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and Princess Margaret Hospitals, Subiaco, Perth, Western Australia, Australia. Preterm birth ... plasia (5). Little is known of how the structure and function of the pre- ... Bartholomew Medical Research Trust and a University of Western Australia.
In Utero LPS Exposure Impairs Preterm Diaphragm Contractility Yong Song1,2, Kanakeswary Karisnan1,2, Peter B. Noble1,2, Clare A. Berry1,2, Tina Lavin1,2, Timothy J. M. Moss3, Anthony J. Bakker1, Gavin J. Pinniger1*, and J. Jane Pillow1,2,4* 1 School of Anatomy, Physiology, and Human Biology, and 2Centre for Neonatal Research and Education, School of Paediatrics and Child Health, University of Western Australia, Crawley, Western Australia, Australia; 3Ritchie Centre, Monash Institute of Medical Research, and Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia; and 4Women and Newborns Health Service, King Edward Memorial and Princess Margaret Hospitals, Subiaco, Perth, Western Australia, Australia

Preterm birth is associated with inflammation of the fetal membranes (chorioamnionitis). We aimed to establish how chorioamnionitis affects the contractile function and phenotype of the preterm diaphragm. Pregnant ewes received intra-amniotic injections of saline or 10 mg LPS, 2 days or 7 days before delivery at 121 days of gestation (term ¼ 150 d). Diaphragm strips were dissected for the assessment of contractile function after terminal anesthesia. The inflammatory cytokine response, myosin heavy chain (MHC) fibers, proteolytic pathways, and intracellular molecular signaling were analyzed using quantitative PCR, ELISA, immunofluorescence staining, biochemical assays, and Western blotting. Diaphragm peak twitch force and maximal tetanic force were approximately 30% lower than control values in the 2-day and 7-day LPS groups. Activation of the NF-kB pathway, an inflammatory response, and increased proteasome activity were observed in the 2-day LPS group relative to the control or 7-day LPS group. No inflammatory response was evident after a 7-day LPS exposure. Seven-day LPS exposure markedly decreased p70S6K phosphorylation, but no effect on other signaling pathways was evident. The proportion of MHC IIa fibers was lower than that for control samples in the 7-day LPS group. MHC I fiber proportions did not differ between groups. These results demonstrate that intrauterine LPS impairs preterm diaphragmatic contractility after 2-day and 7-day exposures. Diaphragm dysfunction, resulting from 2-day LPS exposure, was associated with a transient activation of proinflammatory signaling, with subsequent increased atrophic gene expression and enhanced proteasome activity. Persistently impaired contractility for the 7-day LPS exposure was associated with the down-regulation of a key component of the protein synthetic signaling pathway and a reduction in the proportions of MHC IIa fibers. Keywords: chorioamnionitis; preterm infant; diaphragm; contractile dysfunction; molecular signaling

Chorioamnionitis, an inflammation of the placental and fetal membranes, is implicated in up to 70% of preterm births before 30 weeks of gestation (1). Adverse neonatal outcomes of chorioamnionitis (Received in original form March 7, 2013 and in final form May 23, 2013) * Joint senior authors. This study was supported by National Health and Medical Research Council (NHMRC) grant APP1010665 and a Sylvia and Charles Viertel Senior Medical Research Fellowship (J.J.P.), Women and Infants Research Foundation, the Ada Bartholomew Medical Research Trust and a University of Western Australia Research Development Award (Y.S.), and NHMRC Career Development Fellowship 1,045,824 (P.B.N.). Correspondence and requests for reprints should be addressed to J. Jane Pillow, B.Med.Sci., M.B.B.S., Ph.D., School of Anatomy, Physiology, and Human Biology, University of Western Australia, 35 Stirling Highway, M309, Crawley 6009, Western Australia, Australia. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 49, Iss. 5, pp 866–874, Nov 2013 Copyright ª 2013 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2013-0107OC on March 14, 2013 Internet address: www.atsjournals.org

