Levosimendan protects human hepatocytes from ischemia-reperfusion injury

Abstract

Background: Ischemia-reperfusion injury (IRI) is a major challenge in liver transplantation. The mitochondrial pathway plays a pivotal role in hepatic IRI. Levosimendan, a calcium channel sensitizer, was shown to attenuate apoptosis after IRI in animal livers. The aim of this study was to investigate the effect of levosimendan on apoptosis in human hepatocytes.

Methods: Primary human hepatocytes were either exposed to hypoxia or cultured under normoxic conditions. After the hypoxic phase, reoxygenation was implemented and cells were treated with different concentrations of levosimendan (10ng/ml, 100ng/ml, 1000ng/ml). The overall metabolic activity of the cells was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and aspartate aminotransferase (AST) levels were determined in order to quantify hepatic injury. Fluorescence-activated cell sorting (FACS) analysis was applied to measure necrosis and apoptosis. Finally, Western blotting was performed to analyze apoptotic pathway proteins.

Results: Administration of levosimendan during reperfusion increases the metabolic activity of human hepatocytes and decreases AST levels. Moreover, apoptosis after IRI is reduced in treated vs. untreated hepatocytes, and levosimendan prevents down-regulation of the anti-apoptotic protein Bcl-2 as well as up-regulation of the pro-apoptotic protein BAX.

Conclusion: The present study suggests a protective effect of levosimendan on human hepatocytes. Our findings suggest that treatment with levosimendan during reperfusion attenuates apoptosis of human hepatocytes by influencing BAX and Bcl-2 levels.

Fig 1. Metabolic activity and AST levels of human hepatocytes after treatment with levosimendan.

(A) The metabolic activity of human hepatocytes increased after 24 hour treatment with levosimendan after I/R injury in a dose-dependent manner. (B) After a 48 hour treatment with levosimendan after I/R injury, the metabolic activity is increased but there is no dependency detectable. (C) Normoxic and ischemic/reperfused human hepatocytes were compared regarding metabolic activity after a 24 hour treatment with levosimendan. After I/R the protective effect of levosimendan is amplified compared to normoxic groups. (D) AST levels of ischemic/reperfused hepatocytes are significantly reduced after 48 hour treatment with levosimendan compared to 24h treatment. Human hepatocytes are treated with 10ng/ml (D1), 100 ng/ml (D2) and 1000ng/ml (D3) levosimendan. Saponin served as positive control. Untreated but ischemia/reperfused hepatocytes served as control. (each experiment n = 3), # p<0.05 vs ctr., § p<0.05 vs pos. ctr., * p<0,05.

Fig 2. Total number of apoptotic hepatocytes after treatment with levosimendan.

(A) The number of apoptotic hepatocytes treated under normoxic conditions is reduced in a dose dependent manner compared to the untreated normoxic control group. (B) Under I/R conditions, the number of apoptotic hepatocytes after treatment with levosimendan is reduced compared to the untreated ischemia/reperfused control group, but there is no dose-dependency detectable. Human hepatocytes are treated with 10ng/ml (D1), 100 ng/ml (D2) and 1000ng/ml (D3) levosimendan. (each experiment n = 3–4), # p<0.05 vs ctr., § p<0.05 vs pos. ctr., * p<0,05.

Fig 3. Relative amount of Bcl-2 and BAX after treatment with levosimendan.

(A) Under hypoxic conditions treatment with levosimendan significantly increases the amount of Bcl-2 in a time-dependent manner. (B) Under hypoxic conditions the amount of BAX does not significantly decrease compared to ischemic/reperfused hepatocytes. Human hepatocytes are treated with 1000ng/ml (D3) levosimendan. Untreated but ischemia/reperfused hepatocytes served as positive control. (each experiment n = 2–3), # p<0.05 vs ctr., § p<0.05 vs pos. ctr., * p<0,05.