Impact of anesthesia and storage on posttranslational modifications of cardiac myofilament proteins

Abstract

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.

Keywords: Anesthetic; cardiac myofilaments; posttranslational modifications; storage.

Figure 1

Analysis of Myofilament Phosphorylation by Pro-Q Diamond stain. MyBP-C, myosin-binding protein C; TnT, troponin T; TM, tropomyosin; TnI, troponin I; MLC-2, regulatory myosin light chain; +, Peppermint stick standard. (A) Pro-Q Diamond-stained image specific for phosphorylated proteins. (B) A region of interest from a Coomassie gel showing actin, which was used for normalization. (C) Histograms comparing the relative phosphorylation levels in regard to storage time presented as mean ± SEM, n = 4; P < 0.05. (D) Histograms comparing the relative phosphorylation levels in regard to the anesthetic presented as mean ± SEM, n = 4; P < 0.05.

Figure 2

Representative Pro-Q Images. MyBP-C, myosin-binding protein C; TnT, troponin T; TM, tropomyosin; TnI, troponin I; MLC-2, regulatory myosin light chain. Gel images showing phosphorylation levels at varying storage times in hearts of mice anesthetized with Nembutal, Ket/Xyl, and Avertin.

Figure 3

Analysis of Myofilament Glutathionylation by Western Blot. MyBP-C, myosin-binding protein C; +, GSSG treated; −, DTT treated. (A) Western blot probed for glutathionylated proteins. (B) A region of interest from a Memcode-stained blot showing actin, which was used for normalization. (C) Western blot probed for total MyBP-C. (D) Histogram comparing the relative glutathionylation levels for the respective anesthetic over time presented as mean ± SEM, n = 4; P < 0.05. (E) Histogram comparing the relative glutathionylation levels with each anesthetic at the various time points presented as mean ± SEM, n = 4; P < 0.05. + versus day 60; * versus day 90.

Figure 4

Representative Western Blot Images. MyBP-C, myosin-binding protein C. Western blots of MyBP-C glutathionylation levels at varying storage times in the hearts of mice anesthetized with Nembutal, Ket/Xyl, and Avertin.