Purpose: It is widely accepted that oxidative stress plays a major role in cardiovascular diseases. The aim of this study is to identify novel biomarkers that are released from cardiomyocytes upon oxidative stress before the onset of necrosis.
Methods and Results: Firstly, isolated mouse hearts were subjected to Langendorff perfusion with 1 mM H2O2 for 15 min to induce maximal oxidative stress in the absence of cell death, as detected by lactate dehydrogenase release. Notably, SDS/PAGE of tissue samples displayed the disappearance of a band migrating at 72 kDa in the H2O2 group as compared to the normoxic control. Analysis by mass spectrometry identified two proteins, namely EHD4 and HSPA8. Concomitantly, these proteins were found in the coronary effluent. We confirmed this result by both Western blot and immunohistochemistry. In particular, H2O2-perfused heart cryosections displayed 77% and 70% decrease in immunofluorescence as compared to normoxic-perfused hearts for EDH4 and HSPA8, respectively. EHD4 belongs to the EHD protein family that are involved in intracellular trafficking, while HSPA8 is a chaperonin.
Then, we evaluated another model of oxidative stress caused by single injection of doxorubicin (15 mg/kg). Mice were sacrificed after 3 days. The EHD4 content was reduced in hearts of doxorubicin-treated mice, as compared to hearts of vehicle-treated animals. To confirm that protein release can occur the absence of cell death, we treated an atriomal murine cell line (HL1) with: H2O2 (10-100 μM), doxorubicin (1-6 μM), or H2O2 (10 μM) + doxorubicin (6 μM). We observed that the intracellular EHD4 content was significantly reduced upon the different treatments, as detected by Western blot and immunocytochemistry in the absence of any loss of cell viability, as detected by Hoecst 22358/Propidium Iodide staining. The release of HSPA8 in the tested protocols was less evident.
Conclusions: EHD4 appears as the most prominent protein released by cardiac myocytes undergoing oxidative stress in the absence of cell death. Therefore, EHD4 release should be exploited as a novel plasma biomarker of early oxidative stress, occurring before the onset of necrosis. Further studies will elucidate the mechanisms underlying protein release in viable cardiac myocytes and the effects of specific protein loss on the susceptibility to cell injury.