metallothionein and Cardiomyopathies

Cardiotoxicity is a serious complication of anticancer therapy by anthracycline antibiotics. Except for intercalation into DNA/RNA structure, inhibition of DNA-topoisomerase and histone eviction from chromatin, the main mechanism of their action is iron-mediated formation of various forms of free radicals, which leads to irreversible damage to cancer cells. The most serious adverse effect of anthracyclines is, thus, cardiomyopathy leading to congestive heart failure, which is caused by the same mechanisms. Here, we briefly summarize the basic types of free radicals formed by anthracyclines and the main processes how to scavenge them. From these, the main attention is paid to metallothioneins. These low-molecular cysteine-rich proteins are introduced and their functions and properties are reviewed. Further, their role in detoxification of metals and drugs is discussed. Based on these beneficial roles, their use as a new therapeutic agent against oxidative stress and for cardioprotection is critically evaluated with respect to their ability to increase chemoresistance against some types of commonly used cytostatics.

Topics: Animals; Anthracyclines; Antibiotics, Antineoplastic; Cardiomyopathies; Cardiotonic Agents; Drug Resistance, Neoplasm; Free Radical Scavengers; Free Radicals; Heart Failure; Humans; Metallothionein; Neoplasms; Oxidative Stress

Metallothioneins (MTs) are a class of metal-binding proteins characterized by a high cysteine content and low molecular weight. MTs play an important role in metal metabolism and protect cells against the toxic effects of radiation, alkylating agents and oxygen free radicals. The evidence that individual genetic characteristics of MTs play an important role in physiological and pathological processes associated with antioxidant defense and detoxification inspired targeted studies of genetic polymorphisms in a clinical context. In recent years, common MT polymorphisms were identified and associated with, particularly, western lifestyle diseases such as cancer, complications of atherosclerosis, and type 2 diabetes mellitus along with related complications. This review summarizes all evidence regarding MT polymorphisms of major human MTs (MT1, MT2, MT3 and MT4), their relation to pathological processes, and outlines specific applications of MTs as a set of genetic markers for certain pathologies.

Topics: Cardiomyopathies; Diabetes Mellitus, Type 2; Gene Expression Regulation; Humans; Inflammation; Metallothionein; Multigene Family; Neoplasms; Polymorphism, Single Nucleotide; Protein Isoforms

Cardiac failure is a leading cause for the mortality of diabetic patients, in part due to a specific cardiomyopathy, referred to as diabetic cardiomyopathy, which occurs with or without co-existence of vascular diseases. Although several mechanisms responsible for diabetic cardiomyopathy have been proposed, oxidative stress is widely considered as one of the major causes for the pathogenesis of the disease. Thus, a few laboratories are trying to develop antioxidants used to prevent diabetic cardiomyopathy. Metallothioneins (MTs) are cysteine-rich metal-binding proteins with several biological roles including antioxidant property. We and others have indicated the significant cardiac protection of MT against diabetes using cardiac-specific MT-overexpressing transgenic mice and OVE26MT mice (cross-bred of cardiac MT transgenic mice with genetically engineered diabetic OVE26 mice). Several possible mechanisms responsible for MT's cardiac protection from diabetes were revealed. These include MT's important roles in calcium regulation, zinc homeostasis, insulin sensitization, and antioxidant action. Since MT is ubiquitously expressed in mammalian tissues and is highly inducible by a variety of reagents such as zinc, the clinical potential for inducing cardiac MT as an antioxidant by zinc supplementation to prevent various diabetic complications, including cardiomyopathy, has been explored in diabetic animal models and patients. Since zinc has been therapeutically used for several other non-diabetic diseases in clinics, it provides further potential use of zinc for diabetic patients. Therefore, this review will briefly introduce the biochemical features of MT along with its critical roles in redox homeostasis and antioxidant function in the heart, and then discuss the current research on the prevention of diabetic cardiomyopathy by MT with an emphasis on experimental evidence, possible mechanisms, and clinical implications.

Topics: Amino Acid Sequence; Animals; Cardiomyopathies; Diabetic Angiopathies; Humans; Metallothionein; Mice; Mice, Transgenic; Molecular Sequence Data; Oxidative Stress; Zinc

Diabetic cardiomyopathy has become a major contributor to the increased mortality of diabetic patients. Although the development and progression of diabetic cardiomyopathy are considered to be associated with diabetes-derived oxidative stress, the precise mechanisms for and effectively preventive approaches to diabetic cardiomyopathy remain to be explored. Recent studies showed that reactive oxygen or nitrogen species (ROS/RNS) not only play a critical role in the initiation of diabetic cardiomyopathy, but also play an important role in physiological signaling. Therefore, this review will first discuss the dual roles of ROS/RNS in the physiological signaling and pathogenic remodeling leading to cardiomyopathy under diabetic conditions. The significant prevention of diabetic cardiomyopathy by metallothionein (MT) as a potent and nonspecific antioxidant will be also summarized. It is clearly revealed that although dual roles of peroxynitrite-nitrated proteins have been indicated under both physiological and pathogenic conditions, suppression of nitrative damage by MT in the diabetic heart is the major mechanism responsible for its prevention of diabetic cardiomyopathy. Finally the potential for clinical enhancement of the cardiac MT expression to prevent or delay the occurrence of cardiomyopathy in diabetic patients will also be addressed.

