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In vivo transfer of soluble TNF-alpha receptor 1 gene improves cardiac function and reduces infarct size after myocardial infarction in rats¹

Department of Molecular and Cellular Biology, Division of Molecular and Clinical Gerontology, Medical Institute of Bioregulation, Kyushu University, Beppu, 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. Email: massy{at}tsurumi.beppu.kyushu-u.ac.jp

SPECIFIC AIMS

Soluble TNF- receptor 1 (sTNFR1) is an extracellular domain of TNFR1 and an antagonist to TNF-. Recently, we demonstrated that transfection of sTNFR1 expression plasmid DNA to the heart reduced TNF- bioactivity in the heart and thereby protected the myocardium from acute myocardial infarction (AMI) in vivo. We hypothesized that anti-TNF therapy with sTNFR1 might also have effect on cardiac function since TNF- levels are increased in heart failure. In the present study, we examined effects of sTNFR1 on cardiac function and infarct size after myocardial infarction in rats.

PRINCIPAL FINDINGS

1. Plasmid construction and soluble TNF receptor 1 expression
Gene encoding a soluble form of rat type 1 TNF receptor (extracellular domain of rat type 1 TNF receptor) was produced using reverse transcription polymerase chain reaction (RT-PCR). Produced cDNA was amplified using primers designed to amplify sequences encoding the extracellular domain of rat type 1 TNF receptor. The PCR fragment was gel-purified using a QIAEX II gel extraction kit (QIAGEN, Germany) and cloned into the TA expression vector using pcDNA3.1/V5-His TOPO TA expression kit (Invitrogen). One of the TA expression vectors contained the PCR fragment was designed to express only soluble TNF receptor 1 (sTNFR1 plasmid), but the other TA expression vector contained the PCR fragment designed to express not only sTNFR1 but also V5 epitope and His6 (flag proteins) (sTNFR1-flag plasmid). Male Wistar rats (250–300 g) were subjected to left coronary artery ligation. Rats were assigned in a random blind fashion to one of the following groups: 1) sTNFR1 plasmid administration, 2) sTNFR1-flag plasmid administration, 3) LacZ plasmid administration, 4) saline administration, or 5) sham operated. We administered each plasmid as a naked plasmid (total 150 µg) by direct injection to the left ventricular wall in 3 different sites (from base to apex) immediately after coronary artery ligation was performed. RT-PCR analyses showed that vector-derived sTNFR1 transcripts were present as early as 6 h and as late as 21 days after i.m. injection of sTNFR1 plasmid (Fig. 1 A). These transcripts were absent in both LacZ plasmid-treated rats and saline-treated rats (data not shown). Since the sTNFR1 primer set amplified the sTNFR1 product from cDNA derived from the vector but not from endogenous sTNFR1 cDNA, these findings confirmed successful transfection of the sTNFR1 gene into cardiomyocytes. From 6 h to 21 days after plasmid injection, plasmid-derived proteins (35 kDa) were also expressed in muscle injected with sTNFR1-flag plasmid when anti-V5-HRP antibody was used in order to exclude endogenous sTNFR1 protein (Fig. 1B ).

View larger version (18K): [in this window] [in a new window]   Figure 1. A) RT-PCR analysis after i.m. injection of sTNFR1 plasmid DNA. Detection of vector-derived sTNFR1 transcript by RT-PCR from 6 h to 21 days after plasmid injection. Lane 1: 6 h, lane 2: 7 days, lane 3: 14 days, lane 4: 21 days, lane 5: molecular marker. Figure shows a representative example of multiple analyses (4 experiments at 6 h and 6 experiments at 7, 14, and 21 days). B) Tissues injected with sTNFR1-flag plasmid were lysed, and equal amounts of lysates were fractionated on 12.5% SDS-PAGE. Western blot was probed with anti-V5-HRP antibody. Lane 1: 6 h, lane 2: 7 days, lane 3: 14 days, lane 4: 21 days. Figure shows a representative example of multiple analyses (4 experiments at each time). C) TNF- bioactivity was determined by cytotoxicity assay of WEHI cell line. Obtained values are expressed as means ± SEM(pg/mg, prot., n=6 in LacZ and sTNFR1-treated rats at each time, and n=4 in sham operated rats at each time). a: P < 0.0001, compared with rats receiving LacZ plasmid.

2. TNF- bioactivity and apoptosis
From 1 to 21 days after LCA ligation, TNF- bioactivity in the heart was significantly higher in rats receiving LacZ plasmid than in sham-operated rats (Fig. 1C ). However, TNF- bioactivity in hearts injected with sTNFR1 plasmid was significantly lower throughout the experiment than in hearts injected with LacZ plasmid (Fig. 1C ). No difference was observed between rats treated with LacZ plasmid and those treated with saline (data not shown). From 1 to 21 days after LCA ligation, internucleosomic fragmentation in the heart was prevented by sTNFR1 plasmid treatment (data not shown). Caspase-3 and Bax in the heart were also lower in rats receiving sTNFR1 plasmid than in those receiving LacZ plasmid (data not shown).

