Inhibition of p53 by pifithrin-alpha reduces myocyte apoptosis and leukocyte transmigration in aged rat hearts following 24 hours of reperfusion.

Ischemic heart disease is a common age-related disease. Apoptotic cell death and inflammation are the major contributors to I/R injury. The mechanisms that trigger myocyte apoptosis and inflammation during myocardial I/R (MI/R) remain to be elucidated. Published data from our laboratory demonstrated that pretreatment of MI/R rats with pifithrin-alpha (PFT), a specific p53 inhibitor, reduced myocyte apoptosis and improved cardiac function compared with MI/R rats pretreated with saline at 4 h of reperfusion. In the present study, we investigated the effects of PFT on the occurrence of myocyte apoptosis and leukocyte transmigration in the later period of reperfusion. Aged (20-month-old) male F344 rats were subjected to 30 min of myocardial ischemia via ligature of the LCA, followed by 24 h of reperfusion. Pifithrin-alpha (2.2 mg/kg, intraperitoneally) or saline was administered to rats before ischemia. The results indicate that pretreatment of MI/R rats with PFT significantly decreased the percentage of infarct area to ischemic area (33 +/- 8 vs. 54 +/- 9, P < 0.05) and improved cardiac output (79 +/- 11 vs. 38 +/- 9 mL/min per 100 g body weight, P < 0.05) when compared with rats pretreated with saline at 24 h of reperfusion. The protective effects of PFT may involve the p53/Bax-mediated apoptosis because treatment of MI/R rats with PFT attenuated the ratio of Bax to Bcl2 (0.97 +/- 0.1 vs. 2.1 +/- 0.2, P < 0.05) and reduced myocyte apoptosis. Interestingly, inhibition of p53 transcriptional function by PFT alleviated leukocyte infiltration into the ischemic area of the heart (339 +/- 37 vs. 498 +/- 75 cells/10 high-power fields, P < 0.05). These data suggest that inhibition of p53 transcriptional function by PFT attenuates myocyte apoptosis and alleviates leukocyte transmigration at 24 h of reperfusion. The mechanisms by which p53 modulates leukocyte transmigration require further investigation.

Pifithrin-alpha attenuates p53-mediated apoptosis and improves cardiac function in response to myocardial ischemia/reperfusion in aged rats.

Ischemic cardiovascular disease is a common age-related disease. The p53-dependent cardiac myocyte apoptosis induced by myocardial ischemia/reperfusion (MI/R) is an important feature in the progression of ischemic heart disease. In the present studies, we hypothesized that inhibition of p53-dependent myocyte apoptosis may improve cardiac dysfunction in aged rats after MI/R. A dose (2.2 mg/kg, i.p.) of pifithrin-alpha (PFT), a p53 inhibitor, or saline was administered to 20-month-old male F344 rats, which were subjected to 30 min of myocardial ischemia by ligating the left main coronary artery, followed by release of the ligature and 4 h of reperfusion. Results of our experiments indicate that MI/R induced a significant decrease in cardiac output index (CI) and mean arterial blood pressure (MABP). Administration of PFT to aged rats 40 min before ischemia significantly improved CI and MABP during 3 to 4 h of reperfusion. The improvement of cardiac function was associated with a marked reduction in DNA fragmentation in the area at risk of the heart when compared with aged MI/R rats pretreated with saline. Interestingly, treatment with PFT 10 min after ischemia or 10 min after reperfusion had a similar protective effect on CI and MABP, but this effect did not reach statistical significance when compared with aged MI/R rats pretreated with saline. Treatment with PFT, however, did not influence plasma creatine kinase activity and the number of circulating leukocytes and infiltrated leukocytes in the area at risk of the heart. Moreover, results of Western blot show that pretreatment with PFT significantly attenuated the ratio of Bax to Bcl-2 in the area-at-risk tissue of the heart compared with that of rats pretreated with saline. Our results suggest that pretreatment with PFT significantly improved cardiac function. The mechanism of protective effect of PFT may involve the inhibition of p53 transcriptional function, thereby attenuating the p53/Bax-mediated myocyte apoptosis during the reperfusion period.

Attenuation of antioxidative capacity enhances reperfusion injury in aged rat myocardium after MI/R.

Mortality due to ischemic cardiovascular diseases is significantly higher in elderly than in young adults. Myocardial ischemia-reperfusion (MI/R) can induce oxidative stress and an inflammatory response. We hypothesized that increased vulnerability of aged myocardium to reperfusion injury could be caused by decreased antioxidative capacity, rather than increased oxidant production, after MI/R. Aged (20-mo-old) and young (4-mo-old) male F344BN rats were subjected to 30 min of myocardial ischemia by ligation of the left main coronary artery followed by release of the ligature and 4 h of reperfusion. Four experimental groups were studied: young sham-operated rats, aged sham-operated rats, young rats subjected to MI/R, and aged rats subjected to MI/R. MI/R significantly increased infiltrated leukocyte number and myeloperoxidase (MPO) activity in perinecrotic areas of hearts of young rats compared with aged MI/R rats. These changes in infiltrated leukocyte number and MPO activity were associated with an increase in superoxide generation in perinecrotic areas from hearts of young rats compared with aged rats. Plasma levels of TNF-alpha and IL-1beta were significantly higher in young than in aged MI/R rats. However, plasma 8-hydroxy-2'-deoxyguanosine levels and creatine kinase activity were increased in aged compared with young MI/R rats. Increased reperfusion damage in aged rats was associated with a significant decrease in plasma ratio of GSH to GSSG. Our results suggest that enhanced ischemia-reperfusion injury in aged rat hearts may be related to reduced antioxidative capacity, rather than increased reactive oxygen species production. These findings contribute to a better understanding of effects of aging on oxidative stress and inflammatory responses of the heart after MI/R.

