© 2004 European Society of Cardiology
C-reactive protein co-expresses with tumor necrosis factor-
in the myocardium in human dilated cardiomyopathy
Second Department of Internal Medicine Iwate Medical University School of Medicine, Uchimaru 19-1, Morioka 020-8505, Iwate, Japan E-mail address: m_satoh{at}imu.ncvc.go.jp
* Corresponding author. Tel.: +81 19 651 5111; fax: +81 19 651 0401.
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Abstract |
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 Top Abstract 1. Introduction2. Methods3. Results4. DiscussionReferences |
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Background: C-reactive protein (CRP) has recently been reported to be present in cardiac tissue and to stimulate the production of proinflammatory cytokines. Cardiac expression of tumor necrosis factor-
(TNF-) plays an important role in the pathogenesis of dilated cardiomyopathy (DCM).
Aims: To determine whether CRP co-expresses with TNF- in the myocardium and to examine its association with clinical features in patients with DCM.
Methods and results: Endomyocardial biopsy tissues were obtained from 41 DCM patients and 16 controls by right ventricular endomyocardial biopsy. Levels of CRP and TNF- mRNA were measured by real-time RT-PCR. Immunohistochemistry and in situ hybridization were performed to identify the cellular sources of CRP and TNF-. Both CRP and TNF- mRNA were expressed in myocardium obtained from DCM patients, but not in controls. A positive correlation was found between CRP and TNF- levels. CRP/TNF- double staining was found to be colocalized in the cardiomyocytes of DCM patients. Both forms of mRNA were also expressed in cardiomyocytes. Both CRP and TNF- mRNA levels were negatively correlated with systolic function and positively correlated with left ventricular volume in DCM patients. These mRNA levels were lower in DCM patients treated with a combination of spironolactone and either angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II type 1 receptor blockers (ARBs) than in patients not treated with these drugs.
Conclusion: Cardiac expression of CRP with TNF- may function as a proinflammatory mediator in DCM and may be related to the clinical severity of DCM. Expression of both of these proteins was decreased in DCM patients receiving spironolactone and either ACEIs or ARBs.
Key Words: Immunohistochemistry • Inflammation • In situ hybridization • Myocardium • Polymerase chain reaction • Remodeling
Received May 18, 2004; Revised August 23, 2004; Accepted October 20, 2004
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1. Introduction |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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In previous studies, the immune response against viral replication has been shown to be activated in the myocardium in dilated cardiomyopathy (DCM) and was related to the severity of DCM [1,2]. These reports have suggested that a persistent inflammatory state representing a viral origin may induce advanced myocardial damage resulting in the heart failure with poor prognosis seen in DCM. However, the precise mechanism underlying this persistent inflammatory state remains unclear.
Several epidemiological studies have suggested that elevated baseline serum C-reactive protein (CRP) concentrations with high levels of tumor necrosis factor- (TNF-) or the ability of peripheral blood mononuclear cells to produce TNF- were potent risk factors for new onset congestive heart failure in apparently healthy elderly subjects [3,4]. It has also been reported that higher levels of serum CRP were related to increased risk of mortality in patients with DCM [5]. CRP production may therefore play an important role in human DCM. Although CRP is synthesized by the liver in response to microbial infection, tissue injury, and autoimmune disorders, recent studies have demonstrated local CRP production in extrahepatic sites including vascular smooth muscle cells, respiratory epithelial cells, renal epithelium, and neuronal cells [6–9]. CRP is an inflammatory protein associated with the secretion of various cytokines, including interleukin-6, TNF-, and interleukin-1 [10]. An in vitro model has shown that CRP induced the production of TNF- in human macrophages [11]. Calabró et al. [6] have recently shown that inflammatory cytokines including TNF- induce CRP production from cultured human coronary artery smooth muscle cells. These in vitro models suggest that CRP may be closely linked to TNF- production. Furthermore, the acute-phase reactant protein directly enhanced myocardial expression of inducible nitric oxide synthase (iNOS) which may induce negative inotropic and cytotoxic effects on the myocardium [12]. In postmortem examination, myocardial localization of CRP has been demonstrated in infarcted heart tissue, suggesting that this acute-phase protein may function as a proinflammatory mediator in the heart [13]. We have therefore speculated that cardiac CRP acts synergistically with myocardial TNF- and is thereby involved in the pathogenesis of DCM. The purpose of this study was to determine whether CRP was co-expressed with TNF- in myocardium obtained from patients with DCM and to analyze the relationship between expression levels of these two factors, and left ventricular (LV) function and geometry.
