SGLT2 inhibitor upregulates myocardial genes for oxidative phosphorylation and fatty acid metabolism in Gαq-mice

Figures

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  Fig. 1. ERTU prevents left ventricular (LV) dilation, wall thinning and systolic failure in Gαq mice. Beginning at 4 weeks of age (0 weeks after treatment in all panels), wild type (WT) and cardiac-specific Gαq-overexpressing mice were randomized to chow formulated with ertugliflozin (ERTU; 0.5 mg/g of chow) or control diet without ERTU (CD). Over the subsequent 16 weeks, Gαq mice exhibited decreased heart rate (Panel A), progressive LV dilation (Panels B and C) with LV wall thinning (Panel D) and a decrease in LV fractional shortening (Panel E) as assessed by echocardiography. Treatment with ERTU prevented LV dilation, wall thinning and progressive systolic dysfunction. Data presented as mean ± SEM; n = 10–16 mice/group); * = p < 0.001 Gαq vs. Gαq ERTU. ■ = WT / CD, ● = WT / ERTU, ▼ = Gαq / CD, ▲ = Gαq / ERTU. Individual mouse data at 16 weeks of treatment are shown in Suppl. Fig. 2.

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  Fig. 2. ERTU prevents myocyte apoptosis and cellular remodeling. WT and Gαq mice were sacrificed for histologic analysis after 16 weeks of treatment. Gαq mice exhibited marked myocyte apoptosis, myocyte hypertrophy and interstitial fibrosis, all of which were prevented in ERTU-treated hearts. Panel 2A. Myocyte apoptosis was assessed by TUNEL staining: Apoptotic nuclei (arrows) are shown by green fluorescence in Panels a, e, i, m and q. Nuclei (arrowheads) stained by Hoechst 33258 are shown in blue in Panels b, f, j, n and r. Cardiomyocytes identified by the overlays in Panels d, h, l, p and t allow identification of apoptotic nuclei present in myocytes. Panel 2B. Myocyte hypertrophy was assessed by measurement of cross-sectional areas. Panel 2C. Interstitial fibrosis was assessed by picrosirius red staining. Data are presented as mean ± SEM; n = 3–4 mice/group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 3. ERTU prevents mitochondrial dysfunction and myocardial oxidative stress in Gαq mice. In cardiac mitochondria isolated from LV of Gαq mice after 16 weeks of treatment, ATP production was decreased (Panel A) and H2O2 release was increased (Panel B). Both ATP production and H2O2 release were normalized by ERTU treatment. Consistent with the increased H2O2 release, myocardial oxidative stress, as assessed by immunohistochemical staining for 4-hydroxynonenal (4-HNE), was increased in Gαq mice, and the increase was prevented by ERTU treatment. Shown are representative images of 4-HNE staining (Panel C; scale bar = 25 μm), and the mean scoring for 4-HNE staining (Panel D). Data presented are mean ± SEM; n = 4–6 mice/group for Panels A and B, and 3 mice/group for Panel D.

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  Fig. 4. Effect of ERTU on myocardial high energy phosphates and cardiac contractile reserve. After 16 weeks of treatment, phosphocreatine (PCr) and ATP were measured by 31P NMR simultaneously with contractile function in isolated beating hearts while increasing work demand by raising the pacing rate and the concentration of calcium in the perfusate. Contractile reserve was assessed as the change in rate X pressure product (RPP) with increased work demand. In hearts from Gαq mice (▼, dashed line) RPP failed to increase with increased work demand and was associated with depressed levels of PCr/ATP both with basal and increased work demand. In hearts from Gαq mice, ERTU treatment (▲, solid line) increased RPP and PCr/ATP at all levels of work demand thereby shifting the relationship between RPP and PCr/ATP upward and to the right, indicative of improved energy balance at higher levels of contractile performance. In wild type mice (■, solid line), ERTU (●, dashed line) had little effect. *** = 0.001; ** = 0.01, * = 0.05; data presented as mean ± SEM; n = 5–7 mice/group). See also Table 1.

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  Fig. 5. Gαq over-expression suppresses myocardial gene sets associated with mitochondrial function at 4 and 20 weeks of age. Myocardial gene expression was assessed by mRNA sequencing with gene set enrichment analysis (GSEA). Of the most suppressed Gene Ontology (GO) gene sets, most were related to mitochondrial structure and/or function (see also Suppl. File 1 for full gene set listing and statistics). Further analysis by Hallmark GSEA (shown here) indicated that the gene sets for oxidative phosphorylation (OXPHOS; Panel A; NES -3.26, p < 0.001, q < 0.001) and fatty acid oxidation (FAO; Panel B; NES -2.83, p < 0.001, q < 0.001) were markedly suppressed at both 4 and 20 weeks of age in Gαq mice. Variance-stabilizing-transformed gene expression values were z-score-normalized (to a mean of zero and standard deviation of one) within each row with blue, white and orange indicating z-scores of ≤−2, 0, and ≥+2, respectively (n = 4 mice/group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 6. ERTU treatment up-regulates myocardial genes for oxidative phosphorylation and fatty acid metabolism. After 16 weeks of treatment with ERTU, the most enriched GO gene sets were for mitochondrial structure and function (see also Suppl. File 2 for full gene set listing and statistics). Hallmark GSEA showed that ERTU treatment markedly enriched gene sets for oxidative phosphorylation (OXPHOS; Panel A; NES 2.97, p < 0.0001, q < 0.0001) and fatty acid metabolism (FAO; Panel B; NES 2.61, p < 0.0001, q < 0.0001). Variance-stabilizing-transformed gene expression values were z-score-normalized (to a mean of zero and standard deviation of one) within each row with blue, white and orange indicating z-scores of ≤−2, 0, and ≥+2, respectively (n = 4–5 mice/group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 7. Kegg pathway depiction of mitochondrial genes for oxidative phosphorylation arranged by mitochondrial complex. Panel A depicts the change in gene expression for Gαq vs. wild type mice at 20 weeks of age. Panel B depicts the changes in gene expression for mice at 20 weeks of age treated with ERTU vs. control diet for 16 weeks. Blue, white and orange indicate log2 fold change values of ≤−1, 0 and ≥+1, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 8. A. Kegg pathway depicting mitochondrial genes for fatty acid degradation for Gαq vs. wild type mice at 20 weeks of age. Blue, white and orange indicate log2 fold change values of ≤−1, 0 and ≥+1, respectively. B. Kegg pathway depicting mitochondrial genes for fatty acid degradation for Gαq vs. wild type mice at 20 weeks of age treated with ERTU vs. control diet for 16 weeks. Blue, white and orange indicate log2 fold change values of ≤−1, 0 and ≥+1, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)