Abstract
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.
Footnotes
This work was supported in part by the National Institutes of Health National Heart, Lung, and Blood Institute [Grant HL085061]; the Edna Ittner Research Foundation; the American Diabetes Association [Grant 1-06-RA-11]; and a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan [Grant 18790619].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- SR
- sarcoplasmic reticulum
- RyR2
- type 2 ryanodine receptor
- DMEM
- Dulbecco's modified Eagle's medium
- RCS
- reactive carbonyl species
- ROS
- reactive oxygen species
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- MS
- mass spectrometry
- MS/MS
- tandem mass spectrometry
- TOF
- time of flight
- GA
- glycolaldehyde
- STZ
- streptozotocin
- MALDI
- matrix-assisted laser desorption ionization
- MGO
- methylglyoxal
- SERCA2
- sarco(endo)plasmic reticulum Ca2+-ATPase
- Py
- pyridoxamine
- Ag
- aminoguanidine
- cRyR2
- control type 2 ryanodine receptor
- dRyR2
- diabetic type 2 ryanodine receptor
- Ins-DRyR2
- insulin-treated diabetic type 2 ryanodine receptor.
- Received February 20, 2012.
- Accepted May 25, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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