Hypoxia-Inducible Factor 1-Alpha and Glucose Metabolism during Cardiac Remodeling Progression from Hypertrophy to Heart Failure

Figures

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  Figure 1. Bodyweight and post mortem cardiac morphology at different euthanasia moments (2, 6, 18 weeks [W] and heart failure [HF]). Sham: operated control (blue); AoS: supravalvar aortic stenosis (red). (A) BW: body weight. (B) LV/TB: left ventricle corrected by tibia (TB). (C) RV/TB: right ventricle corrected by TB; (D) AT/TB: atrium corrected by TB; (E) Lung/TB: lung corrected by TB. Data are expressed as mean ± SD and analyzed by ANOVA and Tukey. p < 0.05. * AoS vs. respective Sham. Distinct letters signify statistical difference between moments (n = 12–18 animals for each group).

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  Figure 2. Heart rate and structural echocardiographic data at different euthanasia moments (2, 6, 18 weeks [W] and heart failure [HF]). Sham: operated control (blue); AoS: supravalvar aortic stenosis (red). (A) HR: heart rate; (B) LA/AO: left atrial diameter (LA) to aortic diameter (AO) ratio; (C) LVDD: left ventricle (LV) diastolic diameter; (D) LVWDT: LV posterior wall diastolic thickness; (E) RWT: LV relative wall thickness. Data are expressed as mean ± SD and analyzed by ANOVA and Tukey. p < 0.05. * AoS vs. respective Sham. Distinct letters signify statistical difference between moments (n = 12–18 animals for each group).

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  Figure 3. Functional echocardiographic data at different euthanasia moments (2, 6, 18 weeks [W] and heart failure [HF]). Sham: operated control (blue); AoS: supravalvar aortic stenosis (red). (A) EF (%): left ventricle (LV) ejection fraction in percentage. (B) Meso (%): LV shortening of mesocardium in percentage. (C) LVPW: LV posterior wall shortening velocity. (D) E wave: early diastolic mitral inflow velocity. (E) A wave: late diastolic mitral inflow velocity. (F) E/A: ratio between E wave and A wave. Data are expressed as mean ± SD and analyzed by ANOVA and Tukey. p < 0.05. * AoS vs. respective Sham. Distinct letters signify statistical difference between moments; (n = 12–18 animals for each group).

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  Figure 4. Hypoxia-inducible factor-1α (HIF-1α) protein expression evaluated by Western blot. (A) Quantification of HIF-1α. (B) Representative bands of protein. Sham: operated control; AoS: supravalvar aortic stenosis; HF: heart failure; Sham2: Sham 2 weeks; Sham6: Sham 6 weeks; Sham18: Sham 18 weeks; ShamHF: Sham HF; AoS2: AoS, 2 weeks; AoS6, 6 weeks; AoS18, 18 weeks; AoSHF, HF. ISC: Internal control of the sample; AU: arbitrary unity. Data are expressed as mean ± SD and analyzed by ANOVA and Tukey, p < 0.05. * AoS vs. respective Sham. Distinct letters signify statistical difference between moments (n = 8 animals for each group).

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  Figure 5. Glucose and insulin data. (A) Blood glucose. (B) Serum insulin. Group abbreviations are as defined in Figure 4. Data are expressed as mean ± SD and analyzed by ANOVA and Tukey in (A). Data expressed in median (Min and Max) and analyzed by ANOVANP and Dunn in (B). p < 0.05. * AoS vs. respective Sham. Distinct letters signify statistical difference between moments; (n = 8–12 animals for each group).

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  Figure 6. Cardiac insulin signaling and glucose transporters 1 and 4 at different euthanasia moments (2, 6, 18 weeks [W] and heart failure [HF]) evaluated by Western blot. Sham: operated control (blue); AoS: supravalvar aortic stenosis (red). Quantification of (A) pIR: phosphorylated insulin receptor, (B) Total IR: total insulin receptor, (C) pIR/Total IR: pIR/Total IR ratio, (D) pAKT: phosphorylated protein kinase B, (E) Total AKT: total protein kinase B, (F) pAKT/Total AKT: pAKT/Total AKT ratio; (G) PI3K: phosphoinositide 3-kinase, (H) GLUT4: glucose transporter 4, and (I) GLUT1: glucose transporter 1. (J) Representative bands of proteins. Data are expressed as mean ± SD and analyzed by ANOVA and Tukey. p < 0.05. * AoS vs. respective Sham. Distinct letters signify statistical difference between moments (n = 8 animals for each group).

