Oxidative stress and diabetic cardiomyopathy

Oxidative Stress and Diabetic Cardiomyopathy 181

Cardiovascular Toxicology Humana Press Volume 1, 2001181

*Author to whom allcorrespondence andreprint requests should beaddressed: Y. James Kang,Department of Medicine,University of LouisvilleSchool of Medicine,511 S. Floyd St., MDR 530,Louisville, KY 40202.E-mail: [email protected]

Cardiovascular Toxicology,vol. 1, no. 3, 181–193, 2001

Oxidative Stress and Diabetic CardiomyopathyA Brief Review

Lu Cai1 and Y. James Kang1,2,3,*Departments of 1Medicine, 2Pharmacology and Toxicology, Universityof Louisville, Louisville, KY 40202; and 3Jewish Hospital Heart and LungInstitute, Louisville, KY 40292

Cardiovascular Toxicology (2001) 01 181–193 $13.25 (http://www.cardiotox.com)© Copyright 2001 by Humana Press Inc. All rights of any nature whatsoever reserved. 1530-7905/01Humana Press

AbstractDiabetes is a serious public health problem. Improvements in the treatment

of noncardiac complications from diabetes have resulted in heart diseasebecoming a leading cause of death in diabetic patients. Several cardiovascularpathological consequences of diabetes such as hypertension affect the heartto varying degrees. However, hyperglycemia, as an independent risk factor,directly causes cardiac damage and leads to diabetic cardiomyopathy. Dia-betic cardiomyopathy can occur independent of vascular disease, although themechanisms are largely unknown. Previous studies have paid little attentionto the direct effects of hyperglycemia on cardiac myocytes, and most studies,especially in vitro, have mainly focused on the molecular mechanisms under-lying pathogenic alterations in vascular smooth-muscle cells and endothelialcells. Thus, a comprehensive understanding of the mechanisms of diabetic car-diomyopathy is urgently needed to develop approaches for the prevention andtreatment of diabetic cardiac complications. This review provides a survey of cur-rent understanding of diabetic cardiomyopathy. Current consensus is that hyper-glycemia results in the production of reactive oxygen and nitrogen species,which leads to oxidative myocardial injury. Alterations in myocardial struc-ture and function occur in the late stage of diabetes. These chronic alterationsare believed to result from acute cardiac responses to suddenly increased glu-cose levels at the early stage of diabetes. Oxidative stress, induced by reactiveoxygen and nitrogen species derived from hyperglycemia, causes abnormalgene expression, altered signal transduction, and the activation of pathwaysleading to programmed myocardial cell deaths. The resulting myocardial cellloss thus plays a critical role in the development of diabetic cardiomyopathy.Advances in the application of various strategies for targeting the prevention ofhyperglycemia-induced oxidative myocardial injury may be fruitful.Key Words: Cardiomyopathy; diabetic complications; hyperglycemia; oxida-tive stress; antioxidants.

Introduction

The occurrence of systemic hyperglycemia in diabetes can ultimately causelong-term damage to multiple organs and lead to severe complications. The micro-vasculature is a key target of hyperglycemic damage. It is thought that damageto small blood vessels can lead to and exacerbate systemic complications. Among

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these complications is the development of cardiovas-cular disease. It has been estimated that 65–70% of dia-betics die of heart disease, making it the major mortalityin both Type 1 and Type 2 diabetes, incurring an esti-mated cost of $10 billion per year. A report from theAmerican Heart Association states that diabetes is acardiovascular disease (1). In a workshop organizedby the European Association for the Study of Diabetes(EASD) and the Juvenile Diabetes Research Founda-tion (JDRF), Robert Goldstein, MD, PhD, Chief Sci-entific Officer of JDRF, pointed out that diabetes isa serious, potentially fatal cardiovascular disease (http://www.jdrf.org/research/feature/res082201.php).

Diabetic cardiovascular injury includes both car-diac and peripheral blood vessels. However, researchhas focused largely on peripheral vascular injury.Retinopathy (disease of the retina that can lead to visionloss), neuropathy (nerve damage), and nephropathy(kidney disease) are all caused, in part, by changesin blood flow or abnormal vessel growth (angio-genesis). Although recent studies have extended ourunderstanding of the pathogenesis of the vascularcomplications of diabetes, the mechanism by whichhyperglycemia causes tissue damage, especially car-diomyopathy, is poorly understood (1–3).

