1 Department of Pharmacology, University of Nebraska Medical Center, Omaha, Nebraska
2 Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana
3 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana
4 Department of Pharmacology, Faculty of Pharmacy, University of Ankara, Tandogan, Ankara, Turkey
5 Krannert Institute of Cardiology, Center for Vascular Biology and Medicine, Indianapolis, Indiana
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ABSTRACT |
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A significant percentage of patients with diabetes (both type 1 and type 2) develop a unique cardiomyopathy that is independent of coronary atherosclerosis (13). This "diabetic cardiomyopathy" as it is termed starts off with asymptomatic left ventricular diastolic dysfunction (slowing of relaxation kinetics). As the disease progresses, systolic function becomes compromised, leading to an increase in incidence of morbidity and mortality (46).
The release of calcium ions from internal sarcoplasmic reticulum via the type 2 ryanodine receptor calcium-release channel (RyR2) is an integral step in the cascade of events leading to cardiac muscle contraction (7). We and others have shown that expression of this protein decreases in hearts of chronic diabetic patients (8,9) as well as in the streptozotocin (STZ)-induced diabetic rats (1013). Using the latter model, we found that in addition to a decrease in expression of RyR2, its functional integrity is also compromised in diabetes (14,15). This dysfunction is manifested as a decrease in RyR2 ability to bind the specific ligand [3H]ryanodine and a slowing in its electrophoretic mobility using denaturing SDS-PAGE.
Two distinct and separate types of post-translational modifications are likely to be induced by diabetes. First, it is well known that metabolic changes brought about by diabetes increase production of reactive oxygen species (e.g., superoxide anions [O·], hydroxy radicals [OH·], lipid peroxides [ROO·], singlet oxygen [1O2], and hydrogen peroxide [H2O2]) as well as reactive nitrogen species (e.g., nitrosonium cation [NO+], nitroxyl anion [NO-], and peroxynitrite [ONOO-] [1618]). These free radical and nonradical species react with several amino acid residues altering their structures and, by extension, the tertiary structures of the parent protein. In the case of RyR2, changes in its tertiary structure will alter its sensitivity to endogenous ligands like calcium and ATP. It is also possible that by altering the oxidative environment, radical species could promote oxidation of sulfhydryl groups leading to formation of additional disulfide bonds on RyR2. In a recent study, we found that in vitro treatment of RyR2 from 6-week diabetic rat hearts with 2 mmol/l dithiothreitol partially restored its ability to bind [3H]ryanodine (19). These data are consistent with the notion that the dysfunction of RyR2 stems from diabetes-induced increases in disulfide bond formation. However, at this time, it is uncertain which of the 90 cysteine residues on each RyR2 monomer are modified and how many of these disulfide bonds are formed intra-monomer and how many are formed inter-monomer.
Second, it is known that elevation in circulating levels of aldose and ketose sugars brought on by diabetes accelerates the formation of Schiff bases on lysine, arginine, or histidine residues (nonenzymatic glycation reactions [16,20]). Over time (2448 h), Schiff bases undergo internal rearrangement to form more stable Amadori products (21). On long-lived proteins such as ryanodine receptors (22), Amadori products rearrange further to form advanced glycation end products (AGEs) (23,24). Once formed, these complex molecules remain attached to the protein throughout its lifetime. Studies have shown that modification of proteins with nonenzymatic glycation products precipitates tissue and ultimately organ dysfunction (25,26).
The present study was initiated to determine whether non-cross-linking AGEs are formed on RyR2 during chronic diabetes and if formation of these post-translational modifications could be minimized or attenuated with 2 weeks of insulin treatment, initiated after 6 weeks of diabetes.
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RESEACH DESIGN AND METHODS |
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Induction and verification of experimental STZ-induced diabetes.
All animal procedures were carried out in accordance with guidelines established by institutional animal care and use committees. Male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) weighing between 180 and 190 g were anesthetized with Brevital (25 mg/kg i.p.) and then injected via a tail vein with a single dose of either STZ in 0.1 mol/l citrate buffer, pH 4.5 (50 mg/kg), or citrate buffer. Three days later, blood glucose levels were determined using a Glucometer II and Glucostix (Peridochrom Glucose GOD-PAP Assay Kit; Roche Molecular Biochemicals, Indianapolis, IN) to establish induction of diabetes. Animals with blood glucose levels <15 mmol/l were injected with a second dose of STZ. Throughout this study, animals were housed in pairs (similar weights to minimize dominance) at 22°C with fixed 12-h light/12-h dark cycles and given free access to food and water. Blood glucose and body weights were monitored weekly.