CLINICAL RELEVANCE The results of this study are novel and important insofar as they will help further the understanding of researchers regarding the susceptibility of preterm infants to respiratory failure in response to chronic inflammation exposure in womb. The cellular and molecular mechanisms elucidated will lay the foundation for designing therapeutic targets for future interventions aiming to recover the function of the fragile preterm respiratory muscle after exposure to inflammation.

include fetal systemic inflammation, lung, brain, and gastrointestinal injury (2–4), and an increased risk of bronchopulmonary dysplasia (5). Little is known of how the structure and function of the preterm diaphragm is affected by chorioamnionitis, despite its obvious role as the primary respiratory muscle and the incidence of respiratory distress in preterm infants. In the setting of severe sepsis in adults, diaphragmatic impairment is acknowledged as a cause of respiratory failure (6). Because preterm infants often breathe against an increased mechanical load, diaphragm integrity is particularly critical for self-sufficient ventilation in preterm infants. Preterm infants exhibit reduced diaphragm contractility, compared with their term gestation counterparts (7, 8). Additional compromise of the functional and phenotypic integrity of the weakened immature diaphragm, as induced by intrauterine exposure to inflammation, could precipitate or accelerate the development of postnatal respiratory failure. A marked and rapid reduction is evident in respiratory skeletal muscle strength with infection in adult animals (9–12). Although the precise mechanisms by which infection impairs muscle function are not fully understood, accelerated proteolysis and reduced protein synthesis likely contribute to muscle protein loss during sepsis in adults (13–15). The key factors in the metabolic response to sepsis include the induction of catabolic agents (e.g., TNF-a, IL-1b, IL-6, and cortisol) and the suppression of the anabolic factor insulin growth factor-1 (IGF-1) (14). The activated ubiquitin– proteasome system (UPP) comprises a primary pathway responsible for the breakdown of accumulated muscle protein (12, 15). UPP E3 ligase (atrogin-1/MAFbx and MuRF1) is up-regulated alongside the induction of skeletal muscle wasting during infections (15, 16). The depression of protein synthesis arises from insulin-resistant or impaired IGF-1/P13K/Akt1 signaling (13, 14). In addition to inflammation-induced muscle wasting, evidence from animal models suggests that both local (17, 18) and systemic (19) inflammation decreases the intrinsic force-producing capacity of skeletal muscle (force loss independent of muscle wasting, i.e., reduced contractility). This effect may be mediated by an increased production of reactive oxygen species (ROS) and their

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effects on myofilaments and/or altered intracellular calcium homeostasis (20, 21). Previous studies investigating diaphragmatic impairment in the context of inflammation were undertaken in adult subjects or during the postnatal period (6, 9, 18). The effects of inflammation on diaphragm structure and function are likely to differ during critical stages of prenatal development. Because prenatal organ development is largely under the control of genetic programming (22), environmental insults encountered in utero may alter gene expression, adversely affect the metabolic and endocrine balance of affected individuals, and subsequently lead to dysfunction later in life (22). Moreover, the premature diaphragm is weaker and more susceptible to injury compared with that at term, owing to differences in fiber type (7, 23), oxidative capacity (24), and the functional immaturity of the immune system’s response to bacterial infection (25). The present study sought to evaluate functional changes in the preterm fetal diaphragm after in utero inflammation, and to explore the underlying mechanisms that may explain any change in function. We hypothesized that antenatal exposure to inflammation promoted structural and physiological changes in the fetal diaphragm, resulting in weakness at birth. To test this hypothesis, we used a well-established preterm ovine model of chorioamnionitis induced by intra-amniotic (IA) injections of LPS (26, 27) to analyze diaphragm contractile properties, fiber type composition, and the activity of protein degradation and synthesis pathways.

