Effects of carvedilol treatment on cardiac cAMP response element binding protein expression and phosphorylation in acute coxsackievirus B3-induced myocarditis
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The role of β-adrenergic stimulation on viral myocarditis has been investigated in animal models of viral myocarditis. Excess stimulation of β-adrenergic receptors by catecholamines causes phosphorylation/activation of cAMP response element binding protein (CREB) by the cAMP signaling pathway. CREB as an important regulator of gene expression mediates the cardiovascular remodeling process and promotes anti-inflammatory immune responses. However, the CREB expression and phosphorylation have not been studied, and the effects of carvedilol (a nonselective β-adrenoceptor antagonist) on the CREB has not been investigated in the setting of acute viral myocarditis.
This study was therefore designed to examine the effects of carvedilol on the transcriptional factor CREB in a murine model of acute viral myocarditis. In a coxsackievirus B3 murine myocarditis model (Balb/c), effects of carvedilol on plasma noradrenaline, heart rate and blood pressure, myocardial histopathological changes and fibrosis, cardiomyocyte apoptosis, cardiac CREB and phosphorylated CREB, cytokine levels, and viral RNA were studied.
The expression and phosphorylation of CREB were decreased with concomitant increase of IL-6 and TNF-α in murine coxsackievirus-induced acute viral myocarditis. The levels of IL-6 and TNF-α were correlated with the expression of CREB or phosphorylated CREB. Carvedilol increased the cardiac CREB expression and phosphorylation and decreased the plasma catecholamine levels and the production of IL-6 and TNF-α with amelioration of acute viral myocarditis.
These results show that CREB may be involved in the pathophysiology of viral myocarditis and carvedilol exerts some of its beneficial effects by increasing the CREB expression and phosphorylation.
KeywordsViral myocarditis cAMP response element binding protein Carvedilol
Viral myocarditis is a frequent cause of cardiac failure, especially in young adults . Acute inflammation of the heart appears to be a major cause of sudden, unexpected death in persons younger than 40 years old  and often follows viral infection . Enteroviruses, particularly the coxsackievirus B3 (CVB3), have been identified as the predominant cause of viral myocarditis in both animal models and humans . Despite the well-characterized molecular structure of coxsackieviruses  and the successful use of common  or novel immunization procedures [7, 8, 9] in animal models, immune suppression trials  to reverse underlying active myocardial inflammation have been largely disappointing, and no specific treatment for viral myocarditis are in clinical use today. Therefore, elucidation of the fundamental mechanisms involved in the development of myocarditis, and the progression from virus infection to heart failure is important.
In patients with heart failure, plasma epinephrine and norepinephrine levels were elevated. Excess stimulation of the β-adrenergic receptor by catecholamines may contribute to the pathogenesis of congestive heart failure of various causes . The cardiac responsiveness to β-adrenergic stimulation in heart failure is associated with gene expression alterations in myocardial regulatory proteins which are involved in the cAMP-dependent signal transduction [12, 13, 14, 15]. The transcriptional control by the cAMP-response element binding protein (CREB) is a major mechanism regulating the gene expression by the cAMP-dependent signaling pathway [16, 17, 18, 19, 20]. CREB plays an important role in the regulation of a number of genes involved in the maintenance of normal cellular function [16, 17, 18, 19, 20]. Several studies suggested CREB as an important regulator of gene expression involved in the pathophysiology of heart failure [21, 22, 23, 24]. Transgenic mice with cardiac myocyte-specific expression of dominant negative CREB showed impaired contractile response to isoproterenol stimulation as well as dilatation of the 4 chambers of the heart, mimicking idiopathic dilated cardiomyopathy . Moreover, evidence from an increasing number of studies has shown that CREB mediates the cardiovascular remodeling process , including inflammation , cell migration , and apoptosis [28, 29]. However, CREB in viral myocarditis has not been studied. The role of β-adrenergic stimulation on viral myocarditis has also been investigated in animal models of viral myocarditis. We and other investigators have recently reported that the circulating plasma levels of catecholamines were significantly increased in murine viral myocarditis [30, 31]. These results implicated that the transcriptional factor CREB may be also involved in the pathophysiology of viral myocarditis. Therefore, in the present study, we examined the role of the transcriptional factor CREB in murine coxsackievirus-induced acute viral myocarditis.