Insulin treatment protocols.
Six weeks after the STZ injection, diabetic animals were randomly divided into two subgroups. One of the subgroups was placed on an insulin regimen for 2 weeks. For these animals, insulin doses were individually adjusted to maintain the euglycemic state and varied between 12 and 16 units/kg s.c., given once per day between 9:00 and 11:00 A.M. The other subgroup of diabetic animals continued as nontreated diabetics for 2 additional weeks. Diabetic animals whose body weight fell below 90% of initial starting body weight during the in vivo protocol were killed and eliminated from the study. This experimental protocol afforded 8-week age-matched control (8C), 8-week STZ-induced diabetic (8D), and 6-week STZ-induced diabetic/2-week insulin-treated (6D-2I) animals.
Sample collection.
At the end of the in vivo procedure, animals were anesthetized with a single lethal dose of Brevital (75 mg/kg i.p.). Abdominal cavities were opened, and blood samples were collected via left renal arteries (27) for analysis of whole blood glucose, insulin, and HbA1c content. Chest cavities were then opened and hearts were removed and quick-frozen by embedding in crushed dry ice. Frozen hearts from each of 8C, 8D, and 6D-2I were then divided into two subgroups of three and six hearts each. Hearts in the smaller subgroup were used to determine levels of mRNA encoding RyR2, whereas the larger subgroup of six hearts was used for determination of RyR2 protein and function.
Quantitation of mRNA encoding RyR2 in 8C, 8D, and 6D-2I rat hearts.
A detailed procedure for determining RyR2 mRNA content is described elsewhere (14,15). Briefly, total RNA was extracted separately but simultaneously from 8C, 8D, and 6D-2I rat hearts using Quick Prep total RNA extraction kits (Amersham Pharmacia Biotech, Piscataway, NJ). At the end of the procedure, extracted RNA was suspended in 1 ml of slightly alkaline diethylpyrocarbonate-treated water (pH 7.5), and concentrations were determined spectrophotometrically at 260 nm. Thereafter, equivalent amounts of RNA from each of 8C, 8D, and 6D-2I were used to synthesize first-strand cDNA. PCRs were then carried out to amplify cDNAs encoding RyR2 using ß-actin as an internal reference. Primers for RyR2 were as follows: sense (5'-GTGTTTGGATCCTCTGCAGTTCAT-3') and anti-sense (5'-AGAGGCACAAAGAGGAATTCGG-3'); whereas those for ß-actin were as follows: sense (5'-CGTAAAGACCTCTATGCCA-3') and anti-sense (5'-AGCCATGCCAAATGTCTCAT-3').
Quantitation of the mRNA encoding receptor for AGEs in 8C, 8D, and 6D-2I rat hearts.
RT-PCR was carried out to determine the levels of mRNA encoding the receptor for AGEs (RAGE) as well as a major spliced variant of RAGE in 8C, 8D, and 6D-2I. A single pair of primers was used to determine RAGE as well as its splice variant. This primer pair was selected from sequences (GenBank accession number L33413 for RAGE) 5' and 3' of the intron that generates the variant and are as follows: sense (5'-AGGTGGAACAGTCGCTCC-3') and anti-sense (5'-CACAGCTGAGGTCGGAAC-3').
Determination of amount of RyR2 protein in 8C, 8D, and 6D-2I rat hearts.
As described previously, two sequential steps were used to determine the relative levels of RyR2 protein in 8C, 8D, and 6D-2I rat hearts (14,15). First, membrane vesicles were prepared simultaneously from control, STZ-induced diabetic, and insulin-treated rat hearts (three hearts per preparation x two preparations), and their protein content was determined using the method of Lowry et al. (28). Second, 100 µg total protein from each vesicle preparation was solubilized in gel dissociation medium containing 10 mg/ml dithiothreitol and electrophoresed for 3.5 h at 150 V using denaturing 420% linear gradient polyacrylamide gels. Various amount of purified RyR2 protein (50350 ng) were simultaneous run in parallel lanes on the same gel to serve as a calibration curve. Gels were then stained with Coomassie blue dye, destained overnight, and dried between two sheets of cellophane. The intensity of the RyR2 band in each vesicle preparation was then determined by interpolation on the calibration curve derived from various RyR2 concentrations. Western blot analyses were also carried out as described previously (14,15) to confirm relative levels of RyR2 protein in each vesicle preparation. For these experiments, ß-actin served as an internal control to correct for sample loading.