Topics: Cardiomyopathies; Diabetes Mellitus; Diabetic Angiopathies; Humans; Metallothionein; Nitrates; Nitrosation; Reactive Nitrogen Species; Reactive Oxygen Species

Pathogenesis of diabetic cardiomyopathy (DCM) is a complicate and chronic process that is secondary to acute cardiac responses to diabetes. One of the acute responses is cardiac cell death that plays a critical role in the initiation and development of DCM. Besides hyperglycemia, inflammatory response in the diabetic heart is also a major cause for cardiac cell death. Diabetes or obesity often causes systemic and cardiac increases in tumor necrosis factor-alpha, interleukin-18 and plasminogen activator inhibitor-1. However, how these cytokines cause cardiac cell death remains unclear. It has been considered to relate to oxidative and/or nitrosative stress. We have demonstrated that metallothionein as a potent antioxidant or stress protein significantly protected the heart from oxidative damage and cell death caused by these cytokines, leading to effective prevention of DCM. The direct link of the inhibition of oxidative stress and damage to the prevention of cardiac cell death was defined by addition of superoxide or peroxynitrite specific inhibitor to completely prevent cytokine-induced cardiac cell death. Cardiac cell death is induced by the inflammatory cytokines that is increased in response to diabetes. Inflammatory cytokine-induced cardiac cell death is mediated by oxidative stress and is also the major initiator for DCM development.

Topics: Animals; Cardiomyopathies; Cell Death; Diabetes Mellitus; Humans; Inflammation; Interleukin-18; Metallothionein; Myocardium; Obesity; Oxidative Stress; Tumor Necrosis Factor-alpha

Zinc (Zn) is an essential mineral that is required for various cellular functions. Its abnormal metabolism is related to certain disorders such as diabetic complications. Oxidative stress has been considered as the major causative factor for diabetic cardiomyopathy. Zn has a critical antioxidant action in protecting the heart from various oxidative stresses. Zn deficiency was found to be a risk factor for cardiac oxidative damage and supplementation with Zn provides a significant prevention of oxidative damage to the heart. Diabetes causes a significant systemic oxidative stress and also often is accompanied by Zn deficiency that increases the susceptibility of the heart to oxidative damage. Therefore, there is a strong rationale to consider the strategy of Zn supplementation to prevent or delay diabetic cardiomyopathy. This short article collects the preliminary evidence, based on our own studies and those by others, for a preventive effect of Zn supplementation on diabetes-induced injury to the heart in animals and under in vitro conditions. Possible mechanisms by which Zn supplementation prevents diabetic heart disease are discussed. They include an antioxidant action of Zn, insulin function and metallothionein induction. In the final section, the future of Zn supplementation for diabetic patients is also briefly discussed. Although Zn supplementation has not been clinically used to prevent diabetic complications, because several issues need to be addressed, the fact that Zn supplementation is being used clinically for other disorders encourages us to explore its direct clinical application for the prevention of diabetic cardiomyopathy.

Topics: Animals; Antioxidants; Biological Transport; Cardiomyopathies; Cell Line; Coloring Agents; Diabetes Complications; Diabetes Mellitus, Experimental; Dietary Supplements; Glutathione; Glutathione Peroxidase; Humans; Metallothionein; Nitric Oxide; Nitric Oxide Synthase Type III; Oxidative Stress; Reactive Oxygen Species; Superoxide Dismutase; Tetrazolium Salts; Thiazoles; Time Factors; Triglycerides; Zinc

Topics: Animals; Cardiomyopathies; Glutathione; Heart; Metallothionein; Mice; Oxidative Stress

Nuclear factor-E2-related factor 2 (Nrf2) and metallothionein have each been reported to protect against chronic intermittent hypoxia- (IH-) induced cardiomyopathy. Sulforaphane-rich broccoli sprout extract (BSE) and zinc can effectively induce Nrf2 and metallothionein, respectively, to protect against IH-induced cardiomyopathy via antioxidative stress. However, whether the cardiac protective effects of the combination of BSE and zinc can be synergistic or the same has not been evaluated. In this study, we treated 8-week-old C57BL/6J mice with BSE and/or zinc during exposure to IH for 8 weeks. Cardiac dysfunction, as determined by echocardiography, and pathological remodeling and abnormalities, including cardiac fibrosis, inflammation, and oxidative damage, examined by histopathology and western blotting, were clearly observed in IH mice but were not significant in IH mice treated with either BSE, zinc, or zinc/BSE. Furthermore, the effects of the combined treatment with BSE and zinc were always greater than those of single treatments. Nrf2 function and metallothionein expression in the heart increased to a greater extent using the combination of BSE and zinc than using BSE or zinc alone. These findings for the first time indicate that the dual activation of Nrf2 and metallothionein by combined treatment with BSE and zinc may be more effective than monotherapy at preventing the development of IH-induced cardiomyopathy.