3. Effects of sTNFR1 on cardiac function and infarct size
To examine whether sTNFR1 improved cardiac function, we measured the LV end-diastolic dimension (LVDd), the LV end-systolic dimension (LVDs) and fractional shortening (FS) by echocardiography. From 7 to 21 days after LCA ligation, LVDd and LVDs in rats treated with sTNFR1 plasmid were significantly lower than those in rats treated with LacZ plasmid (data not shown). From 1 to 21 days after LCA ligation, FS was significantly higher in rats treated with sTNFR1 plasmid than in those treated with LacZ plasmid (data not shown). Twenty-one days after LCA ligation, LV end diastolic pressure (LVEDP) was also significantly lower in rats treated with sTNFR1 plasmid than in those treated with LacZ plasmid. However, LV systolic pressure (LVSP) did not differ significantly between them (Table 1 ). sTNFR1 expression plasmid treatment significantly reduced the area of myocardial infarction at 21 days after coronary artery ligation (Table 1) . No difference was observed between rats treated with LacZ plasmid and those treated with saline (data not shown).

CONCLUSIONS AND SIGNIFICANCE

Transgenic mice with cardiac-specific overexpression of TNF- tend to present myocarditis, cardiomegaly, cardiac dysfunction, and congestive heart failure. Anti-TNF- therapy with adenovirus-mediated sTNFR1 improves cardiac function in transgenic mice with cardiac-specific overexpression of TNF-. Clinical data also show that the circulating level of TNF- is elevated after heart failure. In rat models with myocardial infarction, levels of TNF- gene expression and TNF- protein were elevated from the early stage of infarction and persisted in the heart over time. Plasma concentrations of TNF- are also persistently elevated in patients with a prior history of myocardial infarction. TNF- not only shows a negative inotropic effect on cardiomyocytes but also induces apoptosis of cardiomyocytes. Reduction of apoptosis of cardiomyocytes may reduce the area of myocardial infarction. In postmyocardial infarction it is speculated that elevated levels of TNF- bioactivity contribute not only to LV dysfunction but also to infarct size. The present study showed for the first time that delivery of sTNFR1 gene significantly reduced LVDd and LVEDP, and significantly increased FS in rats with postmyocardial infarction. Delivery of the sTNFR1 gene from early stage of infarction significantly reduced infarct size. In fact, internucleosomic fragmentation the heart was prevented by treatment of sTNFR1 plasmid Caspase-3 and Bax in the heart were also lower in rats receiving sTNFR1 plasmid than in those receiving LacZ plasmid. These findings suggest reduction of apoptosis in ischemic rat heart with sTNFR1 plasmid. We have shown that delivery of the sTNFR1 gene from the early stage of infarction improved LV function until 21 days after myocardial infarction in rats. It is possible that sTNFR1 might directly improve cardiac function. However, reduction of infarct size is also thought to contribute in part to improved LV function. These findings suggest potential importance for therapeutic use of sTNFR1 in treatment of myocardial infarction patients who usually suffer from heart failure. Recently, etanercept, a soluble p75 TNF receptor (soluble TNF receptor 2; sTNFR2) fusion protein, resulted in a significant improvement in LV structure and function in patients with advanced heart failure. More recently, E. S. Chung, et al. reported that short-term TNF antagonism with infliximab caused no improvement and high doses (10 mg/kg) adversely affected the clinical condition of patients with moderate to severe chronic heart failure. Infliximab is a human-murine chimeric monoclonal antibody that specifically and potently binds to and neutralizes the soluble TNF homotrimer and its membrane-bound precursor. Favorable results with therapeutic agents in experimental models of heart failure and/or ischemic heart might not be replicated in controlled clinical trials. It is possible that anti-TNF therapy with sTNFR1, sTNFR2 (etanercept), or antibody to TNF (infliximab) might have different effects on heart failure and/or ischemic heart disease. Soluble TNF receptor 1, as used in the present study, blocks binding of TNF to TNF receptor 1 which contains death domain (Fig. 2 ). Blocking binding of TNF to TNF receptor 1 might be more important for improving cardiac function and/or reducing infarct size. Further studies are called for to examine possible long-term benefit of anti-TNF therapies of various types in the clinical arena. In conclusion, in a rat model of myocardial infarction, the level of TNF- bioactivity in the heart increased from the early stage of infarction and remained elevated, which seemed to contribute in part to impairment of LV function and to increased infarct size (Fig. 2) . Suppression of TNF- bioactivity from the early stage of infarction with sTNFR1 plasmid improved cardiac function and reduced infarct size in rats with postmyocardial infarction (Fig. 2) .

View larger version (14K): [in this window] [in a new window]   Figure 2. Schematic diagram of hypothesized soluble TNF receptor 1 involvement in cardiac function and reduction of infarct size after myocardial infarction.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1148fje;

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