Obesity is associated with increased myocardial oxidative stress - Nature

International Journal of Obesity (1999) 23, 67±74 ß 1999 Stockton Press All rights reserved 0307±0565/99 $12.00 http://www.stockton-press.co.uk/ijo

Obesity is associated with increased myocardial oxidative stress HK Vincent*1 SK Powers1, DJ Stewart1, RA Shanely1, H Demirel1 and H Naito1 1

Departments of Exercise and Sport Sciences and Physiology, Center for Exercise Science, University of Florida, Gainesville, FL 32611, USA

OBJECTIVE: To determine: 1) whether obesity predisposes the myocardium to oxidative stress as evidenced by higher tissue levels of myocardial lipid peroxidation, and 2) what cellular mechanisms are responsible for this predisposition. DESIGN: Comparative, descriptive study of the myocardial tissue of lean and obese Fatty Zucker animals. ANIMALS: 12 month old lean ( 7 =fa; n 6; mean body weight 590 g) and obese (fa=fa; na 7; mean body weight 882 g) male Fatty Zucker rats. MEASUREMENTS: Basal lipid peroxidation (assessed using thiobarbituric reactive acid substances (TBARS) and cumene hydroperoxide equivalents), oxidative and antioxidant enzyme activities (citrate synthase (CS), superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT), thiol content, heat shock protein expression (HSP72=73) and TBARS concentrations following an iron-mediated challenge in vitro. RESULTS: Compared to lean, lipid peroxidation was greater (P < 0.05) in the left ventricle (LV) from obese rats as indicated by higher levels of lipid hydroperoxides (mean 11.48 vs 13.7 cumene hydroperoxide equivalents (CHPE)=mg lipid) and TBARS (mean 11.1 vs 13.9 nMol=mg lipid.). The activity of the manganese isoform of superoxide dismutase in the LV was higher (P < 0.05) in obese animals, compared to controls (mean 135 vs 117 U=mg protein). In contrast, LV catalase and glutathione peroxidase activities did not differ (P > 0.05) between groups. Also, LV levels of HSP 72 (inducible) and 73 (constitutive) did not differ (P > 0.05)( between lean and obese animals. Following an iron-stimulated oxidative challenge in vitro, TBARS concentration was signi®cantly greater (P < 0.05) in LV of obese rats compared to the lean (mean 12.7 vs 16.7 nMol=mg lipid). CONCLUSIONS: These results support the notion that obesity predisposes the myocardium to oxidative stress. However, the postulate that obesity is associated with elevated myocardial antioxidant enzyme activities and HSPs was only partially supported by these ®ndings. Keywords: obesity; myocardium; lipid peroxidation; antioxidant enzymes; reactive oxygen species (ROS)

Introduction Obesity is a growing epidemic in af¯uent nations, with the estimated prevalence range from 10 to 50% or more in the adult population.1 Obese patients are frequently characterized by several complications including hypertension, diabetes, hyperinsulinaemia and hypertriglyceridaemia. Together, these factors increase the mechanical and metabolic loads on the myocardium, thus increasing myocardial oxygen consumption. A potentially negative consequence of elevated myocardial metabolism is the production of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide and the hydroxyl radical, during mitochondrial respiration. Production of ROS at high

Correspondence: *Heather K. Vincent, Center for Exercise and Sport Sciences, 25 Florida Gym, University of Florida, Gainesville, FL 32611, USA. Received 11 February 1998; revised 29 June 1998; accepted 24 July 1998 Abbreviations: ROS reactive oxygen species; LV left ventricle; HSP heat shock protein; TBARS thiobarbituric acid reactive substances; SOD superoxide dismutase; GPX glutathione peroxidase; CAT catalase; CS citrate synthase; PUFA polyunsaturated fatty acid

