Dapagliflozin Prevents Heart Failure With Preserved Ejection Fraction by Inhibiting the Sodium/Hydrogen Exchanger

Figure 1. Oral Dapa Prevented HypoMg-Induced HFpEF (A) Cardiac diastolic function was evaluated with E/e’ from echocardiography. Seven to 25 mice were tested per group. (B) Representative mouse echocardiographic images of pulsed-wave Doppler (marked E waves) and tissue Doppler (marked e’ waves) and the mean ± SD of E/e’ for each group. (C) The ratio of S-Glu-cMyBPC/total cMyBPC was significantly decreased by Dapa treatment. (D) Total cMyBPC was significantly increased by Dapa treatment. In (C) and (D), 4 mice were tested per group; protein molecular weight was labeled in kDa. Dapa treatment started after 3 weeks of HypoMg diet in half of the HypoMg group and lasted for 3 weeks. Red bars in figures are mean ± SD. One-way analysis of variance with Tukey's multiple comparisons test was used for statistical analysis, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Dapa = dapagliflozin; E/e’ = the ratio between mitral peak early filling velocity E and longitudinal tissue velocity of the mitral anterior annulus e’; HypoMg = hypomagnesemia; MyBPC3 = cardiac myosin binding protein C 3; S-Glu-cMyBPC = S-glutathionylated cardiac myosin binding protein C.

Figure 2. Dapa Prevented the Low-Mg Diet Induced HypoMg (A) Serum Mg2+ levels. (B) Urine Mg2+ extrusion. Dapa treatment was the same as in Figure 1. Seven to 8 mice were tested for each group. Red bars in figures are mean ± SD. One-way analysis of variance with Tukey's multiple comparisons test was used for statistical analysis. ∗∗P < 0.01, ∗∗∗∗P < 0.001. Abbreviations as in Figure 1.

Figure 3. Dapa Prevented HypoMg-Induced Elevation of TRPM7 Expression, MitoROS Overproduction, and (C) NOX4 Expression in Ventricles (A) TRPM7 expression. (B) mitoROS overproduction. (C) NOX4 expression. The mouse treatments were the same as in Figure 1. Four to 5 mice were tested per group in (A) and (C) and protein molecular weight was labeled in kDa. MitoROS levels of isolated cardiomyocytes were measured with MitoSox Red fluorescence by confocal imaging (Z-stack); 35 to 62 cardiomyocytes from 3 mice were tested per group. Red bars in figures are mean ± SD. One-way analysis of variance with Tukey's multiple comparisons test was used for statistical analysis, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. ΔMFI = the fluorescent intensity difference between a cell and the background in the same image; mitoROS = mitochondrial ROS; NOX4 = NADPH oxidase 4; TRPM7 = transient receptor potential melastatin 7; other abbreviations as in Figure 1.

Figure 4. Dapa Treatment Alleviated HypoMg-Induced Cardiac Inflammation Protein expression of (A) MCP-1, (B) CD68, (C) NLRP3, (D) IL-1β, and (E) IL-18 in the mouse ventricular tissues. (F) Representative Western blot bands with protein molecular weight in kDa. (G) Proinflammatory macrophages labeled with CCR2 but not Timd4 (CCR2+/Timd4−) in the total CD11b+F4/80+-labeled macrophages. (H) Proresolving macrophages labeled with Timd4 but not CCR2 (CCR2−/Timd4+) in the total CD11b+F4/80+-labeled macrophages. The mouse treatments were the same as in Figure 1. Four mice were tested for each group in (A)-(E). Six to 7 mice were tested per group in (G) and (H). Red bars in figures are mean ± SD. One-way analysis of variance with Tukey's multiple comparisons test was used for statistical analysis, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. CD68 = cluster of differentiation 68; CCR2+ = C-C chemokine receptor 2; IL-1β = interleukin-1β; IL-18 = interleukin-18; MCP-1 = monocyte chemoattractant protein-1; NLRP3 = NLR family pyrin domain-containing protein 3; Timd4 = T-cell immunoglobulin and mucin domain-containing 4; other abbreviations as in Figure 1.

