Sepsis is a systemic inflammatory response syndrome and is accompanied with multiorgan system dysfunction. Cardiovascular dysfunction is a common event in sepsis and associated with an increase in mortality in patients suffering from sepsis (1). Treatment with lipopolysaccharide (LPS), a structural component of the outer membrane of Gram-negative bacteria, has been used as an experimental model to investigate cardiac dysfunction in septic condition (2). Transcriptional factor nuclear factor κB (NFκB) has been suggested to play a central role in LPS-induced cardiac toxicity, although its mechanism is not fully understood at this moment (3, 4). Recently, various heat shock proteins (HSPs) have been associated with LPS-induced cardiac toxicity as regulatory factors of NFκB signaling. For example, HSP70 and HSP20 were reported to suppress LPS-induced cardiac toxicity by inhibiting nuclear accumulation and DNA binding of NFκB (3, 5, 6).
Co-chaperone cytoplasmic constitutive active/androstane receptor retention protein (CCRP) is a member of the heat shock protein (HSP) 40 family. CCRP is highly conserved from fly to human and is ubiquitously expressed in various tissues including liver, lung, and heart (7, 8). This protein was originally found to sequester nuclear receptor constitutive active/androstane receptor (CAR), also known as NR1I3, in the cytoplasm of human hepatoma-derived HepG2 cells, and then it was named CCRP (7, 9). In mouse livers, CCRP was demonstrated to regulate the phenobarbital activation of CAR-mediated transcription of the Cyp2b10 gene at multiple steps, such as nuclear CAR accumulation, histone demethylation, and RNA polymerase II recruitment (10). In addition to CAR, several research groups further reported that CCRP regulates other nuclear receptors in cellular localization, gene activation, and so on in cultured cell systems (11–14). For example, our recent molecular-based work demonstrated that CCRP facilitates an interdomain interaction within glucocorticoid receptor (GR) and regulates GR in protein complex conformation and gene activation (15). However, physiological roles of CCRP are still largely unknown in vivo. Utilizing our CCRP knockout (KO) mice, we searched a new physiological role of CCRP and found that CCRP KO mice developed more severe cardiac dysfunction after LPS treatment. Given this fortuitous finding, here we have investigated mechanisms by which CCRP protected mice from LPS-induced cardiac toxicity.
MATERIALS AND METHODS
LPS (serotype 0127:B8, L3129) and ammonium pyrrolidinedithiocarbamate (PDTC, P8756) were purchased from Sigma-Aldrich (St. Louis, Mo). Mouse anti-TNFα antibody (AB-410-NA) was purchased from R&D Systems (Minneapolis, Minn). PBS was used to dissolve these chemicals. Aliquots of LPS solution at the concentration of 1 mg/mL were made and kept at −20°C without repeated freezing and thawing. For PDTC solution, powder PDTC was solved in PBS to make fresh solution immediately before each use.
CCRP KO mice were generated and maintained as previously described (10). The 10 to 16 weeks’ old male CCRP wild-type (WT) and KO mice were used for all the experiments. Experiments were performed in accordance with protocols approved by Animal Care and Use Committee of NIEHS. All the animals survived during LPS challenge experiments.
CCRP WT and CCRP KO mice were intraperitoneally injected with vehicle PBS or LPS (10 mg/kg BW). For PDTC pretreatment study, vehicle PBS or PDTC (10 or 20 mg/kg BW) was intraperitoneally injected 1 h before LPS injection based on a previous report (16). For anti-TNFα antibody pretreatment study, vehicle PBS or anti-TNFα antibody (100 μg/mouse) was intravenously injected 30 min before LPS injection based on previous reports (17, 18). Electrocardiography (ECG) data were recorded noninvasively in conscious mice routinely for indicated time periods using the ECGenie apparatus (Mouse Specifics, Inc., Boston, Mass). Briefly, mice were removed from their cages and placed on the ECG recording platform that is enclosed on four sides. An array of ECG electrodes are embedded in the floor of the platform and spaced to facilitate contact between the electrodes and mouse paws to provide a lead I, II, or III ECG in mice. Mice freely move on the platform until sufficient ECG data are collected. Data from continuous recordings of 20 to 30 ECG signals were used in the analyses. Raw ECG signals were analyzed using e-MOUSE software (Mouse Specifics). Heart rate was determined from R–R intervals. The software also determined PR, QRS, and QT cardiac intervals.
