Protease enzymes generated from injured cells and leukocytes are the primary cause of myocardial cell damage following ischemia/reperfusion (I/R). The inhibition of protease enzyme activity via the administration of particular drugs may reduce injury and potentially save patients' lives. The aim of the current study was to investigate the cardioprotective effects of treatment with recombinant human secretory leukocyte protease inhibitor (rhSLPI) on
Despite advances in knowledge and technology regarding health, myocardial ischemia remains the most common cause of mortality worldwide and the number of people succumbing to myocardial ischemia is predicted to increase over the coming decades (
Secretory leukocyte protease inhibitor (SLPI) is an 11.7 kDa long cationic non-glycosylated protein that belongs to the Whey Acidic Protein (WAP) family (
Schneeberger
Therefore, the aim of the current study was to investigate the effect of rhSLPI on isolated adult rat ventricular myocytes (ARVMs) subjected to simulated I/R and to investigate the cardioprotective effects of rhSLPI on an
All basic chemicals were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). M199 medium was obtained from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). For SDS-PAGE and western blot analysis, 30% polyacrylamide solution was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA) and the polyvinylidenedifluoride (PVDF) membrane was purchased from GE Healthcare Life Sciences (Little Chalfont, UK). The antibodies for the dual phosphorylated (p)-Thr180/Tyr182 form of p38 mitogen-activated protein kinase (MAPK; cat. no. sc-17852-R), total p38 MAPK (cat. no. sc-728), p-Akt (cat. no. sc-293125) and total Akt (cat. no. sc-8312) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Enhanced chemiluminescence (ECL) solution was purchased from GE Healthcare Life Sciences and collagenase type II was purchased from Worthington Biochemical Corporation (Lakewood, NJ, USA). rhSLPI was purchased from Sino Biological, Inc. (Beijing, China).
Adult male Wistar rats (8 weeks old) weighing 200–250 g (n=6) and adult male C57BL/6 mice (8 weeks old) weighing 25–30 g (n=18) were obtained from the National Animal Center, Salaya Campus, Mahidol University (Bangkok, Thailand). All animals were maintained under environmentally controlled conditions (a temperature of 22±1°C, humidity of 45–60% and 12-h light/dark cycle) at the Center for Animal Research, Naresuan University (Phitsanulok, Thailand). All protocols used in the current study were approved by the animal ethics committee of the Center for Animal Research, Naresuan University (approval no. NU-AE550732).
The Wistar rats were euthanized by intraperitoneal injection (IP) with pentobarbital sodium (Nembutal® Sodium Solution CII; 100 mg/kg; Akorn, Inc., Lake Forest, IL, USA) and lithium heparin (150 U; Government Pharmaceutical Organization, Bangkok, Thailand). Adult rat ventricular myocytes (ARVMs) were isolated from the hearts using collagenase-based enzymatic digestion. Hearts were excised and initially perfused for 10 min with a modified Krebs solution (solution A) containing 130 mM NaCl, 4.5 mM KCl, 1.4 mM MgCl2, 0.4 mM NaH2PO4, 0.75 mM CaCl2, 4.2 mM HEPES, 20 mM taurine, 10 mM creatine and 10 mM glucose at pH 7.3 at 37°C. Hearts were then perfused with a calcium-free solution containing 100 µM EGTA for 5 min (solution B), followed by perfusion with solution A containing 100 µM CaCl2 and 0.4 mg/ml Type II collagenase for 30 min at 37°C. Following enzymatic perfusion, ventricles were cut into small pieces, which were incubated in 20 ml collagenase solution. Ventricles were incubated with 100% O2 for a further 7 min at 37°C and underwent regular gentle triturating. Isolated cardiomyocytes were separated from undigested ventricular tissue using a cell strainer. Subsequently, isolated myocytes were allowed to pellet at the bottom of the tube (sedimentation by gravity) and the supernatant was removed and replaced with solution A containing 1% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) and 500 µM CaCl2. Isolated cardiomyocytes were then allowed again to sediment at the bottom of the tube (sedimentation by gravity) and the supernatant was then removed and replaced with 10 ml solution A containing 1 mM CaCl2. The cell pellet was washed with M199 culture medium containing 100 IU/ml penicillin/streptomycin. Myocytes were resuspended in M199 supplemented with 2 mM creatine, 2 mM carnitine and 5 mM taurine, and then seeded on pre-laminin-coated 6-well plates (15 µg/ml laminin). Myocytes were allowed to adhere for 2 h in an incubator containing 5% CO2 at 37°C. The culture medium was replenished with fresh modified M199 medium, prior to further experiments.
sI was performed following a modified protocol (
The viability of ARVMs was assessed using the trypan blue dye exclusion method. Following reperfusion, the culture medium was removed and cells were incubated in 0.4% trypan blue solution for 1–2 min at room temperature. Following incubation, the numbers of trypan blue-positive and negative cells were counted under a light microscope in 5 different microscopic fields. The percentage of surviving cells from the total amount of cells was then calculated and the relative percentage of cell viability was compared with the control group.
