5 Results
5.1 Analysis of AGAT-related gene expression in the heart
5.1.3 Differential gene expression in AGAT knockout mice
The number of differentially expressed genes between the groups was evaluated for each comparison as shown in Figure 8. After correction for multiple testing, the FDR was set to ≤ 0.05.
Figure 8: Number of differentially expressed genes between the groups in murine heart tissue
Expression profiling was performed using the Affymetrix Mouse GeneChip 1.0 ST Array. Each line indicates the comparison of two groups and the number of significantly regulated genes.
Significance level: False discovery rate (FDR) ≤ 0.05. WT: wild-type; AGAT-/-: AGAT knockout; n:
number of animals.
Wild-type versus AGAT knockout mice
Of the 19,954 probes on the array that were included in the gene expression analysis, 485 were significantly regulated (FDR ≤ 0.05) in left ventricular heart tissue of AGAT-/- mice. Table 12 shows the top 20 genes that are differentially expressed between WT and AGAT-/- littermates. The fold change (FC) describes the change in gene expression in AGAT-/- compared to WT mice. Expectably, a significant down-regulation of the Agat gene was observed in AGAT-/- mice (P = 1.18×10-10, FC = -2.19). As already described, the knockout leads to a shortened instable mRNA and accordingly, the expression was significantly lower but not completely absent in AGAT-/- mice. The first two genes within the analysis, sodium voltage-gated channel alpha subunit 4 (Scn4a) and sodium voltage-gated channel beta subunit 4 (Scn4b) were down-regulated more than 3-fold respectively (Scn4a P = 3.02×10-13, FC = -3.22; Scn4b P = 1.47×10-12,
785 8
485
55 484
Wild-type (WT) n = 7
AGAT -/-n = 7
AGAT -/-+ Homoarginine
n = 5
AGAT -/-+ Creatine
n = 4
FC = -3.74). Both are described to be involved in cardiac rhythmicity (Lau et al., 2009, Li et al., 2013).
Table 12: Top 20 differentially expressed genes between wild-type and AGAT knockout mice in heart tissue
Significance level: False discovery rate (FDR) ≤ 0.05. FC: fold change.
Gene Gene name P-value FC
Scn4a sodium channel, voltage-gated, type IV, alpha 3.02×10-13 -3.22
Scn4b sodium channel, type IV, beta 1.47×10-12 -3.74
Tmod4 tropomodulin 4 8.93×10-11 -1.76
Tmem150c transmembrane protein 150C 9.97×10-11 -2.96
Fah fumarylacetoacetate hydrolase 1.08×10-10 -2.36
Agat L-arginine:glycine amidinotransferase 1.18×10-10 -2.19 Lgi1 leucine-rich repeat LGI family, member 1 2.07×10-10 -3.1
Lad1 ladinin 2.63×10-10 2.17
Stom stomatin 1.12×10-9 -1.41
Zfp106 zinc finger protein 106 1.31×10-9 1.49
Egf epidermal growth factor 6.62×10-9 -1.55
Ndrg4 N-myc downstream regulated gene 4 9.12×10-9 1.44 Vwa8 von Willebrand factor A domain containing 8 1.2×10-8 -1.35
Ano5 anoctamin 5 1.78×10-8 -2.27
Slc16a7 solute carrier family 16 member 7 1.97×10-8 1.7 Ivd isovaleryl coenzyme A dehydrogenase 2.54×10-8 -1.41 Hn1 hematological and neurological expressed sequence 1 2.6×10-8 1.41 Nr0b2 nuclear receptor subfamily 0, group B, member 2 3.93×10-8 1.47 Slc22a3 solute carrier family 22 (organic cation transporter),
member 3 3.99×10-8 -1.66
Acsm5 acyl-CoA synthetase medium-chain family member 5 4.32×10-8 -1.88
Subsequently performed pathway analyses (Wikipathways, WebGestalt) within that cluster of 485 genes revealed that regulated genes were especially enriched in pathways involved in energy metabolism such as fatty acid biosynthesis (adjusted P (adj. P) = 4.07×10-9), mitochondrial LC-fatty acid beta-oxidation (adj.