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  Figure 7. Myocardial glycolysis and glycogen at different moments of euthanasia (2, 6, 18 weeks [W] and heart failure [HF]) evaluated by Western blot and enzymatic activity assay. Sham: operated control (blue); AoS: supravalvar aortic stenosis (red). Quantification of (A) HKII: hexokinase II, (B): HK: activity of hexokinase II, (C) PFKB2: phosphofruct kinase 2, (D) PK: pyruvate kinase, and (E): cardiac glycogen. (F) Representative bands of proteins. Data are expressed as mean ± SD and analyzed by ANOVA and Tukey. p < 0.05. * AoS vs. respective Sham. Distinct letters signify statistical difference between moments; (n = 8 animals for each group).

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  Figure 8. Myocardial aerobic and anaerobic metabolism at different euthanasia moments (2, 6, 18 weeks [W] and heart failure [HF]) evaluated by Western blot and enzymatic activity assay. Sham: operated control (blue); AoS: supravalvar aortic stenosis (red). Quantification of (A) PDH: pyruvate dehydrogenase, (B) CS: citrate synthase activity, (C) LDH: lactate dehydrogenase, (D) LDH: LDH activity, and (E) cardiac lactate concentration. (F) Representative bands of proteins. Data are expressed as mean ± SD and analyzed by ANOVA and Tukey. p < 0.05. * AoS vs. respective Sham. Distinct letters signify statistical difference between moments; (n = 8 animals for each group).

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  Figure 9. Overview of results observed during the evolution process of cardiac remodeling from hypertrophy to heart failure. Expr.: expression. Protein abbreviations are as defined in Figure 4, Figure 5, Figure 6, Figure 7 and Figure 8. Different symbols mean statistical difference between AoS vs. Sham (↑ increase or ↓ decrease) or between the AoS groups († vs. AoS2; # vs. Aos6; *** vs. Aos2, 6, 18; ** vs. Aos 2, 18; * vs. Aos6, 18; Ω vs. Aos2, 6), p < 0.05.

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  Figure 10. Scatter plot of canonical variables in aortic stenosis groups. AoS: supravalvar aortic stenosis; HF: heart failure; • AoS2, 2 weeks; • AoS6, 6 weeks; • AoS18, 18 weeks; • AoSHF, HF; HIF-1α: factor inducible hypoxia. (A) Insulin signaling and glucose transporters 1 and 4 (total and phosphorylated insulin receptor, total and phosphorylated protein kinase B, phosphoinositide 3-kinase and glucose transporter 1 and 4 expression); p < 0.027. (B) Glycolysis (hexokinase activity, hexokinase II, phosphofruct kinase 2 and pyruvate kinase expression); p < 0.018. (C) Anaerobic pathways (lactate dehydrogenase expression and activity); p < 0.024. (D) Aerobic pathways (pyruvate dehydrogenase expression and citrate synthase activity); p < 0.957. Canonical correlations: insulin signaling and glucose transporters 1 and 4, 0.671; glycolysis, 0.625; anaerobic pathways, 0.675; aerobic pathways, 0.054. 𝑢1 = 1.00 (HIF-1α). Insulin signaling and glucose transporters 1 and 4: A𝑣1 = 0.40 pIR + 0.23 total IR + 0.56 pAKT − 0.01 total AKT + 0.54 PI3K + 0.74 GLUT1 + 0.26 GLUT4. Glycolysis: A𝑣1 = 0.32 HKII + 0.66 HK + 0.81 PFK2 + 0.28 PK. Anaerobic pathways: A𝑣1 = 0.45 LDH − 0.99 LDH activity. Aerobic…