EDITORIALS the investigators did demonstrate that Th2-associated gene expression markers were higher in one subgroup. Work is still needed to identify other, possibly novel, molecular pathways involved. It is ultimately through identifying causative molecular pathways in these unsupervised analyses that we will transform the care of patients with asthma. In asthma and beyond, achieving precision medicine in pulmonary disease will hinge not only on our ability to identify reproducible disease subgroups but also on the underlying molecular perturbations driving these distinct ’types. Understanding the underlying pathobiology is essential both for deﬁning disease endotypes and for appropriately targeting therapies to individual patients. It is through this integrative approach that we as a ﬁeld will be able to remodel the classiﬁcation and care of patients with pulmonary disease. n Author disclosures are available with the text of this article at www.atsjournals.org. Katrina Steiling, M.D., M.Sc. Department of Medicine Boston University School of Medicine Boston, Massachusetts and Bioinformatics Program Boston University Boston, Massachusetts Stephanie A. Christenson, M.D. Department of Medicine University of California, San Francisco San Francisco, California
References 1. National Research Council (US) Committee on a Framework for Developing a New Taxonomy of Disease. Toward precision medicine: building a knowledge network for biomedical research and a new taxonomy of disease. Washington, DC: National Academies Press; 2011. 2. Collins FS, Varmus H. A new initiative on precision medicine. N Engl J Med 2015;372:793–795. 3. Ofﬁce of the Press Secretary. Remarks by the President on precision medicine. 2015 Jan 30 [accessed 2015 Mar 26]. Available from: https://www.whitehouse.gov/the-press-ofﬁce/2015/01/30/remarkspresident-precision-medicine 4. Ofﬁce of the Press Secretary. Fact sheet: President Obama’s precision medicine initiative. 2015 Jan 30 [accessed 2015 Mar 26]. Available from: https://www.whitehouse.gov/the-press-ofﬁce/2015/01/30/factsheet-president-obama-s-precision-medicine-initiative 5. Sørlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Jeffrey SS, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 2001;98:10869–10874.
6. Perou CM, Sørlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, et al. Molecular portraits of human breast tumours. Nature 2000;406:747–752. 7. Rouzier R, Perou CM, Symmans WF, Ibrahim N, Cristofanilli M, Anderson K, Hess KR, Stec J, Ayers M, Wagner P, et al. Breast cancer molecular subtypes respond differently to preoperative chemotherapy. Clin Cancer Res 2005;11:5678–5685. 8. Wenzel SE. Asthma phenotypes: the evolution from clinical to molecular approaches. Nat Med 2012;18:716–725. 9. Moore WC, Bleecker ER, Curran-Everett D, Erzurum SC, Ameredes BT, Bacharier L, Calhoun WJ, Castro M, Chung KF, Clark MP, et al.; National Heart, Lung, Blood Institute’s Severe Asthma Research Program. Characterization of the severe asthma phenotype by the National Heart, Lung, and Blood Institute’s Severe Asthma Research Program. J Allergy Clin Immunol 2007;119:405–413. 