LPS (from Escherichia coli 055:B5), TNF-α, IL-6, KN-93 and β-actin antibody (cat no. A5316) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Y-27632 and fasudil were obtained from Selleck Chemicals (Houston, TX, USA). Antibodies against: Drp1 (cat no. 8570), phosphorylated Drp1 (p-Drp1) Ser616 (cat no. 4494), p-Drp1 Ser637 (cat no. 4867), calcium/calmodulin-dependent protein kinase type II (CaMK II) (cat no. 4436), phosphorylated c-Jun N-terminal kinase (p-JNK) (cat no. 4668), Ras homolog gene family member A (RhoA) (cat no. 2117), Ras-related C3 botulinum toxin substrate 1 (Rac1)/cell division control protein 42 homolog (Cdc42) (cat no. 4651) and voltage dependent anion channel (VDAC, cat no. 4866) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The LPS ELISA kit (cat no. CSB-E14247r) was purchased from Cusabio Biotech Co., Ltd. (Wuhan, Hubei, China) and the TNF-α and IL-6 ELISA kits (cat nos. BMS622 and BMS625, respectively) were purchased from eBioscience; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). MitoTracker TM Red CMXRos was purchased from Thermo Fisher Scientific, Inc.

A total of 32 male Sprague-Dawley rats (230–250 g, 7–8 weeks old) were purchased from the Experimental Animal Center of Zhejiang University (Hangzhou, China) and cared for in compliance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA). Four rats per cage were housed in a controlled room (20–24°C, 40–60% humidity) and 12/12-h light/dark cycle. All rats were allowed free access to water and food. The rats were randomly divided into 4 groups (n=8 each), and injected intraperitoneally with either 0.9% NaCl (for control group) or LPS (5, 10 or 20 mg/kg in 0.9% NaCl) in order to establish the sepsis model as previously described (15). All experimental protocols were approved by the Ethics Committee on Animal Experimentation of Zhejiang University (Hangzhou, China).

H9C2 rat cardiomyocyte cell line was purchased from the Chinese Academy of Sciences (Shanghai, China). Cells were incubated in completed Dulbecco's modified Eagle's medium (DMEM; Genom Biomedical Technology Corporation, Hangzhou, China) containing 10% fetal bovine serum (Sijiqing Corporation, Hangzhou, China) in a humidified 5% CO₂ environment. Following further incubation with serum-free medium for 12 h at 37°C, cells were divided into the following groups: i) Control group, untreated cells; ii) LPS groups, cells were treated with 0.01, 0.1 or 1 µg/ml LPS; iii) TNF-α groups, cells were treated with 5, 10 or 20 ng/ml TNF-α; iv) IL-6 groups, cells were treated with 5, 10 or 20 ng/ml IL-6; v) TNF-α + KN-93 group, cells were incubated with a CaMK II inhibitor, 1 µM KN-93, for 1 h at 37°C, and then co-incubated with 20 ng/ml TNF-α for 6 or 48 h at 37°C; vi) TNF-α + Y-27632/fasudil group, cells were incubated with Rho kinase (ROCK) inhibitors 1 µM Y-27632 or1 µM fasudil for 1 h at 37°C, and then co-incubated with 20 ng/ml TNF-α for 6 or 48 h at 37°C; and vii) KN-93/Y-27632/fasudil alone group, cells were incubated with 1 µM KN-93, 1 µM Y-27632 or 1 µM fasudil for 7 or 49 h at 37°C. Protein levels were determined 6 h post-treatment of TNF-α, and cellular viability was evaluated 48 h post-treatment of TNF-α.

Rat blood samples were collected in clot activator-coated test tubes. Serum was extracted via centrifugation at 1,000 × g for 10 min at room temperature and then stored at −80°C until analysis. Subsequently, the serum LPS levels were determined using ELISA kits in accordance with the manufacturer's protocol (Cusabio Biotech Co., Ltd.). Plasma TNF-α and IL-6 levels were determined using ELISA kits in accordance with the manufacturer's protocol (eBioscience; Thermo Fisher Scientific, Inc.).

H9C2 cells were seeded into 96-well plates (1,000 cells/well). Cells were washed and supplemented with serum-free DMEM medium 48 h after the aforementioned treatments. CCK-8 solution was then added to the culture medium at a dilution of 1:10 for 2 h at 37°C. A highly water-soluble tetrazolium salt, WST-8, which is utilized in CCK-8 solution, can receive two electrons from viable cells to generate an orange or purple formazan dye. The quantity of WST-8 formazan dyes, which are directly proportional to the number of living cells, was subsequently analyzed using a microplate reader (Tecan Infinite M200; Tecan Group Ltd., Mannedorf, Switzerland) at a wavelength of 450 nm.

