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Department of Experimental Medicine and Oncology (M.A., R.M., I.V., P.B., O.D.), University of Turin, 10125 Turin, Italy; Department of Clinical Pathophysiology (M.G.C., G.B.), University of Turin, 10126 Turin, Italy; Department of Animal and Human Biology (G.A.), University of Turin, 10123 Turin, Italy; and Department of Clinical and Biological Sciences (S.G.), University of Turin, San Luigi Hospital, 10043 Orbassano, Italy
Address all correspondence and requests for reprints to: Professor Giuseppe Boccuzzi, Department of Clinical Pathophysiology, University of Turin, Via Genova 3, 10126 Turin, Italy. E-mail: giuseppe.boccuzzi{at}unito.it.
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Abstract |
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B and activator protein-1, and profibrogenic growth factors (connective tissue growth factor and TGFβ1) were determined in the left ventricle of treated and untreated streptozotocin-diabetic rats. Structural and ultrastructural changes, and the contractile force developed by electrically driven papillary muscles, under basal conditions and after stimulation with isoproterenol, were also evaluated. Oxidative stress induced by hyperglycemia increased AGEs and AGE receptors and triggered a cascade of signaling, eventually leading to interstitial fibrosis. DHEA treatment, by improving oxidative balance, counteracted the enhanced AGE receptor activation and increase of profibrogenic factors and restored tissue levels of collagen I, collagen IV, and fibronectin to those of control animals. Moreover, DHEA completely restored the contractility of isolated papillary muscle. Oxidative stress led to cardiac fibrosis, the most important pathogenetic factor of the heart’s impaired functional integrity in diabetes. Structural and ultrastructural changes and impairment of muscle function induced by experimental diabetes were minimized by DHEA treatment. |
Introduction |
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B (NFB) and activating protein-1 (AP-1), a further element that increases ECM production (10). Thus, strategies preventing the detrimental effects of AGEs offer promising prospects of improving heart function in diabetic patients. It has been reported that the interruption of free-radical overproduction counteracts AGE formation (11). However, the conventional antioxidants used to prevent oxidative damage in diabetes have not given substantial results (12) because these scavenger species react in a stoichiometric manner. Dehydroepiandrosterone (DHEA) is a compound of physiological origin that possesses multitargeted antioxidant properties (13, 14); this multifunctional steroid has been found to prevent the tissue damage induced by hyperglycemia in several in vivo and in vitro models (15, 16, 17). Moreover, recent reports show that the human heart synthesizes DHEA, that its production is suppressed in the failing heart, and that plasma levels of the sulfate conjugate of DHEA decrease in patients with chronic heart failure in proportion to the severity of the condition (18). DHEA is also reported to counter the impairment of cardiac myogenic factors and switch in myosin gene expression in the heart of rats after 3 wk induction of diabetes (19).
The study evaluated whether continuation of hyperglycemia to 6 wk also triggers the appearance of myocardial fibrosis, the most important pathogenetic factor in the heart’s impaired functional integrity in diabetes (20). It also examined the downstream signaling activated by oxidative stress in the left ventricle of streptozotocin (STZ) diabetic rats, and the effects of DHEA treatment on AGE receptor activation, biochemical and structural changes in the left ventricle, and myocardial dysfunction.
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Materials and Methods |
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Animal treatment
Male Wistar rats (Harlan-Italy, Udine, Italy) weighing 200–220 g were cared for in compliance with the Ethical Guidelines of the Italian Ministry of Health Guidelines (No. 86/609/EEC) and Principles of Laboratory Animal Care (National Institutes of Health, No. 85-23, revised 1985). The scientific project, including animal care, was supervised and approved by the local ethical committee. Animals were provided with Piccioni pellet diet (Gessate Milanese, Italy) and water ad libitum. Hyperglycemia was induced through a single iv injection of STZ. DHEA was administered for 6 wk in the diet [0.02% (wt/wt)], provided by the suppliers of the pellets. After 6 wk, the rats were anesthetized with Zoletil 100 (Tiletamine-Zolazepam; Virbac, Carros, France) and killed; the blood and heart were removed.
Tissue extracts
The subcellular fractions were prepared by the method of Meldrum et al. (21). Protein content was determined using the Bradford assay (22) and samples were stored at –80 C until use.
Oxidative biochemical parameters
Reactive oxygen species (ROS) were measured using 2',7'-dichlorofluorescein diacetate as probe (23). The antioxidant level was evaluated in terms of reduced and oxidized glutathione content by the method of Owens and Belcher (24). The difference between total glutathione and reduced glutathione (GSH) content represents the oxidized glutathione (GSSG) content; the ratio between GSSG content and GSH is considered a good indicator of antioxidant status.