Topics: Animals; Antioxidants; Brassica; Cardiomyopathies; Drug Therapy, Combination; Humans; Hypoxia; Metallothionein; Mice; Mice, Inbred C57BL; NF-E2-Related Factor 2; Oxidative Stress; Plant Extracts; Sleep Apnea, Obstructive; Zinc

Engineered cardiac tissues (ECTs) serve as robust in vitro models to study human cardiac diseases including cardiac toxicity assays due to rapid structural and functional maturation and the ability to vary ECT composition. Metallothionein (MT) has been shown to be cardioprotective for environmental toxicants including heavy metals. To date, studies on the role of cardiomyocyte (CM)-specific MT expression and function have occurred in dissociated single cell assays or expensive in vivo small animal models. Therefore, we generated 3D ECTs using neonatal mouse ventricular cells from wild-type (WT) and the CM-specific overexpressing MT-transgenic (MT-TG) to determine the effect of MT overexpression on ECT maturation and function. Because Cadmium (Cd) is an environmentally prevalent heavy metal toxicant with direct negative impact on cardiac structure and function, we then determined the effect of MT overexpression to reduce Cd mediated CM toxicity within ECTs. We found: (1) structural and functional maturation was similar in WT and MT-TG ECTs; (2) Cd exposure negatively impacted ECT cell survival, maturation, and function; and (3) MT-ECTs showed reduced Cd toxicity as defined by reduced cleaved caspase 3, reduced Bax/Bcl2 ratio, reduced TdT-mediated dUTP nick-end labeling positive cells, reduced CM loss after Cd treatment, and delayed onset of cardiac dysfunction after Cd treatment. Thus, neonatal murine ECTs can serve as a robust in vitro model for heavy metal toxicity screening and as a platform to evaluate the role cardioprotective mechanisms, such as the MT-TG model, on environmentally relevant toxicants.

Topics: Animals; Animals, Newborn; Cadmium; Cardiomyopathies; Heart Ventricles; Metallothionein; Mice; Mice, Transgenic; Models, Biological; Myocytes, Cardiac; Tissue Engineering; Transgenes

We reported previously that nuclear factor erythroid 2-related factor 2 (Nrf2) and metallothionein (MT) play critical roles in preventing intermittent hypoxia (IH)-induced cardiomyopathy. In addition, positive feedback regulation between Nrf2 and MT is required for the efficient compensative responses of the heart to IH. As an activator of Nrf2, sulforaphane (SFN) has attracted attention as a potential protective agent against cardiovascular disease. Here, we investigated whether SFN can up-regulate cardiac Nrf2 expression and function, as well as MT expression, to prevent IH-induced cardiomyopathy, and if so, whether Nrf2 and MT are indispensable for this preventive effect. Nrf2-knock-out (Nrf2-KO) or MT-KO mice and their wild-type (WT) equivalents were exposed to IH for 4 weeks with or without SFN treatment. SFN almost completely prevented IH-induced cardiomyopathy in WT mice, and this preventive effect was abolished in Nrf2-KO mice but retained in MT-KO mice. In IH-exposed WT mice, SFN induced significant increases in the expression levels of Nrf2 and its downstream antioxidant target genes, as well as those of MT, but these effects were not seen in IH-exposed Nrf2-KO mice. By contrast, KO of MT did not affect the ability of SFN to up-regulate the expression of Nrf2 and its downstream antioxidant targets. These results suggest that SFN-induced MT expression is Nrf2-dependent, and SFN prevents IH-induced cardiomyopathy in a Nrf2-dependent manner, for which MT is dispensable. This study provides important information that is relevant to the potential use of SFN to prevent IH-induced cardiomyopathy.

Topics: Animals; Cardiomyopathies; Cardiotonic Agents; Gene Expression Regulation; Hypoxia; Isothiocyanates; Male; Metallothionein; Mice; Mice, Knockout; NF-E2-Related Factor 2; Sulfoxides

