Regulation Of Na～+/K～+-ATPase Activity And α-subunits Expression By Mechanical Strain In Cultured Maxillofacial Skeletal Muscle Cells In Vitro

Maxillofacial skeletal muscle played an important role in generating,developing,treating and retention of malloclusion. During functional orthopedic therapy, not only the remodeling of hard tissue but also the adaptation of the soft tissue of maxillofacial system would happen in a new mechanical environment. It was a crucial factor for a successful outcome that the structure and function of these muscles were remodeled adaptively. The Na~+/K~+-ATPase, which maintains the Na~+ and K~+ gradients across the plasma membrane, could play a major role in modulation of skeletal muscle excitability and contractility. And it was also one of the marker enzymes that sensed the mechanical strain and then adapted to the stimuli.To investigate the effect of mechanical strain on Na~+/K~+-ATPase function and the expression of its functional isoform -α subunits after a SD rats maxillofacial skeletal muscle cell culture model was builted.The mechanism of regulation by mechanical strain should also be determined.Firstly, Skeletal muscle cultures were prepared from maxillofacial skeletal muscle from 2 to 3 days neonatal SD rats as described by Blau's method. The biochemical characteristics were studied to investigate the effect of cell fusion and mytube formation on Na~+/K~+~ ATPase functional activities.The results indicated that Cell fusion and myotube formation could enhance the Na~+/K~+-ATPase activities.6 to 7 days later afterplating,the cell fusion was finished. The Na+/K+-ATPase activities would not change again.The proliferation, growth and differentiation of myoblast could be regulated by modulating the foetus Bovine serum concentration in the medium.Secondly,Cells were collected at different time course after strain loading by "Forcel" four point bending device. Na+/K+—ATPase activities and intracellular Na+ concentration were determined to investigated the effect of strain on Na+/K+—ATPase activities and the mechanisms underlying the regulation. Na+/K+-ATPase activity was significantly increased above the baseline activity after stretch for 12 hours. Intracellular Na+was elevated at the beginning of stretch, but return to control levels when the treatment continued after stretched for 12 hours. Blocking the entry of Na+ with Gd3+ did not affect the stretch-induced increase in Na pump activity. Intracellular Na+ may not play a key role in stretch-induced stimulation.Thirdly, total RNA and protein extracted from different experimental groups was used to study the effects of strain on the expression of mRNA and protein of Na+/K+—ATPase subunits (di and 02) treated by mechanical strain. The quantity of mRNA in groups was analyzed with quantitative real-time PCR with the special primers of up- and down-regulated genes; and the changes of protein were analysed with Wester Blot analyse at different time course after cyclic stretch. The results indicated Cyclic stretch stimulates the mRNA expression of both ap and ai - subunits of Na+/K+-ATPase. The sensitivity of the two genes to the degree and duration of stretch was different. The stretch-induced increase of mRNA might be a result of increased transcription. Cyclic, mechanical stretch also induced a significant upregulation of both the a.\- and 0.2 -subunits. This upregulation was differentially affected by Gd3+, a blocker of SAC, nonselective cation channels; Blocking of CX2 upregulation by Gd3+ was consistent with the roleof stretch activation of ionic fluxes in regulation of ci2-protein expression. Since the upregulation of the aj-subunit was not Gd3+ sensitive, it might be likely caused by a direct effect of membrane strain on the pump, it blocked the upregulation of ci2 but not of a\. It showed that both of the a subunits had different regulation mechanisms under mechanical strain.Based on these findings, Mechanical strain could inhance the expression of mRNA and protein of Na+/K+—ATPase asubunites (al and a2) ,and could also change the activities of Na+/K+~ ATPase. There were different mechanisms controlling the expression of the subunit isoforms. The correlated enzyme protein of maxillofacial skeletal muscle would remodel to adapt to the stretch stimuli.,but there were different regulation mechanisms between ai and 012 subunits of Na+/K+—ATPase. So it could provide us the theoretical basis to intervent the skeletal muscle remodeling clinically by using different methods and Measures.