- Total knee arthroplasty (TKA) is the most common and cost-effective treatment for older adults with long?standing osteoarthritis.
- During recovery from TKA the surgical leg is swollen, very weak and has limited range of motion about the knee joint. This requires the patient to use crutches or a front-wheeled walker for mobility. During the early period of recovery muscle atrophy occurs, which for older adults is likely to remain permanent, and may impact long-term recovery.
- Most orthopedic surgeons and health care professionals will agree that identifying every potential contributing factor causing post-operative changes will greatly improve treatment including enhanced physical therapy strategies to maximize recovery of functional mobility and quality of life. This includes determining the extent to which tourniquet use during TKA impacts muscle metabolism at the cellular and molecular level.
In 2008, more than 650,000 primary total knee arthroplasties (TKA) were performed in the United States at a cost of $9 billion, and it is estimated that the number of annual cases will increase to 3.48 million by 2030 (22). Despite the success of TKA in mitigating the chronic pain associated with osteoarthritis (OA) and restoring functional mobility for many older adults, there are residual consequences, such as persistent muscle atrophy, which may limit the ability of patients to achieve long-term functional mobility and quality of life compared with healthy peers (28).
Using magnetic resonance imaging, we have measured a 14% reduction in quadriceps volume within 2 wk of TKA (9). This corresponds to a loss rate of 1% per day. In contrast, at 6 wk after surgery, we have measured a total change in quadriceps volume of −18% in the same patients (9). In other words, approximately 80% of the muscle atrophy occurred within the first 14 d of surgery (Fig. 1). Muscle atrophy approaching 1% per day suggests that active catabolic processes may be taking place, as opposed to a reduction in the rate of muscle protein synthesis at rest and in response to protein ingestion, which are estimated to decrease by as much as −60% and −50%, respectively (35). Our interpretation is that muscle atrophy after TKA is influenced by several factors ranging from postoperative reductions in ambulation and energy intake to local and systemic effects of inflammation triggered by surgery, which in our view is exacerbated by ischemia-reperfusion (IR) injury (Fig. 2). We also suspect that obesity, reduced activity levels caused by OA-induced knee pain, and the older age of this patient cohort make muscle cells particularly sensitive to IR injury.
Insight into the mechanisms of atrophy after TKA is essential, considering that muscle loss in older patients is likely permanent (10). Age-associated muscle dysfunction results from reduced muscle mass, strength, and functional capacity (i.e., sarcopenia). Sarcopenia occurs with normal healthy aging and is associated with elevated risk for falls, physical disability, and loss of independence (10). The estimated health care cost caused by sarcopenia in the United States in 2000 was $18.5 billion (15). Reducing the prevalence of sarcopenia by 10% could save $1.1 billion per year (15). Whereas sarcopenia progresses at a rate of 1% per year in healthy older adults, surgeries such as knee replacement accelerate sarcopenia (9). Although eliminating knee pain with surgical remediation (i.e., TKA) is the most appropriate course of action and clinically necessary, available evidence suggests that long-term functional gains are hampered by the inability to regain muscle mass (40). Our view is that minimizing or preventing muscle loss during this critical “14-d window” allows for maximal recovery. Based on work from our laboratory and others, we have developed a potential rationale for our hypothesis that tourniquet-induced IR injury occurring in older adults during and after TKA alters cell and tissue metabolism in a way that may contribute to muscle atrophy (Fig. 3).
Cells respond to acute oxygen deprivation by activating an evolutionarily conserved molecular cascade that works to stabilize the microenvironment until oxygen concentration is normalized. The series of molecular events begins with immediate reductions in cell metabolism, favoring decreased energy demand, as well as activation of cell signaling pathways that regulate proteins and gene clusters associated with the cell stress response(s). The degree of cell stress increases as the duration of O2 deprivation continues. Once O2 levels are reestablished, a second stage of injury occurs during reperfusion when metabolic by-products that have accumulated during ischemia are released systemically, and oxygen-rich blood is made available to the previously anoxic cells and reactive oxygen species (ROS) generation temporarily exceeds the capacity of endogenous antioxidants to neutralize them. The combined effect of ischemia (or anoxia) followed by reperfusion is referred to as ischemia-reperfusion injury.
