Development of a Diagnostic Method for Detecting Increased Muscle Protein Degradation in Patients with Catabolic Conditions
Biruh T. Workeneh*,
Helbert Rondon-Berrios*,
Liping Zhang,
Zhaoyong Hu,
Gashu Ayehu*,
Arny Ferrando*,
Joel D. Kopple,,
Huiyun Wang,
Thomas Storer,||,
Mario Fournier¶,
Seoung Woo Lee**,
Jie Du and
William E. Mitch
* Medicine and Surgery, University of Texas Medical Branch, Galveston, and Nephrology Division, Baylor College of Medicine, Houston, Texas; Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center and || El Camino College, Torrance, California; David Geffen School of Medicine at UCLA and the UCLA School of Public Health and ¶ Department of Medicine, Cedars Sinai Medical Center, Los Angeles, California; and ** Division of Nephrology and Hypertension, Department of Internal Medicine, Inha University College of Medicine, Inchon City, Korea
Address correspondence to: Dr. William E. Mitch, Division of Nephrology, M/S: BCM 285, One Baylor Plaza, Alkek N-520, Houston, TX 77030. Phone: 713-798-8350; Fax: 713-798-5010; E-mail: mitch{at}bcm.edu
Received for publication February 9, 2006.
Accepted for publication August 20, 2006.
Muscle atrophy in catabolic illnesses is due largely to acceleratedprotein degradation. Unfortunately, methods for detecting acceleratedmuscle proteolysis are cumbersome. The goal of this study wasto develop a method for detecting muscle protein breakdown andassess the effectiveness of anticatabolic therapy. In rodentmodels of catabolic conditions, it was found that acceleratedmuscle protein degradation is triggered by activation of caspase-3.Caspase-3 cleaves actomyosin/myofibrils to form substrates forthe ubiquitin-proteasome system and leaves a characteristic14-kD actin fragment in the insoluble fraction of a muscle lysate.Muscle biopsies were obtained from normal adults and three groupsof patients: 14 who were undergoing hip arthroplasty, 28 hemodialysispatients who were participating in exercise programs, and sevenseverely burned patients. In muscle of patients who were undergoinghip arthroplasty, the 14-kD actin fragment level was correlated(r = 0.787, P < 0.01) with the fractional rate of proteindegradation. In muscle of hemodialysis patients who were undergoingendurance exercise training, the 14-kD actin fragment decreasedto values similar to levels in normal adults; strength trainingdid not significantly decrease the actin fragment. Severelyburned patients had increased muscle protein degradation andactin fragment levels, but the two measures were not significantlycorrelated. The experimental results suggest that the 14-kDactin fragment in muscle biopsies is increased in catabolicstates and could be used in conjunction with other methods todetect and monitor changes in muscle proteolysis that occurin patients with mild or sustained increases in muscle proteolysis.
The loss of lean body mass in aging, diabetes, uremia, trauma,burns, and immobilization causes increased morbidity and mortality(1). Unfortunately, methods that are used for detecting muscleatrophy are unwieldy, and the problem usually is unrecognizeduntil there is weight loss or visible muscle wasting. Availablemethods for detecting loss of lean body mass or judging theeffectiveness of therapeutic interventions to correct the probleminclude estimating muscle size by nuclear magnetic resonanceimaging (MRI), calculating amino acid/protein turnover duringinfusion of isotopically labeled amino acids, or measuring nitrogenbalance. The MRI method of measuring protein/amino acid turnoverrequires at least two measurements, special expertise, and expensiveequipment. Nitrogen balance does not assess changes in muscleprotein directly. Instead, it measures the net metabolism ofall proteins and requires several days of collections. Our goalis to devise a method that directly evaluates changes in muscleprotein degradation. It should satisfy the following criteria:It detects muscle protein breakdown in several conditions thatcause muscle wasting, it is minimally invasive, it is correlatedwith measured rates of protein catabolism in muscle, and itidentifies an effective intervention that improves muscle proteinmetabolism and function.
