The Effect Of Chronic Electrical Stimulation On The Expression Of MHC Isoforms During Myogenic Differentiation Of C2C12Cells

Background and Objectives:Obstructive sleep apnea-hypopnea syndrome （OSAHS） is a sleep disorder relatedwith breathing, which is characterized by destructured sleep, repetitive episodes of upperairway obstruction causing sleepiness, episodic interruption of ventilation for10seconds ormore, and impaired cognitive function and poor health status. OSAHS is frequentlyassociated with a large variety of conditions, such as hypertension, cardiovascular,neuropsychological or metabolic diseases. The clinical manifestation of OSAHS is mouthand nasal airflow stops but thoracic and abdominal respiratory movements remaining.OSAHS is deemed to be the most important respiratory dysfunction while sleeping. Anassociation between OSAHS and various disorders such as hypertension, cardiovasculardisease, neuro-psychological, genitourinary and metabolic disorders, etc. has been found insound epidemiological studies for several years.Normal sleep is distinguished as rapid eye movement （REM） sleep or no-synchronizedsleep, which occurs cyclically and more frequently during the second half of the night, andnon-REM sleep or synchronized, which is subclasssified into four stages and the amount ofsleep time. There are four stages consist of drowsiness （stage one）, mild sleep （stage two）,and deep sleep （stage three and four）. Decreased heart rate, blood pressure and sympatheticnerve activity occur during non-REM sleep. REM sleep, instead, is characterized byincreased electrical activity of brain and sympathetic nerve outflow, moreover, it isconnected to intermittent and abrupt changes in blood pressure and heart rate. In obstructivesleep apnea, this homeostatic control is severely altered.The critical pathphysiological feature of OSAHS is sleep-related collapse of the upperairway at the level of pharynx. Obstructive apneas and hypopneas occur because duringsleep, the intermittent complete or partial collapse of pharynx respectively. Pharyngeal collapse can occur at the end of expiration or at the beginning of inspiration. The collapseinitially starts in the retropalatal or oropharyngeal areas in most OSAHS patients. This isfollowed by caudal extension of the collapse to the base of the tongue in most patients andfinally to the hypopharyngeal region in the rests patients.The upper airway muscle most frequently studied in the context of OSAHS is theextrinsic protrudor muscle of the tongue, genioglossue. The genioglossue is accessible, isthe major dilator of the upper airway during wakefulness, has been shown to be particularlyimportant in maintaining airway patency during sleep, and the genioglossue is thought to berepresentative of other upper airway muscle with respiratory phasic （inspiratory peak）activity patterns. Clinical and basic research had confirmed that the genioglossusabnormalities can lead to upper airway collapse, and this is one of the most importantetiopathogenisis of OSAHS, therefore, improve the function of genioglossus is a curativemethod for OSAHS treatment.Genioglossus belongs to skeletal muscles whose dysfunction should be associated withabnormal changes in their structure and energetic metabolism. Previously studies indicatedthat a variety external stimulus could change the fiber composition and function of skeletalmuscle. Our previously study showed MHC Ⅱb in genioglossus of rabbits with chronichypoxia was transformed to MHC Ⅱa and MHCⅠ after electrical stimulation especially at alower physiological frequency. Our further study indicated that the activity of SERCA andkinetics of SR Ca²⁺uptake-release of genioglossus were significantly increased afterelectrical stimulation especially at2.5+40Hz. The mechanism of muscle fiber myosinheavy chain transforming induced by electric stimulation is unclear. Calcineurin （CaN） andCa²⁺/calmodulin protien kinase （CaMK） play an important role in myosin heavy chaintransforming.Based on the analysis above, this study was designed to research CaN in myosin heavychain transforming. Provide experimental evidence for utilizing chronic electricalstimulation with lower physiological frequency in clinical treat OSAHS.Methods:1. Establish C2C12myoblast differentiation, C2C12cells were cultured in dulbecco’sminimum essential medium contain10%fetal calf serum （FCS）, then the C2C12cells wereinduced by differentiation medium （DMEM containing2%horse serum）. Three days after HS-induction, C2C12cells were used for further study；2. The differentiating cells were treated with calcineurin （CaN） inhibitor cytosporin-A（CsA,8umol） or calcium/calmodulin-dependent protein kinase （CaMK） inhibitor KN93（6umol） alone, or in combination with chronic electrical stimulation with lower physiologicalfrequency （10+40Hz, CESLPF,1.5h/day for2days）.3. The expression of myosin heavy chain obform mRNA were determined by Reversetranscription-polymerase Chain Reaction （RT-PCR）. The protein of myosin heavy chainisobform in C2C12cells were detected by Western-blot.4. The activity of calcineurin in C2C12cells were analyzed by colorimetric method.Results1. We successfully established the myogenic differentiation model of myoblastC2C12cell in vitro. Myoblast C2C12cells differentiated into myotubes that can beobserved under light microscope;2. Both myosin heavy chain MHC I and MHC IIwere expressed expressing indifferentiated control group. Both CaN inhibitor CsA and CaMK inhibitor KN93couldinhibit the expression of MHC I in differentiated C2C12cells;3. CsA and KN93can significantly reduce the expression of MHC I in C2C12cellsstimulated by10Hz electric stimulation. The expression of MHC I mRNA and protein weresignificantly decreased by CsA or KN93.4. When CESLPF was applied in the presence of inhibitors, it was able to almostcompletely reverse the changes of MHC isoform expression induced by the inhibitors（P>0.05vs control）.5. During the C2C12cells differentiation, the activity of CaN could be inhibited byCsA, but CaN activity can be revered by CESLPF partially. And the CaN activity waspotentiated by CESLPF.Conclusion1. Electric stimulation system and in vitro myogenic cells combined, can wellsimulate the cells in the body in response to the stimulus2. KN93and CSA can inhibit MHCI expression, The CESLPFcan reverse theinhibitory effect, as a manifestation of chronic electrical stimulation of the upregulation ofMHC I expression, inhibition of MHC II expression, namely the muscle fiber type conversion （conversion）, show low physiological frequency electrical stimulation throughthe CaN, CaMK pathway regulation of muscle type conversion.3. The CESLPF can be partially reversed CsA inhibition of CaN activity, and may bepromoted by C2C12muscle cells MHC type conversion of basic biochemistry.4. Our study provide experimental evidence for utilizing chronic electricalstimulation with lower physiological frequency in clinical treat ment of OSAHS.