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Temporally Distinct Regulation of Pathways Contributing to Cardiac Proteostasis During the Acute and Recovery Phases of Sepsis

Crowell, Kristen T.*; Moreno, Samantha§; Steiner, Jennifer L.; Coleman, Catherine S.; Soybel, David I.*,†,‡; Lang, Charles H.*,†

doi: 10.1097/SHK.0000000000001084
Basic Science Aspects
Editor's Choice

Background: Cardiac dysfunction is a common manifestation of sepsis and is associated with early increases in inflammation and decreases in myocardial protein synthesis. However, little is known regarding the molecular mechanisms regulating protein homeostasis during the recovery phase after the removal of the septic nidus. Therefore, the purpose of this study was to investigate diverse signal transduction pathways that regulate myocardial protein synthesis and degradation.

Methods: Adult male C57BL/6 mice were used to identify potential mechanisms mediating the acute (24 h) effect of cecal ligation and puncture as well as long-term changes that manifest during the chronic (10 days) recovery phase.

Results: Sepsis acutely decreased cardiac protein synthesis that was associated with reduced phosphorylation of S6K1/S6 but not 4E-BP1. Sepsis also decreased proteasome activity, although with no change in MuRF1 and atrogin-1 mRNA expression. Sepsis acutely increased apoptosis (increased caspase-3 and PARP cleavage), autophagosome formation (increased LC3B-II), and canonical inflammasome activity (increased NLRP3, TMS1, cleaved caspase-1). In contrast, during the recovery phase, independent of a difference in food consumption, global protein synthesis was increased, the early repression in proteasome activity was restored to basal levels, whereas stimulation of apoptosis, autophagosome formation, and the canonical inflammasome pathway had abated. However, during recovery there was a selective stimulation of the noncanonical inflammasome pathway as evidenced by activation of caspase-11 with cleavage of Gasdermin D.

Conclusions: These data demonstrate a temporally distinct homeostatic shift in the cardiac proteostatic response to acute infection and recovery.

*Department of Surgery, Penn State College of Medicine, Hershey, Pennsylvania

Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania

Department of Nutritional Sciences, Penn State University, University Park, Pennsylvania

§Franklin and Marshal College, Lancaster, Pennsylvania

Address reprint requests to Charles H. Lang, PhD, Penn State College Medicine, Hershey, PA. E-mail: chl1@psu.edu

Received 13 October, 2017

Revised 2 November, 2017

Accepted 6 December, 2017

Authors’ contributions: All authors conceived and designed the study; collected, analyzed, and/or interpreted the data; and drafted and approved the final manuscript.

This work was supported by a grant from GM 38032 (CHL), as well as by postdoctoral fellowship awards F32 GM112401 (KTC), F32 AA023422 (JLS), and by an American Heart Association Summer Undergraduate Research Fellowship (14UFEL3900000) to Samantha Moreno.

The authors report no conflicts of interest.

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INTRODUCTION

The immune and metabolic alterations produced acutely by sepsis affect essentially all tissues and organ systems. However, the sepsis-induced changes in myocardial function are particularly relevant in the progression to septic shock and the ultimate recovery of the patient from the septic episode (1). Between 40% and 60% of all septic patients manifest with some type of cardiac dysfunction, with in vivo and in vitro preclinical studies demonstrating that sepsis negatively impacts a number of determinants of cardiac contractility and function (2). Moreover, sepsis-induced myocardial dysfunction is a key determinant of increased mortality in this condition and the ability to recover from the initial insult (3).

Although the etiology of this cardiac dysfunction is likely multifactorial, inflammation-enhanced oxidative stress seems to represent a central physiological process (4). The early sepsis-induced increase in proinflammatory cytokines in blood and heart is also associated with a reduction in global protein synthesis in this tissue (5), although the underlying mechanism has not been determined. Moreover, acting through a cytokine-mediated mechanism(s), sepsis also impairs the ability of nutrient signals to increase myocardial protein synthesis (6) potentially impairing the recovery of this organ. In addition to the synthesis of new proteins, cellular protein homeostasis is governed by alterations in protein degradation via a number of mechanisms, including the ubiquitin-proteasome pathway (UPP), apoptosis, and autophagy (7). Because of the low mitotic potential of cardiomyocytes, proteostasis is of particular importance in maintaining normal heart function.