Ability of RyR2 from 8C, 8D, and 6D-2I rat hearts to bind [3H]ryanodine.
The functional integrity of RyR2 from each of 8C, 8D, and 6D-2I was assessed from its ability to bind [3H]ryanodine (14,15). For this, 100 µg/ml of membrane vesicle protein from 8C, 8D, and 6D-2I were incubated in binding buffer containing 200 µmol/l free calcium (500 mmol/l KCl, 20 mmol/l Tris-HCl, 300 µmol/l CaCl2, 0.1 mmol/l EGTA, 6.7 nmol/l [3H]ryanodine, pH 7.4) for 2 h at 37°C. After incubation, vesicles were filtered and washed, and the amount of [3H]ryanodine bound to RyR2 was determined by liquid scintillation counting. Nonspecific binding was determined simultaneously by incubating vesicles with 1 µmol/l unlabeled ryanodine.
The affinities of ryanodine for RyR2 from 8C, 8D, and 6D-2I were determined using binding assays. These experiments were conducted as described above except that increasing concentrations of unlabeled ryanodine (0300 nmol/l) were also added to the samples. Equilibrium dissociation constant (Kd) values were ascertained using the Cheng-Prusoff relationship (29) given by the following:
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Binding affinity experiments were also carried out to determine whether the calcium sensitivity of RyR2 is altered by diabetes. For this, 100 µg/ml vesicle protein from 8C, 8D, and 6D-2I were incubated in binding buffer (500 mmol/l KCl, 20 mmol/l Tris-HCl, 0.1 mmol/l EGTA, and 6.7 nmol/l [3H]ryanodine, pH 7.4) containing various amounts of free calcium (13,000 µmol/l) for 2 h at 37°C. After incubation, vesicles were filtered and washed, and the amount of [3H]ryanodine bound to RyR2 was determined using liquid scintillation counting. Nonspecific binding was determined simultaneously by incubating vesicles with 1 µmol/l unlabeled ryanodine.
Determination of glycation products on RyR2 from 8C, 8D, and 6D-2I rat hearts.
Membrane vesicles prepared from control, diabetic, and insulin-treated rat hearts were electrophoresed using denaturing SDS-PAGE for 3.5 h (one gel for each membrane preparation from 8C, 8D, and 6D-2I). At the end of this time, the gels were stained with Coomassie blue dye and destained, and the bands corresponding to RyR2 were excised and transferred to 1.5 ml Eppendorff tubes containing distilled deionized water. Two sequential steps were then conducted to determine the presence of glycation products on RyR2.
In-gel protein digestion.
Coomassie-stained RyR2 protein bands from 8C, 8D, and 6D-2I gels were cut into small pieces, destained with 50% acetonitrile/50 mmol/l ammonium bicarbonate, reduced with 10 mmol/l dithiothreitol, and then alkylated with 55 mmol/l iodoacetamide. After alkylation, the gel pieces were digested overnight with trypsin (6 ng/nl; Promega) at 37°C. The next day, the peptides were desalted by eluting from micro C18 ZipTip columns (Millipore, Bedford, MA) with a solution of 50% acetonitrile and 0.1% trifluoroacetic acid. -Cyano-4-hydroxycinnamic acid was used as the matrix.
Mass spectrometry analysis.
First, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrograms were recorded in the positive reflectron mode (Micromass, Manchester, U.K.). Time of flight was measured using the following parameters: 3,400 V pulse voltage, 15,000 V source voltage, 500 V reflectron voltage, 1,950 V multichannel plate (MCP) voltage, and low mass gate of 500 Da. Internal calibration was performed using auto digestion peaks of bovine trypsin (monoisotopic mass [M+H+], m/z 842.5099 and m/z 2211.1045) before analysis of experimental samples. Second, liquid chromatography-mass spectrometry analyses of digested RyR2 peptides from 8C and 8D were also performed using a capillary liquid chromatography system coupled with a quadruple time of flight (Q-TOF) mass spectrometer (Micromass) fitted with a Z-spray ion source. Samples were desalted and concentrated using an online pre-column (C18, 0.3 mm i.d., 5 mm length). Separation of the peptides was carried out on a reverse-phase capillary column (Pepmap C18, 75 mm i.d., 15 cm length; LC Packings, San Francisco, CA) running at 300 nl/min. The mobile phase consisted of a linear gradient from 100% solution A (0.1% formic acid/3% acetonitrile/96.9% H2O, vol/vol) to 70% solution B (0.1% formic acid/2.9% H2O/97% acetonitrile, vol/vol) for 50 min followed by a 10-min gradient to 100% solution B. Mass spectra were acquired in positive ion mode.
