Sustained Elevation of Circulating Growth and Differentiation Factor ...

Clinical InvestigationsAcute muscle loss in critically ill patients causes weaknessranging from mild loss of strength and musclewasting to profound muscle weakness. Severe weaknessin this context is known as ICU-acquired paresis (ICUAP),which is associated with significant mortality and morbidity(1). Although it is a common problem, affecting at least 25% ofpatients admitted to critical care (2, 3), our understanding ofthe underlying pathological mechanisms is limited. Sepsis, systemicinflammatory response syndrome, immobility, and hyperglycemiaare all thought to be major etiological risk factorsfor the development of ICUAP (1, 4–7). However, elucidationof the molecular mechanisms involved in human cases is difficult,and evidence has often been contradictory. In part, thisis because data are often obtained on ICU from heterogenousgroups of patients with ICUAP with a range of reasons for ICUadmission and varying lengths of stay.As in other disease processes, muscle mass in critically ill patientsis determined by the balance between dynamic processesmediating muscle breakdown (atrophy, apoptosis, and autophagy)and synthesis (protein synthesis and satellite cell recruitment).Myostatin, a member of the transforming growth factorβ(TGF-β) superfamily, is a well-recognized negative regulatorof muscle mass (8), which has been implicated in the developmentof ICUAP. Elevation of both myostatin messenger RNA(mRNA) and protein was observed in the muscle of critically illpatients (9) and increased circulating levels of myostatin wereobserved in patients with muscle wasting associated with chronicobstructive pulmonary disease (10), cardiac failure (11), andliver failure (12). Other members of the TGF-β family also regulatemuscle phenotype. Growth and differentiation factor-15(GDF-15) is a member of this family that, under normal conditions,is not expressed highly in most tissues (13). However,expression is increased in response to oxidative stress, hypoxia,inflammation, and tissue damage in liver, brain, lung, and vasculartissue (13, 14). These factors are all thought to be importantin the development of ICUAP (7). GDF-15 is increasinglyimplicated in several disease processes, including cardiovasculardisease (14), multiple cancers (15–17), pulmonary artery hypertension(18), and importantly cachexia (19). Furthermore, in amouse model of cardiac hypertrophy, GDF-15 transgenic micewere resistant to cardiac hypertrophy and exposure to circulatingGDF-15 limited cardiac myocyte hypertrophy (20).Insulin-like growth factor-1 (IGF-1) drives many hypertrophypathways. Suppression of IGF-1 was observed in variousanimal models of sepsis and immobility (7) and in patients withsevere ICUAP (21). Although both increased myostatin and decreasedIGF-1 have been implicated in the development of ICU-AP, the relative pattern of change in the circulating levels of thesemediators during the acute development of muscle wasting hasnot been established. Therefore, to identify potential circulatingfactors driving acute muscle wasting, we studied a homogenouscohort of patients undergoing high-risk elective cardiothoracicsurgery who were at risk of prolonged critical care admissionand ICUAP. We hypothesized that the acute insult of surgerywould result in an acute rise in the known circulating drivers ofmuscle breakdown (myostatin) and suppression of those promotingmuscle hypertrophy (IGF-1) and that the dynamic patternof these changes would differ between those patients whodeveloped muscle wasting and those who did not. Furthermore,although a direct effect of GDF-15 on skeletal muscle has notbeen reported previously, the observations discussed above ledus to hypothesize that circulating GDF-15 would rise followingthe acute stress of cardiothoracic surgery and be associated withmuscle wasting. We went on to test our hypothesis further, byexposing cultured myotubes to GDF-15 in vitro.METHODSPatient Recruitment and Study DesignWritten consent was obtained from all those involved in thisprospective observational study (local research ethics committeeapproval 10/H0504/9). Adults undergoing elective cardiac surgeryat the Royal Brompton Hospital were screened for inclusionin the study. The principle inclusion criteria were a high-riskelective procedure requiring postoperative admission to adultcritical care, defined by the surgical team and by the patients’EuroSCORE (22–24). Exclusion criteria included pre-existingmuscle or neuromuscular disease, malignancy, or contraindicationto serial blood sampling. Patients were only included if theywere stable preoperatively and not suffering from acute illness orrequiring emergency surgery. Muscle mass was assessed by measuringthe cross-sectional area