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2. Methods |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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2.1. Subjects
Endomyocardial tissues were obtained from 41 patients with DCM by right ventricular endomyocardial biopsy [32 males and 9 females; mean age, 49±3 years; mean LV ejection fraction (LVEF), 34±1%; mean LV end-systolic diameter (LVESD), 50.8±1.1 mm]. Exclusion criteria included concomitant diseases, such as chronic infection, pulmonary disease, myocarditis, myocardial infarction, cancer, diabetes mellitus, pregnancy, severe liver dysfunction, renal dysfunction, or connective tissue disease. All patients underwent coronary angiography at the time of biopsy to exclude ischemic heart disease and other secondary cardiac diseases. The clinical diagnosis of DCM was made according to the World Health Organization/International Society and Federation of Cardiology Task Force criteria [14]. The DCM patients were receiving loop diuretics (n=25), digitalis (n=16), spironolactone (n=14), angiotensin-converting enzyme inhibitors (ACEIs; enalapril, 2.5 to 5 mg/day, n=16; benazepril, 5 mg/day, n=3; temocapril, 5 mg/day, n=1), angiotensin II type 1 receptor blockers (ARBs; candesartan, 4 to 8 mg/day, n=9; valsartan, 40 to 80 mg/day, n=3; losartan, 50 mg/day, n=1), or β blockers (n=15). Most of these drugs had been prescribed for more than 3 months.
Control myocardial tissue samples were obtained by right ventricular endomyocardial biopsy from 16 subjects (9 males and 7 females; mean age 44±12 years) with a suspected cardiac disorder on the basis of ECG abnormality and echocardiographic changes, such as a slight increase in wall thickness without LV dysfunction. As the resulting pathology findings showed no evidence of myocardial disease and there was no medical history of infectious illness (such as myocarditis, pneumonia, or sepsis), these subjects were designated as controls. This study protocol was approved by our hospital ethics committee, and written informed consent was obtained from all subjects.
2.2. Extraction of RNA
Total RNA was extracted from endomyocardial tissues by the acid guanidinium thiocyanate-phenol-chloroform method and treated with DNase I (GIBCO BRL) [15].
2.3. Sequences of primers and probes
The published sequences for human CRP and TNF- were used for construction of primers and TaqMan probes [16,17]. The following primers and probes were used: for human CRP—forward primer 5'-TGT ACA AGC TGG GAG TCC GC-3', reverse primer 5'-CAA AGT TCC CAC CGA AGG AA-3', and TaqMan probe 5'-TCA GGG ATC GTG GAG TTC TGG GTA GAT G-3'; for TNF-—forward primer 5'-CTT CTC CTT CCT GAT CGT GG-3', reverse primer 5'-GCT GGT TAT CTC TCA GCT CCA-3', and TaqMan probe 5'-CAG GCA GTC AGA TCA TCT TCT CGA AC3'. For all samples, glyceraldehyde3-phosphate dehydrogenase (GAPDH) mRNA was amplified using TaqMan GAPDH control reagents as an internal control (PE Biosystem, Foster City, CA, USA).
2.4. Real-time polymerase chain reaction
We analyzed CRP and TNF- mRNA expression levels using a quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method as previously described [1,2]. The cDNA was synthesized and amplified from 100 ng of total RNA and 10-fold serial dilutions of human control RNA (PE Biosystem) by RT-PCR using Taq Man EZ RT-PCR kit (PE Biosystem). The cDNA products were synthesized at 60 °C for 30 min and amplified with 40 cycles of PCR, with each cycle consisting of denaturation at 94 °C for 20 s, and annealing and extension at 62 °C for 1 min. A quantitative PCR method was developed using detection and 5' nuclease assay by an ABI PRISM 7700 sequence detector (PE Biosystem). To improve the accuracy of the real-time PCR method for quantification of CRP, TNF-, and GAPDH mRNAs, amplifications were performed in triplicate for each RNA sample. To account for variations in input RNA and RT efficiency, CRP and TNF- levels were normalized to GAPDH expression in each sample. To account for PCR amplification of contaminating genomic DNA, a control without RT was included.
2.5. Immunohistochemistry
CRP/TNF- double staining was performed on serial paraffin sections to determine the cellular sources of CRP and TNF-. Monoclonal anti-CRP antibody (clone CRP-8, Sigma) and monoclonal antibody to human TNF- (HyCult Biotechnology) were used as primary antibodies. The tissue sections were deparaffined and thoroughly dehydrated. After inhibition of endogenous peroxidase and blocking of nonspecific reactions, monoclonal anti-CRP antibody was applied. Biotinylated mouse immunoglobulin was used as a secondary antibody. Peroxidase-labeled streptavidin (Histofine, MAX-PO kit, Nichiren) was applied and visualized using diaminobenzidine as a chromogen.