Because anesthesia has significant impacts on cardiac functions, including heart rate (19), echocardiographic measurements were performed on conscious mice before and 6 h after an intraperitoneal injection with LPS (10 mg/kg BW) using a Vevo 770 ultrasound biomicroscopy system (VisualSonics, Toronto, Canada). Briefly, mice were removed from their cages and placed on a platform in a slight upward position (head up) under a slight angle. Each hand and leg of the mouse was gently immobilized by clips connecting to elastic strings that can be fixed to the plexiglass platform. The chest fur was removed by hair removing gel. Then a layer of preheated ultrasound gel was applied to the chest. To make echocardiographic measurements, the ultrasound probe was positioned with a 90° angle between the probe and the heart for imaging the long axis of the heart. Once the mouse was calm, then M-mode and B-mode images were obtained. It took 5 to10 min per mouse to make a measurement. Immediately after the measurement, mice were placed back to their original cage. Fraction shortening was calculated from left ventricular dimensions as previously described (20).
Cell culture, transfection, and treatment
Rat cardiomyocyte-derived cell line H9c2 cells were purchased from American Type Culture Collection (ATCC) (Manassas, Va) and cultured in Dulbecco's modified Eagle's medium (30–2002, ATCC) supplemented with 10% fetal bovine serum in a humidified 5% CO2 incubator at 37°C. H9c2 cells were transiently transfected with mCCRP-V5 expression vector by reverse transfection technique using FuGENE 6 (Promega, Madison, Wis) according to the manufacturer's protocol (7). The cells were incubated in the same growth condition described above. After 40 h from the transfection, cells were treated with vehicle PBS or LPS at a concentration of 1 μg/mL for indicated time periods.
Nuclear and cytoplasmic proteins from heart tissues were obtained according to a previous report with minor modifications: buffer A contained 0.1% NP-40 instead of IGEPAL CA-630 (16). Nuclear and cytoplasmic proteins were extracted from H9c2 cells using NE-PER kit (PIERCE, Rockford, Ill) according to the manufacturer's protocol.
Western blot analysis
Proteins were separated with 8.5% SDS-PAGE and transferred to polyvinylidene difluoride membrane. After blocking with 5% nonfat dry milk containing TBS-0.1% Tween20 (TBST) buffer, membrane was probed with anti-p65 antibody (1:5,000, C-20X; Santa Cruz, Santa Cruz, Calif), anti-CCRP antibody (1:5,000, generated as previously described, 7), anti-HSP90 antibody (1:1,000, 610419; BD Transduction Laboratories, San Jose, Calif), anti-HDAC1 antibody (1:1,000, #2062; Cell Signaling Technology, Beverly, Mass), or anti-βACTIN antibody (1:1,000, C4; Santa Cruz) in the 5% milk or 5% BSA containing TBST buffer for overnight at 4°C and then with proper secondary HRP-conjugated antibody (1:5,000, sc-2314 and sc-2004; Santa Cruz) for 1 h at room temperature. Protein bands on membrane were visualized using chemiluminescence detection reagents with various sensitivity to obtain desired band intensity (Advansta, Menlo Park, Calif).