The mechanism for intermittent hypoxia (IH)-induced cardiomyopathy remains obscure. We reported the prevention of acute and chronic IH-induced cardiac damage by selective cardiac overexpression of metallothionein (MT). Herein we defined that MT-mediated protection from IH-cardiomyopathy is via activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a critical redox-balance controller in the body. For this, mice were exposed to IH for 3 days (acute) or 4 or 8 weeks (chronic). Cardiac Nrf2 and MT expression in response to IH were significantly increased acutely yet decreased chronically. Interestingly, cardiac overexpression (Nrf2-TG) or global deletion of the Nrf2 gene (Nrf2-KO) made mice highly resistant or highly susceptible, respectively, to IH-induced cardiomyopathy and MT expression. Mechanistically, 4-week IH exposure significantly decreased cardiac Nrf2 binding to the MT gene promoter, and thus, depressed both MT transcription and translation in WT mice but not Nrf2-TG mice. Likewise, cardiac MT overexpression prevented chronic IH-induced cardiomyopathy and down-regulation of Nrf2 likely via activation of a PI3K/Akt/GSK-3β/Fyn-dependent signaling pathway. These results reveal an integrated relationship between cardiac Nrf2 and MT expression in response to IH -- acute compensatory up-regulation followed by chronic down-regulation and cardiomyopathy. Cardiac overexpression of either Nrf2 or MT offered cardioprotection from IH via complicated PI3K/Akt/GSK3B/Fyn signaling. Potential therapeutics may target either Nrf2 or MT to prevent chronic IH-induced cardiomyopathy.

Topics: Animals; Cardiomyopathies; Gene Expression Regulation; Glycogen Synthase Kinase 3 beta; Hypoxia; Male; Metallothionein; Mice; Mice, Transgenic; NF-E2-Related Factor 2; Oxidation-Reduction; Oxidative Stress; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-fyn; Signal Transduction

Aims. Repetitive brief ischemia and reperfusion (I/R) is associated with left ventricular dysfunction during development of ischemic cardiomyopathy. We investigated the role of zinc-donor proteins metallothionein MT1 and MT2 in a closed-chest murine model of I/R. Methods. Daily 15-minute LAD-occlusion was performed for 1, 3, and 7 days in SV129 (WT)- and MT1/2 knockout (MT(-/-))-mice (n = 8-10/group). Hearts were examined with M-mode echocardiography and processed for histological and mRNA studies. Results. Expression of MT1/2 mRNA was transiently induced during repetitive I/R in WT-mice, accompanied by a transient inflammation, leading to interstitial fibrosis with left ventricular dysfunction without infarction. In contrast, MT(-/-)-hearts presented with enhanced apoptosis and small infarctions leading to impaired global and regional pump function. Molecular analysis revealed maladaptation of myosin heavy chain isoforms and antioxidative enzymes in MT1/2(-/-)-hearts. Despite their postponed chemokine induction we found a higher total neutrophil density and macrophage infiltration in small infarctions in MT(-/-)-hearts. Subsequently, higher expression of osteopontin 1 and tenascin C was associated with increased myofibroblast density resulting in predominately nonreversible fibrosis and adverse remodeling in MT1/2(-/-)-hearts. Conclusion. Cardioprotective effects of MT1/2 seem to be exerted via modulation of contractile elements, antioxidative enzymes, inflammatory response, and myocardial remodeling.

Topics: Animals; Apoptosis; Cardiomyopathies; Disease Models, Animal; Echocardiography; Metallothionein; Mice; Mice, Knockout; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Osteopontin; Tenascin

Cardiac-specific metallothionein (MT) overexpression extends lifespan, but the mechanism underlying the effect of MT protection against age-associated cardiovascular diseases (CVD) remains elusive. To elucidate this, male wild-type and two lines of MT-transgenic (MT-TG) mice, MM and MT-1 (cardiac-specific overexpressing MT about 10- and 80-fold, respectively) at three representative ages (2-3, 9-10, and 18-20 months), were utilized. A stable human MT2A overexpressing cardiomyocytes (H9c2MT7) was also introduced.. Histomorphology and echocardiographic analysis revealed that age-associated cardiac hypertrophy, remodeling, and dysfunction were ameliorated in MT-TG mice. Also, aging-accompanied NF-κB activation, characterized by increased nuclear p65 translocation, elevated DNA-binding activity, and upregulation of inflammatory cytokines, was largely attenuated by MT overexpression. Treatment of H9c2 cardiomyocytes with tumor necrosis factor-α (TNF-α), which mimicked an inflammatory environment, significantly increased NF-κB activity, and some age-related phenotypes appeared. The NF-κB activation was further proved to be pivotal for both age-associated and TNF-α-induced nitrative damage to cardiac 2-oxoglutarate dehydrogenase (2-OGD) by virtue of NF-κB p65 gene silencing. MT inhibited NF-κB activation and associated nitrative damage to cardiac 2-OGD in both old MT-TG hearts and TNF-α-treated H9c2MT7 cardiomyocytes; these protective effects were abolished in H9c2MT7 cardiomyocytes by MT-specific gene silencing. Innovation and Conclusion: Together, these findings indicate that the protective effects of MT against age-associated CVD can be attributed mainly to its role in NF-κB inhibition and resultant alleviation of nitrative damage to 2-OGD. Antioxid. Redox Signal. 25, 936-952.