Carvedilol, a third-generation, nonselective β-adrenoceptor antagonist that also possesses α1-adrenergic blocking, antioxidant, antiapoptotic, anti-inflammatory, and antifibrotic properties, has been shown to provide greater benefit than traditional β-adrenoceptor antagonists in chronic heart failure . Recently, our and other studies have demonstrated protective effects of carvedilol in viral myocarditis [30, 31, 33, 34, 35]. The beneficial action of carvedilol in murine viral myocarditis may be at least partly due to its effects of catecholamine reduction [30, 31, 33, 34, 35]. In patients with heart failure, the reducion of catecholamines by β-blockade “restored” the changes of the cardiac regulatory proteins which are involved in the cAMP-dependent signal transduction [32, 36, 37]. Therefore, β-adrenoceptor antagonists may also be useful to “normalize” the altered CREB expression in viral myocarditis. However, its effects on the CREB in experimental viral myocarditis has not yet been investigated. Thus, in the present study, we studied the effects of carvedilol on the transcriptional factor CREB in a murine model of acute viral myocarditis induced by CVB3.
The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, 8th Edition, 2011), and all experiments were carried out in accordance with China Animal Welfare Legislation and were approved by the Wenzhou Medical College Committee on Ethics in the Care and Use of Laboratory Animals. For the survival study, individual mice were monitored daily and were euthanized when they displayed signs of myocarditis-associated morbidity such as excessive weakness and lethargy. All animals were anaesthetized with pentobarbital (100 mg/kg, one dose intraperitoneally) prior to sacrifice. Efficient anaesthesia was monitored through pinching the hind paw, when sufficiently sedated the mice were euthanized through cervical dislocation.
Mouse model of viral myocarditis
Specific pathogen-free inbred, 4-week-old, male Balb/c mice, obtained from Shanghai Laboratory Animal Center, China, were inoculated intraperitoneally with 1.0 × 106 plaque-forming units (pfu) of CVB3 (strain Nancy, ATCC VR-30) diluted in phosphate-buffered saline (PBS) to a final volume of 0.1 ml (n = 60). Group control was inoculated intraperitoneally 0.1 ml with normal saline solution (n = 30). The day of virus inoculation was defined as day 0.
Carvedilol was obtained from Roche China Co. (Shanghai, China). Starting 24 h after infection carvedilol (10 mg/kg per day, n = 30) were administered by gavage for 14 consecutive days, whereas normal control group (n = 30) and myocarditis group mice (n = 30) received the normal saline solution in the same way. Eight surviving mice from each group were killed on day 7 or 14.
Plasma noradrenaline was measured using high-performance liquid chromatography and electrochemical detection. After mice were anesthetized intraperitoneally with pentobarbital (50 mg/kg), the arterial blood samples were taken and centrifuged at 3000 g for 15 min. The plasma were stored at −80°C for subsequent determination of noradrenaline concentration. The experiment was repeated 6 times for each sample.
HR and blood pressure (BP) were measured using a photoelectric tail cuff detection system (softron BP-98A from Japan) on conscious mice that had been pre-warmed for 10 minutes at 37°C in a thermostatically controlled heating cabinet. The values were averaged from at least three consecutive readings on each occasion.
Survival was measured over a 14-day period.
The ratio of heart weight to body weight (HW/BW) was calculated. The heart tissue was fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Several sections of each heart were scored blindly by two observers. The scores assigned to these specific sections were averaged. The extent of cellular infiltration and myocardial necrosis was graded and scored as follows: 0 = no lesion; 1 + = lesions involving <25% of the myocardium; 2 + = lesions involving 25% to 50%; 3 + = lesions involving 50 to 75%; and 4 + = lesions involving 75% to 100%.
Masson trichrome staining
The heart tissue specimen was cut in a 5-mm-thick, fixed in 10% formalin, dehydrated and embedded in paraffin. The slices were stained with Masson’s trichrome stain (GENMED SCIENTIFICS INC. USA). Five visual fields were randomly selected in each slice stained with Masson’s trichrome under a microscope and measured by Image Pro Plus image analysis software. Collagen volume fraction (CVF) obtained from the average ratio of collagen area to total tissue area was used to evaluate the extent of interstitial fibrosis. CVF excluded scars and perivascular collagen areas.
