Glyoxylase-1 combats dicarbonyl stress and right ventricular dysfunction in rodent pulmonary arterial hypertension

Figures

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  FIGURE 1. Bioinformatics analysis of previously identified glycated proteins from murine myocardium suggested a link between protein glycation and mitochondrial metabolic activity. (A) STRING analysis of the previously identified glycated proteins from murine myocardium (17). K-means clustering delineated three clusters of proteins. (B) KEGG pathways, Reactome pathways, and WikiPathways identified in Cluster 1. (C) KEGG pathways and Reactome pathways identified in Cluster 2. No WikiPathways were identified from Cluster 2 proteins. (D) KEGG pathways, Reactome pathways, and WikiPathways identified in Cluster 3. FDR: False discovery rate.

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  FIGURE 2. Exogenous methylglyoxal treatment impaired mitochondrial function when H9c2 cardiomyoblasts were exposed to palmitate. (A) Western blot of total protein glycation (top) and Coomassie brilliant blue (CBB) SDS-PAGE of control and methylglyoxal treated cells subjected to Mito Stress test. (B) Representative Seahorse tracings of cells treated with vehicle or methylglyoxal in the Mito Stress test protocol. Media contained 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine. Quantification of basal oxygen consumption rate (C), maximal oxygen consumption rate (D), and ATP production (E). (F) Western blot of total protein glycation (top) and CBB SDS-PAGE (bottom) of control and methylglyoxal treated cells undergoing fatty acid oxidation assay. Media included basal media supplemented with 0.5 mM carnitine and 169 μM palmitate. (G) Representative Seahorse tracings of cells treated with vehicle or methylglyoxal in the fatty acid oxidation protocol. Quantification of basal oxygen consumption rate (H), maximal oxygen consumption rate (I), and ATP production (J). P-values determined by unpaired t-test.

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  FIGURE 3. AAV9-mediated GLO1 overexpression reduced protein glycation in the RV of MCT rats. (A) Representative Western blot images of protein glycation, GLO1, GLO2, and DJ-1 in RV tissue extracts. Post transfer SDS-PAGE gels were stained with CBB. (B) Quantification of Western blot protein expression of protein glycation, GLO1, GLO2, and DJ-1 in the RV. p-values determined by unpaired t-test because control animals were used as the normalizing standard of 1. (C) Three-dimensional reconstructions of RV sections stained with GLO1 antibody. Yellow areas highlight intramyocardial GLO1 staining. AAV-GFP rats had small areas of intense GLO1 immunoreactivity outside of cardiomyocytes (white arrows) and reduced cardiomyocyte GLO1 staining. AAV-Glo1 animals also had punctate staining of high GLO1 reactivity (white arrows), but intramyocardial staining was also increased (yellow arrow). (D) Representative three-dimensional projections of confocal micrographs of RV cardiomyocytes showing GLO1 localization. AAV-GFP RV cardiomyocytes had reduced intracellular GLO1 levels, but AAV-Glo1 treatment partially restored cardiomyocyte GLO1 levels. p-values determined by Kruskal-Wallis test with Dunn’s…

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  FIGURE 4. AAV-Glo1 Treatment increased mitochondrial density, the abundance of fatty acid handling and metabolic proteins, and prevented lipid accumulation in the RV of MCT rats. (A) Representative three-dimensional confocal micrographs of RV cardiomyocytes stained with the mitochondrial marker TOM20 (green) and wheat germ agglutinin (red) to outline cells. Arrows highlight areas of reduced mitochondrial density. (B) Quantification of mitochondrial density in RV cardiomyocytes. p-values determined by one-way ANOVA with Tukey’s multiple comparisons test. (C) Representative Western blots and quantification (D) of protein abundance of fatty acid handling and metabolizing proteins FABP4, HADHA, and HADHB. p-values determined by unpaired t-test with control values serving as a reference of 1. n = 3 control for HADHB but n = 4 for controls for FABP4 and HADHA. (E) Representative images of RV sections subjected to Oil Red O staining. Arrows highlight intramyocardial fatty acid detection. Scale bar 40 μm. (F) Quantification of Oil Red O staining in RV sections. p-values determined by Kruskal-Wallis test with Dunn’s multiple comparison test.

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  FIGURE 5. AAV-Glo1 had a modest effect on RV hypertrophy but reduced RV fibrosis. (A) Representative images of hematoxylin and eosin-stained RV sections. Scale bar 20 μm. (B) Quantification of RV cardiomyocyte cross-sectional area. Glo1 overexpression reduced RV cardiomyocyte area. (C) Quantification of RV to LV + septum mass. p-values determined by one-way ANOVA with Tukey’s multiple comparisons test (D) Representative images of Masson-Trichrome stained RV sections to delineate fibrosis (marked by arrows). Scale bar 40 μm. (E) Quantification of RV fibrosis area. AAV-Glo1 treatment reduced RV fibrosis. p-values determined by Kruskal-Wallis test with Dunn’s multiple comparison test in (B,E).

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  FIGURE 6. GLO1 overexpression enhanced RV systolic and diastolic function. (A) AAV-Glo1 treatment significantly improved Ees/Ea. AAV-Glo1 non-significantly improved RV ejection fraction (RVEF) (B) and significantly reduced RVEDP (C) compared to AAV-GFP. (D) RV tau was non-significantly reduced by AAV-Glo1. (E) TAPSE was normalized by AAV-Glo1 treatment and (F) RV free wall thickening was augmented, although not statistically different from AAV-GFP animals. p-values determined by using one-way ANOVA with Tukey’s multiple comparisons test in (A–C,E,F) and Kruskal-Wallis test with Dunn’s multiple comparison test in (D).

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  FIGURE 7. GLO1 overexpression did not alter PAH severity. Pulmonary artery acceleration time (A), RV systolic pressure (RVSP) (B), and effective arterial elastance (Ea) (C) were all equivalent in AAV-GFP and AAV-Glo1 rats. (D) Representative images of hematoxylin and eosin stained small pulmonary arterioles. Scale bar 20 μm. (E) Quantification of small arteriole remodeling in lung sections. p-values determined by Kruskal-Wallis test with Dunn’s multiple comparison test in (A,E), Brown-Forsythe and Welch ANOVA with Dunnett multiple comparisons test in (B), and one-way ANOVA with Tukey’s multiple comparisons test in (C).