BALB/c mice inoculated intraperitoneally with coxsackievirus group B type 3 (CVB3) were allocated to five groups; namely, a viral myocarditis group infected with CVB3 alone (control group), an antibody intervention group that received intracardiac anti-MCP-1, an antibody intervention control group that received goat IgG, a tMCP-1 intervention group that received plasmid pVMt expressing tMCP-1, and a tMCP-1 intervention control group that received plasmid pVAX1. There was also a normal control group. The ratio of murine heart weight to body weight, pathological score of myocardial tissue, serum creatine kinase-MB titers and CVB3 loading of myocardial tissue were assessed. The cardiac lesions in mice that received 20, 40 or 60 µg pVMt (P < 0.05) were less severe than those in control mice with untreated viral myocarditis. In addition, fewer mononuclear cells had infiltrated the myocardium of mice who received 40 or 60 µg pVMt intramyocardially (P < 0.01), whereas there was no difference in mononuclear cell infiltration between mice with viral myocarditis and those that received 20 µg pVMt (P > 0.05). There was also no difference between mice that received anti-MCP-1 antibody and those that received 40 µg pVMt in ratio of HW/BW, serum CK-MB titers and pathological score (P > 0.05). This study showed that tMCP-1 can alleviate cardiac lesions and cardiac injury in mice with viral myocarditis via infiltration of mononuclear cells. Thus, tMCP-1 may be an alternative to anti-MCP-1 antibody treatment of viral myocarditis. Further research is required.
C-C chemokine receptor 2
creatine kinase-MB fraction
coxsackievirus group B type 3
heart weight to body weight
interferon-gamma inducible protein
peripheral blood mononuclear cell
50% tissue culture infectious dose
Chemokines provide migrating signals, activate cells and initiate trafficking of inflammatory cells . More interestingly, several conditions, including cardiovascular diseases, allergic inflammatory diseases, transplantation, neuroinflammation, cancer and HIV-associated diseases, are known to be associated with inappropriate activation of the chemokine network [2-4]. Viral myocarditis is a common cardiovascular disease. In a previous study, we found that blocking MCP-1 with its specific antibody significantly reduced infiltration by mononuclear cells and myocardial tissue injury in mice with viral myocarditis [5, 6]. Relationships between the structure and function of MCP-1 provide opportunities to use MCP-1 analogues to interfere with the functioning of wild type MCP-1 [7, 8]. In the present study, we used a megaprimer PCR technique to construct an MCP-1 analogue from which the N-terminal residues 2–8 had been deleted, here denoted tMCP-1. The plasmid pVMt expressing tMCP-1 was then inoculated into the myocardium to interfere with the functioning of MCP-1. We found that, similarly to anti-MCP-1 antibody, tMCP-1 alleviated cardiac lesions and injury in mice with viral myocarditis. Our findings provide a potential target for the prevention and therapy of viral myocarditis.
MATERIALS AND METHODS
Mice, virus and antibody
Four-week-old male BALB/c mice were purchased from the Experimental Animal Center of Zhengzhou University (Zhengzhou, China). CVB3 (Nancy strain) was propagated in HeLa cells and purified by a method previously described by Schmidtke et al.  Virus titers were routinely determined by TCID50 assay of HeLa cell monolayers according to previously published procedures . The aliquots were then stored at −70 °C. Anti-MCP-1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
General outline of experimental procedure
Four-week-old male BALB/c mice were inoculated intraperitoneally with 105 TCID50 CVB3 in 100 μL basal Eagle's medium. The mice were then randomly allocated to five groups, each containing five mice. There was also a normal control group. On the third day and fifth days after CVB3 infection, the mice in the anti-MCP-1 antibody intervention group were inoculated intramyocardially with 50 µg anti-MCP-1, whereas the mice in the antibody intervention control group were inoculated with an equal amount of goat IgG. On the third day after CVB3 infection, the mice in the tMCP-1 intervention group were inoculated myocardially with 20, 40 or 60 µg pVMt, whereas the mice in the tMCP-1 intervention control group were inoculated with plasmid pVAX1. On the ninth day after CVB3 infection, all mice were weighed and blood samples collected from their orbits to measure serum CK-MB titers and isolate PBMCs. The mice were then killed and their hearts removed. After washing blood out of the cardiac chambers, each heart was weighed, and then cardiac CVB3 titer, PS of myocardial tissue and expression of tMCP-1 were assessed.
