Tomoji Hata

Status of myocardial antioxidants in ischemia-reperfusion injury.

BACKGROUND Myocardial ischemia-reperfusion represents a clinically relevant problem associated with thrombolysis, angioplasty and coronary bypass surgery. Injury of myocardium due to ischemia-reperfusion includes cardiac contractile dysfunction, arrhythmias as well as irreversible myocyte damage. These changes are considered to be the consequence of imbalance between the formation of oxidants and the availability of endogenous antioxidants in the heart. OBSERVATIONS An increase in the formation of reactive oxygen species during ischemia-reperfusion and the adverse effects of oxyradicals on myocardium have now been well established by both direct and indirect measurements. Although several experimental studies as well as clinical trials have demonstrated the cardioprotective effects of antioxidants, some studies have failed to substantiate the results. Nonetheless, it is becoming evident that some of the endogenous antioxidants such as glutathione peroxidase, superoxide dismutase, and catalase act as a primary defense mechanism whereas the others including vitamin E may play a secondary role for attenuating the ischemia-reperfusion injury. The importance of various endogenous antioxidants in suppressing oxidative stress is evident from the depression in their activities and the inhibition of cardiac alterations which they produce during ischemia-reperfusion injury. The effects of an antioxidant thiol containing compound, N-acetylcysteine, and ischemic preconditioning were shown to be similar in preventing changes in the ischemic-reperfused hearts. CONCLUSIONS The available evidence support the role of oxidative stress in ischemia-reperfusion injury and emphasize the importance of antioxidant mechanisms in cardioprotection.

Interaction of mRNAs for Angiotensin II Type 1 and Type 2 Receptors to Vascular Remodeling in Spontaneously Hypertensive Rats

We administered angiotensin II (Ang II) receptor type 1 (AT1) blockade (losartan, 40 mg x kg-1 x d-1), type II receptor (AT2) blockade (PD123319, 100 mg x kg-1 x d-1), or angiotensin-converting enzyme (ACE) inhibitor (enalapril, 30 mg x kg-1 x d-1) to spontaneously hypertensive rats (SHR) from 10 to 20 weeks of age. Control SHR and Wister-Kyoto rats (WKY) received a placebo for the same period. At the end of treatment, losartan and enalapril were both found to have significantly reduced the arterial systolic blood pressure and the collagen concentration to the level of WKY, whereas PD123319 had no effect. Enalapril and PD123319 significantly reduced the media cross-sectional area of the aorta in comparison to that of untreated SHR, which was still larger than that of the WKY; however, losartan did not change it. Using reverse transcription-polymerase chain reaction, we next examined the mRNA expressions for ACE, AT1 receptor, and AT2 receptor in experimental animals. We observed significantly enhanced mRNA expression for AT1 and AT2 receptors and ACE in untreated SHR compared with WKY. The AT1 mRNA level was also significantly decreased in the SHR treated with either losartan or enalapril, whereas the AT2 mRNA level was significantly decreased in the SHR treated with either PD123319 or enalapril in comparison to untreated SHR. The level of ACE mRNA was significantly decreased only in the SHR treated with enalapril. These results indicate that AT1 receptor, but not AT2 receptor, plays a crucial role in the remodeling of matrix tissue, while AT2 receptor may play a role in the development of hypertrophy of smooth muscle in aorta in SHR, and that the reduction of hypertrophy of smooth muscle does not fully account for the suppression of hypertension.

