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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 13, 2005 as doi:10.1096/fj.05-4020fje. |
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Department of Molecular and Cellular Biology, Division of Molecular and Clinical Gerontology, Medical Institute of Bioregulation, Kyushu University, Beppu, Oita, Japan
1Correspondence: 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
The Src homology domain 2 (SH2) -containing tyrosine phosphatase-1 (SHP-1) plays a key role in apoptosis and decreases phosphorylation of Akt. Apoptosis of cardiomyocytes is thought to contribute to the increased area of acute myocardial infarction (AMI) and Akt activation exerts a powerful cardioprotective effect after ischemia. Thus, a therapeutic strategy designed to inhibit expression of SHP-1 would be beneficial in AMI. In the present study, we examined the effects of SHP-1 on infarct size in AMI using small interfering RNA (siRNA) targeting the SHP-1 gene.
PRINCIPAL FINDINGS
1. We confirmed ischemia of the regional myocardia by monitoring the change of the ST segment level on the electrocardiogram and by observing the change in the color of the myocardia
Male Wistar rats (250–300 g) were subjected to left coronary artery ligation as described previously. Rats were assigned in a random blind fashion to one of four groups: 1) SHP-1 siRNA vector administration, 2) scramble siRNA vector administration, 3) saline administration, or 4) sham operated. We administered each vector (total 100ug) by direct injection to the left ventricular wall in three different sites (from base to apex) immediately after the coronary artery ligation was performed.
2. The amount of SHP-1 mRNA increased in the myocardium injected with the SHP-1 siRNA vector and the scramble siRNA vector, compared with the amount in sham-operated rats
Throughout the experiment, the SHP-1 siRNA vector treatment significantly reduced SHP-1 mRNA (data not shown). The amount of SHP-1 mRNA in muscles was not significantly different between treatment with the scramble siRNA and treatment with saline throughout the experiment (data not shown).
3. Western blot analysis also showed that SHP-1 was higher in both the myocardium injected with the SHP-1 siRNA vector and that injected with the scramble siRNA vector, compared with the amount in sham-operated rats (Fig. 1
)
Throughout the experiment, the SHP-1 siRNA vector treatment reduced SHP-1 (Fig. 1)
. In contrast, the amount of SHP-2 did not differ between rats treated with the SHP-1 siRNA vector and those treated with the scramble siRNA vector throughout the experiment (Fig. 1)
. Since SHP-1 decreases the phosphorylation of Akt and Akt activation exerts a powerful cardioprotective effect after ischemia, we examined the phosphorylation of Akt in the myocardium injected with the SHP-1 siRNA vector. Throughout the experiment, the SHP-1 siRNA vector induced a greater level of phosphorylation of Akt than the scramble siRNA vector (Fig. 1)
.
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4. Fas-R was immunoprecipitated from the hearts to determine the association of SHP-1 with Fas-R
The siRNA vector treatment significantly reduced the amount of SHP-1 that bound to Fas-R (data not shown). Because SHP-1 is required for FAS-mediated cytotoxic signaling that causes intracellular acidification and apoptosis, we proposed that the reduction in SHP-1 recruitment has an anti-apoptotic effect. Apoptosis was confirmed by DNA fragmentation using 1.5% TraviGel 500 gel electrophoresis (data not shown). The internucleosomic fragmentation in cardiomyocytes was prevented by the treatment of the SHP-1 siRNA vector (data not shown). A caspase activity assay in the myocardium showed that the caspase activity was significantly lower in rats receiving the SHP-1 siRNA vector than in those receiving the scramble siRNA vector (data not shown).
5. The SHP-1 siRNA vector markedly reduced the area of myocardial infarction 2 days after coronary artery ligation (Fig. 2 a, b), whereas no difference was observed between rats treated with the scramble siRNA vector and those treated with saline
To examine whether the SHP-1 siRNA vector improved cardiac function, we measured the LV end-diastolic dimension (LVDd), the LV end-systolic dimension (LVDs) and fractional shortening (FS) by echocardiography. One and 2 days after the LCA ligation, the LVDd was significantly lower and the FS was significantly higher in rats treated with SHP-1 siRNA vector than in those treated with the scramble siRNA vector (data not shown).
