E2f1 deletion attenuates infarct-induced ventricular remodeling without affecting O-GlcNAcylation

Abstract

Several post-translational modifications figure prominently in ventricular remodeling. The beta-O-linkage of N-acetylglucosamine (O-GlcNAc) to proteins has emerged as an important signal in the cardiovascular system. Although there are limited insights about the regulation of the biosynthetic pathway that gives rise to the O-GlcNAc post-translational modification, much remains to be elucidated regarding the enzymes, such as O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which regulate the presence/absence of O-GlcNAcylation. Recently, we showed that the transcription factor, E2F1, could negatively regulate OGT and OGA expression in vitro. The present study sought to determine whether E2f1 deletion would improve post-infarct ventricular function by de-repressing expression of OGT and OGA. Male and female mice were subjected to non-reperfused myocardial infarction (MI) and followed for 1 or 4 week. MI significantly increased E2F1 expression. Deletion of E2f1 alone was not sufficient to alter OGT or OGA expression in a naïve setting. Cardiac dysfunction was significantly attenuated at 1-week post-MI in E2f1-ablated mice. During chronic heart failure, E2f1 deletion also attenuated cardiac dysfunction. Despite the improvement in function, OGT and OGA expression was not normalized and protein O-GlcNAcyltion was not changed at 1-week post-MI. OGA expression was significantly upregulated at 4-week post-MI but overall protein O-GlcNAcylation was not changed. As an alternative explanation, we also performed guided transcriptional profiling of predicted targets of E2F1, which indicated potential differences in cardiac metabolism, angiogenesis, and apoptosis. E2f1 ablation increased heart size and preserved remote zone capillary density at 1-week post-MI. During chronic heart failure, cardiomyocytes in the remote zone of E2f1-deleted hearts were larger than wildtype. These data indicate that, overall, E2f1 exerts a deleterious effect on ventricular remodeling. Thus, E2f1 deletion improves ventricular remodeling with limited impact on enzymes regulating O-GlcNAcylation.

Keywords: Glycosylation; Heart failure; Hexosamine biosynthetic pathway; Metabolism.

Fig. 1

E2F1 is upregulated during heart failure. Male mice aged 10–16 weeks were subjected to sham or myocardial infarction surgery for 1 or 4 week. E2f1 mRNA expression was assessed in whole sham (n = 4) and MI (n = 4) hearts 1 week following MI (a; p = 0.0044 vs Sham). A separate cohort of sham (n = 6) and MI (n = 5) mice was used to spatially determine E2f1 mRNA expression in infarcted (I), non-infarcted (N), and right ventricle (RV) regions of sham and infarcted mice at 1-week post-MI (b). Western blot of E2F1 protein expression and densitometric analysis (c; p = 0.0389 vs Sham). E2f1 mRNA expression in whole Sham and MI hearts 4 weeks following MI (d; p = 0.0133 vs sham). Region-specific expression of E2f1 mRNA in sham (n = 6) and MI (n = 5) hearts 4 week after MI (e). E2F1 immunoblot and densitometric analysis of whole sham and MI hearts 4 weeks following MI (f). An unpaired Student’s t test was used to determine significance between Sham and MI groups. Oneway ANOVA followed by a Newman–Keuls multiple comparison test analysis was used to determine significance between region-specific expression of E2f1

Fig. 2

E2F1 deficiency in naïve mice does not affect O-GlcNAcylation or cardiac mass. Cardiac tissue from naïve, male E2f1^+/+(n = 4) and E2f1^−/−(n = 4) mice was harvested at 10–16 weeks of age. E2f1 mRNA expression in E2f1^+/+ and E2f1^−/− hearts (a; p < 0.00001). Expression of markers of HBP and metabolism (b). Gravimetric analysis of heart weight to body weight (c) and heart weight to tibia length (d). Cardiac expression of OGT (e), OGA (f), total protein O-GlcNAcylation (g). An unpaired Student’s t test was used to determine significance between E2f1^+/+ and E2f1^−/− groups

Fig. 3

E2f1 deficiency attenuates cardiac dysfunction in male mice 1-week post-MI. Male (blue) and female (red) E2f1^+/+(n = 25) and E2f1^+/+(n = 26) were subjected to echocardiography after 1-week post-MI. Representative m-mode images of E2f1^+/+ and E2f1^−/− hearts (a). Ablation of E2f1 attenuated acute heart failure as indicated by reduction in ventricular end-diastolic (b; p = 0.0024) and end-systolic (c; p = 0.0020) volumes and a concomitant improvement of ejection fraction (d; p = 0.0109). Gravimetric analysis of wet versus dry lung weight indicated no differences in pulmonary edema between E2f1^+/+ and E2f1^−/− groups (e). An unpaired Student’s t test was used to determine significance between E2f1^+/+ and E2f1^−/− groups