MATERIALS AND METHODS Animals and Experimental Design All experiments were approved by the Animal Ethics Committee of the University of Western Australia. Date-mated ewes with singleton fetuses were randomly assigned to a treatment group receiving an IA injection of LPS (10 mg Escherichia coli 055:B5; Sigma Chemical Company, St. Louis, MO) at 114 days (7-d LPS) or 119 days (2-d LPS) of gestational age (GA), or to a control group receiving IA saline at equivalent time points. Preterm fetal lambs were delivered surgically via hysterotomy at 121 days of GA (term ¼ 150 d of GA), and were killed immediately with pentobarbitone (150 mg/kg, intravenously; Pitman-Moore, Sydney, NSW, Australia). The right hemidiaphragm was removed for analyses of contractile function. The left costal hemidiaphragm was used for biochemical and molecular experiments, or embedded in optimal cutting temperature (OCT) compound for histological staining. Plasma was obtained from the umbilical artery to assess systemic responses to IA LPS exposure.

Muscle Contractile Properties The muscle preparation and contractile property measurements were performed as described previously (7). The detailed protocol is described in the online supplement.

Western Blot Analysis The whole-cell lysate was prepared as described previously (24). Cytosolic and nuclear protein fractions were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Billerica, MA). Western blotting was performed as previously described (24). Detailed information for the antibodies used and for the quantification and normalization methods is described in the online supplement.

IL-1b and IL-6 Plasma Levels The sheep ELISA assays for IL-1b and IL-6 were developed in our laboratory. The detailed procedure is described in the online supplement.

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previously (7, 24). The proteolytic gene (calpain I, calpain II, caspase-3, MAFbx, MuRF1, E2, C8, and ubiquitin) primers used were optimized and validated by us previously (24). Cytokine gene (IL-1b, IL-6, and TNF-a) primers were described previously by other investigators (28, 29).

Myosin Heavy Chain Fiber-Typing and Myofiber Cross-Sectional Areas Each OCT-embedded diaphragm was sectioned and stained with antibodies specific to laminin (1:250; Abcam, Waterloo, NSW, Australia), myosin heavy chain (MHC) Type I (1:50; Novocastra, Newcastle, UK), or MHC Type IIa (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The detailed protocol is described in the online supplement.

Biochemical Analysis The activities of calpain (Abcam), caspase-3 (Sigma, Castle Hill, NSW, Australia), and 20S proteasome (the chymotrypsin-like peptidase; Enzo Life Sciences, Farmingdale, NY) were measured fluorometrically in crude extracts using commercial kits, according to the manufacturer’s instructions.

Data Analysis Sigmaplot (version 12.0; Systat Software, Inc., San Jose, CA) was used for statistical analyses. Differences among multiple groups were assessed using one-way ANOVA, with a Tukey honestly significant difference test implemented as post hoc analysis. Nonparametric data were analyzed using ANOVA on ranks. The Pearson correlation index was calculated to determine the association among different variables, using linear regression analysis. P , 0.05 was considered statistically significant. Data are presented as mean (SD) or median (range), unless specified otherwise.

RESULTS Animal Characteristics

For the animals used in the physiological assessment of contractile function, the control group included three male and five female lambs, with a mean gestational age of 120.7 6 0.5 days. The 2-day LPS group comprised three male and three female lambs, and the 7-day LPS group contained two male and four female lambs, all at 121 days of gestation. No significant differences in body weight (Table 1) or in condition at delivery were evident. Contractile Measurements

Contractile measurements are shown in Table 1. No significant differences were evident in optimal muscle length, time to peak twitch force, half relaxation times, and twitch/tetanus ratio. Peak twitch force was decreased by 29% and 31% after a 2-day and 7-day LPS exposure, respectively (P , 0.05) (Figure 1A). A similar decrease in maximal tetanic force was also observed, with 28% and 33% reductions in the 2-day and 7-day LPS groups, respectively (P , 0.05) (Figure 1B). The normalized force frequency relationship was not different between the control and LPS-treated groups (Figure 1C).