Mitochondria were isolated from rat ventricular myocardium using the Tissue Mitochondria Isolation kit, in accordance with the manufacturer's protocol (eBioscience; Thermo Fisher Scientific, Inc.). Mitochondria in H9C2 cells were isolated using the Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher Scientific, Inc.).

Left ventricular myocardium tissue and H9C2 cells were lysed in ice cold cell lysis buffer containing a protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Inc., Danvers, MA, USA). Following centrifugation at 14,000 × g 4°C for 5 min, the supernatants were collected. The protein concentration in supernatants was determined by using BCA Protein Assay kit (cat. no. P0012; Beyotime Institute of Biotechnology, Haimen, China). Aliquots of total cell extract or mitochondria containing 20 µg protein were loaded on a 10% SDS-PAGE gel. The proteins were transferred to nitrocellulose membranes (Invitrogen; Thermo Fisher Scientific, Inc.). Membranes were blocked with 5% bovine serum albumin (BSA; Shanghai Sangon Biological Engineering Technology & Services Co., Shanghai, China) for 1 h at room temperature, membranes were then incubated with primary antibodies (1:1,000) overnight at 4°C. Following washing, membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (1:1,000; cat. nos. 7074 and 7076; Cell Signaling Technology, Inc.) for 1 h at room temperature. An enhanced chemiluminescence kit (Beyotime Institute of Biotechnology) was used for protein visualization. The membranes were then stripped and re-probed using β-actin and VDAC antibodies, which represent the internal loading controls. The band densities were subsequently determined using Quality One version 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and normalized to β-actin or VDAC. Experiments were performed in triplicate.

Cells were cultured on coverslips and then fixed with PBS containing 4% paraformaldehyde for 15 min at room temperature. Furthermore, cells were blocked with 1% BSA in PBS for 30 min at room temperature, and were then incubated with antibodies against p-Drp1 Ser616 (1:2,000) overnight at 4°C. Following thorough washing, the cells were then incubated with Alexa Fluor 488-conjugated anti-rabbit-IgG (1:1,000; cat. no. 4412; Cell Signaling Technology, Inc.) at room temperature for 1 h. Images of fluorescently-labeled cells were obtained using a Confocal Laser Scanning Biological microscope at magnification, ×40 (FV3000; Olympus Corporation, Tokyo, Japan). Resulting images were analyzed using Image Pro Plus version 6.0 (Media Cybernetics Inc., Rockville, MD, USA).

Following aforementioned treatments, cells were incubated with a mitochondria-specific dye 200 nM MitoTracker TM Red CMXRos for 15 min at 37°C in a 5% CO₂ incubator. Following two washes with PBS, cells were exposed to 4′,6-diamidino-2-phenylindole (5 µg/ml) for 5 min in order to stain the nuclei. Cells were then washed twice and suspended in PBS pH 7.4. Images of fluorescently-labeled cells were examined using a Confocal Laser Scanning Biological microscope at magnification, ×60 (FV1000; Olympus Corporation). The mitochondrial quantity, individual area of each mitochondria and mitochondrial length-width ratio were quantified according to fission criteria as previously described (16) using Image Pro Plus version 6.0. A decrease in mitochondrial area combined with an increase in the number of mitochondria was considered to represent fission criteria.

Data were expressed as mean ± standard error of the mean and analyzed using either the Student's t-test or one-way analysis of variance followed by Student-Newman-Keuls post hoc test. Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used to analyze the data. P<0.05 was considered to indicate a statistically significant difference.

Total Drp1 expression in rat heart tissues did not significantly change 6 h after injection with 5, 10 or 20 mg/kg LPS; however, the expression of mitochondrial Drp1 was revealed to significantly increase in a dose-dependent manner (Fig. 1A–D; P<0.05), suggesting that sepsis may induce mitochondrial translocation of Drp1. Furthermore, the level of p-Drp1 Ser616 significantly increased in the LPS-treated groups compared with the control group (Fig. 1E–G; P<0.05); however, levels of p-Drp1 Ser637 did not significantly change post-treatment with LPS.