IR injury can occur during organ transplantation; limb reattachment; general surgery; stroke and head injury; myocardial, kidney, liver, and intestinal infarction; and, more frequently, as a consequence of tourniquet use during orthopedic surgeries. It is estimated that a tourniquet is used on average 20,000 times per day globally during routine surgeries (27). Tourniquets are used by surgeons to prevent bleeding into the surgical site to maximize visualization of the wound and prevent blood loss. During surgeries involving joint arthroplasty, tourniquet use also helps prevent bleeding from the resurfaced bone, allowing proper bone implant cementing. Orthopedic surgeons routinely use tourniquets during procedures involving the upper and lower extremities. Examples of lower-extremity use are foot and ankle surgeries, anterior cruciate ligament (ACL) reconstruction, meniscal repair, open reduction internal fixation (ORIF) of fractures, and TKA. This review will focus primarily on the effects of tourniquet use in older patients having TKA; however, the potential carryover to many orthopedic settings is significant.
The 2-h Window: Is It Really Benign?
It is accepted generally that tourniquet times of 2 h or less are not associated with permanent damage. This “2-h window” was derived from a considerable body of evidence from animals and clinical studies, which clearly show that tourniquet use produces morphological, electrophysiological, and functional impairments that are significant and increase in severity as tourniquet duration increases but appear, in general, to be reversible with time (12). A review from 2012 entitled “Safe Tourniquet Use: A Review of the Evidence” included eight “high-quality clinical studies” to support the recommendation that, “while early electrophysiological changes and muscle atrophy are detectable, long-term functional differences are rare, and outcomes between standard tourniquet and no-tourniquet groups are equivocal” (see Fitzgibbons et al. (12) and references therein). However, a more critical evaluation of the existing data is needed. Animal data may not translate to humans, and results from a few human studies may not necessarily translate to all settings. This review is not intended as a deconstruction of articles cited by Fitzgibbons et al. (12) but argues that these eight studies should not be used as “evidence” of the safety of tourniquet for all surgeries — in particular, TKA in older adults. For younger adults having ACL reconstruction, ORIF, or meniscectomy, tourniquet-induced IR injury may resolve with time and, therefore, be clinically acceptable. For example, of the eight studies included as evidence to support the clinical view that tourniquet use has no lasting negative impact, Fitzgibbons et al. (12) cite only one study conducted in older TKA patients. Main findings showed that pain was greater at 6 h after TKA in the tourniquet group but were similar at 24 and 48 h. Flexion range of motion was greater in patients without tourniquet at 5 d after TKA but was not different at 10 d and 3 months after TKA. Measures of atrophy, strength, and functional mobility were not performed (38). The remainder of the studies cited were conducted with younger adults during and after ACL reconstruction (6), with meniscectomy patients (39), with young patients having ORIF (19), and with subjects requiring surgery for femoral shaft fractures (control group) and tibial and ankle fractures (tourniquet group) (24) that reported changes in measures of pulmonary function.
Tourniquet use is a specific concern in older adults having TKA because their muscles may be sensitive to tourniquet-induced IR injury, but the literature about this patient population is unclear. There is evidence that tourniquet versus nontourniquet groups do not differ in older adults having TKA (5,38). However, many studies find clinically meaningful differences favoring the no-tourniquet group, such as less pain (4,11), better range of motion (4,11,38), less limb swelling (4), greater strength (8), and faster recovery (11). To gain a better understanding of the changes in skeletal muscle occurring during and after TKA at the molecular and cellular levels, our research group has collected muscle biopsy samples in the operating room before tourniquet inflation (baseline), during maximal ischemia, and during reperfusion, usually within 10 to 25 min. Our most recent data (33) examine changes in cell size and gene expression profiles from muscle samples collected about 2.3 h after TKA.
Our research focuses on mitigating muscle atrophy and functional mobility deficits that result from TKA. This surgery involves replacing the proximal tibial plateau, posterior patella, and distal femur with prosthetic components to eliminate the source of pain caused by chronic OA, which is the leading diagnosis for TKA. Currently, OA affects 60% of adults older than 65 yr in the United States and is the leading cause for hospitalization of adults aged 45 to 84 yr (34). OA of the knee is an insidious condition characterized by progressive loss of cartilage and bone, changes to tendon and ligaments, joint swelling, and painful ambulation. Inevitably, the pain from bone-on-bone contact necessitates surgical remediation via TKA, but the time from onset of symptoms to joint replacement may take years, during which health and activity are impacted negatively. Thus, it is not surprising that patients with OA have about two thirds the muscle mass of older adults without OA (28).