of the right rectus femoris muscleby ultrasound (see below) preoperatively and at day 7 of admissionor at discharge from hospital if earlier than day 7. We obtainedblood for analysis preoperatively (“baseline”), and repeatsamples were taken on the first and second postoperative daysand at day 7 or at discharge if earlier; these latter two time pointswere considered identical for the purposes of analysis: only fourpatients (three in the nonwasting patient group and one in thewasting group) were in fact discharged earlier than day 7.Measurement of Cross-Sectional Area of the RectusFemoris by UltrasoundUltrasound cross-sectional area of the rectus femoris (US RF csa)was measured using a previously described technique (25, 26).B-mode ultrasound imaging with a 10-MHz 12L-RS probe wasused (Logiq E, GE Healthcare, Buckinghamshire, UK). The patientwas positioned supine on the bed with their legs flat withleg muscles relaxed. The anterior superior iliac spine (ASIS) andthe point 60% of the distance from the ASIS to the superiorborder of the patella were identified. The ultrasound probe waspositioned perpendicular to the axis of the leg. Where the entireRF csacould not be visualized in a single ultrasound image a point66% or 80% of the distance between the ASIS and patella wasused. The same point was used for follow-up measurements foreach patient. Three separate consecutive images were taken andthe RF csaestimated using Image-J software (National Institutesof Health). The average of these three measurements was used.Inadequate ultrasound images were defined as those where theedges of the RF muscle could not be defined well enough to calculatethe RF csa. This occurred where the patient was very edematousand in these cases the patient data were excluded (n = 3).Critical Care Medicine www.ccmjournal.org 983

Bloch et alPatient Classification and Definition of QuadricepsWastingPatients were classified as those who developed muscle wastingand those who did not based on the coefficient of variation(CV) of repeated measurements. To determine CV, the first tenstudies were repeated at two separate occasions on the sameday. These measurements were then plotted on a Bland-Altmanplot (Supple mental Fig. 1, Supplemental Digital Content1, http://links.lww.com/CCM/A568). This analysis determinedthat change in muscle loss of greater than 9.24% represented asignificant change above the variation expected from repeatedmeasures. Therefore, those with greater than 9.24% muscleloss were categorized as having muscle wasting.Blood Sample HandlingPlasma was separated from blood collected into EDTA sampletubes centrifuged at 1,500 × g (3,500 rpm) for 10 minutes,within 2 hours of collection. Samples were stored at –80°C untilthey were processed.Sample AnalysisPlasma levels of myostatin (Immundiagnostik, Bensheim, Germany)and GDF-15 (R&D systems, Abingdon, UK) and IGF-1(Mediagnost, Reutlingen, Germany) were all quantified usingcommercially available enzyme-linked immunosorbent assaykits. Results were analyzed as per the manufacturers’ guidelines.Determination of the Effect of GDF-15 onMyotube SizeC2C12 myoblasts (1 × 10 5 per well) were seeded in a six-wellplate in growth medium (Dulbecco’s modified eagle medium[DMEM]) (Invitrogen, Paisley, UK) supplemented with 10%fetal calf serum (GE Healthcare). The following day the cellswere transfected with lipofectamine (Invitrogen) as previouslydescribed (27) with pCAGGS-green fluorescent protein(0.5 μg). The cells were allowed to differentiate, forming myotubes,in differentiation medium (DMEM supplemented with2% horse serum) (GE Healthcare) for 7 days. On the eighthday of differentiation, the cells were treated with fresh differentiationmedium containing either the control vehicle (0.1%bovine serum albumin with 20 mM HCl) or 50 ng/mL GDF-15(R&D Systems) and cultured for a further 2 or 4 days.Fluorescence images were taken 48 and 96 hours after treatment.Myotubes were identified in randomly selected fields.The width of each myotube was measured by determiningthe shortest distance across the cell at five randomly selectedpoints along the length of the tube excluding branching regionsof the cell, using ImageJ software. The experiment wasrepeated twice, and each repeat showed a similar pattern: 43control and 52 GDF-15-treated myotubes were measured inthe first experiment and 25 control and 19 GDF-15-treatedmyotubes in the second at 48 hours. At 96 hours, 47 controland 46 GDF-15-treated myotubes were measured in the firstexperiment and 24 control and 27 GDF-15-treated myotubesin the second. The data from the two experiments were combinedfor each time point.Data and Statistical AnalysisData are quoted as mean ± sd unless otherwise stated. Rawmyostatin, GDF-15, and IGF-1 levels were nonparametricallydistributed