levels can exceed the antioxidant capacity of the cell, resulting in oxidative stress. Oxidative stress is associated with cellular damage including oxidation of cell membranes and proteins in conjunction with disturbances of cellular redox homeostasis.2 ± 4 Ultimately, these disturbances can cause cardiac contractile dysfunction and subsequent arrhythmias and infarction.5,6 Nonetheless, it is currently unknown if obesity predisposes the myocardium to oxidative injury. This forms the basis of these experiments. Given that ROS are potentially harmful to cells, it is not surprising that tissues contain endogenous defenses against these species. Cells contain both enzymatic and non-enzymatic antioxidants that form the ®rst line of defense against ROS.7 Also, there is growing speculation that heat shock proteins (HSPs) of the 70 kDa family (that is, HSP72, HSP73) are associated with cellular protection against oxidative injury by stabilizing proteins and assisting in the refolding of damaged proteins.8 Previous studies have reported that myocardial levels of both enzymatic (for example, superoxide dismutase, SOD) and non-enzymatic (for example, glutathione) antioxidants are elevated following chronic or repeated exposure to ROS.9,10 Furthermore, exposure to heat and=or ROS has been shown to increase the expression of HSP72

Obesity and myocardial oxidative stress HK Vincent et al

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in cardiac myocytes.9,11,12 It follows that if obesity is associated with an increased ROS production in cardiac myocytes, the myocardium could respond by increasing the levels of HSP72 and enzymatic and non-enzymatic antioxidant defenses. Currently, there are no published reports regarding the effects of obesity on myocardial antioxidant capacity. Therefore, the purpose of this study was to examine the effects of obesity on myocardial antioxidant capacity and oxidative damage. Speci®cally, these experiments examined obesity-related changes in myocardial antioxidant enzyme activities, concentrations of nonprotein thiols, levels of HSP72 and HSP73, and lipid peroxidation in lean and obese Zucker rats. We tested the hypothesis that obesity is associated with increased myocardial lipid peroxidation. Furthermore, we postulated that obesity will promote an increase in both myocardial antioxidant enzyme activity and the levels of HSP72.

Methods Animals

These experiments were approved by the University committee for the use of animals in research and followed the guidelines established by the American Physiological Society. Male obese Zucker rats and age matched lean Zucker rats were acquired at 2 months of age from a licensed laboratory animal vendor and maintained at the university animal facility for an additional 10 months prior to the study. This strain was chosen for its strength as a model of human obesity.31 Animals were housed one to a cage. Ambient temperature was sustained at approx 21 C and animals were maintained on a 12 h light : 12 h dark photoperiod. At 12 months of age, lean ( 7 =fa) (n 6) and obese (fa=fa) (n 7) male Fatty Zucker rats were anaesthetized with an intraperitoneal injection of xylazine (2 ± 10 mg=kg) and ketamine (60 ± 80 mg=kg). After the animals reached a surgical plane of anaesthesia, the hearts were quickly removed and the left ventricle dissected into sections and frozen in liquid nitrogen. Ventricular samples were stored frozen at 7 80 C until assay. Antioxidant and oxidative enzyme analysis

Left ventricular (LV) samples were homogenized in ice-cold 100 mM phosphate buffer with 0.05% bovine serum albumin (pH 7.4, 1 : 100 wt=vol). Samples were subjected to two 15 s of homogenization by a blade (Ultra-Turrax). Homogenates were then centrifuged (3 C) for 10 min at 400 g. The resulting supernatant was decanted and stored on ice until use. Supernatants were assayed for the Krebs cycle enzyme, citrate synthase (CS, E.C. 4.1.3.7), glutathione peroxidase (GPX, E.C. 1.11.1.9), SOD (E.C. 1.15.1.1) and catalase (CAT, E.C. 1.11.1.6). SOD, GPX and CAT were

measured using the methods described by Srere,14 Flohe and Gunzler,9 Oyanagui15 and Aebi,16 respectively. In our laboratory, the coef®cients of variation are 3, 3, 4 and 5% for the CS, SOD, GPX and CAT assays, respectively. All assays were performed in duplicate at 25 C and samples from both lean and obese animals were assayed on the same day to reduce interassay variation. Myocardial water=dry mass ratio and lipid content

