The Impaired Bioenergetics of Diabetic Cardiac Microvascular Endothelial Cells

Figures

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  Figure 1. Comparison of WT and db/db mice. (A) A db/db mouse in comparison to an age-matched C57BL/6 WT mouse; (B) Body weight (g) in 6-month-old WT mice and db/db mice; results are shown as mean ± SEM (n = 3, * p < 0.05); (C) Blood glucose levels (mg/dl) in 6-month-old WT mice and db/db mice fed with standard chow, (n = 3, **p < 0.01); (D) Green fluorescence intensity was used as a measure of relative intracellular ROS in WT CMECs and diabetic CMECs.

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  Figure 2. Assessment of oxygen consumption rate (OCR) in WT and diabetic CMECs. (A) Schematic representation of the protocol employed in data collection and calculations of mitochondrial respiration; (B) Normalized OCR data; (C) Parameters of mitochondrial respiration function calculated from the OCR tracing in (B). Results are normalized to total cellular protein and shown as mean ± SEM (n = 8), NS p > 0.05, *p < 0.05, **p < 0.01.

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  Figure 3. Assessment of extracellular acidification rate (ECAR) in WT and diabetic CMECs. (A) Schematic representation of the protocol employed in data collection and calculations of glycolytic function. (B) Normalized ECAR data. (C) Parameters of glycolytic function calculated from the ECAR tracing in (B). Results are normalized to total cellular protein and shown as mean ± SEM (n = 12), ns p > 0.05, **p < 0.01.

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  Figure 4. Comparison of relative mRNA levels of genes related to (A) fatty acid β-oxidation (Sirt1, Acox1, Acox3, Hadha and Hadhb), (B) TCA cycle (Idh3a and Ogdh), and (C) ETC (Sdhd and Uqcrq) in WT-CMECs and db/db-CMECs. The amount of mRNA was normalized using β-actin. TCA, tricarboxylic acid cycle; ETC, electron transport chain. Results are shown as mean ± SEM (n = 3), ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (D, E) Comparison of relative protein levels of enzymes related to fatty acid β-oxidation (Hadha and Acox1) and ETC (Uqcrq) in WT CMECs and diabetic CMECs. The amount of protein was normalized using GAPDH. Results are shown as mean ± SEM (n = 3), *P < 0.05. (F) Comparison of the relative copy number of mitochondria (MT)-encoded NADH-ubiquinone oxidoreductase chain 6 (MT-ND6) between WT CMECs and diabetic CMECs. The amount of mitochondrial DNA was normalized to the nuclear-encoded control gene DNA polymerase-β (PolB). Results are shown as mean ± SEM (n = 3), ns p > 0.05.

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  Figure 5. Comparison of cardiac angiogenesis, endothelial proliferation, and apoptosis between WT-hearts and db/db-hearts. (A) Immunofluorescent staining of lectin to detect capillary density in WT-hearts and db/db-hearts; (B) Comparison of Lectin-positive cells per high power field (HPF) between WT-hearts and db/db hearts (ns, P >0.05, n=7); (C) Immunofluorescent staining of Ki67 and CD31 to detect proliferative endothelial cells in WT-hearts and db/db-hearts; (D) The comparison of the percentage of Ki67+ endothelial cells between WT and db/db hearts (***P < 0.001, n=7); (E) Immunofluorescent staining of TUNEL and CD31 to detect apoptotic endothelial cells in WT-hearts and db/db hearts; (F) The comparison of the percentage of TUNEL-positive endothelial cells between WT and db/db hearts (****P < 0.0001, n=6).

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  Figure 6. A schematic model for mechanism CMEC dysfunction in diabetes. High glucose-induced ROS accumulation in diabetic cardiac microvascular endothelial cells disrupts normal ATP production by inhibiting both mitochondrial electron transport system and glycolysis, therefore inhibiting CMEC proliferation and inducing apoptosis.