Proteins regulating cap-dependent translation are downregulated during total knee arthroplasty

Abstract

Total knee arthroplasty (TKA) utilizes a tourniquet to reduce blood loss, maintain a clear surgical "bloodless" field, and to ensure proper bone-implant cementing. In 2007, over 600,000 TKAs were performed in the United States, and this number is projected to increase to 3.48 million procedures performed annually by 2030. The acute effects of tourniquet-induced ischemia-reperfusion (I/R) on human skeletal muscle cells are poorly understood and require critical investigation, as muscle atrophy following this surgery is rapid and represents the most significant clinical barrier to long-term normalization of physical function. To determine the acute effects of I/R on skeletal muscle cells, biopsies were obtained at baseline, maximal ischemia (prior to tourniquet release), and reperfusion (following tourniquet release). Quadriceps volume was determined before and 2 wk post-TKA by MRI. We measured a 36% decrease in phosphorylation of Akt Ser(473) during ischemia and 37% during reperfusion (P < 0.05). 4E-BP1 Thr(37/46) phosphorylation decreased 29% during ischemia and 22% during reperfusion (P < 0.05). eEF2 Thr(56) phosphorylation increased 25% during ischemia and 43% during reperfusion (P < 0.05). Quadriceps volume decreased 12% in the TKA leg (P < 0.05) and tended to decrease (6%) in the contralateral leg (P = 0.1). These data suggest cap-dependent translation initiation, and elongation may be inhibited during and after TKA surgery. We propose that cap-dependent translational events occurring during surgery may precipitate postoperative changes in muscle cells that contribute to the etiology of muscle atrophy following TKA.

Figures

Fig. 1.

Study design. Bilateral midthigh MRI was performed 2 wk prior to and 2 wk post-TKA by the same technician. Arrows indicate skeletal muscle biopsies. A baseline muscle biopsy was acquired in the OR ∼1 h after a single-injection femoral nerve block was given and following anesthesia but before inflation of the tourniquet (see Table 1 for details). 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.

Fig. 2.

Increased gene expression of eIF4G and the activating transcription factor 4 (ATF4) transcription target growth arrest and DNA damage 45A (GADD45A). Transcripts for eukaryotic initiation factor 4 gamma 1 (eIF4G1) increased 19% (P = 0.048) (A), eIF4G2 increased 19% (P = 0.04) (B), and eIF4G3 increase 28% (P = 0.01) (C) from baseline to reperfusion. The transcription target of ATF4, GADD45A mRNA, significantly increased during ischemia 37% (P = 0.02) and showed a trend to increase, by 30% (P = 0.11) above baseline, during reperfusion (D). Results were normalized to GAPDH. Data are expressed as mean fold change ± SE (n = 12). *P ≤ 0.05 vs. baseline.

Fig. 3.

Dephosphorylation of Akt and 4E-BP1 during ischemia and reperfusion. Phosphorylation status of Akt at Ser473 was significantly decreased by 36% (P = 0.01) during ischemia and 37% (P = 0.02) during reperfusion (A). 4E-BP1 Thr36/47 was hypophosphorylated during ischemia by 29% (P = 0.04) and 22% (P = 0.046) during reperfusion (B). 4E-BP1 hypophosphorylation is indicative of blunted cap-dependent translation, which will block protein synthesis. Phosphorylation status was made relative to total protein. Actin served as a loading control. Data are expressed as means ± SE (n = 13). *P ≤ 0.05 vs. baseline.

Fig. 4.

Increased Mnk1 and eIF4E total protein during ischemia. Mnk1 total protein content significantly increased 54% (P = 0.03) during ischemia and returned to baseline during reperfusion (A). eIF4E total protein content significantly increased 51% (P = 0.03) during ischemia and returned to baseline during reperfusion (B). Actin served as a loading control. Data are expressed as means ± SE (n = 11). *P ≤ 0.05 vs. baseline.

Fig. 5.

Increased phosphorylation of eukaryotic elongation factor-2 (eEF2) during ischemia and reperfusion. Phosphorylation status of eEF2 Thr36 significantly increase by 25% (P = 0.04) during ischemia and 43% (P = 0.01) during reperfusion compared with baseline. Phosphorylation status was made relative to total protein. Actin served as a loading control. Data are expressed as means ± SE (n = 11). *P ≤ 0.05 vs. baseline.

Fig. 6.

Downstream targets of eIF2α Ser51. Total protein content of ATF4 increased 96% during ischemia (P = 0.01) and returned to baseline during reperfusion. A: total protein content of GADD34 increase 83% during ischemia (P = 0.03) and returned to baseline during reperfusion (B). Actin served as a loading control. Data are expressed as means ± SE (n = 13). *P ≤ 0.05 vs. baseline.

Fig. 7.

Representative MRI cross section showing acute quadriceps muscle atrophy 2 wk post-TKA. Representative cross section of the midthigh before (Pre) and post-TKA (2 wk) of the (TKA) leg and the contralateral, nonoperative, (Con) leg. Compared with before surgery, midthigh quadriceps volume in the TKA leg (TKA) decreased 12% 2 wk after surgery (P = 0.012). Similar muscle atrophy (6% decrease) was observed in the nonoperative (Con) lower extremity 2 wk after surgery but was not significant (P = 0.11). Before surgery, midthigh quadriceps volume was smaller, by 7% in the operative vs. nonoperative lower extremity (P = 0.05). Pre- and post-TKA images are obtained at the exact midthigh region of interest. Differences in appearances of the TKA leg are due to issues related to reduced range of motion postoperatively (knee flexion contracture), preventing full extension of the knee. RF, rectus femoris; VL, vastus lateralis; VI, vastus intermedius; and VM, vastus medialis.

Fig. 8.

Ischemia-reperfusion inhibits multiple proteins regulating cap-dependent translation. The schematic diagram of proteins regulating signaling pathways controlling cap-dependent mRNA translation initiation and elongation. Ischemia-reperfusion inhibits cap-dependent translation initiation via inhibition of Akt-mTOR pathway and availability of 4E-BP1 to bind to and inhibit eIF4E association with eIF4G to form an active mRNA cap-binding complex (eIF4F). Ischemia-reperfusion also activates endoplasmic reticulum (ER) stress via eIF2α, which inhibits cap-dependent translation initiation. ATF4 and GADD34, each downstream components of eIF2α, are proteins involved in recovery from cell stress and are upregulated during ischemia and provide inhibitory feedback onto eIF2α. Alterations due to ischemia/reperfusion (I/R) additionally stimulate mRNA for all three isoforms of eIF4G. Together, these data may potentially provide some insight into the dramatic quadriceps atrophy (−12%) occurring within 2 wk post-TKA.