The role of redox signaling in cardiac hypertrophy induced by experimental hyperthyroidism.

This study was conducted to test whether oxidative stress activates the intracellular protein kinase B (AKT1) signaling pathway, which culminates with cardiac hypertrophy in experimental hyperthyroidism. Male Wistar rats were divided into four groups: control, vitamin E, thyroxine (T(4)), and T(4)+vitamin E. Hyperthyroidism was induced by T(4) administration (12 mg/l in drinking water for 28 days). Vitamin E treatment was given during the same period via s.c. injections (20 mg/kg per day). Morphometric and hemodynamic parameters were evaluated at the end of the 4-week treatment period. Protein oxidation, redox state (reduced glutathione, GSH/glutathione dissulfide, GSSG), vitamin C, total radical-trapping antioxidant potential (TRAP), hydrogen peroxide (H2O2), and nitric oxide metabolites (NO(X)) were measured in heart homogenates. The p-AKT1/AKT1 ratio, p-glycogen-synthase kinase (GSK)3B/GSK3B ratio, FOS, and JUN myocardial protein expression were also quantified by western blot after 4 weeks. Increases in biochemical parameters, such as protein oxidation (41%), H2O2 (62%), and NO(X) (218%), and increase in the left ventricular end-diastolic pressure were observed in the T(4) group. T(4) treatment also caused a decrease in GSH/GSSG ratio (83%), vitamin C (34%), and TRAP (55%). These alterations were attenuated by vitamin E administration to the hyperthyroid rats. Expression of p-AKT1/AKT1, p-GSK3B/GSK3B, FOS, and JUN were elevated in the T(4) group (by 69, 37, 130, and 33% respectively), whereas vitamin E administration promoted a significant reduction in their expression. These results indicate that oxidative stress plays an important role in cardiac hypertrophy, and suggest redox activation of AKT1 and JUN/FOS signaling pathways with H2O2 acting as a possible intracellular mediator in this adaptive response to experimental hyperthyroidism.

Activation of apoptotic processes during transition from hypertrophy to heart failure in guinea pigs.

Changes in oxidative stress and apoptotic process were studied during the progression of a compensated hypertrophy to a decompensated heart failure in guinea pigs. Banding of the ascending aorta resulted in heart hypertrophy. At 10 wk, ventricle-to-body weight ratio and thickness of the interventricular septum as well as the left ventricular wall were increased significantly. Although fractional shortening and ejection fraction were decreased, there were no signs of heart failure. Furthermore, there was no increase in wet-to-dry weight ratios for the lungs and liver at this stage. However, at 20 wk, heart failure was characterized by a significant depression in heart function as indicated by a decrease in fractional shortening, and ejection fraction and a lesser increase in wall thickness from diastole to systole. Animals also showed clinical signs of heart failure, and the wet-to-dry weight ratios of the lungs and liver were significantly higher. Cardiomyocyte oxidative stress was significantly higher in the 20-wk aortic-banded group. The ratio of Bax to Bcl-xl showed an increase at 10 wk, and there was a further increase at 20 wk. Mitochondrial membrane potential in the aortic-banded animals was significantly decreased at 10 and 20 wk. Cytochrome c levels were higher in the cytosol compared with the mitochondria, leading to a considerable increase in the expression of p17 subunit of caspase-3. At 20 wk, both early and late stages of apoptosis were observed in isolated cardiomyocytes. It is suggested that an increase in oxidative stress initiates mitochondrial death pathway during the hypertrophic stage, leading to apoptosis and heart failure at a later stage.

Growth hormone in vascular pathology: neovascularization and expression of receptors is associated with cellular proliferation.

