Post-TAC
Parameter Wild Type G6PD Deficient
Sham TAC Sham TAC
Beginning Body Mass (g) 25.5±0.4 25.6±0.4 25.6±0.4 25.5±0.3 Terminal Body Mass (g) 29.4±0.5 29.5±0.5 29.9±0.5 28.4±0.4 Tibia Length (mm) 19.4±0.1 19.5±0.1 19.7±0.1 19.7±0.1 LV Mass (mg) 87±4 131±4* 88±4 137±3* LV Mass/Body Mass (mg/g) 2.96±0.18 4.46±0.15* 2.94±0.18 4.85±0.14* RV Mass (mg) 20.4±3.6 29.2±3.0 21.2±3.6 30.6±2.8* Atrial Mass (mg) 4.9±2.4 11.9±2.0* 5.5±2.4 15.8±1.8* Data were obtained using mice at 6 weeks after Sham or TAC surgery with a 28 gauge needle; *P <0.05 vs. Sham; #P <0.05 vs. WT; Sham n=7/group, TAC=10-12/group. LV, left ventricle; RV, right ventricle.
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Table S5. Echocardiography - 6 Weeks Post-TAC
Parameter Wild Type G6PD Deficient
Sham TAC Sham TAC
Fractional Shortening % 42.8±2.8 29.0±2.4* 44.0±2.8 24.2±2.2* Stroke Volume (µL) 33.4±2.2 27.9±1.8 32.8±2.2 29.1±1.7 AWT;d (mm) 1.07±0.07 1.18±0.06 1.03±0.07 1.15±0.06 AWT;s (mm) 1.48±0.09 1.62±0.07 1.47±0.09 1.57±0.07 End Diastolic Diameter (mm) 3.32±0.19 3.53±0.16 3.26±0.19 3.83±0.14* End Systolic Diameter (mm) 1.92±0.22 2.54±0.18* 1.85±0.22 2.94±0.17* PWT;d (mm) 1.08±0.07 1.22±0.06 1.10±0.07 1.29±0.06* PWT;s (mm) 1.55±0.09 1.51±0.08 1.53±0.09 1.53±0.07 Relative Wall Thickness 0.66±0.07 0.70±0.06 0.67±0.07 0.67±0.05 End Diastolic Area (mm2) 8.27±1.03 9.89±0.86 8.40±1.03 10.21±0.79 End Systolic Area (mm2) 4.34±0.93 6.62±0.78 4.58±0.93 7.24±0.71* Area Fractional Shortening % 47.9±4.3 35.2±3.6* 46.3±4.3 30.2±3.3* Isovolumetric Contraction Time (ms) 14.2±2.9 10.3±2.5 10.1±2.9 17.0±2.3 Isovolumetric Relaxation Time (ms) 22.0±4.3 23.4±3.6 20.7±4.3 22.1±3.4 Ejection Time (ms) 30.6±3.0 36.6±2.5 30.3±3.0 36.5±2.4 Myocardial Performance Index 1.25±0.33 1.16±0.28 1.13±0.33 1.23±0.27 Heart Rate (bpm) 632±17 611±14 609±17 585±13 Data were obtained using mice anesthetized with 1% isoflurane at 6 weeks after Sham or TAC surgery with a 28 gauge needle; *P <0.05 vs. Sham; #P <0.05 vs. WT; Sham n=7/group, TAC=10-12/group. AWT;d, anterior wall thickness at diastole; AWT;s, anterior wall thickness at systole; PWT;d, posterior wall thickness at diastole; PWT;s, anterior wall thickness at systole; bpm, beats per minute.
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Table S6. LV PCR Data - 6 Weeks Post-TAC
Parameter Wild Type G6PD Deficient
Sham TAC Sham TAC
ANP (Ct) 22.6±0.4 17.2±0.4* 22.2±0.4 16.8±0.3* MHCβ (Ct) 23.5±0.2 20.3±0.2* 23.0±0.2 20.0±0.2* MHCα (Ct) 16.6±0.3 16.4±0.2 16.4±0.3 16.3±0.2 G6PD (Ct) 28.6±0.3 27.5±0.3* 30.6±0.3# 29.6±0.3*# 18s (Ct) 8.34±0.30 7.43±0.25 7.52±0.30 7.31±0.24 Data were obtained using left ventricular myocardium from mice at 6 weeks after Sham or TAC surgery with a 28 gauge needle; *P <0.05 vs. Sham; #P <0.05 vs. WT; Sham n=7/group, TAC=10-11/group. 18s, ribosomal RNA; ANP, atrial natriuretic peptide; Ct, cycle at which threshold is crossed; MHC, myosin heavy chain; G6PD, glucose 6-phosphate dehydrogenase.
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Table S7. Macronutrient Composition of Diets for the High Fructose/TAC Experiment
Starch 6 4 57 12 0 20 Fructose 6 4 9 0 60 20 Values are energy contribution to the diet (% of total). Diets were matched for vitamin and mineral content.
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Table S8. Morphometric Data - 17 Weeks Post-TAC
Parameter Wild Type G6PD Deficient
Sham TAC Sham TAC
High Starch Diet
Initial Body Mass (g) 24.0±0.4 24.1±0.2 24.1±0.3 24.1±0.4
Terminal Body Mass (g) 33.6±1.0 32.4±1.0 33.8±1.0 32.5±1.0
Atrial Mass (mg) 6.5±1.1 10.2±1.1* 6.2±1.2 12.9±1.1*
Atria/Tibia (µg/mm) 323±54 506±52* 309±57 642±52*
Data were obtained using mice at 17 weeks after Sham or TAC surgery with a 27 gauge needle; *P <0.05 vs. TAC; #P <0.05 vs. WT; Sham n=12-13/group, TAC n=14-15/group. LV, left ventricle; RV, right ventricle.