Fig. 4

Loss of E2F1 reduces cardiac size without affecting cardiomyocyte hypertrophy after infarction. mRNA expression of markers of cardiac hypertrophy (a). Representative images of WGA-stained E2f1^+/+ and E2f1^−/− heart sections (b). Gravimetric analysis of heart size revealed reduced heart weight to tibia length (c) in E2f1^−/− mice. Cardiomyocyte cross-sectional area was measured in the border zone of the infarct (“BZ”; panel d), in the infarct zone (“IZ”; panel e), and remote zone (“RZ”; panel f). An unpaired Student’s t test was used to determine significance between E2f1^+/+ and E2f1^−/− groups

Fig. 5

Capillary density is increased acutely after MI in the remote zone of E2f1 deficient hearts. mRNA expression of Pdgfb (a). Representative images of isolectin-stained hearts (b). Capillary density in border zone (“BZ”, panel c), infarct zone (“IZ”; panel d), and remote zone (“RZ”; panel e). Bcl2 mRNA expression (f). Representative TUNEL-stained sections (g). Quantification of TUNEL positive cells (h). Representative images for cardiac fibrosis (i) and quantification (j). An unpaired Student’s t test was used to determine significance between E2f1^+/+ and E2f1^−/− groups

Fig. 6

E2f1 deficiency promotes durable improvement in ventricular function in male mice. Male (blue) and female (red) E2f1^+/+ (n = 15) and E2f1^+/+ (n = 17) were subjected to echocardiography after 4 weeks of non-reperfused myocardial infarction. Representative m-mode images of E2f1^+/+ and E2f1^−/− hearts (a). Ablation of E2f1 attenuated chronic heart failure as evidenced by reduction in ventricular end-diastolic (b, p = 0.0349) and end-systolic (c, p = 0.0195) volumes and a concomitant attenuation of ejection fraction (d, p = 0.0098) compared to E2f1^+/+. Gravimetric analysis of wet versus dry lung weight indicated increased water retention in the lungs between E2f1^+/+ and E2f1^−/− groups (e, p = 0.0116). An unpaired Student’s t test was used to determine significance between E2f1^+/+ and E2f1^−/− groups

Fig. 7

E2f1 deficiency promotes cardiomyocyte hypertrophy in the remote zone of the failing heart. Gene expression of markers of cardiac hypertrophy (a). Representative images of WGA-stained E2f1^+/+ (n = 13) and E2f1^−/− (n = 14) heart sections (b). Gravimetric analysis of heart size; heart weight to tibia length (c). Cardiomyocyte cross-sectional area was measured in the border zone (d), infarct zone (e), and remote zone (f; p = 0.0252). An unpaired Student’s t test was used to determine significance between E2f1^+/+ and E2f1^−/− groups

Fig. 8

Cardiac capillary density and fibrosis are not improved during chronic heart failure in E2f1 deficient hearts. mRNA expression of Pdgfb at 4-week post-MI (a). Representative images of isolectin-stained of E2f1^+/+ and E2f1^−/− heart sections 4-week post-MI (b). Capillary density in border zone (“BZ”; c), infarct zone (“IZ”; d), and remote zones (“RZ”; e). Bcl2 mRNA (f). Representative TUNEL-stained of E2f1^+/+ and E2f1^−/− heart sections 4-week post-MI (g). Quantification of TUNEL-positive cells (h). Representative images for cardiac fibrosis (i) and quantification (j). An unpaired Student’s t test was used to determine significance between E2f1^+/+ and E2f1^−/− groups

Fig. 9

E2F1 ablation promotes expression of markers of metabolism but does not alter protein O-GlcNAcylation. Cardiac tissue harvested from 1-week post-MI E2f1^+/+ and E2f1^−/− hearts were probed for O-GlcNAc enzymes and markers of metabolism. Immunoblot for OGT (a), OGA (b), and protein O-GlcNAcylation (c). Expression of metabolic transcripts via RT-PCR (d). An unpaired Student’s t test was used to determine significance between E2f1^+/+ and E2f1^−/− groups

Fig. 10

E2F1 ablation induces OGA expression in the failing heart. Cardiac tissue harvested from 4-week post-MI E2f1^+/+ (n = 13) and E2f1^−/− (n = 14) mice were probed for O-GlcNAc enzymes and markers of metabolism. Immunoblot for OGT (a), OGA (b), and protein O-GlcNAcylation (c). Expression of metabolic transcripts via RT-PCR (d) An unpaired Student’s t test was used to determine significance between E2f1^+/+ and E2f1^−/− groups