Dapagliflozin Attenuates Heart Failure With Preserved Ejection Fraction Remodeling and Dysfunction by Elevating β-Hydroxybutyrate–activated Citrate Synthase

FIGURE 1.. β-OHB level and CS activity are decreased in 2-Hit–treated blood and heart. (A) The blood β-OHB level in the rats (n = 5). B, The β-OHB level in hearts (n = 5). C, Immunoblotting analysis of protein levels of CS. D, The CS activity in hearts and blood (n = 5). Data are expressed as means ± SEM, and n represents number of animals, *P < 0.05 versus control; #P < 0.05 versus 2-Hit; ns, P > 0.05 versus control or 2-Hit.

FIGURE 2.. Administration of DAPA attenuated hypertension and improved cardiac dysfunction with 2-Hit strategy HFpEF in rats. (A), A schematic representation of the 2-Hit strategy. B, FBG levels (n = 6). C, SBP was measured every week by tail-cuff method (n = 6). D, M-mode echocardiography of LV chamber, and measurements of EF%, FS% and transmitral E/A ratio (n = 6). Data are expressed as means ± SEM and analyzed using repeated-measures 2-way analysis of variance and Sidak's multiple comparisons test (B–C), and n represents number of animals, *P < 0.05 versus control; #P < 0.05 versus 2-Hit; ns, P > 0.05 versus control or 2-Hit. EF%, ejection fraction; FBG, fasting blood glucose; FS%, fractional shortening; SBP, systolic blood pressure.

FIGURE 3.. DAPA alleviated HFpEF cardiac hypertrophy, inflammation, fibrosis, and oxidative stress. A, TRITC-labeled WGA staining of heart sections (left), and quantification of cross-sectional area of myocytes (200 cells counted per heart, right, n = 6), qPCR analysis of the mRNA levels of ANF in the heart (n = 5). B, The heart weight/body weight, heart weight/TL ratios (n = 6). C, Masson's trichrome staining of heart sections (left), and quantification of myocardial interstitial and perivascular collagen deposition (right, n = 6). D, Immunohistochemical staining of cardiac myofibroblasts with anti-α-SMA antibody (left), and quantification of α-SMA–positive cells (right, n = 6). E, Representative images of immunohistochemical staining of macrophages with anti-Mac-2 antibody (left), and quantification of Mac-2–positive area (right, n = 6). F, DHE staining of heart sections (left), quantification of DHE fluorescence intensity (right, n = 6). Scale bar: 50 μm. G, Representative immunoblots of p-ERK, ERK, TGF-β1, p-P65, P65, NOX1 in heart tissues from each group (left). Quantification of the relative protein levels by densitometry (right, n = 4). Data are expressed as means ± SEM, and n…

FIGURE 4.. Administration of DAPA-activated 5′-AMP–activated protein kinase (AMPK) and improved energy metabolism by inhibiting myocardial FA uptake in HFpEF. A, Representative PET images of axial (top), coronal (middle), and sagittal (bottom) cardiac sections with [18F]-FDG in rats and quantification of the maximum LV SUV (right, n = 3), showing myocardial glucose uptake. B, Representative PET images: Axial (top), coronal (middle), and sagittal (bottom) cardiac sections with [18F]-FTHA in rats and quantification of the maximum LV SUV (right, n = 3), showing myocardial FA uptake. C, Representative images of TUNEL (red), α-actinin (green), and DAPI (blue) staining of the myocardial sections (left), and quantification of the TUNEL-positive nuclei (right, n = 4). Scale bar: 5 mm or 20 μm. D, Immunoblotting analysis of the protein levels of p-AMKP (T172), GAPDH as an internal control (left), and the quantification of the relative protein levels (right, n = 4). E, Measurement of ATP contents (n = 5). Data are expressed as means ± SEM, and n represents number of animals, *P < 0.05 versus control; #P < 0.05 versus 2-Hit; ns, P > 0.05 versus control or 2-Hit. SUV, standard uptake values.