Vascular tumours are common lesions of the skin and subcutaneous tissue, but also occur in many other tissues and internal organs. The well-differentiated tumours consist of irregular anastomosing, blood-filled vascular channels that are lined by variably atypical endothelial cells. The less differentiated tumours may show solid strands and sheets, resembling carcinoma or lymphoma. Several growth factors, including basic fibroblast growth factor, transforming growth factors and vascular endothelial growth factor, play a role in tumour angiogenesis. Growth hormone (GH) is mitogenic for a variety of vascular tissue cells, including smooth muscle cells, fibroblasts and endothelial cells and exerts its regulatory functions in controlling metabolism, balanced growth and differentiated cell expression by acting on specific membrane-bound receptors, which trigger a phosphorylation cascade resulting in the modulation of numerous signalling pathways and of gene expression. Essential to the initiation of a cellular response to GH, the presence of receptors for this hormone may predict the adaptation of tumour cells resulting from GH exposure. To address the site/mode of action through which GH exerts its effects, a well characterized monoclonal antibody, obtained by hybridoma technology from Balb/c mice immunized with purified rabbit and rat liver GH-receptor (GHR) and directed against the hormone binding site of the receptor, was applied, using the ABC technique to determine GHR expression in a panel of vascular tumours. The GHR was cloned from a rabbit liver cDNA library with the aid of an oligonucleotide probe based on a 19 residue tryptic peptide sequence derived from 5900 fold purified rabbit liver receptor. A total of 64 benign and malignant vascular tumours were obtained from different human organ sites, including the chest wall, skin, axillary contents, duodenum, female breast, abdomen, stomach, colon, lymph node, bladder, body flank and neck regions. The tumours were of the following pathological entities: Haemangioma (n = 12); haemangioendothelioma (n = 10); Castleman's disease (n = 3), haemangiopericytoma (n = 4); angiosarcoma, (n = 11), Kaposi's sarcoma with focal infiltration by lymphoma, HIV +ve (n = 7), Kaposi's sarcoma (n = 17). The endothelial cell marker CD-31 was used to establish endothelial cell characteristics and microvascular density. To delineate tumour cell growth, immunohistochemical analysis of cycling nuclear protein and of proliferating cell nuclear antigen, using Ki-67 and PCNA polyclonal antibodies respectively, was used to demonstrate proliferative indexes. Results show that, compared to their normal tissue counterparts, nuclear and cytoplasmic expression of GHR consistently result in strong receptor immunoreactivity in the highly malignant angiosarcomas and Kaposi's sarcomas and was localized in the cell membranes and cytoplasm, but strong nuclear immunoreactivity was also identified. The presence of intracellular GHR is the result of endoplasmic reticulum and Golgi localization. Nuclear localization is due to identical nuclear GHR-binding protein. Furthermore, there was a positive correlation of GHR immunoreactivity with neoplastic cellular proliferation and cycling, as measured by Ki-67 and PCNA. In conclusion, this study shows that GHR expression in vascular tumours is a function of malignancy and cancer progression. Malignant cells, which are highly expressive of the receptor, have a greater proliferation rate and thereby also higher survival rate compared to tumours expressing lower or minimal receptor level. The presence of GHR in endothelial cells of vascular neoplasm indicates that they are target cells and GH is of importance in the proliferation of vascular tumour angiogenesis. GH is necessary not only for differentiation of progenitor cells, but also for their subsequent clonal expansion and maintenance. The results support the hypothesis that GH is involved in the paracrine-autocrine mechanism, acting locally in regulating vascular tumour growth and will be useful for site-specific studies of the evolution of vascular cancers. The use of anti-GHR antibodies to block tumour progression is an intriguing possibility.

Significance of changes in TNF-alpha and IL-10 levels in the progression of heart failure subsequent to myocardial infarction.

We tested whether a decrease in the ratio of interleukin-10 (IL-10) to tumor necrosis factor-alpha (TNF-alpha) correlates with the decrease in cardiac function in heart failure. It has been suggested that TNF-alpha plays a role in the progression of heart failure, and the effect of TNF-alpha in many tissues is modulated by IL-10. Any relation of these two cytokines to heart failure has never been examined. Cardiac function was assessed by echocardiographic and hemodynamic techniques in coronary artery-ligated rats at 1, 4, 8, and 16 wk after myocardial infarction (MI). Membrane-bound and soluble fractions of TNF-alpha and IL-10 proteins, the ratio of TNF-alpha to IL-10, and TNF-alpha and IL-10 mRNA levels were analyzed. Losartan was used to modify cardiac function in rats 4 wk after MI to further validate the relation between the IL-10-to-TNF-alpha ratio and cardiac function. Cardiac function deteriorated with time in all coronary artery-ligated groups, with severe failure at 16 wk after MI. Membrane-bound and soluble TNF-alpha protein fractions were increased 1 and 4 wk after MI, whereas TNF-alpha mRNA was increased 4 and 8 wk after MI. Membrane-bound IL-10 protein and mRNA levels were decreased 4, 8, and 16 wk after MI. The decrease in the IL-10-to-TNF-alpha protein ratio in all coronary artery-ligated groups correlated with the depressed cardiac function. Losartan improved cardiac function, membrane-bound and soluble TNF-alpha and IL-10 protein levels, the ratio of IL-10 to TNF-alpha, and IL-10 mRNA. This study suggests that a decrease in IL-10 and IL-10-to-TNF-alpha ratio correlates with depressed cardiac function.