The mechanisms that cause loss of muscle mass in most catabolicconditions involve activation of the ubiquitin-proteasome system(UPP) (2,3). In addition, an initial, rate-limiting step inmuscle protein loss is activation of caspase-3 (4). Using animalmodels of catabolic conditions, we found that activated caspase-3is capable of cleaving actomyosin to create substrates thatare degraded rapidly by the UPP. Caspase-3–mediated proteincleavage leaves a characteristic "footprint" in the myofibrilfraction of muscle, a 14-kD actin fragment. In muscles of rodentswith catabolic conditions, the level of the 14-kD actin fragmentis closely associated with measured rates of protein degradation(4–7). We evaluated the actin fragment level in musclebiopsies from patients with different catabolic conditions todetermine whether it yields a method for diagnosing the presenceof accelerated muscle proteolysis and monitoring the effectivenessof therapy that is designed to reduce muscle protein losses.
The studies were approved by the Institutional Review Boardsof the University of Texas Medical Branch (UTMB) and Harbor-UCLAMedical Center. Biopsies of chronic hemodialysis patients andnormal adults were performed at the Harbor-UCLA Medical Center.Other biopsies and protein kinetic studies were performed inthe UTMB General Clinical Research Center and Blocker Burn Unit.Informed consent was obtained from all patients or their families.
Patient Groups
We studied normal individuals and three groups of patients withmuscle wasting. Fourteen patients who were aged 56 ±12 yr (±SE) and had severe degenerative hip disease hadtheir leg muscle mass measured by dual energy x-ray absorptiometry(DEXA; Hologic, Natick, MA). During hip replacement surgery,the rate of muscle protein breakdown was calculated from theturnover of amino acids that were labeled with stable isotopeson the basis of dilution of intracellular amino acid enrichmentfrom unlabeled amino acids that arose from protein breakdown.Muscle biopsies were obtained approximately 1 to 1.5 h intothe arthroplasty procedure to calculate fractional protein breakdown(8).
There were four groups of maintenance hemodialysis patients(4 h of hemodialysis three times a week) plus normal adultsof similar age, gender, and racial/ethic backgrounds (Table 1).The hemodialysis patients were stable enough to participatein an exercise program that was always supervised by an experiencedtrainer. They had evidence of abnormalities in factors thatregulate muscle protein metabolism (9). The patients were randomlyassigned to one of three types of exercise training or to notraining. Seven group 1 patients had a muscle biopsy beforeand after approximately 18 wk of endurance training using acalibrated, electrically braked, cycle ergometer (Ergoline 800;SensorMedics Corp., Yorba Linda, CA) (10). Seven group 2 patientshad biopsies before and after approximately 18 wk of leg strengthtraining using LifeFitness leg extension/curl and LifeFitnessleg press/calf extension apparatus (LifeFitness, Schiller Park,IL). Six group 3 patients had biopsies before and after approximately18 wk of both endurance and strength training. Endurance trainingwas done during the first 90 min of each hemodialysis treatment,and strength training was immediately before a hemodialysis.Eight group 4 patients had no exercise training but had musclebiopsies before and after approximately 18 wk. Finally, sixnormal adults with no exercise training had two biopsies separatedby an average of 9.5 wk.
Table 1. Characteristics of MHD patients and normal adultsa
In hemodialysis patients, biopsies were obtained from the rightvastus lateralis muscle, 10 cm cephalad to the superior borderof the patella and 1 to 2 cm anterior to the mid-lateral line.A Bergstrom or U.C.H. muscle biopsy needle (Popper & Sons,New York, NY) was used between 9 and 11 a.m. midweek, 1 d aftera hemodialysis treatment; the participants fasted overnight.
Seven patients in the UTMB Blocker Burn Unit were studied aftertheir clinical condition had stabilized. Their age was 44 ±8 yr, and the average burn was 68 ± 6% of body surfacearea (average of 28 ± 10% third-degree burns) (11,12).Because of severe injury, protein degradation and muscle biopsies(Bergstrom needle) were obtained at variable times after theinjury (average 12 d; range 6 to 30 d). Leg protein metabolismwas measured using labeled amino acids with arteriovenous balanceand the three-pool model to calculate protein synthesis anddegradation (13).