Following injection with LPS (20 mg/kg), the serum level of LPS was revealed to significantly increase 1 h post-injection, and increase ~4.5-fold 6 h post-injection compared with the control (Fig. 2A). Furthermore, LPS administration significantly increased the TNF-α and IL-6 concentration in rat serum samples (Fig. 2B and C). The concentration of serum TNF-α reached high level at 1.5 h after LPS injection, no significant difference was identified when compared with that of 6 h post-treatment. The concentration of serum IL-6 was highest at 6 h of post-treatment. Treatment with LPS (0.01–1 µg/ml), TNF-α (10–20 ng/ml) and IL-6 (10–20 ng/ml) for 48 h resulted in a significant decrease in the viability of cultured H9C2 cardiomyocytes (Fig. 3). LPS (1 µg/ml), 20 IL-6 (ng/ml) and TNF-α (20 ng/ml) were then used for subsequent experiments.

H9C2 cells were treated with 1 µg/ml LPS, 20 ng/ml IL-6 and 20 ng/ml TNF-α to investigate the effects of inflammatory cytokines on the expression and mitochondrial translocation of Drp1. Cells were also incubated with serum (1:100) obtained from three rats 1 h post-LPS injection. Compared with the control group, administration of LPS and IL-6 did not exhibit a significant effect on the expression of total Drp1 and mitochondrial Drp1 (Fig. 4; P>0.05). However, following treatment with TNF-α (20 ng/ml) and serum from LPS-injected rats, the level of mitochondrial Drp1 increased ~2-fold compared with the control; however, total Drp1 expression did not significantly change post-treatment (Fig. 4; P<0.01).

The mechanism underlying TNF-α-induced phosphorylation of Drp1 remains to be elucidated. Incubation of H9C2 cells with TNF-α (20 ng/ml) was revealed to significantly enhance the expression of CaMK II compared with control (Fig. 5; P<0.01 vs. control); however, the level of p-JNK did not significantly change following treatment with TNF-α (Fig. 5; P>0.05 vs. control). Furthermore, the level of p-Drp1 Ser616 was significantly increased following treatment with TNF-α (Fig. 6; P<0.01 vs. control). In addition, it was demonstrated that treatment with KN-93, an inhibitor of CaMK II, did not significantly inhibit the TNF-α-induced increase in the levels of mitochondrial Drp1 and p-Drp1 Ser616 (Fig. 6A-D; P>0.05 vs. control), nor significantly inhibit the TNF-α-induced decrease in cell viability (Fig. 6E; P>0.01 vs. TNF-α). Therefore, these findings suggest that CaMK II and JNK are not involved in TNF-α-induced phosphorylation of Drp1.

The present investigated whether the three most prevalent members of the Rho protein family (RhoA, Cdc42 and Rac1) are involved in TNF-α-induced Drp1 phosphorylation. Following treatment with TNF-α (20 ng/ml), the expression of Rac1/Cdc42 did not exhibit a significant change; however, the expression of RhoA in H9C2 cells treated with TNF-α increased ~2.3-fold compared with the control (Fig. 5; P<0.01 vs. control). In addition, it was demonstrated that administration of Y-27632 and fasudil ROCK inhibitors in TNF-α treated cells significantly attenuated the TNF-α-induced increase in the levels of p-Drp1 Ser616 and mitochondrial Drp1 (Fig. 7; P<0.01 vs. TNF-α). However, when administered to non-TNF-α treated cells, neither Y-27632 nor fasudil demonstrated a significant effect regarding the levels of p-Drp1 Ser616 or mitochondrial Drp1 compared with the control (Fig. 7; P>0.01 vs. control). These results were confirmed by performing immunofluorescence analysis to determine the level of p-Drp1 Ser616 (Fig. 8).

Analysis using confocal laser microscopy demonstrated that treatment with TNF-α increased mitochondrial fragmentation by significant enhancement of the total number of mitochondria per cell and significant reduction of mitochondrial area (Fig. 9A–C); however, the mitochondrial length-width ratio did not significantly change post-treatment (Fig. 9D). Treatment with both Y-27632 or fasudil alone was revealed to significantly attenuate the increase in mitochondria per cell otherwise induced by treatment with TNF-α (Fig. 9A; P<0.05). Furthermore, treatment with Y-27632 alone was revealed to significantly attenuate the decrease in mitochondrial area otherwise induced by treatment with TNF-α (Fig. 9B; P<0.05). Thus, administration of Y-27632 and fasudil was revealed to attenuate the TNF-α-induced increase in mitochondrial fragmentation (Fig. 9A–D), and significantly attenuate the decrease in cell viability otherwise induced by treatment with TNF-α (Fig. 9E; P<0.01).