Oxidative stress has been linked both to the onsetof diabetes and its complications (4–8). Recent stud-ies demonstrate that oxidative damage induced byreactive oxygen species (ROS) and reactive nitro-gen species (RNS) derived from hyperglycemia playsa critical role in diabetic injury in multiple organs.Thus, the relationship between oxidative stress anddiabetic cardiomyopathy is a major focus of currentresearch.

This review provides a brief survey of our currentunderstanding of the pathogenesis of hyperglycemia-induced cardiomyopathy. It focuses on hyperglyce-mia-derived ROS and RNS and their causal relationto diabetic cardiomyopathy. Several good reviews onrelated topics of diabetic vascular diseases are avail-able (9–11) and are not covered in this review.

Diabetic Cardiomyopathy

Definition and GeneralCharacteristics of Diabetic CardiomyopathyCardiomyopathies were previously defined as “heart

muscle diseases of unknown cause” and were differ-

entiated from specific heart muscle disease of knowncause. Cardiomyopathies are now defined as diseasesof the myocardium associated with cardiac dysfunc-tion and classified by the dominant pathophysiologyor, if possible, by etiological/pathogenetic factors (12,13). These include dilated cardiomyopathy, hyper-trophic cardiomyopathy, restrictive cardiomyopathy,arrhythmogenic right ventricular cardiomyopathy,and specific cardiomyopathy. The term “specific car-diomyopathy” is now used to describe heart musclediseases that are associated with specific cardiac orsystem disorders. Diabetic cardiomyopathy, as onetype of specific cardiomyopathy, is a diabetes-relatedmyopathy and is characterized mainly by impaireddiastolic function (2,3,12,13).

Rubler et al. first recognized the existence of diabe-tic cardiomyopathy in diabetic patients with conges-tive heart failure. None of their patients had evidenceof valvular, congenital, hypertensive, alcohol-relatedheart disease, or significant coronary atherosclerosis(14). After a myocardial infarction, diabetic patientsare twice as likely to die and three times as likely toprogress to congestive heart failure than are nondia-betic patients (15). The Framingham study (16) of292 diabetic and 4900 nondiabetic subjects and arecent study (17) of 1810 diabetic patients and 944age-matched controls further demonstrated signifi-cantly increased incidence of heart failure in diabetics,irrespective of coronary artery diseases and hyperten-sion. Moreover, diabetes accelerates heart failure toa greater extent in women than in men. Taken together,these studies show that diabetes mellitus affects car-diac structure and function independent of blood pres-sure or coronary artery disease, although hypertensioncan accelerate myocardial damage in diabetic patients(2,3,18–20).

The development of diabetic cardiomyopathyis associated with defects in cellular organelles suchas myofibrils, mitochondria, sarcoplasmic reticulum,and sarcolemma. In addition, the calcium-handlingproperties of the diabetic heart are altered as a con-sequence of changes in myocardial metabolism,and these then determine the derangement of pro-cesses involved in cardiac contraction and relax-ation. This derangement will eventually lead to heartdysfunction and heart failure (10,21). Figure 1 brieflysummarizes the developing stages of diabetic cardio-myopathy.



Metabolic Changes in the Diabetic Myocardium

Cardiac function demands a high amount of energysupplied as adenosine triphosphate (ATP). Undernormal physiological conditions, the adult heart usesa combination of glucose (carbohydrates), free fattyacids (FFA), pyruvate, and ketone bodies as fuel sources(21,22). In the fetus, the major source of reducingequivalents that generate energy is mitochondrialacetyl-CoA, which is produced from pyruvate via cyto-solic glycolysis. Shortly after birth, cardiac mitochon-dria use FFA as the major energy source, typicallyproviding 60–70% of the heart’s energy needs (21,22).

In uncontrolled diabetes, the use of glucose de-creases, and FFA oxidation can provide from 90% to100% of the heart’s ATP requirements (21). As longago as 1912, the inability of the diabetic heart to oxi-dize sugar was the most prominent feature of the dis-order noted (21). Glucose metabolism can be roughlydivided into three categories: (1) glucose transportand phosphorylation, (2) glycolysis (i.e., metabolismfrom glucose-6-phosphate to pyruvate and lactate),and (3) glucose oxidation. All aspects of glucose metab-olism with the exception of glycolysis are inhibited indiabetes; the diabetic heart mostly relies on the oxida-tion of lipids for its energy support (21).