The following is a brief review of the cell physiology of ischemia and reperfusion injury, along with results from our studies with older adults during and immediately after TKA surgery showing that cell signaling of muscle cells is altered. Included are brief reviews of the mechanisms governing muscle protein synthesis and degradation as well as cell and endoplasmic reticulum (ER) stress responses. To supplement this brief review, readers are encouraged to take advantage of the many excellent published reviews that cover relevant areas: immune and complement activation of IR injury (3), signaling pathways controlling translation (36), mitochondrial ROS generation (31), ER stress and mitochondrial involvement in cell survival (26), ER stress in skeletal muscle (7), clinical perspective on safe tourniquet use (12), and cell biology of IR injury (18).
Under normoxic conditions, basal cell metabolism relies primarily on complete oxidation of long-chain fatty acids in the mitochondria through beta-oxidation generating acetylCoA followed by citric acid cycling, electron transport chain pumping of hydrogen ions, and adenosine triphosphate (ATP) synthase transferring an inorganic phosphate to adenosine diphosphate (ADP) to form ATP. Oxygen’s role is to accept electrons from the electron transport chain, which combine with two hydrogen ions (H+) to form molecular water, allowing the flow of electrons (and H+ pumping) to proceed. When blood flow is cut off, oxygen tension and myoglobin saturation fall, resulting in tissue ischemia/anoxia regionally depending on local metabolic and cellular adjustments. It has been shown that, in human skeletal muscle, molecular oxygen (O2) levels decrease by 50% within 5 min of ischemia and are reduced further after 15 and 30 min, reaching levels of 28% and 19% of baseline, respectively (29).
Several mechanisms are available in muscle cells to buffer ATP levels. The phosphagen system is the immediate go-to source for energy via the coupled creatine kinase reaction (Creatine phosphate + ADP + H+ → Creatine + ATP) and adenylate kinase (referred to as myokinase in muscle cell metabolism) activity (2ADP → AMP + ATP). Nuclear magnetic resonance spectroscopy has shown that phosphocreatine (PCr) concentrations fall by one half within 14 min of ischemia (25). This and another study (29) have shown that ATP concentration remains steady throughout 2.5 h of ischemia, whereas the concentrations of ADP, adenosine monophosphate (AMP), inosine monophosphate (IMP), and inorganic phosphate (Pi) are elevated. However, other researchers have measured a decrease in ATP concentration in skeletal muscle under ischemic conditions (17). In general, once ATP levels are depleted, cell death and necrosis result.
The phosphagen system is sensitive to alterations in the concentrations of substrate and products and acts in synergy with glycogenolysis during ischemia. AMP generated by the adenylate kinase reaction increases the activity of phosphorylase, which breaks down glycogen for ATP production (limited to the cytosol during O2 lack). In addition to stimulating phosphorylase, AMP increases the activity of phosphofructokinase by allosteric regulation and enhances the activity of the purine nucleotide cycle (PNC). The PNC converts AMP to IMP, which combines with the amino acid aspartate to form ribose 5-P adenylosuccinate and the anaplerotic (increase in citric acid cycle intermediates) formation of fumarate by adenylosuccinase. The PNC is highly active in Type II muscle fibers during intense muscle activity, which parallels metabolic activity during O2 lack.
During the shift from oxidative to O2-depleted energy metabolism, the cell becomes acidic because of the accumulation of protons (H+) from excessive reliance on glycolytic ATP hydrolysis to meet energy demands. In one study, glucose levels decrease significantly within 15 min of ischemia and drop to less than 50% of basal levels within 30 min, reaching nadir levels of approximately 35% of basal within 45 min (20). Lactate concentrations from this study showed inverse reciprocal increases, greater than 150% of baseline at 15 min and greater than 250% at the end of the ischemic period, approximately 75 min, indicating a rapid increase in anaerobic glycolysis. In response, the cell removes protons from the cytoplasm by coexport with lactate and by increasing Na+-H+ exchange. Thus, there is an accumulation of intracellular Na+, which in the ischemic heart has been shown to increase linearly by fourfold within 30 min (31). Na+ accumulation also may occur because of a decrease in efflux secondary to reduced ATP availability to drive Na+/K+ exchange via the Na+/K+ ATPase pump. Intracellular concentrations of Ca2+ are kept several orders of magnitude lower than the sarcoplasmic reticulum (SR) (ER) and extracellular levels under homeostatic conditions. This is accomplished by several pumps, including the SR Ca2+ATPase, which pumps Ca2+ into the SR; the sarcolemmal Ca2+ATPase, which pumps Ca2+ out of the cell; and the Na+-Ca2+ exchanger, which exchanges intracellular Ca2+ with extracellular Na+. These pumps rely on a constant supply of energy in the form of ATP, which may be limited during ischemia (31). The accumulation of positively charged ions in the cytoplasm attracts negatively charged chloride ions. The combined increase in ion concentration within the cytoplasm causes bulk movement of water from the cell exterior to the cell interior by osmosis, facilitated by ubiquitously expressed aquaporin channels within the sarcolemma. As sarcoplasmic levels of lactate accumulate, it progressively inhibits glycolytic ATP production and exacerbates acidosis, further impairing the ability of the above-mentioned energy-requiring pumps to maintain ion gradients, resulting in cell swelling and rupture.