FIGURE 5.. Administration of DAPA rescued the HFpEF phenotype by increasing β-OHB levels and activating CS in rats (A), The blood β-OHB level in the rats (n = 6). B, The β-OHB level in hearts (n = 6). C, Representative immunoblots of BDH1, SCOT, and ACAT1 in heart tissues from each group. D, Quantification of the relative BDH1, SCOT, ACAT1 protein levels by densitometry (n = 4). E, The acetyl-CoA level in hearts (n = 5). F, The CS activity in hearts (n = 5). G, Immunoblotting analysis of protein levels of CS, mitochondrial complex I (NDUFB8), complex II (SDHB), complex III (UQCRC2), complex IV (MTCO2), and complex V (ATP5A) (left), and quantification of the relative protein level (n = 4). Data are expressed as means ± SEM, and n represents number of animals, *P < 0.05 versus control; #P < 0.05 versus 2-Hit; ns, P > 0.05 versus control or 2-Hit.

FIGURE 6.. Administration of DAPA inhibited the progression of established HFpEF cardiac structural and functional disorders (A), SD and 2-Hit rats at 6 weeks were administered with DAPA or vehicle control for additional 6 weeks. The following experiments were performed up to the end of 12 w. B, FBG levels (n = 6). C, SBP was measured every week by tail-cuff method (n = 6). D, M-mode echocardiography of left ventricular chamber, and measurement of EF%, FS% transmitral E/A ratio (n = 6). E, The heart mass/body mass, heart mass/TL ratios (n = 6). F, TRITC-labeled WGA staining of heart sections (left), and quantification of cross-sectional area of myocytes (200 cells counted per heart, right, n = 6), qPCR analysis of the mRNA levels of ANF in the heart (n = 5). G, Masson's trichrome staining of heart sections (left), and quantification of myocardial interstitial and perivascular collagen deposition (right, n = 6), qPCR analysis of the mRNA levels of collagen I in the heart (n = 5). H, Immunohistochemical staining of cardiac myofibroblasts with anti-α-SMA antibody (left), and quantification of α-SMA–positive cells (right, n = 6). I, Representative images of immunohistochemical staining of…

FIGURE 7.. Administration of DAPA improved energy metabolism by inhibiting myocardial FA uptake and energy pathway activation in response to established HFpEF progression. A, Representative PET images of axial (top), coronal (middle), and sagittal (bottom) cardiac sections with [18F]-FDG in rats and quantification of the maximum LV SUV (right, n = 3) showing myocardial glucose uptake. B, Representative PET images: Axial (top), coronal (middle), and sagittal (bottom) cardiac sections with [18F]-FTHA in rats and quantification of the maximum LV SUV (right, n = 3) showing myocardial FA uptake. C, Representative images of TUNEL (red), α-actinin (green), and DAPI (blue) staining of the myocardial sections (left) and quantification of the TUNEL-positive nuclei (right, n = 4). Scale bar: 5 mm or 20 μm. D, Immunoblotting analysis of the protein levels of p-AMKP (T172) and CS, GAPDH as an internal control (left), and the quantification of the relative protein levels (right, n = 4). E, Measurement of ATP contents (n = 5). F, The blood β-OHB level in the rats (n = 6). G, The β-OHB level in hearts (n = 6). H, The acetyl-CoA level in hearts (n = 5). I, The CS activity in hearts (n = 5). Data are…

FIGURE 8.. DAPA attenuated a working model of HFpEF remodeling and dysfunction by elevating β-hydroxybutyrate (β-OHB)–activated CS, 2-Hit initiated hypertension, inflammation, oxidative stress, and induced cardiac hypertrophy and fibrosis. These lead to cardiac remodeling in HFpEF. Subsequently, the elevation of β-hydroxybutyrate by DAPA attenuated these effects. A, The β-OHB level in blood samples of normal controls (n = 15), HFpEF patients (n = 15), HFpEF patients with DAPA (n = 15), and HFrEF patients with DAPA (n = 15). B, The acetyl-CoA level in blood samples of normal controls (n = 15), HFpEF patients (n = 15), HFpEF patients with DAPA (n = 15), and HFrEF patients with DAPA (n = 15). C, The CS activity in blood samples of normal controls (n = 15), HFpEF patients (n = 15), HFpEF patients with DAPA (n = 15), and HFrEF patients with DAPA (n = 15). D, A schematic illustration of DAPA attenuates HFpEF remodeling and dysfunction by elevating β-hydroxybutyrate-activated CS. Data are expressed as means ± SEM, and n represents number of patients, *P < 0.05 versus control; #P < 0.05 versus HFpEF patients; ns, P > 0.05 versus control or HFpEF patients.