Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection (1). Approximately 60% of septic patients suffer from cardiac dysfunction, with a significantly higher mortality rate than those without myocardial dysfunction (2). Although many therapeutic measures have been used in septic patients, including fluid resuscitation, antimicrobial therapy, and vasoactive medications, there is no specific and effective treatment for the sepsis-induced cardiac dysfunction (2).
The pathophysiology of sepsis-induced cardiac dysfunction is complex and involves a multitude of factors (3), such as lipopolysaccharide (LPS), autonomic deregulation, mitochondrial dysfunction, pro-inflammatory cytokines, oxidative stress, impaired calcium handling, and myocardial apoptosis. Wencker et al. (4) demonstrated that very low rate of myocyte apoptosis could cause a lethal heart failure and inhibition of cardiomyocyte death largely prevented the development of cardiac dysfunction in a transgenic mice that express a conditionally active caspase exclusively in the myocardium. Although Takasu et al. (5) observed that cardiomyocyte apoptosis was rare in septic patients, other researchers reported that myocardial apoptosis and caspase-3 activation were remarkably increased in cecal ligation and puncture (CLP)-treated mice (6, 7) and caspase-3 knock-down attenuated contractile dysfunction in sepsis-induced adult rat ventricular myocytes (ARVMs) (8). Therefore, inhibition of cardiomyocyte apoptosis may be a feasible therapeutic strategy for the treatment of sepsis-induced cardiac dysfunction.
The alpha-1 adrenergic receptor (α1-AR) is a G protein coupled receptor activated by endogenous catecholamines. In the past few decades, α1-AR has been thought to be located on the surface of cell membrane. Recently, α1-AR was found to be present in the nucleus of cardiomyocytes (9) and its activation contributed to crucial adaptive processes in the heart, including hypertrophy under stress, inhibiting cardiomyocyte death, and increasing cardiac contractility, as it was reviewed (10). Many studies have shown that α1-AR-mediated cell survival depends on the activation of extracellular signal-regulated kinase1/2 (ERK1/2) signaling. In the neonatal rat cardiomyocytes, α1-AR activation by phenylephrine (PE) induced ERK1/2 phosphorylation and inhibited cardiomyocyte apoptosis induced by deoxyglucose (11). In addition, A61603, an α1A-AR subtype specific agonist, could reduce myocardial apoptosis induced by doxorubicin in mice via increasing ERK1/2 phosphorylation (12). On the other hand, our previous study demonstrated that α1-AR activation by PE inhibited tumor necrosis factor (TNF-α) expression and improved cardiac function in LPS-challenged mice (13). However, it is unclear whether PE suppresses sepsis-induced myocardial apoptosis. Therefore, in the present study, we designed the experiment in vivo and in vitro to explore whether the activation of α1-AR by PE suppresses sepsis-induced cardiomyocyte apoptosis. Our data demonstrated that intracellular α1-AR activation by PE inhibited sepsis-induced cardiomyocyte apoptosis and cardiac dysfunction via activating ERK1/2 signal pathway.
MATERIALS AND METHODS
Male Sprague–Dawley rats (8–10 weeks old) were obtained from Guangdong Medical Laboratory Animal Center. Animals were kept at 24 ± 2°C and acclimatized to laboratory conditions for at least 7 days. All procedures were performed in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (National Institutes of Health Publication No. 85-23, revised 1996) and was approved by the Animal Care and Use Committee at Jinan University School of Medicine. It was demonstrated that female rats had improved left ventricle adaptation to the pressure-loading effects of PE and recovered the inotropic cardiac function earlier than males after septic shock induction (14). In order to exclude the effect of gender, we used male rats in this study.