Measurement of the 14-kD Actin Fragment
Muscle samples were stored at –80°C. At least 30 mgof muscle was weighed and placed in PBS that contained CompleteProtease Inhibitors (Roche, Indianapolis, IN) in a ratio of1:30 (1 mg of muscle in 30 µl of PBS). After homogenizationon ice for 3 min (VWR High Viscosity Mixer at 300 rpm; VWR,West Chester, PA), the samples (approximately 100 µl)were centrifuged at 3300 x g for 10 min at 4°C. Pelletswere weighed and resuspended in 2x Laemmli sample buffer (1mg of insoluble tissue with 10 µl of 2x Laemmli samplebuffer). Samples were boiled for 20 min, and 20 µl wasseparated on a 15% SDS gel. After transfer to a 0.2-µmProtran 83A nitrocellulose membrane (Whatman, Sanford, ME),membranes were incubated with an affinity-purified, anti-actinantibody (1:500 dilution) that recognizes the carboxy-terminal11 amino acids of -actin (Sigma-Aldrich, St. Louis, MO) at 4°Covernight (4). After 1 h of incubation with IR Dye-800–labeledgoat anti-rabbit secondary antibody (1:5000 dilution; LiCor,Lincoln, NE), blots were washed with 0.1% Tween 20 in PBS beforebeing scanned using an Infra-red Laser Scanner (Odyssey, LiCor).The 14-kD actin fragment was quantified using the National Institutesof Health Image J program. It also can be detected by a horseradishperoxidase–conjugated anti-rabbit secondary antibody followedby enhanced chemiluminescence. However, the sensitivity of thismethod is lower compared with the laser scanner. With 30 mgof muscle, we were able to repeat the assay at least twice.
Reproducibility of Measurements of the 14-kD Actin Fragment in Muscle
There was insufficient muscle from individual patients to testthe intra-assay reproducibility by assaying a muscle biopsyrepeatedly. Therefore, we divided a normal rat muscle into sixsamples and detected the 14-kD actin fragment in six differentassays. The assay coefficient of variation was 11.8%. We alsomeasured the actin fragment density in biopsies from six normaladults; the average value in these normal muscles was 39.5 ±6.6 (SE), and the muscle level that was obtained before andafter an average of 9.5 wk differed by an average of 4.4% (NS).Regarding the assay sensitivity, we assessed different amountsof muscle from a normal adult. The 14-kD actin fragment wasreadily detected by the Infrared scanner when the pixel valuewas at least 50% above background values. With a minimum of500 µg of muscle from a hemodialysis patient, we obtainedthis degree of sensitivity, but for repeated testing, we routinelybegan the assay with at least 30 mg of muscle. We also testedthe reproducibility of the method by assaying muscles from twohemodialysis patients plus a normal muscle sample on seven differentWestern blots. Each patient sample was normalized for the valuefrom the same normal adult that was on each blot. This procedurewas used because it is the same as what we used to assay samplesfrom the various groups of hemodialysis patients. In this reproducibilityevaluation, the coefficients of variation were 9.5 and 10.9%.
The 14-kD Actin Fragment Level Is Correlated with Protein Degradation in Muscle
Muscle size in the unaffected leg of hip replacement patients(determined by DEXA) was greater versus values in the affectedleg. The muscle mass of the unaffected leg was significantlygreater than the muscle mass in the affected leg (unaffectedleg lean mass 8896 ± 688 g; difference in lean mass ofthe affected leg –966 ± 202 g; P < 0.001). Likewise,the muscle mass in the unaffected leg corrected for body weightwas significantly greater than in the affected leg (unaffectedleg lean mass/kg body weight 0.108 ± 0.005; differencein lean mass/kg body weight in the affected leg –0.012± 0.001; P < 0.001). A representative Western blotof the 14-kD actin fragment in muscle of patients who were undergoinghip arthroplasty was increased in comparison with the intensityof the band in a muscle of a normal adult (Figure 1A). In 14patients, the rate of muscle protein degradation was highlycorrelated with the intensity of the 14-kD actin fragment (r= 0.787; Figure 1B).
Figure 1. The level of the 14-kD actin fragment in muscle correlates with measured muscle protein degradation. (A) Representative Western blot of the 14-kD actin fragment in muscle biopsies that were obtained from a normal adult and one of 14 patients who underwent hip arthroplasty. Equal amounts of the insoluble myofibril tissue were separated by 15% SDS-PAGE, and the 14-kD actin fragment was detected by an anti-actin antibody. (B) The rate of protein degradation and the level of the 14-kD actin fragment were examined by linear regression and found to be significantly correlated (r = 0.787). The rate of protein degradation was measured with labeled amino acids (8), and the level of the actin fragment was quantified as in A.