Released-Lactate Dehydrogenase (LDH) enzyme activity was measured by the LDH SCE mod. liquiUV kit (cat. no. 12214; HUMAN Diagnostics Worldwide, Wiesbaden, Germany). A total of 10 µl culture medium was mixed with 1,000 µl LDH activity assay buffer and incubated at 37°C for 5 min. Then, 250 µl substrate reagent was added. The solution was mixed and after 1 min, absorbance was measured at a wavelength of 340 nm. The mean absorbance change per minute (ΔA/min) was measured to evaluate released-LDH activity using the following formula: LDH activity (U/I)=ΔA/min × 20,000.
ARVMs were cultured with modified M199 medium in 24-well black plates and maintained at 37°C in an atmosphere containing 5% CO2 and 95% O2. The culture medium was removed and the cells were washed with phosphate-buffered saline (PBS). Subsequently, cells were incubated with M199 medium containing 25 µM carboxy-H2DCFDA (Sigma-Aldrich; Merck KGaA) in a dark room for 30 min at 37°C. The medium containing carboxy-H2DCFDA was discarded and ARVMs were washed once with PBS. For rhSLPI treatment, 500 µl M199 medium containing various concentrations of rhSLPI (0, 200, 400, 600, 800 and 1,000 ng/ml) was added and incubated for 1 h at 37°C. Subsequently, cells were incubated with 250 µM H2O2 for 30 min at 37°C. The control cells were incubated with M199 medium without treatment. ROS activity was determined by measuring fluorescence intensity using an EnSpire Multimode Plate Reader (PerkinElmer, Inc., Waltham, MA, USA) at an excitation wavelength of 498 nm and emission wavelength of 522 nm.
The
ARVMs were washed twice in ice-cold PBS prior to the addition of 200 µl 2× SDS-sample buffer containing β-mercaptoethanol. Cells were scraped and samples were transferred to pre-cooled micro-centrifuge tubes. For protein extraction, hearts from the I/R, I/R + 400 ng/ml rhSLPI group and I/R + 1,000 ng/ml rhSLPI groups (each, n=3) were perfused on a Langendorff perfusion system, subjected to 30 min stabilization, perfused with KHB containing 400 ng/ml and 1,000 ng/ml rhSLPI or KHB alone as a vehicle for 30 min prior to 10 min without perfusion (global ischemia). At the end of the global ischemia, hearts were rapidly snap frozen. Approximately 50 mg of heart samples were weighed and homogenized in 500 µl homogenization buffer (20 mM Tris pH 6.8, 1 mM sodium orthovanadate, 5 mM sodium fluoride, and 1 cOmplete™, Mini Protease Inhibitor Cocktail Tablet; Sigma-Aldrich; Merck KGaA). Heart homogenates were centrifuged at 13,148 × g for 10 min at 4°C. Supernatants were collected and an equal volume of 2X SDS-PAGE sample buffer containing 10% (v/v) β-mercaptoethanol and bromophenol blue dye was added. Samples were boiled for 10 min and stored at −80°C prior to analysis. Extracted proteins (30 µg/lane) were separated on 12% SDS-polyacrylamide gels (prepared from a 30% polyacrylamide solution) and transferred to PVDF membranes that were blocked for 1 h with 5% non-fat milk and 1% BSA in Tris-buffered saline (pH 7.4) containing 0.1% Triton X-100. Membranes were then probed overnight at 4°C with primary antibodies against total p38, diphospho-p38, total Akt and p-Akt (all 1:1,000). Following washing and exposure for 1 h at room temperature to horseradish peroxidase-conjugated secondary antibodies, antibody-antigen complexes were visualized using ECL, revealed as bands corresponding to the detected proteins of interest. Band densities were detected by Gel DocXR+ Imaging System, quantified using Image Lab™ software (version 6.0; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and compared, providing information on the relative abundance of the protein of interest.