The LV water and dry mass composition was assessed using a freeze drying technique. Brie¯y, the wet weight of a segment of LV tissue was determined and the tissue was rapidly frozen in liquid nitrogen. The frozen tissue was then freeze dried incorporating a vacuum pump with a pressure of 1072 to 1073 mmHg. The measurement of dry mass was terminated after the same weight was recorded three times in succession following a 48 h interval. Water mass was calculated from the difference of the wet mass and the post-freeze drying mass. Lipid content of the myocardium was assessed using a chloroform : methanol extraction technique described by Burdon and Knippenberg.13 Brie¯y, heart samples (approx 0.5 g) were homogenized in a methanol : chloroform mix (2 : 1 v=v) for 2 min at room temperature. Samples were centrifuged for 4 min at 400 g. The supernatants were decanted and the pellet was resuspended and re-extracted with 38 ml methanol : chloroform : 0.2 N HCl (2 : 1 : 0.8 v=v). The two phases were separated by centrifugation at 400 g for 4 min at room temperature. The supernatants were pooled and the phases were separated by a third centrifugation at 400 g for 2 min. The lower chloroform phase was removed and neutralized by dropwise addition of 0.2 N methanolic NH4OH. Lastly, 10 ml benzene were added to the samples to remove water, and samples were allowed to concentrate in a rotary evaporator at 30 C. The mass of the residue in the tube was recorded as lipid mass per unit mass heart tissue and expressed as percent of wet mass. Lipid peroxidation

Basal myocardial levels of lipid peroxidation were determined in samples of LV using two separate methods: 1) measurement of tissue malondialdehyde concentrations via the thiobarbituric reactive substances (TBARS) technique; and 2) measurement of lipid hydroperoxides. TBARS content was determined by the spectrophotometric method of Ohkawa et al.17 in which malondialdehyde reacts with thiobarbituric acid and forms a pink chromagen that is detected at 532 nm. Tissue lipid hydroperoxides were assayed using the ferrous-oxidation=xylenol orange (FOX) method described by Hermes-Lima et al.18 Lipid peroxide values were calculated as cumene hydroperoxide equivalents (CHPE) per mg of lipid.


Cellular thiol content

We assessed the non-protein thiol (NP-SH) levels as an estimate of glutathione content in both lean and obese LV using the technique described by Jocelyn.33 Thiols are important determinants of cellular redox status; speci®cally, glutathione is the largest determinant of myocyte redox status, and is important in the recycling of vitamin E.2 HSP analysis

HSPs of the 70 kDa family have been shown to play a protective role during myocardial injury. Therefore, LV levels of HSP72 and HSP73 were assessed by

performing polyacrylamide gel electrophoresis and western blotting techniques.19 In brief, LV samples were homogenized in a buffer containing 10 mM Tris, 10 mM NaCl, 0.1 mM EDTA, 15 mM mercaptoethanol at pH 7.6. Samples (30 mg) were loaded into wells of a 4% polyacrylamide stacking gel=12% separating gel (Bio-Rad Mini Protean II Gel apparatus). After separation, proteins were transferred onto nitrocellulose membranes using the Bio-Rad mini-protean II gel transfer system at a constant voltage of 20 V for 36 min. Membranes were subsequently blocked for 2 h with 3% bovine serum albumin (BSA). Blots were incubated for 2 h with primary monoclonal antibodies (1 : 1000 in phosphate buffered saline (PBS)) for the

Figure 1 Oxidative and antioxidant enzyme activity in the left ventricles of lean and obese Zucker rats. Values are means ( s.e.m.). *Denotes signi®cance at the P < 0.05 level.

