Contributed equally
The present study aimed to investigate the effects of acute sepsis on diaphragm contractility and relaxation, via examining the Ca2+-uptake function of sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA), and the protein levels of SERCA1, SERCA2 and the ryanodine receptor (RyR) of the sarcoplasmic reticulum (SR). A sepsis rat model was established through cecal ligation and puncture (CLP). A total of 6 and 12 h following CLP, the isometric contractile and relaxation parameters of the diaphragm were measured. In addition, Ca2+ uptake and release from the SR, and the protein expression levels of SERCA1, SERCA2 and RyR in diaphragm muscle tissue were investigated. At 6 and 12 h post-CLP, the diaphragm half-relaxation time was prolonged and the maximum rate of tension decline was decreased and the Ca2+-uptake function of SERCA was markedly reduced. The maximum rate of twitch force development, the maximal twitch and tetanic tension, and the release function of SR were decreased at 12 h post-CLP. A total of 12 h following CLP, the protein expression levels of SERCA1 were significantly downregulated, and its activity was significantly reduced; conversely, the protein levels of SERCA2 remained unaltered. The present findings indicated that at the acute stage of sepsis induced by CLP the contractile and relaxation functions of the diaphragm were significantly compromised. The impairments in relaxation may be a result of the impaired uptake function of the SR and the downregulation in SERCA1 protein expression. Conversely, the compromised contractility may be a result of the impaired release function of the SR and the downregulation in RyR protein levels. This could provide some new insights into the treatment of sepsis. In acute stages of sepsis, the improvement of SERCA function could reduce the disequilibrium of calcium homeostasis to improve the critical illness myopathy and respiratory failure.
Severe sepsis commonly leads to muscle dysfunction and multiple organ failure (
Ca2+ signaling is essential for physiological muscle function. The intracellular concentration of Ca2+ in myocytes is critical for muscle contraction and relaxation, and the regulation of intracellular Ca2+ levels in skeletal muscle cells serves a central role in diaphragm function (
Sepsis-induced impairments in diaphragm contractility and relaxation have been studied in the late phases of sepsis; however, during the acute phase of sepsis, the alterations in diaphragm function, and Ca2+ release and uptake functions of the SR, have yet to be elucidated. In addition, the exact mechanisms underlying the sepsis-induced Ca2+ overload and the compromise in diaphragm function remain to be explored, and previous studies have not focused on the Ca2+ release and uptake functions of the SR which are essential for physiological muscle contraction. The present study aimed to examine the alterations in diaphragm function during acute sepsis. Furthermore, it aimed to investigate the sepsis-induced compromise of Ca2+ release and uptake, and explore the roles of RyR and SERCAs in the SR and the mechanisms underlying the dysfunction of diaphragm contraction and relaxation during the early phase of sepsis.
A total of 40 male Sprague-Dawley rats weighing 215–240 g and aged five weeks were purchased from the Shanghai Animal Research Centre (Shanghai, China). Upon arrival at the animal house, rats were maintained in cages (5 rats/cage) in an environmentally-controlled room (temperature, 21–24°C; relative humidity, 40–60%), under a 12 h light/dark cycle. A 2-week acclimation period, during which standard rat food and water were offered
The cecal ligation and puncture (CLP) procedure, as described by Chaudry
Rats were weighed and anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight). A 2-cm lower midline abdominal incision was made, the cecum was exposed and ligated at a site distal to the ileocecal valve. The cecum was then punctured through both walls with an 18-gauge hollow needle, and was gently compressed until feces were extruded into the peritoneal cavity. Subsequently, the cecum was returned to the peritoneal cavity, and the peritoneal and skin incisions were closed. Rats were resuscitated via a subcutaneous bolus injection of sterile normal saline (30 ml/kg body weight) in the back. Following recovery from anesthesia, rats moved around freely in the cages. Rats in the sham group underwent the laparotomy procedure without ligation and puncture. As septic rats exhibit a decrease in food intake, feeding was restrained following surgery for all rats, in order to minimize the variation between groups (
The surviving rats in the sham and CLP groups were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight) after 6 h (6 h CLP group) and 12 h (sham group and 12 h CLP group) following CLP. Subsequently, the chest and upper abdomen were opened through a median incision of 4–5 cm under sterile conditions, and the thoracic cavity was opened through the incision at the edges of the two clearly exposed costal arches. Strips from the left diaphragm were rapidly removed from each rat, for the assessment of isometric contractile characteristics, while leaving the central tendon and rib cage intact. The right hemidiaphragm was removed, instantly frozen in liquid nitrogen, and stored at −80°C for subsequent use in western blot analysis. A small piece of tissue (~10×4 mm) was immediately obtained from the right hemidiaphragm and immediately homogenized as described below for the SR functional assays.