All values are presented as the mean ± standard error of the mean. All comparisons were assessed for significance using one-way analysis of variance, followed by the Tukey-Kramer test. Statistical tests were performed using GraphPad Prism version 5 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.
The results demonstrated that rhSLPI-treatment administered 2 h prior to sI or during sI significantly reduced sI/R-induced cell death. Pretreatment of ARVMs with 1, 10, 100 and 1,000 ng/ml rhSLPI significantly increased cell viability compared with the untreated sI/R group (P<0.05;
By contrast, rhSLPI-treatment at the onset of reperfusion (
To identify the maximum rhSLPI concentration required to induce cardioprotection in ARVMs without inducing any toxicity, ARVMs were treated with 0, 200, 400, 600, 800 and 1,000 ng/ml rhSLPI 2 h prior to sI or immediately following the onset of sI. Following treatment, all groups were subjected to sI for 20 min followed by 2 h reperfusion. Cell viability and cell injury were determined.
The results indicated that following 2 h reperfusion, the percentage of viable cells in the groups treated with rhSLPI either 2 h prior to sI or at the onset of sI increased compared with the sI group. Pretreatment of ARVMs with 200, 400, 600, 800 and 1,000 ng/ml rhSLPI significantly increased the viability of ARVMs exposed to sI/R injury compared with the sI group (P<0.05;
The toxicity of rhSLPI was determined by culturing ARVMs with 0, 200, 400, 600, 800 and 1,000 ng/ml rhSLPI for 24 h and then measuring cell viability and cell injury. The results indicated that treatment with all concentrations of rhSLPI did not reduce cell viability (
To further investigate the cardio-protective effects of rhSLPI treatment during sI/R injury, intracellular ROS generation was measured following H2O2 challenge.
The results demonstrated that exposure to H2O2 significantly increased intracellular ROS levels compared with the control group (P<0.05;
To determine the cellular signaling in response to rhSLPI treatment in ARVMs during sI/R injury, western blot analysis was performed to determine the phosphorylation of p38 MAPK and Akt.
The results demonstrated that the phosphorylation of p38 MAPK was significantly increased following sI (P<0.05;
In addition, treatment of ARVMs with rhSLPI at the onset of sI reduced the phosphorylation of p38 MAPK in a dose-dependent manner (
Hearts isolated from adult male C57BL/6 mice were perfused on a Langendorff system with buffered solutions containing 400 or 1,000 ng/ml rhSLPI during I/R injury (
The results indicate that following 30 min global ischemia and 2 h reperfusion, the infarct size was 48.40±4.23% (
To further investigate the cardioprotective effects of rhSLPI pretreatment on
The present study demonstrated that rhSLPI exhibits a protective effects on
During myocardial I/R injury, levels of pro-inflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin (IL) 6, IL-8, and mitochondrial pyruvate carrier-1 (MPC1), are upregulated and secreted from adjacent cardiomyocytes, resulting in leukocyte infiltration (
Previous studies have determined the effects of protease inhibitors on ischemic injury. In a rabbit model of heterotopic cardiac transplantation, the serine elastase inhibitor elafin decreased post-cardiac transplant coronary arteriopathy and decreased myocardial necrosis following transplantation (
Studies have investigated the effects of SLPI on inflammation. SLPI is a protein involved in downregulating macrophage responses against bacterial lipopolysaccharides (LPS) by inhibiting NF-κB activation (
The excessive formation of ROS during I/R injury induces cardiac cell death directly by inducing cell membrane and protein damage (
Myocardial I/R injury is mediated by numerous signaling pathways, including p38 MAPK. Myocardial I/R injury activates p38 MAPK, which induces cardiac cell death (
Akt is a serine/threonine protein kinase and serves an important role in cell survival (
Although it has been demonstrated that various treatments/interventions administered during reperfusion are effective at reducing infarct size, the results of the current study indicated that administering rhSLPI at the onset of reperfusion failed to protect ARVMs from sI/R induced cell death and cell injury. These results were similar to those of previous studies investigating the effects of treatments or interventions administered prior to ischemia, which may protect the heart from I/R injury (
There were a number of limitations of the current study. Treatment of ARVMs by rhSLPI or treatment of
In conclusion, the results of the current study indicate that treatment with rhSLPI exhibits cardioprotective effects against myocardial I/R injury in
The authors wish to acknowledge the support from the Franco-Thai Scholarship program in 2015 and-2016 for authors SK and SBL. The authors would also like to thank the Newton Fund in cooperation with The Royal Golden Jubilee PhD Program for providing a PhD placement scholarship for EP, SK and MM. The authors are grateful to the Center for Animal Research, Naresuan University for their excellent technical assistance.