Involvement of mitogen-activated protein kinases in adriamycin-induced cardiomyopathy.

The current study investigated the phosphorylation of mitogen-activated protein kinases (MAPKs) as well as pro- and anti-apoptotic proteins in adriamycin (ADR)-induced cardiomyopathy (AIC) and heart failure in rats. Modulatory effects of antioxidant probucol on the activation of MAPKs were also examined. Male rats were administered with ADR (15 mg/kg body wt ip, over 2 wk) with and without probucol (120 mg/kg body wt for 4 wk ip). Hearts from these animals were studied at 1- to 24-h as well as at 3-wk posttreatment durations. In the 3-wk group, ADR depressed cardiac function, increased left ventricular end-diastolic pressure (LVEDP), and caused dyspnea and mortality. These changes were prevented by probucol. Phosphorylation of extracellular signal-regulated kinase (ERK)1/2, in the early stage of AIC, showed a biphasic response, with a maximum increase to 513% seen at 4 h, followed by a decrease to 66.8% at 3 wk after the last injection of ADR. Phosphorylation of p38 and c-Jun NH(2)-terminal kinases (JNKs) showed a steady increase through 2, 4, and 24 h and 3 wk (116% to 148%). In gene microarray analysis at 3 wk (heart failure stage), mRNA expression for both ERK1/2 and p38 kinases was decreased, whereas JNK mRNA was undetectable. Probucol completely prevented these MAPK changes. Activation of caspase-3 as well as the increase in the ratio of Bax to Bcl-xl were seen at early time points (1-24 h) as well as in the heart failure stage (3 wk). It is suggested that a transient increase in ERK1/2 at a shorter interval indicate an early adaptive response, and failure of this response corresponded with heart failure. In contrast, a gradual and persistent increase in p38 and JNK MAPKs as well as in caspase-3 and the Bax-to-Bcl-xl ratio may contribute in the initiation of apoptosis and progression of heart failure. Because probucol modulated changes in cellular signaling pathways and cardiac function, it is likely that oxidative stress plays a key role in AIC and heart failure.

Cytokines are not upregulated in adriamycin-induced cardiomyopathy and heart failure.

Heart failure due to a variety of causes is accompanied by an upregulation of cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6). Adriamycin-induced cardiomyopathy (AIC) and heart failure is an important clinical problem. The current study investigated the expression of these cytokines in AIC and heart failure in rats. Both early and late stages of AIC was produced in rats. Myocardial gene expressions for TNF-alpha, IL-1beta and IL-6 were examined with DNA microarrays and RT-PCR. Protein levels of these cytokines in both the plasma and the myocardium were also examined by ELISA. In the early stage, myocardial mRNA expression of IL-1beta showed significant increase at 4 and 24 h, peaking at 4 h, while TNF-alpha did not change and IL-6 was undetectable. The protein levels of these three genes did not show any upregulation in the plasma or the heart. In the late stage, heart failure was confirmed by clinical signs as well as homodynamic changes. In this stage, plasma protein levels for TNF-alpha, IL-1beta and IL-6 were not changed. However, myocardial TNF-alpha mRNA expression and protein levels were significantly decreased, while both IL-1beta mRNA and protein levels were not different compared to the control group. IL-6 mRNA expression was undetectable in both normal and adriamycin-treated hearts while its protein level was not changed by adriamycin. Positive control using lipopolysaccharides (LPS) treatment (0.5 mg/kg body weight) for 2 h resulted in a significant increase in these three cytokines in the heart and plasma. These data suggest that an upregulation of cytokines may not be involved in AIC. Heart failure may in fact be accentuated by a downregulation of myocardial TNF-alpha.

Antioxidant enzyme gene expression in congestive heart failure following myocardial infarction.