TABLE 1. DESCRIPTIVE DATA AND TWITCH PARAMETERS FOR SALINE-TREATED AND LPS-TREATED PRETERM LAMBS Saline (n ¼ 8)

Two-D LPS (n ¼ 6) Seven-D LPS (n ¼ 6)

RNA Isolation, Reverse Transcription, and Quantitative PCR

Body weight, kg 2.48 6 0.34 29.6 6 3.15 Lo, mm TTP, ms 161.9 6 107.2 1/2 RT, ms 193.1 6 76.0 Twitch/tetanus ratio 0.51 6 0.06

Detailed methods regarding RNA purification, reverse transcription, and quantitative PCR conditions used by our laboratory were described

Definition of abbreviations: Lo, optimal muscle length; TTP, time to peak twitch force; 1/2 RT, half relaxation time.

2.63 30.3 115.2 220.3 0.50

6 6 6 6 6

0.15 3.15 14.4 42.9 0.07

2.50 28.2 234.7 258.8 0.53

6 6 6 6 6

0.16 1.87 143.7 60.9 0.09

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IIa fiber CSAs were not significantly different between experimental groups (Figure 3C). Cytokine Response

A 12-fold increase in plasma IL-6 protein levels was evident after the 2-day exposure to LPS (P , 0.05; Figure 4A). The IL-6 concentration after the 7-day treatment was not significantly different from control values. A similar pattern was observed in systemic IL1b, although the difference was not significant (Figure 4A). Consistent with the maximal induction of plasma cytokines after 2-day LPS exposure, the cytokine mRNA expression in the diaphragm was up-regulated in the 2-day LPS group, with a 4-fold and 2-fold change relative to the control group for IL-1b and IL-6, respectively (P , 0.05) (Figure 4B). TNF-a mRNA was unaltered by LPS exposure. Molecular Signaling

To identify signal transduction cascades involved in intra-amniotic LPS-induced diaphragm dysfunction, we evaluated several key intracellular mediators of anabolic (Akt, mammalian target of rapamycin [mTOR], p70S6K, and 4E-BP1) and catabolic (FOXO1 and NF-kB) pathways (Figure 5). LPS exposure did not change the phosphorylation state of Akt or 4E-BP1 and total mTOR protein. However, 7-day LPS exposure resulted in a decreased

Figure 1. Fetal diaphragm contractile properties, maximal twitch and tetanic force, and force frequency relationships. Peak twitch force (A), maximal tetanic force (B), and normalized force frequency relationship (C) were determined for saline (n ¼ 8), 2-day LPS (n ¼ 6), and 7-day (n ¼ 6) LPS exposure groups. Values represent means 6 SEMs. *P , 0.05 and **P , 0.01, compared with the control group.

The fatigue protocol reduced maximum tetanic force by approximately 65%, although no significant difference was evident in the fatigue index between groups (Figure 2A). The stretchinduced muscle damage protocol resulted in an approximately 15% decrease in maximum tetanic force, which was not altered by previous exposure to LPS (Figure 2B). The unloaded shortening velocity in fetal diaphragms was also unaffected by LPS exposure (Figure 2C). MHC Proteins and Fiber Cross-Sectional Areas

The MHC fiber typing of preterm diaphragms in the control group showed that 15% of total muscle fibers were slow Type I, and 67% were fast Type IIa fibers (Figure 3A). Exposure to LPS 7 days before the study caused a 19% decrease in MHC Type IIa fiber proportions (P , 0.05) (Figure 3B). The apparent lower proportion of Type IIa fibers in the 2-day LPS group did not reach statistical significance (P ¼ 0.190). The percentage of slow Type MHC I fibers was similar in the LPS and control groups (Figure 3B). The total fiber cross-sectional area (CSA), including all different MHC type fibers, was unchanged by LPS exposure (P ¼ 0.136) (Figure 3C). Moreover, MHC Type I and

Figure 2. Susceptibility to fatigue and muscle damage, and unloaded shortening velocity of the fetal diaphragm. Fatigue index (A), percentage muscle damage after stretch protocol (B), and unloaded shortening velocity (V0) (C) were determined for saline (n ¼ 8), 2-day LPS (n ¼ 6), and 7-day LPS (n ¼ 6) exposure groups. Values represent means 6 SEMs.