Age-related difference in myocardial function and inflammation in a rat model of myocardial ischemia-reperfusion.

OBJECTIVE: Aging is associated with a reduced tolerance to myocardial ischemia reperfusion injury when compared to the young adult. However, there is very little information in the literature regarding age-related changes in myocardial function and inflammation during myocardial ischemia-reperfusion (MI/R) in vivo. METHODS: We examined age-related differences in myocyte apoptosis and the inflammatory response in a rat model of myocardial ischemia-reperfusion (MI/R). The aged (19 months) and young (4 months) male F344 BN rats were subjected to 30 min of myocardial ischemia by ligating the left main coronary artery, followed by release of the ligature and 4 h of reperfusion. Four experimental groups, e.g. young sham control, aged sham control, young rats subjected to MI/R, and aged rats subjected to MI/R, were studied. RESULTS: MI/R induced a 78% increase in circulating leukocytes and a 30% increase in superoxide generation in the ischemic region of the heart of young rats, when compared to aged rats. Moreover, the arrhythmia scores were higher in young rats than in aged rats (P=0.058) following MI/R. There was no difference in hemodynamics between young sham and aged sham rats. However, the cardiac index was decreased by 34% at 3 h of reperfusion and by 33% at 4 h of reperfusion in aged rats, when compared to young rats following MI/R. Furthermore, stroke volume index was decreased by 54, 56, and 65% at 2, 3, and 4 h of reperfusion in aged rats, respectively, when compared that of young rats subjected to MI/R. There was an enhanced myocyte apoptosis, as indicated by ELISA and TUNEL staining in the myocardium of aged rats compared to young rats following MI/R. Interestingly, RT-PCR analysis indicated that MI/R significantly increased the ratio of Bax mRNA to Bcl-2 mRNA in aged rats compared to that of young rats (3.51 vs. 0.74). CONCLUSION: MI/R is associated with an increase in circulating leukocytes and generation of superoxide in the peri-ischemic areas of the heart of young rats, compared to aged rats. However, MI/R induces a significant decrease in cardiac index and stroke volume index in aged rats, when compared to young rats following MI/R. Furthermore, aged rats exhibit an increase in the ratio of Bax mRNA to Bcl-2 mRNA and cardiomyocyte apoptosis following MI/R, which may explain, at least in part, the enhanced myocardial dysfunction.

L-NAME enhances microcirculatory congestion and cardiomyocyte apoptosis during myocardial ischemia-reperfusion in rats.

Besides necrosis, apoptosis is the other major mode of cardiomyocyte loss in ischemic cardiovascular disease. In the present study, we examined the hypothesis that nitric oxide (NO) protects myocardial function by improving myocardial microcirculation and attenuating cardiomyocyte apoptosis in a rat model of myocardial ischemia/reperfusion (MI/R). The left main coronary artery of anesthetized male rats was ligated for 40 min, followed by 4 h reperfusion. Four groups of animals were studied: sham operated control + saline; sham operated control + N(W)-nitro-L-arginine methyl ester (L-NAME); MI/R + saline; MI/R + L-NAME (10 mg/kg, iv, 10 min prior to reperfusion). Results show that MI/R caused a decrease in mean arterial blood pressure (MABP), cardiac index (CI), and stroke volume index (SVI). Inhibition of NO synthesis by L-NAME attenuated plasma NO levels, but increased MABP and SVR in sham control rats and rats subjected to MI/R, and further depressed left ventricular function in rats subjected to MI/R as indicated by decreased CI and SVI. Furthermore, administration of L-NAME to rats subjected to MI/R enhanced cardiomyocyte apoptosis as indicated by a significant increase in DNA fragmentation compared to rats with MI/R alone. Histological study revealed that L-NAME caused arterial constriction and congestion of red blood cells in arteries and capillaries in the peri-ischemic areas of the hearts in rats subjected to MI/R and, interestingly, also in the sham control rats. Data suggest that the mechanism of increased reperfusion injury may be attributable to a "no-reflow" phenomenon induced by L-NAME, resulting in increased cardiomyocyte apoptosis secondary to ischemia and enhanced cytochrome-c release from mitochondria. In addition, cardiac injury may be increased due to the augmented oxygen consumption of cardiomyocytes caused by the increased SVR and afterload. These results suggest that endogenous NO may act to improve myocardial microvascular perfusion, reduce SVR, and limit cardiomyocyte apoptosis, thereby, attenuating myocardial dysfunction induced by MI/R.