Metabolic changes during cardiac regeneration in the axolotl

Figures

• 

  FIGURE 1. Heart regeneration. (A) Heart rate measured by doppler echocardiography as an average of three cardiac cycles. n = 4. (B) Ventricular fractional area change (VFAC) measured by B‐mode echocardiography as an average of three cardiac cycles. n = 4. VFAC. (C) Cardiac output measured by B‐mode echocardiography as an average of three cardiac cycles. n = 4. (D) Non‐contracting fraction based on echocardiography. The non‐contracting fraction was calculated from area‐tracings as an average across three cardiac cycles. Representative images of echocardiography analysis are shown in Supplementary Figure 1. (E) Infarction fraction based on quantitative stereological analysis of Masson's Trichrome stained cryosections. 10 μm sections obtained at a 150 μm distance were collected throughout the entire ventricle of each heart. (F) Masson's Trichrome stain of cryosections at 4, 14, 30, 60, and 120 dpi. Yellow frame encompasses infarction area. (A–C) was measured in the same four animals that were used for analysis at 120 dpi in (D) and (E). Statistical analysis performed by repeated measures one‐way ANOVA with Dunnet's post hoc test comparing all other time points to baseline in (A–C). *p < 0.05,…

• 

  FIGURE 2. Systemic metabolic changes after cryoinjury. (A) Oxygen consumed during undisturbed closed respirometry. Oxygen was measured using Unisense OXY meter. (B) Plasma lactate assayed with colorimetric microplate assay. All error bars represent standard deviation. Red dotted lines indicate baseline levels. Asterisk represents statistically significant difference from baseline values in one‐way ANOVA with repeated measures followed by Šidák post hoc tests. *p < 0.05, **p < 0.005, ***p < 0.0001.

• 

  FIGURE 3. Heart regeneration under hyperoxia. (A) Two types of general hyperoxia treatments were used: constant saturation of housing water with pure O2 (constant normobaric hyperoxia) or intermittent hyperbaric O2 treatment (HBOT) where animals were exposed to a daily regimen of 2 h 3 atm with complete O2 saturation of housing water. (B–C) Intracardiac plasma pO2 during normoxia, hyperoxia, and HBOT exposure in sham and cryo‐injured animals at 4 dpi. (D) Intracardiac hemoglobin O2‐saturation during normoxia and hyperoxia in sham and cryoinjured animals at 4 dpi. Values for both the uninjured and injured zone of the heart are provided for cryo‐injured animals. (E,F) Non‐contracting fraction measured by echocardiography in the course of 70 days post cryo‐injury in two rounds of experiments including axolotls exposed to normoxia and hyperoxia or HBOT, respectively. (G,H) Infarction fraction measured by quantitative histology at the termination of hyperoxia and HBOT experiments at 73 dpi where there was no significant difference in the remaining fraction of infracted myocardium. Error bars represent standard deviation. For figure elements (B–F), asterisk indicates significant difference in…

• 

  FIGURE 4. Autoradiography. (A) Representative images of 18F‐FDG autoradiography imaged cryosections at baseline, 4, 14, 30, 60, and 120 dpi. (B) Representative images of 14C‐acetate autoradiography imaged cryosections at baseline, 4, 14, 30, 60, and 120 dpi. (C) Average 18F‐FDG signal in uninjured myocardium. (D) Maximum 18F‐FDG signal in uninjured myocardium. (E) Average 18F‐FDG signal across entire ventricle including infarction area. (F) Average 14C‐acetate signal in uninjured myocardium. (G) Maximum 14C‐acetate signal in uninjured myocardium. (H) Average 14C‐acetate signal across the entire ventricle including infarction area. (I) Average signal of 14C‐acetate in different zones (zone 1 closest to infarction, zone 4 furthest away. All error bars represent standard deviation. Asterisks indicate statistical significance in one way ANOVA with Šidák post hoc test. *p < 0.05, **p < 0.005, and ***p < 0.0001. 4* indicates significant difference from zone 4. n = 3 in baseline group and n = 4 in all other groups. Corresponding brightfield images of the sections are shown in Supplementary Figure 3.

