Laboratory of Structure and Functions of Muscle Proteins, Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Russia.
Pushchino State Institute of Natural Science, Pushchino, Russia.
Alcohol Clin Exp Res. 2017 Oct;41(10):1686-1694. doi: 10.1111/acer.13476. Epub 2017 Sep 21.
Proteolysis can proceed via several distinct pathways such as the lysosomal, calcium-dependent, and ubiquitin-proteasome-dependent pathways. Calpains are the main proteases that cleave a large variety of proteins, including the giant sarcomeric proteins, titin and nebulin. Chronic ethanol feeding for 6 weeks did not affect the activities of μ-calpain and m-calpain in the m. gastrocnemius. In our research, changes in μ-calpain activity were studied in the m. gastrocnemius and m. soleus of chronically alcohol-fed rats after 6 months of alcohol intake.
SDS-PAGE analysis was applied to detect changes in titin and nebulin contents. Titin phosphorylation analysis was performed using the fluorescent dye Pro-Q Diamond. Western blotting was used to determine μ-calpain autolysis as well as μ-calpain and calpastatin contents. The titin and nebulin mRNA levels were assessed by real-time PCR.
The amounts of the autolysed isoform (78 kDa) of full-length μ-calpain (80 kDa) increased in the m. gastrocnemius and m. soleus of alcohol-fed rats. The calpastatin content increased in m. gastrocnemius. Decreased intact titin-1 (T1) and increased T2-proteolytic fragment contents were found in the m. gastrocnemius and m. soleus of the alcohol-fed rats. The nebulin content decreased in the rat gastrocnemius muscle of the alcohol-fed group. The phosphorylation levels of T1 and T2 were increased in the m. gastrocnemius and m. soleus, and decreased titin and nebulin mRNA levels were observed in the m. gastrocnemius. The nebulin mRNA level was increased in the soleus muscle of the alcohol-fed rats.
In summary, our data suggest that prolonged chronic alcohol consumption for 6 months resulted in increased autolysis of μ-calpain in rat skeletal muscles. These changes were accompanied by reduced titin and nebulin contents, titin hyperphosphorylation, and development of hindlimb muscle atrophy in the alcohol-fed rats.
蛋白水解可以通过几种不同的途径进行，如溶酶体、钙依赖性和泛素-蛋白酶体依赖性途径。钙蛋白酶是切割包括巨大肌节蛋白肌联蛋白和原肌球蛋白在内的多种蛋白质的主要蛋白酶。6 周的慢性乙醇喂养并未影响腓肠肌中的μ-钙蛋白酶和 m-钙蛋白酶的活性。在我们的研究中，在慢性酒精喂养大鼠摄入酒精 6 个月后，研究了腓肠肌和比目鱼肌中 μ-钙蛋白酶活性的变化。
SDS-PAGE 分析用于检测肌联蛋白和原肌球蛋白含量的变化。使用荧光染料 Pro-Q Diamond 进行肌联蛋白磷酸化分析。Western 印迹法用于确定 μ-钙蛋白酶自解以及 μ-钙蛋白酶和钙蛋白酶抑制剂的含量。实时 PCR 用于评估肌联蛋白和原肌球蛋白 mRNA 水平。
腓肠肌和比目鱼肌中全长 μ-钙蛋白酶（80 kDa）的自解同工型（78 kDa）的量增加。腓肠肌中的钙蛋白酶抑制剂含量增加。在慢性酒精喂养大鼠的腓肠肌和比目鱼肌中发现完整肌联蛋白-1（T1）减少，T2 蛋白水解片段增加。腓肠肌中肌联蛋白含量减少。在慢性酒精喂养组大鼠的腓肠肌和比目鱼肌中，T1 和 T2 的磷酸化水平增加，腓肠肌中的肌联蛋白和 nebulin mRNA 水平降低。比目鱼肌中的 nebulin mRNA 水平增加。
总之，我们的数据表明，慢性酒精摄入 6 个月导致大鼠骨骼肌中 μ-钙蛋白酶的自解增加。这些变化伴随着肌联蛋白和 nebulin 含量减少、肌联蛋白过度磷酸化以及慢性酒精喂养大鼠后肢肌肉萎缩的发展。