H9c2 cells were treated with PBS or LPS (1 μg/mL) for 60 min and fixed in ice-cold methanol for 5 min at room temperature. After cells were washed in PBS, blocking and permeabilization were conducted by treating with PBS containing 0.4% Triton X-100, 1% BSA, and 4% goat serum for 20 min. Anti-p65 antibody (1:100, C-20X; Santa Cruz) diluted in PBS was applied to cells for 1 h at room temperature, followed by three rinses with PBS for 5 min each. Then, anti-rabbit Alexa Fluor 594 (1:2,000; Invitrogen, Carlsbad, Calif) diluted in PBS was applied as a secondary antibody for 30 min at room temperature. After washing by three rinses with PBS for 5 min each, coverslips were mounted in PBS containing DAPI and cells were observed using a ZEISS LSM 710 confocal microscopy (Carl Zeiss Microscopy, Thornwood, NY) fitted with the appropriate filters.
Measurement of TNFα protein levels
TNFα levels in the sera were determined by ELISA using mouse TNFα ELISA Max Set Deluxe Kits (BioLegend, San Diego, Calif) according to the manufacturer's protocol.
Quantitative PCR analysis
Total RNAs were extracted using Trizol (Invitrogen) according to a manufacturer's protocol. cDNAs were synthesized from extracted RNA using SuperScript first strand synthesis system (Invitrogen) with random hexamers as primers. Real-time PCR was performed using TaqMan probe (Applied Biosystems, Foster City, Calif) to evaluate expression levels of Tnfα. The RNA levels of target gene were normalized against the β-Actin levels using the comparative CT (ΔΔCT) method.
Data are presented as means ± SD. Statistical comparisons between groups were performed using unpaired t test or analysis of variance (ANOVA) with Graphpad Prism 6 (Graphpad Software, San Diego, Calif). Differences among means were considered significant at P < 0.05.
Decreases in heart rate was observed in LPS-treated CCRP KO mice
To examine if CCRP protects cardiac functions from LPS-induced toxicity, we measured heart rates of CCRP-WT and CCRP KO mice with and without LPS treatment. First, baseline of heart rate was examined in nontreated animals. WT and KO mice showed heart rates of 734.0 ± 43.8 and 734.4 ± 47.2, respectively (n = 6, mean ± SD). PR intervals were 28.3 ± 3.4 and 29.6 ± 1.2, QRS duration was 10.4 ± 2.8 and 10.9 ± 1.3, and QT intervals were 40.4 ± 3.9 and 41.9 ± 1.5 in WT and KO mice, respectively (n = 6, mean ± SD). There were no statistically significant differences in heart parameters, heart rate, PR, QRS, and QT cardiac intervals, between WT and KO. Heart parameters of WT and KO mice are very similar to those of C57BL/6 previously reported (21). Next, heart rates were examined in WT and KO mice treated with PBS or LPS for 6 h (Fig. 1). In WT mice, LPS treatment did not change heart rates. In sharp contrast, heart rates of KO mice were significantly decreased at 5 h from LPS injection and kept diminishing up to 6 h, compared with those of PBS-treated animals at each time point. Heart rates at 6 h from the treatment in each group were 715.0 ± 46.9 in WT-PBS, 704.5 ± 17.8 in WT-LPS, 709.7 ± 30.1 in KO-PBS, and 510.7 ± 59.5 in KO-LPS (n = 3, mean ± SD). In addition, significantly prolonged PR intervals were also observed only in LPS-treated KO mice. PR interval in each group was 29.3 ± 0.7 in WT-PBS, 32.2 ± 2.3 in WT-LPS, 28.6 ± 3.2 in KO-PBS, and 40.2 ± 2.8 in KO-LPS (n = 3, mean ± SD). Prolongation of PR interval in LPS-treated KO mice is thought to reflect slowing of heart rate.
