Combined therapy with dapagliflozin and entresto offers an additional benefit on improving the heart function in rat after ischemia-reperfusion injury
Sheung‐Fat Ko, Pei‐Hsun Sung, Chih Chao Yang, John Y. Chiang, Hon Kan Yip
Abstract
This study tested whether combined dapagliflozin and entresto treatment would be superior to either one alone for preserving the left-ventricular ejection-fraction (LVEF) in rat after ischemia-reperfusion (IR) injury. Cell culture using H9C2 cells and IR injury in rat with dapagliflozin-entresto treatment were conducted in the present study. In vitro flow-cytometric result showed that the intracellular and mitochondrial reactive oxygen species and mitochondrial permeability transition pore, and protein levels of oxidative-stress/DNA-damaged markers [NADPH-oxidase-1 (NOX-1)/NOX-2/oxidized-protein/γ-H2A-histone-family member X (γ-H2AX)] were significantly higher in hydrogen peroxide (H2O2) (300μM)-treated H9C2 cells as compared with the controls that were significantly reversed in sacubitril/valsartan and dapagliflozin therapy in the same H2O2-treated condition, whereas the protein expressions of antioxidants [Sirtuin-1 (SIRT1)/SIRT3/superoxide dismutase/catalase/glutathione peroxidase) exhibited an opposite pattern among the groups (all p<0.001). Adult-male-Sprague-Dawley rat (n=40) were equally categorized into group 1 (sham-operated control), group 2 (IR), group 3 (IR+dapagliflozin/20mg/kg/orally at 3h and post-days 1/2/3 after IR), group 4 (IR+entresto/100mg/kg/orally at 3h and post-days 1/2/3 after IR) and group 5 (IR+dapagliflozin+entresto) and the hearts were harvested by day 3 after IR. The 3rd day’s LVEF was highest in group 1, lowest in group 2 and significantly higher in group 5 than in groups 3/4, but it was similar between the latter two groups (p<0.001). The protein expressions of oxidative-stress (NOX-1/NOX-2/oxidized protein), fibrotic (transforming-growth factor-ß/phosphorylated-Smad3), apoptotic [mitochondrial-Bax/cleaved-caspase-3/cleaved-poly (ADP-ribose) polymerase], mitochondria/DNA damaged (cytosolic-cytochrome-c/γ-H2AX), pressure-overload/heart-failure [brain natriuretic peptide (BNP)/ß-myosin heavy chain] and autophagic (ratio of meiotic cyclins CLB3-II/CLB3-I) biomarkers, and the upstream (high-mobility group box 1/Toll-like receptor-4/MyD88/phosphorylated-nuclear factor-κB and downstream [interleukin (IL)-1ß/IL-6/tumor necrosis factor-α] inflammatory signalings revealed an antithetical features of LVEF among the groups (all p<0.0001). The cellular levels of inflammatory (myeloperoxidase+/CD68+), pressure-overload/heart-failure (BNP+) and DNA-damage (γ-H2AX+) biomarkers as well as infarct area demonstrated an opposite pattern of LVEF among the groups (all p<0.0001). Incorporated entresto-dapagliflozin treatment was superior to either one alone on protecting the heart against IR injury.