Identification of glycation products on RyR2 from 8C, 8D, and 6D-2I rat hearts.
Mass data files obtained from MALDI-TOF analyses of RyR2 from 8C, 8D, and 6D-2I were then search using an in-house PERLscript algorithm for M+H+ peaks corresponding to theoretical RyR2 peptides with one lysine or arginine miscleavage (or no miscleaved peptide with a histidine residue within) that have been modified with a single N-(carboxymethyl)-lysine, imidazolone A, imidazone B, pyrraline, or 1-alkyl-2-formyl-3,4-glycosyl pyrrole molecule (AFGP) functionality.
Data analyses.
Differences between values from each of 8C, 8D, and 6D-2I rat hearts were evaluated by one-way ANOVA followed by the Newman-Keuls test. The data shown are means ± SE. Results were considered significantly different at P < 0.05.
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RESULTS |
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At the beginning of the study, the average whole blood glucose level of all animals was 4.2 ± 0.2 mmol/l. For those animals injected with citrate buffer only (controls), mean blood glucose levels remained essentially unchanged throughout the 56-day study. However, blood glucose levels of animals injected with 50 mg/kg STZ increased to 20.1 ± 0.4 mmol/l after 3 days and increased progressively thereafter to 29.4 mmol/l. Daily insulin treatment initiated after 6 weeks of diabetes normalized blood glucose levels to near control values. Heart weight to body weight ratios were also significantly higher in 8D when compared with 8C (4.5 vs. 2.7 mg/g). This ratio returned to near control values with insulin treatment (3.6 mg/g). Other characteristics of the animals used in this study are listed in Table 1.
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Calcium sensitivity of [3H]ryanodine binding by RyR2 from 8C, 8D, and 6D-2I rat hearts.
Experiments were also conducted to determine whether the sensitivity of RyR2 to calcium alters with diabetes. The response of RyR2 to calcium concentration was evaluated from its ability to bind [3H]ryanodine, because the amount of [3H]ryanodine bound is a direct measure of the "openness" of these channels (32). In this study, [3H]ryanodine bound was normalized per microgram RyR2 protein for each of the different preparations. As shown in Fig. 5 (), the amount of [3H]ryanodine bound to control RyR2 was biphasic as the concentration of calcium varied from 1 to 3,000 µmol/l. Between concentrations of 1 and 100 µmol/l calcium, [3H]ryanodine binding increased linearly and then plateaued between 200 and 500 µmol/l, reaching a maximum of 441.4 ± 16.3 pmol [3H]ryanodine bound/µg RyR2 protein. As the concentration of buffered free calcium increased beyond 500 µmol/l, the amount of [3H]ryanodine bound to RyR2 from 8C decreased, suggesting that the channel becomes deactivated. RyR2 from 8D also exhibited the typical biphasic response to calcium, but with three notable differences. First, the maximum amount of [3H]ryanodine bound to RyR2 from 8D was 46.9% less than that bound by 8C (234.4 ± 10.5 pmol [3H]ryanodine bound/µg RyR2 compared with 441.4 ± 16.3 pmol [3H]ryanodine bound/µg RyR2). Second, peak [3H]ryanodine binding occurred at 100 µmol/l calcium for RyR2 from 8D compared with 500 µmol/l for RyR2 from 8C. Third, concentrations of calcium >100 µmol/l rapidly deactivated or closed RyR2 from 8D, as indicated from its decreased ability to bind [3H]ryanodine. These data show for the first time that diabetes alters the sensitivity of RyR2 to calcium.
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Trypsin digestion of RyR2 from 8C, 8D, and 6D-2I rat hearts and determination of peptide masses.
Membrane vesicles prepared from hearts of 8C, 8D, and 6D-2I were electrophoresed using denaturing SDS-PAGE. At the end of the separation, the gels were stained with Coomassie blue dye and destained, and the bands corresponding to RyR2 were excised, washed, and digested overnight with trypsin. The M+H+ values of the peptides were then determined using MALDI-TOF and nanoflow-ESI-Q-TOF. It should be mentioned that although the complete cDNA encoding rat RyR2 is not available, this protein is highly conserved among species, and its amino acid sequence is expected to be similar to those published for mouse, rabbit, and human RyR2, which all share >92% homology. In addition, to avoid ambiguity in our analyses, only consensus RyR2 sequences from mice, rabbits, and humans were investigated, because these said sequences are likely to be present on rat RyR2.
