Limits...
Cardiac oxidative stress in a mouse model of neutral lipid storage disease.

Schrammel A, Mussbacher M, Winkler S, Haemmerle G, Stessel H, Wölkart G, Zechner R, Mayer B - Biochim. Biophys. Acta (2013)

Bottom Line: Systemic deletion of the gene encoding adipose triglyceride lipase (ATGL), the enzyme that catalyzes the rate-limiting step of triglyceride lipolysis, results in a phenotype characterized by severe steatotic cardiac dysfunction.Investigating the effect of oxidative and inflammatory stress on nitric oxide/cGMP signal transduction we observed a ~2.5-fold upregulation of soluble guanylate cyclase activity and a ~2-fold increase in cardiac tetrahydrobiopterin levels.Upregulation of soluble guanylate cyclase and cardiac tetrahydrobiopterin might be regarded as counterregulatory mechanisms in cardiac ATGL deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Institute of Pharmaceutical Sciences, University of Graz, Universitätsplatz 2, 8010 Graz, Austria. Electronic address: astrid.schrammel-gorren@uni-graz.at.

Show MeSH

Related in: MedlinePlus

Effects on NO/cGMP signaling. (A) 10,000 g supernatants of hearts from WT (open bars), ATGL(−/−) (solid bars), WT/MHC-A35 (striped bars), and ATGL(−/−)/MHC-A35 mice (gray bars) were prepared and assayed for Ca2 +-dependent [3H]l-citrulline formation. Data are mean values ± S.E.M. of 4 experiments performed in duplicate. (B) Cardiac sGC activity was measured in cytosols stimulated with 0.1 mM DEA/NO with and without YC-1 (0.1 mM). The inset depicts basal enzyme activity assayed in the presence of MnCl2. Data are mean values ± S.E.M. of 5 experiments performed in duplicate. *p < 0.05 ATGL(−/−) vs WT. (C) cGMP levels were determined by radioimmunoassay. Data represent mean values ± S.E.M. of 5–7 experiments performed in duplicate. (D) Phosphorylation of VASP at serine 239 was quantified by Western blot. Data are presented as the ratio of phosphorylated to total VASP protein and represent mean values ± S.E.M. of 6 individual experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3795454&req=5

f0025: Effects on NO/cGMP signaling. (A) 10,000 g supernatants of hearts from WT (open bars), ATGL(−/−) (solid bars), WT/MHC-A35 (striped bars), and ATGL(−/−)/MHC-A35 mice (gray bars) were prepared and assayed for Ca2 +-dependent [3H]l-citrulline formation. Data are mean values ± S.E.M. of 4 experiments performed in duplicate. (B) Cardiac sGC activity was measured in cytosols stimulated with 0.1 mM DEA/NO with and without YC-1 (0.1 mM). The inset depicts basal enzyme activity assayed in the presence of MnCl2. Data are mean values ± S.E.M. of 5 experiments performed in duplicate. *p < 0.05 ATGL(−/−) vs WT. (C) cGMP levels were determined by radioimmunoassay. Data represent mean values ± S.E.M. of 5–7 experiments performed in duplicate. (D) Phosphorylation of VASP at serine 239 was quantified by Western blot. Data are presented as the ratio of phosphorylated to total VASP protein and represent mean values ± S.E.M. of 6 individual experiments.

Mentions: The potential consequences of oxidative stress for cardiac NO/cGMP signaling in ATGL deficiency were studied by measuring various parameters characteristic for different steps of the signaling cascade. As shown in Fig. 4A, cardiac NOS activity determined as conversion of l-arginine to l-citrulline was not affected by any genetic manipulation. Ca2 +-independent activity was not detectable (data not shown), arguing against significant iNOS expression in any experimental group. Protein expression of eNOS and nNOS was not affected (data not shown). However, activity of sGC, the major downstream target of NOS-derived NO, was significantly increased in ATGL(−/−) hearts (Fig. 4B). Basal cGMP formation measured in the presence of Mn2 + was increased from 0.023 ± 0.002 to 0.077 ± 0.011 nmol × mg− 1 × min− 1 (Fig. 4B; inset). Upon stimulation with the NO donor DEA/NO or co-stimulation with the NO-sensitizing drug YC-1 [23], the rates of cGMP formation were enhanced ~ 2.6- and ~ 2.4-fold, respectively. Thus, sGC activity was significantly increased at different activation states of the enzyme. Cardiac-specific overexpression of ATGL resulted in sGC activities identical to that of WT animals (Fig. 4B). Quantification of cardiac cGMP by radioimmunoassay showed that, despite the significantly higher sGC activity in ATGL(−/−) hearts, cGMP levels were virtually identical in all experimental groups (Fig. 4C). Similarly, cGMP-dependent phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at serine 239, which represents a reliable biochemical marker of the NO/cGMP pathway [24], was not affected (Fig. 4D).