Glucose uptake is mediated by transporter protein(GLUT). GLUT4 is found primarily in insulin-sen-sitive tissue such as fat and skeletal muscle and is thepredominant isoform of cardiac muscle. Decreasedglucose transport is directly related to the decreasein abundance of GLUT4 mRNA and its protein (21,23,24). In experimentally induced diabetic animalmodels, the levels of GLUT4 mRNA and protein werereduced by about 50%, and both basal and the insu-lin-stimulated glucose transport were significantlyreduced in their cardiomyocytes (21,22).

In mammalian tissues, the phosphorylation of intra-cellular glucose to glucose-6-phosphate (Glu-6-P)is facilitated by four distinct hexokinase (HK) isoen-zymes, designated as HK I, II, III, and IV. Amongthese, HK II is the major glycolytic enzyme in insu-lin-sensitive tissues such as adipose tissue, skeletalmuscle, and heart. Decreased glucose uptake in dia-betic animals has been linked to decreased HK IImRNA and protein (21); yet, the overexpression ofHK II does not improve or affect glucose phospho-rylation (25,26).

The rate-limiting reaction in glycolysis is the con-version of fructose-6-phosphate to fructose-1,6-

Fig. 1. Stages of diabetic cardiac effects.

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diphosphate catalyzed by phosphofructokinase (PFK).However, this aspect of glucose metabolism maynot be significantly altered by diabetes because aslight decrease or no change in PFK activity has beenobserved in diabetic hearts (21). The reaction of pyru-vate, CoA, and NAD to form acetyl-CoA and NADHis catalyzed by pyruvate dehydrogenase complex(PDHc), a multienzyme complex located in the mito-chondria. Regulation of PDHc that controls the entryof glucose into the citric acid cycle is significantlyaltered by diabetes, with an increase in PDH kinaseactivity leading to a decrease of PDH in the activeform. Decreased acetyl-CoA carboxylase activity asa result of AMP-activated protein kinase that leadsto a reduction in malonyl-CoA has been noted in dia-betic cardiomyocytes, which results in increasedfatty acid oxidation (21,27).

Because of the change of the normal pathwaysof glucose metabolism, the polyol pathway of glu-cose metabolism in the diabetic heart is dramaticallyincreased (28–31). In the polyol pathway, glucose isreduced to sorbitol by aldose reductase in the pres-ence of NADPH, and sorbitol is then oxidized bysorbitol dehydrogenase to fructose at the cost ofNAD+.

Because fatty acid oxidation is the least efficientmetabolic pathway for oxygen, ischemic cardiovas-cular alterations are common, along with the accu-mulation of lactate and acid metabolites that, in turn,cause myocardial deterioration (21,32). However,FFA uptake and �-oxidation in the diabetic heart isnot always altered (33). Therefore, further studies arerequired to comprehensively understand the effectof diabetes on myocardial FFA metabolism.

Because of the abnormal metabolism of glucosein diabetics, other concomitant metabolic abnormal-ities occur, such as altered glycogen metabolism (21).Cardiomyocyte glycogen stores are dramaticallyincreased, and the concentration correlates with theduration and severity of the disease (21). The ele-vated circulating concentration of ketone body indiabetes could be one of the causes for diabetic myo-cardial dysfunction. It is apparent that ketone bodiescan inhibit glycolysis and glucose oxidation in theheart, even at low concentrations, and can also stimu-late glycogen synthesis (21). The response of the heartto adenosine in diabetes is altered with a decreasedadenosine kinase (AK) activity and depressed AKmRNA expression (34).

Diabetes also causes inhibition of mitochondrialfunction, resulting in altered calcium handling. In addi-tion, diabetes decreases creatine kinase activity andmRNA expression in the heart (21,35,36). Becausecreatine kinase is important in maintaining normalATP levels, it has been proposed that impaired energysupply via the creatine kinase reaction contributes tothe development of diabetes-induced cardiomyopa-thy. In the adult heart, lipoprotein lipase (LPL) is pro-duced in myocytes and is significantly elevated indiabetic hearts (37). This enhanced LPL pool in thediabetic heart results in the increased uptake of FFAby myocytes. This abnormally high level of FFA inthe diabetic heart leads to a number of metabolic,morphological, and mechanical changes and, even-tually, to cardiomyopathy (27,37).