Adult rat ventricular myocyte isolation
ARVMs were obtained by enzymatic dissociation procedure (15). Briefly, the rat was injected intraperitoneally with 1.0 mL heparin (100 IU, Sigma-Aldrich, St. Louis, Mo) once the rat was anesthetized with isoflurane. After 15 min, the chest of the rat was opened and the heart was harvested and hanged on the Langendorff perfusion apparatus (Radnoti, Covina, Calif). First, the heart was perfused with the perfusion buffer (120.4 mM NaCl, 14.7 mM KCl, 0.6 mM Na2HPO4, 0.6 mM KH2PO4, 4.6 mM NaHCO3, 1.2 mM MgSO4 7H2O, 5.5 mM glucose, 10 mM Na-HEPES, 30 mM taurine, and 10 mM BDM) for 5 min at 37°C at 4 mL/min, and then with collagenase II (1.0 mg/mL) for 6 min. At the end of digestion procedure, live rod-shaped myocytes were collected. Cardiomyocytes (5 × 105/mL) were plated on laminin-coated culture dishes, and then incubated in culture medium supplemented with bovine serum albumin (0.2%), creatine (5 mM), taurine (5 mM), L-carnitine (2 mM), HEPES (25 mM), butanedione monoxime (10 mM), and 0.1% penicillin-streptomycin at 37°C under a 5% CO2–95% air atmosphere. All reagents were purchased from Sigma-Aldrich (St. Louis, Mo).
Treatment of adult rat ventricular myocytes
After 24 h culture, ARVMs were treated with vehicle or α1-AR agonist phenylephrine (PE, 0.2, 2, and 20 μM) for 15 min and followed by normal saline or LPS (10 ng/mL) for the indicate time. In the separate experiments, the ARVMs were pre-incubated with 2 μM prazosin (a membrane permeable α1-AR antagonist), 20 μM CGP12177A (a membrane impermeable α1-AR antagonist), or 2 μM U0126 (a highly selective inhibitor of ERK1/2) for 30 min before treatment with PE or/and LPS. All reagents were purchased from Sigma-Aldrich (St. Louis, Mo).
Rat treatment and CLP model
The male Sprague–Dawley rats were divided into four groups: sham, CLP, CLP+PE, and sham+PE groups. Sepsis was induced by CLP described previously (16). Briefly, animals were fasted for 12 h before surgical procedure. A midline laparotomy was performed under sufficient anesthesia by isoflurane inhalation. The cecum was carefully isolated to avoid any damage to blood vessels, and ligated at 1.5 cm from the tip of the cecum. Then, the cecum was punctured five times with an 18-gauge needle in the middle of the ligated cecum. A small amount of fecal materials was extruded after removing the needle, the cecum was returned to the abdomen, and the incision was closed in layers. In the sham group, the same surgery was made without CLP. All animals received 0.9% NaCl solution (10 mL/kg, subcutaneously) for intraoperative fluid loss and buprenorphine for postoperative analgesia. Three hours after sham operation or CLP, normal saline or PE were injected subcutaneously. Survival was monitored every 12 h during the next 7 days.
Heart preparation and perfusion
After anesthesia with isoflurane (Sigma-Aldrich, St. Louis, Mo), the heart of rat was removed from the chest and placed in the ice-cold K-H solution (6.896 g/L NaCl, 0.35 g/L KCl, 0.296 g/L MgSO4, 0.277 g/L CaCl2, 2.1 g/L NaHCO3, 0.16 g/L KH2PO4, 2.18 g/L Glucose) quickly. A dissection of the aorta was made and a perfusion needle was inserted into the aorta and connected to the Langendorff system (Radnoti, Covina, Calif). The heart was perfused at least 30 min at a flow of 10 mL/min, and the maximum rate of left ventricular pressure rise (+dP/dt), the maximum rate of left ventricular pressure decrease (−dP/dt) were recorded.
Mean arterial pressure measurement
After an intraperitoneal injection with 4,000 U heparin, the rat was anesthetized and fixed in a supine position. A minor incision in the middle of the neck was made and the carotid artery was isolated. A 24-gauge catheter (Becton, Dickinson and Company, Franklin Lakes, NJ) was inserted into the carotid artery and connected to the system (BL-420F, Techman, Chengdu, China). Mean arterial pressure (MAP) was then detected
The level of TNF-α was determined using a rat TNF-α enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, Minn) according to the manufacturer's instructions.
