Changes Of Excitation-contraction Units In Cyclic-strain-loaded Cultured Myotubes In Vitro And In Superficial Masseter Of Rats After Functional Mandible Advancement In Vivo

Forces are employed in the functional orthopedic procedure treatment everywhere. The uniqueness of functional appliances lies in their mode of force application. They do not produce any mechanical forces in a similar manner to conventional appliances, but transmit, eliminate, and guide nature forces (e.g. muscle activity) by changing the position of mandible. The muscles are the most important mediums for functional orthopedic forces. Masseter is the major one, of which tension, excitability, and contractibility changes adaptively. Dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs) are two kinds of Ca²⁺ ion channels lying in sarcolemma and sarcoplasmic reticulum respectively. They play crucial roles in excitation-contraction coupling by acting as signal transmitters and Ca²⁺ ionchannels formation proteins. So they are named as an unit -excitation-contraction unit. Investigation the changes of dihydropyridine receptors and ryanodine receptors in cyclic-strain-loaded cultured myotubes and masseter will be helpful to interpret how orthopedic force was sensed and translated into the changes that modified skeletal muscle phenotype, and what was the molecular mechanisms involved in maxillofacial skeletal muscle remodeling during functional orthopedic therapy.Objectives: The purpose of this study is to investigate the changes of dihydropyridine receptors α₁ subunit and ryanodine receptors in cyclic-strain-loaded cultured myotubes and in superficial layer masseter of rats after the functional mandibular advancement, then to discuss the molecular mechanisms of maxillofacial skeletal muscle remodeling during functional orthopedic therapy in vitro and in vivo.Methods: (1) Skeletal muscle cultures were prepared from maxillofacial skeletal muscle from 1² days neonatal SD rats as described by Blau's method. Cells were planted on plates at a density 4.0×10⁵/plate. A new cyclic strain loading apparatus - "Forcel" four point bending device was used for mechanically loading. Cells were collected at 1,4,8,12,24 hours after strain loading. The mRNA expression of DHPRai subunit and RyRs was determined by quantitative real-time PCR. ( 2 ) 32 30³5 days old rats ,male Sprague-Dawley rats were randomly divided into 4 experimental groups (5 rats each) and 4 control groups (3 rats each). In the experimental groups, removable functional appliances were used to create forward mandibular advancement. The rats were killed after 7, 14, 21, and 28 days. Superficial layer masseter of each rat was collected for DHPRai subunit and RyRs mRNAanalysis by quantitative real-time PCR and protein analysis for DHPRai subunit by western blot.Results: ( 1 ) Maxillofacial skeletal muscle cells showed typical characteristics of myoblasts. mRNA analysis shows that 2000ustrain, 0.5Hz, different sustained strain produce no significant change on the mRNA expression of DHPRai subunit and RyRs in myotubes. Mean values of every experimental group is slight lower than its control groups insignificantly.(2) mRNA and protein expression of DHPRai subunit in superficial layer masseter of mandible advanced rats was detected a significantly increase on day 14, then back to normal level on day 21. mRNA expression of RyRs was increased significantly on day 7 and back to normal level on day 14. There is a different time relation between the two elements in E-C unit.Conclusion: (1) There is no identified relationship between Ca2+ ion concentration change in cyclic-strain-loaded cultured myotubes and E-C unit. (2) mRNA and protein expression of DHPRai subunit and mRNA expression of RyRs in superficial layer masseter of mandible advanced rats adaptively changed. So we conclude that the E-C unit may be an important part of skeletal muscle remodeling during functional orthopedic therapy. The results of animal experiments were not coincided with cultured myotubes experiments in vitro. The absence of nerve and different fluid environment might be the key to answer why mRNA expression of DHPRai subunit and RyRs showed no significant response to cyclic strain. The results were also not coincided with eccentric contraction-induced skeletal muscle injury experiments. Soft forces and intermittent forces used in mandible advancement which weredifferent from injury experiments might be the reason.The study confirmed that soft and intermittent forces, which might produce minimum muscle injury, were strongly recommended to be used in functional orthopedic treatment, and step-by-step bite reconstruction was recommended in dealing with severe class II malocclusions as well. It also implied successful adaptation need proper nerve and body liquid condition.