DHEA content
Plasma levels of DHEA were measured by the RIA method (25).
AGE detection (pentosidine) with HPLC-mass spectrometry
Cytosol fractions were treated with hydrochloric acid (26) and then injected in a Thermo-Finnigan Surveyor instrument (Thermo-Finnegan, Rodano, Mi, Italy) equipped with autosampler and PDA-UV 6000 LP detector. Mass spectrometry analyses were performed using an LCQ Deca XP plus spectrometer (Thermo-Finnegan, with electrospray interface and ion trap as mass analyzer, using a Varian Polaris C18-A column (150 x 2 mm, 3 µm particle size), flow rate of 200 µl min–1 and gradient mobile phase [95/5 to 0/100 (vol/vol) 5 mM heptafluorobutanoic acid in water/methanol].
Western blot
Protein levels of RAGE, galectin-3, fibronectin, collagen I and IV (total extracts), inhibitory-B (IB)-
, TGFβ, CTGF (cytosol), and NFB-p65 (nuclear extract) were quantified using Western blot analysis (27) followed by densitometry of the bands.
Quantitative real-time RT-PCR
Gene transcripts for TGFβ1, CTGF, β1-adrenergic receptor (adrenoceptor), and cyclophilin were quantified by PCR. All experiments were performed on at least three independent cDNA preparations. PCR products were electrophoresed on 2% agarose gels and amplification products were stained with GelStar nucleic acid gel stain (FMC BioProducts, Rockland, ME). Gels were photographed and analyzed with 1D Image Analysis software (Kodak, Rochester, NY).
AP-1 detection
An ELISA kit (Active Motif, Vinci Biochem, Italy) was used to detect AP-1 binding activity. Nuclear tissue extracts containing 100–120 µg of protein were used for analyses.
Histological staining and immunohistochemistry
Six-micrometer paraffin sections of left ventricle were used to stain for collagen I, collagen IV, and fibronectin with immunohistochemistry using primary polyclonal goat antirat antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and the avidin biotin complex staining system (Santa Cruz Biotechnology).
Electron microscopy
For ultrastructural analysis, samples were fixed as described in the online supplemental data. Thin sections (70 nm) were cut using a Ultracut UCT (Leica, Québec, Canada), stained with uranyl acetate and lead citrate, and examined in a JEM-1010 transmission electron microscope (JEOL, Tokyo, Japan) equipped with a Mega-View-III digital camera and a Soft Imaging System (SIS, Münster, Germany) for computerized acquisition of images.
Isolated papillary muscle and contractility determination
Papillary muscles were stimulated at constant frequency (120 beats/min) with a pair of electrodes connected to a 302 T Anapulse stimulator via a 305-R stimulus isolator (W. P. Instruments, New Haven, CT) operating in constant current mode. Isometric twitches were evaluated by a Harvard transducer (60-2997), visualized on a Tektronix 2211 digital storage oscilloscope and continuously acquired and recorded in a Power Mac computer, using the Labview Software (National Instruments Corp., Austin, TX). The same software was used to measure developed peak mechanical tension (Tmax), maximum rate of rise and fall of developed mechanical tension (+dT/dtmax and –dT/dtmax), time to peak mechanical tension, and duration of contraction. To study responsiveness to β1-adrenergic stimulation, the positive inotropism caused by isoproterenol (1 µM) was compared.
Statistical analysis
All results are presented as means ± SD. Differences between means were analyzed for significance using one-way ANOVA with the Bonferroni post hoc test (28).
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Results |
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). Body weight of STZ-diabetic rats was significantly lower than controls (P < 0.05), whereas the weight of STZ plus DHEA rats was similar to controls and significantly higher than the STZ group (P < 0.05). After 6 wk, the heart to body weight ratio in STZ diabetic rats was markedly higher than in the control group; DHEA treatment maintained the ratio at the control value. The DHEA plasma level was double the control value in both normal and STZ-treated rats undergoing DHEA treatment.