P = 6.78×10-8), fatty acid beta-oxidation (adj. P = 1.06×10-7) and glycogen metabolism (adj. P = 8.83×10-5). In particular, these energy-related genes revealed a down-regulation in AGAT-/- mice. More detailed, at the level of individual genes, important genes of the fatty acid oxidation such as acetyl-CoA C-acyltransferase 2 (Acaa2), long chain fatty acid CoA ligase 1 (Acsl1) or acyl-CoA dehydrogenase short chain (Acads) were down-regulated in response to the AGAT knockout. In addition, a relation to the cardiovascular system was found by an enrichment of genes involved in cardiac calcium regulation (adj. P = 6.51×10-6). The top ten pathways including significantly regulated genes are illustrated in Figure 9.
Figure 9: Wikipathways pathway analysis within the 485 significantly regulated genes between wild-type and AGAT knockout mice in heart tissue
The top ten pathways are shown and the involved genes are listed in the bars. The direction of regulation is indicated by up and down arrows respectively. Significance level: Adjusted P-value
≤ 0.05.
Effects of creatine supplementation
AGAT catalyzes the first step in creatine biosynthesis, so that AGAT-/- mice are deficient of creatine. In order to investigate the effects of creatine supplementation, differential gene expression analysis was performed between WT and AGAT-/-Cr. The FC refers to the first named mouse group. Besides a 2-fold down-regulation of Agat only seven other genes remained significantly differentially expressed (FDR ≤ 0.05) between WT and AGAT-/-Cr (Table 13). This indicates that only 2% of AGAT-dependent deregulated genes remained differentially expressed after creatine supplementation and gave first evidence that gene expression in AGAT-/- mice can be rescued by creatine supplementation. In order to further investigate this assumption, the comparison of AGAT-/- and AGAT-/-Cr mice was conducted. The analysis revealed 484 significantly regulated genes between the groups. Crucially, most of the genes
Fatty Acid Biosyn thesis
Mitochondrial LC-Fatty Acid Beta-Oxidation
Fatty Acid Beta-Oxidation Myo
metrial Relaxa tion
and Contraction Pathways Calcium
Regulation in the Cardiac Cell
Regulation of Act in Cytoske
leton
Glycogen Metabolism
Striated Muscle Cell Contraction Chemo
kine signaling pathway
Senescence and Autophagy 10-9
10-8
10-7
10-6
10-5
10-4
10-3
10-2
10-1
adjusted P-value
Acta1 ↓ Acta2 ↑ Myl1 ↓ Myl9 ↑ Myom2 ↑
Cd44 ↑ Cdkn1a ↑ Cxcl14 ↓ Igf1r ↑
Irf7 ↑ Uvrag ↑ Adcy5 ↑
Ccl11 ↓ Ccl7 ↓ Cxcl14 ↓
Gnb3 ↓ Grk5 ↑ Ptk2b ↑ Rock1 ↑ Gys1 ↓
Pgm2 ↓ Phkb ↓ Phkg2 ↑
Pygb ↓ Chrm2 ↑
Egf ↓ Ezr ↑ Fgf16 ↑
Fgf2 ↓ Fgf6 ↓ Myl1 ↓ Pak6 ↓ Pip5k1b ↑
Rock1 ↑ Adcy5 ↑
Cacna1s ↓ Camk2a ↓ Camk2b ↓ Chrm2 ↑ Gnb3 ↓ Grk5 ↑ Pkia ↓ Prkcq ↓ Rgs2 ↑ Acta1 ↓
Acta2 ↑ Adcy5 ↑ Atf5 ↑ Camk2a ↓ Camk2b ↓ Corin ↑ Gnb3 ↓ Grk5 ↑ Pkia ↓ Prkcq ↓ Rgs2 ↑ Acaa2 ↓
Acsl1 ↓ Acss2 ↓ Decr1 ↓ Ech1 ↓ Echdc2 ↓ Echdc3 ↓ Hadh ↓
Acads ↓ Acsl1 ↓ Cpt2 ↓ Eci1 ↓ Hadh ↓ Slc25a20 ↓
Acads ↓ Acsl1 ↓ Acss2 ↓ Cpt2 ↓ Crat ↓ Decr1 ↓ Eci1 ↓ Hadh↓
Slc25a20 ↓
also occur in the comparison of WT versus AGAT-/- mice but are regulated in the other direction respectively.