Construction of pVMt coding tMCP-1 and expression of tMCP-1 in eukaryocytes
MCP-1 encoding gene was obtained from the cardiac tissue of mice by RT-PCR using the following primers: forward: 5′-CGGAATTCGCCACCATGCAGGTCCCTGTCAT-3′; reverse: 5′-GCTCTAGACTAGTTCACTGTCACACT-3′. The PCR products of MCP-1 were purified and dissolved in water. The PCR products and plasmid pVAX1 were digested with EcoRI and XbaI in the designated buffer solution (Tris-HCl). The digested products were then purified and connected by T4 ligase. A recombinant expressing MCP-1, here denoted pVM, was then transformed into competent Escherichia coli DH5α cells. The plasmids of individual resistant colonies were isolated and the inserts were sequenced. Based on the construction of pVM, megaprimer PCR was performed to construct pVMt expressing tMCP-1. The procedure involved two rounds of PCR that utilized two ‘‘flanking” primers A and B and one internal primer MP containing the desired base pairs. The primer MP was complementary with the flanking sequence of 5′-GTAGCAGCAGGTGAGCTGAGCCAACACGTG-3′ encoding N-terminal 2–8 amino acids of MCP-1. The first round PCR (PCR1) was performed using the primers MP and A (5′-GTAGCAGCAGGTGAGC TGAGCCAACACGTG-3′). The products of the first round PCR were purified and used as the mega-primer (M) for the second round PCR (PCR2). The second round of PCR was performed using the primers M and B (5′-GCTCTAGACTAGTTCACTGT CACACT-3′), the products of which were the sequence coding tMCP-1. Additionally, the products of PCR2 were cloned into plasmid pVAX1 and identified by sequencing. The recombinant expressing tMCP-1 was denoted pVMt. The pVMt was transfected into murine muscle cell line C2C12 by a lipofectamine eukaryotic transfection kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The culture supernatant of C2C12 was harvested 24, 48 and 72 hr after transfection and the amounts of tMCP-1 in the supernatant of C2C12 quantified by ELISA according to the manufacturer's instructions.
Preparation of peripheral blood mononuclear cells
Blood was collected from the mice with viral myocarditis and the PBMCs isolated by a density gradient method as described previously .
Chemotaxis assays were performed as described previously . The culture supernatant of C2C12 was added to the bottom chamber of a chemotaxis chamber. In some cases, 10 ng/mL of MCP-1 (Promega, Madison, WI, USA) and different concentrations of tMCP-1 were added to the bottom chamber as stated in Table 1. PBMCs from mice with viral myocarditis (105 cells in 50 µL) were placed in the upper chemotaxis chamber and the wells incubated in a humidified incubator at 37 °C for 8 hr. Triplicate wells were used for each data point. At the end of incubation, the transwell filters were removed, and the lower surfaces swabbed. The swabbed cells together with the cells in the lower chamber were counted. The chemotaxis index was calculated as the ratio of the number of cells chemoattracted by the treated media to the number attracted by the control media.