Despite recognizing that recovery from sepsis occurs over weeks or months and impairs quality-adjusted survival in humans (8), the large majority of published studies focus on the acute (<48 h postinfection) phase of sepsis. Whereas, there is limited information pertaining to the temporal progression by which protein homeostasis in heart is restored and changes that may develop during convalescence. The present study uses a murine model of cecal ligation and puncture (CLP) to study the acute phase of sepsis (24 h) as well as a CLP model that incorporates surgical removal of the punctured cecum to eliminate the septic nidus and the administration of antibiotics to investigate the convalescence or recovery phase (10 days) of sepsis (9, 10). Therefore, to investigate the aforementioned gaps in knowledge, we assessed temporal changes in the sepsis-induced signal transduction pathways regulating myocardial protein synthesis (i.e., protein translation initiation and elongation), ubiquitin-proteasome degradation, autophagy, and apoptosis.

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MATERIALS AND METHODS

Animal care

Adult male C57BL/6 mice at 10 weeks of age (26.9 ± 1.9 g; Charles River Laboratories, Wilmington, Mass) were used in all experiments described below. Mice were individually housed in polycarbonate cages with corncob bedding throughout the 1-week acclimation period and the subsequent experimental protocol. Standard rodent chow (Teklad Globak 2019, Harlan Teklad, Boston, Mass) and water were provided ad libitum, and mice were maintained in a controlled environment (23°C; 30%–70% humidity; 12 h:12 h light:dark cycle) in the animal care facility at the Pennsylvania State University (PSU) College of Medicine that is specific-pathogen free. All experiments were approved by the Institutional Animal Care and Use Committee at the PSU College of Medicine (#46-946-17) and adhered to National Institutes of Health (NIH) guidelines.

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Experimental design

Mice were randomly assigned to either a septic group or a control group that was time-matched. This study used two different models, one designed to mimic the acute phase of peritonitis and the other the recovery phase. Although these two models are distinct (e.g., differing in nutritional intake and antibiotic treatment), they have been purposely designed to recapitulate the two time points after sepsis that were being studied.

Polymicrobial peritonitis was induced using CLP, and separate groups of mice were studied in the acute phase of sepsis (i.e., 24 h post-CLP) and during the recovery phase (i.e., day 10 post-CLP), as previously described (9, 10). Briefly, mice were anesthetized with isoflurane (3%–4% induction with 2%–3% maintenance; Vedco, St. Joseph, Mo) in 100% oxygen, and a warming blanket used to maintain body temperature. Isoflurane was selected because of its rapid induction time and the need for short-term anesthesia (<10 min) during the CLP procedure. The abdomen was shaved and a midline laparotomy was performed after cleaning with betadine. The cecum was ligated and punctured through-and-through using a 25-g needle. Cecal material was extruded from the puncture site to ensure patency. The cecum was returned to the abdominal cavity and the rectus abdominis was closed using 5-0 silk and the skin closed with metal wound clips. One milliliter of warm sterile saline (0.9%) containing 0.05 mg/kg of the analgesic buprenorphine (Reckitt Benckiser Pharmaceuticals, Richmond, Va) was administered subcutaneously 30 min before the start of surgery to all mice. Sham-control mice underwent the same surgical procedure except the cecum was not ligated or punctured. Mice were subsequently transferred to a warming pad until they regained consciousness. As septic mice consume little or no food during the first night after CLP (9), both control and septic mice were fasted to ensure the same basal nutritional state. Mice were euthanized at 24 h after laparotomy ± CLP and this is referred to as the “acute sepsis” group.