Topics: Aging; Animals; Apoptosis; Cardiomyopathies; Cytokines; Energy Metabolism; Enzyme Activation; Gene Expression; Gene Knockdown Techniques; Heart Function Tests; Inflammation Mediators; Ketoglutarate Dehydrogenase Complex; Male; Metallothionein; Mice; Mice, Transgenic; NF-kappa B; Nitrites; Phenotype; Signal Transduction; Tumor Necrosis Factor-alpha; Ventricular Remodeling

Obstructive sleep apnea (OSA) causes chronic intermittent hypoxia (IH) to induce cardiovascular disease, which may be related to oxidative damage. Metallothionein (MT) has been extensively proved to be an endogenous and highly inducible antioxidant protein expressed in the heart. Therefore, we tested the hypotheses that oxidative stress plays a critical role in OSA induced cardiac damage and MT protects the heart from OSA-induced cardiomyopathy. To mimic hypoxia/reoxygenation events that occur in adult OSA patients, mice were exposed to IH for 3 days to 8 weeks. The IH paradigm consisted of alternating cycles of 20.9% O₂/8% O₂ F(I)O₂ (30 episodes per hour) with 20s at the nadir F(I)O₂ for 12 h a day during daylight. IH significantly increased the ratio of heart weight to tibia length at 4 weeks with a decrease in cardiac function from 4 to 8 weeks. Cardiac oxidative damage and fibrosis were observed after 4 and 8 weeks of IH exposures. Endogenous MT expression was up-regulated in response to 3-day IH, but significantly decreased at 4 and 8 weeks of IH. In support of MT as a major compensatory component, mice with cardiac overexpression of MT gene and mice with global MT gene deletion were completely resistant, and highly sensitive, respectively, to chronic IH induced cardiac effects. These findings suggest that chronic IH induces cardiomyopathy characterized by oxidative stress-mediated cardiac damage and the antioxidant MT protects the heart from such pathological and functional changes.

Topics: Animals; Cardiomyopathies; Hypoxia; Male; Metallothionein; Mice; Myocardium; Oxidation-Reduction; Oxidative Stress; Sleep Apnea, Obstructive

We have demonstrated that zinc supplementation provides cardiac protection from diabetes in mice, but its underlying mechanism remains unclear. Since zinc mimics the function of insulin, it may provide benefit to the heart via stimulating Akt-mediated glucose metabolism. Akt2 plays an important role in cardiac glucose metabolism and mice with Akt2 gene deletion (Akt2-KO) exhibit a type 2 diabetes phenotype; therefore, we assumed that no cardiac protection by zinc supplementation from diabetes would be observed in Akt2-KO mice. Surprisingly, despite Akt2 gene deletion, zinc supplementation provided protection against cardiac dysfunction and other pathological changes in Akt2-KO mice, which were accompanied by significant decreases in Akt and GSK-3β phosphorylation. Correspondingly, glycogen synthase phosphorylation and hexokinase II and PGC-1α expression, all involved in the regulation of glucose metabolism, were significantly altered in diabetic hearts, along with a significantly increased expression of Akt negative regulators: PTEN, PTP1B, and TRB3. All these molecular, pathological, and functional changes were significantly prevented by 3-month zinc supplementation. Furthermore, the stimulation of Akt-mediated glucose metabolic kinases or enzymes by zinc treatment was metallothionein-dependent since it could not be observed in metallothionein-knockout mice. These results suggest that zinc preserves cardiac function and structure in Akt2-KO mice presumably due to its insulin mimetic effect on cardiac glucose-metabolism. The cardioprotective effects of zinc are metallothionein-dependent. This is very important since zinc supplementation may be required for patients with Akt2 gene deficiency or insulin resistance.

Topics: Animals; Blood Glucose; Cardiomyopathies; Cell Cycle Proteins; Diabetes Mellitus, Experimental; Gene Expression Regulation; Glycogen Synthase; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hexokinase; Insulin; Male; Metallothionein; Mice; Mice, Knockout; Myocardium; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Transcription Factors; Zinc

Increasing evidence from in vivo and in vitro studies has indicated that MT exerts protective effects against DOX-induced cardiotoxicity; however the underlying precise mechanisms still remain an enigma. Therefore, the present study was designed using MT knockout mice in concert with genomic approaches to explore the possible molecular and cellular mechanisms in terms of the genetic network changes. MT-I/II null (MT⁻/⁻) mice and corresponding wild-type mice (MT+/+) were administrated with a single dose of DOX (15 mg/kg, i.p.) or equal volume of saline. Animals were sacrificed on the 4th day after DOX administration and samples were collected for further analyses. Global gene expression profiles of cardiac mRNA from two genotype mice revealed that 381 characteristically MT-responsive genes were identified between MT+/+ mice and MT⁻/⁻ mice in response to DOX, including fos, ucp3, car3, atf3, map3k6, etc. Functional analysis implied MAPK signaling pathway, p53 signaling pathway, Jak-STAT signaling pathway, PPAR signaling pathway, Wnt signaling pathway, etc. might be involved to mediate the protection of DOX cardiomyopathy by MT. Results from the present study not only validated the previously reported possible mechanisms of MT protection against DOX toxicity, but also provided new clues into the molecular mechanisms involved in this process.