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. STZ-diabetic rats showed double the ROS content, evaluated in the cytosolic fraction of the left ventricle vs. controls. The GSSG to GSH ratio increased in the STZ group, indicating a decrease in GSH level and thus an imbalance in the oxidative state in the heart tissue. In the DHEA plus STZ rats, the level of ROS significantly fell vs. the STZ group, and the GSSG to GSH ratio also decreased, indicating a return toward normal conditions. The transcription factor AP-1 was examined at the end of the experimental time in the nuclear extract of rat ventricle. Figure 1B shows a significant increase in activation of AP-1 in STZ-diabetic rats vs. controls; activation of AP-1 was significantly suppressed (P < 0.01 vs. STZ) in STZ rats receiving DHEA. NFB activation and its inhibitor IB- were detected, respectively, in nuclear extract and the cytosolic fraction (Fig. 2). NFB activation increased in the STZ group, as did the level of IB- in the cytosol. A significant reduction of NFB activation in DHEA-treated STZ rats, and free IB- in the cytosol from this group, were observed.
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). The receptors of AGE, RAGE, and galectin-3 were detected in total extract of the left ventricle. Figure 3A shows the content of RAGE, evaluated by Western blot. In the STZ group, RAGE increased vs. controls; treatment of STZ rats with DHEA produced a significant reduction in RAGE (P < 0.05 vs. STZ). Galectin-3 was also high in STZ-diabetic rats, whereas it was significantly reduced (P < 0.05 vs. STZ, data not shown) in DHEA-treated STZ rats.
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, A and B, respectively). DHEA treatment of STZ rats was associated with a reduction in TGF-β1 protein and gene expression. The results for CTGF protein (Fig. 4C) and gene expression (Fig. 4D) paralleled these findings.
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reports the content of collagen I (Fig. 3B), collagen IV (Fig. 3C), and fibronectin (Fig. 3D). The levels of these proteins significantly increased in STZ rats vs. controls (P < 0.05). Six weeks of DHEA treatment of STZ rats greatly reduced the deposition of these compounds (P < 0.05).
Immunohistochemistry and histological analysis
After 6 wk hyperglycemia, immunohistochemical analysis showed matrix deposition in left ventricles isolated from diabetic rats, as shown in Fig. 5. Protein accumulation was associated with significantly increased staining for collagen I (Fig. 5A), collagen IV (Fig. 5B), and fibronectin (Fig. 5C) vs. control rats (a of each panel). When DHEA was given to STZ diabetic rats, the sections of left ventricle (Fig. 5Cd) showed reduced staining for collagen I, collagen IV, and fibronectin vs. diabetic rats (Fig. 5Cc).
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). Basal contractility (Table 2A) was weaker than controls in papillary muscles from STZ-treated rats, in terms of maximum developed mechanical tension (Tmax: 31.9 ± 4.3 vs. 7.5 ± 2.3 mN/mm2; control and STZ-treated samples, respectively; n = 6 and 4; P < 0.01 by ANOVA), +dT/dtmax, and –dT/dtmax. No significant difference was found for time to peak mechanical tension or duration of contraction. Treatment with DHEA completely countered the effects of STZ. However, DHEA alone did not affect the contractile properties of papillary muscles. Moreover, papillary muscles from STZ-treated rats showed a reduced response to β-adrenergic stimulation, which was 75% stronger in control preparations. As shown in Table 2B, the inotropic effect of isoproterenol (1 µM) was significantly higher in control papillary muscles (224.9 ± 17.1% of basal value, n = 6) than in preparations obtained from STZ-treated rats (130.6 ± 6.9% of basal value, n = 4; P < 0.01 by ANOVA). These differences were also evident for +dT/dtmax, –dT/dtmax, and time to peak mechanical tension (TPT), whereas no significant difference was found for the duration of contraction. Papillary muscles treated with DHEA were significantly protected from the effects of STZ. However, the response to isoproterenol was not affected by treatment with DHEA alone.
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Electron microscopy
Figure 6 shows the results of electron microscope observations. Figure 6, A and B, shows the ultrastructure of control myocardium. Cardiomyocytes show regularly organized cytoplasm with numerous parallel myofibrils and Z lines. In STZ-treated rats not receiving DHEA, low-power electron microscope examination (Fig. 6C) revealed widespread signs of tissue degeneration, dense packages of collagen fibrils between cardiomyocytes (asterisk), and large areas of cytoplasm had lost their regular myofibrillar organization (asterisk, in Fig. 6D); mitochondria showed signs of degeneration, chiefly desgregation of the cristae and mitochondrial membrane damage (Fig. 6D, arrow). The heart morphology of STZ rats treated with DHEA (Fig. 6, E and F) was similar to controls: fibrils with Z lines regularly organized in the cardiomyocytes and only occasional mitochondria, indicating the massive damage observed in the STZ group.