A separate cohort of mice was used to examine changes 10 days post-CLP, and this group is referred to as “sepsis-recovery.” Control mice had their surgery performed 1 day after septic mice. This staggered approach permitted us to determine the food consumed by septic mice each day and then provide control animals with the same amount of food. Hence, over the 10-day course of the study, septic and control mice had the same nutritional intake and are considered pair-fed. For these sepsis-recovery mice, antibiotic (0.5 mg meropenem; Fresenius Kabi, Lake Zurich, Ill) and buprenorphine were injected subcutaneously (total volume 1 mL of saline) every 12 h starting 24 h post-CLP (i.e., day 1) and continued for the next 5 days. Mice were anesthetized at 48 h post-CLP and the original incision was reopened. The ischemic portion of the cecum distal to the original suture ligation was then resected and the abscess surrounding the cecum was excised. The peritoneal cavity was washed with warmed saline (5 mL) to remove the debris and abscess. The abdominal incision was again closed and resuscitation provided as above, and mice were observed through day 10. Control mice received the same volume of fluid resuscitation with buprenorphine but without antibiotic. It is noteworthy that the two control groups (e.g., acute sepsis and sepsis-recovery) used in this study are not equivalent because of differences in the nutritional state of mice and the time postsurgery when mice were euthanized. These differences may account for the observed differences between the two control groups for some of the endpoints determined (discussed later).

In general, mice were monitored approximately every 8 h and were weighed daily. If mice were observed to be moribund, immobile, unable to consume food and/or water, or had a rectal temperature less than 30oC for more than 8 h, mice were euthanized by certified research personnel that had successfully completed appropriate institutional animal training. Mice not meeting these humane endpoints were deeply anesthetized with isoflurane and euthanized by cardiac excision and exsanguination. There were no deaths in the control group (n = 10) during the 10-day experimental protocol. For the septic mice (n = 30), there were no deaths within the first 24 h, 2 deaths within 48 h, 6 deaths within 72 h, 8 deaths within 96 h, and 13 deaths after 120 h. No mice died after day 5 and the overall 10-day mortality was 43%. We believe this model is more representative of the recovery phase than a chronic form of septic disease because the nidus of the infection (i.e., cecum) was removed, the peritoneal cavity irrigated, and a 5-day course of antibiotics administered. As a result, food consumption of sepsis-recovery mice on days 5 to 10 did not differ from food consumption determined under basal presurgical conditions, and there is no difference in body weight between time-matched control mice and sepsis-recovery mice on days 9 and 10. Furthermore, the plasma IL-1β concentration had returned to basal control levels, and concentrations of TNFα and IL-6 were reduced 97% to 99.5% from concentrations observed 24 h post-CLP (9, 10). Finally, data from preliminary studies indicated there were no additional deaths when septic mice were observed between 10 and 21 days post-CLP (unpublished observation).

Cardiac ventricular tissue was excised at either 24 h or 10 days post-CLP. The heart was freeze-clamped to the temperature of liquid nitrogen and stored at −70°C until analyzed. Because of the extensive nature of the endpoints to be assessed (below), no tissue was processed for histological examination. This approach has the advantage of permitting systematic multiparameter analysis of mRNA and protein content and enzymatic activity from the same tissue that was necessary to assess molecular markers of myocardial protein balance.

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Protein synthesis

In vivo protein synthesis was measured using the nonradioactive SUnSET method, as modified in our laboratory (9, 10). Puromycin (0.04 μmol/g body weight, dissolved in sterile saline) was administered by intraperitoneal injection 30 min before euthanasia (isoflurane as described above). At this relatively low dose, puromycin is incorporated into elongating peptide chains and the relative global rate of protein synthesis is estimated by determining the synthesis of puromycin-labeled peptides by Western blotting (Kerafast, Boston, Mass).

