Prolongation of relaxation is a hallmark of diabetic cardiomyopathy. Most studies attribute this defect to decreases in sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2a) expression and SERCA2a-to-phospholamban (PLB) ratio. Since its turnover rate is slow, SERCA2a is susceptible to posttranslational modifications during diabetes. These modifications could in turn compromise conformational rearrangements needed to translocate calcium ions, also leading to a decrease in SERCA2a activity. In the present study one such modification was investigated, namely advanced glycation end products (AGEs). Hearts from 8-week streptozotocin-induced diabetic (8D) rats showed typical slowing in relaxation, confirming cardiomyopathy. Hearts from 8D animals also expressed lower levels of SERCA2a protein and higher levels of PLB. Analysis of matrix-assisted laser desorption/ionization time-of-flight mass data files from trypsin-digested SERCA2a revealed several cytosolic SERCA2a peptides from 8D modified by single noncrosslinking AGEs. Crosslinked AGEs were also found. Lysine residues within actuator and phosphorylation domains were cross-linked to arginine residues within the nucleotide binding domain via pentosidine AGEs. Two weeks of insulin-treatment initiated after 6 weeks of diabetes attenuated these changes. These data demonstrate for the first time that AGEs are formed on SERCA2a during diabetes, suggesting a novel mechanism by which cardiac relaxation can be slowed during diabetes.

Decrease in cardiac contractility is a hallmark of chronic diabetes. Previously we showed that this defect results, at least in part, from a dysfunction of the type 2 ryanodine receptor calcium-release channel (RyR2). The mechanism(s) underlying RyR2 dysfunction is not fully understood. The present study was designed to determine whether non-cross-linking advanced glycation end products (AGEs) on RyR2 increase with chronic diabetes and if formation of these post-translational complexes could be attenuated with insulin treatment. Overnight digestion of RyR2 from 8-week control animals (8C) with trypsin afforded 298 peptides with monoisotopic mass (M+H⁺) ≥500. Digestion of RyR2 from 8-week streptozotocin-induced diabetic animals (8D) afforded 21% fewer peptides, whereas RyR2 from 6-week diabetic/2-week insulin-treated animals generated 304 peptides. Using an in-house PERLscript algorithm, search of matrix-assisted laser desorption ionization-time of flight mass data files identified several M+H⁺ peaks corresponding to theoretical RyR2 peptides with single N^ε-(carboxymethyl)-lysine, imidazolone A, imidazone B, pyrraline, or 1-alkyl-2-formyl-3,4-glycosyl pyrrole modification that were present in 8D but not 8C. Insulin treatment minimized production of some of these nonenzymatic glycation products. These data show for the first time that AGEs are formed on intracellular RyR2 during diabetes. Because AGE complexes are known to compromise protein activity, these data suggest a potential mechanism for diabetes-induced RyR2 dysfunction.

The present study was undertaken to assess the effects of exercise training (ExT) initiated after the onset of diabetes on cardiac ryanodine receptor expression and function. Type 1 diabetes was induced in male Sprague-Dawley rats using streptozotocin (STZ). Three weeks after STZ injection, diabetic rats were divided into two groups. One group underwent ExT for 4 wk while the other group remained sedentary. After 7 wk of sedentary diabetes, cardiac fractional shortening, rate of rise of left ventricular pressure, and myocyte contractile velocity were reduced by 14, 36, 44%, respectively. Spontaneous Ca²⁺spark frequency increased threefold, and evoked Ca²⁺release was dyssynchronous with diastolic Ca²⁺releases. Steady-state type 2 ryanodine receptor (RyR2) protein did not change, but its response to Ca²⁺was altered. RyR2 also exhibited 1.8- and 1.5-fold increases in phosphorylation at Ser²⁸⁰⁸and Ser²⁸¹⁴. PKA activity was reduced by 75%, but CaMKII activity was increased by 50%. Four weeks of ExT initiated 3 wk after the onset of diabetes blunted decreases in cardiac fractional shortening and rate of left ventricular pressure development, increased the responsiveness of the myocardium to isoproterenol stimulation, attenuated the increase in Ca²⁺spark frequency, and minimized dyssynchronous and diastolic Ca²⁺releases. ExT also normalized the responsiveness of RyR2 to Ca²⁺activation, attenuated increases in RyR2 phosphorylation at Ser²⁸⁰⁸and Ser²⁸¹⁴, and normalized CaMKII and PKA activities. These data are the first to show that ExT during diabetes normalizes RyR2 function and Ca²⁺release from the sarcoplasmic reticulum, providing insights into mechanisms by which ExT during diabetes improves cardiac function.