10. Fitzpatrick AM, Teague WG, Meyers DA, Peters SP, Li X, Li H, Wenzel SE, Aujla S, Castro M, Bacharier LB, et al.; National Institutes of Health/National Heart, Lung, and Blood Institute Severe Asthma Research Program. Heterogeneity of severe asthma in childhood: conﬁrmation by cluster analysis of children in the National Institutes of Health/National Heart, Lung, and Blood Institute Severe Asthma Research Program. J Allergy Clin Immunol 2011;127:382–389, e1–e13. 11. Haldar P, Pavord ID, Shaw DE, Berry MA, Thomas M, Brightling CE, Wardlaw AJ, Green RH. Cluster analysis and clinical asthma phenotypes. Am J Respir Crit Care Med 2008;178:218–224. 12. Siroux V, Basagaña X, Boudier A, Pin I, Garcia-Aymerich J, Vesin A, Slama R, Jarvis D, Anto JM, Kauffmann F, et al. Identifying adult asthma phenotypes using a clustering approach. Eur Respir J 2011;38:310–317. 13. Lotvall ¨ J, Akdis CA, Bacharier LB, Bjermer L, Casale TB, Custovic A, Lemanske RF Jr, Wardlaw AJ, Wenzel SE, Greenberger PA. Asthma endotypes: a new approach to classiﬁcation of disease entities within the asthma syndrome. J Allergy Clin Immunol 2011;127:355–360. 14. Busse W, Corren J, Lanier BQ, McAlary M, Fowler-Taylor A, Cioppa GD, van As A, Gupta N. Omalizumab, anti-IgE recombinant humanized monoclonal antibody, for the treatment of severe allergic asthma. J Allergy Clin Immunol 2001;108:184–190. 15. Nair P, Pizzichini MM, Kjarsgaard M, Inman MD, Efthimiadis A, Pizzichini E, Hargreave FE, O’Byrne PM. Mepolizumab for prednisone-dependent asthma with sputum eosinophilia. N Engl J Med 2009;360:985–993. 16. Woodruff PG, Boushey HA, Dolganov GM, Barker CS, Yang YH, Donnelly S, Ellwanger A, Sidhu SS, Dao-Pick TP, Pantoja C, et al. Genome-wide proﬁling identiﬁes epithelial cell genes associated with asthma and with treatment response to corticosteroids. Proc Natl Acad Sci USA 2007;104:15858–15863. 17. Woodruff PG, Modrek B, Choy DF, Jia G, Abbas AR, Ellwanger A, Koth LL, Arron JR, Fahy JV. T-helper type 2-driven inﬂammation deﬁnes major subphenotypes of asthma. Am J Respir Crit Care Med 2009;180:388–395. 18. Corren J, Lemanske RF, Hanania NA, Korenblat PE, Parsey MV, Arron JR, Harris JM, Scheerens H, Wu LC, Su Z, et al. Lebrikizumab treatment in adults with asthma. N Engl J Med 2011;365:1088–1098. 19. Baines KJ, Simpson JL, Wood LG, Scott RJ, Gibson PG. Transcriptional phenotypes of asthma deﬁned by gene expression proﬁling of induced sputum samples. J Allergy Clin Immunol 2011; 127:153–160, e1–e9. 20. Yan X, Chu J-H, Gomez J, Koenigs M, Holm C, He X, Perez MF, Zhao H, Mane S, Martinez FD, et al. Noninvasive analysis of the sputum transcriptome discriminates clinical phenotypes of asthma. Am J Respir Crit Care Med 2015;191:1116–1125.
Copyright © 2015 by the American Thoracic Society
Muscle Weakness in Critical Illness Skeletal muscle weakness is a major complicating factor of critical illness, especially when combined with prolonged mechanical ventilation. In particular, diaphragm muscle weakness in critically ill patients can lead to dependence on mechanical 1094
ventilation with associated morbidity. In this issue of the Journal, Hooijman and colleagues (pp. 1126–1138) explore the pathophysiology of diaphragm muscle weakness at the singlemuscle-ﬁber level by measuring biomechanical properties (1).