For double staining, monoclonal anti-CRP antibody was first stained as mentioned above. Thereafter, the sections were incubated with monoclonal antibody to human TNF- at 4 °C overnight, followed by incubation for 30 min with rabbit antimouse immunoglobulin and alkaline phosphatase antialkaline phosphatase, and visualized with FastBlue (Histofine, SAP-AP kit, Nichiren). The specificity of the immunohistochemistry was confirmed by substituting the primary antibodies with mouse IgG1 isotype control (Dako) on sections from patients with DCM.
2.6. In situ hybridization
In situ hybridization was performed on serial paraffin sections to identify the cellular sources of CRP and TNF- mRNA expression. Antisense oligonucleotide probes (CRP: 5'-CCT CAG GCG GAG TCC CTA GC-3', TNF-: 5'-CCC TCT GGG GTC TCC CCC GAC TCC GG-3') were used for in situ hybridization [16,17]. The probes were labeled with a 3'-biotinylated tail (Britati tail). For every specimen, we used 20-base poly-T oligonucleotide probe (Research Genetics) to examine the retention of mRNA in biopsy samples. Hybridization was performed with a MicroProbe staining system (Fisher Scientific). Tissue sections were placed on Probe ON Plus microscopic slides (Fisher Scientific) and were rapidly dewaxed, cleared with alcohol, rehydrated with Tris-based buffer, pH 7.4 (Universal Buffer, Research Genetics), and then digested with pepsin (1.25 mg/mL; Research Genetics) for 3 min at 105 °C. The probes were applied in a formamide-free diluent, and the slides were heated to 105 °C for 3 min, cooled to room temperature, and allowed to hybridize at 40 °C for 30 min. The sections were then washed with 2x SSC buffer (300 mmol/L NaCl and 30 mmol/L trisodium citrate, pH 7.0) at 45 °C and detected with alkaline phosphatase-conjugated streptavidin (Research Genetics). After hybridization, the products were washed in AP chromogen buffer, pH 9.5 (Research Genetics) at room temperature and were visualized with fast red. The slides were counterstained with hematoxylin, air dried, and then coverslipped for microscopic examination.
2.7. Serum CRP levels
Venous blood samples were obtained from DCM patients and controls at the time of biopsy. Serum CRP levels were measured by latex photometric immunoassay (cut off value <0.1 mg/dL; LPIA-CRP, Mitsubishi Chemical).
2.8. Statistical analysis
All values are presented as mean±standard error. Statistically, the differences in clinical parameters of DCM patients were analyzed by unpaired t-test. Pearson's correlation coefficients were used to examine the relationship between mRNA levels and clinical parameters. A value of P<0.05 was considered statistically significant.
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3. Results |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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3.1. Real-time PCR and serum CRP levels
Both CRP and TNF-
mRNAs were expressed in myocardium obtained from patients with DCM (mean levels of CRP/GAPDH ratio and TNF-/GAPDH ratio in patients with DCM: 1.19±0.11 and 1.23±0.07, respectively). As shown in Fig. 1, a weak positive correlation was found between CRP and TNF- mRNA levels in patients with DCM (r=0.56, P<0.001). Neither CRP nor TNF- mRNA was detected in controls. In 16 of 41 DCM patients, the CRP titer was <0.1 mg/dL. In the other 25 DCM patients, CRP titer was 0.3±0.1 mg/dL (range 0.1 to 1.7 mg/dL). There was no significant correlation between serum CRP and myocardial CRP mRNA levels in DCM patients (r=0.26, P=0.30). Serum CRP titer was <0.1 mg/dL in all of the controls.
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3.2. Immunohistochemistry
CRP/TNF-
double staining was positive in 15 patients with DCM. CRP/TNF- double staining showed colocalization of CRP and TNF- proteins in the cytoplasm of cardiomyocytes from DCM patients (Fig. 2a). Immunostaining of CRP was also observed in microvascular endothelial cells and endocardial endothelium from DCM patients (Fig. 2a and c). There was no evidence of nonspecific immunostaining in myocardium obtained from patients with DCM (Fig. 2b and d). In the other 26 patients with DCM, CRP and/or TNF- staining was not found. Neither CRP nor TNF- immunostaining was present in any specimens from control subjects. Both CRP and TNF- mRNA levels were higher in CRP/TNF- double staining-positive patients with DCM than in negative patients with DCM (CRP/GAPDH ratio: 1.73±0.11 vs. 0.71±0.07, P<0.001; TNF-/GAPDH ratio: 1.47±0.11 vs. 1.02±0.07, P<0.001).