Actin Fragment Level Predicts a Benefit of a Therapeutic Intervention
To compare changes in the actin fragment in muscles of the fourgroups of hemodialysis patients, we normalized its density tothat of an internal control, the value from a muscle biopsyof a normal adult. As shown in Table 2, the amounts of the 14-kDactin fragment in the initial muscle biopsy, corrected by theinternal control, were remarkably similar in the four groupsof hemodialysis patients. By ANOVA, there were no statisticaldifferences in the initial amounts of the actin fragment amongthe four groups of hemodialysis patients. The overall averageof the actin fragment level in muscles of hemodialysis patientswas 37.7% higher than the average value in muscles of normaladults of similar ages, gender, and racial/ethnic distributionas the hemodialysis patients.
Table 2. The influence of various types of exercise on the level of the 14-kD actin fragment in muscle of hemodialysis patientsa
To determine whether the amount of the 14-kD actin fragmentchanges in response to various types of exercise training, weexamined muscle biopsies that were obtained before and after18 wk of thrice-weekly regimens of endurance training, strengthtraining, a combination of endurance and strength training,or no training (10). After 18 wk of endurance training, thelevel of the 14-kD actin fragment in all seven participantswas reduced; the average decrease was 40% (P < 0.01; Table 1).Notably, this level of the 14-kD actin fragment that was obtainedafter endurance training was comparable to values that werefound in muscle of normal adults (Figure 2A). In the hemodialysispatients who were assigned to strength training, there was nochange in the 14-kD actin fragment (three values decreased,three values increased, and one did not change) (Figure 2B).In the muscle of patients who were assigned to strength plusendurance training, there was a 9.8%, statistically significantdecrease in the amount of the 14-kD actin fragment in muscle.Finally, there was no significant change in the actin fragmentin muscles of hemodialysis patients who did not undergo exercisetraining or in muscles that were obtained from normal adults.These results are consistent with earlier reports. Storer andcolleagues (14,15) reported that hemodialysis patients who underwent8 wk of endurance training had significant improvement in legmuscle endurance capacity, strength, and performance and decreasedfatigability. Endurance training also was associated with anincrease in the IGF-1 receptor mRNA and a decrease in myostatinmRNA. This pattern of changes in mRNA would be consistent withan increase in the insulin/IGF-1 signaling pathway, and, experimentally,this signaling pathway will decrease actin cleavage and proteindegradation in muscle (4–7).
Figure 2. Changes in the level of the 14-kD actin fragment in muscle of hemodialysis patients who were undergoing exercise training. (A) Representative Western blot of the 14-kD actin fragment in muscle biopsies of one normal adult (N) and two of seven dialysis patients before (B) and after (A) 18 wk of endurance training (ET). (B) The same as in A except that the patients underwent 18 wk of strength training (ST).
Actin Fragment Level Is Increased in Muscle of Severely Burned Patients
Western blots of the 14-kD actin fragment in muscle of a burnedpatient and in muscle of a normal adult are shown in Figure 3.Earlier, Biolo et al. (12) and Ferrando et al. (13) reportedthat such patients have a marked increase in protein degradation.The intensity of this band in each of the seven patients withan average of 68% total body burns was significantly greaterthan that in muscle biopsies of normal adults. However, we didnot find a significant correlation between the intensity ofthe actin band and the rate of protein degradation.
Figure 3. Representative Western blot showing that the 14-kD actin fragment in muscle of one of seven burned patients. The actin fragment was increased compared with the level in muscle of normal adults.
Our goal is to devise a method for detecting the presence ofaccelerated muscle protein degradation in humans. In rodentmodels of catabolic conditions, we found a characteristic, 14-kDactin fragment in the insoluble, myofibril fraction of homogenizedmuscle; it was closely related to the rate of protein degradation(4). In this study of patients who were undergoing hip replacementsurgery, we found that the amount of the 14-kD actin fragmentin muscle was significantly correlated (r = 0.787, P < 0.01)with the measured rate of protein degradation. Hemodialysistherapy, another catabolic condition (16–19), also yieldedinformation that supports this thesis; the amount of the actinfragment in muscle of hemodialysis patients changed in concertwith established physiologic and biologic (e.g., mRNA of myostatin/IGF-1)functions, thereby predicting the effectiveness of therapeuticinterventions that are used to improve muscle performance (10,14,15).Therefore, detecting the 14-kD actin fragment in a muscle biopsymight satisfy two requirements for a diagnostic test: It iscorrelated with the measured rate of muscle protein degradation,and it could be used to monitor a therapeutic intervention thatchanges muscle proteolysis.