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Further association analysis demonstrated that the mRNA expression of MAFbx was positively correlated with the nuclear/ cytosolic NF-kB protein content ratio (r ¼ 0.570, P , 0.05) and UPP activity (r ¼ 0.761, P , 0.001) (Figure E1).

DISCUSSION

Figure 3. Diaphragm muscle fiber types and measurements of crosssectional area (CSA). (A) Muscle fiber sections were stained with laminin (red) or MHC (green) antibody. Graphs show percentages of MHC Type I and MHC Type IIa fibers (B) and muscle-fiber cross-sectional areas (C) in the control (n ¼ 4), 2-day (n ¼ 4), and 7-day (n ¼ 6) LPS exposure groups. Values represent means 6 SEMs. *P , 0.05. MHC, myosin heavy chain.

phosphorylation of p70S6K (P , 0.05). The FOXO signaling pathway was not altered in response to LPS. In contrast, LPS resulted in a significant decline in nuclear NF-kB after 7 days (P , 0.05), although no effect was evident after 2 days (P ¼ 0.136) (Figure 5D). A significant association was observed between the dynamic pattern of NF-kB signaling and IL-1b gene expression in fetal diaphragms (r ¼ 0.481, P , 0.05) (Figure E1 in the online supplement). Proteolytic Pathways

The key components of the three major protein degradation pathways (calpain, caspase-3, and UPP) were analyzed for gene expression levels (Figure 6) and enzyme activities (Figure 7). LPS slightly promoted the gene expression of MAFbx, E2, C8, and ubiquitin after a 2-day exposure, whereas expression was reduced after a 7-day LPS exposure. In contrast, a decreasing pattern of gene expression was observed in MuRF1 in response to increasing durations of LPS exposure. LPS did not affect caspase-3, calpain I, or calpain II mRNA levels. In accordance with the proteolytic gene expression data, 20S proteasome activity was markedly higher in the 2-day LPS group compared with the control group. No significant changes in calpain and caspase-3 activities were detected across different groups.

We show that IA LPS impaired preterm (121 d) diaphragmatic contractility after a 2-day and 7-day in utero exposure. These changes are consistent with the documented effects on respiratory and limb muscles in animals injected with LPS (10, 11). The phenotypic change was characterized by a preferential reduction in the proportions of MHC Type IIa muscle fibers. Short-term (2-d) LPS exposure was associated with a transient activation of inflammatory signaling and the NF-kB pathway, with the subsequent promotion of atrophic gene expression and increased proteasome activity. A more prolonged (7-d) exposure involved an attenuation of the protein synthesis pathway. We propose that the difference in regulatory mechanisms between the 2-day and 7-day LPS exposure groups represents a progressive change in signaling behavior associated with either increasing duration of LPS exposure, or the temporal nature of the fetal response to LPS determined by gestational age (and the diaphragm’s developmental stage) at the time of LPS exposure. Our observation of decreased diaphragm muscle force after 2-day and 7-day in utero LPS exposure supports our hypothesis that antenatal inflammation impairs preterm respiratory muscle function. A 2-day and 7-day LPS exposure reduced both diaphragm peak specific twitch force and maximal specific force by approximately 30%. This indicates that diaphragm muscle strips from the LPS group were intrinsically weaker than similar sized strips from the control group. Because the strips varied in size, absolute muscle mass, as an indicator of muscle atrophy, could not be ascertained in this study. However, decreased specific force is commonly associated with inflammatory diseases and their associated cytokine secretion (21). Cytokines can compromise muscle contractile function via the modulation of calcium transients (30), decreasing the sensitivity of myofilaments to calcium activation (21), both of which would result in decreased specific force. Surprisingly, reduced force-generating capacity was not accompanied by increased fatigability. Increased skeletal muscle fatigability has been demonstrated in LPS-treated adult rats (31) and during sepsis (32). The discordant results may be attributable to different compositions of muscle fiber types in preterm muscles compared with adult muscles. Preterm diaphragm muscle consists of a majority of oxidative glycolytic (Types I and IIa) fibers that are less fatigable, compared with Type IIb/x fibers, which are found at a higher proportion in adult muscles (33). Muscle MHC isoform is an important determinant of the contractile properties of individual myocytes (34). Our finding that MHC Type IIa was the predominantly expressed isoform (67%), whereas MHC Type I represented 15% of the total fibers, is consistent with the fiber composition reported in lamb diaphragms at 127 days of GA (35). From late gestation to term, a significant increase in the expression of MHC Types I and IIa, a decrease in MHC Type IIb/x, and an almost complete loss of embryonic/ neonatal MHC expression occurred (35). After a 7-day LPS exposure, the muscle fiber Type IIa proportion was decreased by 19%, although Type I remained unchanged. Altered conformations or reduced absolute numbers of contractile proteins may also decrease the number of cross-bridges available to generate force, and may thus result in a decrease in peak twitch force and maximal tetanic force. However, the reduced specific force after 2 days and 7 days of LPS exposure preceded any significant loss of contractile proteins, and was not accompanied by decreased myofiber CSAs. Thus, MHC Type IIa protein loss is not the sole factor