Cardiac oxidative stress in a mouse model of neutral lipid storage disease.

Schrammel A, Mussbacher M, Winkler S, Haemmerle G, Stessel H, Wölkart G, Zechner R, Mayer B - Biochim. Biophys. Acta (2013)

Effects on NO/cGMP signaling. (A) 10,000 g supernatants of hearts from WT (open bars), ATGL(−/−) (solid bars), WT/MHC-A35 (striped bars), and ATGL(−/−)/MHC-A35 mice (gray bars) were prepared and assayed for Ca2 +-dependent [3H]l-citrulline formation. Data are mean values ± S.E.M. of 4 experiments performed in duplicate. (B) Cardiac sGC activity was measured in cytosols stimulated with 0.1 mM DEA/NO with and without YC-1 (0.1 mM). The inset depicts basal enzyme activity assayed in the presence of MnCl2. Data are mean values ± S.E.M. of 5 experiments performed in duplicate. *p < 0.05 ATGL(−/−) vs WT. (C) cGMP levels were determined by radioimmunoassay. Data represent mean values ± S.E.M. of 5–7 experiments performed in duplicate. (D) Phosphorylation of VASP at serine 239 was quantified by Western blot. Data are presented as the ratio of phosphorylated to total VASP protein and represent mean values ± S.E.M. of 6 individual experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3795454&req=5

f0025: Effects on NO/cGMP signaling. (A) 10,000 g supernatants of hearts from WT (open bars), ATGL(−/−) (solid bars), WT/MHC-A35 (striped bars), and ATGL(−/−)/MHC-A35 mice (gray bars) were prepared and assayed for Ca2 +-dependent [3H]l-citrulline formation. Data are mean values ± S.E.M. of 4 experiments performed in duplicate. (B) Cardiac sGC activity was measured in cytosols stimulated with 0.1 mM DEA/NO with and without YC-1 (0.1 mM). The inset depicts basal enzyme activity assayed in the presence of MnCl2. Data are mean values ± S.E.M. of 5 experiments performed in duplicate. *p < 0.05 ATGL(−/−) vs WT. (C) cGMP levels were determined by radioimmunoassay. Data represent mean values ± S.E.M. of 5–7 experiments performed in duplicate. (D) Phosphorylation of VASP at serine 239 was quantified by Western blot. Data are presented as the ratio of phosphorylated to total VASP protein and represent mean values ± S.E.M. of 6 individual experiments.
Mentions: The potential consequences of oxidative stress for cardiac NO/cGMP signaling in ATGL deficiency were studied by measuring various parameters characteristic for different steps of the signaling cascade. As shown in Fig. 4A, cardiac NOS activity determined as conversion of l-arginine to l-citrulline was not affected by any genetic manipulation. Ca2 +-independent activity was not detectable (data not shown), arguing against significant iNOS expression in any experimental group. Protein expression of eNOS and nNOS was not affected (data not shown). However, activity of sGC, the major downstream target of NOS-derived NO, was significantly increased in ATGL(−/−) hearts (Fig. 4B). Basal cGMP formation measured in the presence of Mn2 + was increased from 0.023 ± 0.002 to 0.077 ± 0.011 nmol × mg− 1 × min− 1 (Fig. 4B; inset). Upon stimulation with the NO donor DEA/NO or co-stimulation with the NO-sensitizing drug YC-1 [23], the rates of cGMP formation were enhanced ~ 2.6- and ~ 2.4-fold, respectively. Thus, sGC activity was significantly increased at different activation states of the enzyme. Cardiac-specific overexpression of ATGL resulted in sGC activities identical to that of WT animals (Fig. 4B). Quantification of cardiac cGMP by radioimmunoassay showed that, despite the significantly higher sGC activity in ATGL(−/−) hearts, cGMP levels were virtually identical in all experimental groups (Fig. 4C). Similarly, cGMP-dependent phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at serine 239, which represents a reliable biochemical marker of the NO/cGMP pathway [24], was not affected (Fig. 4D).

Bottom Line: Systemic deletion of the gene encoding adipose triglyceride lipase (ATGL), the enzyme that catalyzes the rate-limiting step of triglyceride lipolysis, results in a phenotype characterized by severe steatotic cardiac dysfunction.Investigating the effect of oxidative and inflammatory stress on nitric oxide/cGMP signal transduction we observed a ~2.5-fold upregulation of soluble guanylate cyclase activity and a ~2-fold increase in cardiac tetrahydrobiopterin levels.Upregulation of soluble guanylate cyclase and cardiac tetrahydrobiopterin might be regarded as counterregulatory mechanisms in cardiac ATGL deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Institute of Pharmaceutical Sciences, University of Graz, Universitätsplatz 2, 8010 Graz, Austria. Electronic address: astrid.schrammel-gorren@uni-graz.at.

Show MeSH
Related in: MedlinePlus