Utter Megan S, Warren Chad M, Solaro R John
Department of Physiology and Biophysics, Center for Cardiovascular Research, College of Medicine, University of Illinois at Chicago, Chicago, Illinois.
Department of Physiology and Biophysics, Center for Cardiovascular Research, College of Medicine, University of Illinois at Chicago, Chicago, Illinois
Physiol Rep. 2015 May;3(5). doi: 10.14814/phy2.12393.
Although high fidelity measurements of posttranslational modifications (PTMs) of cardiac myofilament proteins exist, important issues remain regarding basic techniques of sample acquisition and storage. We investigated the effects of anesthetic regimen and sample storage conditions on PTMs of major ventricular sarcomeric proteins. Mice were anesthetized with pentobarbital (Nembutal), ketamine/xylazine mixture (Ket/Xyl), or tribromoethanol (Avertin), and the ventricular tissue was prepared and stored for 1, 7, 30, 60, or 90 days at -80°C. Myofilament protein phosphorylation and glutathionylation were analyzed by Pro-Q Diamond stain and Western blotting, respectively. With up to 7 days of storage, phosphorylation of troponin T was stable for samples from mice anesthetized with either Nembutal or Ket/Xyl but not Avertin; while myosin-binding protein C (MyBP-C) phosphorylation was reduced at 7 days with Nembutal and Ket/Xyl, though generally not significant until 90 days. Tropomyosin and regulatory myosin light chain phosphorylation were stable for up to 7 days regardless of the anesthetic regimen employed. In the case of Troponin I, by 7 days of storage there was a significant fall in phosphorylation across all anesthetics. Storage of samples from 30 to 90 days resulted in a general decrease in myofilament phosphorylation independent of the anesthetic. S-glutathionylation of MyBP-C presented a trend in reduced glutathionylation from days 30-90 in all anesthetics, with only day 90 being statistically significant. Our findings suggest that there are alterations in PTMs of major myofilament proteins from both storage and anesthetic selection, and that storage beyond 30 days will likely result in distortion of data.
尽管存在对心肌肌丝蛋白翻译后修饰（PTM）的高保真测量方法，但在样本采集和储存的基本技术方面仍存在重要问题。我们研究了麻醉方案和样本储存条件对主要心室肌节蛋白PTM的影响。用戊巴比妥（戊巴比妥钠）、氯胺酮/赛拉嗪混合物（氯胺酮/赛拉嗪）或三溴乙醇（阿佛丁）对小鼠进行麻醉，然后制备心室组织并在-80°C下储存1、7、30、60或90天。分别通过Pro-Q Diamond染色和蛋白质印迹法分析肌丝蛋白的磷酸化和谷胱甘肽化。储存长达7天时，用戊巴比妥钠或氯胺酮/赛拉嗪麻醉的小鼠样本中肌钙蛋白T的磷酸化是稳定的，但用阿佛丁麻醉的小鼠样本则不然；而肌球蛋白结合蛋白C（MyBP-C）的磷酸化在使用戊巴比妥钠和氯胺酮/赛拉嗪麻醉7天时有所降低，不过通常直到90天才显著。无论采用何种麻醉方案，原肌球蛋白和调节性肌球蛋白轻链的磷酸化在长达7天内都是稳定的。就肌钙蛋白I而言，储存7天时，所有麻醉剂处理后的样本磷酸化均显著下降。样本从30天储存到90天导致肌丝磷酸化普遍降低，与麻醉剂无关。MyBP-C的S-谷胱甘肽化在所有麻醉剂处理下均呈现出从第30天到90天谷胱甘肽化减少的趋势，只有第90天具有统计学意义。我们的研究结果表明，储存和麻醉剂选择都会导致主要肌丝蛋白PTM发生改变，并且储存超过30天可能会导致数据失真。