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3.3. In situ hybridization
To define the localization of CRP and TNF-
mRNA expression in myocardium, we carried out in situ hybridization using CRP and TNF- oligonucleotide probes. Both CRP and TNF- mRNAs in in situ hybridization were identified in 18 DCM patients, but not in controls. As shown in Fig. 3a and b, CRP and TNF- mRNAs were mainly localized to cardiomyocytes in the myocardium obtained from DCM patients. Poly-T massage was also identified in all specimens (Fig. 3c).
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3.4. Comparison of clinical data
Both CRP and TNF-
mRNA levels were correlated negatively with LVEF in DCM patients (CRP vs. LVEF: r=–0.57, P<0.001; TNF- vs. LVEF: r=–0.69, P<0.001; Fig. 4a and b). A weak positive correlation was also found between these mRNA levels and LVESD in DCM patients (CRP vs. LVESD: r=0.49, P=0.003; TNF- vs. LVESD: r=0.51, P<0.001; Fig. 4c and d). Serum CRP levels were not correlated with LVEF and LVESD (serum CRP vs. LVEF: r=–0.33, P=0.11; serum CRP vs. LVESD: r=0.28, P=0.18).
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In DCM patients receiving combination treatment with spironolactone and either ACEIs or ARBs (n=10), both CRP and TNF-
mRNA levels were significantly lower than in patients not receiving this drug combination (CRP/GAPDH ratio: 0.80±0.13 vs. 1.36±0.13, P=0.02; TNF-/GAPDH ratio: 0.92±0.09 vs. 1.37±0.08, P=0.03). CRP mRNA levels in DCM patients treated with spironolactone (n=14) tended to be lower than in patients not receiving either drug (n=27), but this difference was not significant (CRP/GAPDH ratio: 0.96±0.14 vs. 1.37±0.15, P=0.06). |
4. Discussion |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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The most important findings of this study are (1) CRP immunostaining was colocalized with TNF-
in cardiomyocytes; (2) CRP immunostaining was also found in endocardial endothelium and microvascular endothelial cells; (3) both CRP and TNF- mRNAs were expressed in cardiomyocytes; (4) CRP mRNA levels were closely correlated with TNF- mRNA levels in myocardial tissues and were related to LV systolic dysfunction and dimension of DCM; (5) CRP and TNF- levels were relatively low in DCM patients receiving combination treatment with spironolactone and either ACEIs or ARBs.
4.1. Expression of CRP in cardiac tissues
Although CRP was believed to be produced exclusively by hepatocytes during the acute-phase response, there is evidence of local CRP production at extrahepatic sites including vascular smooth muscle cells, respiratory epithelial cells, renal epithelium, and neuronal cells [6–9]. This study has demonstrated that CRP immunostaining can be found in failing cardiac tissues including cardiomyocytes, endocardial endothelium, and microvascular endothelial cells. In situ hybridization has also shown that expression of CRP mRNA was localized in cardiomyocytes. Myocardial CRP mRNA levels were not correlated with serum CRP levels. These findings were associated with significant myocardial production of CRP in patients with DCM. No reports have yet shown evidence of locally produced CRP in cardiac tissues; however, Nijimeijer et al. [13] have demonstrated the localization of CRP in cardiomyocytes of autopsy cases due to myocardial infarction. Although the exact origin of CRP in DCM has not been confirmed, as demonstrated in endothelial cells and macrophages [11,18], local expression of CRP may stimulate local cytokine production and thus enhance the smoldering myocardial inflammation induced by viral replication as has been suggested in the pathogenesis of DCM.
This study has also demonstrated for the first time that CRP immunostaining was found in endocardial endothelium and microvascular endothelial cells. This acute-phase protein has been reported to impair vascular endothelial expression of nitric oxide synthase [19,20] and to up-regulate endothelial expression of chemokines and adhesion molecules [21,22]. As endocardial endothelium and vascular endothelium share these paracrine systems, cardiac endothelial dysfunction may create functional and structural cardiac maladaption, leading to disease progression [23].