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

Increased circulating and cardiac TNF‐alpha levels during myocardial ischemia have been found in both experimental animals and patients with ischemic heart disease and advanced heart failure. Soluble TNF‐alpha receptor 1 (sTNFR1) is an antagonist to TNF‐alpha. In the present study, we examined whether sTNFR1 improves cardiac function in rats after myocardial infarction. Male Wistar rats were subjected to left coronary artery (LCA) ligation. Immediately after the ligation, a total of 200 μg of either the sTNFR1 or LacZ plasmid was injected into three different sites in the left ventricular wall. From 1 to 21 days after LCA ligation, TNF‐alpha bioactivity in the heart was higher in rats receiving LacZ plasmid than in sham‐operated rats, whereas sTNFR1 plasmid significantly suppressed the increase. The LV diastolic dimension was significantly lower, and the fractional shortening was significantly higher in rats treated with the sTNFR1 plasmid than in those treated with the LacZ plasmid. At 21 days after LCA ligation, the LV end‐diastolic pressure was also significantly lower in the rats treated with the sTNFR1 plasmid. In addition, the sTNFR1 expression plasmid had significantly reduced the infarct size. In conclusion, TNF‐alpha bioactivity in the heart increased during the early stage of infarction and remained elevated. This elevation seemed partially responsible for the impairment of LV function and the increased infarct size. Suppression of TNF‐alpha bioactivity from the early stage of infarction with the sTNFR1 plasmid improved cardiac function and reduced infarct size.

Molecular Mechanism of Angiotensin II Type I and Type II Receptors in Cardiac Hypertrophy of Spontaneously Hypertensive Rats

We administered angiotensin (Ang) II receptor type 1 (AT1) blockade (losartan; 10 or 40 mg/kg per day), type II receptor (AT2) blockades (PD123319; 100 mg/kg per day), or angiotensin-converting enzyme (ACE) inhibitor (enalapril; 30 mg/kg per day) to spontaneously hypertensive rats (SHR) from 10 to 20 weeks of age. At the end of the treatment, high doses of losartan and enalapril significantly reduced the arterial systolic blood pressure compared with the untreated SHR to the level of WKY rats. But low doses of losartan and PD123319 were without effect. High doses of losartan and enalapril also significantly reduced both the left ventricular (LV) weight and the ratio of LV to body weight compared with the untreated SHR, which were still larger than that of WKY rats. However, the collagen concentration of SHR treated with high doses of losartan or enalapril was completely reduced to the level of WKY rats. Using reverse transcription polymerase chain reaction, we examined the mRNA expression for ACE, AT1, and AT2 in experimental animals. The enhanced AT1 mRNA expression was significantly decreased in the SHR treated with a high dose of losartan or PD123319 compared with the untreated SHR. The level of ACE mRNA was also decreased in the SHR treated with a high dose of losartan or enalapril. The level of AT2 mRNA was not significantly different between the Wistar-Kyoto rats and the SHR; however, this expression was decreased significantly after the treatment with a high dose of losartan or PD123319. These results indicate that AT1 receptor and ACE, but not AT2 receptor, play a crucial role in the remodeling of matrix tissue but a smaller role in the development of the hypertrophy of LV myocyte in SHR and that the LV/body weight changes do not fully account for the complete suppression of hypertension.

Local delivery of soluble TNF-alpha receptor 1 gene reduces infarct size following ischemia/reperfusion injury in rats.

Apoptosis in the myocardium is linked to ischemia/reperfusion injury, and TNF-alpha induces apoptosis in cardiomyocytes. A significant amount of TNF-alpha is detected after ischemia and reperfusion. Soluble TNF-alpha receptor 1 (sTNFR1) is an extracellular domain of TNF-alpha receptor 1 and is an antagonist to TNF-alpha. In the present study, we examined the effects of sTNFR1 on infarct size in acute myocardial infarction (AMI) following ischemia/reperfusion. Male Wistar rats were subjected to left coronary artery (LCA) ligation. After 30 min of LCA occlusion, the temporary ligature on the LCA was released and blood flow was restored. Immediately after reperfusion, a total of 200 μg of sTNFR1 or LacZ plasmid was injected into three different sites of the left ventricular wall. At 6 h, 1 and 2 days after reperfusion, the TNF-alpha bioactivity in the myocardium was significantly higher in rats receiving LacZ plasmid than in sham-operated rats, whereas sTNFR1 plasmid significantly suppressed the increase in the TNF-alpha bioactivity. The sTNFR1 plasmid significantly reduced DNA fragmentation and caspase activity compared to the LacZ plasmid. Finally, the sTNFR1 expression-plasmid treatment significantly reduced the area of myocardial infarction at 2 days after ischemia/reperfusion compared to LacZ plasmid. In conclusion, the TNF-alpha bioactivity in the heart increased from the early stage of ischemia/reperfusion, and this increase was thought to contribute in part to the increased area of myocardial infarction. Suppression of TNF-alpha bioactivity with the sTNFR1 plasmid reduced the infarct size in AMI following ischemia and reperfusion (Mol Cell Biochem 266: 127–132, 2004)