Pathological Changesin the Diabetic MyocardiumAlthough there are numerous studies on morpho-

logical alterations in the diabetic heart (21), it maybe concluded that there is no single characteristicpathology of diabetic lesions. The most consistentfindings are myocyte hypertrophy, interstitial fibro-sis, increased periodic acid–Schiff’s reagent- (PAS-)positive material, and intramyocardial microangio-graphy. Therefore, diabetic cardiomyopathy may bebetter defined at functional or biochemical levels(38). Kawaguchi et al. (39) compared the histologi-cal alterations of diabetic hearts with hypertensionand found four major changes, including (1) the thick-ening of the capillary basement membrane, (2) theaccumulation of toluidine-blue-positive materials, (3)the small size of myocytes (cell atrophy), and (4) inter-stitial fibrosis. Hypertension alone does not causethese changes, but it amplifies the thickening ofthe capillary basement membrane. Therefore, it wasassumed that alterations in myocardial cells and cap-illaries of diabetic hearts leads to interstitial fibrosisand, ultimately, to ventricular systolic and diastolicdysfunction (39). In addition, mitochondrial swellingand an increase in lipid droplets were also observedin diabetic cardiomyocytes by ultrastructural obser-vation; both changes could be attenuated with insu-lin treatment (40).

Impaired myocardial blood flow is another phenom-enon that leads to a decrease in cardiac flow reservein diabetic patients compared to that of matched con-trols, even in the absence of obvious heart disease.



This impaired blood flow currently is thought torelate to impaired endothelial-dependent vasodila-tion, although the mechanism of endothelial dysfunc-tion in the diabetic patient is not fully understood (38).Acute hyperglycemia may impair endothelial-derivedvasodilation in healthy humans. Elevated blood glu-cose by itself may be one of the important factors forthe impaired vascular response and may contributeto the lack of hyperkinetic response and the diastolicdysfunction seen in diabetes mellitus (21).

There are several other cardiovascular pathologi-cal consequences of diabetes, including (1) acceler-ated atherosclerosis leading to coronary and cerebralartery disease, (2) systemic hypertension, (3) micro-circulatory injury, leading to chronic multifocalmyocyte damage and cardiomyopathy, and (4) neu-ropathy, especially autonomic dysfunction. Thesepathological conditions may affect the diabetic heartto varying degrees; however, hypertension is defi-nitely a cocontributor to diabetic cardiomyopathy(3,39,41,42).

Functional Changesin the Diabetic MyocardiumAlterations in intracellular cations are directly

related to the altered electrical activities of diabetichearts. Sodium is often accumulated with a decreasein potassium and calcium in the diabetic heart, assummarized in Table 1. Sodium accumulation maybe related, in part, to the diabetes-caused conforma-

tional change of Na+-K+ ATPase. This change mayincrease in high-affinity sodium-binding sites, result-ing in high levels of sodium in the cell (21,43). Thedecrease in calcium concentration is the result of im-paired Ca2+ uptake by sarcolemma. This decrease maybe ameliorated by the accumulation of Na+ throughthe Ca2+/Na+ -exchanging mechanism, which couldalso be impaired by diabetes (21).

Decreased calcium uptake, decreased calcium bind-ing to the sarcolemma, decreased calcium intake byvesicles of the sarcoplasmic reticulum, and decreasedmyofibrillar Ca2+-ATPase activity have been shownin diabetic rat models. Insulin reverses many of thesealterations. Although it is not clear how importantany one of these elements is in diminishing cardiacfunction in diabetes, the presence of abnormalitiesin calcium uptake and �-adrenergic stimulation, cor-rectable by insulin administration, is of interest tothe overall understanding of myocardial dysfunctionin diabetes (21).

Diabetic hearts exhibit decreased responsivenessto stimulation by �-adrenoceptor (�-AR) agonists,which may be the result of changes in the expressionor signaling of �-AR, or both (44). The alteration of car-diac �1-adrenoceptor (�1-AR) signaling in strepto-zotocin (STZ)-induced diabetic rats has been observedon the surface of isolated cardiac myocytes. Insulintreatment partially restored �-AR mRNA and proteinlevels. These data suggest that the decreased respon-siveness of diabetic hearts to the stimulation of �-ARagonists may be the result of a decrease in �-AR expres-sion (45). All of these abnormalities in intracellularcations and responses to �-AR stimulation will resultin the functional alteration of the heart.