|Untreated cell culture supernatant||3||1.09 ± 0.03|
|10 ng/mL tMCP-1||3||1.03 ± 0.05† ‡|
|10 ng/mL MCP-1||3||4.28 ± 0.40|
|10 ng/mL MCP-1 + 10 ng/mL tMCP-1||3||2.98 ± 0.23‡|
|10 ng/mL MCP-1 + 15 ng/mL tMCP-1||3||2.45 ± 0.41‡|
|10 ng/mL MCP-1 + 20 ng/mL tMCP-1||3||1.68 ± 0.09‡|
|10 ng/mL MCP-1 + 25 ng/mL tMCP-1||3||1.10 ± 0.08† ‡|
Transfer of antibody and plasmids into the murine myocardiums
Mice were injected intracardially with anti-MCP-1 antibody, goat IgG, pVAX1 or pVMt by the method of Lim et al. . Briefly, mice were anesthetized by intraperitoneal injection of 0.75% pentobarbital sodium (50 mg/kg). The skin was dissected and a 2–3 cm incision made longitudinally through the left chest wall to allow visualization of the beating heart through the pleura. Then anti-MCP-1 antibody, goat IgG, pVAX1 or pVMt was injected between the second and third rib with a 1 mL syringe, the depth being controlled to ≤2.5 mm. After injection, the skin was closed.
CVB3 loading in myocardial tissue
CVB3 loading in myocardial tissue was determined by TCID50 assay of HeLa cell monolayers according to previously published procedures . Briefly, the myocardial tissue was weighed, ground, and suspended in 10 times its volume of Eagle minimum essential medium. The myocardial tissue suspension was frozen at −70 °C for 15 min and then thawed at room temperature for 20 min three times. Finally, the myocardial tissue suspension was centrifuged at 2500 g for 30 min. The supernatant was harvested and diluted to varying degrees (10-1 to 10-10) for infection of HeLa cell monolayers. The TCID50 of CVB3 was calculated by the Reed–Muench method .
For histological analysis, the hearts were fixed in 10% neutral buffered formalin, sectioned into 5 mm slices and stained with hematoxylin and eosin. Two investigators who were blind to the experimental treatment assessed the severity of myocardial necrosis and number of infiltrating mononuclear cells in five fields for each sample. The number of infiltrating mononuclear cells is presented as the mean cell count in five fields. The myocardial lesions were given a PS by grading them from 0 to 4 according to the extent of myocardial necrosis and infiltration of inflammatory cells . Score 0 indicates no myocardial lesions. Scores 1–4 denote myocardial necrosis and infiltration of inflammatory cells affecting <25%, 25–50%, 50–75% and >75% of the myocardium, respectively.
The amount of CK-MB in serum
Matrix liquid (2.5 mL) was added to 100 µL serum and incubated at 37 °C for 10 min, after which colorimetry was performed for to measure amount of CK-MB.
The obtained experimental data is presented as mean ± SE and SPSS version 10.0 software was used to perform Student's t-test.
The impact of tMCP-1 on the chemoattractant activity of MCP-1
To investigate the biological activity of tMCP-1, pVMt was transfected into the C2C12 cell line. The tMCP-1 concentrations in the culture supernatant of C2C12 were 1002 ± 12, 1133 ± 11 and 1098 ± 14 pg/mL, 24, 48 and 72 hr after transfection, respectively, indicating that pVMt can express tMCP-1 in eukaryocytes.
To further investigate the effect of tMCP-1 on MCP-1, a chemotaxis assay was performed. As shown in Table 1, varying concentrations of tMCP-1 were used as the chemotatic media for the chemotaxis assay, together with 10 ng/mL of MCP-1. It was found that the cells were less strongly chemoattracted to tMCP-1 than to MCP-1 alone. Additionally, 25 ng/mL tMCP-1 blocked the chemoattractant activity of 10 ng/mL MCP-1 to PBMCs of mice with viral myocarditis.