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Figure 4. Systemic and local cytokine response. (A) Plasma IL-1b and IL-6 concentrations in 2-day (n ¼ 7) and 7-day (n ¼ 7) LPS exposure groups relative to saline control (n ¼ 5). Values represent means 6 SEMs. *P , 0.05. (B) Diaphragm IL-1b, IL-6, and TNF-a mRNA expression after intra-amniotic LPS exposure. Values represent medians (with 25th and 75th centiles). *P , 0.05 and #P , 0.05, compared with the control and LPS 7-day groups, respectively. Horizontal dashed lines indicate the mean or median of the reference (saline control) group.

accounting for the loss of contractile force deficit, particularly in the 2-day LPS group. Proinflammatory cytokines are primary mediators that trigger the development of muscle fiber injury (36, 37). Muscle fiber injury is consistent with our finding that the early activation of

systemic and local cytokines (IL-6 and IL-1b) occurs in parallel with impaired contractile function. Cytokines and other proinflammatory mediators released from distant organs can enter the circulation and act upon the diaphragm in an endocrine fashion in the setting of sepsis. Local proinflammatory cytokine

Figure 5. Activity of signaling molecules after LPS exposure. (A) Western blots illustrate the expression of signaling molecules, using representative samples from each group. Graphs show p-Akt/total Akt protein (B), mTOR protein content (C), p-p70S6/total p70S6 kinase (D), p-4E-BP1/total 4EBP1 protein (E), nuclear/cytosolic FOXO protein (F), and nuclear/cytosolic NF-kB protein (G) in the costal diaphragm of control animals (n ¼ 5), or after exposure to LPS for 2 days (n ¼ 7) or 7 days (n ¼ 7). Values represent means 6 SEMs. *P , 0.05. Cyto, cytosolic; p, phosphorylated.

