SDS-PAGE and Densitometric Analysis of Myofibrillary Proteins: Actin and Myosin in Skeletal Muscle of Fluoride Intoxicated Rats

Abstract

Aim: The present study was undertaken to evaluate the contractile proteins; Actin and Myosin in the skeletal muscle of rats exposed to three different concentrations of fluoride during 40 days of fluoride toxicity. Changes in muscle proteins of control and test rats have been examined using Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Densitometric analysis. The percentage decrease in both Actin and Myosin proteins was observed for all treated groups. Study Design: 8 weeks old, Wistar albino rats of both sexes were used for the experiments. The rats were divided into three groups (Group I, Group II and Group III) of six animals each. The first group served as control group and was administered 1 ml deionized water /kg bw/ day. Sodium fluoride was given to Group II and Group III at the dose rate of 300 mg NaF/ kg bw/day and 600 mg NaF / kg bw /day orally for 40 days. Animals were housed in polypropylene cages lined with husk and kept in light/dark condition (12h light/ 12 h dark) .The animals had free access to water and were supplied with standard pellet diet. Methodology: At the end of the experimental period, rats were fasted overnight and anaesthetized  using ether anaesthesia. Skeletal muscle tissue were dissected out in ice-cold saline. The tissues were weighed and 10% tissue homogenate was prepared with the 0.1 M Tris-HCl buffer, pH 7.5 using a ultrasonic homogenizer. After centrifugation at 2000 rpm for 10 minutes, the clear supernatant was used for the analysis of contractile proteins. Results: SDS-PAGE and Densitometric analysis revealed that intensities of Actin and Myosin protein bands were reduced due to fluoride intoxication. The intensity of Myosin in Control group was found to be 2.57, 2.00 in Group II and 0.41 in Group III. The intensity of Actin in Control group was 3.041, 2.11 in Group II and 0.30 in Group III. The 600 mg NaF fluoride treated group exhibited more prominent decrease that the intensity of Myosin was decreased to 0.41 and Actin was decreased to 0.30. In the present study the percentage decrease of both Actin and Myosin proteins showed that Actin was 46% decreased in 300 ppm group and 58% decreased in 600 ppm group. Similarly, Myosin was 41% decreased in 300 ppm group and 87% decreased in 600 ppm group. Conclusion: The results of polyacrylamide gel electrophoresis (SDS-PAGE) and densitometric analysis demonstrated that fluoride disturbed the contractile pattern of skeletal muscle proteins. The results suggested that skeletal muscle damage, induced by fluoride, is probably due to its proteolytic action on myofibrils, which are responsible for the maintainence of the cellular architecture.