Direct Cardiac Actions of Sodium-Glucose Cotransporter 2 Inhibition Improve Mitochondrial Function and Attenuate Oxidative Stress in Pressure Overload-Induced Heart Failure

Figures

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  Figure 1. EMPA treatment attenuated remodeling of the LV, improved cardiac function and increased mitochondrial respiration. (A) Left, Masson staining of cross sections of hearts and Right (the collagen fibers were stained blue), quantitative analysis of left ventricle (LV) mass and heart collagen volume. (B) Transthoracic echocardiography showed the ejection fraction (EF), fraction shortening (FS), isovolumetric relaxation time (IVRT) and myocardial performance index (MPI) of hearts from different groups. (C) Cardiac function assessed by direct intraventricular pressure measurements. (D) Mitochondrial respiration and peroxide production in hearts from different groups. Original magnification × 100, Results are expressed as mean ± SEM, n = 5–7, *p < 0.05 vs. corresponding sham group, †p < 0.05 vs. corresponding TAC vehicle group. One-way ANOVA and Tukey post hoc test. EMPA, empagliflozin; SEM, standard error of the mean; TAC, transverse aortic constriction; LV, left ventricle; HR, heart rate; EDPVR, end diastolic pressure volume relationship; LVESP, left ventricular end-systolic pressure; OCR, oxygen consumption rate.

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  Figure 2. EMPA treatment attenuated cardiomyocyte overproduction of reactive oxygen species (ROS) induced by TAC. (A) Left, Mitosox staining of cardiomyocytes in sham, sham + EMPA, TAC and TAC + EMPA groups and Right, quantitative results. (B) Representative blots of NRF-2 and HO-1, and quantitative results. (C) The relative mRNA levels of genes related to endogenous antioxidants. Results are expressed as mean ± SEM, n = 5–7, *p < 0.05 vs. corresponding sham group, †p < 0.05 vs. corresponding TAC vehicle group. One-way ANOVA and Tukey post hoc test. EMPA, empagliflozin; SEM, standard error of the mean; TAC, transverse aortic constriction; NRF-2, the nuclear factor erythroid 2–related factor 2; HO-1, heme oxygenase-1; GCLM, glutamate-cysteine ligase modifier subunit; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  Figure 3. EMPA treatment increased mitochondrial biogenesis. (A) Left, Transmission electron microscopy showing the morphology of mitochondria in sham, sham + EMPA, TAC and TAC + EMPA groups and Right, quantitative analysis of mitochondrial size and counts. Results are expressed as mean ± SEM, n = 3–5, *p < 0.05 vs. corresponding sham, †p < 0.05 vs. corresponding TAC. (B) Left, Representative blots of mitochondrial biogenesis-related proteins and Right, quantitative results. (C) Relative mRNA levels of PGC1α, NRF1, TFAM and COX1. Results are expressed as mean ± SEM, n = 5–7, *p < 0.05 vs. corresponding sham group, †p < 0.05 vs. corresponding TAC vehicle group. One-way ANOVA and Tukey post hoc test. EMPA, empagliflozin; SEM, standard error of the mean; TAC, transverse aortic constriction; PGC1-α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; NRF-1, nuclear respiratory factor 1; TFAM, mitochondrial transcription factor A; COX1, cyclooxygenase1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  Figure 4. EMPA treatment attenuated apoptosis in TAC-induced HF. (A) TUNEL staining of heart sections in sham, sham + EMPA, TAC and TAC + EMPA groups. (B) Immunohistochemistry results of cleaved caspase-3 in different groups. (C) Left, representative blots of proteins related to cellular survival and apoptotic pathways and Right, quantitative results. (D,E) Increased Cyt C released from mitochondria into cytoplasm after TAC is attenuated by EMPA treatment. Results are expressed as mean ± SEM, n = 5–7, *p < 0.05 vs. corresponding sham group, †p < 0.05 vs. corresponding TAC vehicle group. One-way ANOVA and Tukey post hoc test. EMPA, empagliflozin; SEM, standard error of the mean; TAC, transverse aortic constriction; ERK, extracellular signal-regulated kinase; Cyt C, cytochrome C; COX IV, cytochrome c oxidase, complex IV in the mitochondrial respiratory chain; DAPI, 4',6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  Figure 5. EMPA treatment enhanced autophagy in failing hearts. (A) Transmission electron microscopy (TEM) showed autophagosomes in the hearts. Red arrows point to autophagosomes. (B) EMPA activated autophagy pathway by inhibiting mTOR pathway. (C) RT-PCR results showed that the mRNA expression of Beclin1, Atg7 and LC3 were increased by EMPA treatment after TAC. Results are expressed as mean ± SEM, n = 5–7, *p < 0.05 vs. corresponding sham group, †p < 0.05 vs. corresponding TAC vehicle group. One-way ANOVA and Tukey post hoc test. EMPA, empagliflozin; SEM, standard error of the mean; TAC, transverse aortic constriction; AMPK, AMP-activated protein kinase; mTOR, mammalian target of rapamycin; Ulk, Unc-51 like autophagy activating kinase; Atg7, autophagy related 7; LC3, light chain 3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  Figure 6. Acute treatment of hearts from TAC groups with EMPA improved mitochondrial function. (A) Baseline and maximal oxygen consumption rate in heart tissues treated with vehicle or EMPA. (B) Baseline and maximal peroxide production in heart tissues treated with vehicle or EMPA. Results are expressed as mean ± SEM, n = 7, *p < 0.05 vs. vehicle group. Student t test. EMPA, empagliflozin; SEM, standard error of the mean; TAC, transverse aortic constriction; OCR, oxygen consumption rate.

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  Figure 7. Empagliflozin improves mitochondrial function, restores redox homeostasis and reduces apoptosis in heart failure. EMPA, empagilflozin; NRF-1, nuclear respiratory factor 1; TFAM, mitochondrial transcription factor A; mtDNA, mitochondrial DNA; ROS, reactive oxygen species; AMPK, adenosine monophosphate-activated protein kinase; mTOR, mammalian target of rapamycin.