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Figure 6. Gene expression of key components in proteolytic pathways. Graphs show calpain I (A), calpain II (B), caspase-3 (C), E2 (D), C8 (E), ubiquitin (F), MAFbx (G), and MuRF1 (H) mRNA expression in 2-day (n ¼ 7) and 7-day (n ¼ 7) LPS exposure groups, relative to the saline control group (n ¼ 5). Values represent medians (with 25th and 75th centiles). Horizontal dashed lines indicate medians of the reference (saline control) group. *P , 0.05, compared with the LPS 7-day group.

expression may lead to fiber weakness via the promotion of protein degradation (38), the suppression of anabolic processes (39), and the induction of oxidative stress (40). An elevation of circulating cytokines has recently been shown to play a key role in LPS-induced diaphragm weakness in mice (41). Thus, the local up-regulation of IL-6 and IL-1b expression we observed was likely induced by circulating proinflammatory mediators in a synergistic manner. IL-6 and IL-1b function as catabolic factors to stimulate muscle weakness and induce contractile dysfunction (39, 42, 43). In addition to IL-6 and IL-1b, TNF-a is an important mediator of adult muscle dysfunction. However, we found that local TNF-a mRNA was unchanged by antenatal fetal exposure to LPS. The lack of TNF-a response to IA LPS in the liver, placental membranes, and jejunum of fetal lambs was reported previously (44). Furthermore, Ikegami and colleagues (45) showed that preterm lambs did not develop an inflammatory response to TNF-a, known as a potent inducer of inflammation in adult sheep. This

unique differential response of the fetus to TNF-a is probably attributable to poorly developed innate immune systems in preterm compared with adult subjects (46, 47). It follows that the predominant signaling pathways in the development of fetal diaphragm dysfunction after IA LPS exposure may diverge from those defined in the adult diaphragm muscle. Aberrant NF-kB signaling is triggered by inflammatory stimuli, and is implicated in muscle atrophy (48, 49). Normally, NFkB is sequestered into the cytoplasm of nonstimulated cells, and is subsequently translocated into the nucleus to promote gene expression after the NF-kB pathway is activated. Indeed, we observed a consistent change between NF-kB signaling activity and cytokine response in fetal diaphragms after antenatal LPS exposure. The mechanistic link between NF-kB signaling activity and cytokine response was supported further by the significant correlation between IL-1b mRNA expression levels and nuclear/ cytoplasmic NF-kB protein ratios (Figure E1).

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Figure 7. Biochemical activity of calpain, caspase-3,th and UPP pathways. Graphs show the response of calpain (A), caspase-3 (B), and UPP (C) pathways in the control (n ¼ 5), 2-day (n ¼ 7), and 7-day (n ¼ 7) LPS exposure groups. Values represent means 6 SEMs. *P , 0.05. UPP, ubiquitin–proteasome system; RFU, relative fluorescence units; AFU, arbitrary fluorescence units.

NF-kB is a transcriptional factor that accelerates muscle protein loss by regulating the expression of multiple atrophic genes and by activating the proteasome system (48, 50). We observed an up-regulation of multiple atrophy genes (MAFbx, E2, C8, and ubiquitin) associated with increased activity of the UPP pathway after a 2-day LPS exposure. Moreover, the expression of MAFbx, the gene specific to muscle atrophy, correlated with both NF-kB signaling and UPP activity. Other proteolytic enzyme systems, such as caspase-3 and calpain, may also contribute to muscle proteolysis under catabolic conditions by breaking down the contractile proteins and releasing the protein elements to be targeted by UPP. Because we detected no change in the gene expression and activity of caspase-3 and calpain, we propose that UPP activation within the preterm diaphragm is mainly responsible for the loss of muscle protein in response to 2-day LPS exposure in utero. Thus, UPP activation is probably mediated by inflammatory cytokine signaling and subsequent NF-kB signaling, similar to the regulatory mechanism in an adult animal model of inflammation-associated diaphragm muscle weakness (48). Compared with the 2-day LPS group, a 7-day LPS exposure resulted in a marked reduction of cytokine response, NF-kB signaling, and UPP activity. The diminished response of the cellular events beyond 2-day LPS exposure suggested an ability of the preterm diaphragm to resolve inflammation. Although the precise