Functional abnormalities are often detected byvarious noninvasive approaches, including electro-cardiogram, echocardiography, radionuclide ventric-ulography, stress testing, and by invasive approachessuch as catheterization. These abnormalities includea shorter left ventricular ejection time (LVET), anincreased pre-ejection period (PEP) presented as anincrease in PEP/LVET ratio, reduced ejection frac-tion, increased wall stiffness, decreased fractionalshortening, decreased rate of LV filling, and increasedaction potential duration (17,21). Heart rate changewas also directly associated with plasma glucose level(46). These findings reflect that diabetic cardiomy-opathy takes the form of diastolic and/or systemic(dilated) left ventricular dysfunction (47,48). Left

Table 1Summary of Cationic

Changes in Diabetic Myocardium

Decreased myosin Ca2+-ATPase activity:shift from V1 to V3 myosin isoform

Decreased sarcoplasmic reticulum (SR) Ca2+ pumpactivities: accumulation of long-chain acylcarnitines

Decreased mitochondrial Ca2+ uptakeDecreased number of sarcolemmal Ca2+-channelsImpaired function and decreased number

of �- and �-ARDecrease in sarcolemmal Na+-K+ ATPase

and Ca2+-ATPase at protein and mRNA levelsDecrease in sarcolemmal Na+-Ca2+ exchange protein

and mRNADecreased sarcolemmal content of phosphatidylethanol-

amine and diphosphatidylglycerol

Source: From refs. 21, 36, and 43.

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ventricular diastolic dysfunction (LVDD) may rep-resent the first stage of diabetic cardiomyopathy andis a frequent finding in many studies of cardiac func-tion in diabetic subjects without symptoms and signsof heart disease. Diastolic dysfunction independentof ischemic heart disease has been clinically proven.Therefore, diastolic dysfunction is one of the mostprominent mechanical defects associated with diabe-tic cardiomyopathy and is characterized by decreasedcompliance and slower rates of myocardial relax-ation (38,47,49).

Myocardial diastolic abnormalities reflect the in-creased interstitial connective tissue resulting froman altered extracellular matrix (21). In one study,diabetes markedly prolonged both the contractionand relaxation phases (50). Contractile dysfunctionseen in the diabetic heart is the result, in part, of abnor-malities of the myocyte. These abnormalities occurafter only 4–6 d of diabetes, suggesting a rapid alter-ation in the processes of myocyte shortening andrelengthening, which likely includes impaired Ca2+

sequestration or extrusion (50).In the late stage of diabetes, systolic (dilated) dys-

function and altered electrical properties in diabetichearts may occur, and ischemia and infarction maytake place in human diabetics. These are often asso-ciated with some degree of myocardial failure. Inexperimental diabetic animals, increased LV massand cavity dimensions (diastolic length), prolongedisovolumic relaxation time, increased atrial contribu-tion to diastolic filling, and elevated LV end-diastolicpressure and chamber stiffness have been observedby echocardiography (51,52).

In summary, the pathology of the diabetic heartincludes coronary atherosclerosis, ischemic heartdisease, microvascular injury, multifocal myocytol-ysis, fibrosis, and, eventually, cardiomyopathy. Hyper-tension, commonly seen in association with diabetes,may accelerate the process of cardiomyopathy. Meta-bolic derangement may lead to myocyte toxicity,increased atherosclerosis, and cardiac dysfunction(53). As shown in Fig. 1, typical changes in mem-brane and contractile function usually occur daysafter diabetic development, whereas morphologicalchanges and heart dysfunction take months to years.The chronic alterations at the late stages of diabetesare, however, believed to result from acute cardiacresponses to suddenly increased glucose levels atthe early stage (36,54). Importantly, oxidative stress

has been proposed as the major pathogenic effect ofhigh levels of glucose.