Methods for diagnosing and monitoring accelerated muscle proteindegradation in patients include MRI assessment of changes inmuscle mass or measurement of nitrogen balance or protein turnoverby an isotope method. Only the latter directly evaluates muscleprotein metabolism, but it is expensive and clinically cumbersomeand requires extensive analysis. A practical method is neededto detect muscle wasting and to monitor therapy that is directedat minimizing this problem (1,16,17,20). Our earlier studiesshowed that the 14-kD actin fragments that are released duringaccelerated muscle atrophy are degraded rapidly by the ATP-dependentUPP (4). Consequently, the amount of the 14-kD actin fragmentin the soluble fraction of a muscle cell lysate is very lowand highly variable unless a proteasome inhibitor is present.Therefore, we explored events that occur before the degradationof the 14-kD actin fragment. We found the 14-kD actin fragmentin the insoluble fraction of muscle, presumably because it isprotected by the myofibril complex from additional proteolysis.
Experimentally, caspase-3 generates the 14-kD actin fragment(4). However, measuring caspase-3 activity in muscle samplesis not useful as a diagnostic method because we and others havefound that caspase-3 activity in muscle samples is low and the"signal to noise" ratio is high even in muscles of rodents withdemonstrably high rates of protein degradation. Wei et al. (21)reported difficulty in detecting changes in caspase-3 activityin muscle of septic rats. Sepsis activates the UPP in muscle,so their results might be related to degradation of caspase-3by the ATP-dependent UPP (22). In contrast, the 14-kD actinfragment is detectable in the insoluble fraction of muscle ofseptic rats (data not shown), emphasizing why we assessed thisproduct of caspase-3 rather than measuring caspase-3 activity.
Especially important is our finding that the amount of the 14-kDactin fragment is significantly correlated with the rate ofmuscle protein degradation in patients who undergo hip replacement.The decrease in leg muscle mass measured by DEXA before surgeryand the increase in muscle protein degradation measured duringsurgery presumably reflect disuse atrophy plus inflammationfrom osteoarthritis as well as the influence of surgery. Weemphasize the detection of protein degradation because the actinfragment reflects muscle proteolysis but recognize that decreasedmuscle protein synthesis could contribute to the loss of legmuscle mass. In the hip surgery patients, protein degradation,expressed as fractional breakdown, was measured using isotope-labeledamino acids, but this requires arterial blood sampling and measurementof amino acid enrichment in these samples (12,13). The correlationthat we found (Figure 1B) indicates that muscle proteolysiscan be detected by the presence of the actin fragment in themyofibril complex of muscle.
Another important finding is that changes in the level of the14-kD fragment can be used to monitor the effectiveness of atreatment for muscle wasting. In hemodialysis patients, we useddifferent types of exercise to improve muscle function and thebalance between muscle protein synthesis and breakdown (10,23,24).We found that endurance training reduced the level of the actinfragment in muscle of hemodialysis patients to values that werefound in the muscle of normal adults (Table 2). This is consistentwith the improvement in muscle strength, power, and fatigabilitythat these patients experienced with endurance training (10).Preliminary analyses of their responses to endurance exercisealso show that these patients had a significant decrease inmyostatin mRNA (P = 0.006) (14).
A potential mechanism for the decrease in muscle protein degradationwith endurance training is stimulation of the phosphatidylinositol3-kinase/Akt (PI3K/Akt) pathway. Sakamoto et al. (25) foundthat treadmill exercise or mechanical stretch stimulates thePI3K/Akt pathway in muscle. This is relevant because we havefound that an increase in the PI3K/Akt pathway suppresses caspase-3and reduces the amount of the 14-kD actin fragment (4–7).These experimental results indicate how endurance exercise couldreduce the level of the 14-kD actin fragment.