The present study was supported by The Royal Golden Jubilee PhD Program (grant no. PHD/0043/2555) - joint funding between the Thailand Research Fund, Naresuan University and National Research Council of Thailand (NRCT)-Naresuan University (grant no. R2558B06).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
The present study was approved by the animal ethics committee of the Center for Animal Research, Naresuan University (approval no. NU AE550732).
EP and SK conceived and designed the experiments. EP, JS, SBL, JN, HN, MM and SK performed the experiments and analyzed the data. EP, MM and SK wrote, re-checked, and proofread the paper.
Not applicable.
The authors declare that they have no competing interests.
Optimization of the cardioprotective dose of rhSLPI for ARVMs subjected to sI/R. ARVMs were cultured with various concentrations of rhSLPI and treated over three different periods, including (A and B) 2 h prior to sI, (C and D) at the onset of sI and (E and F) at the onset of reperfusion. Following rhSLPI treatment, all groups were subjected to 20 min sI followed by 2 h reperfusion. The percentage of viable cells and released LDH activity in all groups were determined. The results are presented as the mean ± standard error of the mean (n=3). *P<0.05 vs. control group; #P<0.05 vs. sI group. rhSLPI, recombinant human secretory leukocyte protease inhibitor; sI, stimulated ischemia; ARVM, adult rat ventricular myocytes; LDH, lactate dehydrogenase; sI/R, stimulated ischemia/reperfusion.
Determination of the maximum rhSLPI concentration required to provide cardioprotection in ARVMs subjected to sI/R. The percentage of (A) viable cells and (B) LDH activity following pretreatment of ARVMs with various concentrations of rhSLPI. The percentage of (C) viable cells and (D) LDH activity following treatment of ARVMs with various concentrations of rhSLPI during sI. The percentage of (E) viable cells and (F) LDH activity following treatment of ARVMs with various concentrations of rhSLPI without sI. The results are presented as the mean ± standard error of the mean (n=3). *P<0.05 vs. control group; #P<0.05 vs. sI group. rhSLPI, recombinant human secretory leukocyte protease inhibitor; ARVM, adult rat ventricular myocytes; sI, stimulated ischemia; sI/R, stimulated ischemia/reperfusion; LDH, lactate dehydrogenase.
Cellular ROS levels following treatment of ARVMs with rhSLPI. ARVMs were incubated with carboxy-H2DCFDA and treated with 0, 200, 400, 600, 800 and 1,000 ng/ml rhSLPI. Following incubation, H2O2 was applied to each group and ROS production was determined using the EnSpire Multimode Plate Reader. Data are presented as the mean ± standard error of the mean (n=3). *P<0.05 vs. control group; #P<0.05 vs. H2O2 treated group. ROS, reactive oxygen species; ARVM, adult rat ventricular myocytes; rhSLPI, recombinant human secretory leukocyte protease inhibitor; A.U., arbitrary unit.
Cellular signaling in response to pretreatment of ARVMs with rhSLPI, subjected to ischemia/reperfusion. ARVMs were treated with 200, 400, 600, 800 and 1,000 ng/ml rhSLPI for 2 h prior to sI. The phosphorylation of (A) p38 MAPK and (B) Akt was determined by western blot analysis. Each bar graph represents the phosphorylation ratio (p-/t-) of p38 MAPK and Akt. *P<0.05 vs. control group; #P<0.05 vs. sI group. ARVM, adult rat ventricular myocytes; rhSLPI, recombinant human secretory leukocyte protease inhibitor; sI, stimulated ischemia; MAPK, mitogen-activated protein kinase; p-phosphorylated; t-, total.
Cellular signaling following treatment of ARVMs with rhSLPI during sI. ARVMs were treated with 200, 400, 600, 800 and 1,000 ng/ml rhSLPI at the onset of sI. The activation of (A) p38 MAPK and (B) Akt was determined by western blot analysis. Each bar graph represents the phosphorylation ratio (p-/t-) of p38 MAPK and Akt. *P<0.05 vs. control group; #P<0.05 vs. sI group. ARVM, adult rat ventricular myocytes; rhSLPI, recombinant human secretory leukocyte protease inhibitor; sI, stimulated ischemia; MAPK, mitogen-activated protein kinase; p-, phosphorylated; t-, total.
The effect of rhSLPI treatment on the myocardial infarct size of
Signaling in response to treatment of