• 

  FIGURE 5. Metabolomics PCA scores plot. (A) All detected features in positive and negative ionization mode following pre‐processing and normalization. (B) All annotated and identified metabolites following normalization. QC = quality controls. Quality controls are the same pooled sample injected for each fifth sample. QCs are in the center of the PCA scores plot, and with low variation, indicating that the analysis was not affected by instrumental errors. The circle indicates Hotelling's T 2. n = 4 in all groups.

• 

  FIGURE 6. Nucleotide metabolism. (A) Pathway map of purine metabolism. Metabolites in purple were detected by metabolomics in cardiac tissue samples. (B–G) Metabolic profiles of d‐ribose‐5‐phosphate, inosine monophosphate (IMP), xanthosine monophosphate (XMP), adenine, deoxyadenosine monophosphate (dAMP), and uric acid, respectively. (H) Pathway map of pyrimidine and beta‐alanine metabolism. Metabolites in purple were detected by metabolomics in cardiac tissue. (I–P) Metabolic profiles of glutamine, cytidine monophosphate (CMP), uridine, cytidine, spermidine, and carnosine, respectively. All error bars represent standard deviations. Asterisk indicates statistical significance in an unpaired t‐test comparing baseline and 4 dpi samples. *p < 0.05, **p < 0.005, and ***p < 0.0001. n = 4 in all cases.

• 

  FIGURE 7. Glutathione metabolism. (A) Pathway map of glutathione metabolism and connection to other pathways. (B–D) Metabolomics profile of reduced glutathione, oxidized glutathione, and ophthalmic acid at baseline at 4 dpi. Asterisk indicates p‐value <0.05 in unpaired t‐test comparing baseline at 4 dpi samples. All error bars represent standard deviations. Asterisk indicates statistical significance in an unpaired t‐test comparing baseline and 4 dpi samples. *p < 0.05 and **p < 0.005. n = 4 in all cases.

• 

  FIGURE 8. mTOR activity in the heart after cryo‐injury. (A–E) Immunofluorescence labeling of Ps6 (Serine‐6‐phosphorylated ribosomal protein, a known marker of mTOR activity). (A) 7 μm cryosections stained for Ps6 (FITC, Green), α‐actinin (Cy5, Red), and DAPI nuclear stain (Blue). Areas that are not α‐actinin‐positive represent the remaining injury. (B) Same images as above without red channel to show Ps6 activity in the entire ventricle. (C) Higher magnification images corresponding to yellow brackets in (A). (D) Semi‐quantitative analysis of Ps6 positivity. The fractional area positive for Ps6 above a standardized threshold and laser intensity measured across three sections from each animal in QuPath. All error bars represent standard deviation. Asterisk indicates p‐value <0.05 in one‐way ANOVA with Šidák post hoc test comparing each group to baseline. (E) Percentage of animals in each group where in all three sections Ps6 activity was found to be localized in and around the infarction area and/ or the epicardium. Brightfield images of neighboring sections in Supplementary Figure 5.

• 

  FIGURE 9. Enzyme histochemistry stains. Deposition of blue product from nitroblue tetrazolium chloride reaction indicates enzymatic activity in the presence of substrate (here either LDH, SDH, or G6PD). The red lines indicate the injured area recognized based on morphology of the hematoxylin counter stain (counter stain appears faint in the images due to the much higher saturation of enzyme specific stain). (A) LDH activity enzyme stain, (B) SDH activity enzyme stain, and (C) G6PD activity enzyme stain. (D–F) Each dot represents a replicate (heart) in which three sections all show staining pattern where signal is most prominently localized to the designated area (infarction, myocardium, or epicardium) for LDH, SDH, and G6PD, respectively.