β1-adrenoceptor stimulation promotes LPS-induced cardiomyocyte apoptosis through activating PKA and enhancing CaMKII and IκBα phosphorylation
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Caspase activation and cardiomyocyte apoptosis have been implicated in lipopolysaccharide (LPS)-induced cardiac contractile dysfunction. We have recently demonstrated that β1-adrenoceptor (AR) activation by endogenous norepinephrine contributes to cardiomyocyte apoptosis in endotoxemic mice. Here, we further investigated the molecular mechanisms for the enhancing effect of β1-AR activation on LPS-induced cardiomyocyte apoptosis.
The adult mouse ventricular myocytes were exposed to LPS, dobutamine, protein kinase A (PKA) inhibitor or/and nifedipine, an L-type Ca2+ channel blocker. Male BALB/c mice were treated with LPS or/ and β1-AR antagonist, atenolol. Cardiomyocyte apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assay and apoptosis-associated molecules were detected.
LPS induced apoptosis in adult mouse ventricular myocytes, dobutamine (DOB), a β1-AR agonist, promoted apoptosis, caspase-8, 9 and 3 activation and increased cytosolic Ca2+ concentration in LPS-challenged cardiomyocytes. DOB also up-regulated TNF-α expression, decreased Bcl-2 levels, promoted Bax translocation to mitochondria, mitochondrial membrane potential loss and cytochrome c release as well as IκBα, p38 MAPK, JNK and Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation in LPS-treated cardiomyocytes. PKA inhibitor abolished the effects of DOB on caspase-9 activation, Bcl-2 levels as well as JNK and p38 MAPK phosphorylation, but not on IκBα phosphorylation, TNF-α expression and caspase-8 activation in LPS-stimulated cardiomyocytes. Pretreatment with nifedipine not only significantly blocked the enhancing effects of DOB on LPS-induced elevation in cytosolic Ca2+ concentration and CaMKII phosphorylation in cardiomyocytes, but also partly reversed the effects of DOB on caspase-9 and caspase-3/7 activities in LPS-treated cardiomyocytes. Furthermore, atenolol suppressed TNF-α expression, JNK, p38 MAPK and CaMKII phosphorylation, increased Bcl-2 expression, and inhibited cytochrome c release and cardiomyocyte apoptosis in the myocardium of endotoxemic mice.
β1-AR activation promotes LPS-induced apoptosis through activating PKA, increasing CaMKII phosphorylation as well as enhancing IκBα phosphorylation and TNF-α expression in cardiomyocytes.
KeywordsCardiomyocyte Apoptosis CaMKII Phosphorylation Adult Mouse Cardiomyocytes Adult Mouse Ventricular Myocytes
calmodulin-dependent protein kinase II
Dulbecco’s modified Eagle’s medium
extracellular signal-regulated kinase
c-jun NH2-terminal kinase
mitogen-activated protein kinase
protein kinase A
tumor necrosis factor-α
terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling
voltage-dependent anion channel
Myocardial dysfunction is a common complication that greatly contributes to the mortality of sepsis in both patients and experimental models of lipopolysaccharide (LPS)-induced endotoxemia [1,2]. Accumulating evidence has indicated that cardiomyocyte caspase activation plays an important role in myocardial dysfunction in patients and various animal models of sepsis, such as endotoxemia [3-6]. LPS can activate caspases in cardiomyocytes in vivo and in vitro [5,7,8], and caspase-3 activation not only induces end-stage nuclear apoptosis, cleavage of cardiac myofilament proteins and sarcomere disorganization , but also blunts contractile response of ventricular myocytes to norepinephrine . On the contrary, caspase-3 inhibition ameliorates LPS-induced myocardial contractile dysfunction [10,11]. Clearly, it is very important to identify and block the initiating apoptotic stimuli or inhibit apoptotic pathways in cardiomyocytes for treating sepsis-induced myocardial dysfunction.
