慢性电刺激对成肌分化的C2C12细胞肌型转换的影响

目的研究钙调神经磷酸酶(calcineurin,CaN)在低频复合生理频率慢性电刺激(chronic electrical stimula-tion with lower physiological frequency,CESLPF)对骨骼肌细胞肌球蛋白重链(myosin heavy chain,MHC)亚型表达的影响。方法以体外2%马血清诱导小鼠成肌母细胞C2C12成肌分化为模型。实验分3组:对照组、CsA组和KN93组。对照组为正常分化的细胞;CsA组和KN93组在细胞诱导分化3 d后,分别用CaN抑制剂环孢菌素A(CsA,8μmol)、Ca2+/钙调蛋白依赖的蛋白激酶(CaMK)抑制剂KN93(6μmol)单一或联合CESLPF(10+40 Hz,1.5 h/d,共2 d)处理。通过RT-PCR、Western blot检测观察成肌分化过程中肌球蛋白重链亚型的表达水平及其转换情况,采用比色法检测CaN的活性。结果在成功构建的体外成肌分化模型上发现,CsA和KN93能够抑制C2C12细胞MHCⅠ的mRNA及蛋白的表达。与分化对照组MHCⅠmRNA相对表达量(0.79±0.04)相比,CsA组(0.62±0.03)和KN93组(0.69±0.05)能够抑制C2C12细胞MHC I型纤维的表达(与对照组比P<0.05);对照组、CsA和KN93处理组MHC I蛋白相对水平分别是(0.91±0.05)、(0.36±0.01)和(0.66±0.02)。对照组MHCⅡb相对表达量为(0.53±0.01),CsA(0.63±0.02)和KN93(0.75±0.03)促进MHCⅡb型纤维的表达(与对照组比较,P<0.05);CESLPF能够逆转上述改变(与对照组比较,P>0.05)。CsA能够抑制CaN的活性表达;CESLPF能够显著提高CaN活性(P<0.05),但仍显著低于正常水平(P<0.05)。结论 CaN和CaMK是骨骼肌细胞MHCⅡ型向MHCⅠ型转变的重要调控酶,CESLPF可能是经CaN通路和非CaN通路联合作用促进肌型转换,该结果为临床应用CESLPF治疗OSAS提供了实验依据。

Effect of chronic electrical stimulation on transformation of myosin heavy chain isoforms in differentiated C2C12 cells

Objective To study the effect of chronic electrical stimulation with lower physiological frequency(CESLPF) on expression of myosin heavy chain(MHC) isoforms in differentiated C2C12 cells.Methods Three days after a C2C12 cell differentiation model was induced by 2% horse serum(HS),expression level of MHC isoforms in differentiated C2C12 cells treated with calcineurin(CaN) inhibitor cytosporin-A(CsA,8 μmol) or calcium/calmodulin-dependent protein kinase(CaMK) inhibitor KN93(6 μmol) alone or in combination with CESLPF(10+40 Hz,1.5 h/d for 2 d) was measured by RT-PCR and Western blotting,respectively.Activity of CaN was detected by colorimetry.Results CsA and KN93 could inhibit the expression of MHC I mRNA and protein in C2C12 cell differentiation model in vitro.The expression level of MHC Ⅰ mRNA and protein was higher in control group than in CsA and KN93 treatment group(0.79±0.04 vs 0.62±0.03 and 0.69±0.05,0.91±0.05 vs 0.36±0.01 and 0.66±0.02,P<0.05).The expression level of MHC Ⅱb mRNA was lower in control group than in CsA and KN93 treatment group(0.53±0.01 vs 0.63±0.02 and 0.75±0.03,P<0.05).CESLPF was able to reverse the expression of MHC isoforms(P>0.05).CsA could suppress while CESLPF could increase the CaN activity which was still significantly lower than its normal level(P<0.05).Conclusion CaN and CaMK are the important regulatory enzymes for the transformation of MHC Ⅱ to MHC Ⅰ.CESLPF can promote the transformation of MHC Ⅱ to MHC Ⅰ through CaN-dependent and CaN-independent pathway.Our results provide evidence for the application of CESLPF in treatment of obstructive sleep apnea syndrome.