Isolated left diaphragm strips from the rats in each group (width, ~5 mm) were immediately immersed in temperature-controlled organ chambers (volume, 40 ml) at 37°C filled with Krebs solution. Strips were fixed at the rib cage and, using a 3–0 silk suture, were suspended at the central tendon from a force displacement transducer (ALC-AF; Shanghai Alcott Biotech Co., Ltd., Shanghai, China). The organ bath was continuously perfused with a 95% O2 and 5% CO2 mixture. The modified Krebs solution contained 137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM KH2PO4, 12 mM NaHCO3, and 6.5 mM glucose; the pH was adjusted to 7.4–7.5 through aeration with CO2. Strips were placed between 2 platinum stimulating electrodes, and 12 µM d-tubocurarine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to the bath to abolish neuromuscular transmission (
A total of 2 twitches (frequency, 0.1 Hz; pulse duration, 0.2 msec) were delivered at Lo to determine the following parameters: Peak twitch tension, time to peak tension, maximum rate of twitch force development (+dF/dt), and maximum rate of tension decline (−dF/dt). Half-relaxation time was measured during the decline phase as the time required for the tension to reach 50% of maximal value. Average values of 3 experiments were calculated and used for further analysis.
The maximal tetanic tension was recorded as the maximal tension of the diaphragm strips that was elicited following stimulation at 120 Hz.
Diaphragm strips were stimulated (pulse duration, 0.2 msec; train duration, 250 msec) at various frequencies, ranging from 10–120 Hz over 10 Hz increments using 2 min intervals, and the force-frequency relationship of the muscle was determined.
The fatigue properties of the muscle were determined using 330-msec stimulations (pulse duration, 0.2 msec; train duration, 670 msec) repeated every sec at 25 Hz for 5 min. The ratio of the contraction force obtained following 1 sec over that obtained following 120 sec of stimulation was determined and defined as the fatigue index (FI).
Following the assessment of the aforementioned parameters, the strips were blotted dry and weighed. All force values were normalized to cross-sectional areas, calculated by dividing the muscle weight by the muscle length and specific density, as previously described (
The functional characteristics of the SR were assessed using crude muscle homogenates. Following weighing, the right hemidiaphragm was placed in ice-cold homogenization buffer containing 250 mM sucrose, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), 0.2% sodium azide and 0.2 mM phenylmethylsulfonyl fluoride (pH 7.5). The tissue was minced and homogenized on ice using a rotor tissue homogenizer (Tissue-Tearor 985370; BioSpec Products, Inc., Bartlesville, OK, USA) with 15 sec bursts at 15,000 rpm repeated 3 times in 10 volumes of fresh buffer. The duration of tissue extraction and homogenization was typically 10 min. The homogenates were centrifuged at 1,600 × g for 15 min at 4°C. The supernatants were harvested, rapidly frozen in liquid nitrogen and stored at −80°C until use in the SR function assay. Protein concentration was determined using a bicinchoninic acid (BCA) protein assay.
The kinetics of Ca2+ uptake were evaluated by Ca2+ imaging using the fluorescent Ca2+-binding dye fura-2 (pentapotassium salt; Sigma Aldrich; Merck KGaA, Darmstadt, Germany) on a Varioskan Flash microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA), as previously described (
Diaphragm tissue samples isolated at 6 and 12 h following the induction of sepsis or the sham operation were used for western blot analysis. Tissues were homogenized (Tissue-Tearor 985370; BioSpec Products, Inc., Bartlesville, OK, USA) on ice for 5 20-sec bursts at intervals of 20 sec in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing 1 mM phenylmethylsulfonyl fluoride (Amresco, Solon, OH, USA). The obtained homogenate was centrifuged at 15,000 × g for 15 min at 4°C. Protein concentrations were determined using a BCA protein assay kit (Beyotime Institute of Biotechnology). Equal amounts (50 µg) of extracted protein samples were separated by 12% SDS-PAGE and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). Following blocking with 5% non-fat milk for 1 h at room temperature with agitation, membranes were probed at 4°C overnight with the following primary antibodies: Anti-RyR (ab2868; 1:5,000; Abcam, Cambridge, UK), anti-SERCA1 (ab2819; 1:1,000; Abcam), anti-SERCA2 (ab2861; 1:1,000; Abcam), and anti-GAPDH (KC-5G4; 1:5,000; KangChen Biotech, Shanghai, China). The membranes were then incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibodies (A0216; 1:5,000; Beyotime Institute of Biotechnology) and protein bands were visualized by enhanced chemiluminescence (EMD Millipore, Billerica, MA, USA). Blots were semi-quantified (Image Pro Plus version 6.0, Media Cybernetics, USA) by densitometry and protein expression was normalized to GAPDH as an internal reference.