Digestion of RyR2 from 8C with trypsin afforded 298 peptides with M+H+ values between 500 and 3000 Da. Peptides with M+H+ >3000 Da were not detected probably because of their low ionization efficiencies and large size (unable to escape from the gel). Of these 298 peptides, at least 136 corresponded to non-miscleaved consensus trypsin peptides from mouse, rabbit, and human RyR2 proteins (140 or 46.7% from mouse RyR2, 144 or 48.3% from rabbit RyR2, and 136 or 45.6% from human RyR2). As shown in the middle panel of Fig. 6A, these peptides spanned the entire range of rabbit RyR2, with minimum variability (≤595 parts per million, right panel). Digestion of RyR2 from 8D with trypsin afforded 20.8% fewer peptides compared with 8C. This reduction in peptides suggests that changes are occurring to RyR2 in such a manner as to compromise trypsins ability to digest it. Because trypsin cleaves at lysine and arginine residues, it is logical to conclude that modifications are occurring at these residues.
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Digestion of RyR2 from 6D-2I with trypsin afforded 304 peptides with M+H+ values ranging from 700 to 3000 Da. Of these, 124 corresponded to the masses of trypsin peptides from rabbit RyR2 (127 from mouse RyR2 and 118 from human RyR2). Interestingly, the M+H+ values from 6D-2I that corresponded to peptides from RyR2 were also not wholly a subset of the M+H+ values obtained from 8C (left panel of Fig. 6C). These data clearly suggest that 2 weeks of insulin treatment was able only to partially attenuate or minimize post-translational modifications on critical lysine and arginine residues on RyR2.
Identification of glycation products on RyR2.
As mentioned above, the MALDI-TOF mass data files generated for RyR2 from 8D and 6D-2I were searched using an in-house PERLscript algorithm for M+H+ values corresponding to theoretical RyR2 peptides with one miscleavage (or theoretical trypsin digested peptide containing a histidine residue within) modified with a single N-(carboxymethyl)-lysine, imidazolone A, imidazone B, pyrraline, or AFGP. It should be pointed out that modifications found naturally in 8C were not included in the analysis since we were only interested in those modifications that resulted from diabetes. The structures of these non-cross-linking AGEs and their delta masses (
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Modifications were not always seen on peptides from both diabetic and insulin-treated RyR2. Several peptides from insulin-treated RyR2 contained modifications that were either less prominent or absent from RyR2 of 8D as well as RyR2 from 8C. As shown in Fig. 9, a M+H+ of 1653.67 Da was prominent in insulin-treated diabetic RyR2, but not present in control and diabetic RyR2. Analysis of the theoretical mass files suggests that this M+H+ could have resulted from a single imadazolone B (+142.03 Da) adduct on the lysine residue on the peptide 2186EITFPKMVANCCR2198. A comprehensive listing of prominent consensus RyR2 peptides, the theoretical masses of the modification, and the AGEs involved are given in Table 2. We have also listed Schiff base and Amadori adducts (peptides with masses of 162.02) that were detected because they are important intermediate products (last two rows of Table 2).
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DISCUSSION |
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A principal finding of the present study is that RyR2 from 8-week STZ-induced diabetic rat hearts contains several non-cross-linking AGEs, including N-(carboxymethyl)-lysine, imidazolone A, imidazone B, pyrraline, and AFGP. These post-translational products were detected by digesting RyR2 with trypsin, determining the masses of the resultant peptides using MALDI-TOF mass spectrometry and then searching the mass files for modifications using an in-house PERLscript program. These data are the first to show that AGEs are formed on intracellular RyR2 during chronic diabetes. This strategy also allowed us to determine not only the nature of the modifications but also the specific amino acids involved in the modifications.
In a previous study, we confirmed using a modified Langendorff procedure that hearts from 8-week STZ-induced diabetic rats show typical diabetic cardiomyopathic changes (15). In the present study, we show that the functional integrity of RyR2 from these hearts was compromised, as assessed from its decreased ability to bind [3H]ryanodine, its altered sensitivity to calcium, and its slowed SDS-PAGE electrophoretic mobility. Taken together, these data suggest a relationship between a diabetes-induced decrease in activity of RyR2 and its AGE content. However, formation of AGEs on RyR2 is not the only contributor to its dysfunction and, by extension, the diabetic cardiomyopathy. In a previous study, we also showed that the dysfunction of RyR2 stems in part from a diabetes-induced increase in its disulfide bond content (19).
