Alterations in Glucose Metabolism During the Transition to Heart Failure: The Contribution of UCP-2

Figures

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  Figure 1. Basal expression levels of UCP-2 in adult rat ventricular cardiomyocytes and cardiac non-myocytes and UCP-2 expression in cardiomyocytes after siRNA treatment. (A–B) Real-time RT–PCR cycle thresholds (Ct) for the housekeeping gene HPRT (A), UCP-2 (B) and UCP-2 after normalization to HPRT (Ct) expression (C) in cardiomyocytes (CMC) and cardiac non-myocytes (CNM). Data are from n = 4 preparations and statistically analyzed using unpaired t-test (* p = 0.02959). (D) Relative UCP-2 mRNA expression in cells exposed to small inhibitory (siRNA) against UCP-2 or scrambled RNA (scRNA) for 24 h. Data are from n = 8 cultures out of 4 individual preparations and statistically analyzed using unpaired t-test (* p = 0.011). (A–D) Data expressed as 25%, 50%, and 75% quartiles with whiskers representing the total range, and dots representing the individual data points. (E) Original Western blot showing expression of UCP-2 in cardiomyocytes after 3 or 5 days in the presence of scRNA or siRNA directed against UCP-2. Rec. UCP-2 = control peptide.

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  Figure 2. Effects of UCP-2-silencing or genipin treatment on cell shortening (A,D), contraction velocity (B,E), and relaxation velocity (C,F) after hypertrophic stimulation. (A–C) Cardiomyocytes were exposed to scRNA or siRNA and incubated with angiotensin-II (Ang-II 0.1–10 µM) or vehicle (Ctrl). Data are means ± SD. from 189–252 cells (Ctrl), 36 cells (Ang-II 0.01 and 0.1 µM), 144 cells (1 µM) or 153–198 cells (10 µM). (D–F) Cardiomyocytes were exposed to genipin (10 µM) or vehicle (control with DMSO) and incubated with Ang II (0.1–10 µM) or vehicle. Data are means ± S.D. from n = 108 (Ctrl), 36 (Ang-II 0.01 and 0.1 µM), 126 (Ang-II 1 and 10 µM). A one-way ANOVA with a subsequent Student–Newman–Keuls test for post hoc analysis was performed (* p < 0.05 vs. Ang-II-free; # p < 0.05 vs. siRNA- or genipin-treated cells).

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  Figure 3. Effect of UCP-2 silencing or inhibition on mitochondrial function, glucose metabolism, and viability upon Ang-II stimulation. (A–B) LV mitochondria were isolated and ROS formation (A) or respiration (B) was determined in the absence (control with DMSO) or presence of genipin (Gen, 10 µM) from n = 4 (A) or n = 5 (B) hearts. Statistical analysis was performed with unpaired t-test (p > 0.05). (C) mRNA expression of Glut-4 in cells exposed to siRNA directed against UCP-2 or scrambled RNA (scRNA). Statistical analysis was performed with a Mann–Whitney test, from n = 12 cultures out of 6 individual preparations (* p = 0.038). (D) Original Western blot showing expression of Glut-4 in cardiomyocytes exposed to siRNA against UCP-2 (siR) or scRNA (scR). (E) Quantification of Western blots as shown in (D) normalized to GAPDH and compared to the basal expression in cultures exposed to scRNA. Data from n = 6 cultures out of 3 individual preparations. Statistical analysis was performed with unpaired t-test (* p = 0.030). (F) Glucose uptake of cardiomyocytes exposed to siRNA directed against UCP-2, normalized to the controls. Data are from n = 29 cultures out of 6 individual preparations and…