and so the median with interquartile ranges wasused to describe the data. Repeated-measures Friedman’s testwith Dunn’s correction for comparison of all time points tothe preoperative baseline was used for within group analysis.GDF-15 levels were also analyzed using regression with robustvariances to determine the “treatment” effect. This is reportedas the ratio of the geometric mean between wasting and nonwastingpatients. Student t tests (for normally distributed data)or Mann-Whitney U tests were used where appropriate for betweengroup analyses. Statistical analysis and figure constructionwere carried out using GraphPad PRISM 5 (GraphPadSoftware, San Diego, CA).RESULTSPatients and Data CollectionA total of 50 patients meeting the inclusion criteria were approached:five declined consent and in three patients we wereunable to achieve good quality US RF csaimages. Of the remaining42 patients, 23 developed muscle wasting following surgery(Fig. 1). Baseline demographic data, patient comorbidities,and critical care data are shown in Table 1. The different surgicalprocedures undergone are shown in Supplemental Table 1(Supplemental Digital Content 1, http://links.lww.com/CCM/A568). The nonwasting and wasting patient groups were wellmatched for age, sex, body mass index, and EuroSCORE (Table1). Total bypass time for both groups is also shown. Not allpatients underwent bypass as one patient in the nonwastinggroup underwent an off-pump coronary artery bypass procedureand three patients underwent transcutaneous aortic valveinsertion; in the wasting group, two patients had off-pumpprocedures. Importantly, the circulating plasma levels for themediators measured in these patients were within the rangeof the rest of the group. Length of stay in ICU was short witha median stay of only 1 day for the nonwasting patients andFigure 1. Change in ultrasound cross-sectional of the area rectusfemoris (US RF csa) from preoperative baseline to day 7 in high-risk electivecardiac surgery patients who did not develop muscle wasting (nonwastingn = 19, p = 0.14 paired Student t test) and those who did develop musclewasting (> 9.24% loss, wasting n = 23, p ≤ 0.0001 paired Student t test).Change in US RF csaexpressed as percentage change from baseline. Nonwastingpatients change in US RF csa1.3% ± 5.9 and in the wasting group24.6% ± 13.7 (mean ± sd).984 www.ccmjournal.org April 2013 • Volume 41 • Number 4

Table 1. Demographic Data, Comorbidities, and Critical Care Data for Nonwastingand Wasting PatientsClinical InvestigationsNonwastingPatients(n = 19)WastingPatients(n = 23)p (t Test,Mann-WhitneyU Test # )Demographic dataAge (yr) 65.7 ± 17.2 62.0 ± 16.2 0.47Sex (m/f) 9/10 12/11Body mass index (kg/m 2 ) a 26.6 ± 4.6 24.7 ± 4.1 0.18EuroSCORE 5.3 ± 1.8 5.5 ± 2.6 0.76Preoperative left ventricular ejection fraction (%) b 60.8 ± 15.0 56.6 ± 16.4 0.41Preoperative creatinine (μmol/L) a 81.3 ± 23.6 77.3 ± 17.1 0.52Preoperative creatinine clearance (mL/min) a 84.7 ± 36.1 86.8 ± 34.7 0.66Preoperative comorbidities (n)Ischemic heart disease 10 8Structural heart disease 9 11Dysrhythmia 2 3Systemic hypertension 7 5Pulmonary hypertension 1 2Hypercholesterolemia 7 10Congenital cardiac disease 3 6Diabetes 3 1Obstructive lung disease 3 2Statin therapy 8 10Angiotensin-converting enzyme inhibitor or receptor blocker therapy 3 4Critical care dataTotal bypass time (min) c 115 ± 39 117 ± 35 0.91Total cross-clamp time c 89 ± 43 86 ± 28 0.87ICU length of stay, days (median) 2.5 (1) 2.5 (2) 0.62#Hospital length of stay, days (median) 11.2 (8) 10.5 (9) 0.44#Mean blood glucose during critical care admission (mmol/L) 7.48 7.35 0.56IV insulin therapy while in critical care (n) 10 10Mean insulin dose during critical care admission, if treated withIV insulin (units/hr)2.16 1.91 0.46Exposure to neuromuscular blocking agents (n) 0 0Exposure to corticosteroids (n) 3 4Data presented as mean ± sd unless otherwise stated. neuro muscular blocking agents are given other than at the time of surgery.aWeight unavailable for two patients in the wasting group; therefore, n = 21.bPreoperative ejection fraction not available for one patient in each group.cFour patients in the nonwasting and two patients in the wasting group did not undergo bypass.Critical Care Medicine www.ccmjournal.org 985