Caspase activity assay
The caspase-3, -8, and -9 activities were determined spectrophotometrically using specific assay kits (BioVision, Milpitas, Calif). Briefly, protein (100 μg) was collected and resuspended in 50 μL cell lysis buffer, then incubated on ice for 10 min. The reaction buffer (50 μL) and 1 mM enzyme substrate (5 μL) were added to each sample, and then incubated for 2 h at 37°C. The relative fluorescence unit (RFU) was read in fluorometer equipment (Tecan safire 2, Tecan, Männedorf, Switzerland) with a 400 nm excitation filter and 505 nm emission filter. Data was shown as the ratio normalized to control or sham group.
Cardiomyocytes and heart samples were homogenized in RIPA lysis buffer (Bioteke Co, Beijing, China) containing 100 μM phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich, St. Louis, Mo). The protein in mitochondria, cytoplasm, or nucleus was prepared using a mitochondria isolation or nucleus extraction kit (Thermo scientific, Rockford Ill) and the concentration of samples was determined using the BCA protein assay kit (Pierce, Rockford, Ill). Equal amounts of protein from each sample were loaded and separated by SDS-PAGE (8%–12%). Proteins were transferred to PVDF membrane (Merk Millipore, Darmstadt, Germany). Nonspecific binding was blocked by incubation of membranes with 5% (w/v) nonfat milk (Sigma-Aldrich) for 1 h. Blots were incubated with the designated antibody (p-cTnI/cTnI, p-IκBα/IκBα, TNF-α, NF-κB, p-ERK/ERK, p-JNK/JNK, p-p38MAPK/p38MAPK, Bax, cytochrome c, Bcl-2 antibodies. Cell Signaling, Danvers, Mass) at 1:1,000 dilution overnight at 4°C. The blots were then washed with TBS-0.1% Tween 20 (Sigma-Aldrich) and incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG (Promega, Madison, Wis). After washing, blots were visualized with an enhanced chemiluminescence detection kit (Thermo scientific, Rockford Ill). We stripped the blots with antibody for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Cell Signaling, Danvers, Mass), voltage-dependent anion-selective channel (VDAC, Cell Signaling, Danvers, Mass) and Laminin B1 (Cell Signaling, Danvers, Mass) as housekeeping controls.
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assay
The TUNEL assay was carried out using an In-Situ Cell Death Detection Kit (Roche Applied Science, Indianapolis, Ind) to identify the myocardial apoptosis. Briefly, tissue sections (5 μm) from frozen heart were permeabilized with 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, Mo) for 10 min at room temperature and stained with 1:200 dilution of anti-cardiac troponin I (cTnI; Abcam, Cambridge, UK) at 4°C overnight and then added 0.1 mL TUNEL reaction buffer according to the manufacturer's protocol. Then, the tissue was washed three times and incubated with secondary antibody conjugated with Alexa Fluor dyes (Invitrogen, Life Technologies, Grand Island, NY). Nuclei were visualized with DAPI (4’, 6-diamidino-2-phenylindole) for 15 min. The immunofluorescence was analyzed by confocal microscopy (LSM510META, Carl Zeiss, Jena, Germany).
All data were expressed as the mean ± standard error of the mean (SEM) and analyzed using SPSS13.0 software. One-way analysis of variance (ANOVA) and Bonferroni post hoc test were used for the normally distributed data. Non-parametric Mann–Whitney U tests were used for data that was not normally distributed. Survival was estimated using the Kaplan–Meier method and Log-rank analysis for the comparison among groups. P < 0.05 was considered to be statistically significant.