Increased oxidative stress and reduction in antioxidant enzymes have been suggested to be involved in the pathophysiology of congestive heart failure subsequent to myocardial infarction (MI). The objective of the present study was to characterize changes in the mRNA abundance and protein levels for the enzymatic antioxidants, superoxide dismutase (SOD), glutathione peroxidase (GSHPx) and catalase during the sequelae of congestive heart failure in rats. MI was produced by the ligation of the left coronary artery and hearts from controls and 1, 4 and 16 week PMI groups were analyzed. Losartan treatment (2 mg/ml in drinking water, daily) was started at 4 weeks and continued for 12 weeks. The mRNA levels for SOD were reduced by about 40% at 1-week PMI, were near to the control levels at 4-week PMI and at 16 weeks PMI, the levels were reduced by about 73% below the controls. GSHPx mRNA levels remained unchanged at all time points. The mRNA levels for catalase remained unchanged at 1 and 4 weeks PMI and were significantly reduced by about 44% at 16 weeks PMI as compared to the controls. The protein levels for MnSOD, CuZnSOD, GSHPx at 1 and 16 weeks remained unchanged in treated and untreated PMI groups. However, the protein levels for catalase was significantly increased in the control and PMI groups treated with Losartan. It is concluded that changes in the SOD and catalase activities during severe heart failure correlated with changes in mRNA for these enzymes. The precise mechanism/s for the improvement in antioxidant reserve and protein levels after Losartan treatment is/are unclear at this time.

Apoptosis in adriamycin cardiomyopathy and its modulation by probucol.

The dose-dependent cardiomyopathy and heart failure due to adriamycin have been shown to be due to increased oxidative stress and loss of myocytes. We examined the incidence of myocardial apoptosis as well as changes in the expression of apoptotic regulatory gene products in an established animal model of adriamycin cardiomyopathy. Rats were treated with adriamycin (cumulative dose, 15 mg/kg), and the hearts were examined for apoptosis as well as expression of Bax, caspase 3, and Bcl-2 at 0, 4, 10, 16, and 21 days after the treatment. A significant increase in the incidence of apoptosis was seen at 4 days, followed by a decline at 10 and 16 days of posttreatment. At 21 days, the number of apoptotic cells increased again and included cells of the conducting system. Expression of Bax corresponded to these biphasic changes, whereas the converse was true for the expression of Bcl-2. The latter peaked at 10 days followed by a decline at 16 and 21 days. The Bax/Bcl-2 ratio also correlated with the incidence of apoptosis. Expression of caspase 3 correlated with increased apoptosis, but only at early time points. Probucol (cumulative dose, 120 mg/kg), a known antioxidant as well as promoter of endogenous antioxidants, significantly reduced the incidence of apoptosis as well as expression of Bax. Adriamycin-induced hemodynamic changes were also prevented by probucol. These data suggest that adriamycin-induced apoptosis is mediated by oxidative stress and may play a role in the development of heart failure.

Modulation of oxidative stress by a selective inhibition of angiotensin II type 1 receptors in MI rats.

OBJECTIVES: To examine whether blocking of the renin-angiotensin system (RAS) at the angiotensin II type 1 (AT1) receptor site is accompanied by changes in the oxidative stress parameters. BACKGROUND: Congestive heart failure in rats after myocardial infarction (MI) has been shown to correlate with a decrease in antioxidant enzyme activities and an increase in oxidative stress. Inhibition of the RAS with captopril improves cardiac function and survival in MI rats with a reduction in oxidative stress. METHODS: Myocardial infarction in rats was produced by ligation of the left coronary artery. At four weeks after surgery, animals from the sham as well as MI groups were treated with losartan (2 mg/ml in drinking water daily). At 16 weeks after surgery, the animals were examined for hemodynamic function and the hearts were analyzed for antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase and catalase) and oxidative stress (lipid hydroperoxides, reduced and oxidized glutathione and redox ratio). RESULTS: Congestive heart failure was characterized by dyspnea, depressed hemodynamic function and presence of lung and liver congestion. This was also associated with a decrease in the myocardial catalase (-25%), glutathione peroxidase (-38%) and superoxide dismutase (-42%) activities. An increase in oxidative stress in these hearts was indicated by an increase in lipid hydroperoxides (+67%) and reduction in the redox ratio (-75%). Hemodynamic function was better maintained and there were no indications of dyspnea or lung or liver congestion in the losartan-treated MI rats. In these animals, myocardial oxidative stress was markedly reduced and glutathione peroxidase and catalase activities were significantly improved compared with the untreated MI group. CONCLUSIONS: Blocking of RAS at the AT1 receptor site without the inhibition of angiotensin-converting enzymes modulates heart failure after MI, and this beneficial effect is associated with a decrease in oxidative stress. This study suggests a newer role for losartan in the treatment of heart failure.