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Western blot analysis

Briefly, a portion of heart was homogenized in ice-cold homogenizing buffer (in mM): 20 HEPES (pH 7.4), 2 EGTA, 0.2 EDTA, 100 KCl, 50 β-glycerophosphate, 50 NaF, 0.5 sodium orthovanadate, 1 Benzamidine, 0.1 PMSF, and 1 DTT (9, 10). The protein concentration of each tissue homogenate was quantified (Bio-Rad Protein Assay, Hercules, Calif) and SDS-PAGE was performed using equal amounts of total protein per sample. In addition, each PDVF membrane was stained with Ponceau S (Aqua Solutions, Deer Park, Tex) to verify equal protein loading. Blots were then blocked in 5% (w/v) nonfat dry milk and incubated overnight with primary antibody at 4°C. Antibodies (Cell Signaling, Beverly, Mass, unless otherwise noted) included 4E-BP1 (Bethyl Laboratories, Montgomery, Tex; A300-501), 4E-BP1 (Ser65; #9451), S6K1 (Santa Cruz Biotechnology, Santa Cruz, Calif; sc-230), ribosomal protein S6 kinase 1 (S6K1; Thr 389; #9205), rpS6 (#2217), rpS6 (Ser240/244; #2215), eukaryotic elongation factor (eEF2; #2332), eEF2 (Thr56; #2331), total eIF2Bε (#3595) (p62 #5114), light-chain 3B (LC3B; #3868), autophagy-related protein (Atg) 12 (#4180), beclin (#3738), cleaved caspase-3 (#9661), cleaved nuclear poly (ADP-ribose) polymerase (PARP; #9542), activating transcription factor 4 (ATF4; courtesy Dr. Michael Killberg), c-Jun N-terminal kinase (JNK; #9252), JNK (Thr183/Tyr185; #9251), NLR pyrin domain containing 3 (NLRP3; #15101), adaptor protein containing a C-terminal caspase-recruitment domain (ASC; aka TMS1; #13833), caspase-1 (p10; Santa Cruz, Calif; sc-514), Gasdermin (Santa Cruz; sc-393656), caspase-11 (#14340), calpain (Abcam, Cambridge, Mass; ab28258), and calpastatin (Abcam; ab88065). Blots were then washed with an appropriate horseradish peroxidase conjugated secondary antibody and developed with enhanced chemiluminescence (ECL) reagents (Pierce Chemical, Rockford, Ill) according to manufacturer's instruction. Blots were imaged using the FluorChem (ProteinSimple, San Jose, Calif) and densities in the linear range were quantified using Image J (NIH, Bethesda, Md). Each Western blot included samples from mice in the acute sepsis and sepsis-recovery groups as well as samples from the respective time-matched control mice.

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Proteasome assay

A portion of heart was homogenized and reconstituted in cold assay buffer (Proteasome Activity Fluorometric Assay Kit; BioVision; Milpitas, Calif) and the assay was performed exactly as described (9, 10). The protein content was assayed in a sample aliquot (BioRad; Hercules, Calif). Each sample was measured in the presence and absence of the provided proteasome inhibitor to account for nonproteasomal degradation of the substrate. Proteasome activity (nmol/min/mg protein) was calculated by the change in the fluorescence signal where 1 unit of activity equals 1.0 nmol of the fluorophore 7-amino-4-methylcoumarin (AMC) per minute.

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RNA extraction and real-time quantitative PCR

Total RNA was extracted using Tri-reagent (Molecular Research Center, Inc., Cincinnati, Ohio) and RNeasy mini kit (Qiagen, Valencia, Calif) after manufacturers’ protocols. On-column DNase I treatment was used to remove residual DNA contamination. RNA was eluted from the column with RNase-free water and an aliquot was used for quantitation (NanoDrop 2000; Thermo Fisher Scientific, Waltham, Mass). RNA quality was analyzed on a 1% agarose gel. Total RNA (1 μg) was reverse transcribed using superscript III reverse transcriptase (Invitrogen, Carlsbad, Calif). Real-time quantitative PCR was performed using 25 ng of cDNA in a StepOnePlus system using TaqMan gene expression assays (Applied Biosystems, Foster City, Calif) using primers as previously described by our laboratory (9, 10). The comparative quantitation method 2-ΔΔCt was used in presenting gene expression of target genes in reference to the endogenous control. Samples for determination of cardiac mRNA content in mice from both acute and recovery sepsis were run simultaneously.

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Plasma CK-MB and cTnI

The plasma concentration of the MB fraction of creatine kinase (CK-MB) was determined on a VITROS 5,600 automated biochemical analyzer (Ortho-Clinical Diagnostics, NY) and the plasma analysis for cardiac troponin I (cTnl) was performed by ELISA (Life Diagnostics, West Chester, Pa).