With strength training, the hemodialysis patients had littleor no decrease in the level of the actin fragment (Table 2).The underlying mechanism for this result is not clear becausestrength training can increase muscle regeneration and hypertrophy.However, the severity of training that is needed to elicit theseresponses is vigorous (26). The patients whom we studied wererelatively deconditioned, limiting the intensity of their resistancetraining (10). Preliminary analyses of the influence of strengthor strength plus endurance training suggest that IGF-1 mRNAin muscle increases by 71% and IGF-1 protein by 41% (15). Thesegrowth factor responses should increase muscle mass, but innormal adults, strength/resistance training does not suppressprotein degradation in muscle and may even increase it (27,28).If similar responses occur in hemodialysis patients, then strengthtraining would not decrease the muscle level of the actin fragment.Another factor is that both strength and endurance trainingcan cause changes in muscle proteins and accrual of new protein.Strength training reportedly leads to muscle hypertrophy, whereasendurance training without causing hypertrophy induces proteinremodeling, altering the proportions of type I and type II myosinheavy chains and increasing the levels of enzymes, includingthose that are involved with oxidative phosphorylation (29–32).Finally, the combination of strength and endurance trainingcould activate different intracellular signaling processes orstimulate specific cell types (e.g., mature muscle cells versussatellite cells), resulting in different responses in termsof protein expression and exercise capacity (25,26,33,34). Regardless,our results indicate that endurance training can benefit hemodialysispatients who exhibit increased muscle proteolysis. The complexityof responses to strength training does not suggest an explanationfor why it did not reduce the presence of the 14-kD actin fragmentin muscle.
To evaluate the assay in a condition with extreme loss of muscle,we studied patients with burn injuries that are severe enoughto stimulate protein losses that amount to 20 to 25 g/m2 perd (12). The amount of the 14-kD actin fragment in muscle ofburned patients was uniformly greater than that of normal adults,but this change was not statistically correlated with the degreeof protein degradation in muscle. A potential reason for thisfinding is that severe loss of myofibrils could limit the protectiveeffect of muscle tissue against degradation of the actin fragmentby the UPP (4). For example, we found a significant decreasein the amount of the actin band in cultured muscle cells thatwere treated with 100 nM staurosporine for 24 h (data not shown).Therefore, when there is a high rate of muscle proteolysis,the assay may not provide an accurate estimate of muscle proteinbreakdown. Another explanation is that a severe burn injurynot only increases proteolysis in muscle but also stimulatesprotein breakdown in other organs, including skin and bone (12,35).The three-pool method that we used to measure overall proteindegradation in muscle of burned patients includes values frommuscle, bone, and skin (13) and would overestimate the levelof proteolysis in muscle, whereas the actin fragment evaluatesprotein degradation only in muscle. Future studies should bebased on a more direct measurement of the rate of protein degradationand the amount of the 14-kD actin fragment in muscle of patientswho are in the early stage of a serious burn injury.
In our research study of a relatively small group of 49 patientswith various catabolic conditions, we found that the assay forthis marker of accelerated muscle protein degradation can detectan increase in muscle protein degradation and a positive responseto therapy. Because it is relatively simple and cost-effective,this method could be used to detect the presence of muscle proteincatabolism and to monitor therapy. Additional testing of theclinical usefulness of the method will require a larger numberof patients with other types of catabolic conditions. At thisstage, our results indicate that detection of the 14-kD actinfragment should be used to complement results from other measuresof muscle protein metabolism.
Acknowledgments
This study was supported by National Institutes of Health grantsR01 DK37175, R01 GM57295, R01 HL70762, R01 DK54457, and P50DK64233.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
-actin (Sigma-Aldrich, St. Louis, MO) at 4°C overnight (4). After 1 h of incubation with IR Dye-800–labeled goat anti-rabbit secondary antibody (1:5000 dilution; LiCor, Lincoln, NE), blots were washed with 0.1% Tween 20 in PBS before being scanned using an Infra-red Laser Scanner (Odyssey, LiCor). The 14-kD actin fragment was quantified using the National Institutes of Health Image J program. It also can be detected by a horseradish peroxidase–conjugated anti-rabbit secondary antibody followed by enhanced chemiluminescence. However, the sensitivity of this method is lower compared with the laser scanner. With 30 mg of muscle, we were able to repeat the assay at least twice. 