4.2. Co-expression of TNF- and CRP
We found a close relationship between mRNA levels of TNF- and CRP, and gained immunohistochemical confirmation of the colocalization of these molecules in cardiac tissue. Additionally, an in situ hybridization method showed that expression of CRP and TNF- mRNAs was mainly localized in cardiomyocytes. Several reports have shown that CRP colocalized with complement and activated it in jeopardized cardiomyocytes in infarcted human myocardium, suggesting that accumulation of CRP may enhance inflammatory reaction in the ischemic myocardium [13,24]. An in vitro model has shown that exogenous application of CRP directly induced the production of TNF- in human macrophages, suggesting that CRP may increase TNF- production [11]. One recent study has shown that TNF- increases CRP production from cultured human vascular tissues in a dose-dependent fashion [6]. These observations suggest that myocardial co-expression of CRP and TNF- may create a positive vicious loop of a persistent inflammatory state in the myocardium in DCM.
4.3. Clinical implications
This study has also found that myocardial expression levels of both CRP and TNF- mRNA is related to the severity of LV dysfunction and geometry in DCM. Our previous studies have demonstrated that myocardial TNF- production is consistently co-expressed with its converting enzyme, toll-like receptor and iNOS, and that this activation may be related to the clinical severity of DCM [1,2,25]. In several in vitro studies, CRP has activated a host immune response that is critical for up-regulating the synthesis of inflammatory mediators and endogenous proteins, such as inflammatory cytokines and iNOS [10–12]. CRP enhances iNOS expression and subsequent NO synthesis in cytokine-stimulated rat cardiomyocytes, and these may in turn induce negative inotropic and cytotoxic effects on the myocardium [12]. In addition, CRP directly increases the expression of chemokines and adhesion molecules in human cultured cells [21,22]. From these reports, it appears that cardiac-produced CRP may play an important role in a continuous immune response through cytokine cascade in the failing heart. These observations suggest that high levels of CRP in the heart may be linked to activated TNF- cascade and to the induction of LV functional and structural abnormality in human DCM.
Another important finding of this study was a significant decrease in the expression of CRP and TNF- mRNA levels in DCM patients treated with a combination of spironolactone and either ACEIs or ARBs, when compared with patients not receiving these drugs. We were unable to determine whether the cardiac effects were due to direct or indirect unloading resulting from spironolactone and either ACEIs or ARBs. However, in a rat model, aldosterone treatment activated nuclear factor-
B (NF-B) that was an increased gene expression of chemokine and proinflammatory cytokines, suggesting that aldosterone may directly activate an inflammatory phenotype in the heart [26]. In addition, spironolactone has down-regulated the renin-angiotensin aldosterone system (RAAS) and prevented the appearance of an inflammatory phenotype in this model [26]. Another recent study has shown that ARB suppressed the NF-B signaling pathway while inducing inhibitor B and suppressing plasma CRP concentrations [27]. Combined treatment with spironolactone and either ACEIs or ARBs may therefore down-regulate activation of the RAAS in the failing heart, and thus decrease inflammatory phenotype, such as expression of CRP and TNF-. Further prospective studies will be needed to confirm this hypothesis.
4.4. Limitations of the study
It remains to be elucidated whether expression of CRP and TNF- in biopsy samples from the right side of the heart directly reflect that in the left side of the heart. However, our previous study has shown a similar pattern of TNF- in both ventricles in DCM [1]. Moreover, in classical studies, no significant histological and histochemical differences were observed between both ventricular biopsy samples obtained from patients with suspected congestive cardiomyopathy [28,29]. Immunohistochemistry and in situ hybridization suggest local CRP production in cardiomyocytes. However, this does not exclude the classical pathway of hepatic CRP synthesis in response to microbial stimulation in patients with a failing heart. This study could not confirm whether a concomitant mechanism, such as the expression of chemokines, adhesion molecules, or iNOS through CRP synthesis, was responsible for clinical severity in DCM. Therefore, this study is solely descriptive, and further studies will be needed to establish a causal relationship between these phenomena. Although CRP and TNF- levels were found to be lower in DCM patients treated with spironolactone and either ACEIs or ARBs, these treatments were not randomized for prospective analysis, and no data were available at the initiation of the study on CRP and TNF- mRNA levels or evolution of CRP mRNA. Therefore, this study could not confirm the capacity of these medications to decrease CRP and TNF- levels in the myocardium.
4.5. Conclusions
In conclusion, this study has demonstrated that co-expression of CRP with TNF- in cardiac tissue may function as a proinflammatory mediator and contribute to the progression of left ventricular dysfunction and remodeling in patients with DCM. In addition, CRP and TNF- levels were found to be lower in DCM patients receiving spironolactone and either ACEIs or ARBs.
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