American Journal of Respiratory and Critical Care Medicine Volume 191 Number 10 | May 15 2015
EDITORIALS These authors clearly demonstrate contractile weakness in diaphragm muscle ﬁbers from critically ill patients that is associated with both atrophy of muscle ﬁbers and reduced force per cross-sectional area (speciﬁc force). The sarcomere is the basic structural unit of skeletal muscle ﬁbers, comprising thin (actin) ﬁlaments that are anchored at the Z-discs bounding both sides of the sarcomere and emanate toward the midline, where they interdigitate with thick (myosin) ﬁlaments. Actin and myosin ﬁlaments are aligned in a highly organized, crystalline structure with a ﬁxed 6:1 stoichiometry. From the thick ﬁlaments, myosin heads (myosin heavy chains [MyHCs]) bind to the actin ﬁlament following Ca21 activation to form cross bridges, which are the essential units of force generation and contraction in muscle ﬁbers. The MyHC is both a structural protein and an enzyme responsible for ATP hydrolysis during cross-bridge cycling between attached and detached states. In adult skeletal muscle, there are four isoforms of MyHC that vary in their structural and enzymatic properties. These four MyHC isoforms comprise four different muscle ﬁber types that vary in their contractile and fatigue properties: slow-twitch, fatigueresistant (type I); fast-twitch, fatigue-resistant (type IIa); fasttwitch, fatigue-intermediate (type IIx); and fast-twitch, fatigable (type IIb). Regardless of ﬁber type, the force vectors generated by cross bridges pulling on the thin ﬁlaments within a sarcomere are toward the midline, so the total force generated by a muscle ﬁbers depends on the number of cross bridges formed in parallel per half sarcomere, which in turn depends on the MyHC content per half sarcomere and the level of Ca21 activation (2). Thus, the mechanobiology of skeletal muscle ﬁbers is highly dependent on sarcomeric structure and can be readily interpreted in this context (3,4). In 1957, Huxley (5) introduced a simple two-state model for cross-bridge cycling between attached and detached states that was essentially adopted by Hooijman and colleagues (1) in the present study. This model still provides a valuable conceptual framework for assessing the mechanobiology of skeletal muscle ﬁbers. In this two-state model, two apparent rate constants, one for cross-bridge attachment (fapp) and a second for cross-bridge detachment (gapp), deﬁne the cross-bridge cycle. When myoplasmic [Ca21] increases, the MyHC binds to the actin ﬁlament leading to cross-bridge attachment and force generation (described by fapp). When myoplasmic [Ca21] decreases, and after MyHC-mediated ATP hydrolysis, cross bridges detach to a non-force-generating state (described by gapp). According to the Huxley model, Brenner (6,7) proposed that the force generated by a muscle ﬁber is determined by the following equation: force = n $ afs $ F, where n is the number of myosin heads in parallel per half sarcomere (MyHC content per half sarcomere), afs is the steady-state fraction of strongly bound cross bridges in the force-generating state, and F is the force generated per cross bridge. Accordingly, as observed by Hooijman and colleagues (1), the decrease in force generated by diaphragm muscle ﬁbers of critically ill patients may have resulted changes in any one of these parameters. For example, they found that the diaphragm ﬁbers of critically ill patients were atrophied compared with those of controls. With a decrease in ﬁber cross-sectional, n is reduced with fewer myosin heads in parallel per half sarcomere. However, Hooijman and colleagues (1) also found that the speciﬁc force of diaphragm ﬁbers of Editorials
critically ill patients was reduced, suggesting that the mechanobiology underlying diaphragm muscle ﬁber weakness was more complex than just atrophy alone. These investigators concluded that together, their results indicated that the reduced speciﬁc force of diaphragm muscle ﬁbers of critically ill patients was caused by a reduction in the number of cross bridges during maximum Ca 21 activation. Other than the atrophy associated decrease in the myosin heads in parallel per half sarcomere, however, what else is affected in critically ill patients? One possibility was that the reduction in MyHC content per half sarcomere was disproportionately greater than the decrease in ﬁber cross-sectional area. In this respect, it was noteworthy that Hooijman and colleagues (1) found that the MyHC concentration of ﬁbers did not differ between control and critically ill patients. However, they did not directly measure MyHC content per half sarcomere, which involves normalizing MyHC concentration for half-sarcomere volume (2,8,9). In animal studies, a variety of conditions reduce MyHC content per half sarcomere and thereby decrease speciﬁc force (10,11). Associated with muscle ﬁber atrophy, the authors reported that the ubiquitin–proteasome pathway was activated in diaphragm ﬁbers of critically ill patients. They