PE inhibits LPS-induced cardiomyocyte apoptosis via activating intranuclear α1-AR
To assess the effect of cardiomyocyte α1-AR activation on LPS-induced myocardial apoptosis, we examined the caspase-3 activity in isolated ARVMs treated with LPS at various concentrations for 12 or 24 h. LPS (10, 100, or 1,000 ng/mL) induced caspase-3 activation in cardiomyocytes at 24 h, but not 12 h (Fig. 1A). PE (0.2, 2, or 20 μM) treatment significantly inhibited caspase-3 activation 24 h after 10 ng/mL LPS administration (Fig. 1B). PE (20 μM) alone had no significant effect on caspase-3 activation in cardiomyocytes.
Many researches have shown that α1-AR is located in the inner nuclear membrane of cardiomyocytes (9). To identify whether the inhibitory effect of PE on LPS-induced caspase-3 activation was dependent on the intranuclear α1-AR activation, a membrane permeable α1-AR antagonist prazosin (which can block the intranuclear signal) (17) and a membrane impermeable α1-AR antagonist CGP12177A (which cannot block the intranuclear signal) (17) were used, respectively. Figure 1C and D showed that prazosin abolished the inhibitory effect of PE on the caspase-3 activation 24 h after LPS exposure, but not CGP12177A. In addition, prazosin and CGP12177A did not significantly affect LPS-treated cardiomyocyte caspase-3 activation. Prazosin or CGP12177A alone had no significant effect on caspase-3 in cardiomyocytes.
α1-AR activation by PE inhibits death receptor-related apoptotic proteins in LPS-treated cardiomyocytes
Studies showed that death receptor apoptotic pathway induced by TNF-α-caspase-8 played a critical role in LPS-induced cardiomyocyte apoptosis (18). Therefore, we determined cardiomyocyte caspase-8 activity and TNF-α expression after 10 ng/mL LPS exposure. As shown in Figure 2A, at 24 h after LPS administration, caspase-8 activity was increased compared with control cells. PE treatment significantly inhibited caspase-8 activation induced by LPS. The α1-AR antagonist prazosin significantly reversed the inhibitory effect of PE on caspase-8 activation in LPS-treated ARVMs. Similarly, PE markedly decreased TNF-α expression 6 h after LPS exposure and prazosin significantly reversed the inhibitory effect of PE on TNF-α expression induced by LPS (Fig. 2B). Prazosin alone had no significant effect on the caspase-8 activation and TNF-α expression in ARVMs.
α1-AR activation by PE inhibits mitochondria-related apoptotic pathway in LPS-treated cardiomyocytes
It is well known that the mitochondria apoptotic pathway also mediates cardiomyocyte apoptosis (19). We further observed the effects of PE on caspase-9 activation and cytoplasmic Cyt c content 24 h after LPS administration. As shown in Figure 2A and E, caspase-9 activity and cytoplasmic Cyt c content were elevated in LPS-treated ARVMs, and 0.2 μM PE decreased LPS-induced caspase-9 activation and Cyt c release in ARVMs. The α1-AR antagonist prazosin significantly reversed the inhibitory effect of PE on caspase-9 activation and Cyt c release in LPS-treated ARVMs.
Some studies demonstrated that the anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax were involved in maintaining the stability of mitochondria (19). We further observed the effects of PE on the Bcl-2 and Bax protein 24 h after LPS administration. LPS decreased Bcl-2 and increased mitochondrial Bax at 24 h after LPS exposure. Treatment with PE significantly increased Bcl-2 level and decreased mitochondrial Bax protein content in LPS-treated ARVMs. Furthermore, the α1-AR antagonist prazosin significantly reversed the above effects of PE. Prazosin alone had no significant effect on Bcl-2 and Bax levels in ARVMs (Fig. 2, C and D).
