Empagliflozin improves post-infarction cardiac remodeling through GTP enzyme cyclohydrolase 1 and irrespective of diabetes status

Figures

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  Figure 1. Experimental design. (a) The study consort flow diagram. (b) Experimental design representative scheme; the time interval where rats were treated with EMPA is shown in red. EMPA: empagliflozin; MI: myocardial infarction; STZ: streptozotocin.

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  Figure 2. Empagliflozin therapy improves cardiac remodeling. (a) Representative images of whole heart from infarcted hearts harvested 4 weeks post-surgery. (b) Heart weight-to-tibia length ratio. (c,d) Echocardiographic analysis of FS and EF. (e,f) Left; Representative transversal histology sections from Masson trichrome-stained myocardium of the indicated groups taken for infarct size measurements (tissue fixation 4 weeks after MI). Collagen-rich areas (scar tissue) are colored in blue and healthy myocardium in red. Scale bar: 0.5 cm. (Right). Quantification of the infarct size. *p < 0.05, ***p < 0.001. In the lower part of each bar, number of animals analyzed per group.

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  Figure 3. Empagliflozin therapy prevents fibrosis and hypertrophy. (a,b) Quantitative real-time PCR analysis of Col1a1 and Col3a1. (c) Representative LV sections stained with Masson; Scale bars: 25 μm. (d) Fibrosis-associated collagens. (e,f) Quantitative real-time PCR analysis of ratio Myh7/Myh6 and Nppa. ***p < 0.001.

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  Figure 4. Empagliflozin modulates mRNA and protein levels of cGCH1. (a) Cardiac mRNA expression of cGCH1. (b) Representative western blot analysis of cGCH1 in border zone tissue and quantitative analysis. (c,d) Representative immunohistochemical cGCH1 staining in border zone; Scale bar: 50 μm. (e) Cardiac GCH1 activity. (f) Tetrahydrobiopterin levels. *p < 0.05, ***p < 0.001.

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  Figure 5. Empagliflozin modulates NOS activity. (a) Representative western blot bands showing the activation of nNOS. (b) Representative western blot bands showing the activation of eNOS. (c) Representative western blot bands showing the modulation of iNOS. (d) NO levels. (e) O2.- levels. (f) Nitrotyrosine levels. ***p < 0.001.

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  Figure 6. Empagliflozin prevents cardiac hypertrophy. (a) BH4 levels. (b) Quantitative analysis of in vitro cell size measurements of adult cardiomyocytes. (c) Leucine uptake in cardiomyocytes. All quantifications derive from six independent experiments/group. The results are displayed as scatter plots and summarized with boxplots. **p < 0.01, ***p < 0.001.

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  Figure 7. Empagliflozin modulates NOS activity and cardiac GCH1. (a,b) A representative western blot analysis of nNOS and eNOS activation. (c) NO levels. (d) O2.- levels. (e) Nitrotyrosine levels. (f) Confocal microscopy images following immunofluorescence analysis performed with cardiomyocytes expressing tubulin (green). Scale bar in image 4, also applicable to 1, 2 and 3, was 40 μm whereas in image 5 and 6, was 10 μm. (g) Model of Empagliflozin action in the myocardium. All quantifications derive from six independent experiments/group. The results are displayed as scatter plots and summarized with boxplots. **p < 0.01, ***p < 0.001.