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Discussion |
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- to the β-myosin heavy chain isoform and that DHEA treatment, by preventing activation of oxidative pathways, counteracts the switch in myosin gene expression (19).
Here we observe that oxidative stress and AGE hyperproduction are maintained through 6 wk hyperglycemia and, in addition, profibrogenic gene expression, and ECM deposition increase, eventually leading to cardiac dysfunction. Besides activation of the NFB pathway, we also found an increase in AP-1 activity (30): activation of both transcription factors up-regulates several genes correlated to fibrosis, such as fibronectin, TGFβ-1, and CTGF (31). The findings of the immunohistochemical analysis, showing matrix deposition in the left ventricle of STZ-diabetic rats with increased staining for collagen I, collagen IV, and fibronectin, in agreement with the increased protein levels of collagen I, collagen IV, and fibronectin, are in line with the up-regulation of these genes. Moreover, the levels of growth factors TGFβ1 and CTGF were increased, in keeping with other studies (32, 33) that have reported fibronectin to be elevated in the kidney, heart, and retina of diabetic rats; in human mesangial cells, hyperglycemia has also been found to induce expression of CTGF, which precedes accumulation of ECM (34).
We also detected an increase in pentosidine and AGE receptors, RAGE, and galectin-3 in the heart of diabetic animals. AGEs, whose formation is closely correlated to oxidative stress (35) and which accumulate in the tissue of diabetic patients (36), participate in diabetes-induced structural myocardial changes (5). Pentosidine accounts for a small proportion of AGEs, but its presence in vivo closely reflects the degrees of protein glycation and oxidation (37), and it is used as a marker of AGE biogenesis (38, 39, 40). Besides their well-known direct toxicity, AGEs also exert their detrimental effect by interacting and up-regulating their receptors (12). It has been reported that AGEs, via RAGE, induce a phenotype transition in renal tubular epithelial cells, through the TGFβ1 and CTGF pathways; this transition leads to a profibrotic feature (41). Moreover, galectin-3 must be activated for TGFβ1 to stimulate myofibrillar activation and procollagen production and has recently been proposed as a key step in the induction of tissue fibrosis (42). Collectively, these findings show that, by activating AGE receptors, after 6 wk the diabetic condition induces ECM accumulation and interstitial fibrosis in the left ventricle of STZ rats.
Conversely, in STZ rats treated with DHEA, both pentosidine content and AGE receptors, galectin-3, and RAGE, remain close to levels observed in control animals. Moreover, by preventing the redox imbalance, DHEA counteracts activation of both AP-1 and NFB as well as the subsequent increase in TGFβ-1 and CTGF, which are involved in ECM production and ECM accumulation. By counteracting the increase of profibrogenic factors in cardiac tissue of diabetic rats, DHEA treatment restores tissue levels of collagen I, collagen IV, and fibronectin to control levels, preventing the development of fibrosis. Studies in vitro have shown that DHEA attenuates collagen type I synthesis at the transcriptional level in cardiac fibroblasts (43).
The electron microscope findings are in keeping with biochemical and histochemical findings: the myocardial ultrastructure of STZ-diabetic rats showed large areas of collagen accumulation between myocardiocyte cell membranes. DHEA treatment minimized the ultrastructural damage induced by STZ: electron microscope observations showed only occasional disruption of myofibrillar organization and mitochondrial cristae damage; the large collagen packages between myocardiocytes, very widespread in STZ-treated animals, were rare in the STZ plus DHEA tissue.
The structural damage observed could only in part explain the changes in myocardial functioning revealed by measurement of contractile activity. Basal contractility of the papillary muscles was decreased in STZ rats; this affected not only the maximal developed tension but also the maximum rate of rise and fall of the developed tension, which in vivo observations have shown to be a sign of diastolic dysfunction (1). Moreover, in accordance with studies showing a decreased response to β-adrenergic stimulation (44), we observed that the inotropic effect induced in the papillary muscles by isoproterenol was significantly reduced in STZ-treated rats. Alongside Tmax, significant differences were also found between control and STZ-treated preparations for +dT/dtmax and –dT/dtmax, suggesting that several mechanisms controlling intracellular calcium handling are affected by diabetic injury. Taken together, these findings are in line with the reduced expression of β1-adrenoreceptors observed in diabetic rats, reported in other studies (45), and confirmed here.