α1-AR activation by PE enhances ERK phosphorylation, suppresses the phosphorylation of IκBα, p38MAPK and JNK as well as NF-κB activation in LPS-treated cardiomyocytes
It is well recognized that MyD88-dependent NF-κB activation involves the LPS-induced TNF-α expression in cardiomyocytes. Thus, we investigated the effects of PE on LPS-induced IκBα phosphorylation and NF-κB activation. As shown in Figure 3A, LPS (10 ng/mL) stimulation caused an increase in IκBα phosphorylation, which was prevented by PE pretreatment. Furthermore, LPS significantly increased nuclear NF-κB level and reduced cytosolic NF-κB level in ARVMs compared with controls, which was prevented by PE pretreatment (Fig. 3, B and C). In contrast, prazosin administration abolished the effects of PE on IκBα phosphorylation and NF-κB activation in LPS-challenged ARVMs. However, prazosin did not affect IκBα phosphorylation and NF-κB activation in ARVMs.
It was reported that MAPK pathway played an important role in myocardial apoptosis (20). We further examined the effects of PE on LPS-induced p38MAPK, JNK, and ERK phosphorylation. Results showed that JNK and p38MAPK phosphorylation were significantly increased in LPS-treated ARVMs. Treatment with PE significantly inhibited LPS-induced JNK and p38MAPK phosphorylation. In addition, the α1-AR antagonist prazosin significantly reversed the inhibitory effect of PE on JNK and p38MAPK phosphorylation (Fig. 3, D and E).
As shown in Figure 3F, LPS at dose of 10 ng/mL failed to significantly elevate ERK1/2 phosphorylation compared with control cells, whereas PE markedly increased the phosphorylation of ERK1/2 both in LPS-stimulated and control ARVMs, which was prevented by prazosin. Prazosin alone did not alter ERK1/2 phosphorylation in LPS-stimulated ARVMs.
U0126 reverses the inhibitory effects of PE on caspase-3, -8, and -9 activation as well as IκBα, p38MAPK, and JNK phosphorylation in LPS-treated cardiomyocytes
The previous studies demonstrated that TNF-α expression was decreased by inhibiting p38MAPK phosphorylation via ERK1/2 activation in LPS-treated neonatal rat cardiomyocytes (13). It was also reported that ERK1/2 activation inhibited JNK and p38MAPK phosphorylation in ICE-6 cells (21). To demonstrate the relationship between ERK1/2 activation and the phosphorylation of IκBα, p38MAPK, and JNK, we used U0126 to inhibit ERK1/2 activation. As shown in Figure 4, PE suppressed caspase-3, -8, and -9 activation in LPS-treated ARVMs. PE also inhibited LPS-induced IκBα, p38MAPK, and JNK phosphorylation 30 min after LPS exposure. U0126 pretreatment significantly increased caspase-3, -8, and -9 activities, IκBα, p38MAPK, and JNK phosphorylation in PE plus LPS-stimulated ARVMs. Exposure of control or LPS-treated ARVMs to U0126 had no effect on caspase-3, -8, and -9 activation as well as the phosphorylation of IκBα, p38MAPK, and JNK. These results suggest that activation of ERK1/2 induced by PE inhibits IκBα, p38MAPK, and JNK phosphorylation and in turn suppresses apoptosis in LPS-treated ARVMs.
PE inhibits sepsis-induced cardiac dysfunction and improves survival in septic rats
To determine whether α1-AR activation suppresses myocardial apoptosis via modulating ERK1/2–IκBα/p38MAPK/JNK signaling pathway in vivo, we first observed different doses of PE on the survival rate of septic rats. As depicted in Figure 5A, subcutaneous injection of 5 mg/kg PE 3 h after CLP significantly improved the survival rate, while 10 mg/kg or 20 mg/kg PE had no obvious impact on the survival in CLP rats (P > 0.05). Thus, we used 5 mg/kg PE in the following experiments in vivo. We observed the effect of PE on the sepsis-induced cardiac function and the mean artery pressure (MAP). As it was shown in Figure 5B–D, the cardiac systolic (+dP/dt) and diastolic function (−dP/dt) were obviously inhibited 20 h after CLP operation. The cTnISer23/24 phosphorylation was significantly increased in septic rats compared with sham-operated rats. The MAP was markedly decreased in CLP group than those in sham-operated group. Treatment with PE significantly attenuated the cardiac dysfunction, but had no obvious effect on the MAP in septic rats. Treatment with PE alone did not affect cardiac function in sham-operated rats. There was no significant difference in +dP/dt, -dP/dt, cTnISer23/24 phosphorylation and MAP (all P > 0.05) between the PE and sham-operated groups.