Hyperglycemiaand Accumulation of ROS and RNS

ROS Formationby High Levels of Glucose In VitroIn vitro cell-free studies have shown that ROS

formation is caused directly by autoxidation of glu-cose. Glucose nonenzymatically reacts with proteinamino groups, forming a Schiff base that may rear-range to form Amadori products. This reaction iscalled glycation and generates ROS (55). In addition,Amadori products subsequently degrade into �-keto-aldehyde compounds such as 3-deoxyglucosone (3-DG), which is a potent crosslinker responsible forthe polymerization of proteins to form advancedglycation end products (AGEs) (55,56). All of thesecharacteristics of glucose autoxidation contribute tothe formation of ROS, including hydrogen peroxide,superoxide, hydroxyl radicals, and protein-reactiveketoaldehyde compounds (56,57).

We have performed a study incubating lipopro-tein and bovine serum albumin (BSA) in phosphate-buffered saline (PBS) with high levels of glucose for72 h and 96 h and found a significant increase in lipidperoxidation measured by thiobarbituric acid reac-tive substances (TBARS) assay and the productionof fragmented protein with increased carbonyl con-tent (Cai and Kang, unpublished observation). Finottiet al. (58) also showed that high levels of glucosewere associated with increased carbonyl content witha strong oxidative radical signal measured by elec-tron paramagnetic resonance (EPR) spectra. Incu-bation of native DNA with high levels of glucose inthe presence of Cu,Zn-SOD (superoxide dismutase)caused significant oxidative DNA damage (55). Ithas been suggested that free radicals and hydrogenperoxide are slowly produced by glucose autoxi-dation and are substantial causes of the structuraldamage (56).

ROS Formation by High Levelsof Glucose in Cultured CellsStudies using cultured cells have shown that ROS

formation under various experimental conditions isa major cellular response to high levels of glucose



(59–62). In rat and mouse mesangial cells, highlevels of glucose rapidly produced cytosolic ROS,whereas neither L-glucose nor 3-O-methyl-D-glucoseincreased cytosolic ROS (63). A significant increasewas observed at glucose concentrations as high as10 mM(62). Furthermore, an inhibitor of glucose trans-port cytochalasin B effectively inhibited ROS gener-ation by high levels of glucose, suggesting that glucoseuptake is required for ROS generation in the cells(63). In cultured aortic smooth-muscle cells (SMCs)and human umbilical vein endothelial cells (HUVECs),short-term (48 h) and long-term (14 d) exposure to30 mM glucose and free fatty acid (palmitate) resultedin a significant increase in intracellular ROS forma-tion (64,65). Hydrogen peroxide generation in cul-tured cells exposed to high levels of glucose has beenobserved (66). We found ROS formation in cardiacmyoblasts exposed to high levels of glucose as earlyas 3 h, reaching a peak at 6 h after the exposure (Caiand Kang, unpublished observation).

Advanced glycation end products have been shownto stimulate ROS and RNS formation. The forma-tion and accumulation of AGE adducts in variousdiabetic tissues are associated with altered proteinstructure and function and are able to activate cellu-lar responses that generate ROS (60,67). One mecha-nism by which AGEs exert their effects on cellularfunction is their interaction with specific receptors(RAGE). The interaction of AGE with RAGE acti-vates different signal transduction pathways, includ-ing mitogen-activated protein kinase (MAPK) andprotein kinase C pathways, and generates oxidativestress that results in cellular damage and inflamma-tion (68).

ROS Formationby Hyperglycemia in the Heartof Diabetic Patients and Animal ModelsA significant increase in oxidative damage (lipid

peroxidation), measured by TBARS assay, was ob-served in the hearts of diabetic rats (69). Productionof hydroxyl radicals was detected in diabetic ratsinduced by STZ using a quantitative assay that trappedhydroxyl radicals with salicylic acid as 2,3- and 2,5-dihydroxybenzoic acid (70). In the heart, hydroxylradical production and blood glucose concentra-tion were directly correlated with the amount of STZinjected into rats, up to 60 mg/kg body weight (70).In the cardiac artery wall, increased hydroxyl radi-

cals were also observed in 6-mo-old diabetic mon-keys (71). Using fluorescent probes myocytes iso-lated from STZ-induced diabetic mice were used todetect hydrogen peroxide and hydroxyl radical, andincreased ROS were observed relative to that of con-trol mice (72).