Table 13: Differentially expressed genes between wild-type and creatine-supplemented AGAT knockout mice in heart tissue
Significance level: False discovery rate (FDR) ≤ 0.05. FC: fold change.
Gene Gene name P-value FC
Agat L-arginine:glycine amidinotransferase 2.39×10-8 -1.99 Mcm8 minichromosome maintenance deficient 8 (S. cerevisiae) 4.13×10-7 -1.52
Pde1c phosphodiesterase 1C 7.98×10-7 -1.38
Mertk c-mer proto-oncogene tyrosine kinase 9.01×10-7 -1.43
Hmcn1 hemicentin 1 8.26×10-6 -1.46
Cds2 CDP-diacylglycerol synthase 2 9.07×10-6 -1.3
Rasgef1b RasGEF domain family, member 1B 1.25×10-5 -1.56
Gm14085 predicted gene 14085 1.37×10-5 1.37
Effects of homoarginine supplementation
Additional to creatine deficiency, AGAT-/- mice are unable to produce homoarginine, which itself is a biomarker for poor prognosis in CVD (Atzler et al., 2015). AGAT-/- mice exhibit chronotropic, inotropic and lusitropic deficits in vivo that are completely rescued by homoarginine supplementation (Faller et al., 2017). Therefore, it was of interest to find out whether transcriptomic signatures might explain these observations. The analysis of WT and AGAT-/-HA mice showed 785 (FDR ≤ 0.05) significantly regulated genes. Table 14 represents the selection of the 20 most significant genes. The comparison of these genes with the 485 differentially expressed genes between WT and AGAT-/- mice revealed that only 204 genes (i.e. 42%) could be restored by homoarginine supplementation, whereas a large proportion remained regulated or demonstrated regulation by homoarginine supplementation only.
Table 14: Top 20 differentially expressed genes between wild-type and homoarginine-supplemented knockout mice in heart tissue
Significance level: False discovery rate (FDR) ≤ 0.05. FC: fold change.
Gene Gene name P-value FC
Mthfd2 methylenetetrahydrofolate dehydrogenase (NAD+
dependent), methenyltetrahydrofolate cyclohydrolase
4.37×10-13 4.59 Scn4a sodium channel, voltage-gated, type IV, alpha 6.63×10-11 -2.81 Agat L-arginine:glycine amidinotransferase 4.92×10-10 -2.32 Wfdc1 WAP four-disulfide core domain 1 6.87×10-10 -1.75
Myot myotilin 7.11×10-10 -2.88
Tmem150c transmembrane protein 150C 8.76×10-10 -2.94 Lgi1 leucine-rich repeat LGI family, member 1 9.99×10-10 -4.27
Lad1 ladinin 2.23×10-9 2.42
Fah fumarylacetoacetate hydrolase 3.36×10-9 -2.33
Slc16a7 solute carrier family 16 (monocarboxylic acid transporters), member 7
4.54×10-9 1.75
Stom stomatin 7.04×10-9 -1.46
Asns asparagine synthetase 7.21×10-9 2.97
Atf5 activating transcription factor 5 7.45×10-9 2.02 Slc7a5 solute carrier family 7 (cationic amino acid transporter,
y+ system), member 5
3.38×10-8 1.75
Amot angiomotin 5.25×10-8 -1.52
Scn4b sodium channel, type IV, beta 6.13×10-8 -3.34
Tmod4 tropomodulin 4 6.27×10-8 -1.74
Aldh1l2 aldehyde dehydrogenase 1 family, member L2 6.98×10-8 1.98 Ttc12 tetratricopeptide repeat domain 12 7.23×10-8 1.38 Ivd isovaleryl coenzyme A dehydrogenase 1.11×10-7 -1.53