Figure 1.
qPCR analysis confirms the expression pattern of several genes detected in the microarray analysis.
mRNA expression of tRNA splicing endonuclease 3 (Tsen15; panel a), Growth arrest specific 5 (Gas5; panel b), karyopherin alpha 2 (Kpna2; panel c) and suppressor of cytokine signaling 4 (Socs4; panel d) was increased in mutant mice. mRNA analysis of ribonuclease, RNase A family 4 (Rnase4; panel e) and oxoglutarate-dehydrogenase like (Ogdhl; panel f) confirmed transcriptional downregulation in whole heart RNA from the mutant animals. Data represent the mean ± SEM, with n indicated within bars. *p<0.05 versus wild-type mice.
Figure 2.
Purine nucleoside phosphorylases are upregulated in the hearts of VAChT KDHOM mice.
mRNA expression of purine nucleoside phosphorylase (Pnp; panel a) and purine nucleoside phosphorylase 2 (Pnp2, panel b) were upregulated. Pnp/Pnp2 protein content appears to upregulated in VAChT KDHOM animals as compared to wild-type mice (panel c). Data represent the mean ± SEM, with n indicated within bars. *p<0.05 versus wild-type mice.
Figure 3.
VAChT KDHOM cardiomyocytes show increased levels of ROS.
Isolated cardiomyocytes loaded with a MitoSOX superoxide indicator reveal greater ROS levels in mutant myocytes (sample image; panel a). A robust, significant increase in fluorescence was observed in the KD cardiomyocytes as compared to wild-type control cells (panel b). Data represent the mean ± SEM, with n indicated within bars. *p<0.05 versus wild-type mice. Scale bar = 10 µm.
Figure 4.
The transcription of genes related to fatty acid biosynthesis is upregulated.
mRNA expression of ATP citrate lyase (ACLY, panel a), Acetyl-CoA carboxylase (ACC; panel b) and fatty acid synthase (FAS; panel c) was increased expression in VAChT KDHOM mice. Data represent the mean ± SEM, with n indicated within bars. *p<0.05 versus wild-type mice.
Figure 5.
There are no alterations in the protein levels of enzymes involved in lipid biosynthesis.
Immunoblotting analysis of ATP citrate lyase (ACLY, panel a), Acetyl-CoA carboxylase (ACC; panel b) and fatty acid synthase (FAS; panel c) revealed no differences in the protein levels of these enzymes in VAChT KDHOM mice. Data represent the mean ± SEM, with n indicated within bars. *p<0.05 versus wild-type mice.
Figure 6.
Chronic treatment with isoproterenol (ISO)-induces cardiac remodeling in wild-type mice.
Two week ISO treatment led to a significant increase in heart weight/tibia length ratio as compared to saline-treated control mice (panel a). The expression of β-myosin heavy chain (β-MHC, panel b) and atrial natriuretic factor (ANF, panel c) were significantly upregulated in ISO-treated mice. Data represent the mean ± SEM, with n indicated within bars. *p<0.05 versus wild-type mice.
Figure 7.
ISO treatment does not lead to the same transcriptional alterations observed in VAChT KDHOM mice.
mRNA expression of tRNA splicing endonuclease 3 (Tsen15; panel a), Growth arrest specific 5 (Gas5; panel b), karyopherin alpha 2 (Kpna2; panel c), suppressor of cytokine signaling 4 (Socs4; panel d), ribonuclease, RNase A family 4 (Rnase4; panel e) and oxoglutarate-dehydrogenase like (Ogdhl; panel f) were not significantly altered in the ISO-treated mice as compared to saline-treated controls. Data represent the mean ± SEM, with n indicated within bars. *p<0.05 versus wild-type mice.
Figure 8.
Expression of genes related to fatty acid synthesis is increased following isoproterenol (ISO) treatment.
mRNA expression of purine nucleoside phosphrylases (Pnp and Pnp2; panels a and panel b) were not altered following ISO treatment. ATP citrate lyase (ACLY; panel c), acetyl-CoA carboxylase (ACC; panel d) and fatty acid synthase (FAS; panel e) expression were all significantly increased in ISO-treated mice as compared to saline-treated controls. Data represent the mean ± SEM, with n indicated within bars. *p<0.05 versus wild-type mice.