Bloch et al2 days for those who developed muscle wasting (p = 0.62,Mann-Whitney U test). Length of stay in hospital was alsosimilar in the two groups (median 8 for nonwasting and 9 forwasting patients, p = 0.44, Mann-Whitney U test). One patientdied at 21 days postoperatively in the wasting group; no patientsdied in the nonwasting group.Change in US RF csaThe mean change in US RF csafrom baseline over the weekfor the wasting group (n = 23) was a loss of 24.6% ± 13.7%(Fig. 1). The mean change in the nonwasting group (n = 19)was not significant (1.3% ± 5.9%).MyostatinBaseline plasma myostatin did not differ significantly betweenthe groups (p = 0.1). Unexpectedly, plasma myostatin concentrationfell significantly in both groups following surgery.In both groups, plasma myostatin concentration returned tobaseline at day 7 (Fig. 2).Growth and Differentiation Factor-15Baseline plasma GDF-15 also did not differ significantly betweenthe groups (p = 0.52). In contrast to myostatin, GDF-15 levels increased dramatically after surgery (p < 0.001 forboth groups). In the nonwasting patients, the increase inGDF-15 returned to baseline by day 7 (p > 0.05); however, inthe wasting group, the level remained significantly elevated(p < 0.01; Fig. 3). The ratio of the geometric mean of GDF-15 for nonwasting subjects to wasting subjects was 1.17 (0.97,1.41), suggesting that the geometric mean for the nonwastingsubjects was on average higher than that for the wastingsubjects over the week after cardiac surgery although the ratiofailed to achieve statistical significance of p = 0.10. This togethersuggests a higher relative exposure to circulating GDF-15Figure 3. Plasma growth and differentiation factor-15 (GDF-15) concentration(pg/mL) in nonwasting (n = 19) and wasting patients (thosewith > 9.24% muscle loss: n = 23) preoperatively (PO), on day 1 (D1),day 2 (D2), and day 7 (D7). Data presented as box and Whisker plotswith median, interquartile ranges, and 5–95% percentiles. *p < 0.05;**p < 0.01; ***p < 0.001; ns = not significant; repeated-measures Friedman’swith Dunn’s correction for comparison with preoperative baseline.in the wasting patients in comparison those in whom musclewasting did not develop.Insulin-Like Growth Factor-1Baseline plasma IGF-1 did not differ significantly betweenthe groups (p = 0.22). The pattern of change in IGF-1 levelsafter surgery was the same in both groups, the IGF-1 plasmaconcentration fell significantly at days 1 and 2 after surgery inboth groups (p < 0.001 for both). In the nonwasting group, theIGF-1 concentration returned to baseline by day 7 (p > 0.05).However, in wasting patients, it remained significantly reducedat day 7 (p < 0.01, Fig. 4).Figure 2. Plasma myostatin concentration (ng/mL) in nonwasting (n =19) and wasting patients (those with > 9.24% muscle loss: n = 23) preoperatively(PO), on day 1 (D1), day 2 (D2), and day 7 (D7). Data presentedas box and Whisker plots with median, interquartile ranges, and 5–95%percentiles. *p < 0.05; **p < 0.01; ***p < 0.001; ns = not significant;repeated-measures Freidman’s test with Dunn’s correction for comparisonwith preoperative baseline.Figure 4. Plasma insulin-like growth factor-1 (IGF-1) concentration(ng/mL) in nonwasting (n = 19) and wasting patients (those with> 9.24% muscle loss: n = 23) preoperatively (PO), on day 1 (D1),day 2 (D2), and day 7 (D7). Data presented as box and Whisker plotswith median, interquartile ranges, and 5–95% percentiles. *p < 0.05;**p < 0.01; ***p < 0.001; ns = not significant; repeated-measures Freidman’stest with Dunn’s correction for comparison with preoperativebaseline.986 www.ccmjournal.org April 2013 • Volume 41 • Number 4

Bloch et alsepsis, showed acute elevation of myostatin mRNA, followedby suppression over the next week (40).Our clinical data show the association of a more prolongedexposure to GDF-15 with the development of muscle wastingfollowing cardiac surgery; therefore, it is possible that thisassociation may represent a novel mechanism for acute musclewasting. GDF-15 was first described as macrophage inhibitorycytokine-1 and has hallmark characteristics of the TGF-β familyof proteins (41). It is secreted as a propeptide and cleaved toform a dimeric mature protein of 224 amino acids (14). GDF-15 expression increased in response to stress, hypoxia, andinflammation in vitro (14, 17) and in a variety of animal andhuman chronic disease settings (13, 17, 18). All the patients inthis study underwent the significant acute inflammatory insultof cardiac surgery and as expected circulating GDF-15 roseacutely in all patients.To our knowledge, a direct effect of GDF-15 on skeletalmuscle has not previously been demonstrated. GDF-15 inducedsignificant fiber atrophy of cultured C2C12 myotubes.This result is consistent with data from cardiac and cancer celllines, and models in which GDF-15 limited hypertrophy andpromoted apoptosis (16, 20). Limiting cardiac hypertrophyfor a prolonged period may be beneficial. Mice deficient inGDF-15 show both increased hypertrophy and loss of cardiacfunction in response to pressure overload compared with wildtypemice (20). Beneficial effects of GDF-15 in cardiac tissuealso include the protection of the myocardium from infiltrationof polymorphonuclear leukocytes and protection againstmyocardial rupture