All data were obtained from three separate experiments and are expressed as the mean ± standard error of the mean. The statistical significance of the differences between groups was assessed using an unpaired Student's t-test for pair-wise comparisons or one-way analysis of variance followed by a Bonferroni post hoc test for multiple comparisons. The Ca2+ curves were fitted smoothly by the values of the time points using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Statistical analysis was performed using SPSS software version 20.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.
No differences in body weight were detected between rats belonging to the sham and the CLP groups (data not shown). The width of the diaphragm strip that was used for the assessment of muscle contractility ranged between 4 and 6 mm for all groups. In addition, no differences were observed in total protein concentration in muscle tissue samples between groups (data not shown). Septic rats demonstrated symptoms including chills, hair erection, ocular exudates and diarrhea, which were not present among sham-operated rats. Septic rats became lethargic and spontaneous activity was progressively reduced during the experiments. A single case of mortality occurred in the 12 h CLP group (mortality rate, 8.3%), and the rat was eliminated from the study; however, no cases of mortality occurred in the 6 h CLP and sham groups.
The contractile characteristics of diaphragm tissue samples isolated from surviving rats were investigated in the present study and are presented in
Force-frequency curves were plotted for the diaphragm strips in response to stimuli of various frequencies, following normalization (
A schematic representation of the Ca2+ uptake and release imaging process is included in
Sepsis is a severe systemic inflammatory response syndrome which is responsible for the high rates of mortality of patients in intensive care units. Critical illness myopathy (CIM) may occur in critically ill patients and may be a result of sepsis; its morbidity may reach 60–80% (
The CLP rat model has been extensively applied in sepsis-associated research, due to its high similarity to the pathophysiology of human sepsis. In the present study, CLP was performed according to standard procedures, as described by Rittirsch
The contractile characteristics of the diaphragm during sepsis have been previously investigated: Peritoneal cavity inflammation has been demonstrated to reduce the maximal force of diaphragm contraction (
Previous research has suggested several mechanisms underlying sepsis-induced diaphragm dysfunction, including increased cytokine release, oxygen free radical generation and mitochondrial damage (
In the present study, Ca2+ imaging using a fluorescent dye was used to investigate SR function and SERCA activity, via measuring Ca2+ release and uptake in diaphragm muscle tissue homogenates. The present results revealed that the maximum rate of Ca2+ uptake was decreased 6 and 12 h following the induction of sepsis; however, the maximum rate of Ca2+ release and SERCA activity were significantly suppressed only in the 12 h CLP group compared with in sham-operated rats. Ca2+ release and uptake in the SR of myocytes may be reflected by twitch kinetics (
The activation of RyRs, which are Ca2+ release channels of the SR, lead to a massive Ca2+ release that initiates muscle contraction. The primary type of RyR expressed in diaphragm muscle fibers is RyR1, which serves a critical role in excitation-contraction coupling; RyR2 is absent in skeletal muscle, and sepsis has not been revealed to affect RyR3 contents, which constitute only ~3% of the total RyRs (
Ca2+ released through RyRs is subsequently reuptaken into the SR via SERCA, upon which muscle relaxation occurs. In the adult rat diaphragm, the proportion of Type I (slow-twitching) and II (fast-twitching) muscle fibers are ~33 and 67%, respectively (
In conclusion, the results of the present study indicated that during the acute stage of CLP-induced sepsis, the contractile and relaxation functions of the diaphragm appeared to decline. The dysfunctions in relaxation may be a result of the reduced Ca2+ uptake function of the SR and the downregulation in SERCA1 expression, whereas the impairments in contractility may be associated with a compromise in the SR Ca2+ release function and the decreased RyR expression levels in diaphragm muscle tissue.
