Transgenic overexpression of GTP cyclohydrolase 1 in cardiomyocytes ameliorates post-infarction cardiac remodeling

Figures

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  Figure 1. Wild-type (WT) mice developed cardiac remodeling and lost GTP cyclohydrolase 1 (GCH1) proteins following myocardial infarction (MI). (A) Time-dependent changes in mouse hearts after MI or sham surgery (n = 6–8 mice/group). Top: representative images of mouse hearts at baseline (0), 1, 2, 4, 8, and 12 weeks after operation. The vertical scale bar indicates 4 mm, and the horizontal scale bar indicates 2 mm. Bottom: the area of the hearts after MI or sham surgery; (B) The anterior wall thickness of the left ventricule after MI or sham surgery (n = 8–10 mice/group); (C) The thickness of interventricular septum following MI or sham surgery (n = 8–10 mice/group); (D) Representative PCR bands showing the expression of GCH1 mRNA and the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), at 0, 1, 2, 4, 8, and 12 weeks after MI; (E) GCH1 mRNA normalized to GAPDH (n = 6 mice/group); (F) Representative Western blot bands showing the expression of cardiac mouse GCH1 proteins and GAPDH proteins in WT mice following MI; (G) Expression of mouse GCH1 proteins normalized to GAPDH proteins (n = 6 mice/group). *P < 0.05 versus sham WT groups at corresponding time points (A,B and C)…

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  Figure 2. Effects of GTP cyclohydrolase 1 (GCH1) overexpression on cardiac remodeling 4 weeks after myocardial infarction (MI). (A) Heart rate; (B) The anterior wall thickness of the left ventricle (LV) at end diastole: (C) The anterior wall thickness of the LV at end systole; (D) The interventricular septum thickness of the LV at end diastole: (E) The interventricular septum thickness of the LV at end systole; (F) LV end-diastolic diameter; (G) LV end-systolic diameter; (H) Fractional shortening; (I) Figure legend: sham WT (wild-type), MI WT, sham Tg (GCH1 transgene), and MI Tg; (J) representative M-mode echocardiograms showing anterior wall, LV chamber, and interventricular septum in the mice 4 weeks after MI. *P < 0.05 versus sham WT groups; †P < 0.05 versus MI WT groups (n = 8–12 mice/group).

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  Figure 3. Effects of GTP cyclohydrolase 1 (GCH1) overexpression on myocardial infarct size and interstitial fibrosis after myocardial infarction (MI). (A) Representative Masson’s trichrome-stained cross section of the heart showing infarct area with fibrosis. The scale bar shows 2 mm, and the arrows indicate infarct area with fibrosis. BZ, border zone; IZ, infarct zone; LV, left ventricle; RV, right ventricle; RZ, remote zone; (B) Infarct size expressed as a percentage of infarct circumference/total circumference of the LV; (C) LV internal diameters at the papillary muscle levels; (D) Masson’s trichrome-stained myocardium showing interstitial fibrosis. The scale bar shows 400 µm, and the arrows indicate fibrosis; (E) Interstitial fibrosis expressed as a percentage of LV area; (F) Myocyte cross-sectional area. Mouse hearts were stained with Masson’s trichrome in wild-type (WT) and transgenic (Tg) mice 4 weeks after MI or sham surgery. *P < 0.05 versus sham WT groups; †P < 0.05 versus MI WT groups; #P < 0.05 versus sham Tg groups (n = 8–10 mice/group).

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  Figure 4. Effects of GTP cyclohydrolase 1 (GCH1) overexpression on microRNA-21 and p38 mitogen-activated protein kinase (MAPK) after myocardial infarction (MI). (A) Representative transverse sections of fresh mouse hearts showing the positon of tissue sampling for measurements of microRNA-21 and p38 MAPK 4 weeks after (MI) or sham surgery. The drawing areas indicate the position of tissue sampling, and the scales indicate 1 mm. (B) The expression of microRNA-21 mRNA; (C) representative Western blot bands showing the expression of phosphorylated p38 (p-p38) MAPK, total p38 MAPK, and GAPDH; (D) The ratio of phosphorylated p38 (p-p38) MAPK/p38 MAPK. *P < 0.05 versus sham WT groups; †P < 0.05 versus MI WT groups; #P < 0.05 versus sham Tg groups (n = 8–10 mice/group).

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  Figure 5. GTP cyclohydrolase 1 (GCH1) overexpression improves cardiac function 4 weeks after myocardial infarction (MI). (A) Left ventricular (LV) end-systolic pressure; (B) The rate of LV pressure rise (+dP/dt); (C) The LV end-diastolic pressure-volume relationship; (D) Heart weight normalized to body weight; (E) LV weight normalized to body weight: (F) Lung weight normalized to body weight. Figure legend: sham WT, wild-type mice receiving sham surgery; MI WT, wild-type mice undergoing myocardial infarction; sham Tg, transgenic GCH1 mice receiving sham surgery; MI Tg, transgenic GCH1 mice undergoing myocardial infarction. *P < 0.05 versus sham WT groups; †P < 0.05 versus MI WT groups (n = 8–10 mice/group).