The effects of tMCP-1 on the pathogenesis of viral myocarditis
To determine appropriate doses of tMCP-1 with which to inoculate mice with viral myocarditis, mice were injected intracardially with 20, 40 or 60 µg pVMt on the third day after CVB3 infection. Serum CK-MB titers and PS were less in mice that received 20, 40 or 60 µg pVMt (P < 0.05) than in mice in the viral myocarditis group infected with CVB3 alone. In addition, significantly fewer mononuclear cells infiltrated the myocardium in mice that received 40 and 60 µg pVMt than in mice in the viral myocarditis group infected with CVB3 alone (P < 0.01). However, there were no significant differences between any groups of mice in titers of CVB3 (P > 0.05) (Table 2, Fig. 1).
|Groups||n||CK-MB(U/L)||Titer of CVB3||PS (lgTCID50)||Infiltrating mononuclear cells in myocardium (cells/field)|
|Untreated control group||5||118.66 ± 9.82||5.23 ± 1.17||2.52 ± 0.56||17.32 ± 1.53|
|20 µg pVMt||5||100.55 ± 7.89 *||4.77 ± 1.05||2.12 ± 0.65 *||15.63 ± 0.89|
|40 µg pVMt||5||78.78 ± 8.66**||3.79 ± 0.79||1.77 ± 0.46 *||8.55 ± 1.65**|
|60 µg pVMt||5||80.52 ± 9.37**||4.85 ± 0.95||1.83 ± 0.71*||9.32 ± 1.21**|
|Normal control||5||26.53 ± 5.89**||ND||ND||ND|
Comparison of the effects of anti-MCP-1 antibody and tMCP-1 on the pathogenesis of viral myocarditis
On Days 3 and 5 after CVB3 infection, the mice in the anti-MCP-1 antibody intervention group were inoculated intramyocardially with 50 µg anti-MCP-1, whereas the mice in the antibody intervention control group were inoculated with an equal amount of goat IgG. On the third day after CVB3 infection, the mice in the tMCP-1 intervention group were inoculated intramyocardially with 40 µg pVMt, whereas the mice in the tMCP-1 intervention control group received an equivalent amount of plasmid pVAX1. Nine days after CVB3 infection, HW/BW, CK-MB, PS and the number of infiltrating monocytes in the myocardiums of mice in all groups were assessed. No significant differences were found between the anti-MCP-1 antibody intervention group and tMCP-1 intervention group in any of these variables (P > 0.05) (Table 3, Fig. 2).
|Variable||Anti-MCP-1 antibody intervention group||tMCP-1 intervention group||P-value|
|HW (mg)||88.20 ± 2.03||89.10 ± 2.12||>0.05|
|BW (mg)||11.24 ± 1.41||12.25 ± 1.31||>0.05|
|HW/BW (×103)||7.61 ± 1.41||7.56 ± 1.46||>0.05|
|CK-MB (U/L)||76.74 ± 8.65||78.78 ± 8.66||>0.05|
|PS||1.72 ± 0.46||1.87 ± 0.48||>0.05|
|Infiltrating mononuclear cells in myocardium||10.58 ± 1.76||8.55 ± 1.65||>0.05|
The structure of chemokines comprises three distinct domains: (i) a highly flexible N-terminal domain, which is constrained by disulfide bonding between the N-terminal cysteine(s); (ii) a long loop that leads into three antiparallel β-pleated sheets; and (iii) an α-helix that overlies the sheets . Structure-function studies have revealed that the N-terminal region is important for receptor binding and activation . CCR2, the major receptor of MCP-1, is a G-protein-coupled receptor containing seven transmembrane domains and is expressed on monocytes, memory T cells, B cells and basophils in humans [16, 17]. After integrated MCP-1 binds CCR2, the signal transduction pathway is activated and cell migration accompanied by upregulation or activation of adhesion molecules, desensitization or internalization of receptors, cytoskeleton rearrangement, and so on occurs [18, 19]. If the structure of MCP-1 is altered, the interaction between MCP-1 and CCR2 is affected. The product of truncation of N-terminal residues 2–8 of human MCP-1 ([1 + 9–76]hMCP-1) (7ND) does not stimulate chemotaxis but retains high affinity for CCR2 . The binding affinity of a mutant containing a proline substitution for isoleucine at position 5 (I5P), which would be expected to disrupt the putative 310 helix observed in the crystal structure of MCP-1, does