The authors would like to thank Dr Tao Lang and Dr Min Li for their excellent technical assistance and strong support. The present study was supported by the National Natural Science Foundation of China (grant no. 81171845) and the Shanghai Natural Science Foundation (grant no. 14ZR1438100).
Force-frequency relationship of diaphragm skeletal muscle strips isolated from rats in the various treatment groups (n=12 rats/group). Sham group, rats underwent a sham operation; 6 h CLP group, rats underwent CLP and were assessed 6 h post-surgery; 12 h CLP group, rats underwent CLP and were assessed 12 h post-surgery. The force-frequency curve of 12 h CLP rats exhibited a significant downward shift compared with the sham group, indicating that the force of contraction following stimulation of varying frequency was significantly decreased. Data are presented as the mean ± standard error of the mean. #P<0.05 vs. sham group. CLP, cecal ligation and puncture.
Ca2+ kinetics in the SR of diaphragm skeletal muscle isolated from rats in the various treatment groups (n=12 rats/group). Sham group, rats underwent a sham operation; 6 h CLP group, rats underwent CLP and were assessed 6 h post-surgery; 12 h CLP group, rats underwent CLP and were assessed 12 h post-surgery. (A) Fluctuations in free Ca2+ concentration in crude muscle homogenates measured with the Ca2+-binding dye Fura-2. The maximal rate of SR Ca2+ uptake and release were calculated using this curve. (B) Fitted curves demonstrating the kinetics of Ca2+ release and uptake in the sham and CLP groups. (C) Peak rates of SR ATP-induced Ca2+ uptake and AgNO3-stimulated Ca2+ release in diaphragm muscle tissue samples. (D) SERCA activity in diaphragm tissue. Data are presented as the mean ± standard error of the mean. **P<0.01 vs. sham group; ##P<0.01 vs. 6 h CLP group. SR, sarcoplasmic reticulum; CLP, cecal ligation and puncture; ATP, adenosine triphosphate; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; [Ca2+], Ca2+ concentration.
Expression of proteins associated with Ca2+ release and uptake in diaphragm skeletal muscle isolated from rats in the various treatment groups (n=12 rats/group), as assessed using western blot analysis. Sham group, rats underwent a sham operation; 6 h CLP group, rats underwent CLP and were assessed 6 h post-surgery; 12 h CLP group, rats underwent CLP and were assessed 12 h post-surgery. Representative blots are included for SERCA1, SERCA2 and RyR. Densitometric analysis revealed that SERCA1 and RyR protein expression levels were significantly downregulated 12 h post-CLP. Data are presented as the mean ± standard error of the mean. **P<0.01 vs. sham group; ##P<0.01 vs. 6 h CLP group. CLP, cecal ligation and puncture; SERCA, sarco/endoplasmic reticulum Ca2+-adenosine triphosphatase; RyR, ryanodine receptor.
Contractile parameters of the diaphragm for rats in the sham, 6 h CLP and 12 h CLP groups.
Sham | 6 h CLP | 12 h CLP | |
---|---|---|---|
Pt (mN mm−2) | 3.49±0.66 | 3.38±0.81 | 2.45±0.65 |
Po (mN mm−2) | 8.98±1.87 | 8.87±1.51 | 6.75±1.54 |
+dF/dt (mN mm−2 s−1) | 0.35±0.05 | 0.34±0.06 | 0.23±0.05 |
−dF/dt (mN mm−2 s−1) | 0.31±0.05 | 0.26±0.07 |
0.17±0.04 |
1/2 RT (ms) | 8.41±0.46 | 9.12±0.74 |
10.05±0.84 |
TPT (ms) | 18.15±0.58 | 18.18±0.76 | 22.49±1.12 |
FI (%) | 83.1±5.9 | 79.2±9.2 | 81.7±10.5 |
Sham group (n=12), rats underwent a sham operation; 6 h CLP group (n=12), rats underwent CLP and were assessed 6 h post-surgery; 12 h CLP group (n=12), rats underwent CLP and were assessed 12 h post-surgery. Data are expressed as the mean ± standard error of the mean.
P<0.05
P<0.01 vs. sham
P<0.01 vs. 6 h CLP. CLP, cecal ligation and puncture; Pt, peak twitch tension; Po, peak tetanic tension; +dF/dt, maximum rate of twitch force development; -dF/dt, maximum rate of tension decline; 1/2 RT, half-relaxation time; TPT, time to peak tension; FI, fatigue index.