Abstract: Objective To investigate the protective effect and mechanism of klotho protein on hypoxia/reoxygenation-induced injury in H9C2 cells line. Methods H9C2 cells were cultured and divided into control group, hypoxia/reoxygenation group (cells were treated with 1% O2 for 6 h hypoxia and then reoxygenation in 5% CO2 and 95% air for 2 h) and klotho protein intervention group (10 μg/ml). Intracellular calcium level was determined indirectly by microplate reader; CCK8 and TUNEL assay was used to detect the cell viability and cell apoptosis respectively. Spectrophotometry was used to evaluate the activity of Na+/ K+ - ATPase (NKA) and the reverse mode of Na+/Ca2+-exchange (NCX). Results Hypoxia/reoxygenation (6h/2h) treatment of H9C2 cells in vitro could significantly reduce its cell viability (p <0.01), increase intracellular Ca2+ ([Ca2+]i) and apoptosis rate (p＜0.01). Moreover, the activity of Na+/ K+ - ATPase (NKA) was decreased and the the activity of reverse mode of Na+/Ca2+-exchange (NCX) was increased in cultured H9C2 cells treating with hypoxia/reoxygenation. However, the klotho protein administration observably attenuated all these changes (p＜0.01). Conclusions klotho protein administration can attenuate hypoxia/reoxygenation induced intracellular calcium overload and high apoptosis rate in cultured H9C2 cells.

Key words: klotho protein, H9C2 cells, myocardial protection, Na+/K+-ATPase, Na+/Ca2+-exchanger