mechanisms remain unclear, negative regulators such as suppressor of cytokine signaling 1 (SOCS1), IL-1 receptor–associated kinase M (IRAK-M), and SH2-containing inositol 5’-phosphatase (SHIP) are proposed to play a key role, along with the downregulation of Toll-like receptor–4 on cell-surface and gene reprogramming (51). The administration of LPS could also reduce skeletal muscle protein synthesis in neonatal animals (52, 53) through the suppression of the anabolic cascade Akt/mTOR and its downstream effectors (p70S6K and 4E-BP1), to impede the efficiency of translation initiation (54, 55). A 7-day LPS exposure in utero did not change Akt/mTOR activity, but significantly decreased the activity-related phosphorylation of p70S6K. The activation of p70S6 kinase is essential to maintain normal muscle fiber mass in vivo, whereas the attenuation of p70S6K signaling could interfere with the translation of mRNAs into the 59 terminal oligopyrimidine tract and with the accretion rates of protein synthesis (56). These data imply that the alteration of p70S6K activity in response to fetal inflammation contributes to muscle protein loss, independently of Akt/ mTOR regulation. However, the direct measurement of in vivo protein synthesis rates is needed to support this possibility. In addition, the nuclear translocation of cytoplasmic FOXO is a common mechanism in disused muscle atrophy via the decreased activity of Akt (57). Unsurprisingly, the level of translocated FOXO remained unchanged, which is consistent with its upstream regulator Akt, further excluding the role of Akt/FOXO in cell signaling and increased protein breakdown. We note, however, that the reduced specific force after 2 days and 7 days of LPS exposure preceded any significant loss of contractile proteins, and was not accompanied by signs of atrophy. Thus, contractile protein loss is not the sole factor accounting for the loss of contractile force deficit, particularly in the 2-day LPS group. Using a sheep model of chorioamnionitis, we showed previously that IA LPS caused systemic oxidative stress at 7 days after in utero exposure, but not after a 2-day exposure (58). Cytokines and LPS are well-known to prime the increase in the ROSinduced production of neutrophils through the activation of nicotinamide adenine dinucleotide phosphate reduced (59, 60). The increase in systemic and local cytokine response after a 2-day LPS exposure in the present study contrasts with our previous observation that a 7-day LPS exposure was required to increase oxidant activity (58). Together, these data suggest that the oxidant response after LPS is mediated by inflammation. Redox disturbance is a known modulator of disused muscle atrophy through activating multiple proteolytic systems (61). A recent in vitro study also revealed that oxidants depressed protein synthesis by reducing the phosphorylation of mTOR substrates (4EBP1 and p70S6K) (62). Moreover, ROS exert a direct effect on muscle contractile function via altering myofibrillar Ca21 sensitivity and cross-bridge kinetics, leading to muscle dysfunction (20). Therefore, ROS may feasibly have contributed to fetal diaphragm weakness in the present study, particularly in the 7-day LPS group, by a direction modulation of muscle function and/or an indirect activation of signaling pathways. We argue that the difference in regulatory mechanisms between the 2-day and 7-day LPS exposure groups represents a progressive change in signaling behavior, associated with either increasing durations of LPS exposure or the temporal nature of the fetal response to LPS, as determined by gestational age (and developmental stage of the diaphragm) at the time of LPS exposure. In conclusion, a brief (2-d or 7-d) in utero exposure to an inflammatory stimulus impairs the function of the preterm diaphragm. The dysfunction resulting from 2-day LPS is strongly related to proinflammatory signaling, the activated NF-kB pathway, and the 20S proteasome system. In contrast, 7-day LPS exposure directly affects the key component of signal transduction

Song, Karisnan, Noble, et al.: Preterm Diaphragm Weakness

pathways regulating protein synthesis. Overall, IA LPS appears to trigger a complex series of effects consisting of impaired contractile function, an early inflammatory response accelerating proteolysis, and secondary changes to the protein synthesis pathway, leading to muscle weakness. The contribution of diaphragm dysfunction to respiratory insufficiency in the preterm infant after a proinflammatory exposure warrants further investigation. Author disclosures are available with the text of this article at www.atsjournals.org.

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