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Academic year: 2022



Testo completo



Keshore R. Bidasee, Sarah Ingersoll, and Chun Hong Shao

Dept. of Pharmacology, University of Nebraska Medical Center, 985800 Nebraska Medical Center, Omaha, NE


Diabetes mellitus (DM) is a major metabolic illness affecting populations worldwide. In America more than 17 million individuals are inflicted with this syndrome, which is clinically characterized as having fasting blood glucose levels of (7mmol/L).786 In addition to frank DM, a significant number of individuals also exhibit lesser degrees of impaired glucose regulation. DM is classified into three types, Type 1 (account for about 10% of all cases), Type 2 of cases) and gestational diabetes

of cases). Type 1 DM arises when beta cells of the pancreas are unable to produce insulin. This defect can occur from chemical toxicity, lymphocytic infiltration following viral infection (autoimmune reaction) or from tumors.787 Usually children and young adults are the ones inflicted with Type 1 DM. Type 2 DM results either from defects in beta cell signaling, insulin receptor signaling, glucose transport proteins, or combinations thereof. While genetics is an important predisposition, factors such as obesity and a sedentary lifestyle also increase risk for developing Type 2 diabetes. Although initially thought to occur only in adult life, recent data suggest that young obese children are also developing Type 2 diabetes.

Gestational diabetes occurs in about 5% of all pregnancies. For the mother, gestational diabetes increases risk of preeclampsia, cesarean section and future risk of Type 2 diabetes. For the fetus or neonate, the disorder is


associated with higher rates of perinatal mortality, macrosomia, birth trauma, hyperbilirubinemia and neonatal hypoglycemia.788

Persistent elevation in circulating glucose levels lead to the progressive loss of function of many components of the cardiovascular system, including the heart itself. This “diabetic cardiomyopathy” which occurs independent of coronary arteriosclerosis and/or hypertension, starts of as an asymptomatic slowing in relaxation kinetics.789 As the syndrome progresses, systolic function also becomes compromised, increasing morbidity and mortality. To date, it is well accepted that the etiology underlying diabetic cardiomyopathy (DC) is multifactorial.790 At the molecular level, DC reflects alterations in expression of several proteins. For example, increased myocardial stiffness has been attributed to increases in expression of extracellular fibronectin and collagen IV.791 Changes in expression of complement and associated intracellular signal transduction proteins are also responsible in part for decrease in autonomic function.792,793 Diabetes also decreases synthesis and release of thyroid hormones, and 794 This in turn decreases the ATPase activity of myosin heavy chain resulting in a decrease in the extent of myocyte shortening. Expression of several proteins intimately involved in excitation-contraction coupling is also altered in the heart during diabetes.

The UK Prospective Diabetes Study (UKPDS) and the Diabetes Control and Complications Trial (DCCT) clearly demonstrated that complications resulting from both Type 1 and Type 2 patients can be minimize with tight glycemic control (metformin/sulfonylureas/insulin).795,796 Insulin pumps have made this goal more achievable for type 1 diabetic patients. Also, islet cell transplantation could become another widely use treatment strategy for regulating blood glucose levels in Type 1 diabetics if stem cell technology is able to reduce dependency on viable of pancreatic tissues. However, for the vast majority of patients, tight glycemic control is often difficult to achieve and can lead to more severe hypoglycemia. Persistent elevation in blood glucose levels lead to failure of several components of the cardiovascular system that requires treatment with therapeutics in order to manage the symptoms and improve quality of life. Among the drugs used are angiotensin-converting enzyme inhibitors (ramipril/perindopril), angiotensin receptor blockers (losartan, irbesartan), diuretics (thiazides) and lipid lowering drugs (chlorthalidone, atorvastatin).797-801 While some of these drugs appear to exert secondary beneficial effects on the heart by reducing inflammation and triggering reverse remodeling, their primary mode of action is to reduce peripheral resistance and/or load. Carvedilol, a

blocker widely used to treat DC also has blocking and antioxidant properties (10 x greater than that of vitamin E).802 Multi-drug therapies in combination with diet and exercise are currently being explored


to reduce cardiovascular complications.803 However, economic cost and compliance may be inhibitory factors against this treatment strategy.

Although, current pharmacotherapeutics improve cardiovascular hemodynamics, the beneficial effect on the diabetic heart per se remains modest. As such, heart failure continues to persist in the diabetic population.

Two immediate challenges therefore are (i) to identify additional mechanisms that contribute to loss in left ventricular function, and (ii) identify/develop newer therapeutic strategies that can minimize these changes.


Decrease in the ability of the heart to effectively contract is one of the major causes for the increased incidence of morbidity and mortality in diabetic patients. Studies suggest that this defect is due in part to alterations in function of several proteins involved in excitation-contraction coupling.