The duration of ischemia is the primary factor mediating the magnitude of cellular injury; however, reperfusion with oxygen-rich blood exacerbates initial damage caused by ischemia. Although reperfusion is obligatory for cell survival, it creates two immediate challenges for the patient. First, metabolic by-products that accumulated during the ischemic phase are now disseminated systemically via circulation, which is of considerable concern to physicians because this may lead to remote organ involvement/injury. Second, the previously anoxic cells and tissues are now exposed to oxygen, which can lead to oxidative damage in myocytes by promoting formation of ROS generation (18).
Under basal conditions, most of the oxygen entering cells are reduced to molecular water in the mitochondrial matrix. However, highly reactive superoxide anion (O2−·) can be formed when a single electron attaches itself to the antibonding orbital of molecular oxygen (32). A primary site of O2−· production during IR is the mitochondrial respiratory chain, particularly NADH-ubiquinone oxidodehydrogenase (complex I) and ubiquinone—cytochrome c oxidoreductatse (complex III). Superoxide also is formed enzymatically by the membrane-bound enzymes nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidases and xanthine oxidase and uncoupled nitric oxide synthase. If not enzymatically converted to hydrogen peroxide (H2O2) by endogenous superoxide dismutase (SOD), superoxide will react with cellular proteins, membrane phospholipids, RNA, and DNA, contributing to cellular injury. Superoxide dismutase enzymes exist in two forms: copper zinc (CuZn) SOD expressed in the cytosol and interstitial space and manganese (Mn) SOD located in the matrix of the mitochondria. SOD represent the first line of defense against oxygen radicals and under resting and exercise conditions are capable of mediating ROS production. Once generated, H2O2 can diffuse easily through cell membranes (e.g., mitochondrial, nuclear envelope, out of the cell) and react with transition metals Fe2+ and Cu2+ to form the highly reactive hydroxyl radical (·OH), unless it is reduced to H2O and O2 by catalase or glutathione peroxidase (32). Under basal conditions, endogenous antioxidant enzymes can keep pace with ROS formation. However, after ischemia, when metabolic by-products dilate blood vessels and oxygen is made available to the extremity, local ROS generation is accelerated at a rate that outpaces the capacity for antioxidant enzymes to neutralize them and damage occurs.
Muscle Protein Synthesis During TKA-Induced IR Injury
Protein turnover is regulated in skeletal muscle cells by transcriptional and translational signaling mechanisms. A balance between anabolic and catabolic processes is coordinated primarily through the phosphoinositol 3-kinase/Akt/forkhead boxO3a (FOXO3a) pathways. During anabolic conditions, such as with insulin-like growth factor-1 stimulation, Akt activation of the mammalian target of rapamycin complex 1 (mTORC1) stimulates the activation of proteins controlling translation initiation and elongation. Translation initiation is the processes whereby mRNA destined for expression as a protein is attached to a mature ribosomal complex. Elongation denotes the process of peptide elongation when each amino acid is attached to the next via disulfide bonds according to the nucleotide sequence within the mRNA. In all cells, the process of protein synthesis is highly regulated and requires significant energy, accounting for 20% to 30% of total energy consumption of a cell (36). The stoichiometry works out to two ATP hydrolyzed for each amino acid activated and bound to t-RNA and two GTP for synthesis of each peptide bond formed during elongation. Thus, for every 1 mole of amino acids incorporated into polypeptides, 4 moles of ATP is used. Under conditions that significantly reduce ATP supply, energetic processes such as protein synthesis are inhibited until O2 supply is normalized.