RNA interference targeting SHP-1 attenuates myocardial infarction in rats

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. Here we report that siRNA targeting SHP‐1 reduced infarct size in a rat model of AMI. Upon injection into the ischemic left ventricular wall, the vector‐based siRNA significantly suppressed the increase in the SHP‐1 mRNA and the SHP‐1 protein levels. The siRNA vector also significantly reduced the SHP‐1 that bound to Fas‐R. The SHP‐1 siRNA vector increased phospho‐Akt and reduced DNA fragmentation and caspase activity compared with the scramble siRNA vector. Finally, the area of myocardial infarction was significantly smaller with the SHP‐1 siRNA vector than with the scramble siRNA vector at 2 days after LCA ligation. In conclusion, SHP‐1 in the heart increased from the early stage of AMI, and this increase was thought to contribute to the increased area of myocardial infarction. Suppression of SHP‐1 with the SHP‐1 siRNA vector markedly reduced the infarct size in AMI.

Interleukin 1α-induced expression of manganous superoxide dismutase reduces myocardial reperfusion injury in the rat

We investigated the effects of pretreatment with interleukin(IL)-1α on the expression of manganous (Mn) superoxide dismutase (DOD) mRNA and reperfusion-induced arrhythmias and the size of myocardial infarct in rats. Male Wistar rats received 10 mg intraperitoneal injetions of human recombinant IL-1α. Their hearts were thereafter isolated at 6, 12, 24, 36 h. A Northern analysis showed that Mn-SOD mRNA was mainly expressed in the heart and slightly in kidney, but not in any other organs. The expression of Mn-SOD mRNA peaked at 6 h after the injection of IL-1α. The Mn-SOD protein content was most increased 12 h after injection. In the isolated heart model, the rats were pretreated with Il-1α 24 h earlier and their hearts were perfused by the Langendorff method. After 20 min of ischemia which was induced by a ligation of a coronary artery, reperfusion-induced arrhythmias were observed. There we no significant differences in the incidence of ventricular arrhythmias between the IL-1α pretreated and the untreated hearts. IL-1α pretreatment significantly reduced the mean duration of the ventricular arrhythmias and also delayed the onset of arrhythmias. The effect of IL-1α pretreatment was also investigated in a 30-min model of ischemia followed by a 3-min reperfusion in anesthetized rats. The infarct size expressed as as percentage of the area at risk was significantly reduced in the IL-1α pretreated hearts compared with the untreated hearts. The left ventricular systolic pressure increased significantly in rat hearts pretreated with IL-1α. Our results therefore showed that the pretreatment with IL-1α induced the overexpression of Mn-SOD mRNA in the rat hearts and also suggested that pretreatment with IL-1α 24 h before ischemia reduced the risk of ischemia-reperfusion injury.

In vivo gene transfer of soluble TNF-alpha receptor 1 alleviates myocardial infarction.

Apoptosis is the major independent form of cardiomyocyte cell death in acute myocardial infarction (AMI). TNF‐α release early in the course of AMI contributes to myocardial injury, and TNF‐α induces apoptosis in cardiomyocytes. Soluble TNF‐alpha receptor 1 (sTNFR1) is an antagonist to TNF‐α. However, the effect of sTNFR1 on AMI remains unclear. Here we report that direct injection of an sTNFR1 expression plasmid DNA to the myocardium reduces infarct size in experimental rat AMI. Treatment with sTNFR1 expression plasmid DNA reduced the TNF‐α bioactivity in the myocardium and the apoptosis of cardiomyocytes. These findings suggest that the anti‐TNF‐α therapy by sTNFR1 can be a new strategy for treatment of AMI.