A second major finding of the present study is that non-cross-linking AGEs were also found on RyR2 from 6D-2I rat hearts. We also found that whereas insulin treatment was able to restore the calcium sensitivity of RyR2, RyR2 from 6D-2I bound 11.5% less [3H]ryanodine per microgram of protein when compared with RyR2 from age-matched control animals (297.8 fmol [3H]ryanodine/µg RyR2 compared with 336.6 fmol [3H]ryanodine/µg RyR2). These data suggest that 2 weeks of insulin treatment, initiated after 6 weeks of diabetes was able to only partially prevent and/or reverse formation of these post-translation products on RyR2. Because the turnover rate of RyR2 is slow (8 days [22]) and, once formed, AGEs remain chemically linked to the protein throughout its lifetime, it is likely that the RyR2 analyzed may have consisted of a mixture of newly synthesized as well as not yet degraded AGE-modified RyR2.
In the present study, we also found that mRNA levels encoding RAGE as well its major spliced variant decreased after 8 weeks of diabetes. This result was surprising because we anticipated that mRNA levels for RAGE would increase after 8 weeks of diabetes. In a previous study, we found that membrane vesicles from 6-week STZ-induced diabetic rat hearts contain fluorophores with wavelength maxima (max) were similar to those for AGEs (19). The latter suggested that in our STZ-induced diabetic rat model (50 mg/kg i.v. injected), AGEs may be formed on RyR2 as early as 6 weeks. If the latter holds true, then it is likely that the decrease in transcription of RAGE reflects receptor desensitization. Transcription of these proteins was restored to near control values with 2 weeks of insulin treatment, initiated after 6 weeks of diabetes.
In addition to these non-cross-linking AGEs, we also detected several RyR2 peptides from insulin-treated animals with mass changes of 162.02 Da, consistent with modification by either a single Schiff base or an Amadori product. These data are clearly important because they also show for the first time the presence of AGE precursor molecules on RyR2. Although mass spectrometry is unable to differentiate between these two glycation products, this modification is more likely to have resulted from an Amadori product rather than a Schiff base. The latter is more labile and therefore likely degraded during the isolation and sample preparation procedures. Why these modifications were found in insulin-treated but not diabetic and control samples remains to be ascertained.
It is also of importance to note that the AGEs appeared to be found on "hot spots" on RyR2. Four modifications were detected between amino acids 10001700, three between amino acids 3400 and 3600, and three between amino acids 44004700 (Table 2). We speculate that because modifications are more likely to occur on regions of RyR2 that are exposed to higher concentrations of reducing sugars (i.e., the cytoplasmic side), then these "hot spots for glycation" are not likely to constitute the pore-forming and/or transmembrane segment of the channel or reside within the lumen of the sarcoplasmic reticulum.
In conclusion, data from the present study suggest that loss in cardiac function induced by diabetes probably stems in part from a dysfunction of RyR2 (seen as a decrease in its ability to bind the specific ligand [3H]ryanodine, alterations in its sensitivity to calcium, and a slowing in its electrophoretic mobility). In addition, this dysfunction could result from diabetes-induced formation of non-cross-linking AGEs on RyR2. Our data also show that 2 weeks of insulin treatment, initiated after 6 weeks of diabetes, only partially restored and/or prevented loss in RyR2 activity. Analysis of RyR2 from 6D-2I also showed that it contained several AGE adducts. Because RyR2 is a long-lived protein (8 days) and AGE complexes remain on a protein throughout its lifetime, these data may help explain why congestive heart failure still persists in diabetic patients who are in compliance with insulin and/or oral hypoglycemic therapies.
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ACKNOWLEDGMENTS |
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Address correspondence and reprint requests to Keshore R. Bidasee, Department of Pharmacology, University of Nebraska Medical Center, 986260 Nebraska Medical Center, Omaha, NE 68198-6260. E-mail: kbidasee{at}unmc.edu
Received for publication January 26, 2003 and accepted in revised form March 31, 2003
6D-2I, 6-week streptozotocin-induced diabetic/2-week insulin-treated animals; 8C, 8-week control animals; 8D, 8-week streptozotocin-induced diabetic animals; AFGP, 1-alkyl-2-formyl-3,4-glycosyl pyrrole molecule; AGE, advanced glycation end product; Kd, equilibrium dissociation constant; M+H+, monoisotopic mass; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; RAGE, receptor for AGEs; RyR2, type 2 ryanodine receptor calcium-release channel; STZ, streptozotocin
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REFERENCES |
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