Nitric oxide (NO) can interact with superoxide toform peroxynitrite. Peroxynitrite, a very active radi-cal similar to hydroxyl radical, interacts with cyto-plasmic proteins to form nitrotyrosine, which has beenconsidered an index of RNS-induced oxidative dam-age under in vivo conditions (72,73). In the heartsof STZ-induced diabetic mice, a significant increasein nitrotyrosine concentrations in myocytes wasobserved (72). In addition, heart tissues from dia-betic patients (both with and without hypertension)also show a strong immunochemistical staining of3-nitrotyrosine (73).

In summary, ROS and RNS formation by highlevels of glucose have been observed under differ-ent conditions, including in vivo diabetic hearts. Thepathways by which high levels of glucose induceROS and RNS are complex and incompletely under-stood; Fig. 2 outlines several of these pathways. Inaddition, the enhanced polyol pathway of glucosemetabolism results in the oxidation of sorbitol tofructose, coupled to the reduction of NAD+ to NADH(28–31). The increased ratio of NADH/NAD+ en-hances ROS and RNS production by several path-ways, including the increased activation of xanthineoxidase, autooxidation of NADH, and inactivationof Cu,Zn-SOD. Hink et al. (74) reported that endo-thelial NO synthase dysfunction, coupled with acti-vation of an NAD(P)H-dependentoxidase, was largelyresponsible for enhanced superoxide production. Theformation of ROS and RNS results in oxidative dam-age, which may act as the major mechanism of dia-betic cardiomyopathy.

The Role of Oxidative Stressin Diabetic Cardiomyopathy

The term “oxidative stress” is defined as a seriousimbalance between the production of reactive freeradicals and antioxidants (11). Oxidative damagecaused by ROS and RNS has been shown to relatedirectly to multiple complications of diabetes (4,6,7,11). Blocking ROS formation has been shown toprevent hyperglycemic damage (31).

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Mitochondrial dysfunction is closely related toROS formation and has been considered to play acritical role in the development of diabetic cardio-myopathy (40,75). Coenzyme Q is an important com-ponent in mitochondrial energy metabolism and isalso a potent antioxidant. In heart mitochondria ofdiabetic rats, the concentration of �-tocopherol wasincreased; however, the concentrations of CoQ9 andCoQ10 were decreased and lipid peroxidation wasdecreased (75). Abnormal activities of (Na,K)-ATPaseand calcium ATPase have been observed, and the sup-plementation of antioxidants prevented the diabetes-induced elevation of oxidative stress and inhibiteddecreases of myocardial (Na,K)-ATPase and cal-cium ATPase (6). Diabetes depresses cardiac creat-

ine kinase activities (Table 1). This change was alsoassociated with the formation of ROS (5). The sulf-hydryl group of this enzyme is most likely oxidized,because the addition of N-acetyl-cysteine (NAC), DL-dithiothreitol (DTT), or �-glutamylcysteine ester sig-nificantly reversed the depression (76). In addition,a reduction in the number of calcium channels, alter-ations in heart sarcolemmal Ca2+-ATPase and pumpactivity (77), and Na+-Ca2+-exchange activity havebeen associated with ROS.

Another type of oxidative damage is caused byoxysterols, oxidative derivatives of cholesterol, whichare known to be highly cytotoxic. In STZ-induceddiabetic rats, two oxysterols, 7-�-hydroxycholesteroland 7-ketocholesterol, were significantly increased

Fig. 2. Scheme of possible mechanisms by which hyperglycemia induces ROS and RNS and the subsequent effectsleading to cardiomyopathy.



in the heart, along with a significant increase in myo-cardial oxidative damage as demonstrated by elevatedTBARS (69,78). Fibrillar collagen plays an impor-tant role in determining the structural integrity of themyocardium (79). The amount of extracellular col-lagen is determined by the balance between synthesisand degradation, which is mediated by matrix metal-loproteinases (MMPs) and their inhibitors (TIMPs).MMP activities increase in the failing myocardiumof patients and animal models of myocardial remod-eling and heart failure (8,80). Collagen synthesis andthe derangement of MMP regulation are consideredto be critical factors for diabetic cardiac dysfunction(8,80,81). MMP activity is significantly increased indiabetic rat tissues and in the endothelial cell culturesexposed to high levels of glucose (8). The enhancedcollagen synthesis and MMP activity are related toROS formation, because if ROS formation is blocked,the enhanced expression of MMP by high levels ofglucose can be prevented (8,80,81). Taken together,these data indicate that oxidative stress from hyper-glycemia-induced ROS and RNS is involved in thepathogenesis of diabetic cardiomyopathy.