Detection of apoptosis
Apoptosis was detected in myocardial tissue sections using the terminal transferase-mediated DNA nick end labelling (TUNEL) assay. Apoptotic cells were identified using an in situ cell death detection kit, pod (Roche, Switzerland). The paraffin sections of myocardial tissue (3 μm-thickness) were deparaffinized for 5 min in xylone twice, and then rehydrated in a series of serially diluted ethanol solutions (100%, 95%, 90%, 80%, 70%, and then H2O). Tissue sections were then treated with proteinase K (final concentration of 20 ug/ml in 10 mM Tris–HCl, pH 7.4–7.8) for 30 min at 22°C. A positive control was prepared by incubating a tissue section with DNase I (1 U/ml in 50 mM Tris–HCl, pH7.5, 1 mg/ml BSA) for 10 min before proteinase K treatment. The paraffin sections incubated with 50 μl TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT) for 60 minutes at 37°C in a humidified chamber in the dark. A negative control was prepared without TUNEL enzyme. Tissue sections were washed three times with PBS and the TUNEL-positive apoptotic cardiomyocyte nuclei were examined under a fluorescence microscope using an excitation wavelength in the range of 450–550 nm and a detection wavelength in the range of 515–565 nm. Tissue sections were then treated with converter-POD in a humidified chamber for 30 min at 37 μ. and incubated with 50 ul DAB for 10 min. Positive staining cells were manually counted in 10 randomly selected fields of each slide by microscope (200× magnification field). Cell death was expressed as the average percentage of total cells counted.
Assay of myocardial virus concentration
For the infectivity assay, portions of the heart were weighed and homogenized aseptically in 2 mL PBS. After a 15-minute centrifugation at 1500 g, virus titers in the supernatants were determined by a plaque assay method, as previously described . In brief, VERO (African green monkey kidney) cells suspended (1 × 106/mL) in Eagle’s minimal essential medium (EMEM) with 5% FCS plus 100 μg/mL each penicillin and streptomycin were placed in six-well plates and allowed to grow for 2 or 3 days at 37°C in 5% CO2. After adsorption, the cells were overlaid with 3 mL EMEM containing 5% FCS and 1% methylcellulose. After a 2-day incubation at 37°C in a humidified atmosphere containing 5% CO2, the cells were fixed with acetic acid and methanol (1:3) and stained with 1% crystal violet; plaques were then counted with an inverted microscope. The myocardial virus titer was expressed as log pfu/mg of heart.
Western blot analysis for CREB and phosphorylated CREB
Heart tissues were homogenized and dissolved with RIPA buffer [150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris (pH 7.4)] containing 1 M PMSF (Phenylmethanesulfonyl fluoride). Protein concentrations were determined with the BCA Protein Assay Kit. Total protein (50 ug per lane) was separated on 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred to a PVDF membrane (BioRad, Hercules, CA). Membranes were incubated with primary antibodies according to the manufacturer’s protocol, subsequently washed with PBS-T (phosphate-buffered saline Tween-20), and were incubated with an appropriate secondary antibody dependent to primary antibody. Results were scanned and quantified using densitometry and ImageQuant software and normalized to the housekeeping protein GAPDH. Phosphorylated CREB (pCREB), CREB and GAPDH antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The experiment was repeated 3 times for each sample.
Total RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR)
Primer sequences used for semi-quantitative RT-PCR
Annealing temperature ( ° C)
Fluorescent quantitative real-time PCR
Total RNA, frozen in liquid nitrogen, was extracted from the myocardial samples by the Trizol method (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA concentration were quantified using the spectrophotometer. cDNA was synthesized by reverse transcription using total RNA (2 μg) as a template. The internal control gene used was GAPDH. For PCR amplification, the following mouse-specific sense and antisense primers were used: IL-6, 5′- TGCCTTCTTGGGACTGAT-3′ (forward) and 5′- TAAGCCTCCGACTTGTGA-3′ (reverse); TNF-а, 5′- CCACGCTCTTCTGTCTACTGA-3′ (forward) and 5′- AAGGTACAACCCATCGGCTG-3′ (reverse); GAPDH, 5′-AGGGAAATCGTGCGTGACAT-3′ (forward) and 5′- CATCTGCTGGAAGGTGGACA-3′ (reverse). PCR amplification was performed on a Light Cycler FastStart DNA Master SYBR Green I. The amplification was performed in a Roche Light Cycler 480 instrument under the following condition: initial denaturation at 95°C for 5 minutes, 50 cycles of amplification at 95°C for 10 seconds, annealing at 60°C for 10 seconds, and then extension at 72°C for 10 seconds.
