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. 2012 Aug 15;303(4):R376-86.
doi: 10.1152/ajpregu.00146.2012. Epub 2012 Jul 3.

MAFbx, MuRF1, and the Stress-Activated Protein Kinases Are Upregulated in Muscle Cells During Total Knee Arthroplasty

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Free PMC article

MAFbx, MuRF1, and the Stress-Activated Protein Kinases Are Upregulated in Muscle Cells During Total Knee Arthroplasty

Ashley N Bailey et al. Am J Physiol Regul Integr Comp Physiol. .
Free PMC article

Abstract

Total knee arthroplasty (TKA) is the most common and a cost-effective surgical remediation for older adults with long-standing osteoarthritis. In parallel with the expanding population of older adults, the number of TKAs performed annually is projected to be 3.48 million by 2030. During this surgery, a tourniquet is used to stop blood flow to the operative leg. However, the molecular pathways that are affected by tourniquet use during TKA continue to be elucidated. We hypothesized that components of the catabolic FoxO3a (i.e., MuRF1, MAFbx, and Bnip3) pathway, as well as the cellular stress pathways [i.e., stress-activated protein kinase (SAPK)/JNK and MAPKs], are upregulated during TKA. The purpose of this study was to measure changes in transcripts and proteins involved in muscle cell catabolic and stress-activated pathways. We obtained muscle biopsies from subjects, 70 ± 1.3 yr, during TKA, from the vastus lateralis at baseline (before tourniquet inflation), during maximal ischemia (just before tourniquet release), and during reperfusion. Total tourniquet time was 43 ± 2 min and reperfusion time was 16 ± 1. Significant increases in FoxO3a downstream targets, MAFbx and MuRF1, were present for mRNA levels during ischemia (MAFbx, P = 0.04; MuRF1, P = 0.04), and protein expression during ischemia (MAFbx, P = 0.002; MuRF1, P = 0.001) and reperfusion (MuRF1, P = 0.002). Additionally, stress-activated JNK gene expression (P = 0.01) and protein were elevated during ischemia (P = 0.001). The results of this study support our hypothesis that protein degradation pathways are stimulated during TKA. Muscle protein catabolism is likely to play a role in the rapid loss of muscle volume measured within 2 wk of this surgery.

Figures

Fig. 1.
Fig. 1.
Study design. Arrows indicate skeletal muscle biopsies. A baseline muscle biopsy was acquired in the operating room following anesthesia, immediately prior to tourniquet inflation. The second muscle biopsy was obtained immediately prior to tourniquet deflation. The third muscle biopsy was obtained following deflation of the tourniquet (reperfusion) just prior to the subject leaving the operating room. Please see Table 1 for details of anesthetics used and individual tourniquet (ischemia) and reperfusion times. Average tourniquet time was 43 ± 4 min, and the average reperfusion time was 16 ± 3 min.
Fig. 2.
Fig. 2.
Cytoplasmic and nuclear separation. Representative blots showing adequate separation of cytoplasmic and nuclear fractions were assessed via Western blot analysis detection of α-tubulin, cytoplasmic protein (C), and laminin A/C, nuclear protein (N), in each lane. A single muscle homogenate sample was loaded in duplicate with each gel and used as a between-blot internal loading control. All gels were run in with cytoplasmic and nuclear fractions loaded in adjacent cells, i.e., baseline cytoplasmic next to the baseline nuclear fraction as shown in the representative blot.
Fig. 3.
Fig. 3.
Protein translation downregulation during total knee arthroplasty (TKA). Data represent Akt at Ser-473 in whole muscle fraction (A) (n = 6); 4E-BP1 Thr-36/47 (B); eukaryotic initiation factor 4E (eIF-4E) at Ser-209 in the cytoplasmic (C) and nuclear fraction (D). Representative phosphorylated, total, and loading control (Ponceau) Western blot are included. Data are expressed as means ± SE (n = 10). *P ≤ 0.05 vs. baseline.
Fig. 4.
Fig. 4.
Stress pathway during TKA. Stress-activated protein kinase (SAPK/JNK) protein in the cytoplasmic (A) and nuclear fraction (B). MAPK p-38α protein in the cytoplasmic (C) and nuclear fraction (D). MAPK p-38β protein in the cytoplasmic (E) and nuclear fraction (F). Representative phosphorylated, total, and loading control (Ponceau) Western blot are included. Data are expressed as means ± SE (n = 10). *P ≤ 0.05 vs. baseline.
Fig. 5.
Fig. 5.
Autophagic/lysosomal pathway during TKA. Bcl2 protein in the cytoplasmic (A) and nuclear fraction (B). Bcl2/adenovirus EIB 19-kDa-interacting protein 3 (Bnip3) protein in the cytoplasmic (C) and nuclear fraction (D). Representative phosphorylated, total, and loading control (Ponceau) Western blot images are included. Data are expressed as means ± SE (n = 10). *P ≤ 0.05 vs. baseline.
Fig. 6.
Fig. 6.
E3 ubiquitin proteasomal pathway during TKA. MAFbx protein in the cytoplasmic (A) and nuclear fraction (B). MuRF1 protein in the cytoplasmic (C) and nuclear fraction (D). Representative phosphorylated, total, and loading control (Ponceau) Western blot images are included. Data are expressed as means ± SE (n = 10). *P ≤ 0.05 vs. baseline.
Fig. 7.
Fig. 7.
mRNA expression during TKA. FoxO3a (A) MAFbx (B), MuRF1 (C), SAPK/JNK (D), and p-38α (E). Data are expressed as means ± SE (n = 7). *P ≤ 0.05 vs. baseline.
Fig. 8.
Fig. 8.
Ischemia-reperfusion stimulates multiple catabolic/autophagic cell signaling pathways during total knee arthroplasty (TKA). A schematic diagram of proteins regulating signaling pathways controlling cap-dependent mRNA translation initiation (36) and autophagy/lysosomal and ubiquitin-proteasomal protein degradation. Ischemia-reperfusion inhibits cap-dependent translation initiation via dephosphorylation of Akt leading to inhibition of the Akt-mTOR pathway and the availability of 4E-BP1 to bind to and inhibit eIF4E association with eIF4G to form an active mRNA cap-binding complex (eIF4F). Dephosphorylation of Akt leads to the dephosphorylation and nuclear translocation of FoxO3a, leading to increased transcription of MAFbx, MuRF1, and Bnip3 and subsequent activation of ubiquitin-proteasomal and autophagic/lysosomal protein degradation pathways. Cell stress signaling increases the expression of SAPK/JNK and p38α/β MAPKs, contributing to the nuclear translocation of FoxO3a and protein degradation.

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