One of these proteins is RyR2, the channel through which calcium ions leaves the sarcoplasmic reticulum to effect contractions. Yu and McNeill804 were the first to implicate RyR2 in the etiology of DC when they found that post-rest potentiation (Woodworth staircase: an enhancement in stimulated contraction following a long rest) was significantly reduced in hearts from diabetic rats. They also showed that membrane vesicles prepared from diabetic rat hearts bound significantly less when compared with age-matched, non-diabetic controls805 and suggested that expression of RyR2 is being decreased during diabetes. We and others later showed that expression of RyR2 do indeed decrease in hearts of chronic diabetic patients806,807 as well as in hearts of streptozotocin (STZ)-induced diabetic rats.808-811 However, since critical calcium cycling proteins are usually expressed in amounts that exceed that required for minimal physiologic functioning, it seems unlikely that a decrease in expression of RyR2 per se could be solely responsible for the decrease in post-rest potentiation induced by diabetes. Eisner and co-workers also echoed this view when they suggested that a decrease in RyR2 protein density would be compensated for by an increase in the amount of releasable calcium inside the sarcoplasmic reticulum.812 Moreover, using the streptozotocin-induced diabetic rat model we found that not all hearts with establish DC show a decrease in expression of RyR2 protein (Fig. 21-1, panels A, B and C). Zhong et al.810 also found that loss of function precedes loss of protein expression. Thus, it seems likely that mechanisms other than changes in RyR2 expression are


responsible for decrease in post-rest potentiation and loss in ventricular contractility associated with diabetes.

Figure 21-1. Comparison of basal cardiac function in hearts from 8-week control (8C), 8- week diabetic (8D) and 6-week diabetic/2-week insulin-treated (6D/2I) animals and its correlation with expression and function of type 2 ryanodine receptors (RyR2). A-B.

comparision of in vivo cardiac function. For this, animals were lightly anesthetized with Inactin® (20mg/kg) and a pressure transducer attached to the end of a catheter was inserted into the left ventricle by way of a carotid artery. Basal heart rates, left ventricular pressures, and left ventricular end diastolic pressures were then directly obtained A. The first derivatives, ± dP/dt were also obtained to determine rates of changes B. C. Steady state level of RyR2 protein in hearts from 8C, 8D and 6D/2I. For this, Western blot analyses were carried out using standard procedures employing of membrane protein from 8C, 8D and 6D/2I. D. Comparison of the ability of of membrane protein from 8C, 8D and 6D/2I to bind the specific ligand E. Comparison of the relative affinities of RyR2 from these three groups of animals for ryanodine.811,813,814



Using a lower dose of streptozotocin to induce experimental diabetes (50mg/kg instead of the typical 65mg/kg, IV), we recently discovered that equivalent amounts of RyR2 protein from diabetic rat hearts bind less of the specific ligand when compared with age-matched controls.811 We also found that this defect preceded loss of expression of RyR2.813 Interestingly, while the amount of accessible ryanodine binding sites decreased (lower the affinity of RyR2 for ryanodine did not change. In other experiments, we also found that the electophoretic mobility of RyR2 from diabetic animals on SDS-PAGE gels was slowed, suggesting posttranslational modification.814 Two salient questions that arises from these data are (i) can posttranslational modification decrease RyR2 function, and (ii) if so, what types of modifications are occurring on RyR2 as a result of diabetes. It should also be pointed out that since RyR2 appears to have multiple binding site for ryanodine/ryanoids, a decrease in is possible without significant changes in 811 Studies have also shown that single point mutations on homologous RyR1 can significantly decrease its ability to bind 815 Thus, it seems reasonable to conclude that posttranslational modifications on RyR2 can also decrease RyR2’s ability to bind Moreover, several point mutations on RyR2 are also known to trigger sudden cardiac death in children and young adults as is the case with catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right-ventricular dysplasia/cardiomyopathy (ARVD2).294


It has been known for a long time that the turnover rate of RyR2 is slow (half-life 8 days). It is therefore conceivable that alterations in intracellular milieu brought about by diabetes could trigger posttranslational modifications. Two types of modifications that can be readily be formed on RyR2 as a result of shifts in metabolism and biochemistry are oxidation of sulfhydryl groups on exposed cysteine residues and non-enzymatic glycation of lysine, arginine and histidine residues.


Figure 21-2. Examples of posttranslational modification reactions that can occur on RyR2 during diabetes. A. Select reactions that lead to the formation of nitrothiols, disulfide bonds and sulfenic acid adducts on sulfhydryl moieties of exposed cysteine residues, represented by “R”. B. Reactions between aldose and arginine/lysine residues that lead to the formation of crosslinking (pentosidine) and non-crosslinking advanced glycation end products (AGEs) on RyR2.