Oxidative stress and functional deficit in diabetic cardiomyopathy.

When the equilibrium between free-radical production and cellular antioxidant defences is disturbed in favour of more free radicals, it causes oxidative stress which can promote cellular injury. Oxidative stress has been suggested to play a role in the pathogenesis of diabetic cardiomyopathy. In streptozotocin-induced diabetes, there is a decrease in antioxidant enzyme activities and an increase in myocardial lipid peroxidation. Probucol, an antioxidant, was found to improve cardiac function which may have been due to an increase in myocardial antioxidant enzyme activities and a decrease in lipid peroxidation in the diabetic animals. Some of the beneficial effects of probucol may also be due to an improvement in plasma insulin levels and a decrease in the plasma glucose. The diabetic state is also associated with endothelial dysfunction, retinopathy, neuropathy and renopathy. Some of these secondary complications may also be mediated by oxidative stress. It is suggested that diabetic cardiomyopathy is associated with an antioxidant deficit and that antioxidant therapy may be useful in improving cardiac function in diabetes.

Myocardial oxidative stress and antioxidants in hypertension as a result of nitric oxide synthase inhibition.

Rats were made hypertensive by the administration of the nitric oxide synthase inhibitor nitro-L-arginine (LNA, 2.74 mmol/L) in drinking water for 7 d. Hearts from hemodynamically assessed animals were analyzed for lipid peroxidation (LPO), gamma-glutamylcysteine-synthetase (gamma-GCS), glutathione disulfide reductase (GR), glutathione peroxidase (GSHPx), catalase (CAT), superoxide dismutase (SOD), and total radical trapping potential (TRAP) activities. LNA treatment increased the mean arterial blood pressure by 46% and the heart rate by 22% without changing plasma renin activity. LNA treatment resulted in a 30% increase in LPO. gamma-GCS was reduced by 48% and GR by 36% in the cardiac tissue of hypertensive rats as compared to controls. The activity of nonselenium GSHPx was reduced by 27%, and selenium-dependent GSHPx activity in the heart was not affected by LNA treatment. In hypertensive rats, SOD activity was increased by 16%, and CAT was decreased by 46%. TRAP was lower (27%) in the myocardium of hypertensive rats than in that of controls. These data suggest that LNA-induced hypertension is associated with increased myocardial oxidative stress.

Circaannual changes in antioxidants and oxidative stress in the heart and liver in rats.

Reactive oxygen species are formed in physiological and pathological conditions in mammalian tissues. Because of their high reactivity, they may interact with biomolecules, inducing oxidative injury. Increases in lipid peroxidation can result in oxidative damage to cellular membranes. Protection against oxidative damage is provided by enzymatic and non-enzymatic antioxidant defenses. Antioxidant enzyme activities and lipid peroxidation, as an index of oxidative stress injury, were evaluated in different seasons over one year in the heart and liver of rats, maintained on a 12 h light and dark cycle. Glutathione peroxidase and catalase activities, in both tissues, were maximal in the summer season. Lipid peroxidation in the heart was maximal in the spring as compared to the other seasons and it did not vary in the liver during the year. These findings suggest that any study of antioxidants or oxidative stress must take into account such seasonal variations for a more precise analysis of changes due to any pathological condition.

Adriamycin-induced early changes in myocardial antioxidant enzymes and their modulation by probucol.

BACKGROUND: The clinical usefulness of adriamycin is restricted by the development of congestive heart failure. It has been suggested that probucol, a strong antioxidant, completely prevents adriamycin-induced cardiomyopathy without interfering with its antitumor properties. The present study investigated the effects of adriamycin and probucol on myocardial antioxidant enzyme activities and immunoreactive protein levels in rats. METHODS AND RESULTS: Activities and protein levels of glutathione peroxidase (GSHPx) were significantly decreased from 2 to 24 hours, and those of manganese superoxide dismutase were decreased at 1 and 2 hours after adriamycin treatment. These changes were prevented by probucol. Catalase activity was increased from 2 to 24 hours after adriamycin treatment, but its protein levels were not significantly changed. Copper zinc superoxide dismutase activity and protein level were not changed at any time. Myocardial lipid peroxidation was found to be significantly higher at all time points, and this change was also prevented by probucol. Treatment with probucol alone increased GSHPx activity at 2 weeks, and in these hearts, lipid peroxidation was lower than the control value. Within 24 hours, there was no mortality in any of the groups. CONCLUSIONS: It is suggested that an early and persistent decrease in GSHPx activity and protein may play an important role in the pathogenesis of adriamycin-induced cardiomyopathy, worsening heart failure and mortality.