following infarction (42). However, evenwithin cardiac tissue, GDF-15 is also linked to pathological effects,and its role is likely to be bidirectional depending on thesituation and environment (14).Similarly, GDF-15 may have a protective role by promotingapoptosis in prostate cancer cells (43, 44); conversely, it mayalso enhance metastatic potential and angiogenesis (13). It ispossible that in muscle tissue a similar situation exists suchthat the role of GDF-15 may depend on the molecular environment.However, our findings of an association between GDF-15 and acute muscle wasting following the physiological stressof cardiac surgery are consistent with the understanding thatGDF-15 is released under situations of physiological stress (13,14). The ability of GDF-15 to cause muscle wasting in culturedmyotubes is consistent with the antihypertrophy/proapoptosisrole described above at least under some conditions. Alsocancer-related cachexia in mice resulting from excess GDF-15expression from transplanted prostate cancer cells can be reversedwith GDF-15 antibodies (19). It was postulated that thiswas due to a central antianorexic effect, and reduced food intakewas reversed. However, our data suggest that it is possiblethat GDF-15 also has a direct effect on skeletal muscle, resultingin reduced muscle bulk and lean body mass.A weakness of our data is that we are unable to demonstratea difference between the two groups on the raw values of circulatingfactor’s concentrations alone. This may be due to therelatively small number of patients in each group and the widerange of baseline values. Alternatively, despite the high-risk natureof the cardiac surgery that our patients underwent, the overallclinical outcome was very good, in that ICU stay was shortwith a low mortality. Furthermore, although measurable, thesepatients can be considered to only have mild muscle wastingwith clinically insignificant weakness and therefore differencesbetween the two groups will be harder to detect. Nevertheless,the validity of our approach is supported by the observationthat IGF-1 decreased acutely in all patients and remained suppressedin those who developed muscle wasting, consistent withprior observations (21, 32). A larger prospective study involvingmore patients and patients with clinically obvious ICUAPwould be desirable as a next step to confirm our findings.It is well established that the use of bypass per se may influencecirculating levels of inflammatory molecules (45). However,we do not believe this explains the current data becausebypass time was not different in patients who developed wastingand those who did not (Table 1). In addition, four patientsin the nonwasting group and two in the wasting group did notundergo bypass, and these patients had circulating plasma levelsfor the mediators measured of the same order of magnitudeas the rest of the group.A perfect human model of ICUAP does not exist. However,we believe our experimental paradigm is of interest owing toits prospective nature, the relative homogeneity of the patientcohort and the uniformity of the insult. By contrast, elucidatingmechanisms of ICUAP by studying cases is also problematicbecause ICUAP is a heterogeneous disorder that occursin a very mixed cohort of patients, and the time course of thesyndrome is difficult to define. Our model can be criticized forthe fact that patients only develop subclinical muscle weaknessand therefore it may be difficult to extrapolate findings totrue ICUAP. It is also important to acknowledge that althoughthese patients have all undergone significant physiological insultthe cardiac surgical model may not be generally applicableto ICU patients—in whom, for example, sepsis and prolongedstays on critical care are common. However, the strengths ofthe model are that by studying our patients’ blood profile frombaseline, we are able to demonstrate prospectively the dynamicpattern of change of circulating drivers of muscle metabolism.Furthermore, by identifying prospectively a specific cohort ofpatients with similar clinical experiences, we are able to controlfor many variables and potentially identify specific factors inthe pathophysiology of acute muscle wasting in the critically ill.In conclusion, we have shown that even elective high-riskcardiac surgery with a good clinical outcome is commonly associatedwith measurable loss of quadriceps muscle mass, andour data support the hypothesis that this muscle loss occurs asa result of a dynamic imbalance between drivers of muscle atrophyand hypertrophy. Furthermore, we speculate that GDF-15 may be a clinically relevant cause of skeletal muscle wastingin humans; if so GDF-15 antagonism may prove a useful futuretherapeutic approach.REFERENCES1. 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