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Data and statistical analysis

Data are presented as the mean ± standard error of mean (SEM) with the number of mice in each group presented in the figure legends. Unless otherwise noted, statistical analyses of the data were analyzed on commercial statistical software (SigmaPlot, Systat, San Jose, Calif) using a two-way ANOVA (sepsis × time) with Student–Newman–Keuls (SNK) post hoc test. Difference were considered significant when P < 0.05 and such differences are graphically depicted by letters (e.g., a, b, c) above bars on each graph. There are no differences between group means when bars have the same letter, whereas bars with different letters indicate a statistically significant difference.

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RESULTS

Protein synthesis and mTOR signaling

Cardiac protein synthesis was reduced by 45% during the acute phase of sepsis but increased 30% above time-matched pair-fed control values during the recovery phase (Fig. 1). As the mammalian or mechanistic target of rapamycin complex 1 (mTORC1) integrates various hormonal, nutrient, and energy signals (11), we determined the activity of this kinase by assessing the extent of phosphorylation of two immediate downstream substrates S6K1 and 4E-BP1 (11) (Fig. 2). S6K1 phosphorylation was reduced during the acute phase of sepsis and increased ∼2-fold above control levels during recovery (Fig. 2, A and F). A comparable temporal change was seen with S240/244-phosphorylated S6 (Fig. 2, B and F) and eIF4B (data not shown), both authentic S6K1 substrates. In contrast, there was no consistent sepsis-induced change in S65-phosphorylated 4E-BP1 (Fig. 2, C and F).

Fig. 1

Fig. 1

Fig. 2

Fig. 2

Global protein synthesis can also be regulated by changes in the eIF2/eIF2B system (11) and therefore we assessed the relative amount of the catalytic epsilon (ε)-subunit for the eIF2B complex. Our data (Fig. 2D) indicate the amount of total eIF2Bε was increased similarly during both the acute and recovery phases of sepsis. Protein synthesis can also be regulated at the level of peptide-chain elongation where specifically the upregulation of T56-phosphorylated eEF2 inhibits translation elongation. The extent of eEF2 phosphorylation in heart during acute sepsis was increased, whereas there was no difference in eEF2 phosphorylation between control and septic mice during sepsis-recovery (Fig. 2, E and F).

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Proteolysis

Protein balance can also be modulated by a change in protein degradation, and the UPP seems primarily responsible for catabolic condition-induced enhanced rates of protein breakdown (12). Therefore, we measured the in vitro protease activity of the 20S proteasome core that catalyzes the final step of the UPP. In the acute phase, the 20S proteasome activity was reduced 50% in hearts from septic mice, compared with control values; however, this decrement was not seen during the recovery phase (Fig. 3A). The mRNA content for the muscle-specific ubiquitin-E3 ligases MuRF1 and atrogin-1 was also determined, but no time- or sepsis-induced changes in either ligase were detected (Fig. 3, B and C). Calpains can modify cytoskeletal proteins, among others, by selective and limited proteolytic degradation. Western blot analysis of calpain I indicated there was a main effect of sepsis, but only the increase in calpain I observed during the recovery period was statistically significant (Fig. 3, D and G). We also assessed calpastatin which is an endogenous inhibitor of calpain. Although we did not detect high molecular weight proteins (e.g., 100–120 kDa) as reported by others (13), a major band was visualized at ∼75 kDa as well as multiple bands at lower molecular weights (e.g., ∼50 and 25 kDa) that represent degraded calpastatin. For the 75 kDa form of calpastatin, there was a main time effect with levels being lower in the recovery compared with the acute phase (Fig. 3, E and G). Furthermore, sepsis increased the 75 kDa form of calpastatin during the recovery phase, compared with time-matched control values. In contrast, when the major degradative products of calpastatin (∼50 and 25 kDa) were quantitated there was an 8-fold elevation in hearts from septic mice during recovery, compared with control values (Fig. 3, F and G).