We report here that treatment with DHEA fully protects cardiac tissue from the effects of chronic hyperglycemia, in terms of basal contractility, responsiveness to isoproterenol, and β1-adrenoreceptor expression. However, the basal contractile properties of rat papillary muscle and the sensitivity to β-adrenergic stimulation were not affected by treatment with DHEA alone, suggesting that the action of DHEA is probably due to its direct protective effect against diabetic injury, rather than to stimulation of myocardial contractile activity.
The results of this study show that DHEA prevents interstitial cardiac fibrosis and improves diastolic and systolic functions, which are the prominent features of diabetic cardiomyopathy. Recent observations have shown that DHEA is synthesized in the human heart (46) and that it markedly attenuates production of collagen type 1 by cardiac fibroblasts (43). More interestingly, it has been reported that in man DHEA production is suppressed in the failing heart (46).
Here we show that DHEA improves redox balance and reduces AGE levels. It is well known that on one hand oxidative stress leads to AGE formation (11) and on the other hand AGEs increase oxidative stress through binding to RAGE (47). The doubt remains as to whether DHEA reduces AGE levels and thereby reduces oxidative stress or whether it directly prevents superoxide formation through other mechanisms. We believe that DHEA interrupts this amplifying loop by directly reducing oxidative stress, and we partly base this opinion on previous studies showing that DHEA protects rats against the prooxidant effects of compounds other than glucose, such as carbon tetrachloride (48) and copper (49). We thus suggest that the reduction of radical species level in tissue is the chief mechanism underlying DHEA’s beneficial effects on the diabetic heart and that decreased RAGE activation further contributes by minimizing oxidative stress.
A correlation between systolic and diastolic myocardial dysfunction and oxidative stress has been reported in a highly selected group of uncomplicated type 2 diabetic patients (20), and the greater propensity for oxidative stress after myocardial infarction is associated with the development of heart failure (50). Several explanations have been proposed for the multitargeted antioxidant effects of DHEA, including its effect on catalase expression (51), on the up-regulation of the redox system (52), fatty acid composition of cell membranes, and cytokine production (16), and a high-affinity DHEA receptor activating phosphatidy-linositol 3-kinase/Akt pathway has also been shown to exist (53). However, the precise mechanism remains to be clarified. It is also debated whether the effect of DHEA is due to DHEA itself, its metabolites, or a combination. However, we found negligible variations of both 17β-estradiol and testosterone concentrations in rats treated with 4 mg DHEA (17). Nevertheless, as we report elsewhere, DHEA, but not a variety of other steroids including β-estradiol, androstendiol, and dihydrotestosterone, protects bovine retinal capillary pericytes against glucose-induced lipid peroxidation (54). Although the low endogenous level of DHEA in rodents, compared with primates, is a potential limitation of the relevance of our results, the antifibrogenic effect of DHEA, together with our previous observations on myosin chain synthesis in the heart of diabetic rats, might suggest an additional therapeutic approach to diabetic cardiomyopathy.
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Footnotes |
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Disclosure Statement: All authors have nothing to declare.
First Published Online September 27, 2007
Abbreviations: AGE, Advanced glycated end product; AP-1, activating protein-1; CTGF, connective tissue growth factor; DHEA, dehydroepiandrosterone; +dT/dtmax, maximum rate of rise of developed mechanical tension; –dT/dtmax, fall of developed mechanical tension; ECM, extracellular matrix; GSH, reduced glutathione; GSSG, oxidized glutathione; IkB, inhibitory-B; NFB, nuclear factor-B; RAGE, receptor for advanced glycation end products; ROS, reactive oxygen species; STZ, streptozotocin; Tmax, peak mechanical tension; TPT, time to peak mechanical tension.
Received July 5, 2007.
Accepted for publication September 17, 2007.
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References |
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B-dependent transcription in hepatocytes possibly through antioxidant effect. J Endocrinol Metab 89:3449–3454B and increases TNF- in the heart. J Mol Cell Cardiol 29:2849–2854[CrossRef][Medline]B and AP-1 in increased fibronectin synthesis in target organs of diabetic complications. Am J Physiol Endocrinol Metab 284:E1089–E1097- and β-agonist in streptozotocin-induced diabetes. Can J Physiol Pharmacol 75:781–788[CrossRef][Medline]i2,3. J Biol Chem 277:21379–21388This article has been cited by other articles:
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  J. A. Herlein, B. D. Fink, Y. O'Malley, and W. I. Sivitz Superoxide and Respiratory Coupling in Mitochondria of Insulin-Deficient Diabetic Rats Endocrinology, January 1, 2009; 150(1): 46 - 55. [Abstract] [Full Text] [PDF]
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, P < 0.05; 