Enzyme-linked immunosorbent assay for cytokines in the heart
Cytokine levels were measured with various enzyme-linked immunosorbent assay (ELISA) kits manufactured by Westang Biotech Co Ltd (Shanghai, China) for IL-6, TNF-α, and MCP-1. The sensitivity of the kit is 16 pg/ml for IL-6, 13 pg/ml for TNF-α, and 8 pg/ml for MCP-1. Cytokine levels are expressed as pg/mg of heart. The experiment was repeated 3 times for each sample.
All values were expressed as mean value ± standard error (SE). Survival rate was analyzed by the Kaplan-Meier method. The continuous variables were compared with a Student’s t-test for two groups and with one way analysis of variance (ANOVA) for >2 groups. Bivariate correlations were performed by Pearson’s correlation. A value of P < 0.05 was considered significant.
Plasma noradrenaline levels
Hemodynamics of mice
Seven and 14 days after infection, the myocarditis group showed a reduction in BW and HW and an increase in HW/BW ratio. The HW/BW ratio was significantly lower in the carvedilol group than in the myocarditis group on days 7 and 14 (Figure 3b).
Myocardial histopathology and fibrosis
Virus titer of the heart
Viral genome in the myocardium
Protein assay of CREB and pCREB
Gene expression of cytokines by Semi-Quantitative PCR analysis in the heart
Quantitative PCR analysis of cytokine mRNA expressions in the heart
ELISA analysis of cytokines levels in the heart
Correlation between the expression of CREB, pCREB and the levels of inflammatory cytokines
Two major findings were obtained in the present study. First, the phosphorylation and expression of CREB were decreased with concomitant increase of proinflammatory cytokines in murine coxsackievirus-induced acute viral myocarditis. The levels of IL-6 and TNF-α were correlated with the expression of CREB or pCREB. This finding indicates that the cardiac transcriptional factor CREB may be involved in the pathophysiology of viral myocarditis. Second, carvedilol increased cardiac CREB expression and phosphorylation and decreased the plasma catecholamine levels and the production of IL-6 and TNF-α with amelioration of acute viral myocarditis. These results show that by blocking catecholamine stimulation and increasing the cardiac CREB expression and phosphorylation, carvedilol exerts some of its beneficial effects by suppressing the IL-6 and TNF-α. To the best of our knowledge, this is the first study to investigate the effects of carvedilol on cardiac CREB expression and phosphorylation in viral myocarditis.
CREB expression and phosphorylation in viral myocarditis
It is well-known that stimulation of β-adrenergic receptor by catecholamines results in the elevation in the intracellular concentration of the second messenger cAMP . Increased production of cAMP leads to activation of protein kinase A, which in turn causes phosphorylation/activation of CREB and subsequent gene expression through CRE-mediated transcription . Recent studies have indicated an important role of CREB in the development of the cardiovascular diseases such as heart failure, atherosclerosis, restenosis, and reperfusion injury [21, 22, 23, 24, 25, 26, 27, 28, 29]. Moreover, emerging evidence has revealed specific functions of CREB in immune responses, including regulating NF-kB activation [40, 41], inducing macrophage survival [40, 42, 43], and promoting the proliferation, survival, and regulation of T and B lymphocytes [40, 42, 43]. It has been suggested that CREB is involved in suppression of cytokine gene expression, including IL-6 and TNF-α, and promotes anti-inflammatory immune responses [18, 40, 41, 44, 45, 46, 47, 48]. In the present study, CREB expression and phosphorylation were decreased with concomitant increase of IL-6 and TNF-α. The levels of IL-6 and TNF-α were correlated with the expression of CREB or pCREB. It has been suggested that cytokines exert an important role in the pathophysiology of viral myocarditis [49, 50, 51, 52, 53]. IL-6 and TNF-α are both involved in the pathogenesis of myocarditis and may induce advanced cardiac dysfunction [50, 52, 53]. CREB may thus play an important role in the pathogenesis of viral myocarditis. The reduction in CREB expression and phosphorylation in viral myocarditis may be caused by several mechanisms. First, prolonged explosure to elevated catecholamine levels lead to the desensitization of cardiac β-adrenergic receptors signaling that limit cAMP production and reduce the expression and phosphorylation of CREB [11, 54]. Previous studies have demonstrated that cardiac CREB alteration was associated with catecholamines stimulation time.22,54 CREB mRNA was increased after short-term (30 min) stimulation with forskolin, an activator of cAMP , while prolonged β-adrenergic stimulation in rats by a 4-day infusion of isoproterenol decreased CREB mRNA . Second, a direct proteolytic cleavage of CREB by viruses may lead to the reduction in CREB. A few studies have demonstrated that infection of susceptible cells with members of the picornavirus family results in the rapid inhibition of host cell RNA synthesis [56, 57, 58, 59]. Yalamanchili et al. found that CREB and pCREB were specifically cleaved by the poliovirus-encoded protease 3C pro both in vitro and in virus-infected cells . Therefore, CVB3 may directly cleave the CREB protein and result in the reduction of CREB.