OBJECTIVE Approximately 25% of children and adolescents with type 1 diabetes will develop diastolic dysfunction. This defect, which is characterized by an increase in time to cardiac relaxation, results in part from a reduction in the activity of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2a), the ATP-driven pump that translocates Ca²⁺ from the cytoplasm to the lumen of the sarcoplasmic reticulum. To date, mechanisms responsible for SERCA2a activity loss remain incompletely characterized. RESEARCH DESIGN AND METHODS The streptozotocin (STZ)-induced murine model of type 1 diabetes, in combination with echocardiography, high-speed video detection, confocal microscopy, ATPase and Ca²⁺ uptake assays, Western blots, mass spectrometry, and site-directed mutagenesis, were used to assess whether modification by reactive carbonyl species (RCS) contributes to SERCA2a activity loss. RESULTS After 6–7 weeks of diabetes, cardiac and myocyte relaxation times were prolonged. Total ventricular SERCA2a protein remained unchanged, but its ability to hydrolyze ATP and transport Ca²⁺ was significantly reduced. Western blots and mass spectroscopic analyses revealed carbonyl adducts on select basic residues of SERCA2a. Mutating affected residues to mimic physio-chemical changes induced on them by RCS reduced SERCA2a activity. Preincubating with the RCS, methylglyoxal (MGO) likewise reduced SERCA2a activity. Mutating an impacted residue to chemically inert glutamine did not alter SERCA2a activity, but it blunted MGO9s effect. Treating STZ-induced diabetic animals with the RCS scavenger, pyridoxamine, blunted SERCA2a activity loss and minimized diastolic dysfunction. CONCLUSIONS These data identify carbonylation as a novel mechanism that contributes to SERCA2a activity loss and diastolic dysfunction during type 1 diabetes.

Heart failure and arrhythmias occur at 3 to 5 times higher rates among individuals with diabetes mellitus, compared with age-matched, healthy individuals. Studies attribute these defects in part to alterations in the function of cardiac type 2 ryanodine receptors (RyR2s), the principal Ca²⁺-release channels on the internal sarcoplasmic reticulum (SR). To date, mechanisms underlying RyR2 dysregulation in diabetes remain poorly defined. A rat model of type 1 diabetes, in combination with echocardiography, in vivo and ex vivo hemodynamic studies, confocal microscopy, Western blotting, mass spectrometry, site-directed mutagenesis, and [³H]ryanodine binding, lipid bilayer, and transfection assays, was used to determine whether post-translational modification by reactive carbonyl species (RCS) represented a contributing cause. After 8 weeks of diabetes, spontaneous Ca²⁺ release in ventricular myocytes increased ∼5-fold. Evoked Ca²⁺ release from the SR was nonuniform (dyssynchronous). Total RyR2 protein levels remained unchanged, but the ability to bind the Ca²⁺-dependent ligand [³H]ryanodine was significantly reduced. Western blotting and mass spectrometry revealed RCS adducts on select basic residues. Mutation of residues to delineate the physiochemical impact of carbonylation yielded channels with enhanced or reduced cytoplasmic Ca²⁺ responsiveness. The prototype RCS methylglyoxal increased and then decreased the RyR2 open probability. Methylglyoxal also increased spontaneous Ca²⁺ release and induced Ca²⁺ waves in healthy myocytes. Treatment of diabetic rats with RCS scavengers normalized spontaneous and evoked Ca²⁺ release from the SR, reduced carbonylation of RyR2s, and increased binding of [³H]ryanodine to RyR2s. From these data, we conclude that post-translational modification by RCS contributes to the heterogeneity in RyR2 activity that is seen in experimental diabetes.

In a previous study, we showed that after 6 weeks of streptozotocin-induced diabetes (6D), expression of type 2 ryanodine receptor calcium-release channels (RyR2) did not change significantly in rat hearts. However, the ability of this protein to bind [3H]ryanodine was compromised. Loss in activity therefore resulted from diabetes-induced increases in post-translational modifications on RyR2. In the present study, the effects of diabetes on one type of modification, namely, changes in oxidative state of reactive sulfhydryls was investigated. RyR2 protein from 6D bound 42.3 +/- 7.6 less [3H]ryanodine than RyR2 from controls (6C). The loss in binding was minimized with 2 weeks of insulin treatment initiated after 4 weeks of diabetes (77.8 +/- 5.5% of 6C). Pretreating RyR2 from 6D with 2 mM dithiothreitol in vitro increases [3H]ryanodine binding by 60.8 +/- 5.3%. Dithiothreitol pretreatment of RyR2 from 6C increased [3H]ryanodine binding by 16.8 +/- 4.3%. The reagent pyrocoll interacts with distinct classes of free sulfhydryl groups on 6C RyR2 to induce two major effects. At concentrations < or = 10 microM, it deactivates RyR2 (decreases [3H]ryanodine binding), whereas at higher concentrations it activates them (increases [3H]ryanodine binding). This reagent was unable to activate RyR2 from 6D. Although RyR2 from insulin-treated animals was deactivated by low concentrations of pyrocoll, it was only partially activated at higher concentrations. These data suggest that the dysfunction of RyR2 induced by diabetes may be due in part to formation of disulfide bonds between adjacent sulfhydryl groups and that these changes were attenuated with insulin treatment.