LDH activity was measured by a commercial assay kit according to the manufacturer’s instructions. Fresh heart samples were homogenized in cold assay buffer and centrifuged at 4 °C for 15 min to remove any insoluble material. Tissue supernatants and cell medium were then incubated with the reaction mix, and the absorbance values were measured at 450 nm. Caspase3 activity in the myocardium or cultured cells was measured via detecting the fluorogenic change of Z-DEVD-AMC [26 ]. Serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) were detected by an ADVIA 2400 automatic biochemical analyzer (Siemens, Tarrytown, USA) [25 (link), 26 ]. Circulating OSTN concentrations were measured by a sandwich chemiluminescence enzyme immunoassay, as described previously [26 , 37 (link)]. Briefly, blood samples were quickly stored in microtubes containing EDTA-2Na and then centrifuged at 4 °C for 20 min with the plasma collected. Next, the plasma samples were incubated in pre-coated plates at 4 °C overnight and then probed by an anti-mouse/human OSTN rat antibody. Finally, alkaline phosphatase-conjugated donkey anti-rat antibody was applied to incubate at room temperature for an additional 1 h, and the chemiluminescence signal intensity was measured at 535 nm using a CDP-Star™ substrate with Emerald-II™ enhancer. Cell viability was assessed by the CCK-8 method, as described previously [25 (link), 51 (link)].
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