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  Figure 6. Effects of GTP cyclohydrolase 1 (GCH1) overexpression on intracellular [Ca2+]i after myocardial infarction (MI). (A) Basal Ca2+ concentrations in the absence of isoproterenol; (B) The amplitude of Ca2+ transients elicited by electrical stimulation in the absence of isoproterenol; (C) Time to 50% decay (T50 decay) of Ca2+ transients in the absence of isoproterenol. (D) Original recordings of Ca2+ transients stimulated by electricity in the presence of isoproterenol in cardiomyocytes isolated from transgenic (Tg) and wild-type (WT) mice 4 weeks after MI or sham surgery. The vertical scale bar indicates 0.2 Fura-2 ratio (340/380 nM) unit, and the horizontal scale bar indicates 100 ms; (E) Basal [Ca2+]i in the presence of isoproterenol; (F) Ca2+ transient amplitude in the presence of isoproterenol; (G) T50 decay of Ca2+ transients in the presence of isoproterenol; (H) Figure legend. *P < 0.05 versus sham WT groups; †P < 0.05 versus MI WT groups (n = 63–66 cells/goup).

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  Figure 7. Effects of GTP cyclohydrolase 1 (GCH1) overexpression on sarcoplasmic reticulum (SR) Ca2+ release and Ca2+ handling proteins after myocardial infarction (MI). (A) Original recordings of caffeine-induced Ca2+ release in cardiomyocytes isolated from wild-type (WT) and transgenic (Tg) mice 4 weeks after MI or sham surgery in 0 Na+ and 0 Ca2+ Tyrode buffer under isoproterenol stimulation. The vertical scale bar indicates 0.3 Fura-2 ratio unit, and the horizontal bar indicates 10 s; (B) Basal [Ca2+]I in the presence of isoproterenol; (C) SR Ca2+ release in the presence of isoproterenol (n = 63–66 cells/group); (D) Time to 50% decay (T50 decay) of caffeine-induced Ca2+ transients in the presence of isoproterenol; (E) The expression of ryanodine receptors (RyR2) normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in Tg and WT mice 4 weeks after MI or sham surgery (n = 5 mice/group). Top: Wesern blot bands showing the expression of RyRs and GAPDH. Bottom: the ratio of RyRs/GAPDH; (F) The expression of SR Ca2+-ATPase (SERCA2a) proteins normalized to GAPDH. Top: Western blot bands showing the expression of SERCA2a and GAPDH. Bottom: the ratio of SERCA2a/GAPDH; (G) The expression…

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  Figure 8. Effects of transgenic overexpression of human GTP cyclohydrolase 1 (GCH1) on GCH1 proteins and tetrahydrobiopterin in post-infarction remodeled myocardium. (A) Representative Western blot bands showing the expression of human and mouse GCH1 proteins and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as control in wild-type (WT) and transgenic (Tg) mice 4 weeks after myocardial infarction (MI) or sham surgery; (B) Total (human + mouse) GCH1 proteins normalized to GAPDH; C: cardiac tetrahydrobiopterin concentrations. *P < 0.05 versus sham WT groups; †P < 0.05 versus MI WT groups; #P < 0.05 versus sham Tg groups (n = 5–6 mice/group).

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  Figure 9. GTP cyclohydrolase 1 (GCH1) overexpression differentially regulates nitric oxide synthase (NOS) isoforms in post-infarction remodeled myocardium. (A) Representative Western blot bands showing the expression of dimeric and monomeric neuronal NOS (nNOS) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as loading control in wild-type (WT) and transgenic (Tg) mice 4 weeks after myocardial infarction (MI) or sham surgery (sham); (B) The ratio of nNOS dimers/monomers; (C) Representative Western blot bands of dimeric and monomeric endothelial NOS (eNOS) and GAPDH; (D) The ratio of eNOS dimers/monomers; (E) Western blot bands showing the expression of inducible NOS (iNOS) monomers and GAPDH; (F) iNOS monomers normalized to GAPDH; (G) NOx levels; (H) superoxide levels; (I) representative Western blot bands showing the expresiion of phosphorylated nNOS (p-nNOS), nNOS, and GAPDH; (J) The ratio of p-nNOS/total nNOS; (K) Western blot bands of phosphorylated eNOS (p-eNOS), eNOS, and GAPDH; (L) the ratio of phosphorylated eNOS (p-eNOS)/total eNOS. *P < 0.05 versus sham WT groups; †P < 0.05 versus MI WT groups; #P < 0.05 versus sham Tg groups (n = 5 mice/group).