Researches On The Mechanism Of Manganese Action On The Calcium Homeostasis, Reactive Oxygen Species And Apoptosis In Myocytes

Manganese is an essential trace element found in many enzymes. As is the case of many essential trace elements, excessive manganese is toxic. It has been proven that excessive manganese could cause heart problems. In order to clarify the toxic mechanism, the effects of manganese on rat ventricular myocytes were studied.Manganese could decrease the heart rate and cause the calcium imbalance. But the mechanisms are still unsure. With this aim, we tested the effect of manganese on the calcium channels. The L-type calcium channel current was measured by whole-cell patch clamp recording mode. In the electrophysiology experiments, both 50μM Mn²⁺ and 100μM Mn²⁺ could effectively decrease the channel current amplitude density by 35.7% and 68.2%, respectively. Moreover Mn²⁺ shifted the steady-state activation curve toward more positive potential and the steady-state inactivation curve toward more negative potential. The expression levels ofα_1C/Cav1.2 andα_1D/ Cav1.3 mRNAs were investigated by RT-PCR. Theα_1C/Cav1.2 mRNA with manganese treatment was lower expressed, and the expression was concentration dependent; while theα_1D/ Cav1.3 mRNA was almost unchanged. Fluo-3/AM was utilized for real-time free calcium scanning with laser scanning confocal microscopy (LSCM) and the results showed that Mn²⁺ could elicit a slow and continuous increase of [Ca²⁺]_i in a concentration-dependent manner. These results have suggested that manganese could interfere with the function of the L-type calcium channel, down regulate the expression ofα_1C/ Cav1.2 mRNA, and thus causing long-lasting molecular changes about L-type calcium channel which probably triggered by overloading of calcium in myocytes.There are abundant evidences proved that the mitochondria is the main target of manganese toxicity. And reactive oxygen species could be accumulated under manganese exposure. We also study the relations between ROS generation and the damages to mitochondrial of manganese action on myocytes. Isolated myocytes were incubated in different manganese concentrations for two hours. The reactive oxygen species (ROS) generation, the level of reduced glutathione (GSH) and mitochondrial membrane potential (△ψm) were investigated by flow cytometer (FACScan) and laser scanning confocal microscopy (LSCM). Results showed that the total ROS were increased after manganese treatment. But the hydrogen peroxide (H₂O₂) was the main species elevated while superoxide anion (O₂·) was nearly unchanged. And the generation of H202 became obviously with the higher concentration of manganese. The content of GSH decreased after manganese exposure. When the myocytes were incubated with both manganese and GSH-EE the generation of H202 decreased greatly. Also manganese could induce the decrease of mitochondrial membrane potential (△ψm). In the existence of GSH-EE△ψm were retained. Our results suggested that manganese action may lead to the ROS stress upon myocytes because high acculumation of H₂O₂. The low level of GSH was the main reason to the high content of H₂O₂. GSH could effectively clean the over production of H₂O₂. The mitochondrial membrane potential was affected also by manganese. H₂O₂ and GSH were both involved in the manganese damage to the mitochondrial membrane potential.The imbalance of calcium and dyfunction of mitochondria will most probably induce the cell apoptosis. But till now there is no report on the apoptosis in myocytes under manganese exposure. To find out if manganese could exert apoptic action on myocytes we study this process in neonatal rat ventricular myocytes (NRVM).Firstly, we examined cytotoxicity of manganese on NRVM by MTT assays in vitro. Results showed that after incubation in the different concentrations of manganese for 24h, apparent cytotoxicity was observed at 500μM, 1000μM and 1500μM of manganese and cell viability dropped to 82%±6.13, 78%±5.28 and 66%±4.22 respectively. When cells were treated for 48h all concentrations tested exerted obviously toxic effect, especially from 500μM to 1500μM the cell viability dropped from 67%±4.84 to 37%±3.25. Apoptosis in NRVM was then examined by flow cytometry. Results showed that the percentage of apoptotic cells treated with 500μM of manganese for 24h increased from 4%±0.84 to 7%±1.16. After 48h of incubation, this percentage increased to 11%±0.91. In control group (0μM manganese) there was no significant difference after 24h and 48h of incubation. The morphological changes of NRVM nuclei were visualized with Hoechst33342 after incubation in 500μM of manganese for 48h. Compared to the normal nuclei, typical features of apoptotic nuclei obviously showed fragmentation and condensation. To investigate whether there is any apoptotic genes expression changes involved in the apoptosis, we examined the expression level of Bcl-2, Bax and P53 mRNAs after treatment of 500μM of manganese for 48h. The Bcl-2 mRNA was decreased while the expression of Bax as well as P53 mRNAs was increased obviously. These results suggested that manganese cytotoxicity on NRVM could induce apoptosis in NRVM cells. The apoptosis process might involve and be promoted by the changes of the expression level of P53, Bcl-2 and Bax proteins.Based on above results, it is reasonable to draw conclusion as below: 1) Blocking of L-type calcium channel by manganese induce the dysfunction of myocytes involving the imbalance of calcium and changes on gene expression about the channel protein. 2) Hydrogen peroxide accumulation and decreasing of cellular glutathione involved the manganese damanges on mitochondria. 3) Manganes could induce the apoptosis in myocytes and it has some relations to the dysfunction of mitochondria.