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  Figure 4. Blood pressure and hypertrophy in spontaneously hypertensive rats (SHR). (A) Systolic blood pressure in SHRs at various time-points (1.5 months n = 4; 11.5 months n = 9; all others n = 6). * p < 0.05 vs. 1.5 months. (B) LV weight normalized to tibia length (LVW/TL; 1.5 months: n = 8; 11.5 months: n = 5; all others n = 10). * p < 0.05 vs. 1.5 months; # p < 0.05 vs. 1.5–4.5 months. (C) Ejection fraction (EF) in SHR (n = 5–6). * p < 0.05 vs. 3 months. ANP (D), UCP-2 (E) and PGC-1α (F) mRNA expression in the LV of SHRs (1.5–7.5 months n = 6; 11.5 months n = 12; * p < 0.05 vs. 1.5 months; # p < 0.05 vs. 1.5–7.5 months). (G) Original Western blot showing Glut-4, UCP-2, and GAPDH expression in the LV of SHRs at an age of 1.5, 7.5, and 11.5 months. (H,I) Quantification of protein levels from Western blots of n = 4 independent experiments, normalized to GAPDH (p < 0.05 vs. 1.5 and 11.5 months). (A–F,H,I) Data expressed as 25%, 50%, and 75% quartiles, with whiskers representing the total range and dots representing the individual data points. Statistical analysis was performed with a one-way ANOVA with a subsequent Student–Newman–Keuls test for post hoc analysis.

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  Figure 5. Blood pressure and hypertrophy in 10-months old spontaneously hypertensive rats (SHR) treated with (Spiro) or without (Ctr) spironolactone for the last four months. (A) Systolic blood pressure in SHRs (n = 6). Statistical analysis was performed with unpaired t-test (* p = 0.00035). (B) LV weight normalized to tibia length (LVW/TL; n = 12–16). Statistical analysis was performed with Welch’s t-test (p > 0.05). (C) Ejection fraction (EF) in SHR (n = 10–12). Statistical analysis was performed with a Mann–Whitney test (p = 0.00193). (D–G) mRNA expression of ANP (D), PGC-1α (E), UCP-2 (F) and Glut-4 (G) in the LV of SHRs (n = 12–18). Statistical analysis was performed with unpaired t-test (p > 0.05 (ANP); * p = 0.00664 (PGC-1α); * p = 0.00704 (UCP-2); * p = 0.01404 (Glut-4)). (H, I) Quantification of protein levels from Western blots showing Glut-4, UCP-2, and GAPDH expression in the LV of SHRs at an age of 11.5 months, normalized to GAPDH and compared to the basal expression of the controls. Data are from n = 6 independent experiments and are statistically analyzed using unpaired t-tests. (H) UCP-2 (* p = 0.026) and (I) Glut-4 (* p = 0.015). (J) Original Western blot showing Glut-4,…

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  Figure 6. UCP-2 and Glut-4 expression in LV from healthy donor hearts or explanted hearts of end-stage congestive heart failure (CHF). (A) Original Western blot. (B,C) Quantification of protein levels from Western blots normalized to GAPDH and compared to the expression of donor hearts. Data from n = 4 (Donor) or n = 9 (CHF) hearts are expressed as 25%, 50%, and 75% quartiles, with whiskers representing the total range. The dots represent the individual data points. Statistical analysis with unpaired t-test. (B) UCP-2 (* p = 0.026) and (C) Glut-4 (* p = 0.015). (D) Association between UCP-2 and Glut-4 expression with Pearson’s correlation coefficient (p = 0.03093). Open symbols: donor hearts. Filled symbols: CHF.

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  Figure 7. Final summary. During the adaptive phase of myocardial hypertrophy UCP-2 expression is reduced and Glut-4 expression is increased. We hypothesize that this improves pyruvate import into mitochondria and maintains cardiomyocyte function. During the transition to heart failure (HF), UCP-2 expression is increased and, subsequently, Glut-4 expression reduced. UCP-2 expression would then inhibit pyruvate entry and glucose would be metabolized in a glycolytic pathway leading to decreased ATP levels and lactate accumulation. e- = electron. ETC = electron transport chain.