More reduced ventricular function was observed in LPS-treated CCRP KO mice
A ventricular contractile function was further investigated by echocardiography. Fig. 2A shows representative echocardiographic pictures of conscious WT and KO mice before and after LPS treatment for 6 h. M-mode echocardiography evaluation demonstrated that LPS challenge significantly suppressed left ventricular contractile function in both WT and KO mice as evidenced by reduction in fraction shortening from baseline values (Fig. 2B). Fraction shortening (%) in each group was 54.6 ± 2.9 in WT baseline, 37.5 ± 3.4 in WT-LPS, 54.8 ± 3.2 in KO baseline, and 31.9 ± 4.5 in KO-LPS (n = 5, mean ± SD). Fraction shortening values of LPS-treated KO mice was significantly lower than those of LPS-treated WT. These results indicated that the presence of CCRP also attenuates the reduction of the left ventricular function induced by LPS treatment at least partially.
CCRP prevented LPS-induced p65 nuclear accumulation in H9c2 cells
A central role of NFκB signaling in LPS-induced cardiac dysfunction has been established (3, 4). We hypothesized that CCRP inhibited NFκB signaling activated by LPS and conferred protection of cardiac functions. To examine this hypothesis, we analyzed the effect of CCRP overexpression on LPS-induced nuclear accumulation of p65, an NFκB subunit, in rat cardiomyocyte-derived cell line H9c2. First, western blot analysis confirmed that endogenous CCRP expression in H9c2 cells was undetectable or very low at protein level (Fig. 3A). As expected, treatment of H9c2 cells with LPS at a concentration of 1 μg/mL for 30 and 60 min induced the nuclear translocation of p65. However, overexpression of CCRP clearly decreased p65 nuclear accumulation after LPS treatment (Fig. 3B). Overexpressed CCRP was localized mainly in the cytoplasm and LPS treatment did not affect the expression level and the localization (Fig. 3B). Immunocytochemistry further confirmed an inhibitory effect of CCRP on the p65 nuclear localization induced by LPS (Fig. 3C). In vehicle PBS-treated H9c2 cells, most of p65 was localized in the cytoplasm regardless of CCRP overexpression (Fig. 3C, left panels). The treatment with LPS clearly increased nuclear p65 levels in mock-transfected H9c2 cells, whereas CCRP overexpressing cells showed much lower levels of p65 in nuclear fraction (Fig. 3C). To investigate effects of CCRP on NFκB downstream signaling, proinflammatory cytokine TNFα mRNA levels were determined in H9c2 cells by real-time PCR assays. LPS treatment for 6 h increased TNFα mRNA from 1.0- to 24.8-fold in mock-transfected cells and from 0.2- to 1.4-fold in CCRP-expressing cells (Fig. 3). These results indicate that overexpression of CCRP prevents LPS-induced p65 nuclear accumulation and suppresses NFκB signaling in H9c2 cells.
CCRP protects cardiac functions from LPS-induced toxicity through inhibition of NFκB signaling
The effect of CCRP on p65 nuclear levels was examined in vivo using LPS-treated CCRP WT and KO mice. Western blot analysis demonstrated clear p65 nuclear accumulation in the heart of both WT and KO. As observed with results of H9c2 cell-based experiments, the p65 levels in the nuclei were clearly higher in the hearts of KO mice especially at 1 and 6 h after the injection (Fig. 4A). An asterisk indicates nontarget protein. Because this band seemed at around 80 kDa, it does not seem to be p65 whose size is 65 kDa.
Then, to test a hypothesis that activated NFκB signaling was involved in the decrease in heart rates observed in LPS-treated CCRP KO mice, we pretreated KO mice with PDTC, an NFκB inhibitor, and measured heart rates before and 6 h after LPS treatment. In a preliminary experiment, it was confirmed that PDTC treatment did not affect the heart rate at least under our experimental conditions (data not shown). LPS-induced decreases in heart rate were significantly blocked by pretreatment with PDTC at concentrations of 10 and 20 mg/kg BW in a dose-dependent manner (Fig. 4B). Heart rate at 6 h in each group was 449.5 ± 36.1 in vehicle, 623.7 ± 13.8 in 10 mg PDTC, and 657.5 ± 26.2 in 20 mg PDTC-treated group, whereas heart rates immediately before LPS injection were 742.3 ± 52.3 in vehicle, 770.3 ± 12.6 in 10 mg PDTC, and 745.8 ± 2.9 in 20 mg PDTC-treated group (n = 3, mean ± SD).