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Table S9. Tail Cuff Blood Pressure Data - 10 Weeks Post TAC
Data were obtained using mice at 10 weeks after Sham or TAC surgery with a 27 gauge needle; *P <0.05 vs. TAC; #P <0.05 vs. WT; Sham n=13-16/group, TAC n=15-17/group; bpm, beats per minute.
End Diastolic Area (mm2) 10.5±0.4 10.0±0.4 10.7±0.4 11.1±0.3
End Systolic Area (mm2) 8.2±0.4 7.2±0.4 7.5±0.4 8.7±0.4*#
Area Fractional Shortening % 19.9±±1.1 20.3±±1.1 22.2±±1.2 16.7±±1.1*#
Heart Rate (bpm) 493±12 494±13 498±13 492±12
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Data were obtained using mice anesthetized with 2.5% isoflurane at the given times after Sham or TAC surgery with a 27 gauge needle*P <0.05 vs. TAC; #P <0.05 vs. WT; Sham n=12-16/group, TAC n=15-17/group. AWT;d, anterior wall thickness at diastole; AWT;s, anterior wall thickness at systole; bpm, beats per minute; PWT;d, posterior wall thickness at diastole; PWT;s, anterior wall thickness at systole; bpm, beats per minute.
Data were obtained using left ventricular myocardium from mice at 17 weeks after Sham or TAC surgery with a 27 gauge needle; *P <0.05 vs. TAC; #P <0.05 vs. WT; Sham n=9-13/group, TAC n=12-16/group. 4HA, 4-hydroxyalkenals; ANP, atrial natriuretic peptide; CS, citrate synthase; gww, grams of wet weight tissue; Ct, cycle at which threshold is crossed; MCAD, medium chain acyl-coenzyme A dehydrogenase; MDA, malondialdehyde; MHC, myosin heavy chain; PPIA, peptidylprolyl isomerase A.
Data were obtained using blood serum from mice at 17 weeks after Sham or TAC surgery with a 27 gauge needle; *P <0.05 vs. TAC; #P <0.05 vs. WT; Sham n=12-13/group, TAC n=13-15/group.
Figure S2. Effect of infarction on LV Pressure. A-B. LV end systolic pressure and dp/dt max
were unaffected by G6PD deficiency or myocardial infarction. C. Myocardial infarction
decreased dp/dt min (p=0.030 for main effect), but this parameter not affected by G6PD
deficiency. Data were obtained using mice at 12 weeks after LAD ligation or sham surgery; ǂ
main effect of surgery; Sham n=7-10/group, Infarct n=14-18/group. dP/dt, change in pressure
over time.
Figure S3. A, TAC Experimental Protocol. Ten week-old mice underwent TAC or sham surgery.
TAC was performed using a 28 gauge needle. Echocardiography and euthanization were
performed at 6 weeks. B, Post-TAC Survival. Neither surgery nor G6PD deficiency affected
post-TAC survival over the course of the study (p=0.75). Time 0 of the survival curve begins 1
day after TAC, so the data are representative of survival after complete recovery from the
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surgery. Survival was assessed by Log-Rank Kaplan Meier analysis; n=38 total mice, Sham
n=7/group, TAC n=11-13/group.
Figure S4. A, Fructose/TAC Experimental Protocol. Eight week-old mice underwent sham or
TAC surgery with a 27 gauge needle, and were placed on a high starch or high fructose diet on
the following day. Echocardiography was performed at 16 weeks, and mice were euthanized at
17 weeks for biochemical analysis. B, Post-TAC survival. Post-TAC survival was unaffected by
surgery, G6PD deficiency, or diet over the course of the study (p=0.137). Time 0 of the survival
curve begins 1 day after TAC, so the data are representative of survival after complete recovery
from the surgery. Survival was assessed by Log-Rank Kaplan Meier analysis; n=126 total mice,
n=14-19/group.
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Figure S1.
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Figure S2.
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Figure S3.
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Figure S4.
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Supplementary References
1. Chess DJ, Xu W, Khairallah R, O'Shea KM, Kop WJ, Azimzadeh AM, Stanley WC. The antioxidant tempol attenuates pressure overload-Induced cardiac hypertrophy and contractile dysfunction in mice fed a high-fructose diet. Am J Physiol Heart Circ Physiol. 2008;295:H2223-H2230.
2. O'Shea KM, Chess DJ, Khairallah RJ, Rastogi S, Hecker PA, Sabbah HN, Walsh K, Stanley WC. Effects of adiponectin deficiency on structural and metabolic remodeling in mice subjected to pressure overload. Am J Physiol Heart Circ Physiol. 2010;298:H1639-H1645.
3. Mohazzab KM, Wolin MS. Sites of superoxide anion production detected by lucigenin in calf pulmonary artery smooth muscle. Am J Physiol. 1994;;267(6 Pt 1):L815-L822.
4. Gupte RS, Vijay V, Marks B, Levine RJ, Sabbah HN, Wolin MS, Recchia FA, Gupte SA. Upregulation of glucose-6-phosphate dehydrogenase and NAD(P)H oxidase activity increases oxidative stress in failing human heart. J Card Fail. 2007;13:497-506.