Translation initiation starts with the formation of the ternary initiation complex consisting of eIF4A, eIF4G, and eIF4E (forming the eIF4F complex). The eIF4F complex binds with the 40S ribosomal subunit and stabilizes the preinitiation complex before it binds to the 7-methylguanosine cap (m7G) structure located at the 5′ end of the mRNA transcript. The regulation of eIF4E availability is a primary mechanism for controlling eIF4F complex formation. Formation of eIF4F is blocked by the translational repressor 4E-BP1 (eIF4E-binding protein 1), which inhibits eIF4E from complexing with eIF4A and 4G. Like turning a switch on and off, phosphorylation of 4E-BP1 attenuates its ability to bind eIF4E while dephosphorylation does the reverse, allowing 4E-BP1 to bind eIF4E tightly and inhibit eIF4F complex formation. Thus, under ischemic conditions, 4E-BP1 becomes dephosphorylated, inhibits eIF4F complex formation, and reduces the rate of translation initiation. Additional regulation involves phosphorylation of eIF4E at Ser-209, which decreases the binding affinity of eIF4E for capped mRNA. Stress- and/or cytokine-activated signaling regulates phosphorylation at Ser-209 to a greater extent than anabolic stimuli (36) and suggests multiple levels of local control during cell stress. We have shown that, during TKA phosphorylation of eIF4E at Ser-209, the target of the translational repressor protein 4E-BP1 is increased relative to presurgery (1). 4E-BP1 is a downstream target of mTORC1, which is regulated by Akt. mTORC1 inhibition by downregulation of Akt activates autophagy and inhibits translation initiation. We have demonstrated that, during and after TKA, Akt and 4E-BP1 are hypophosphorylated, indicating a downregulation of translation initiation (1,14,37). In addition to initiation, further regulation of translation occurs via proteins controlling elongation. Eukaryotic elongation factor-2 (eEF2) promotes the translocation of the peptidyl-tRNA from the A-site to the P-site of the ribosome. eEF2 is inhibited by phosphoryation, which is controlled by eEF2 kinase (eEF2K), which itself is controlled by mTORC1. Thus, reduced activity of Akt downregulates mTORC1 signaling and results in decreased phosphorylation of 4E-BP1, which inhibits translation initiation, as previously described, and increases phosphorylation of eEF2, which attenuates translation elongation. We have shown that eEF2 is phosphorylated during and after TKA. Assimilated, during TKA, three significant control points regulating protein synthesis are altered: Akt, 4E-BP1, and eEF2 (1,14,37). These data suggest that, during TKA-induced IR injury, muscle protein synthesis is downregulated by reducing translation initiation and elongation (Fig. 4).
Muscle Protein Degradation and Cell Stress During TKA-Induced IR Injury
The process of muscle protein degradation is initiated, in part, by downregulation of Akt signaling (Akt dephosphorylation). Reduced Akt activity dephosphorylates the transcription factor, FOXO3a, which becomes activated and translocates from the cytosol to the nucleus. Nuclear FOXO3a binds to the promoter region and upregulates the transcription of proteins involved in ubiquination and the 26S proteasome (23). The 26S ubiquitin-proteasomal complex catabolizes soluble and myofibrillar (actin, myosin, and titin, for example) proteins (23). The ubiquitin-proteasomal pathway is upregulated rapidly by two muscle-specific E3 ubiquitin ligases, muscle atrophy F-Box (MAFbx) and muscle RING finger 1 (MuRF1). MAFbx and MuRF1 are transcriptional targets of FOXO3a, which is regulated by Akt activity. The ubiquitin-proteasomal pathway also can be activated by the transcription factor nuclear factor-κB (NF-κB), which is a known target of tumor necrosis factor-α, a cytokine that can bind to muscle cell membrane receptors. We have measured increases in protein and transcripts for MAFbx and MURF1 during TKA, suggesting that Akt downregulation may allow for catabolism to occur (1).
FOXO3a also may be acted on by the stress-activated protein kinase (SAPK)/Janus kinase (JNK) pathway. We have measured an increase in SAPK/JNK transcript levels and protein concentrations in the cytoplasm during TKA (1). We also have measured an increase in p38 mitogen-activated protein kinase (MAPK) during TKA (1). Activation of p38 MAPK has been shown to occur in cardiac cells after IR injury (2). Stimulation of the SAPK/JNK/MAPK pathway during TKA suggests a role for oxidative stress as a potential factor stimulating the activation of the catabolic pathway.