To date LPS-induced cardiomyocyte apoptosis has been attributed to increased production of TNF-α, macrophage migration inhibitory factor and reactive oxygen species [7,12-15]. Although it is well-documented that plasma norepinephrine levels are significantly elevated in the setting of sepsis  and norepinephrine alone can directly stimulate cardiomyocyte apoptosis by activating the β-adrenoceptor (AR) [17,18], little is known about the causative contribution of endogenous norepinephrine in sepsis-induced cardiomyocyte apoptosis. Recently, our preliminary results demonstrated that both exhaustion of cardiac endogenous norepinephrine and blockade of β1-AR significantly suppressed myocardial caspase-3 activation in LPS-challenged mice , suggesting that β1-AR activation by endogenous norepinephrine may be a significant contributor to cardiomyocyte apoptosis during endotoxemia. However, the molecular mechanisms for this enhancing effect of β1-AR activation on cardiomyocyte apoptosis during endotoxemia have not been identified. It is well-known that β1-AR activation induces pro-apoptotic signals through protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation in cardiomyocytes [20,21]. Therefore, we hypothesize that PKA and CaMKII activation mediate the enhancing effect of β1-AR activation on LPS-induced cardiomyocyte apoptosis. Here, we used adult mouse ventricular myocytes and a mouse model of endotoxemia to test this hypothesis. The data demonstrate that cardiomyocyte β1-AR activation is a significant contributor to cardiomyocyte apoptosis during endotoxemia. Stimulation of β1-AR activates PKA, increases CaMKII and IκBα phosphorylation as well as TNF-α expression, in turn enhances caspase-9 and 8 activities, and promotes LPS-induced cardiomyocyte apoptosis.
Materials and methods
Male BALB/c mice were obtained from the medical laboratory animal center of Guangdong province and acclimatized to laboratory conditions for at least 6 days following their arrival. All experiments with animals were conducted in compliance with the guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health, and approved by the Animal Care and Use Committee at Jinan University School of Medicine. Under necessary conditions, the mice were anesthetized with pentobarbital (100 mg/kg), the adequacy of the anesthesia was monitored, and every effort was made to minimize suffering.
Male BALB/c mice were randomized into different groups and received intraperitoneal injection with vehicle or atenolol (Sigma-Aldrich, St Louis, MO, USA) at a dose of 10 mg/kg. LPS, 20 mg/kg; Escherichia coli, 055:B5 (Sigma-Aldrich) or normal saline was administered intraperitoneally 1 hour after treatment with atenolol. At 2, 6 or 12 hours after LPS injection, the serum and hearts of mice were collected for further analysis.
Adult mouse ventricular myocyte isolation and culture
Adult mouse ventricular myocytes were obtained from hearts of male BALB/c mice using Langendorff perfusion apparatus as described previously . Cardiomyocytes (30 to 50 cells/mm2) were plated on fibronectin-coated polystyrene tissue culture dishes. Plated cells were incubated in serum-free culture medium (DMEM) supplemented with HEPES (25 mM), bovine serum albumin (BSA, 0.2%), creatine (5 mM), L-carnitine (2 mM), taurine (5 mM) and 0.1% penicillin-streptomycin at 37°C under a 5% CO2-95% air atmosphere. Two hours after plating, the culture medium was changed to remove unattached dead cells and the viable cardiomyocytes were incubated overnight under the same culture conditions.
Adult mouse ventricular myocytes, cultured for 24 hours, were treated with β1-AR agonist, dobutamine (DOB, 0.02, 0.2, 2 and 20 μM; Sigma-Aldrich) and/or LPS (1, 10, 100 and 1,000 ng/mL; Sigma-Aldrich). To inhibit PKA or L-type Ca2+ channel, KT5720 (5 μM; Sigma-Aldrich) or nifedipine (NIF, 1 μM; Sigma-Aldrich) was added for 1 hour prior to DOB and/or LPS treatment.
Determination of caspase-3/7 activity
The caspase-3/7 activity in cardiomyocytes or myocardium was detected by using the Apo-ONE® homogeneous caspase-3/7 assay kit (Promega Corp., Madison, WI., USA) according to the manufacturer’s instructions.
