Levosimendan reverses the cardiac malfunction and cardiomyocyte ferroptosis under heart failure with preserved ejection via connexin43 signaling activation

Abstract Purpose Recent decades have been witnessing that heart failure with preserved ejection fraction (HFpEF) outweighs heart failure with reduced ejection fraction by degrees, but few drugs were proven to improve long-term clinical outcomes in patients with HFpEF. Levosimendan, a calcium sensitizing cardiotonic agent, has been found to improve decompensated heart failure clinically. However, the protective activities and underlying molecular mechanisms of levosimendan on HFpEF have not been revealed. Methods The double-hit HFpEF C57BL/6N mouse model was established, and levosimendan (3 mg/kg/week) was administered to HFpEF mice aged from 13 to 17 weeks to verify its protective effects on HFpEF. The myocardium was biochemically evaluated by western blot, immunofluorescence, flow cytometry, etc. Results After four-week administration, cardiac hypertrophy, pulmonary congestion, and exercise exhaustion were significantly alleviated. Meanwhile, junction proteins located in endothelial barrier and between cardiomyocytes were improved by levosimendan treatment. Among the gap junction channel proteins concerned, connexin 43, especially expressed on cardiomyocytes, could conduct mitochondrial protection. Furthermore, levosimendan indeed reversed mitochondrial malfunction in HFpEF mice, evidenced by increased mitofilin and decreased ROS, superoxide anion, NOX4 and cytochrome C levels. Interestingly, after levosimendan treatment, myocardium from HFpEF mice showed restricted ferroptosis, indicated by upregulated GSH/GSSG ratio, GPX4, xCT and FSP-1 expression with down-regulated intracellular ferrous ion, MDA and 4-HNE. Conclusion This study confirmed that regular long-term levosimendan administration could benefit HFpEF individuals, particularly those with metabolic syndrome, such as obesity and hypertension, by activating connexin 43-conducted mitochondrial protection and sequential ferroptosis inhibition in cardiomyocytes.