BCL-2 proteins are important for regulating the permeability of the outer mitochondrial membrane. If the outer mitochondrial membrane becomes permeabilized, proteins within the inner mitochondrial membrane leak out and can activate proapoptotic pathways, notably, caspase-3. We have measured a trend (P = 0.054) for BCL-2 protein to accumulate in the cytoplasm during ischemia (1). In addition, we have measured a decrease in nuclear levels of the BCL-2 19-kDA-interacting protein 3 (BNIP3) during TKA. BNIP3 is a pro–cell death member of the BCL-2 family of proteins that govern mitochondrial homeostasis and has been shown to stimulate autophagy in myocardial cells after IR injury. Furthermore, hypoxia and low pH have been shown to activate BNIP3 and initiate cell death pathways in cardiac cells (21). Taken together, the data suggest that, during TKA, altered signaling via the Akt-FOXO3a pathway upregulates MAFbx and MuRF1, which stimulate ubiquitin-proteasomal–dependent protein degradation and may play a role in mitochondrial function via the BNIP3 pro-cell death pathway (Fig. 4). A newly published article by Jawhar et al. (16) shows that, compared with TKA performed without tourniquet, proteolytic activity was elevated in vastus medialis muscle tissue after 60 min of continuous tourniquet application. To our knowledge, besides the studies we have conducted, there is a dearth of studies similar to Jawhar et al. (16) looking at the acute effects of tourniquet-induced anoxia on markers of catabolism and stress-related signaling at the tissue level. Either way, their data (16) corroborate our view that cell catabolism is altered uniquely during TKA under anoxic (tourniquet) conditions.
ER Stress Response During TKA-Induced IR Injury
The ER can be divided into rough and smooth; rough equating to the colocalization of ribosomes within the membrane and smooth representing ER membrane without ribosomes. In skeletal muscle, the smooth ER and the SR are the same and are juxtaposed with mitochondria. The SR functions as a calcium reservoir, with the calcium ATPase pump continuously hydrolyzing ATP to transport cytosolic calcium back into the SR against its electrochemical gradient. The rough ER membrane is contiguous with the nuclear envelope and facilitates the folding of intraorganelle, secretory, and membrane-bound proteins. The ER maintains high Ca2+ concentrations necessary for proper protein folding and protein chaperones to perform properly (26). Disruption in Ca2+ concentrations, because of either sarcolemmal disruption and/or the inability to maintain high calcium levels in the ER, leads to misfolding of proteins, which accumulate in the ER lumen. Alterations in redox state and glucose levels also may cause disruptions in protein folding; in particular, glucose is necessary for protein glycosylation during posttranslational processing/modification. Lack of oxygen also will inhibit the enzyme protein-disulfide isomerase directly, which requires adequate oxygen to function properly, and alter protein folding. The signaling cascade initiated by ER stress is known as the unfolded protein response (UPR).
Unfolded (misfolded) proteins are sensed in the lumen of the ER by the chaperone protein, binding Ig protein (BIP), also known as glucose-regulated protein of molecular weight 78 (GRP78). BIP has a high affinity for hydrophobic segments of the unfolded protein. When the protein is folded properly, these hydrophobic regions are found within the interior. Chaperones other than BIP, along with Ca2+, play a role in sequestering these hydrophobic regions during the folding process. However, if misfolding occurs or Ca2+ homeostasis is not maintained, these hydrophobic regions are exposed and lure BIP away from the ER membrane. Detachment of BIP allows the release of transmembrane proteins such as PKR-like ER kinase/pancreatic eIF2 kinase (PERK/PEK), activating transcription factor 6 (ATF6), and inositol requiring 1α (IRE1) (Fig. 2).
On release from BIP, PERK is released from the ER membrane and binds to and phosphorylates eukaryotic initiation factor 2-alpha (eIF2α) at Ser51. eIF2α phosphorylation at Ser51 inhibits cap-dependent translation initiation. In addition to the critical role 4E-BP1 plays in inhibiting the binding of the 5′ cap of mRNA to the ribosomal complex (described above), translation initiation involves translocation of the initiator methionyl tRNA (met-tRNAi) to the AUG start codon of the mRNA. eIF2 plays a key role in this process when phosphorylated by blocking the GTP exchange for eIF2-GDP, effectively binding eIF2α with the guanine exchange factor, eIF2B, inhibiting the formation of a ternary complex consisting of methionine-tRNA and eIF2 coupled to GTP. Thus, eIF2α phosphorylation at Ser51 is a primary mechanism for downregulating global rates of protein synthesis; we have shown that eIF2α phosphorylation is increased during TKA (14). eIF2α phosphorylation also selectively stimulates the expression of ATF4 and growth and arrest and DNA damage 34 (GADD34). We have measured an increase in eIF2α phosphorylation during TKA (14), as well as increases in ATF4 and GADD34 proteins (37). Each of these proteins (ATF4 and GADD34) is upregulated by eIF2α activity in response to skeletal muscle cell stress. We also have measured increased expression of GADD45A transcripts (mRNA), which is a downstream target of ATF4. GADD34 works to feedback on eIF2α and inhibit its activity. The increase in GADD34 protein may indicate that, during TKA-induced IR injury, appropriate feedback mechanisms are activated to attenuate initial inhibition on protein synthesis during ischemia and reperfusion. Finally, we have measured an increase in glycogen synthase kinase 3β (GSK3β) protein levels during TKA (1). GSK3β can be activated when signaling through Akt is downregulated. GSK3β has been shown to be a negative regulator of eIF2B, which regulates eIF2α phosphorylation and inhibits formation of a ternary complex. GSK3β also is a known regulator of the transcription factor p53, which is activated by cell stress and especially damaged DNA. Collectively, the above results suggest that PERK activation and phosphorylation of eIF2α and downregulation of Akt activity have parallel implications for inhibiting translation initiation at multiple control points during TKA-induced IR injury in response to ER and cell stressors.