Fig. 3

Fig. 3

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Apoptosis

Programed cell death is enhanced in sepsis and can decrease cell number in several tissues (14). A predominant apoptotic signaling cascade involves the activation of caspase-3 and the subsequent cleavage of PARP. Acute sepsis more than doubled the amount of cleaved caspase-3 and cleaved PARP (Fig. 4, A and B); however, there was no difference between septic and control mice during the recovery phase. Likewise, activation of JNK via its phosphorylation increases transcription of a number of proapoptotic genes. JNK phosphorylation on T183/Y185 was increased 2-fold during acute sepsis, whereas there was no difference in JNK phosphorylation between groups during sepsis recovery (Fig. 4C). Finally, the unfolded protein response can lead to apoptosis via activation of the ATF/CHOP pathway. In contrast to the early sepsis-induced increased in caspase-3, cleaved PARP, and phosphorylated JNK, ATF4 did not differ in heart between septic and control mice at either time point assessed (Fig. 4D).

Fig. 4

Fig. 4

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Autophagy

Autophagy is a proteolytic degradative process that removes cytosolic components and its dysregulation can produce cardiomyocyte death and cardiomyopathy (14). During autophagy the cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-II. Hence, the increase in myocardial LC3-II during the acute phase is suggestive of enhance autophagy (Fig. 5, A and E). However, this sepsis-induced increase was not seen during sepsis-recovery. Autophagy is a multiple step process requiring a number of key intracellular proteins, including Atg5/12, p62, and Beclin. In this regard, an increase in Atg5/12 and Beclin and decrease in p62 are often associated with an increase in autophagy. In contrast to the early increase in LC3B-II described above, the relative content for each of these regulatory proteins did not differ between control and septic mice at either time point examined (Fig. 5, B and E). However, for the Atg5/12 complex and p62 there was a time effect as the relative content of these two proteins was lower in the recovery phase than 24-h post-CLP.

Fig. 5

Fig. 5

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Inflammasome

The inflammasome is a multiprotein complex that when activated by either the canonical or noncanonical pathway triggers the inflammatory process and can lead to cell death (15). Protein expression for components associated with the canonical inflammasome pathway, such as TMS1, NLRP3, and cleaved caspase-1, was increased in heart during the acute phase of sepsis but not during the recovery phase (Fig. 6). We were unable to detect cleavage of pro-IL-1β to the mature protein (data not shown), the ultimate end product of increased caspase-1 activity. In contrast, expression levels of caspase-11 together with total and cleaved Gasdermin D, the latter being a marker of noncanonical inflammasome pathway activation, were consistently and markedly increased during the recovery phase but not at 24-h post-CLP (Fig. 7).

Fig. 6

Fig. 6

Fig. 7

Fig. 7

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Cardiac inflammation and damage

We also assessed various markers of inflammation in heart tissue. The mRNA content for IL-1β, IL-6, and TNFα was increased at 24 h post-CLP, but the expression of these cytokines did not differ from control values during sepsis-recovery (Table 1). Finally, the plasma concentrations for CK-MB (primarily found in the heart) and cTnI (a sensitive and specific indicate of myocardial damage) were both increased at the 24-h time point but were not elevated during sepsis-recovery (Table 1). These data collectively suggest the early sepsis-induced increase in cardiac inflammation and damage have largely resolved by day 10.

Table 1

Table 1

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DISCUSSION

Our data demonstrate that sepsis acutely modulates specific protein synthetic and degradative pathways in whole cardiac tissue and suggests compensatory mechanisms are invoked to restore proteostasis during the recovery phase (Fig. 8). On the synthetic side of the protein balance equation, sepsis acutely decreased global protein synthesis. As mTORC1 is a key regulator of protein synthesis (11), we assessed authentic downstream substrates for this kinase. In this regard, the decrease in protein synthesis was associated with a selective reduction in the activity of S6K1 in the absence of a detectable decrement in 4E-BP1 phosphorylation. Moreover, as eEF2 phosphorylation was increased by sepsis, it seems that a sepsis-induced decrease in both translation initiation and elongation may contribute to the early decrease in protein synthesis. It is noteworthy that these results differ from those previously reported where sepsis did not acutely alter cardiac protein synthesis (16, 17). Possible explanations for this difference might include the species used (mouse vs. rat) or, of potentially greater importance, the type and/or severity of the sepsis model (CLP vs. intraabdominal inoculation of purified Escherichia coli and Bacteroides fragilis). In contrast, the recovery phase was characterized by an upregulation of cardiac protein synthesis that occurred with a coordinated increase in S6K1/S6 phosphorylation and the return of eEF2 phosphorylation to baseline. This temporal progression of sepsis-induced changes in cardiac protein synthesis is comparable to that reported for skeletal muscle (9).