Oxidative stress in congestive heart failure.

Heart failure is considered to be a complex clinical syndrome, with alterations in the multiple neurohumoral systems and subcellular cardiac sites that correlate with abnormal cardiac function. Strong evidence for the role of oxidative stress in the pathogenesis of heart failure has been provided by studies on experimental animals as well as humans. This concept is gaining more acceptance due to the fact that during heart failure, changes in different neurohormones, cytokines, nitric oxide, and activated inflammatory cells are closely linked to oxidative stress at the cellular and molecular levels. The present article provides a simple description of oxygen free radicals as well as the antioxidant defense system. Evidence for the role of oxidative stress in the pathogenesis of heart failure is reviewed in a concise manner.

Cholangiocarcinoma and liver cirrhosis in relation to changes due to thioacetamide.

Different doses of thioacetamide (0.05%, 0.1% and 0.15%) were used to induce liver cirrhosis in Wistar rats. Thioacetamide at 0.5% caused cirrhosis by the twelfth week of treatment. A severe bile duct proliferation and cholangiocarcinoma was seen at longer intervals. Animals treated with higher doses (0.1% and 0.15%) of thioacetamide developed more severe intense degenerative changes in the liver and died in the twelfth and eighth week respectively. The serum and tissue contents of Zn and Cu changed in a characteristic fashion that was consistent with the severity of the liver damage. Serum Zn and Cu concentrations were at their lowest in the animals that developed severe degenerative liver and died at higher dose (0.15%) of thioacetamide. This study indicates that treatment of rats with 0.05% thiocetamide is more effective and appropriate for the induction of liver cirrhosis. Continued administration of the drug at this dosage led to the development of further changes in the liver. This model may be suitable for studying these long term changes that occur in the liver and lead to cirrhosis. Events that precede the development of severe bile duct proliferation and cholangiocarcinoma may also be studied.

Adriamycin-induced heart failure: mechanism and modulation.

Adriamycin (doxorubicin) is one of the most effective chemotherapeutic agents against a variety of cancers, but its usefulness is seriously curtailed by the risk of developing heart failure. Available laboratory evidence suggests that an increase in oxidative stress, brought about by increased free radical production and decreased myocardial endogenous antioxidants, plays an important role in the pathogenesis of heart failure. Adriamycin-induced apoptosis and hyperlipidemia may also be involved in the process. Probucol, a lipid-lowering drug and an antioxidant, completely prevents the occurrence of heart failure by reducing oxidative stress as well as by the modulation of apoptosis and high lipid concentrations. Thus, combined therapy with adriamycin and probucol has a high potential for optimizing the treatment of cancer patients.

Effects of probucol on changes of antioxidant enzymes in adriamycin-induced cardiomyopathy in rats.

OBJECTIVE: The clinical usefulness of doxorubicin (adriamycin, ADR) is restricted by the risk of developing congestive heart failure. Probucol has been reported to completely prevent ADR cardiomyopathy without interfering with its antitumor effects. The current study investigated the effects of ADR and probucol on antioxidant enzyme gene expression during adriamycin-induced cardiomyopathy in a rat model. METHODS: The mRNA abundance by Northern and immunoreactive protein levels by Western blotting of myocardial antioxidant enzymes, glutathione peroxidase (GSHPx), manganese superoxide dismutase (MnSOD) and catalase (CAT) were examined in relation to the enzyme activities in hemodynamically assessed control and treated animals. RESULTS: At 3 weeks post-treatment duration, ADR caused heart failure which was prevented by probucol. MnSOD mRNA abundance as well as protein levels were depressed by ADR treatment by 45% and 20%, respectively, and this change was prevented by probucol. However, the mRNA and protein levels of GSHPx and CAT were not significantly changed by ADR or probucol. ADR had no effect on SOD activity but this enzyme activity was increased by probucol and probucol plus ADR. GSHPx enzyme activity was decreased and oxidative stress as indicated by TBARS was increased by ADR and these changes were also modulated by probucol. CONCLUSION: An increase in oxidative stress, GSHPx inactivation and MnSOD downregulation during ADR cardiomyopathy were prevented by probucol treatment.