Topics: Animals; Cardiomyopathies; Creatine Kinase; Doxorubicin; Gene Expression Profiling; Genes; L-Lactate Dehydrogenase; Male; Metallothionein; Mice; Mice, Knockout; Oligonucleotide Array Sequence Analysis; Transcription, Genetic

Antioxidant therapy has shown some promise in critical care medicine in which glutathione depletion and heart failure are often seen in critically ill patients. This study was designed to examine the impact of glutathione depletion and the free radical scavenger, metallothionein (MT), on cardiac function.. Friend virus B and MT transgenic mice were given the glutathione synthase inhibitor buthionine sulfoximine (buthionine sulfoximine [BSO], 30 mmol/L) in drinking water for 2 wks.. Echocardiographic and cardiomyocyte functions were evaluated, including myocardial geometry, fraction shortening, peak shortening, time-to-90% relengthening (TR90), maximal velocity of shortening/relengthening (+/-dL/dt), intracellular Ca2+ rise, sarcoplasmic reticulum Ca2+ release, and intracellular Ca2+ decay rate. Sacro (endo)plasmic reticulum Ca2+-ATPase function was evaluated by 45Ca uptake. Highly reactive oxygen species, caspase-3, and aconitase activity were detected by fluorescent probe and colorimetric assays.. BSO elicited lipid peroxidation, protein carbonyl formation, mitochondrial damage, and apoptosis. BSO also reduced wall thickness, enhanced end systolic diameter, depressed fraction shortening, peak shortening, +/-dL/dt, sarcoplasmic reticulum Ca2+ release, 45Ca uptake, and intracellular Ca2+ decay, leading to prolonged TR90. BSO-induced mitochondrial loss and myofilament aberration. MT transgene itself had little effect on myocardial mechanics and ultrastructure. However, it alleviated BSO-induced myocardial functional, morphologic, and carbonyl changes. Western blot analysis showed reduced expression of sacro (endo)plasmic reticulum Ca2+-ATPase2a, Bcl-2 and phosphorylated GSK-3beta, enhanced calreticulin, Bax, p53, myosin heavy chain-beta isozyme switch, and IkappaB phosphorylation in FVB-BSO mice, all of which with the exception of p53 were nullified by MT.. Our findings suggest a pathologic role of glutathione depletion in cardiac dysfunction and the therapeutic potential of antioxidants.

Topics: Animals; Buthionine Sulfoximine; Cardiomyopathies; Enzyme Inhibitors; Glutathione; Glutathione Synthase; Lipid Peroxidation; Male; Metallothionein; Mice; Mice, Transgenic; Myocardial Contraction; Myocytes, Cardiac; Reactive Oxygen Species; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Ultrasonography

Topics: Angiotensin II; Cardiomyopathies; Diabetic Angiopathies; Humans; Metallothionein; NADP; Oxidative Stress

We evaluated metallothionein (MT)-mediated cardioprotection from angiotensin II (Ang II)-induced pathologic remodeling with and without underlying diabetes.. Cardiac-specific metallothionein-overexpressing transgenic (MT-TG) mice are resistant to diabetic cardiomyopathy largely because of the antiapoptotic and antioxidant effects of MT.. The acute and chronic cardiac effects of Ang II were examined in MT-TG and wild-type (WT) mice, and the signaling pathways of Ang II-induced cardiac cell death were examined in neonatal mouse cardiomyocytes.. Acute Ang II administration to WT mice or neonatal cardiomyocytes increased cardiac apoptosis, nitrosative damage, and membrane translocation of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) isoform p47(phox). These effects were abrogated in MT-TG mice, MT-TG cardiomyocytes, and WT cardiomyocytes pre-incubated with peroxynitrite or superoxide scavengers and NOX inhibitors, suggesting a critical role for NOX activation in Ang II-mediated apoptosis. Prolonged administration of subpressor doses of Ang II (0.5 mg/kg every other day for 2 weeks) also induced apoptosis and nitrosative damage in both diabetic and nondiabetic WT hearts, but not in diabetic and nondiabetic MT-TG hearts. Long-term follow-up (1 to 6 months) of both WT and MT-TG mice after discontinuing Ang II administration revealed progressive myocardial fibrosis, hypertrophy, and dysfunction in WT mice but not in MT-TG mice.. Metallothionein suppresses Ang II-induced NOX-dependent nitrosative damage and cell death in both nondiabetic and diabetic hearts early in the time course of injury and prevents the late development of Ang II-induced cardiomyopathy.