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inducible HSP72 and constitutive HSP73 proteins (Stress Gen, Victoria, Canada). Membranes were subsequently incubated in a conjugated anti-rat IgG alkaline phosphatase second antibody (Stress Gen) at a dilution of 1 : 1000 for approx 30 min or until clarity of the protein bands was achieved. Quanti®cation of the bands from the immunoblots was performed using computerized densitometry (NIH Image). Values are expressed in arbitrary percent units of the total HSP72=73 expression, a modi®cation of HSP analysis performed by Salo et al.12 Oxidative challenge in vitro

To determine whether obesity predisposes the myocardium to oxidative stress, we exposed LV samples from both lean and obese animals to an in vitro oxidative challenge using a modi®ed version of the technique described by Ohkawa et al.17 Brie¯y, LV samples were homogenized in cold 0.9% NaCl (pH 7, 1 : 20 wt=vol), and centrifuged (3 C) for 10 min at 400 g. Supernatants were decanted and added to a reaction mixture containing 400 mM FeCl3. Samples were then incubated for 15 min at 37 C in a shaking water bath. The reaction was stopped with 0.8 M HCl, 12.5% tricarboxylic acid and 1 mM desferrioxamine. A 1.0% solution of thiobarbituric acid was added to each sample and all reaction tubes were boiled for 10 , cooled and centrifuged for 10 min at 1500 g. The supernatant was removed and assayed to determine TBARS concentrations using the protocol of Ohkawa et al.17

activities were signi®cantly greater in obese rats compared to lean (P < 0.05). Finally, no signi®cant differences existed between groups in the mean LV activities of CAT and GPX (P > 0.05). Myocardial water=dry mass ratio and lipid content

There were no signi®cant group differences in LV water and dry mass. The LV from the lean animals was comprised of 66.5% water and 33.5% dry mass, whereas obese LV samples contained 62.7% water and 37.3% dry mass. Lipid content was signi®cantly higher in hearts of obese animals compared to lean (33% and 59% of total wet weight, respectively, P < 0.05). Lipid peroxidation

Figure 2 contains the LV concentrations of TBARs and lipid hydroperoxides. Both TBARS and lipid hydroperoxide concentrations were signi®cantly greater in the LV of obese animals compared to lean. Thiol content

Non-protein thiols were measured as an estimation of reduced glutathione content in the heart. LV levels of non-protein thiols did not differ between lean and obese animals ( > 0.05). Myocardial samples from lean and obese animals contained 14.1 1.2, 16.2 2.0 and mmol thiol=mg protein, respectively.

Statistical Analysis

All dependent variables were subjected to a one-way analysis of variance. Signi®cance was established at P < 0.05. Group differences following the oxidative challenge were detected using a Scheffe post-hoc test. Unless indicated, all data are presented as mean (s.e.m.).

Results Animal body weight

At the time of study, animal mean ( standard error) body weights were 590.2 60.2 g and 881.7 56.2 g for lean and obese rats, respectively. Antioxidant and oxidative enzyme activity

The LV activities of CS, SOD, GPX and CAT are contained in Figure 1. Although LV CS activities tended to be greater in obese compared to lean animals, this difference was not signi®cant (P < 0.05). Total SOD activity did not differ between groups. Similarly, CuZn-SOD (cytosolic isoform) activity did not differ between groups (P < 0.05). However, LV Mn-SOD (mitochondrial isoform)

Figure 2 Thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxide content in left ventricles (LVs) of lean and obese Zucker rats. Values are means ( s.e.m.). *Signi®cant at P < 0.05.


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Oxidative challenge in vitro

Following exposure to an Fe3-stimulated oxidative challenge in vitro, LV homogenates were assessed for TBARS content (Figure 3). Note that the basal level of TBARS was higher in the obese group compared to the lean (7.04 vs 8.38 nMol=mg lipid, P < 0.05). A Scheffe test indicated that following the challenge, LV TBARS content of both groups increased, with the obese group exhibiting a signi®cantly greater increase in TBARS content per unit lipid (12.8 vs 16.7 nMol=mg lipid.) HSP analysis

Figure 4 compares the HSP expression in both lean and obese animals. Obesity did not result in any signi®cant differences (P > 0.05) in LV expression of HSP72 and HPS73.