LPS-induced TNFα contributed to heart rate decreases observed in LPS-treated KO mice
To further investigate the effect of CCRP on NFκB signaling, TNFα levels were determined in the sera of WT and KO mice by ELISA assays. Induced TNFα levels in the sera of LPS-treated KO were twice as high as those in WT at 1, 3, and 6 h after LPS treatment (Fig. 5A). The kinetic pattern of LPS-induced TNFα did not differ between WT and KO. The peak of TNFα levels in the sera was observed at 1 h from the treatment (WT, 4.30 ± 1.17 ng/mL; KO, 9.95 ± 1.66 ng/mL, n = 3, mean ± SD).
Then, we further examined if TNFα induced by LPS treatment was involved in the decrease in heart rates in KO mice. Pretreatment with anti-TNFα antibody significantly restored the heart rate decreased by LPS (Fig. 5B). Heart rate at 6 h in each group was 541.0 ± 40.3 in vehicle and 665.5 ± 19.5 in TNFα antibody, whereas heart rates immediately before LPS injection were 711.8 ± 13.3 in vehicle and 741.3 ± 9.8 in TNFα antibody (n = 4, mean ± SD).
Our present study demonstrated that CCRP protects cardiac functions against LPS-induced toxicity in vivo. LPS-treated CCRP KO mice showed clear decreases in heart rates and more reduced ventricular contractile functions. Although the ventricular contractile function is typically depressed by LPS treatment (20, 22), the effect of LPS on heart rate is complex and not well understood probably due to controversial observations in previous studies: unchanged, decreased, or increased heart rate. The outcome seems to depend on LPS concentration, administration pathway, and so on. According to previous reports that were conducted under similar experimental conditions to ours, an intraperitoneal administration of LPS at a concentration of around 10 mg/kg does not seem to change heart rate in C57BL/6 mice consistently to our observation in CCRP WT mice (20, 22, 23). An intravenous injection of LPS at a concentration of 20 mg/kg B.W in Sprague–Dawley rats decreased heart rate (3). Although differences in experimental conditions between the previous report and ours may make interpretations difficult, excess activation of NFκB signaling and severe inflammation seem to lead to heart rate decreases even in the presence of CCRP. Endogenous CCRP seems to minimize toxic effects of LPS on heart rate at least under these experimental conditions.
In the present study, LPS-induced heart rate decreases in CCRP KO mice were drastically improved by pretreatment with NFκB inhibitor PDTC. In a previous study, inhibition of NFκB signaling by double-stranded decoy DNA that prevents DNA binding of NFκB completely canceled heart rate decreases induced by intravenous injection with LPS at a concentration of 20 mg/kg BW in Sprague–Dawley rats (3). Moreover, TNFα was also demonstrated here to be directly involved in heart rate decreases caused by LPS treatment in CCRP KO mice. Given the fact that transgenic mice overexpressing TNFα in the heart develop a progressive heart failure including the heart rate decrease (24), TNFα seems to be one of most important downstream factors of NFκB signaling for LPS-induced heart rate decreases. CCRP inhibition of LPS-activated NFκB signaling was confirmed by the results that showed that nuclear accumulation of NFκB subunit p65 was inhibited by CCRP in cultured cardiomyocyte H9c2 cells and in the heart. These results can explain marked reduction of LPS-induced TNFα levels by CCRP in cell-based and in vivo studies. Chan et al. previously reported that HSP70 confers cardiac protection, including the maintenance of heart rates, in Sprague–Dawley rats treated with 20 mg/kg LPS intravenously (3). HSP70 inhibits degradation of IκBα that retains NFκB subunits in the cytoplasm, resulting in the repression of NFκB nuclear accumulation as reported for HSP20 (3, 6). In addition to HSP70, Goodwin et al. reported that GR inhibits NFκB phosphorylation and transcriptional activation of its target genes (25). A marked reduction of heart rate in endothelial GR KO mice, but not in control mice, after an intravenous injection with LPS at a concentration of 5 mg/kg in mice was also observed in their study (25). Thus, HSP70 and GR seem to repress NFκB signaling and protect cardiac functions against LPS-induced toxicity in vivo. Because CCRP was previously shown to interact with both HSP70 and GR physically and functionally (9, 11, 15), it is not particularly surprising that these molecules may cooperate to inhibit NFκB signaling.