Topics: Angiotensin II; Animals; Apoptosis; Cardiomyopathies; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Fibrosis; Hypertrophy; Metallothionein; Mice; Mice, Transgenic; Myocardium; Myocytes, Cardiac; NADP; Oxidative Stress; Ventricular Remodeling

Our previous studies showed that transgenic mice that overexpress cardiac-specific metallothionein (MT) are highly resistant to diabetes-induced cardiomyopathy. Zinc is the major metal that binds to MT under physiological conditions and is a potent inducer of MT. The present study therefore explored whether zinc supplementation can protect against diabetic cardiomyopathy through cardiac MT induction.. Diabetes was induced in mice (C57BL/6J strain) by a single injection of streptozotocin. Half were supplemented intraperitoneally with zinc sulfate (5 mg/kg) every other day for 3 months. After zinc supplementation, mice were maintained for 3 more months and then examined for cardiomyopathy by functional and morphological analysis. Significant increases in cardiac morphological impairment, fibrosis, and dysfunction were observed in diabetic mice but not in diabetic mice supplemented with zinc. Zinc supplementation also induced a significant increase in cardiac MT expression. The role of MT in cardiac protection by zinc supplementation was examined in cultured cardiac cells that were directly exposed to high levels of glucose (HG) and free fatty acid (FFA) (palmitate), treatment that mimics diabetic conditions. Cell survival rate was significantly decreased for cells exposed to HG/FFA but did not change for cells exposed to HG/FFA and pretreated with zinc or low-dose cadmium, each of which induces significant MT synthesis. When MT expression was silenced with the use of MT small-interfering RNA, the preventive effect of pretreatment with zinc or low-dose cadmium was abolished.. These results suggest that the prevention of diabetic cardiomyopathy by zinc supplementation is predominantly mediated by an increase in cardiac MT.

Topics: Animals; Cadmium; Cardiomyopathies; Cell Survival; Cells, Cultured; Diabetes Complications; Diabetes Mellitus, Experimental; Fatty Acids, Nonesterified; Glucose; Male; Metallothionein; Mice; Mice, Inbred C57BL; Myocardium; Myocytes, Cardiac; RNA, Messenger; RNA, Small Interfering; Zinc Sulfate

We aimed to test whether attenuation of early-phase cardiac cell death can prevent diabetic cardiomyopathy.. Our previous study showed that cardiac apoptosis as a major early cellular response to diabetes is induced by hyperglycemia-derived oxidative stress that activates a mitochondrial cytochrome c-mediated caspase-3 activation pathway. Metallothionein (MT) as a potent antioxidant prevents the development of diabetic cardiomyopathy.. Diabetes was induced by a single dose of streptozotocin (STZ) (150 mg/kg) in cardiac-specific, metallothionein-overexpressing transgenic (MT-TG) mice and wild-type (WT) controls. On days 7, 14, and 21 after STZ treatment, cardiac apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and caspase-3 activation. Cardiomyopathy was evaluated by cardiac ultrastructure and fibrosis in the diabetic mice 6 months after STZ treatment.. A significant reduction in diabetes-induced increases in TUNEL-positive cells, caspase-3 activation, and cytochrome c release from mitochondria was observed in the MT-TG mice as compared to WT mice. Cardiac protein nitration (3-nitrotyrosine [3-NT]) and lipid peroxidation were significantly increased, and there was an increase in mitochondrial oxidized glutathione and a decrease in mitochondrial reduced glutathione in the WT, but not in the MT-TG, diabetic mice. Double staining for cardiomyocytes with alpha sarcomeric actin and caspase-3 or 3-NT confirmed the cardiomyocyte-specific effects. A significant prevention of diabetic cardiomyopathy and enhanced animal survival were observed in the MT-TG diabetic mice as compared to WT diabetic mice.. These results suggest that attenuation of early-phase cardiac cell death by MT results in a significant prevention of the development of diabetic cardiomyopathy. This process is mediated by MT suppression of mitochondrial oxidative stress.

Topics: Actins; Animals; Antioxidants; Apoptosis; Cardiomyopathies; Caspase 3; Cell Death; Cytochromes c; Diabetes Mellitus, Experimental; Enzyme Activation; Glutathione; Glutathione Disulfide; Lipid Peroxidation; Metallothionein; Mice; Mice, Transgenic; Mitochondria, Heart; Myocardium; Myocytes, Cardiac; Oxidative Stress; Sarcomeres; Survival Analysis; Tyrosine