Determination of mitochondrial membrane potential
Mitochondrial membrane potential was visualized in mouse ventricular myocytes stained with 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) by using a laser scanning confocal microscope, as described previously . JC-1 can be accumulated and aggregates (red fluorescence) in normal mitochondria. The loss of mitochondrial membrane potential prevents JC-1 entry into mitochondria, monomeric JC-1 (green fluorescence) remains in the cytosol. The mouse ventricular myocytes from different treatment groups were washed and incubated with JC-1 at 37°C for 15 minutes, then washed and mounted on the confocal microscopy for imaging. The ratio of monomeric to aggregated JC-1 fluorescence intensity was used to quantify changes in mitochondrial membrane potential, and the increased ratio represents the decrease in mitochondrial membrane potential.
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assay
Cardiomyocyte apoptosis was determined by TUNEL assay using an in situ cell death detection kit (Roche Applied Science, Indianapolis, IN, USA). Briefly, cardiomyocytes were fixed with 4% paraformaldehyde in PBS for 40 minutes at room temperature, washed and permeabilized with 0.1% Triton X-100 for another 5 minutes at 4°C. The fixed cells were incubated with 1:50 dilution of anti-cardiac troponin I (cTnI; Abcam, Cambridge, UK) at 37°C for 1 hour, and subjected to the TUNEL assay according to the manufacturer’s instructions. Then, the cells were washed three times with PBS, incubated with 1:1,000 dilution of secondary antibody conjugated with Alexa Fluor® dyes (Invitrogen, Life Technologies, Grand Island, NY, USA) for 1 hour in the dark. Finally, the cells were incubated with 1:400 dilution of 4',6-diamidino-2-phenylindole (DAPI) for 15 minutes, washed and mounted on coverslips with antifade mounting medium. For in vivo study, tissue sections (5 μm) from frozen cardiac tissues were fixed with 4% paraformaldehyde for 20 minutes and permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate for 2 minutes at 4°C. Triple staining with anti-cardiac troponin I, TUNEL and DAPI was also performed. Fluorescent images were taken in 10 random high-powered fields using laser confocal microscopy. The numbers of TUNEL-positive cardiomyocytes and of total cardiomyocyte nuclei were counted, and the apoptotic index (AI) of cardiomyocytes was expressed as a percentage of TUNEL-positive cells to total cells.
Caspase-8 and caspase-9 activity assay
After indicated treatments, the cardiomyocytes were subjected to caspase-8 and caspase-9 activity measurement using Caspase-Glo assay kit (Promega). Briefly, the plates containing cells were removed from the incubator. Caspase-Glo reagent (100 μl) was added to each well and gently mixed. The plate was incubated for 2 hours at room temperature. Then, the luminescence of each sample was measured in a plate-reading luminometer.
Measurement of TNF-α and nuclear NF-κB activity
Cardiac and serum TNF-α was detected using a mouse TNF-α enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Inc, Minneapolis, MN, USA) according to the manufacturer’s instructions. Cardiomyocyte nuclear NF-κB activity was measured using a NF-kB (p65) Transcription Factor Assay kit (Cayman Chemical, Ann Arbor, MI, USA) as previously described .
Determination of cytosolic Ca2+ concentration
Adult mouse ventricular myocytes were loaded with Ca2+ sensitive indicator fluo-4 AM (2 μM; Invitrogen) in the dark at 37°C for 60 minutes. After the loading, cells were washed three times with dye-free buffer solution for 30 minutes. Fluo-4-loaded cells were excited with an argon laser beam (at a wavelength of 488 nm), and the emitted fluorescence at 520 nm from defined regions within the cells was monitored over time on a confocal laser scanning microscope. The baseline fluo-4 fluorescence intensity at the beginning of each experiment and fluo-4 fluorescence intensity 3 hours after treatment with DOB (0.02 μM), or/and LPS (10 ng/mL) were measured, changes in cytosolic Ca2+ concentration were expressed as a percent relative to the baseline fluorescence intensity, and the data were normalized to controls.
Western blotting analysis
Cytosolic and mitochondrial proteins were harvested from cardiomyocytes or heart homogenates, and protein concentration detected. The levels of specific proteins were determined by western blotting assay, as previously described . The following primary antibodies were used: anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), anti-voltage-dependent anion channel (VDAC), anti-c-jun NH2-terminal kinase (JNK), anti-extracellular signal-regulated kinase (ERK), anti-p38 MAPK (mitogen-activated protein kinase), anti-IκBα, anti-Ca2+/calmodulin-dependent protein kinase II (CaMKII), anti-p-JNK, anti-p-ERK, anti-p-p38 MAPK, anti-p-IκBα, anti-p-CaMKII, anti-Bcl-2, anti-cytochrome c (Cyt c), anti-Bax and anti-TNF-α antibody (Cell Signaling technology, Beverly, MA, USA). GAPDH or VDAC was used to normalize protein loading.