On release from BIP, ATF6 translocates to the Golgi where it is cleaved and then translocates again to the nucleus, where it upregulates a process involving a set of genes controlling selective protein catabolism referred to as ERAD. After it is released from BIP, IRE1α targets XBP1 mRNA via a ribonuclease domain that splices out a 26-nt hairpin from the XBP1 transcript, thus changing its reading frame. This change in reading frame leads to translation of a spliced XBP1 (XBP1s) protein with a C-terminal end that is an active bZIP transcription factor and a stimulator of UPR target genes. Activated IRE1α also stimulates ER membrane-associated initiator caspases that activate terminal apoptotic caspases (30), in particular, caspase 3 (CASP3). Increase in CASP3 activity is significant under these conditions during TKA because it has been shown to play a role in muscle protein degradation via the ubiquitin-proteasome system by targeting actin and myosin, proteins important for muscle contraction. Together, these three branches of the UPR act to alleviate ER stress by downregulating protein synthesis and upregulating chaperones and folding proteins in the ER (7) (Fig. 4).
Cell Swelling and Transcriptional Changes During TKA-Induced IR Injury
A recent finding from our laboratory is that muscle cells tend to swell during ischemia (not significantly so, but a trend, nonetheless) and are significantly swollen after 2 to 3 h. The most obvious cause is the imbibing of water into the cell in response to osmotic changes resulting from shifts in bulk ion movement into the cytosol as the cell attempts to buffer the drop in pH resulting from anaerobic glycolysis. Briefly, the cell exports protons via Na+-H+ exchange, which leads to Na+ accumulation inside the cell. Accumulation of Na+ also may occur if ATP is depleted or is unavailable for use by the Na+/K+ ATPase pump, although it is not clear if the pump begins to decline before ATP levels begin to drop, and there are some data suggesting that acute posttranslational modifications of the pump occur during the ischemic phase (13). As the Na+ ions accumulate, negatively charged chloride ions (Cl−) traverse the cell membrane in an attempt to balance the electrical gradient, which has the effect of driving out potassium ions, while increasing the net ion concentration inside the cell, thus, drawing in more water. Finally, as the intracellular concentration of Na+ increases, the gradient used by the Ca2+-Na+ exchanger is rendered null and Ca2+ accumulates. Similar to Na+, either a decline in ATP levels or posttranslational modifications to the Ca2+ATPase result in inhibition of its pumping ability, and a net increase in Ca2+ occurs. The source of the Ca2+ is the SR/ER membrane, and, as the cytosolic concentration increases, it enters the mitochondrial matrix. Entry of Ca2+ into the cytosol and mitochondria has negative consequences. First, cytosolic Ca2+ can stimulate the calcium-activated calpain system. Calpains are nonlysosomal cysteine proteases that are activated in the presence of cytosolic Ca2+. The calpain proteases exist in an inactive state under basal conditions but when activated can have interactions with the 26S proteasome and Akt and specifically target myofibrillar proteins such as actin and myosin. The rise in cytosolic Ca2+ can result in apoptosis and/or necrosis. Second, the accumulation of cytosolic Ca2+ causes an inward flux into the mitochondrial matrix, which is a potential mechanism for initiating mitochondria-mediated apoptosis, potentially via the mitochondrial permeability transition pore (mPTP). Either way, muscle swelling occurs about 2.3 h after TKA (33), and we were able to measure an increase in cross-sectional area of all three fiber types (Types I, IIa, and IIx) (Fig. 5).