Discussion Overview of principal ®ndings

To our knowledge, this is the ®rst investigation to examine the effects of obesity on myocardial levels of HSPs, antioxidant enzyme activity and oxidative damage. We hypothesized that obesity is associated with an increased myocardial lipid peroxidation and that obesity promotes elevated LV levels of HSPs and antioxidant enzyme activities. Our data support the hypothesis that obesity promotes increased myocardial lipid peroxidation as both lipid hydroperoxides and TBARS were elevated in the LV of obese animals (Figure 2). Our second hypothesis, however, was only partially supported by our data. Despite increases in Mn-SOD activity in the LV of obese animals, obesity

Figure 4 Western blot results for heat shock proteins (HSP) 72 and 73 expression in hearts of lean and obese Fatty Zucker rats. Percent expression of the total HSP72=73 protein in the left ventricles (LVs) of lean and obese animals did not differ (P > 0.05) between groups.

was not associated with an increase in GPX, CuZnSOD and CAT activity. Furthermore, obesity did not alter LV levels of non-protein thiols or HSP72=HSP73. Obesity and myocardial lipid peroxidation

Our data clearly indicate that obesity is associated with increased levels of myocardial lipid peroxidation. There are at least four potential explanations for this observation. Obesity could promote: 1) an elevation in the rate of production of ROS in the myocardium; 2) increased fat deposition which elevates polyunsaturated fatty acids (PUFA) in the heart; 3) a diminished myocardial antioxidant capacity; or 4) some combination of 1 ± 3. A brief discussion of each of these possibilities follows. Obesity and myocardial production of ROS

Figure 3 Left ventricular Thiobarbituric acid reactive substances (TBARS) content in lean and obese animals following an iron-stimulated oxidative challenge in vitro. Values are means ( s.e.m.). *Signi®cantly greater than basal value of respective group (P < 0.05), ** signi®cantly greater than lean post-challenge value (P < 0.05).

Although resting heart rate and blood pressure were not monitored throughout the life span of our animals, it is likely that compared to lean animals, our obese Zucker rats maintained higher resting heart rates and blood pressures. Support for this supposition comes from reports that obese Zucker rats, fed ad libitum throughout life, become hypertensive by approximately 36 weeks of age.20,21 Speci®cally, it is documented that resting heart rates in adult obese Zucker rats are approx 400 bpm (approx 13% higher than lean litter mates) and systolic arterial blood pressures are approx 130 mmHg (approx 15% higher than lean litter mates).20,21 It follows that this obesity-related increase in resting heart rate and systolic blood pressure would elevate the rate pressure product and therefore, increase myocardial oxygen consumption. This increase in myocardial mitochondrial respiration is associated with increased production of ROS.23


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Indeed, leakage of electrons out of the mitochondrial electron transport chain promotes a one-electron reduction of molecular oxygen resulting in the formation of superoxide radicals.3,23,24 Failure of myocardial antioxidants to remove superoxide radicals at the rate that they are produced would lead to the formation of other reactive oxygen species and could explain, at last in part, the observed increases in lipid peroxidation in the LV of our obese animals. Obesity and myocardial fat content

Lipid content was found to be signi®cantly (P < 0.05) higher in hearts from obese animals compared to the lean. Thus, there was increased fat deposition within the heart of the obese Zucker rats. Other reports support this ®nding that obese Zucker rats have an increased fat accumulation in many tissues and organs, and this increase in fat is associated with elevated lipid peroxidation.25 A possible mechanism to explain this observation is that an increased PUFA in tissues promotes lipid oxidation by increasing the amount of substrate available for peroxidation. Human studies investigating the oxidation of serum lipoproteins have shown similar results. That is, lipid peroxidation is positively correlated with serum triglyceride levels and body mass index (BMI).1,26 Hence, it seems likely that increased fat deposition may have contributed to the observed increase in lipid peroxidation in the hearts of the obese animals. Obesity and myocardial antioxidant capacity