The mechanisms of metallothionein prevention of diabetic cardiomyopathy are largely unknown. The present study was performed to test whether inhibition of nitrosative damage is involved in metallothionein prevention of diabetic cardiomyopathy. Cardiac-specific metallothionein-overexpressing transgenic (MT-TG) mice and wild-type littermate controls were treated with streptozotocin (STZ) by a single intraperitoneal injection, and both developed diabetes. However, the development of diabetic cardiomyopathy, revealed by histopathological and ultrastructural examination, serum creatine phosphokinase, and cardiac hemodynamic analysis, was significantly observed only in the wild-type, but not in MT-TG, diabetic mice 2 weeks and 6 months after STZ treatment. Formations of superoxide and 3-nitrotyrosine (3-NT), a marker for peroxynitrite-induced protein damage, were detected only in the heart of wild-type diabetic mice. Furthermore, primary cultures of cardiomyocytes from wild-type and MT-TG mice were exposed to lipopolysaccharide/tumor necrosis factor-alpha for generating intracellular peroxynitrite. Increases in 3-NT formation and cytotoxicity were observed in wild-type, but not in MT-TG, cardiomyocytes. Either urate, a peroxynitrite-specific scavenger, or Mn(111) tetrakis 1-methyl 4-pyridyl porphyrin pentachloride (MnTMPyP), a superoxide dismutase mimic, significantly inhibited the formation of 3-NT along with a significant prevention of cytotoxicity. These results thus suggest that metallothionein prevention of diabetic cardiomyopathy is mediated, at least in part, by suppression of superoxide generation and associated nitrosative damage.

Topics: Animals; Cardiomyopathies; Diabetes Complications; Gene Expression; Heart Diseases; Male; Metallothionein; Mice; Mice, Transgenic; Nitrosation; Superoxides; Tyrosine

Many studies have shown that metallothionein (MT) can be increased significantly by different oxidative insults in multiple organ systems. However, the increase in MT production often fails to protect against oxidative tissue injury. On the other hand, recent studies using a cardiac-specific, MT-overexpressing, transgenic mouse model have shown that MT protects against oxidative heart injury. Thus, the present study was undertaken to test the hypothesis that prior increase in MT levels is required to prevent oxidative injury. Oxidative heart injury was induced by doxorubicin (DOX), an important anticancer drug that causes severe cardiotoxicity through oxidative stress. Cardiac-specific, MT-overexpressing, transgenic mice and wild-type (WT) FVB mice were treated with DOX at 20 mg/kg. Four days after the treatment, MT concentrations were markedly elevated in the WT mouse heart. The elevated MT concentrations were comparable with those found in the transgenic mouse heart, which did not show further MT elevation in response to DOX challenge. Severe oxidative injury occurred in the heart of WT mice, including myocardial lipid peroxidation, morphological changes as examined by electron microscopy, high levels of serum creatine kinase activity, and decreased total glutathione concentrations in the heart. However, all of these pathological changes were significantly inhibited in the MT-transgenic mice. Therefore, this study demonstrates that there is a correlation between MT induction and oxidative stress in the DOX-treated mouse heart. However, MT can protect the heart from oxidative injury only if it is present prior to induction of oxidative stress.

Topics: Animals; Cardiomyopathies; Creatine Kinase; Creatine Kinase, MB Form; Doxorubicin; Drug Administration Schedule; Gene Expression; Genes, Synthetic; Glutathione; Humans; Isoenzymes; Lipid Peroxidation; Male; Metallothionein; Mice; Mice, Transgenic; Organ Specificity; Oxidative Stress; Promoter Regions, Genetic; Recombinant Fusion Proteins; Time Factors

Many diabetic patients suffer from cardiomyopathy, even in the absence of vascular disease. This diabetic cardiomyopathy predisposes patients to heart failure and mortality from myocardial infarction. Evidence from animal models suggests that reactive oxygen species play an important role in the development of diabetic cardiomyopathy. Our laboratory previously developed a transgenic mouse model with targeted overexpression of the antioxidant protein metallothionein (MT) in the heart. In this study we used MT-transgenic mice to test whether an antioxidant protein can reduce cardiomyopathy in the OVE26 transgenic model of diabetes. OVE26 diabetic mice exhibited cardiomyopathy characterized by significantly altered mRNA expression, clear morphological abnormalities, and reduced contractility under ischemic conditions. Diabetic hearts appeared to be under oxidative stress because they had significantly elevated oxidized glutathione (GSSG). Diabetic mice with elevated cardiac MT (called OVE26MT mice) were obtained by crossing OVE26 transgenic mice with MT transgenic mice. Hyperglycemia in OVE26MT mice was indistinguishable from hyperglycemia in OVE26 mice. Despite this, the MT transgene significantly reduced cardiomyopathy in diabetic mice: OVE26MT hearts showed more normal levels of mRNA and GSSG. Typically, OVE26MT hearts were found to be morphologically normal, and elevated MT improved the impaired ischemic contractility seen in diabetic hearts. These results demonstrate that cardiomyocyte-specific expression of an antioxidant protein reduces damage to the diabetic heart.

Topics: Actins; Animals; Antioxidants; Atrial Natriuretic Factor; Base Sequence; Blood Glucose; Blotting, Northern; Cardiomyopathies; Diabetic Angiopathies; Disease Models, Animal; Gene Expression Regulation; Glutathione Disulfide; Insulin; Metallothionein; Mice; Mice, Transgenic; Molecular Sequence Data; Myocardial Contraction; Oligonucleotide Probes; RNA, Messenger; Transcription, Genetic; Triglycerides