PE modulates ERK1/2–IκBα/p38MAPK/JNK signaling pathway and attenuates cardiomyocyte apoptosis in septic rats
As shown in Figure 6A and B, there was a significant increase in cardiomyocyte apoptosis compared with sham group. After treatment with PE, cardiomyocyte apoptosis was decreased compared with CLP group. Meanwhile, caspase-3, -8, and -9 activities as well as mitochondrial Bax, cytosolic TNF-α, and Cyt c levels were markedly increased in septic rats compared with control rats (P < 0.05) (Figs. 6C and 7); Bcl-2 level was significantly decreased in CLP group compared with sham group. Treatment with PE significantly increased Bcl-2 level and decreased mitochondrial Bax, cytosolic TNF-α, and Cyt c content in CLP-challenged rats. The phosphorylation of IκBα, p38MAPK, and JNK in the myocardium was also obviously increased in CLP group compared with sham group. Treatment with PE enhanced myocardial ERK1/2 phosphorylation inhibited the phosphorylation of IκBα, p38MAPK, and JNK in septic rats. In addition, PE alone remarkably increased ERK1/2 phosphorylation in the myocardium of sham-operated rats (Fig. 7).
In recent years, we have investigated the role of different adrenergic receptors in sepsis-induced cardiac dysfunction. We found that the α2-AR blocker yohimbine could prevent LPS-induced cardiac dysfunction in mice (22) via blockade of presynaptic α2A-AR that promotes cardiac norepinephrine release, and yohimbine-elevated cardiac norepinephrine improved cardiac dysfunction, at least in part, via inhibiting cardiomyocyte apoptosis through α1-AR in endotoxemic mice (23). We also demonstrated that α1-AR activation by PE inhibited TNF-α release and improved cardiac function in endotoxemic mice (13). However, the effect of α1-AR activation by PE on sepsis-induced cardiomyocyte apoptosis and the associated mechanisms remains to be elucidated. In the present study, we further investigated the effect of α1-AR agonist, PE, on sepsis-induced cardiomyocyte apoptosis in LPS-treated ARVMs and a rat model of sepsis induced by CLP and underlying mechanisms.
In order to determine the effect of α1-AR activation on cardiomyocyte apoptosis during sepsis, we first isolated ARVMs and investigated the effects of PE, a specific α1-AR agonist, on LPS-induced cardiomyocyte apoptosis. The results showed that LPS increased cardiomyocyte apoptosis, and PE (0.2–20 μM) markedly decreased LPS-induced cardiomyocyte apoptosis. Particularly, the direct effect of low dose of LPS on cardiomyocytes is clinically relevant because nanogram per milliliter of level of LPS was detected in plasma of patients with septic shock (24). Therefore, we chose 10 ng/mL LPS and 0.2 μM PE to treat ARVMs and explored the mechanisms responsible for inhibiting LPS-induced cardiomyocyte apoptosis by α1-AR activation.
It was reported that α1-AR was located in the nucleus of cardiomyocytes and mediated a cardiac protection effect. In order to confirm the signal transduction stimulated by α1-AR activation, a membrane permeable α1-AR inhibitor, prazosin, was used. We found that prazosin successfully reversed the inhibitory effect of PE on cardiomyocyte apoptosis induced by LPS. In contrast, CGP12177A, a membrane impermeable α1-AR inhibitor, did not show this effect. The above results demonstrated that PE suppressed LPS-induced cardiomyocyte apoptosis may via activating intranuclear α1-AR, but this needs to be further determined.