To further elucidate the effects of IR injury after TKA with tourniquet on genomic changes, we performed transcriptional profiling experiments using QuantSeq 3′ mRNA (Lexogen, Greenland, NH) sequencing. Nearly one half of the genes we found to be expressed differentially play a role in cell stress response, including genes involved in MAPK, JNK, Akt, NF-κB, and JAK-STAT signaling. To visualize these results, we mapped our set of differentially expressed genes onto gene regulatory network diagrams we constructed using established annotated pathways (Biocarta and Kegg) or, for less well-characterized genes, relied on literature searches to determine putative interactions (Fig. 6). By constructing these gene regulatory network maps, it became clear that TKA with tourniquet leads to a significant cell stress response. For example, one of the most significantly upregulated genes in this study, PIM-1, is hypoxia inducible, synergizes with MYC, a transcription factor involved in a variety of cell stress responses, is regulated by the JAK/STAT pathway, and regulates FOXO3a activity as well as MAPK activity. Moreover, Akt and JNK interact with FOXO3a and are regulated by inflammatory cytokines CXCL2, CXCL1, and CCL2, which we show in our transcriptional profiling are upregulated. As well, MuRF1, a key regulator of the proteasomal pathway, is regulated by NF-κB (and by FOXO3a), which is regulated by several molecules in our data set, including NFKBIZ, CYR61, KDM6B, MT1M, TNFRSF1A, HES1, and BCL3 (33). Therefore, our gene expression analysis shows that TKA with tourniquet induces expression of the molecular components of muscle atrophy. Therefore, PIM-1 is clearly a critical “node” in the gene regulatory network controlling cell stress response. From a clinical standpoint, this is an important observation because it suggests that older individuals retain the capacity to mount a significant cellular defense response after TKA and tourniquet application (33).
In addition to the insidious loss of muscle mass in older healthy adults of 1% per year, life events, such as major knee surgery (9,28,37,40), accelerate sarcopenia. The clinical view that tourniquet use is benign in older adults having TKA must be reevaluated in light of our recent data showing reductions in anabolic signaling (37) and upregulation of the catabolic FOXO (1) and UPR (14) pathways that precede, by a few hours, muscle cell swelling and gene clusters associated with cell stress (33). Muscle atrophy after TKA occurs at a rate of 1% per day (14% loss within 2 wk) and slows significantly between 2 and 6 wk after TKA (i.e., −14% loss at 2 wk vs -18% at 6 wk) (9). Our interpretation is that muscle atrophy after TKA is an active process involving muscle catabolism rather than purely a reduced rate of muscle protein synthesis at rest and in response to meal (protein) ingestion (35). Moreover, in light of the few benefits of using tourniquet, such as providing a clear surgical field and proper bone implant cementing, the potential positives of not using a tourniquet, such as less pain (4,11), better range of motion (4,11,38), less limb swelling (4), greater strength (8), and faster recovery (11), may benefit from further study at the molecular and cellular level to determine the acute affects of anoxia on muscle metabolism during the first 14 d after surgery and how they may impact long-term physiologic and functional outcomes.
By 2030, the number of older adults having primary TKA in the United States is projected to increase by 673% to more than 3.4 million surgeries performed annually (22). The increase in the number of older adults having TKA surgery presents a significant financial challenge to our health care system, which in 2000 spent $18.5 billion on sarcopenia-related care (15). Thus, devising ways to mitigate the accelerated sarcopenia that occurs primarily within the first 2 wk after surgery (9) is of clinical and fiscal importance. Our approach has been to conduct preliminary proof-of-principle clinical studies to determine if essential amino acid supplementation offsets atrophy in older adults having TKA compared with placebo. Initial results were positive (9), although some muscle atrophy still occurred. We also have examined the potential mechanisms of action that cause the effects of IR injury on muscle metabolism acutely and on functional mobility during rehabilitation. Our investigations have documented effects of tourniquet-induced IR injury on skeletal muscle cell metabolism and signaling and demonstrate the need to reevaluate current clinical understanding that ischemia/anoxia has no lasting negative effects on muscle cells in older adults after TKA.
The author thanks Dr. Adam Chicco for his expertise and comments on the IR section of this article as well as Dr. Jonathan Muyskens and Lisa Strycker for editorial assistance.
This work was supported in part by the National Institutes of Health grants K01-HD57332 (NICHD) and R01-AG046401 (NIA) (ClincialTrials.gov no. NCT02145949) and the Medical Research Foundation, Oregon Health and Science University Foundation.
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