Metabolic reprogramming by immune-responsive gene 1 up-regulation improves donor heart preservation and function

Abstract

Preservation quality of donor hearts is a key determinant of transplant success. Preservation duration beyond 4 hours is associated with primary graft dysfunction (PGD). Given transport time constraints, geographical limitations exist for donor-recipient matching, leading to donor heart underutilization. Here, we showed that metabolic reprogramming through up-regulation of the enzyme immune response gene 1 (IRG1) and its product itaconate improved heart function after prolonged preservation. Irg1 transcript induction was achieved by adding the histone deacetylase (HDAC) inhibitor valproic acid (VPA) to a histidine-tryptophan-ketoglutarate solution used for donor heart preservation. VPA increased acetylated H3K27 occupancy at the IRG1 enhancer and IRG1 transcript expression in human donor hearts. IRG1 converts aconitate to itaconate, which has both anti-inflammatory and antioxidant properties. Accordingly, our studies showed that Irg1 transcript up-regulation by VPA treatment increased nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) in mice, which was accompanied by increased antioxidant protein expression [hemeoxygenase 1 (HO1) and superoxide dismutase 1 (SOD1)]. Deletion of Irg1 in mice (Irg1^-/-) negated the antioxidant and cardioprotective effects of VPA. Consistent with itaconate's ability to inhibit succinate dehydrogenase, VPA treatment of human hearts increased itaconate availability and reduced succinate accumulation during preservation. VPA similarly increased IRG1 expression in pig donor hearts and improved its function in an ex vivo cardiac perfusion system both at the clinical 4-hour preservation threshold and at 10 hours. These results suggest that augmentation of cardioprotective immune-metabolomic pathways may be a promising therapeutic strategy for improving donor heart function in transplantation.

Fig. 1.. Treatment with VPA during cardiac preservation maintains histone acetylation, improved cardiac function, and reduced inflammation and cell death.

(A) Representative Western blot for total acetylated H3K9 in human donor hearts after cold (4°C) preservation for 0 and 8 hours with or without VPA treatment. Quantitative H3K9ac values are expressed as a proportion of total H3 histone expression and then normalized to expression values at 0 hours of preservation with HTK only (n = 4 per group). (B and C) Standard cold (4°C) HTK solution with or without VPA (10 mM) was used to preserve murine donor hearts for 0, 4, 8, or 16 hours. After subsequent ex vivo perfusion with Krebs buffer (n = 5 to 9 per group), cardiac contractility (max dP/dt) (B) and relaxation (min dP/dt) (C) were measured. (D) Schematic for syngeneic heart transplant experiment. Murine donor hearts were isolated and preserved in cold (4°C) HTK solution with or without VPA for 16 hours before sex-matched syngeneic heterotopic cervical heart transplantation. (E and F) Donor heart contractility (max dP/dt) (E) and relaxation (min dP/dt) (F) were measured in vivo 24 hours after transplantation (n = 9 per group). (G) Enzyme-linked immunosorbent assay (ELISA) quantification of serum cardiac troponin T (cTnT), cardiac troponin I (cTnI), IL-6, TNFα, IP-10, and MIG (n = 8 or 9 per group). (H) Representative immunofluorescence staining of CD45⁺ pan leukocyte infiltration (red) in left ventricular tissue of transplanted murine hearts. 4′,6-Diamidino-2-phenylindole (DAPI) was used to visualize nuclei (blue). Scale bars, 50 μm. (I) Quantification of data from (H) (n = 4 per group). (J and K) Quantification (J) of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling–positive cell (TUNEL⁺) images (K) from transplanted murine hearts (green) (n = 4 per group). DAPI was used to visualize nuclei (blue). Scale bars, 50 μm. Data are presented as means ± SD. *P < 0.05 and **P < 0.01, Kruskal-Wallis test followed by Dunn’s multiple comparisons test (A) or Student’s t test (B, C, E to G, I, and J).

Fig. 2.. VPA treatment reduced succinate accumulation in human donor hearts during cold preservation and is associated with increased itaconate availability.

(A to G) Metabolite abundance determined by mass spectrometry in cold-preserved human donor hearts in HTK ± VPA without reperfusion at 0 and 8 hours (n = 4 per group). Succinate (A), itaconate (B), fumarate (C), acetyl-CoA (D), citrate (E), α-ketoglutarate (F), and malate (G) were measured. Abundance was calculated on the basis of one-point calibration using an internal standard control. Data are presented as means ± SD. *P < 0.05 and **P < 0.01 by Kruskal-Wallis test followed by Dunn’s multiple comparisons test.

Fig. 3.. Increased IRG1 expression contributed to improved cardiac preservation quality and cardiomyocyte survival after VPA treatment.