Several studies have demonstrated that death receptor-mediated apoptotic pathway plays an important part in LPS-induced cardiomyocyte apoptosis (18). Therefore, we examined the effects of PE or/and LPS treatment on caspase-8 activation and TNF-α expression in ARVMs. We found that TNF-α expression and caspase-8 activity were significantly increased by 10 ng/mL LPS. Exposure of ARVMs to 0.2 μM PE inhibited LPS-induced caspase-8 activation and TNF-α expression, all of which were abolished by prazosin treatment. These findings suggest that α1-AR activation inhibits LPS-induced cardiomyocyte apoptosis via suppressing the death-receptor pathway. It is well documented that NF-κB and MAPK pathway mediate LPS-induced TNF-α expression in cardiomyocytes (25). Inhibition p38MAPK-dependent TNF-α expression protected against sepsis-induced myocardial apoptosis in CLP-challenged rats (26). Meanwhile, cardiomyocyte apoptosis could be alleviated by inhibiting JNK1/2-mediated IκBα-NF-κB pathway (27). Thus, we examined NF-κB activation as well as IκBα, p38MAPK, and JNK phosphorylation, and found that LPS induced IκBα, p38MAPK, and JNK phosphorylation in ARVMs, all of which were inhibited by PE treatment. These data suggest the possibility that α1-AR activation inhibits LPS-induced cardiomyocyte apoptosis via blocking death receptor-mediated apoptotic pathway by suppressing IκBα, p38MAPK, and JNK phosphorylation. In addition, p38MAPK and JNK also played a pivotal role in mitochondria-mediated apoptotic pathway. Previous studies have demonstrated that the phosphorylation of JNK promotes Cyt c release and decreased Bcl-2 level in LPS-induced cardiomyocyte apoptosis (19), and p38MAPK has been found to mediate ischemia/reperfusion (I/R)-induced mitochondria apoptotic pathway (28). Meanwhile, SB203580 (a p38MAPK inhibitor) significantly inhibited Cyt c release and caspase-3 activation in cardiac I/R injury (28). Thus, it seems reasonable to speculate that α1-AR activation inhibits LPS-induced cardiomyocyte apoptosis via decreasing p38MAPK and JNK phosphorylation, in turn inhibiting Bax translocation to mitochondria, Cyt c release, and caspase-9 activation. To test this hypothesis, the present study further investigated the effect of PE on Cyt c release, caspase-9 activation, Bcl-2, and mitochondria Bax levels in LPS-treated ARVMs. The data showed that pretreatment with PE blocked LPS-induced increase in caspase-9 activation as well as Cyt c release, and also reversed LPS-provoked decrease in Bcl-2 protein level in LPS-treated ARVMs. Taken together, these results suggest that α1-AR activation by PE inhibits LPS-stimulated cardiomyocyte apoptosis also via decreasing p38MAPK and JNK phosphorylation, increasing Bcl-2 levels, suppressing Bax translocation to mitochondria, and in turn Cyt c release and caspase-9 activation.
The signaling mechanisms involved in α1-AR protection effects include ERK1/2 activation. Studies have shown that α1-AR activation inhibited acute doxorubicin -induced cardiomyopathy via increasing ERK1/2 phosphorylation (12). Especially, the activation of ERK reduced cardiomyocyte Bax content, improved Bcl-2 level in an I/R model (29). We showed here that PE dramatically increased ERK1/2 phosphorylation both in LPS-treated ARVMs and control cells, which were prevented by prazosin, suggesting that PE enhances ERK1/2 phosphorylation via activating α1-AR in LPS-treated ARVMs.