Fig. 8

Fig. 8

Alterations in protein breakdown also impact tissue protein balance, and sepsis has consistently been shown to increase the UPP activity in skeletal muscle from rodents and humans (18, 19). In contrast, there is a paucity of data pertaining to sepsis-induced changes in UPP activity in heart. In contradistinction to skeletal muscle, we show that sepsis acutely decreases 20S proteasome activity, a response comparable to the decrease in cardiac 20S B1 and B2 as well as 26S B5 activity seen 8 h after injection of lipopolysaccharide (LPS) (20). Although the sepsis-induced increase in proteasome activity in skeletal muscle is associated with a coordinated increase in the expression of muscle-specific atrogenes (21), we detected no alterations in myocardial MuRF1 or atrogin-1 mRNA content during acute sepsis suggesting an alternative mechanism is operational in cardiac tissue. A similar disconnect between these E3 ligases and UPP activity has been previously reported in skeletal muscle (22).

The activation of calpains can be an important regulator of skeletal muscle breakdown allowing for the release of actin and myosin that are then subsequently degraded by the UPP (23). Others have reported that an acute sepsis-induced increase in cardiac calpain-1 mRNA and protein content was associated with a reduction in the contractile proteins α-actin and heavy-chain cardiac myosin (24). However, no such change in calpain I was detected in the acute phase of the present study. This cardiac response differs from the sustained sepsis-induced increase in the UPP in skeletal muscle that leads to the characteristic atrophic response in this tissue (11). Overall, it seems that myocardial protein content and mass are preserved during early sepsis by the coordinated and proportional change in protein synthesis and protein breakdown.

Apoptotic death of cardiomyocytes is potentially detrimental as the heart has a limited regenerative capacity and cell loss, by any mechanism, can impair myocardial contractility. Several independent laboratories have provided relatively consistent data indicating that sepsis acutely increases cardiac apoptosis as illustrated by the increased presence of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive cells as well as increased protein expression of BAX (bcl-2-like protein 4), cleaved caspase-3, and cleaved PARP (25–28). Furthermore, chemical inhibition of PARP cleavage using 3-aminobenzamide has been reported to prevent apoptosis and mitochondrial damage due to sepsis (28). Sepsis also increases JNK phosphorylation which is mediated by IL-1β and TNFα, both of which are increased in the blood and heart of septic animals and can induce apoptosis and contractile dysfunction (25, 29). Our data obtained 24 h post-CLP are entirely consistent with these early reports. Moreover, our data suggest that the early sepsis-induced increase in apoptosis is ATF4-independent. In contrast, the initial increase in markers of cardiac apoptosis is transient and abates during the recovery phase of sepsis as the relative abundance of cleaved caspase-3 and PARP, and the phosphorylation of JNK did not differ between septic and control mice at day 10. Although the early increase in apoptosis may impair contractile function, apoptotic cell death may limit inflammation thereby minimizing further release of damage-associated molecular patterns and dampening the immune response in heart. Hence, the return of apoptosis to basal control levels during recovery may suggest an amelioration of the inflammatory state, which was confirmed by the return of IL-1β, IL-6, and TNFα mRNA content to control levels.

Autophagy is a type of programmed cell death involving lysosomal degradation and recycling of cellular proteins and organelles. This process can often be cytoprotective serving a prosurvival function in catabolic conditions (14), and inhibition of autophagy increases sepsis mortality (30). Sepsis acutely increases formation of autophagic vacuoles as assessed by electron microscopy (31) and increases LC3-II stained punctate cells in the left ventricle and LC3-II in whole cardiac tissue (14). Increases in LC3-II can indicate either an increase in autophagosome biogenesis or a block in lysosomal degradation. Previous work revealed that both Ras-related protein Rab7 and lysosomal-associated membrane protein (LAMP)-1 were decreased 4 to 24 h after CLP. Moreover, acute sepsis decreased the colocalization of LC3 with LAMP1 suggesting inhibition of autophagosome-lysosome fusion. Finally, degradation of autophagosomes seems decreased as evidenced by an increased number of larger autophagosomes containing mitochondria and partial digestion of autophagosome contents after CLP (14). Although our data do confirm an early sepsis-induced increase in LC3-II, we did not detect concomitant changes in beclin and the Atg5-12 complex, or a decrease in p62 content which would collectively imply an increased in autophagy. Hence, although our data suggest that sepsis increases autophagosome formation, they do not provide unequivocal evidence for an overall change in autophagic flux. As with the abovementioned changes in apoptosis, the sepsis-induced increase in LC3-II was not sustained having returned to basal control levels during the recovery phase.