All data were presented as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) followed by Bonferroni post hoc analysis was performed for comparison among multiple groups. Values of P <0.05 were considered to be statistically significant.
Dobutamine promotes LPS-induced caspase 3/7 activation and nuclear apoptosis in adult mouse cardiomyocytes
Dobutamine enhances activation of caspase-8 and caspase-9 in LPS-treated adult mouse cardiomyocytes
Dobutamine promotes LPS-induced TNF-α release and NF-κB activation, as well as IκBα, JNK and p38 MAPK phosphorylation in adult mouse cardiomyocytes
Dobutamine decreases Bcl-2 levels, increases mitochondrial Bax contents, reduces mitochondria membrane potential and promotes cytochrome c release in LPS-treated adult mouse cardiomyocytes
PKA inhibitor abolishes the effect of DOB on caspase-9 activation and Bcl-2 level as well as JNK and p38 MAPK phosphorylation, but not on IκBα phosphorylation and caspase-8 activation in LPS-stimulated cardiomyocytes
Dobutamine enhances LPS-induced elevation in cytosolic Ca2+ concentration and CaMKII phosphorylation, which were completely blocked by pretreatment with nifedipine
Nifedipine partly abolishes the enhancing effects of dobutamine on caspase-9 and caspase-3/7 activities in LPS-treated cardiomyocytes
As shown in Figure 6C and D, DOB significantly increased caspase-9 and caspase-3/7 activities in LPS-stimulated cardiomyocytes, these effects of DOB were partly reversed with NIF pretreatment. NIF exposure alone failed to affect caspase-9 (data not shown) and caspase-3/7 activities in control and LPS-treated cardiomyocytes.
Blockade of β1-AR decreases myocardial TNF-α expression, JNK, p38 MAPK and CaMKII phosphorylation, restores Bcl-2 level and reduces cytochrome c release and cardiomyocyte apoptosis in LPS-challenged mice
Our previous study demonstrated that systemic administration of 10 mg/kg ATE, a β1-AR antagonist, inhibited myocardial caspase 3/7 activation in LPS-challenged mice . However, indirect effects of peripheral β1-AR blockade on systemic inflammation in endotoxemia cannot be ruled out, as it was reported that peripheral β1-AR blockade attenuated systemic inflammation and improved cardiac dysfunction induced by sepsis . In order to overcome this problem and recognize the role of cardiomyocyte β1-AR activation in cardiomyocyte apoptosis during endotoxemia, we first isolated adult mouse ventricular myocytes and investigated the effects of DOB, a β1-AR agonist, on LPS-induced cardiomyocyte apoptosis. The results showed that LPS increased cardiomyocyte caspase-3/7 activity and apoptosis in a dose-dependent manner. Although 0.02 μM DOB did not significantly affect cardiomyocyte apoptosis, it markedly promoted 10 ng/mL LPS-induced cardiomyocyte apoptosis. Particularly, low nanograms per milliliter of LPS concentrations in plasma occur acutely in patients with septic shock and the direct effects of low doses of LPS on cardiomyocytes are clinically relevant [26,27]. Thus, we used 0.02 μM DOB and 10 ng/mL LPS to treat cardiomyocytes and further explore the mechanisms underlying the enhancing effects of DOB on LPS-induced cardiomyocyte apoptosis.
