Preliminary Study Of Zinc Oxide Nanoparticles Induced Damage In Rat Ventricular Myocytes

In recent years, although the level of medical technology have greatly improved, and therapeutic approaches continue to make new progress, cardiovascular disease（CVD） remains endanger human life and health, high morbidity and mortality make tens of millions of patients die from such disease each year. Overproduction of reactive oxygen species may cause oxidative damage, which is an important mechanism of cardiovascular disease. Oxidation and antioxidant system maintained in dynamic equilibrium is the premise of normal physiological function. When subjected to harmful stimuli, there will be an increase production of oxidative substances, and the antioxidant system cannot clear them to normal levels, thus oxidative damage occurred because of the imbalance of oxidative and antioxidative. As intracellular second messenger, Ca²⁺ plays an important role in regulation of intracellular signaling cascades and other aspects of the physiological functions. Changes of Ca²⁺ distribution and concentration will directly affect myocardial contractility and the regulation of heart rhythm. The influx of Ca²⁺ in myocardial cells mainly through L-type calcium channel（LTCC）, it also act an important role in maintaining normal electro-physiological properties and regulating excitation-contraction coupling.Zinc oxide nanoparticles（Zn O-NPs） are multi-functional inorganic anomaterials, and widely used in food additives, catalysts, coatings, semiconductors, ultra-sensitive molecular sensors, cosmetics industries and biomedical fields because of the unique properties that the conventional zinc oxide doesn’t have. Epidemiologic studies have described the correlation between exposure to fine particular matter in air pollution（d<100nm） and cardiovascular disease. In animal models, inhaled nanoparticles do not remain locally in the lung, but pass into the blood circulation, resulting in distribution to organs such as the heart. Meanwhile cardiac damage markers, immune inflammatory factors in serum increased. Impact of Zn O-NPs on histomorphology is tissue fibrosis. A large number of in vitro experiments shown that, Zn O-NPs can cause vascular endothelial cells to artherial atherosclerotic pathology histological changes. When exposure to other kind cells, Zn O-NPs would lead the elevation of oxide content, mitochondria dysfunction and disturb calcium homeostasis etc. In addition, studies point out that Zn O-NPs alter the electrophysiological properties of neurons, suggesting Zn O-NPs may have some impact on electrophysiological properties in rat ventricular myocytes.In order to preliminary study zinc oxide nanoparticles induced damage in rat ventricular myocytes, we use primary cultured neonatal rat ventricular myocytes as the research material, discussion the morphology, cell viability, ROS generation, intracellular calcium levels, and L-type calcium single-channel dynamic influence affected by Zn O-NPs.Results:1. Using MTT assay to determine cell viability of ventricular myocytes. After cultured with Zn O-NPs for 24 h, the IC50=65.203μg/ml; And after 48 h, the IC50=48.443μg/ml. Result shows that Zn O-NPs can decrease the cell viability. Cell viability decreased with the increasement of Zn O-NPs concentration and prolonged duration, suggesting a dose-dependent and time-dependent manner.2. Using DCFH-DA incubate cell to decide the influence of Zn O-NPs on rat ventricular myocytes after 6 hour handling in ROS generation. Both fluorescence microscopy and flow cytometry analysis results showes that Zn O-NPs can increase intracellular ROS generation.3. Using Fluo-3,AM easter incubate cell to decide the influence of Zn O-NPs on intracellular calcium levels. Result shows that Zn O-NPs can increase intracellular calcium levels, and the increasement is elevated with prolonged duration. Setting Verapamil, Zn O-NPs, Zn O-NPs + Verapamil group, the study found that the effect of Zn O-NPs can increase Ca²⁺ influx through L- type calcium channel.4. Using cell attached patch clamp technique to decide the influence of Zn O-NPs on L-type calcium single-channel. Study shows that Zn O-NPs can increase ventricular myocytes ICa-L, extension the open time, shorten closed time and increase open probability of the single-channel.In conclusion, Zn O-NPs decrease cell viability. And the damage effect on rat ventricular myocytes may be caused by increasing intracellular ROS generation, extended L-type calcium single-channel open time, shorten the closed time, increase the channel open probability, abnormally elevate intracellular calcium levels.