Pyroptosis is a distinct form of cell death defined as being caspase-1-dependent and is enhanced by numerous environmental prompts, including sepsis (32). Caspase-1 plays little role in apoptosis and conversely, the apoptotic caspases, including caspase-3, do not seem to be predominantly involved in pyroptosis (32). What is referred to as the canonical inflammasome pathway includes the NLRP3 complex consisting of NLRP3, the adapter protein TMS1 (ASC1), and caspase-1. Association of caspase-1 with this multiprotein complex allows its processing and activation that in turn facilitates the cleavage of inactive precursors of IL1β and IL-18 to form the mature cytokines (32), and sepsis has been reported to increase IL-1β in muscle and blood (6). The relative importance of this pathway in sepsis is evidenced by the smaller decrement in cardiac contractile function and improved survival of septic mice with a gene deletion in NLRP3 or caspase-1 (15, 33). Moreover, pretreatment of rats with an IL-1 receptor antagonist prevents the sepsis-induced impairment in nutrient-stimulated myocardial protein synthesis (17). However, activation of caspase-1 seems to have other detrimental effects as neutralization of IL-1 and IL-18 during sepsis does not completely mimic the phenotype seen in caspase-1-deficient septic mice (33). Our data from septic mice in the acute phase confirm these early observations and extend them by demonstrating that, similar to the activation of apoptosis and autophagosome formation, these sepsis-induced changes were transient and had resolved during the recovery phase.

A noncanonical NLRP3 pathway has also been described whereby caspase-11 functions as a pattern recognition receptor and is activated by cytosolic LPS (34). Caspase-11 activation results in proteolytic cleavage of the cytosolic protein Gasdermin D (35). The newly generated N-terminal fragment of Gasdermin D associates with the plasma membrane, thereby enhancing pore formation and ultimately causing cell death. Activation of this pathway was seen only during the recovery phase of sepsis as evidenced by the upregulation of caspase-11 and Gasdermin D cleavage. Hence, sepsis regulated both the canonical and noncanonical NLRP3 pathways in a temporally distinct manner, a pattern not previously reported in heart or other tissues. As the caspase-11/Gasdermin D pathway is also necessary for the secretion of HMGB1 and IL-1α (35), our data suggest there is potential for continued release of selected inflammatory cytokines and DAMPs that may slow recovery. The physiological function of the upregulation of this noncanonical pathway remains to be determined.

In this study, we have identified the signal transduction pathways that are upregulated (e.g., apoptosis, autophagosome formation, canonical NLRP3 pathways) or downregulated (protein synthesis and proteasome activity) in heart during the acute phase of CLP-induced sepsis in adult male mice. Although apoptosis, autophagy, and UPP activation returned to baseline control levels during recovery, our work reveals that myocardial protein synthesis is increased and pyroptosis via the noncanonical pathway is activated during convalescence. Hence, there are changes in proteostasis that persist long after the initial septic insult has resolved that may potentially impact heart structure and function. However, we are unable to directly determine the physiological importance of the sepsis-induced changes in signal transduction as the present study was limited by the lack of histological evidence and, more importantly, by any direct assessment of cardiac function. Future studies will be required to characterize both sepsis-induced changes in cardiac function and various aspects of proteostasis to help identify potential mechanisms by which sepsis leads to frailty and deconditioning due to weakening of cardiac reserve in the human population.

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Acknowledgment

We thank Maithili Navaratnarajah and Anne Pruznak for their excellent technical assistance.

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Keywords:

Caspase; mTOR; NLRP3; proteasome; proteostasis

© 2018 by the Shock Society