Efficacy Of All-trans Retinoid Acid In Preventing Nickel Induced Cardiotoxicity In Myocardial Cells

Aims:Due to its electrical property and resistance to corrosion, heavy metal nickel is widely used in electronic and chemical applications and the field of nanotechnology. This study aims to elucidate the cardiotoxicity of nickel and its possible mechanisms, and to find All-trans Retinoic Acid (ATRA)-related drugs to provide prevention and detoxification of nickel-induced toxicity. The results will provide novel theoretical basis and guidance for early prevention of heavy metal pollution in human with direct exposure to hazardous material.Methods:1) Cell Counting Kit-8, flow cytometry, AO/EB staining and Western Blotting were used to detect the in vitro proliferation inhibition property of nickel chloride (NiCl2) on rat myocardial cell line H9c2, as well as cell survival after the combination of different concentrations of ATRA.2) Flow cytometry analysis of reactive oxygen species (ROS) level after NiCl2 treatment in H9c2 cardiomyocytes, and its changes after ATRA combination.3) Western Blotting analysis of MAPK signaling pathway activation in cardiomyocytes after NiCl2 treatment or ATRA combination.4) DAPI staining and measurement of spontaneously contracted primary neonatal rat cardiomyocytes were used to detect NiCl2-induced toxicity and ATRA’s protective effects in cardiac cells.5) Establish a nickel-overdose SD rat model and a chronic nickel exposure rat model to study the cardio-protective effects of ATRA in vivo.Results:1) H9c2 cells treated with NiCl2 for 72 h showed a concentration-dependent inhibition of cell viability, accompanied with induction of cell death. The toxic effects suggest that NiCl2 has potential cytotoxicity in H9c2 myocardial cells. The combination of ATRA reversed cell viability and recovered cell morphology. NiCl2 treatment also led to an activation and cleavage of apoptotic markers PARP and caspase-3, while ATRA combination inhibited Caspase cascade activation.2) In H9c2 cells, treatment with ATRA for longer than 3 h successfully scavenged NiCl2-mediated intracellular ROS production to nearly basal level.3) The phosphorylation level of MEK, ERK and p38 in MAPK pathway were up-regulated by NiCl2 exposure and down-regulated by ATRA co-treatment for 72 h.4) In primary neonatal rat cardiomyocytes, ATRA combination protected cells against NiCl2-induced chromatin condensation, nuclear fragmentation, and loss of spontaneous contraction.5) According to the electrocardiogram (ECG), the acute maximum tolerance dose of NiCl2 is between 2 mg/kg and 3 mg/kg. By consecutive intravenous injection of NiCl2 for 27 days to establish a chronic nickel exposure rat model, ECG showed that NiCl2 induced a continuous retrograde P wave and a sagging ST segment depression. A combinational intragastric administration of 40 mg/kg ATRA, however, partially reversed NiCl2-induced arrhythmia.Conclusions:We demonstrate that NiCl2 induced apoptosis in both rat cardiomyocyte derived H9c2 cells and primary neonatal rat cardiomyocytes. Moreover, NiCl2 altered the systolic function of neonatal rat cardiomyocytes and caused arrhythmia in SD rats. ATRA treatment can efficiently attenuate the above cardiotoxicity of NiCl2 possibly due to its multiple roles in attenuating ROS generation, inhibiting PARP and caspase-3 cleavage, and maintaining regular function of MAPK by decreasing the phosphorylation activation of ERK and p38 induced by NiCl2 treatment. These results not only elucidated the molecular mechanisms of nickel-induced cardiac toxicity, but also offered a new perspective in finding ATRA-related therapy for treatment and prevention of heavy metal-induced cardiotoxicity.