not change . Substitution of tyrosine 13 for an alanine (Y13A) causes almost complete loss of MCP-1 activity but high affinity for CCR2 is preserved . In addition, the binding activity to CCR2 of other mutants of MCP-1 such as K37, K38, Arg24, and Tyr28, is affected to varying degrees by the signal transduction pathway [23, 24]. Because the structure of murine MCP-1 has 66% amino acid identity with human MCP-1 , murine truncated MCP-1 in which N-terminal residues 2–8 (tMCP-1) are deleted was constructed and expressed in C2C12 in this study. We demonstrated that tMCP-1 is expressed in eukaryocytes. Moreover, chemotaxis assay showed that, in mice with viral myocarditis, fewer PBMCs are chemoattracted after tMCP-1 is added than with MCP-1 alone. In addition, 25 ng/mL tMCP-1 blocks the chemoattractant activity of 10 ng/mL MCP-1 to PBMCs of mice with viral myocarditis. We therefore infer that the effects of tMCP-1 and 7ND on MCP-1 are mediated by similar mechanisms . Our findings indicate a potential role for tMCP-1 in alleviating the cardiac lesions of viral myocarditis.
Monocyte chemotactic protein-1 plays an important role in several inflammatory diseases, including rheumatoid arthritis, atherosclerosis and inflammatory bowel disease [27-29]. Our previous study showed that administration of anti-MCP-1 antibody abolishes the effects of MCP-1 on chemotaxis of mononuclear cells in vitro and infiltration of mononuclear cells into the myocardium of CVB3-infected mice in vivo. These results strongly suggest that MCP-1 plays an important role in enhancing the migration and infiltration of mononuclear cells into the myocardiums of CVB3-infected mice. To study the effects of the MCP-1 analogue, tMCP-1, on the pathogenesis of viral myocarditis in vivo, 20, 40 or 60 µg pVMt coding tMCP-1 were injected into the myocardiums of CVB3-infected mice. We found that intramyocardial injection of 20, 40 or 60 µg pVMt resulted in lower serum CK-MB titers and PS than in untreated mice infected by CVB3 alone. There were also fewer mononuclear cells infiltrating the myocardium after intramyocardial injection of 40 or 60 µg pVMt. There were no differences in titers of CVB3 between any groups of mice. It is accepted that both viral infection and cellular immune injury contribute to the pathogenesis of the myocardial damage in viral myocarditis . In this study, we found that tMCP-1 alleviates myocardial damage, but does not induce efficient viral clearance, indicating that its effects are associated with weakened cellular immune responses. Our study also confirmed that there were significantly fewer infiltrating mononuclear cells in myocardial tissue and their numbers were not restored to normal after intracardiac injection of pVMt. This infers that upregulation of other chemokines in myocardial tissue, such as MCP-3, IP-10, and MCP-5, is involved in infiltration by mononuclear cells [5, 6].
Clinically, means of treating viral myocarditis include adrenal cortical hormones, antiviral drugs and specific antibodies. As is well known, specific antibodies are available for treatment of many diseases. However, because they are so expensive, their application is limited. Gene therapy, which has been developed in recent years, has several advantages including that it is low cost and simple to administer. In this study, we substituted pVMt coding tMCP-1 for anti-MCP-1 antibody in mice with viral myocarditis and found no differences between these treatments in HW/BW, PS and serum CK-MB titers in mice with viral myocarditis, which strongly suggests that tMCP-1 is a promising alterative to research for treatment of viral myocarditis. However, how to control the switch of the target gene is so far unknown.
This work was supported by Henan Province Research Project Grant (0496060902).
No authors have any competing financial interests.