PHDs/CPT1B/VDAC1 axis regulates long-chain fatty acid oxidation in cardiomyocytes

Figures

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  Figure 1.. PHD2/3 bind to CPT1B and regulate LCFA uptake and oxidation in primary cardiomyocytes (A) Coomassie-stained SDS-PAGE of FLAG-PHD3 co-immunoprecipitated proteins in NRVMs. PHD3 or LacZ was overexpressed in NRVMs, and co-immunoprecipitation was performed with anti-FLAG agarose beads. Pulled-down proteins were separated with SDS-PAGE and visualized with G-250 Coomassie blue. Protein identities were determined by tandem mass spectrometry analysis (1. Dync1h1; 2. Na+/K+ ATPase α−1; 3. ATP synthase B; 4. GAPDH; 5. ANT; 6. MLP). The respective positions of PHD3 and CPT1B are labeled in the figure. (B) Co-immunoprecipitation and western blot revealed PHD2 and 3 binding to CPT1B. FLAG-PHD2 and FLAG-PHD3 were immunoprecipitated by anti-FLAG beads, and the precipitated proteins were analyzed by western blot with anti-FLAG and anti-CPT1B antibodies. (C) Co-immunoprecipitation and western blot revealed endogenous CPT1B binds to endogenous PHD2 and 3. The precipitated proteins were analyzed by western blot with anti-PHD2, PHD3, or CPT1B antibodies. (D) Oxygen consumption rate (OCR) in primary cardiomyocytes after DMOG or Etomoxir (CPT1 inhibitor, Eto) treatment. OCR was real-time analyzed in…

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  Figure 2.. Global and cardiac-specific loss of PHD2/3 impact cardiac metabolism (A–C) Hyperinsulinemic-euglycemic clamp in conscious D2/3_KO and D2/3_WT mice at 4 weeks after tamoxifen injection (n = 6 per each group). Glucose metabolism is significantly increased in D2/3_KO mice (A). Hepatic glucose production rate is shown in (B). Tissue glucose uptake is shown in (C), showing a predominant increase of glucose uptake in the heart of D2/D3_KO mice, and also in Gastrocnemius muscle at a lower range (C). (D–F) Cardiomyocyte-specific disruption of PHD2/3 was triggered by tamoxifen injection (20 mg/kg/day) for 4 consecutive days in D2/3_HKO mice. Mice from both genotypes were split in two groups randomly and fed with normal chow (CC) or high-fat diet (HFD) for 8–12 weeks (n = 6–11 mice per group). The glucose blood level was monitored at the 8-week time point following PHD2/3 disruption (D). D2/3_HKO mice showed a significantly lower blood glucose level, at both CC and HFD-feeding conditions. (E) Glucose tolerance test at 8 weeks following CC or HFD in D23_HKO and D2/3_HWT mice. Similarly, as expected, HFD-D2/3_HWT mice had a pre-diabetic-specific slower response to glucose, whereas D2/3_HKO…

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  Figure 3.. Cardiac-specific depletion of PHD2/3 worsens hypertrophic cardiomyopathy Following tamoxifen injection, D2/3_HKO and D2/3_HWT mice were fed with HFD or CC for 12 weeks (n = 6–11 mice group). (A and B) Echocardiography analyses were performed at 4 weeks (top panel) and 12 weeks (bottom panel) after HFD or CC. HFD fastens the impairment of cardiac function in D2/3_HKO, showing significant decrease of ejection fraction at 4 weeks after tamoxifen infusion, instead of 12 weeks for CC-fed D2/3_HKO mice. (C and D) Representative pictures of WGA-stained mid-myocardial cross section (top panel), and the resulting quantification (bottom panel; n = 4 per group). Scale bar, 40 μm. (E and F) Representative pictures of Oil Red O-stained cardiac cross-sections at 12 weeks after HFD or CC and subsequent estimation of lipid deposit area (bottom panel, n = 4 per group). Scale bar, 40 μm. Data are presented as means ± SEM. Statistical differences after single-factor ANOVA and post hoc analysis were indicated with asterisks: *p < 0.05 versus the control chow or HFD PHD_HWT control group; **0.01 < p < 0.05 versus the corresponding control group; ***p < 0.001 versus the corresponding control group.