Recently, some studies showed that cardiomyocyte apoptosis was inhibited via increasing ERK1/2 phosphorylation (11, 12), and the apoptosis was also inhibited by decreasing JNK and p38MAPK phosphorylation via ERK1/2 activation in IEC-6 cells (21). Our previous study also found that the myocyte TNF-α expression was decreased by inhibiting p38MAPK phosphorylation via ERK1/2 activation (13). Accordingly, we hypothesized that PE might increase ERK1/2 phosphorylation, which inhibited p38MAPK and JNK phosphorylation and finally inhibited LPS-treated cardiomyocytes apoptosis. To test this hypothesis, we further examined the effect of ERK1/2 inhibitor U0126 on IκBα, p38MAPK, and JNK phosphorylation as well as caspase-3, -8, and -9 activation in LPS-treated ARVMs. We found that PE decreased IκBα, p38MAPK, and JNK phosphorylation in LPS-treated ARVMs, which were reversed by U0126 pretreatment. Moreover, U0126 reversed the inhibitory effects of PE on LPS-induced caspase-3, -8, and -9 activation. Taken together, our data suggest that PE stimulates ERK1/2 phosphorylation, leading to decreased IκBα, p38MAPK, and JNK phosphorylation and cardiomyocyte apoptosis via activating α1-AR in LPS-treated cardiomyocytes.
In order to further clarify the above mechanisms, we observed the effects of PE on cardiac function, myocardial caspase-3, -8, and -9 activation, TNF-α, Bax, Bcl-2, Cyt c content and the phosphorylation of IκBα, p38MAPK, JNK and ERK1/2 in septic rats induced by CLP. The results demonstrated that 5 mg/kg PE not only enhanced ERK1/2 phosphorylation, but also decreased TNF-α, mitochondrial Bax and cytosol Cyt c content, increased Bcl-2 level as well as inhibited myocardial IκBα, p38MAPK and JNK phosphorylation, and reduced caspase-3, -8 and -9 activation and apoptosis in the myocardium of septic rats. PE also enhanced the cardiac systolic and diastolic function and improved the survival in septic rats at a low dose (5 mg/kg) that did not change blood pressure. This was consistent with the previous study, in which low dose of A61603 (α1A-AR subtype specific agonist) prevented acute doxorubicin cardiomyopathy in male mice without affecting blood pressure (12). However, PE at a dose of 10 mg/kg or 20 mg/kg did not significantly improve the survival rate of sepsis rats, and 20 mg/kg PE even may be led to early deaths in septic rats. The reason is, maybe, due to tissue ischemic injury induced by the vasoconstriction effect of α1-AR activation by high dose of PE, this needs to be further investigated.
On the other hand, human α1-AR only account for 11% of cardiac adrenergic receptors in healthy heart, with β-AR at 90%. In heart failure, β1-AR was downregulated, while cardiac α1-AR was upregulated from 11% of total cardiac adrenoceptors to 25% (10, 30). This suggests that α1-AR plays a major role in heart failure. As shown in the ALLHAT (31) and V-HeFT (32) trials, blocking α1-AR signaling with nonselective α1-blockers can cause heart failure and death. Altogether, these studies provide indirect evidence that α1-AR agonists can be used to treat heart failure. In our study, although high dose of PE maybe increased the early mortality of septic rats, we demonstrated that low dose of PE activated myocardial α1-AR, prevented cardiomyocyte apoptosis and cardiac dysfunction and reduced mortality rate in septic rats. Therefore, targeted activation of myocardial α1-AR may have potential clinical value for septic dysfunction.
In conclusion, our results demonstrated that α1-AR activation by PE inhibited cardiomyocyte apoptosis during sepsis. Cardiomyocyte α1-AR activation by PE promoted myocardial ERK1/2 activation, which in turn inhibited IκBα, p38MAPK and JNK phosphorylation to reduce cardiomyocyte apoptosis via inhibiting the death receptor and mitochondria-mediated apoptotic pathways, and then suppressed cardiac dysfunction and improved the survival in septic rats. These findings highlight the importance of myocardial α1-AR activation in the treatment of myocardial dysfunction during sepsis (Fig. 8).
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Keywords:© 2019 by the Shock Society
1 adrenergic receptor">α1 adrenergic receptor; apoptosis; cardiomyocyte; phenylephrine; sepsis