Effects of Carvedilol on CREB expression and phosphorylation in viral myocarditis
In this study, treatment with carvedilol decreased the plasma levels of noradrenaline and increased the expression and phosphorylation of cardiac CREB. As mentioned previously [11, 54], elevated circulating catecholamines may lead to decreased levels and functional activity of cardiac β-adrenergic receptors and thus to marked desensitization of the heart to inotropic β-adrenergic stimulation. Treatment with carvedilol may normalize or remodel signaling through the cardiac β-adrenergic system by reducing desensitization, enhancing catecholamine sensitivity, and raising levels of β-adrenergic receptors [11, 32, 54]. Thus, decreased β-adrenergic stimulation by carvedilol treatment may contribute to the increase in the expression and phosphorylation of CREB. In agreement with previous studies from our and other laboratories [30, 31, 33, 34, 35], we found that carvedilol improved the survival of mice and reduced myocardial inflammation and necrosis in murine viral myocarditis by downregulating the production of IL-6 and TNF-α. In this study, we found that the levels of IL-6 and TNF-α was negatively correlated with the cardiac CREB and pCREB. Moreover, previous studies have found that CREB was involved in suppression of IL-6 and TNF-α gene expression in other systems [18, 40, 41, 46, 47, 48]. Therefore, the reduction in the production of IL-6 and TNF-α may be associated with the increase of CREB in the study.
Recent reports have also emphasized the other cellular mechanisms in murine myocarditis [61, 62, 63, 64, 65]. After viral entry acute injury of the myocytes induced by virus replication leads to myocyte necrosis, exposure of intracellular antigens (e.g., cardiacmyosin), and activation of the host’s immune system, which is characterized by the invasion of natural killer cells and macrophages followed by T lymphocytes. After the acute phase of virus-induced injury, the second phase is characterized by (auto)immune reactions. This subacute phase is defined by activated virus-specificT lymphocytes, which may target the host’s organs by molecular mimicry. Kania and Shi et al. reported that toll-like receptors transmit a cascade of signals to activate nuclear transcription factors, such as nuclear factor κB, and lead to inflammatory cytokine production and immune activation. Cytokine activation (IL-6 and TNF-α) and antibodies to viral and cardiac proteins may aggravate cardiac damage and cause impairment of the contractile function.
The survival rate, HW/BW ratio and myocardial histopathology of infected mice were studied in the present study. However, cardiac function and volume were not studied by echocardiographic examination. In addition, although the cardiac transcriptional factor CREB appears to be involved in the pathophysiology of viral myocarditis, further research to reveal signaling pathways or collaborating proteins that are selectively involved in its proinflammatory or its anti-inflammatory functions is needed.
The expression and phosphorylation of CREB were decreased in murine coxsackievirus-induced acute viral myocarditis. Administration of carvedilol causes an increase in the expression and phosphorylation of CREB together with the reduction in the production of IL-6 and TNF-α. The levels of IL-6 and TNF-α were correlated with the expression of CREB or pCREB. Although the cardiac transcriptional factor CREB appears to be involved in the pathophysiology of viral myocarditis, further research to reveal signaling pathways or collaborating proteins that are selectively involved in its proinflammatory or its anti-inflammatory functions is needed.
LYC designed the whole study, LYC, GLS, CYH, ZND and ZT performed the experiment, LYC and GLS wrote the paper. All authors read and approved the final manuscript.
This study is supported by the National Natural Science Foundation of China (Grant No. 81200165) and the Zhejiang Provincial Natural Science Foundation of of China (Grant No. Y2100551) and the Wenzhou Municipal Science and Technology Commission, China (Grant No. Y20130038).
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