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Figure S1

Supplementary Figures. Figure S1. n.s. n.s. 1.2. n.s. n.s. 1.2. n.s. n.s. 0.9. +/+. 0.9. LVPW, mm. IVS, mm. +/ -. 0.6. 0.6. -/-. 0.3. 0.3. 0. 0. TAC. TAC. *. 4. 60. 6. *. *. n.s. *. n.s. *. n.s. *. 3. 40. 4. LVDd, mm. LVFS, %. LVDs, mm. 2. 20. 2.

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Figure S1

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  1. Supplementary Figures Figure S1 n.s. n.s. 1.2 n.s. n.s. 1.2 n.s. n.s. 0.9 +/+ 0.9 LVPW, mm IVS, mm +/- 0.6 0.6 -/- 0.3 0.3 0 0 TAC TAC * 4 60 6 * * n.s. * n.s. * n.s. * 3 40 4 LVDd, mm LVFS, % LVDs, mm 2 20 2 1 0 0 0 TAC TAC TAC Comparison of echocardiographic data after TAC among wild type, heterozygous knockout, and homozygous knockout mice. Echocardiography was performed after TAC operation in wild type (GCLM+/+), heterozygous knockout (GCLM+/-) and homozygous (GCLM -/-) mice. n = 10 in each experiment. n.s. indicates not statistically significant. *P < 0.05.

  2. Figure S2 A p-Smad3 B * * t-Smad3 † † 10 * 3 † † † 7.5 † † † † † 2 5 p- / t-Smad3 mRNA expression CTGF 1 2.5 0 0 - - + + - - + + - - + + - - + + TGFb1 TGFb1 - + - + - + - + - + - + - + - + GSH-EE GSH-EE +/+ +/+ -/- -/- D C * * † † 4 2 † † † † † † 3 1.5 † † mRNA expression mRNA expression Procollagen III 2 1 Procollagen I 1 0.5 0 0 - - + + - - + + - - + + - - + + TGFb1 TGFb1 - + - + - + - + - + - + - + - + GSH-EE GSH-EE +/+ +/+ -/- -/- Smad3 phosphorylation and expression of CTGF and procollagen in cultured mouse cardiac fibroblasts The cultured cardiac fibroblasts were incubated with TGF-b1 (25 ng/mL) or vehicle in the presence or absence of GSH-EE (2 mmol/L). (A), Smad3 phosphorylation after the incubation for 24 hrs. Upper panel shows representative blotting. (B-D), mRNA expression of CTGF and procollagen I and III after the incubation for 24 hrs and 4 hrs, respectively. The mRNA levels were normalized to GAPDH mRNA expression. n = 10 in each experiment. +/+ denotes GCLM+/+ mice and -/- denotes GCLM -/- mice. *P < 0.05. †P < 0.05. The data were expressed relative to the GCLM+/+ fibroblasts treated with vehicle in the absence of GSH-EE (= 1).

  3. 2 * mRNA expression mRNA expression TGF-bR2 TIMP-2 1 0 TAC Sham +/+ +/+ -/- -/- Figure S3 x 1000 -/- +/+ 9 A B C * 2 6 Vessels / mm2 3 1 0 0 Sham TAC Sham TAC E F D x 1000 60 4 9 * * +/+ * * 45 3 -/- 6 * Vessels / mm2 F/F0 30 GSH / GSSG 2 3 15 1 0 0 0 PBS PBS GSH GSH PBS GSH 0.75 1.0 0.25 0.5 No Treat. Time (second) -/- -/- +/+ +/+ Myocardial expression of TGF-bR2 and TIMP-2 and microvessel density after TAC, transients of intracellular calcium concentration in cultured cardiomyocytes and effect of GSH-EE supplementation on myocardial microvessel density and GSH/GSSG ratio. (A, B), TGF-bR2 and TIMP-2mRNA expression. The expression levels were normalized to GAPDH mRNA expression and are shown relative to the levels in sham-operated GCLM+/+ myocardium (= 1).(C), Microvessel number per mm2 in myocardium after TAC. (D), Representative tracing of transients of intracellular calcium concentration in cultured cardiomyocytes in response to electrical stimulation. (E, F), Effects of GSH-EE supplementation during TAC on myocardial microvascular density and GSH/GSSG ratio. -/- denotes GCLM-/- mice, and +/+ denotes GCLM+/+ mice. n = 6 in each experiment. *P < 0.05.

  4. -/- with GSH-EE -/- with PBS +/+ with no treatment * * * * Figure S4 A B C mmHg mmHg bpm 300 12 800 n.s. n.s. n.s. n.s. 9 600 200 LVEDP Systolic BP at aortic root Heart rate 6 400 100 3 200 0 0 0 TAC TAC TAC D E F mmHg/sec mmHg/sec TAC ms 0 20000 16 * * 15000 12 -5000 Maxdp/dt Tau Mindp/dt 10000 8 -10000 5000 4 * * -15000 0 0 TAC TAC Effects of GSH-EE supplementation on hemodynamic data 4 weeks after TAC operation in GCLM-/- mice. Hemodynamic study was performed in GCLM -/- mice after treatment with GSH-EE or PBS as a placebo and in GCLM+/+ mice with no treatment for 4 weeks after TAC operation. n = 6 in each experiment. n.s. indicates not statistically significant. *P < 0.05.

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