Figure 5. Dapa Prevented HypoMg-Induced Changes in Human Cardiomyocytes Human RL-14 cardiomyocytes were cultured in a normal (0.81 mmol/L Mg2+) or low-Mg (0.04 mmol/L Mg2+) media in the absence or presence of Dapa (5 μmol/L) for 48 hours. (A) Dapa treatment prevented HypoMg-induced reduction of intracellular Mg2+ measured with Mag-Fluo-4 AM fluorescence by confocal imaging; 35 to 42 cells from 3 dishes of cells/group were tested. (B) Dapa treatment suppressed HypoMg-induced TRPM7 overexpression in RL-14 cardiomyocytes with Western blot bands (protein molecular weight in kDa) shown on top; 3 dishes of cells/group were tested. (C) Dapa treatment suppressed HypoMg-induced mitoROS overproduction measured with MitoSox Red fluorescence; 72 to 112 cells from 3 dishes of cells/group were tested. Red bars in figures are mean ± SD. One-way analysis of variance with Tukey's multiple comparisons test was used for statistical analysis. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations as in Figures 1, 3, and 4.

Figure 6. Dapa Inhibited NHE1 Activation or HypoMg Induced Changes in Cardiomyocytes Cell treatments were the same as in Figure 5; cariporide (10 μmol/L) or RA (10 μg/mL) was applied to cells for 48 hours. (A and B) Intracellular pH levels of RL-14 cardiomyocytes were measured with pHrodo Green fluorescence by confocal imaging. (A) Representative fluorescent images of pHrodo Green fluorescence. (B) ΔMFI is the fluorescent intensity difference of pHrodo Green between the cells and the background in the same image, higher fluorescence indicates low acidic pH; 62 to 125 cells from 3 dishes/group were tested. (C and D) mitoROS levels of RL-14 cardiomyocytes were measured with MitoSox Red fluorescence. (C) Representative fluorescent images of MitoSox Red. (D) ΔMFI is the fluorescent intensity difference of MitoSox Red between the cells and the background in the same image; 67 to 145 cells from 3 dishes/group were tested. Red bars in figures are mean ± SD. One-way analysis of variance with Tukey's multiple comparisons test was used for statistical analysis. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. RA = rosmarinic acid; other abbreviations as in Figures 1 and 3.

Figure 7. Dapa Inhibited Changes Mediated by NHE1 Overexpression HypoMg and Dapa treatment were the same as in Figure 5; NHE1 overexpression was achieved as described in the methods section. (A) Intracellular pH levels are shown with representative images of pHrodo Green fluorescence (left) and ΔMFI values (right) with the same methods as stated in Figure 5; 45 to 83 cells from 3 dishes/group were tested. (B) Intracellular Mg2+ was measured with Mag-Fluo-4 AM fluorescence; 18 to 35 cells from 3 dishes/group were tested. (C and D) Protein levels of TRPM7 and NHE1 were measured with stand Western blot and representative Western blot bands are shown with protein molecular weight in kDa; 3 to 4 dishes of cells/group were measured. (E) mitoROS levels of RL-14 cardiomyocytes of RL-14 cardiomyocytes with NHE1 overexpression and Dapa treatment are shown with representative images of MitoSox Red fluorescence (left) and ΔMFI values (right); 47 to 99 cells from 3 dishes/group were tested. (F) mitoROS levels of of RL-14 cardiomyocytes with NHE1 overexpression and TG100-115 treatment (10 mmol/L final concentration) are shown with representative images of MitoSox Red fluorescence (left) and ΔMFI values…

Figure 8. The Mechanism of Dapa Protection Against Cardiac Diastolic Dysfunction and Heart Failure With Preserved Ejection Fraction HypoMg mice had activated NHE1, increased intracellular pH, decreased serum and intracellular Mg2+, TRPM7 overexpression, cardiomyocyte oxidative stress with increased mitoROS production, elevated cMyBPC S-glutathionylation, and cardiac diastolic dysfunction. Dapa treatment improved cardiac diastolic function by inhibiting NHE1 activity, decreasing intracellular pH, increasing serum and intracellular Mg2+, suppressing TRPM7 overexpression, and reducing mitochondrial oxidative stress. Abbreviations as in Figures 1, 3, and 7.