(A) ChIP-qPCR analysis of H3K27 binding to IRG1 enhancer in human donor heart tissue after 0 and 8 hours of cold preservation in HTK ± VPA. The occupancy of H3K27ac was normalized to the total genomic input DNA and compared with immunoglobulin G (IgG) binding (n = 4 per group). (B) IRG1 mRNA transcript expression in human hearts after preservation was determined by normalizing to hypoxanthineguanine phosphoribosyltransferase (HPRT) (n = 4 per group). (C and D) Analysis of cardiac contractility (max dP/dt) (C) and relaxation (min dP/dt) (D) during ex vivo perfusion of murine WT or Irg1^−/− hearts preserved for 16 hours with or without VPA (n = 7 or 8 per group). (E and F) Left ventricular contractility (max dP/dt) (E) and relaxation (min dP/dt) (F) of murine donor hearts preserved with HTK ± VPA were determined in vivo after transplantation into WT mice (n = 9 per group). (G) Representative immunofluorescence staining of fresh-frozen sections from mouse LV for sarcomeric α-actinin⁺ cardiomyocytes (red) and TUNEL⁺ cells (green). DAPI (blue) was used to visualize nuclei. (H) Quantification of data from (G) (n = 4 per group). Data are presented as means ± SD. *P < 0.05 and **P < 0.01 by Kruskal-Wallis test followed by Dunn’s multiple comparisons test (A to D and H) and Student’s t test (E and F).

Fig. 4.. Irg1 mediated a reduction in oxidative stress and an increase in antioxidant protein expression and activation after VPA administration in mice.

(A and B) GSH/GSSG ratios from murine heart tissue preserved for 16 hours using HTK solution with or without VPA without reperfusion (A) or followed by sex-matched syngeneic heart transplantation (B) (n = 4 per group). GSH/GSSG ratio was normalized to amounts in HTK-only preserved hearts. (C) Representative Western blots from LVs of WT or Irg^−/− murine hearts preserved for 16 hours using HTK solution with or without VPA followed by ex vivo perfusion for NRF2, HO1, SOD1, and SOD2. α-Tubulin was used for normalization. (D) Quantification of data from (C) (n = 5 per group). (E) Representative immunofluorescence staining for NRF2 (red) and DAPI (blue) in the LVs of ex vivo perfused WT or Irg^−/− murine donor hearts preserved for 16 hours with or without VPA. Merged images shown with nuclear translocation of NRF2 seen in purple (indicated by white arrows). (F) Quantification of relative NRF2 fluorescence intensity in nuclear regions. Data are presented as means ± SD. Representative images of n = 4 per group. *P < 0.05 and **P < 0.01 by Student’s t test (A and B) or Kruskal-Wallis test followed by Dunn’s multiple comparisons test (D and F).

Fig. 5.. Treatment with exogenous 4-OI improved cardiac function and increased antioxidant activities in mice.

(A) Western blots for NRF2, HO1, SOD1, and α-tubulin (loading control) in murine HL-1 cardiomyocytes with HTK ± 4-OI under normoxic (20% O₂) or hypoxic (1% O₂) conditions for 2 hours. (B) Quantification of data from (A) (n = 6 per group) normalized to α-tubulin and compared with control normoxia cells (Ctr). (C and D) Donor mice were treated with 4-OI or vehicle, and the donor hearts were then HTK-preserved for 16 hours before evaluation of cardiac contractility (max dP/dt) (C) and relaxation (D) (min dP/dt; n = 8 per group). (E) Immunofluorescence staining of murine donor hearts from (C) and (D) for NRF2 (red) and DAPI (blue). NRF2 nuclear translocation shown as purple in merged images (indicated by white arrows). (F) Oxidative DNA damage assessed by immunofluorescence staining for 8-OHdG (red) and DAPI (blue) in the LVs of donor hearts from mice ±4-OI. (G) Quantification of (F) (n = 4 per group). Data are presented as means ± SD. *P < 0.05 and **P < 0.01 by Kruskal-Wallis test followed by Dunn’s multiple comparisons test (B) and Student’s t test (C, D, and G).

Fig. 6.. VPA treatment improved ex vivo pig heart function and reduced cardiac injury.

(A to C) Ex vivo analysis of cardiac function in pig hearts preserved with HTK ± VPA treatment for 4 or 10 hours. Hemodynamic measurements recorded in working mode after 1 hour of perfusion include contractility (max dP/dt) (A), relaxation (min dP/dt) (B), and cardiac output (C) normalized to heart weight in grams (milliliters per minute per gram). (D to H) In hearts preserved for 4 and 10 hours, multiplex ELISA was used to measure arterial perfusate concentrations of TnI (D), TNFα (E), IL-1β (F), IL-2 (G), and IL-6 (H) after 3 hours of perfusion (n = 8 per group). Data are presented as means ± SD. *P < 0.05 and **P < 0.01 by one-way ANOVA with Fisher’s least significant difference multiple comparisons test (A to H).

Fig. 7.. VPA administration reduced cell death, oxidative stress, and inflammation in ex vivo perfused pig hearts.

After 4 or 10 hours of preservation with or without VPA, porcine hearts were perfused ex vivo for 3 hours before analysis. (A and B) Representative images of 8-OHdG fluorescence (A) (red) to assess for oxidative DNA damage with quantification of staining intensity in (B) (n = 4 per group). Immunofluorescence staining for (C) NRF2 (red) to determine NRF2 nuclear translocation (purple and white arrows). Representative images of n = 4 per group. DAPI (blue) was used to assess nuclei in (A) to (C). (D to F) Western blotting was performed for NRF2 (D), cleaved caspase 3 (E), and BCL2 (F). Representative blots shown of n = 4 per group with quantification below by normalization to α-tubulin and standardization to HTK for 4 hours. (G) Pig heart mRNA transcript expression of IRG1 and inflammatory transcripts IL1B, IL6, and TNFA (n = 4 per group). Data are presented as means ± SD. *P < 0.05 and **P < 0.01 by Kruskal-Wallis test followed by Dunn’s multiple comparisons test (B and D to G).