Chronic antisense therapy for angiotensinogen on cardiac hypertrophy in spontaneously hypertensive rats.

OBJECTIVE We examined the effect of the suppression of plasma angiotensinogen (AGT) by the intravenous injection of antisense oligodeoxynucleotides (ODNs) against AGT targeted to the liver on cardiac remodeling in spontaneously hypertensive rats (SHR). The ODNs against rat AGT were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method for regulating liver gene expression. METHODS Male SHR (n = 18), and age-matched male Wistar-Kyoto rats (WKY, n = 6) were used for this study. At 10 weeks of age, the SHR were divided into three groups (each group n = 6), and the systolic blood pressure (SBP) did not significantly change among them. The control SHR and WKY groups received saline, the sense SHR group was injected with the sense ODNs complex and the antisense SHR group was injected with the antisense ODNs complex, from 10 to 20 weeks of age. ASOR-poly(L)lysine-ODNs complex was injected via the tail veins twice a week. RESULTS At the end of the treatment, a reduction of hepatic AGT mRNA, cardiac angiotensin II type 1 receptor mRNA and the plasma AGT concentration was only observed in the antisense-injected SHR but not in the other groups of SHR and WKY. This antisense therapy did not significantly change the mRNA expression for angiotensin converting enzyme, angiotensin II type 2 receptor and AGT in the left ventricle (LV) among all three groups. Although the plasma angiotensin II (Ang II) concentration significantly decreased to the level of WKY after the antisense therapy, the SBP, LV to body weight ratio and % collagen volume fraction also showed a reduction, however, these findings were still larger than in the WKY than in either the sense-injected SHR or control SHR. CONCLUSION The plasma AGT is considered to play a role in the development of cardiac hypertrophy in SHR, but it has not a complete effects on cardiac remodeling even if the plasma Ang II levels are inhibited because of an insufficient suppression of hypertension.

Influence of oxygen free radicals on heart sarcolemmal Na+-ca2+ exchange

Because oxygen free radicals are considered to play an important role in myocardial injury due to ischemia-reperfusion and because sarcolemmal Na+-Ca2+ exchange is markedly altered in ischemic or hypoxic-reperfused hearts, this study was undertaken to investigate the effects of oxygen free radicals on rat heart sarcolemmal Na+-Ca2+ exchange activity. Incubation of sarcolemma for 10 minutes with 2 mM xanthine plus 0.03 U/mL xanthine oxidase inhibited the Na+-dependent Ca2+ uptake activity. This change was associated with significant formation of malondialdehyde. These alterations were significantly prevented by 80 μg/mL superoxide dismutase, a scavenger of superoxide anions, but were completely prevented by a combination of superoxide dismutase (80 μg/mL) and catalase (10 μg/ml). Inhibitory effects on Na+-Ca2+ exchange and increased formation of malondialdehyde were observed with 0.5 mM H2O2 or 0.01 mM H2O2 plus 0.01 mM Fe2+ and were prevented by 10 μg/mL catalase, a scavenger of H2O2, and 2 mM mannitol, a scavenger of hydroxyl radicals, respectively. The depressant effects of all these activated types of oxygen were not associated with any changes in nonspecific Ca2+ uptake, passive Ca2+ permeability or passive Na+ permeability of the sarcolemmal vesicles. The decrease of Na+-dependent Ca2+ uptake by these free radical systems was also evident when the sarcolemmal vesicles (prepared by different methods of isolation from hearts of different animal species) were employed. These results indicate that the sarcolemmal membrane is altered on exposure to oxygen free radicals and this may depress the Na+-Ca2+ exchange system in the myocardial cell under different pathophysiologic conditions.