Several studies have demonstrated that LPS directly induced cardiomyocyte apoptosis via the death-receptor-dependent pathway that activates caspase-8 and mitochondria-dependent apoptotic pathway resulting in caspase-9 activation [28,29]. Therefore, we examined the effects of DOB or/and LPS treatment on the activities of caspase-8 and caspase-9 in adult cardiomyocytes, and found that 10 ng/mL LPS significantly increased caspase-8 activity. However, 10 ng/mL LPS failed to activate caspase-9; this result contrasts sharply with the findings from the previous studies demonstrating that 1 μg/mL LPS caused caspase-9 activation in cardiomyocytes [28,29]. This conflicting result might be associated with the difference in concentrations of LPS used in these studies. Moreover, exposure of cardiomyocytes to 0.02 μM DOB not only promoted caspase-8 activation, but also enhanced caspase-9 activity in LPS-treated cardiomyocytes. These findings suggest that LPS at clinically relevant concentrations induces cardiomyocyte apoptosis mainly via the death-receptor apoptotic pathway, and β1-AR activation promotes LPS-induced cardiomyocyte apoptosis through both the death-receptor and mitochondria-dependent pathways.
Accumulating evidence indicates that TNF-α-mediated caspase-8 activation plays a significant role in LPS-induced cardiomyocyte apoptosis [7,12,30]. The present study demonstrated that LPS increased TNF-α production in adult ventricular myocytes, which was markedly enhanced by DOB treatment. It is well-documented that NF-κB and MAPK activation mediate LPS-induced TNF-α expression in cardiomyocytes [31,32]. Thus, we examined NF-κB activity as well as IκBα, JNK and p38 MAPK phosphorylation, and found that LPS induced NF-κB activation as well as IκBα, JNK and p38 MAPK phosphorylation in cardiomyocytes, all of which were enhanced by DOB treatment.
These data suggest the possibility that β1-AR stimulation promotes LPS-induced TNF-α expression and caspase-8 activation maybe via enhancing IκBα, JNK and p38 MAPK phosphorylation in cardiomyocytes. As β1-AR couples to the stimulatory G protein, resulting in the activation of PKA , and PKA can activate JNK and p38 MAPK in the cardiomyocytes [32,33], we further examined the effects of KT5720, a cell-permeable PKA inhibitor, on cardiomyocytes challenged with LPS and DOB. The results showed that pretreatment with KT5720 abrogated the enhancement effects of DOB on p38 MAPK and JNK phosphorylation, but not on IκBα phosphorylation and caspase-8 activation in LPS-challenged cardiomyocytes. Evidently, β1-AR stimulation enhances LPS-induced TNF-α expression and caspase-8 activation mainly through promoting IκBα phosphorylation, rather than JNK and p38 MAPK phosphorylation in cardiomyocytes under our experimental conditions; this process is independent of PKA activation.
As mentioned above, β1-AR stimulation also strengthens LPS-induced cardiomyocyte apoptosis through activating the mitochondria-dependent apoptotic pathway. We observed that 10 ng/ml LPS treatment alone did not significantly affect cytosolic Cyt c, Bcl-2, mitochondrial Bax protein levels and mitochondrial membrane potential in cardiomyocytes. In contrast, co-stimulation with DOB resulted in a marked decrease in Bcl-2 protein level and mitochondrial membrane potential simultaneously with an increase in cytosolic Cyt c and mitochondrial Bax levels in LPS-treated cardiomyocytes. Previous studies have demonstrated that JNK-dependent activation of the mitochondrial death pathway is involved in β1-AR-stimulated cardiomyocyte apoptosis [33,34] and p38 MAPK has been found to mediate oxidative stress-triggered cardiomyocyte apoptosis . Particularly, activation of p38 MAPK by oxidative stress results in the phosphorylation and degradation of Bcl-2 as well as the inactivation of its anti-apoptotic activity in cardiomyocytes , and p38 MAPK activation can also cause Bax translocation to mitochondria in cardiomyocytes . Thus, it seems reasonable to speculate that β1-AR stimulation strengthens LPS-induced cardiomyocyte apoptosis maybe via increasing JNK and p38 MAPK phosphorylation, subsequently reducing Bcl-2 level, and in turn inducing Bax translocation to mitochondria, Cyt c release and caspase-9 activation. To test this hypothesis, the present study further investigated whether inhibition of PKA abolished the effects of β1-AR stimulation on caspase-9 activation, JNK and p38 MAPK phosphorylation as well as Bcl-2 protein level in LPS-treated cardiomyocytes. The data showed that pretreatment with PKA inhibitor not only blocked DOB-induced increase in caspase-9 activity as well as JNK and p38 MAPK phosphorylation, but also reversed DOB-provoked decrease in Bcl-2 protein level in LPS-treated cardiomyocytes. Taken together, these results suggest that PKA activation by β1-AR stimulation enhances JNK and p38MAPK phosphorylation, reduces Bcl-2 levels, increases Bax translocation to mitochondria, and in turn activates caspase-9 and promotes LPS-stimulated cardiomyocyte apoptosis.
