Kynurenic acid, a key L-tryptophan-derived metabolite, protects the heart from an ischemic damage

Figures

• 

  Fig 1. (A) Creatine and urea levels provided by the metabolomics profile of the plasma (raw data). (B) Venn diagram yielding 26 differentially-expressed cardiac and plasma metabolites at both time points: 1 day and 1 week relative to sham.

• 

  Fig 2. AKI significantly affects the expression of various metabolites related to TRP catabolism. (A-D) Box plot graphs showing the change of relative intensity in AKI-1d (n = 5) and AKI-1w (n = 4) versus the mean intensity value of the sham group (n = 5) per each metabolite. Significance was calculated by one-way ANOVA followed by Tukey’s post-hoc test. X- Denotes mean values, horizontal lines stands for the median values. (E) Schematic illustration of the TRP catabolite pathway demonstrating increased (red), decreased (blue) or unchanged (white) expression in AKI heart (H) and plasma (P) sections at 24 hours versus sham.

• 

  Fig 3. KYNA salvages H9C2 cell viability upon exposure to ischemic conditions. (A) Dose response for H9C2 cell viability grown in the presence of KYNA followed by exposure to normoxic or anoxic conditions, as determined by flow cytometry. Each box represents values derived from three independent experiments. X- Denotes mean values, horizontal lines stands for the median values. (B) Representetative flow cytometry captures showing H9C2 viability. (C) Reduced cell surface expression of GPR35 following exposure to KYNA, determind by flow cytometry (two independent experiments with two technical replicates).

• 

  Fig 4. KYNA rescues H9C2 cells from anoxia-driven cell death and mitochondrial disruption. (A) A PCA analysis demonstrating a clear separation between experimental conditions by the combination of mitochondrial features (24–30 wells per treatment). (B) Representative images of mitochondrial content (MitoTracker Green FM) and function (MitoTracker Deep Red FM) as well as nuclei (Hoechst) and cytoplasm (Calcein-Red-Orange) staining. The merged column represents the merging of Hoechst and the two MitoTracker dyes. (C) Box plot graphs demonstrating median and sample scattering of the values (24–30 wells per treatment) affiliated cell count, mitochondrial fraction and mitochondrial activity after Z-score-based normalization.

• 

  Fig 5. KYNA reduces the anoxic-mediated oxidative damage to the mitochondria of H9C2 cells. (A) Representative images of cytoplasm (Calcein-Green), superoxide levels (MitoSOXTM) and nuclei (Hoechst) staining. (B) Box plots demonstrating median and sample scattering by the combination of Calcein Green and MitoSOX staining after Z-score normalization (30 wells per treatment). The statistical analysis (One-way ANOVA) is given in the text.

• 

  Fig 6. KYNA enhances cardiac recovery following AMI. Female BALB/C mice were treated with KYNA (250 mg/Lit) diluted in their drinking water (n = 9) versus regular tap water (n = 10). (A) KYNA treatment resulted in an elevated LV EF (%), SV (μl), and CO (ml/min) measurements relative to vehicle-treated control animals on day 30 relative to day 1. Statistical difference between day 1 and day 30 was determined by paired two-tailed Student’s T-test. X- Denotes mean values, horizontal lines stands for the median values. (B) Concamitantly, KYNA reduced the percentage of collagen deposition in the heart according to Picro Sirius Red staining. i. representative captures of the LV sections. ii. Box plot demonstrating the collagen content out of the total LV section (n = 6/ arm). Significance was determined by two-tailed Student’s T-test.