Cells contain both enzymatic and non-enzymatic antioxidants that work as a collective unit to remove ROS and other oxidants. Our ®ndings do not support the idea that obesity diminished the activities of primary antioxidant enzymes (that is, SOD, GPX, and CAT). Furthermore, our data do not support the notion that obesity altered myocardial glutathione levels. Nonetheless, it is possible that the chronic oxidative stress placed on the heart of obese animals signi®cantly reduced the vitamin E content of the myocardial membranes. Vitamin E is the most important chain breaking antioxidant in cell membranes and Barclay and Hansel27 have reported signi®cant increases in muscle lipid peroxidation in vitamin E depleted animals. In addition, human studies have reported that reduced tissue vitamin E levels are associated with obesity,28 suggesting that these individuals at greater risk for cardiovascular oxidative injury. However, since vitamin E levels were not measured in our animals, it is unknown as to whether myocardial vitamin E concentrations were compromised in the obese group. This is an important avenue for future research. In an effort to further evaluate the antioxidant capacity of the LV in both lean and obese animals, we examined the ability of myocardial homogenates to defend against a Fe3-stimulated oxidative challenge. Exposure of heart homogenates to this in

vitro oxidative challenge resulted in increased lipid peroxidation in both lean and obese animals; however, the myocardial lipid peroxidation was two-fold greater in obese animals. This observation suggests that the myocardial antioxidant capacity is compromised in obese animals, and=or that there is more lipid substrate available for peroxidation. Since this Fe3catalyzed reaction primarily produces hydroxyl radicals, it appears that myocardial tissue from the obese group had fewer defense mechanisms against hydroxyl-mediated damage. Our data do not provide a de®nitive explanation as to why myocardial homogenates from obese animals have a lower ability to defend against this hydroxyl challenge compared to lean animals. However, one possibility is that myocardial vitamin E levels were lower in obese animals compared to lean. Indeed, it is known that a reduction in membrane vitamin E content compromises the cell's ability to defend against hydroxyl radicals.23 Another potential explanation is as follows. As mentioned previously, if obesity results in an increase in myocardial PUFA, increased PUFA in the homogenate would promote lipid oxidation by increasing the amount of substrate available. Although the in vitro oxidative challenge used in these experiments can provide useful information regarding the oxidative capacity of tissues it is important to recognize the limitations of this technique. One shortcoming to this type of in vitro challenge is that the preparation necessitates disruption of the intact myocytes which alters the normal geometric relationships between cellular antioxidants. Hence, exposure of myocardial homogenate to this type of oxidative challenge provides a non-physiological evaluation of the antioxidant capacity of the intact cardiac myocyte. Nonetheless, the fact that an oxidative challenge results in greater lipid peroxidation in myocardial homogenates suggests that obesity predisposes the myocardium to oxidative injury. Obesity is associated with an up-regulation of Mn-SOD

Our hypothesis that obesity would be associated with elevated LV antioxidant enzyme activities was only partially supported by our data. The rationale for this hypothesis evolved from the idea that obesity is associated with increases in ROS production in cardiac myocytes and that the myocardium would respond to this oxidative challenge by increasing enzymatic antioxidants. Hence, it is surprising the obesity was linked with the increase of only one antioxidant enzyme, Mn-SOD. The fact that the mitochondrial isoform of SOD (Mn-SOD) was elevated in the LV of obese animals suggests that one source of ROS in obesity is the formation of superoxide radicals during mitochondrial respiration. If this is the case, Mn-SOD activity would be up-regulated in the mitochondria to protect against superoxide formation. However, despite the increase in Mn-SOD activity, both lipid hydroperoxides and