On the other hand, it was reported that sustained (24-hour) β1-AR stimulation activates the PKA-independent Ca2+/ CaMKII signaling pathway and subsequently induces cardiomyocyte apoptosis [24,33,38]. β1-AR stimulation increases cytosolic Ca2+ concentration and activates CaMKII in cardiomyocytes, blockade of L-type calcium channel with NIF or inhibition of CaMKII activity fully attenuates cardiomyocyte apoptosis induced by β1-AR stimulation, and overexpression of active CaMKII leads to cardiomyocyte apoptosis through increased cytochrome c release [24,33,39]. Accordingly, we observed the effects of NIF on cytosolic Ca2+ concentration, CaMKII, caspase-3/7 and caspase-9 activation in DOB- and LPS-treated cardiomyocytes, and found that LPS not only increased cytosolic Ca2+ concentration, but also elevated CaMKII phosphorylation in cardiomyocytes, both of which were enhanced by DOB treatment. Furthermore, NIF pretreatment completely eliminated the effects of DOB on cytosolic Ca2+ concentration and CaMKII phosphorylation, and partly reversed the influence of DOB on caspase-9 and caspase-3/7 activities in LPS-treated cardiomyocytes. These data revealed that β1-AR activation enhanced LPS-induced cardiomyocyte apoptosis, at least in part, via increasing cytosolic calcium concentration and activating CaMKII. However, Zhang, et al. recently demonstrated that PKA inhibition alleviated cardiomyocyte apoptosis induced by β1-AR stimulation, by preventing cytosolic and sarcoplasmic reticulum Ca2+ overload and CaMKII activation . Thus, it is worthy to note here that PKA-dependent activation of CaMKII may mediate the promoting effect of β1-AR stimulation on LPS-induced cardiomyocyte apoptosis. Future studies need to clarify this issue.
In order to confirm the current in vitro observations, we further examined the effect of ATE, a β1-AR antagonist, on myocardial apoptosis-associated molecules in LPS-challenged mice. Consistently, LPS challenge markedly upregulated myocardial TNF-α protein expression, increased myocardial JNK, p38 MAPK and CaMKII phosphorylation, reduced Bcl-2 protein level, and led to an increase in cytochrome c release and cardiomyocyte apoptosis in mice, all of which were reversed by pretreatment with ATE at a single dose of 10 mg/kg. In addition, ATE did not significantly affect serum TNF-α level in LPS-challenged mice. These findings confirm that β1-AR activation augments cardiomyocyte apoptosis during endotoxemia by upregulating TNF-α expression, enhancing JNK, p38 MAPK and CaMKII activation, reducing Bcl-2 protein level and increasing cytochrome c release in the myocardium.
Cardiomyocyte β1-AR activation strengthens LPS-induced cardiomyocyte apoptosis through activating PKA and increasing cytosolic Ca2+ concentration and CaMKII activation as well as TNF-α expression in cardiomyocytes
Cardiomyocyte β1-AR stimulation enhances LPS-induced cardiomyocyte IκBα phosphorylation and caspase-8 activation in a PKA-independent manner
PKA activation by β1-AR stimulation augments JNK and p38 MAPK phosphorylation, reduces Bcl-2 level, and subsequently enhances Bax translocation to mitochondria, cytochrome c release and caspase-9 activation in LPS-challenged cardiomyocytes
Cardiomyocyte β1-AR activation plays an important role in cardiomyocyte apoptosis during endotoxemia
This study was supported by grants from the National Natural Science Foundation of China (81372028, 81170222), the Guangdong Natural Science Foundation (S2011020005408) and Project of Department of Education of Guangdong Province (number 2013KJCX0019). The funders had no role in study conception and design, data collection and analysis, preparation of the manuscript or decision to publish.
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