found that MuRF-1 and MAFbx levels were increased in diaphragm ﬁbers of critically ill patients, consistent with activation of proteolytic pathways and a negative protein balance. The authors suggest that the common denominator in the critically ill patients is that they were exposed to mechanical ventilation. In animal studies, mechanical ventilation alone is associated with diaphragm muscle ﬁber atrophy and weakness (12–14) and increased MuRF-1 and MAFbx expression with activation of the ubiquitin–proteasome pathway (15,16). The present study extended this previous work by showing that MuRF-1 knockout mice were protected against mechanical ventilation-induced diaphragm contractile weakness. Unfortunately, the authors did not examine the biomechanical properties of single diaphragm muscle ﬁbers in these mice. However, these results using MuRF-1 knockout mice generally supported their overall hypothesis that atrophy and contractile weakness of the diaphragm muscle of critically ill patients is caused in part by activation of the ubiquitin–proteasome pathway, negative contractile protein, and a reduction in the number of cross bridges contributing to force generation. In their study, Hooijman and colleagues (1) explored whether a reduction in the number of cross bridges during maximum Ca21 activation could be caused by a decrease in afs, the fraction of myosin heads that are in a strongly bound forcegenerating state. Such a decrease in afs could result from a change in either cross-bridge attachment (increased fapp) or detachment (decreased gapp) rate constants, as described by: afs = fapp/( fapp 1 gapp). To assess afs, the investigators measured the rate constant for force redevelopment (ktr), which provides information on the cumulative effect of fapp and gapp: ktr = fapp 1 gapp (cross-bridge cycling rate). Although afs is certainly affected by cross-bridge cycling rate (ktr), the authors incorrectly assumed that ktr “reﬂects” the fraction of strongly bound cross bridges. Not surprisingly, it has 1095
EDITORIALS been shown that ktr is sensitive to the level of Ca21 activation that directly affects fapp and thereby stiffness and afs. Unfortunately, the investigators only examined ktr during maximum Ca21 activation, so possible changes in Ca21 sensitivity of diaphragm muscle ﬁbers of critically ill patients were not evaluated. In their study, Hooijman and colleagues (1) measured muscle ﬁber stiffness to estimate the number of attached cross bridges during maximum Ca21 activation. However, they did not measure muscle ﬁber stiffness during a rigor (ATP-free) condition, which reﬂects the maximum number of attached strongly bound cross bridges. Normalizing muscle ﬁber stiffness during Ca21 activation to the maximum rigor stiffness provides a direct measure of afs (2). To estimate F, Hooijman and colleagues (1) calculated the tension/ stiffness ratio of diaphragm muscle ﬁbers. During maximum Ca21 activation, the authors found that the tension/stiffness ratio was similar between diaphragm ﬁber of critically ill patients and controls. This would not be surprising if the Ca21 sensitivity of diaphragm ﬁber activation was unaffected in critically ill patients, an important measure not assessed in the present study. If MyHC content per half sarcomere was reduced by proteolytic activation in diaphragm ﬁbers of critically ill patients compared with controls, then both force and stiffness would be reduced proportionately and the force/stiffness ratio would be unaffected. F is an important measure and is more directly related to muscle ﬁber force normalized for MyHC content per half sarcomere (2). Unfortunately, the authors did not measure MyHC content per half sarcomere. Hooijman and colleagues (1) also examined the ultrastructure of diaphragm muscle ﬁbers using electron microscopy in one critically ill patient and one control subject. Compared with the normal alignment of contractile proteins in the control ﬁbers, there was evidence of disarray in diaphragm ﬁbers from the critically ill patient. Although the authors appear to suggest muscle ﬁber damage as a possible mechanism for the reduced speciﬁc force (force per cross-sectional area) of diaphragm ﬁbers from critically ill patients, it is unclear how activation of proteolytic pathways is linked to ultrastructural damage. Unfortunately, this analysis was only cursory. In summary, the study by Hooijman and colleagues (1) provides important novel insight into the mechanobiological adaptations of diaphragm muscle ﬁbers in critically ill patients. This information suggests that activation of the ubiquitin–proteasome pathway and the resulting effect on contractile protein expression in diaphragm muscle ﬁbers affects the number of strongly bound cross bridges contributing to force generation. This indicates protein balance as an important therapeutic target to ameliorate diaphragm muscle weakness in critically ill patients. n Author disclosures are available with the text of this article at www.atsjournals.org.
