Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC146
Carbonylation contributes to RyR2 dysregulation and dyssyncronous sarcoplasmic reticulum Ca2+ release in a rat model of type 1 diabetes mellitus
C. Tian1, C. Shao1, O. Shoquin1, C. J. Moore1, W. S. Chen2, J. Singh3, A. Dsouza3, K. R. Bidasee1
1. Departments of Pharmacology and Experimental Neuroscience and Environmental Agricultural and Occupational Health, Nebraska Centre for Redox Biology, Lincoln, Nebraska, United States. 2. Department of Physiology and Biophysics, Libin Cardiovascular Institute of Alberta, University of Calgary, Licoln, Alberta, Canada. 3. School of Forensic and Investigative Science, University of Central Lancashire, Preston, United Kingdom.
The force of contraction of the heart is reduced during type 1 diabetes mellitus (T1DM) . Studies attribute this defect in part to dyssynchronous Ca2+ release from sarcoplasmic reticulum (SR) arising from variations in activities of type 2 ryanodine receptors (RyR2). To date, mechanism(s) underlying RyR2 dysregulation during T1DM remain poorly defined. The streptozotocin-induced (45-50 mg/kg; ip, n>40) rat model of T1DM in combination with echocardiography, ex vivo haemodynamics, video detection, electron and confocal microscopy, Western blots, ligand binding and lipid bilayer assays, mass spectrometry and site-directed mutagenesis were used to evaluate whether changes in dyad junction architecture and/or carbonylation are contributing factors . The study had the relevant ethical clearances from the Ethics Committees of the collaborating Universities. The results show significant reduction (Student’s t-test; p<0.05) and abnormal ventricular and myocyte contractions after 8 weeks of T1DM. Typically, mean cardiac fractional shortening was reduced by 25.2 ± 3.2% and extent of myocyte shortening by 30.3 ± 6.7%, respectively. Diabetic myocytes showed increased 4.4-fold spontaneous Ca2+ and dyssynchronous-evoked Ca2+ releases from the SR.. Electron microscopic analyses revealed no significant disruption in dyad junction architecture. RyR2 protein remained unchanged although total [3H]-ryanodine binding was reduced by 45.6± 4.2% (t-test; p<0.05). Trypsin digestion and mass spectroscopy revealed carbonyl adducts on R1611, K2190, R4462 and R4683 of RyR2. Mutating impacted residues singly and in combination to glycine, tyrosines or tryptophans to mimic charge neutralization and increase in bulk induced by carbonylation afforded two distinct channel phenotypes in lipid bilayer assays. The results show channels with 2 to 9 fold increases in responsiveness to low cis (cytoplasmic) Ca2+ and channels with >10-fold reduction in responsiveness to low cis Ca2+. Insulin-treatment minimized adduct formation, RyR2 dys-regulation and cardiac function loss. In conclusion, the results show that dyssynchronous- evoked Ca2+ release from SR during T1DM stems in part from carbonylation-induced alterations in responsiveness of RyR2 to influxed Ca2+, thus, providing new mechanistic insights into the pathogenesis of diabetic cardiomyopathy.
Where applicable, experiments conform with Society ethical requirements