It is worthwhile to note that the contractile func-tion of the heart dictates its high metabolic demand,and the mitochondrial respiratory chain is the pri-mary energy-releasing system in these cells. Throughthis respiratory chain, a series of oxidation–reductionreactions continually takes place in cardiomyocytes.Therefore, an efficient antioxidant system includingSOD, catalase, glutathione peroxidase (GSHpx), glu-tathione (GSH), and �-tocopherol would be criticalto myocardium. However, in experimental animalmodels, the activities of all of these antioxidants inthe heart are lower than in other organs (82,83). Fur-thermore, hyperglycemia can impair and decreaseantioxidant capacity in the heart of diabetes (84–86),making it more vulnerable to ROS- and RNS-induceddamage.

Taken together, both the increased ROS and RNSand decreased antioxidant capacity in diabetic myo-cardium contribute significantly to oxidative stressand result in myocardial damage at molecular andcellular levels, which, in turn, causes cardiac morpho-logical and functional abnormalities. Cell death asa comprehensive consequence of these myocardialresponses to hyperglycemia is an important determi-nant of cardiac remodeling because it causes a lossof contractile units, compensatory hypertrophy of

myocardial cells, and reparative fibrosis (54). Apop-totic cell death associated with oxidative stress inmultiple organs of diabetes mellitus has been docu-mented (87–89). Recent in vivo studies also demon-strated the induction of myocardial cell apoptosis inexperimental diabetic rats (90), mice (72), and dia-betic patients (73). Heart specimens from diabeticpatients (with or without hypertension) showed anincrease in apoptosis in myocytes, endothelial cells,and fibroblasts (73). The increased cell death wasassociated with ROS and RNS formation (72,73,90).It has been reported that FFA oxidation is signifi-cantly increased in diabetic cardiomyopathy, and arecent study demonstrated that FFA-induced celldeath is mediated by ROS-dependent pathways (91).Enhanced apoptotic cell death through exposure tohigh levels of glucose in murine blastocytes (92) andhuman umbilical vein endothelial cells (66) could besuppressed by antioxidants. Kajstura et al. (72) foundthat overexpression of insulinlike growth factor-Iinhibits myocardial cell death through suppressionof angiotensin-mediated oxidative stress. These stud-ies demonstrated that oxidative stress plays a cri-tical role in the induction of myocardial cell death byhyperglycemia, leading to diabetic cardiomyopathy.These studies also suggest that enhancing cardiacantioxidants or supplementing exogenous antioxi-dants might be helpful in preventing diabetic cardi-omyopathy (11).

Conclusion

Diabetic cardiomyopathy is a critical complicationof diabetes and a leading cause of death. A compre-hensive understanding of mechanisms underlying thepathogenesis of diabetic cardiomyopathy is urgentlyneeded. The link between hyperglycemia and oxida-tive stress in diabetes has been established. The roleof oxidative stress in the development of diabetic car-diomyopathy is under extensive investigation, althoughother potential mechanisms such as the impact of pro-tein glycation on the heart are also being extensivelystudied. One of the extreme consequences of hyper-glycemia-induced oxidative stress is myocardial celldeath. With this regard, apoptosis has been studied indiabetic cardiomyopathy. It is important to understandthe signaling pathways and molecular mechanismsby which hyperglycemia-induced oxidative stressleads to cell death and myocardial pathogenesis.

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Some experimental data suggest that the appropriateapplication of antioxidants can prevent diabetic car-diomyopathy. Therefore, the use of antioxidant ther-apy in the prevention and treatment of diabetic cardiaccomplications may be fruitful.

Acknowledgments

The research work cited in this review was sup-ported in part by National Institutes of Health grantsHL63760 and HL59225 and research grants fromthe Jewish Hospital Foundation, Louisville, KY (toYJK), and University of Louisville School of Medi-cine (to LC). The authors greatly appreciate Dr. Zhan-xiang Zhou, Dr. Xiuhua Sun, Dr. Yan Li, Mr. DonMosley, and Ms. Naira Metreveli for their valuablediscussion and technological assistance.

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Oxidative stress and diabetic cardiomyopathy

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