Caspase activation and mitochondrial cytochrome C release during hypoxia-mediated apoptosis of adult ventricular myocytes.

Oxygen deprivation for prolonged periods leads to cardiac cell death and ventricular dysfunction. The ability to prevent myocardial cell death would be of significant therapeutic value in maintaining cardiac function after injury. While caspases have been suggested to play a critical role in apoptosis, their involvement during hypoxic injury has not been formally determined. In this report, we show that adult ventricular myocytes subjected to hypoxia for 1 h undergo a three-fold increase (P<0.05) in the incidence of apoptosis as determined by TUNEL analysis and Hoechst 33258 nuclear staining. Western blot analysis of hypoxic myocytes revealed a 10-fold increase in the proteolytic processing of caspase 3 to p17 with a concomitant cleavage of the caspase 3 substrate PARP from 116 kd to p85 kd compared to normoxic controls. Defects in mitochondrial membrane integrity were also observed as evidenced by the translocation of cytochrome c from the mitochondrial to cytosolic compartment of hypoxic cells. Pretreatment of ventricular myocytes with the peptide-caspase inhibitor known to block caspases related to caspase 1 (Ac-YVAD-CHO) attenuated cytochrome c release, processing of caspase 3, and apoptosis. While the caspase inhibitor (Ac-DEVD-CHO) which blocks caspases related to caspase 3, suppressed the cleavage of PARP and apoptosis, it had no effect on cytochrome c release by mitochondria. The data provide direct evidence for the proteolytic activation of caspases during hypoxia-mediated apoptosis of adult ventricular myocytes. Furthermore, the data suggest a hierarchical scheme for caspase activation with mitochondrial cytochrome c release occurring proximally to DEVD-CHO-inhibitable caspases.

Metallothioneins, oxidative stress and the cardiovascular system.

Metallothioneins (MTs) discovered four decades ago as metal binding low molecular weight thiol proteins, in recent years, have generated immense interest due to their involvement in various physiological and pathophysiological events. In spite of the recent advances in the knowledge base in MTs, no specific study on the isolation or characterization of MTs isoforms and their precise function in the heart has been conducted. Although most stress conditions in the heart do not produce much change in myocardial MTs, TNF-alpha and IL-6 do induce MT in the heart to the extent comparable with the liver. Cardiotoxicity of anticancer drugs and vulnerability of the heart to other oxidative stress conditions may partially be due to lack of inducibility of MT in the heart by these interventions. A clear understanding of induction of MTs in the heart may help in the development of better approaches to modulate the pathogenesis of cardiomyopathies and heart failure.

Effects of methionine on endogenous antioxidants in the heart.

The deficiency of methionine, an essential amino acid, is associated with cardiovascular lesions. Because different types of cardiac pathologies are caused by a decrease in antioxidants, we examined the effects of methionine on myocardial antioxidant enzymes in hemodynamically assessed rats that were treated with methionine (10 mg/ml) in drinking water for 12, 24, and 48 h. Glutathione peroxidase (GSHPx) activity was significantly increased to 150.5 +/- 12.2 and 191.7 +/- 13.7% of the control value at 12 and 24 h, respectively, followed by a decline to 120 +/- 24.6% at 48 h. The mRNA levels of GSHPx at these time points were 151.2 +/- 12.0, 218.7 +/- 35.3, and 173.5 +/- 25.2%, respectively. Superoxide dismutase (SOD) activity was 144.3 +/- 3.7, 114.3 +/- 10.1, and 143.1 +/- 11. 2% at 12, 24, and 48 h, respectively. Catalase (Cat) activity was 272.4 +/- 5.4, 237.8 +/- 16.6, and 224.1 +/- 17.3% of the control value. The expression of Cat and SOD mRNA was unchanged at 12, 24, and 48 h. The lipid peroxidation was decreased by 24.4 +/- 11.2, 54. 9 +/- 0.1, and 6.4 +/- 2.1% at 12, 24, and 48 h, respectively. Methionine had no effect on the ventricular or aortic pressures, heart rate, and myocardial glutathione levels at any of the time points. The study shows that methionine has a significant effect on the myocardial antioxidant enzyme activities, and only changes in GSHPx enzyme activity correlated with the mRNA changes. These antioxidant changes may have a role in the beneficial effects of methionine in pathological rather than physiological conditions.