TBARS content were signi®cantly higher in the LV of obese animals, suggesting that an increase in Mn-SOD activity alone, may not be adequate to protect myocardial lipid against oxidation. The dismutation of superoxide to H2O2 by SOD, has been shown to be a stimulus for the co-upregulation in GPX activity and an increase of cellular levels of glutathione.23 GPX, in association with reduced glutathione, catalyzes the reduction of H2O2 and organic peroxides to H2O and alcohol.2,7,29 However, myocardial GPX did not differ between lean and obese animals. Furthermore, LV non-protein thiol concentrations did not differ between lean and obese animals. Since glutathione is the dominant non-protein thiol in cells,2 we interpret this ®nding as evidence that glutathione was not increased in the hearts of the obese animals. This is important because glutathione plays a major role in the maintenance of intracellular redox status and antioxidant vitamin levels (recycling of vitamins E and C), and is a substrate for GPX.2 Therefore, the failure to increase both myocardial GPX activity and glutathione levels may have been a factor in the observed increase in lipid peroxidation in the LV of obese animals. HSP and obesity

To our knowledge, this study is the ®rst to examine the basal levels of HSPs in the hearts of lean and obese animals. The rationale for investigating the relationship between obesity and myocardial HSP expression is that HSPs have been shown in our laboratory and others, that HSP72 expression increases following acute or chronic stress upon the heart.8,10 ± 12,32 Previous studies have reported the induction of HSP72 in response to ischemia,11 acute exercise8 and regular exercise training.10 A common stimulus among these conditions is oxidative stress. When ROS are generated at greater rates by acute or chronic stimuli, HSP expression is induced.12 We postulated that the chronic overload and subsequent oxidative stress placed on the myocardium of obese animals due to the increased rate-pressure product would increase the rate of ROS formation and stimulate up-regulation of HSP72. Regardless, our data do not support the hypothesis that obesity is associated with increases in myocardial levels of HSP72 or HSP73. The failure of myocardial HSP72 to increase in the obese animals may be due to an inadequate stimulus to promote the expression of HSP72. Furthermore, obesity did not promote any translational changes in the constitutively expressed protein, HSP73. It is possible that an aerobic muscle, such as the heart, requires an overload stimulus greater than obesity alone to promote up-regulation of HSPs of the 70 kD family. Exercise training studies have revealed that high-intensity exercise (generating heart rates greater than twice the resting values, and blood pressures at least 30% greater than at rest) can upregulate HSP70,8 but this stimulus is indeed greater

than the increased heart rate and blood pressure observed in obese, sedentary animals. Alternatively, the expression of HSP72 or 73 may not have been altered in this chronic stress condition of obesity due to inactivation of transcription factors (such as heat shock factor 1 (HSF1)).8 Recent data suggest that under acute stress conditions, HSPs bound to HSF1 have greater af®nity for damaged proteins. HSF1 molecules trimerize and initiate HSP synthesis. HSF1 trimers remain activated until a new equilibrium between HSPs, damaged proteins and HSF1 molecules is reached. Thus a demand for HSP72=73 is created until the damage is repaired.8 In chronic, lifelong stress such as obesity, however, it is possible that the myocardium is better able to control ROS-mediated damage, though not eliminate it, by elevating other defenses such as Mn-SOD, or perhaps other mitochondrial-related protective proteins such as HSP60.30 It appears that chronic obesity in sedentary animals does not generate suf®cient cellular damage of proteins for HSF1-initiated HSP synthesis. Thus, it is likely that HSP transcription factors are inactivated despite the chronic stress of obesity.

Conclusion In summary, obesity is associated with increased myocardial oxidative stress. This may be due to 1) a compromised antioxidant defense as evidenced by a failure of the up-regulation of the antioxidant defense in the obese animals (enzymes, thiols and HSPs) with the exception of Mn-SOD, 2) an increased availability of polyunsaturated lipid substrate in the myocardium, or 3) a combination of 1 and 2. Both may lead to increased basal levels of lipid peroxidation, and increased peroxidation in response to an oxidative challenge in vitro in obese animals. Both possibilities are future avenues for research. Acknowledgements

This work was supported, in part, by a grant from the American Heart Association ± Florida Af®liate (SKP). References

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