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  Figure 4.. Loss of PHD2/3 transiently increases CPT1B expression, leading to an increase in carnitine-conjugated LFCA in the heart of D2/3_HKO mice (A) Representative western blot for D2/3_HKO cardiac lysates collected at 0, 1, 2, and 4 weeks after tamoxifen infusion. The CPT1B protein level was transiently increased at 1 week after tamoxifen treatment, whereas the Hexokinase-2 (HK-2) protein level started to gradually increase. (B) Relative level of CPT1B level of transcript over the time after PHD2/3 disruption (n = 4 per group). (C) Western blot for PHD2/3_HWT and PHD2/3_HKO cardiac lysates collected after 4 weeks of tamoxifen infusion. (D) Quantitative analysis of CPT1B protein level was performed and normalized with actin protein level (n = 4 per group). (E) CPT1B catalytic activity in D2/3_HWT and D2/3_HKO mouse hearts by DTNB colorimetric assay (n = 10 per group), showing no difference regardless the genotype. (F) Liquid chromatography-mass spectrometry analysis of relative abundance of carnitine-conjugated LCFA in the heart of D2/3_HKO mouse (n = 4 per group), suggesting an accumulation of carnitine-conjugated LCFA and acetyl-carnitine in the heart of PHD2/3_HKO mice. Data are…

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  Figure 5.. CPT1B binds to VDAC1, which may be involved in LCFA oxidation (A) CPT1B/VDAC1 binding domain identification. Full-length and C-terminal-truncated CPT1B were overexpressed with VDAC1 in 293T cells. Immunoblot for VDAC1 after anti-FLAG co-immunoprecipitation demonstrates that both CPT1B1–616 and CPT1B1–451 bind to VDAC1, whereas CPT1B1–219 lost its binding capacity to VDAC1. (B) Recombinant PHD2 interacts with GST-CPT1B220–451 in vitro. (C) Both VDAC1 and C-terminal-truncated VDAC1 bind to CPT1B. Immunoblot for CPT1B following co-immunoprecipitation with full-length or C-terminal-truncated (1–240) VDAC1 in 293T cells, which validates CPT1B/VDAC1 complex formation. (D) Competitive co-immunoprecipitation between CPT1B and C-truncated CPT1B. FLAG-tagged CPT1B was co-overexpressed in NRVMs, aside with a gradient expression level of GFP-tagged CPT1B1–616, which significantly decreases the amount of pull-down VDAC1. (E and F) VDAC1 knockout induced by Sh-RNA interference leads to a decrease in respiratory capacity (E), leading to a significant decrease in Maximal oxygen consumption rate (F). (G) Oxidative respiration was challenged in isolated NRVMs overexpressing CPT1B, CPT1B1–616, or…

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  Figure 6.. CPT1B binds to VDAC1 in an oxygen- and PHD-dependent manner, which can be involved in LCFA metabolism in primary cardiomyocytes (A and B) Representative co-immunoprecipitation of CPT1B and overexpressed VDAC1-GFP in 293T cells (A) or endogenous VDAC1 in NRVMs (B), and resulted quantification (bottom panel, n = 4). (C) LC-MS/MS spectrum for prolyl-hydroxylation of GST-CPT1B220–451 catalyzed by recombinant PHD2 in vitro. P295 residue was identified as the hydroxylation site in CPT1B. (D) Co-immunoprecipitation of VDAC1-GFP with FLAG-CPT1B-WT or CPT1B-P295A from PBS- or DMOG-pre-treated 293T cells, showing a better preservation of CPT1B-295A binding capacity to VDAC1 in DMOG pre-treated 293T cells. (E) OCR measurement by Sea Horse flux analyzer in D2/3_HKO NMCMs following PHD2/3 depletion and CPT1B-WT or CPT1B-P295 overexpression. (F) Estimated maximal oxygen consumption rate. Diagrams are shown as an average of 4 experiments ± SEM. Statistically significant difference is indicated by asterisks: *p < 0.05, ***p < 0.001, ****p < 0.0001.

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  Figure 7.. Working model: PHD2/3, CPT1B, and VDAC1 may work together in cardiomyocytes under normoxic condition PHD2/3 catalyze prolyl-4-hydroxylation on CPT1B P295 residue, which promotes CPT1B/VDAC1 complex formation and facilitates LFCA mitochondrial uptake. Hypoxia inhibits PHD2/3 enzymatic activity, resulting in the disruption of CPT1B/VDAC1 complex and inhibition of LCFA mitochondrial uptake and metabolism.