Oxidation of cysteine residues on type 2 ryanodine receptors

Studies have shown that the activity of RyR2 is dependent on the oxidative state of several sulfhydryl groups (SH).816 When the more reactive of these “exposed” sulfhydryl groups are oxidized, this usually trigger channel activation (increases increases binding). As the concentration of oxidants increases, other sulfhydryl groups also become oxidized, triggering a decrease in channel activity (decreases binding). Studies have also shown that shifts in metabolism and biochemistry brought diabetes increases intracellular levels of reactive oxygen species (ROS) including superoxide anions hydroxy radicals lipid peroxides singlet oxygen and hydrogen peroxide 817 We recently found that expression of nitric oxide synthases (eNOS and iNOS) also increase in the heart during diabetes, suggesting an increase in production of nitric oxide (unpublished data). NO in turn can react with ROS increasing production of several reactive nitrogen species including nitrosonium cation nitroxyl anion and peroxynitrite species. ROS and nitrogen species are also known to react rapidly reacts with the sulfhydryl moiety on cysteine residues forming nitrothiols, disulfide bonds and sulfenic acid derivatives (Fig. 21-2 A).

In a recent study, we found that when RyR2 from diabetic rat hearts were treated with 2mM dithiothreitol, its ability to bind was partially restored.818 These data suggest that the reduced ability of RyR2 to bind stems in part from increased formation of disulfide bonds (S-S). Since there are several classes of reactive sulfhydryl groups on RyRs, further experiments were conducted to ascertain which class of sulfhydryl groups might be involved in disulfide bond formation. For this we synthesized the pyrrole sulfhydryl reagent, pyrocoll (5H,10H-Dipyrrolo[1,2- a:1',2'-d]pyrazine-5,10-dione). At nM concentrations, this drug interacts with one class of free sulfhydryl groups triggering channel deactivation

(decreases RyR2 ability to bind At higher

concentrations, pyrocoll reacts with a second class of SH groups, triggering instead channel activation. Interesting, we found that pyrocoll at concentrations was unable increase binding to RyR2 from diabetic rat hearts, suggesting that a class of SH groups that trigger channel activation are not available. We have yet to determine the location of these specific SH groups (cysteine residues) on RyR2.


Non-enzymatic glycation of arginine, histidine and lysine residues on RyR2

Studies have also shown that glucose-6-phosphate levels increases in myocytes during chronic diabetes. High levels of this aldose sugar, as well as other reactive aldehydes formed as a result of diabetes will accelerate the formation of Schiff bases on lysine, arginine, and histidine residues (non- enzymatic glycation reactions). Over time, these Schiff bases undergo internal rearrangement to form more stable Amadori products. On long-lived proteins, Amadori products further undergo a series of oxidation, reduction, elimination and cyclization reactions to form advanced-glycation end products (AGEs, Fig. 21-2 B).819 Once formed, these complexes remain attached to the protein throughout its lifetime. Studies have also shown that modification of proteins with AGEs ultimately lead to organ dysfunction.

There are two major types of AGEs, those that are crosslinked (formed between adjacent amino acid residues), and those that are non-crosslinking.

Also, some AGEs fluoresce while other do not.

In a recent study using matrix-assisted laser desorption ionization mass spectrometry in conjunction with an in-house PERL algorithm, we found that formation of non-crosslinking AGEs increases on RyR2 during diabetes and production of these complexes were attenuated with insulin-treatment814 (also see Fig. 21-3 A). More recently, we also found that formation of pentosidine-type crosslinking AGEs also increases on RyR2 during diabetes (unpublished data). As an example, pentosidine AGEs were formed between arginine 870 and lysine 748 (see Fig. 21-3A, top left). Interestingly, this crosslinking AGE adduct was found in the highly conserved N-terminal, regions in which mutations trigger malignant hyperthermia (MH), central core disease (CCD), CPVT and ARDV2. Since RyR2 undergo conformation rearrangement when translocating calcium ions from the lumen of the SR to the cytosol, it seems likely crosslinking of the domains could significantly compromise function. Fig. 21-3 B show the approximate locations of select AGEs adducts identified thus far on RyR2.


Figure 21-3. Amino acid residues on RyR2 that are modified by advanced glycation end products and their relative locations on the 3D structure of RyR2. A. Specific arginine, histidine or lysine residues on RyR2 that are modified by defined advanced glycation end products. Bold letters indicate the amino acid residue that is modified. The chemical structures of four structurally diverse AGEs are also shown. B. The approximate location of the amino acids that are modified by AGEs (blue filled circles). The green filled circles represent the locations of previously identified residues.



In conclusion, we have shown that RyR2 becomes dysfunctional during diabetes. We have also identified two types of posttranslational modifications that occur on RyR2 during diabetes. Treatment with insulin reduced posttranslational modifications on RyR2 as well as partially attenuated loss of function induced by diabetes. Although suggestive, these data do not establish that posttranslational modifications are responsible for the loss of RyR2 activity and by extension, the decrease in ventricular contractility seen during diabetes. Experiments using site-directed mutagenesis studies, as well as treatment with drugs capable of preventing AGEs formation and oxidation of amino acid residues are ongoing.


This work was supported in part by grants from the National Institutes of Health (RO1-HL66898). The authors thank Drs. Terrence Wagenknecht and Manjuli Sharma for providing the 3D structure of RyR2. The authors would also like to thank Drs. Kaushik Patel, Mu Wang and Henry R. Besch, Jr. for helpful discussions.


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