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CONCLUSIONS AND SIGNIFICANCE
It is known that the attenuation of cardiomyocyte apoptosis reduces infarct size in vivo. The Src homology domain 2 (SH2) -containing tyrosine phosphatase-1 (SHP-1), a cytoplasmic protein tyrosine phosphatase, plays a negative regulatory role in signal transduction pathways by dephosphorylation of the receptors to which it binds. SHP-1 also has a key role in apoptosis in many cells, and death receptors such as TNFR1 and FAS-R bind SHP-1 and block anti-apoptotic signals in neutrophils. Thus, SHP-1 is thought to play an important role in apoptosis and/or the blocking of survival factors. It is known that SHP-1 protein levels are up-regulated after cerebral ischemia, which may contribute to increased cerebral infarction. However, there have been no studies that have examined whether SHP-1 levels are increased in AMI. In the present study, we have shown for the first time that SHP-1 protein levels are up-regulated in AMI. We thus hypothesized that in AMI, the increase in SHP-1 might stimulate apoptosis of cardiomyocytes, which in part contribute to increased infarct size. The in vivo regulation of various genes by RNAi has been achieved using these vectors or viruses, or by using a high pressure technique. In the present study, we used a vector-based siRNA targeting SHP-1, which was able to achieve a stable expression of siRNA, to reduce SHP-1. The myocardium injected with the SHP-1 siRNA vector showed a significant decrease in SHP-1 mRNA as early as 1 day after injection, and continuing until 2 days. Successful transfection also resulted in a reduction in SHP-1 protein expression in the myocardium injected with the SHP-1 siRNA vector. In the present study, association of SHP-1 with Fas-R was suppressed with SHP-1 siRNA vector. SHP-1 is activated in the Fas receptor-mediated signaling in hematopoietic cells. In addition, SHP-1 is required for the FAS-mediated cytotoxic signaling that causes intracellular acidification and apoptosis in breast adenocarcinoma cells, and recruitment of SHP-1 to Fas-R blocks anti-apoptotic signals in neutrophils. Thus, reduced SHP-1 recruitment to Fas-R may have had an anti-apoptotic effect on cardiomyocytes in the present study. In fact, Fas is known to induce apoptosis in cardiomyocytes, possibly due to stimulation of Fas-R, and Fas is a critical mediator of acute myocardial infarction (AMI) due to ischemia-reperfusion in vivo . In the present study, the internucleosomic fragmentation in the heart was prevented by treatment with the SHP-1 siRNA vector. The caspase-3 activity in the heart was also lower in rats receiving the SHP-1 siRNA vector than in those receiving the scramble siRNA vector until 2 days after the vector injection. Caspase-3 has been reported to be involved in apoptotic cell death in ischemic rat hearts. Thus, these findings indicate that apoptosis in the ischemic rat heart is reduced by SHP-1 siRNA vector. Akt activation exerts a cardioprotective effect after ischemia that probably reflects its ability to both inhibit cardiomyocyte death and improve the function of surviving cardiomyocytes. Transplantation of mesenchymal stem cells overexpressing Akt into the ischemic rat myocardium can repair infarcted myocardium, prevent remodeling and nearly normalize cardiac performance. In the present study, the SHP-1 siRNA vector significantly increased phosphorylation of Akt throughout the experiment. In the present study, treatment with the SHP-1 siRNA vector suppressed the increase in SHP-1 in myocardial tissue, which was thought to reduce the apoptosis of cardiomyocytes and activate Akt. These effects may have contributed to the reduction of the area of myocardial infarction. In conclusion, the present study showed for the first time the potential therapeutic value of the anti-SHP-1 therapy in treating AMI.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4020fje;
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