As shown by greater induced levels of serum TNFα in LPS-treated CCRP KO mice, CCRP would attenuate LPS-induced systemic inflammation as well as the activation of NFκB signaling in the heart. We observed a great gap in timing when serum TNFα was highest and when LPS-treated mice showed decreased heart rate. A possible explanation of this discrepancy is that cytokine induction including TNFα at early time points mediates further downstream signal, such as an increase in nitric oxide level, which has been suggested to be corresponding for LPS-induced heart rate decrease through hemodynamic changes (3, 26). Because the production of nitric oxide is regulated by the protein level of nitric oxide synthase (NOS), it takes several h to increase plasma nitric oxide after LPS treatment in vivo(27). In addition to indirect effects through systemic nitric oxide, the local levels of cytokines and nitric oxide in the cardiomyocytes also seem to be involved in the heart rate decrease. For example, cardiomyocyte-specific overexpression of TNFα or NOS2 results in decreased heart rate in vivo(24, 28). From a previous report representing that cardiomyocyte-specific NOS3-overexpressing mice showed hyperreactivity to the vagus nerve stimulation by carbachol that negatively regulates heart rate, increased nitric oxide in the cardiomyocytes may reinforce slowing of heart rate through the nerve system (29). Together with our current findings with H9c2 cells, these reports emphasize the importance of CCRP expressing in cardiomyocytes for the prevention of NFκB signaling to protect cardiac functions. For further understanding of cardioprotection by CCRP, tissue- and cell-specific CCRP functions must be investigated in the future as well as corresponding NFκB downstream targets for heart rate dysregulation in sepsis. To explore corresponding downstream factor(s) for LPS-induced heart rate decreases in KO mice, treatment with cytokines and NOS inhibitors will help us in the future.
Although we used CCRP KO mice to investigate its function, CCRP is expressed and probably confers cardioprotection against LPS in normal animals and human. Even in the presence of CCRP, however, high concentration of LPS could decrease heart rate (3). When heart rate decrease is accompanied with the impairment of ventricular contractile function, cardiac output would be reduced. The reduced tissue perfusion as a result in such a condition increases the severity of organ dysfunction and the mortality in sepsis. Previous studies suggested a possibility that induction of HSPs by heat shock contributes to the prevention of organ dysfunction and the reduction of the mortality in LPS-treated rats (3). However, from a view of clinical reality, heat shock treatment of patients with severe sepsis would not be appropriate. Recently, geranylgeranylacetone, an antiulcer drug, has been focused on due to its ability to induce HSPs via the activation of heat shock factor (HSF), an HSP-regulating transcriptional factor (30). Interestingly, pretreatment with 200 mg/kg BW geranylgeranylacetone was reported to induce HSP70 in multiple tissues and improve the survival rate after LPS administration at a concentration of 20 mg/kg BW in rats (31). Like other heat shock proteins, such as HSP70, CCRP is expected to be transcriptionally regulated by HSF because its protein level is elevated by heat shock (9). Thus, geranylgeranylacetone is expected to induce CCRP as well as other HSPs. Although further investigations are needed, the induction of multiple HSPs by drugs, such as geranylgeranylacetone, could be one of therapeutic methods in patients suffering from sepsis.
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