Gary C. Sieck, Ph.D. Department of Physiology and Biomedical Engineering Mayo Clinic Rochester, Minnesota
References 1. Hooijman PE, Beishuizen A, Witt CC, de Waard MC, Girbes ARJ, Spoelstra-de Man AME, Niessen HWM, Manders E, van Hees HWH, van den Brom CE, et al. Diaphragm muscle ﬁber weakness and ubiquitin–proteasome activation in critically ill patients. Am J Respir Crit Care Med 2015;191:1126–1138. 2. Geiger PC, Cody MJ, Macken RL, Sieck GC. Maximum speciﬁc force depends on myosin heavy chain content in rat diaphragm muscle ﬁbers. J Appl Physiol (1985) 2000;89:695–703. 3. Gransee HM, Mantilla CB, Sieck GC. Respiratory muscle plasticity. Compr Physiol 2012;2:1441–1462. 4. Sieck GC, Ferreira LF, Reid MB, Mantilla CB. Mechanical properties of respiratory muscles. Compr Physiol 2013;3:1553–1567. 5. Huxley AF. Muscle structure and theories of contraction. Prog Biophys Biophys Chem 1957;7:255–318. 6. Brenner B, Eisenberg E. Rate of force generation in muscle: correlation with actomyosin ATPase activity in solution. Proc Natl Acad Sci USA 1986;83:3542–3546. 7. Brenner B. Effect of Ca21 on cross-bridge turnover kinetics in skinned single rabbit psoas ﬁbers: implications for regulation of muscle contraction. Proc Natl Acad Sci USA 1988;85:3265–3269. 8. Ottenheijm CA, Heunks LM, Sieck GC, Zhan WZ, Jansen SM, Degens H, de Boo T, Dekhuijzen PN. Diaphragm dysfunction in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2005;172:200–205. 9. Levine S, Nguyen T, Kaiser LR, Rubinstein NA, Maislin G, Gregory C, Rome LC, Dudley GA, Sieck GC, Shrager JB. Human diaphragm remodeling associated with chronic obstructive pulmonary disease: clinical implications. Am J Respir Crit Care Med 2003;168:706–713. 10. Geiger PC, Cody MJ, Macken RL, Bayrd ME, Sieck GC. Effect of unilateral denervation on maximum speciﬁc force in rat diaphragm muscle ﬁbers. J Appl Physiol (1985) 2001;90:1196–1204. 11. Geiger PC, Cody MJ, Han YS, Hunter LW, Zhan WZ, Sieck GC. Effects of hypothyroidism on maximum speciﬁc force in rat diaphragm muscle ﬁbers. J Appl Physiol (1985) 2002;92:1506–1514. 12. Le Bourdelles G, Viires N, Boczkowski J, Seta N, Pavlovic D, Aubier M. Effects of mechanical ventilation on diaphragmatic contractile properties in rats. Am J Respir Crit Care Med 1994;149:1539–1544. 13. Sassoon CS, Caiozzo VJ, Manka A, Sieck GC. Altered diaphragm contractile properties with controlled mechanical ventilation. J Appl Physiol (1985) 2002;92:2585–2595. 14. Powers SK, Shanely RA, Coombes JS, Koesterer TJ, McKenzie M, Van Gammeren D, Cicale M, Dodd SL. Mechanical ventilation results in progressive contractile dysfunction in the diaphragm. J Appl Physiol (1985) 2002;92:1851–1858. 15. Shanely RA, Zergeroglu MA, Lennon SL, Sugiura T, Yimlamai T, Enns D, Belcastro A, Powers SK. Mechanical ventilation-induced diaphragmatic atrophy is associated with oxidative injury and increased proteolytic activity. Am J Respir Crit Care Med 2002;166:1369–1374. 16. Sassoon CSh, Zhu E, Fang L, Sieck GC, Powers SK. Positive endexpiratory airway pressure does not aggravate ventilator-induced diaphragmatic dysfunction in rabbits. Crit Care 2014;18:494.
Copyright © 2015 by the American Thoracic Society
Understanding the Global Burden of Pediatric Sepsis Sepsis is common, costly, and deadly. It is also preventable and treatable, but success is predicated on recognizing the disease and understanding its true burden. We now have reasonably reliable estimates of the mortality of adult sepsis, at least in the developed world and over a short 1096
time window (1, 2). We also know that the costs of treating established sepsis in the intensive care unit (ICU) are substantial (3, 4). However, we have a surprisingly limited understanding of the true burden of disease and particularly as it affects children. In this issue of the
American Journal of Respiratory and Critical Care Medicine Volume 191 Number 10 | May 15 2015