[Mechanism by which disulfiram improves cardiac function in HFpEF rats via the GSDMD/Drp1 signaling pathway]

Objective: To investigate the role of the gasdermin D (GSDMD)/dynamin-related protein 1 (Drp1) signaling pathway in cardiac injury and mitochondrial dynamic regulation in a rat model of heart failure with preserved ejection fraction (HFpEF). Methods: Twenty-four 8-week-old male Sprague-Dawley rats weighing 200-240 g were selected and randomly divided into four groups, with six rats per group: control group (regular chow and drinking water), HFpEF group (fed with 60% high-fat diet plus drinking water containing 0.5 g/L N^ω-nitro-L-arginine methyl ester for 8 consecutive weeks), disulfiram group (intragastric administration of disulfiram at 50 mg·kg^-¹·d^-¹ on the basis of HFpEF modeling), and mitochondrial division inhibitor 1 (Mdivi-1) group (intragastric administration of Mdivi-1 at 30 mg·kg^-¹·d^-¹ on the basis of HFpEF modeling). H9C2 cardiomyoblasts were used for in-vitro experiments and assigned to four groups: normal cell group (cultured in routine complete medium with equal-volume vehicle), model group (treated with 12.5 mmol/L high glucose combined with 0.2 mmol/L palmitic acid for 12 h), disulfiram intervention group (40 μmol/L disulfiram added simultaneously with modeling), and Mdivi-1 intervention group (20 μmol/L Mdivi-1 added simultaneously with modeling). Echocardiography was performed to assess cardiac structure and function of rats in each group; a non-invasive blood pressure monitoring system was used to measure arterial blood pressure of rats; an exhaustive treadmill exercise test was performed to evaluate exercise tolerance of rats; HE, Masson, and wheat-germ agglutinin staining were used to assess myocardial histomorphology, collagen deposition, and cardiomyocyte cross-sectional area; Western blot was used to detect the expression levels of GSDMD, cleaved GSDMD, Drp1, mitofusin (MFN) 1 and MFN2 in rat myocardial tissues and H9C2 cardiomyoblasts; enzyme-linked immunosorbent assay (ELISA) was used to measure N-terminal pro-B-type natriuretic peptide and inflammatory factors, including interleukin (IL)-1β and IL-18 in rat myocardium; co-immunoprecipitation assay was performed to identify the interaction between GSDMD and Drp1 in myocardial tissues of HFpEF rats; immunohistochemical staining was used to determine myocardial GSDMD and Drp1 expression in rats. Results: Compared with the control group, the HFpEF group displayed a higher body weight ((364.50±20.98) g vs. (256.50±6.77) g), blood pressure (systolic blood pressure: (162.99±5.61) mmHg vs. (125.33±7.15) mmHg; diastolic blood pressure: (119.78±4.58) mmHg vs. (81.01±3.85) mmHg; 1 mmHg=0.133 kPa), left ventricular end-diastolic anterior wall thickness ((0.38±0.03) cm vs. (0.29±0.03) cm), left ventricular end-diastolic posterior wall thickness ((0.32±0.01) cm vs. (0.25±0.02) cm), left ventricular end-diastolic internal diameter ((0.78±0.03) cm vs. (0.67±0.01) cm), and the ratio of early diastolic mitral inflow velocity to early diastolic mitral annular velocity (24.65±0.95 vs. 18.97±0.68). The HFpEF group also presented more severe myocardial fibrosis, larger cardiomyocyte cross-sectional area, elevated myocardial expression of GSDMD, cleaved-GSDMD and Drp1, as well as an increased phosphorylated-Drp1 (Ser616)/total Drp1 ratio. Higher myocardial levels of N-terminal pro-B-type natriuretic peptide ((112.45±12.18) ng/L vs. (42.38±6.86) ng/L), IL-1β ((60.14±6.28) ng/L vs. (32.31±5.68) ng/L) and IL-18 ((82.52±7.11) ng/L vs. (56.77±5.02) ng/L) were observed in the HFpEF group than in the control group (all P<0.05). By contrast, the exhaustive treadmill running distance ((111.54±13.15)m vs. (268.75±11.79)m), ratio of early-to-late diastolic mitral inflow peak velocity (1.25±0.09 vs. 1.71±0.12) and the myocardial expression levels of MFN1 and MFN2 of HFpEF group were lower than the control group (all P<0.05). Co-immunoprecipitation assay confirmed a physical interaction between GSDMD and Drp1 in myocardial tissues of HFpEF rats. Compared with the HFpEF group, rats in the disulfiram and Mdivi-1 groups showed ameliorated cardiac diastolic dysfunction, cardiomyocyte hypertrophy, myocardial fibrosis, disordered myocardial mitochondrial dynamics and improved exercise tolerance (all P<0.05). Myocardial levels of GSDMD, Drp1, and the phosphorylated-Drp1 (Ser616)/total Drp1 ratio were lower in the disulfiram group than in the HFpEF group (all P<0.05). However, no statistically significant differences in myocardial GSDMD and cleaved-GSDMD expression were found between the Mdivi-1 group and the HFpEF group (all P>0.05). In-vitro experiments showed that, compared with the normal cell group, H9C2 cardiomyoblasts in the model group displayed higher expression of GSDMD, cleaved-GSDMD, Drp1, and phosphorylated-Drp1 (Ser616), as well as an increased phosphorylated-Drp1 (Ser616)/total Drp1 ratio. These parameters were significantly reduced in the disulfiram-intervention group relative to the model group (all P<0.05). Conclusions: A direct interaction exists between GSDMD and Drp1 in HFpEF. Drp1 may act downstream of GSDMD. The GSDMD/Drp1/mitochondrial fission axis serves as a key pathway mediating inflammation and energy-metabolism disorders in the pathogenesis of HFpEF. Interventions with disulfiram and Mdivi-1 can ameliorate cardiac pathological alterations in HFpEF rats by regulating this pathway, which provides novel potential therapeutic targets for HFpEF.