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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 1, 2002 as doi:10.1096/fj.01-0894fje. |
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receptor 1 alleviates myocardial infarction1
Department of Molecular and Cellular Biology, Division of Molecular and Clinical Gerontology, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumihara, Beppu, Oita, 874-0838, Japan
2Correspondence: Department of Molecular and Cellular Biology, Division of Molecular and Clinical Gerontology, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumihara, Beppu, Oita, 874-0838, Japan. E-mail: massy{at}tsurumi.beppu.kyushu-u.ac.jp
SPECIFIC AIMS
Inflammation plays a critical role in acute myocardial infarction (AMI) and tumor necrosis factor 
(TNF-) is a potent inflammatory trigger. TNF- increases in AMI and contributes to myocardial injury. TNF- also induces apoptosis in cardiomyocytes. Soluble TNF- receptor 1 (sTNFR1) is an antagonist to TNF-. However, the effect of sTNFR1 on AMI remains unclear. Accordingly, we hypothesized that transfection of sTNFR1 expression plasmid DNA to the myocardium would reduce the apoptosis of cardiomyocytes and thereby protect myocardium from infarct in vivo.
PRINCIPAL FINDINGS
1. The gene encoding a soluble form of the rat type 1 TNF receptor (extracellular domain of the rat type 1 TNF receptor) was produced using reverse transcription polymerase chain reaction (RT-PCR). The PCR fragment was gel-purified using a QIAEX II gel extraction kit and cloned into the pCRII TA cloning vector. The recombinant pCRII plasmid was digested with HindIII and NotI, and the 727-base pair fragment was ligated to HindIII/NotI-digested pcDNA3.1 plasmid (pcDNA-sTNFR1 plasmid). The pcDNA3.1 plasmid alone was used as a control vector (pcDNA-null plasmid). Male Wistar rats (250–300 g) were subjected to left coronary artery ligation. Rats were assigned in a random blind fashion as follows: 1) pcDNA-sTNFR1 plasmid administration, 2) pcDNA-null plasmid administration, 3) saline administration, or 4) sham operated. We administered each plasmid as a naked plasmid (total 150 µg) by direct injection to the left ventricular wall in three different sites (from base to apex) immediately after coronary artery ligation. The sTNFR1 expression plasmid treatment significantly reduced the area of myocardial infarction 2 days after coronary artery ligation (Fig. 1
A, B), whereas no difference was observed in rats treated with pcDNA-null plasmid or saline (data not shown).
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2. The cardiomyocytes transfected with the sTNFR1 expression plasmid cannot retain the sTNFR1 produced by the gene since the plasmid DNA does not encode the intracellular domain of the TNFR1. A TNF- bioactivity assay confirmed that bioactivity in the myocardium significantly increased in rats receiving pcDNA-null plasmid compared with that in sham-operated rats, whereas treatment with the pcDNA-sTNFR1 plasmid significantly reduced it (Fig. 2
). No difference was observed between rats treated with pcDNA-null plasmid and those treated with saline (data not shown).
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3. The pcDNA-sTNFR1 plasmid gene delivery significantly reduced the percentage of TUNEL-positive staining of cardiomyocytes in the area at risk compared with the pcDNA-null plasmid (32.5±4.8% in pcDNA-sTNFR1 plasmid-treated group and 58.3±6.4% in pcDNA-null plasmid-treated group, n=6 in each group, P<0.01). Apoptosis was also confirmed by DNA fragmentation by 1.5% TraviGel 500 gel electrophoresis, which showed that DNA ladder was prevented by treatment with the pcDNA-sTNFR1 plasmid (data not shown). Caspase-3 activity was determined by Enzchek Caspase-3 assay kit from Molecular Probes. A caspase activity assay in the myocardium showed that caspase activity was significantly lower in rats receiving the pcDNA-sTNFR1 plasmid than those receiving pcDNA-null plasmid (11.03±0.79 in pcDNA-sTNFR1 plasmid-treated group and 16.28±0.95 in the pcDNA-null plasmid-treated group). Values showed the pmol of DEVD-AMC cleavage per min per mg protein and are expressed as mean ± SE n = 6 in each group, P < 0.01.
CONCLUSION AND SIGNIFICANCE
It is known that TUNEL-positive staining of cardiomyocytes is increased in ischemia/reperfusion injury. Hypoxia not only causes necrosis of the myocardium but also activates the suicide program of neonatal cardiac myocytes. The present study showed that sTNFR1 gene delivery reduced the number of these apoptotic cells. Given that apoptosis is the major independent form of cardiomyocyte cell death in AMI, with necrosis running a close second, it is possible that reduction in the apoptosis of cardiomyocytes reduced the area of myocardial infarction. TNF- bioactivity in the myocardium significantly increased in rats receiving pcDNA-null plasmid and saline compared with that in sham-operated rats, whereas treatment with the pcDNA-sTNFR1 plasmid significantly reduced the bioactivity. These findings agree those from previous studies: TNF- increased in AMI and contributed to myocardial injury and sTNFR1 antagonized TNF-. Inflammation plays a critical role in AMI, and TNF- is a potent inflammatory trigger. TNF- also induces apoptosis in cardiac myocytes. In the present study, the cardiomyocytes transfected with the sTNFR1 expression plasmid could not retain the sTNFR1 produced by the gene since the plasmid DNA does not encode the intracellular domain of the TNFR1. It is speculated therefore that the cardiomyocytes transfected with the sTNFR1 expression plasmid secreted the sTNFR1, which neutralized and/or stabilized TNF- and reduced the TNF- bioactivity. We showed that treatment with the sTNFR1 expression plasmid suppressed the increase in the TNF- bioactivity in myocardial tissue and reduced the apoptosis of cardiomyocytes, effects that could contribute to the reduction of the area of myocardial infarction. In an in vitro study, anti-TNF- antibody has been reported to neutralize local TNF- release from cardiac myocytes after ischemia and improve myocardial recovery during reperfusion, which is thought to be an anti-inflammatory effect of the antibody. It may be possible that sTNFR1 can have such an anti-inflammatory effect, which might contribute to the reduction of the infarct area seen in the present study. In conclusion, the present study showed for the first time the potential therapeutic value in the use of sTNFR1 in AMI. However, our study was limited to the period comprising only 2 days after the AMI. Therefore, to elucidate what the possible long-term benefit of anti-TNF therapies of this type offer in the clinical arena, further longer term studies are called for.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0894fje; to cite this article, use FASEB J. (July 1, 2002) 10.1096/fj.01-0894fje 
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