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1.
Heart Fail Rev ; 19(1): 101-12, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23430128

ABSTRACT

Efficient and rhythmic cardiac contractions depend critically on the adequate and synchronized release of Ca(2+) from the sarcoplasmic reticulum (SR) via ryanodine receptor Ca(2+) release channels (RyR2) and its reuptake via sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a). It is well established that this orchestrated process becomes compromised in diabetes. What remain incompletely defined are the molecular mechanisms responsible for the dysregulation of RyR2 and SERCA2a in diabetes. Earlier, we found elevated levels of carbonyl adducts on RyR2 and SERCA2a isolated from hearts of type 1 diabetic rats and showed the presence of these posttranslational modifications compromised their functions. We also showed that these mono- and di-carbonyl reactive carbonyl species (RCS) do not indiscriminately react with all basic amino acid residues on RyR2 and SERCA2a; some residues are more susceptible to carbonylation (modification by RCS) than others. A key unresolved question in the field is which of the many RCS that are upregulated in the heart in diabetes chemically react with RyR2 and SERCA2a? This brief review introduces readers to the field of RCS and their roles in perturbing SR Ca(2+) cycling in diabetes. It also provides new experimental evidence that not all RCS that are upregulated in the heart in diabetes chemically react with RyR2 and SERCA2a, methylglyoxal and glyoxal preferentially do.


Subject(s)
Diabetic Cardiomyopathies , Myocardial Contraction/physiology , Myocardium/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Animals , Diabetic Cardiomyopathies/metabolism , Diabetic Cardiomyopathies/pathology , Diabetic Cardiomyopathies/physiopathology , Humans , Myocardium/pathology , Protein Carbonylation
2.
Mol Cell Biochem ; 376(1-2): 121-35, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23354458

ABSTRACT

Recently, we reported an elevated level of glucose-generated carbonyl adducts on cardiac ryanodine receptor (RyR2) and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) in hearts of streptozotocin(STZ)-induced diabetic rats. We also showed these adduct impaired RyR2 and SERCA2 activities, and altered evoked Ca(2+) transients. What is less clear is if lipid-derived malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) also chemically react with and impair RyR2 and SERCA2 activities in diabetes? This study used western blot assays with adduct-specific antibodies and confocal microscopy to assess levels of MDA, 4-HNE, N (ε)-carboxy(methyl)lysine (CML), pentosidine, and pyrraline adducts on RyR2 and SERCA2 and evoked intracellular transient Ca(2+) kinetics in myocytes from control, diabetic, and treated-diabetic rats. MDA and 4-HNE adducts were not detected on RyR2 and SERCA2 from either control or 8 weeks diabetic rats with altered evoked Ca(2+) transients. However, CML, pentosidine, and pyrraline adducts were elevated three- to five-fold (p < 0.05). Treating diabetic rats with pyridoxamine (a scavenger of reactive carbonyl species, RCS) or aminoguanidine (a mixed reactive oxygen species-RCS scavenger) reduced CML, pentosidine, and pyrraline adducts on RyR2 and SERCA2 and blunted SR Ca(2+) cycling changes. Treating diabetic rats with the superoxide dismutase mimetic tempol had no impact on MDA and 4-HNE adducts on RyR2 and SERCA2, and on SR Ca(2+) cycling. From these data we conclude that lipid-derived MDA and 4-HNE adducts are not formed on RyR2 and SERCA2 in this model of diabetes, and are therefore unlikely to be directly contributing to the SR Ca(2+) dysregulation.


Subject(s)
Aldehydes/metabolism , Diabetes Mellitus, Experimental/metabolism , Malondialdehyde/metabolism , Myocytes, Cardiac/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Aldehydes/chemistry , Animals , Arginine/analogs & derivatives , Arginine/chemistry , Arginine/metabolism , Calcium/metabolism , Cyclic N-Oxides/pharmacology , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/physiopathology , Diabetic Cardiomyopathies/metabolism , Echocardiography/methods , Guanidines/pharmacology , Lysine/analogs & derivatives , Lysine/chemistry , Lysine/metabolism , Male , Malondialdehyde/chemistry , Myocytes, Cardiac/drug effects , Norleucine/analogs & derivatives , Norleucine/chemistry , Norleucine/metabolism , Protein Carbonylation , Pyridoxamine/pharmacology , Pyrroles/chemistry , Pyrroles/metabolism , Rats , Rats, Sprague-Dawley , Ryanodine Receptor Calcium Release Channel/chemistry , Sarcoplasmic Reticulum/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/chemistry , Spin Labels
3.
J Appl Physiol (1985) ; 114(5): 665-74, 2013 Mar 01.
Article in English | MEDLINE | ID: mdl-23288552

ABSTRACT

Individuals working in commercial hog confinement facilities have elevated incidences of headaches, depression, nausea, skeletal muscle weakness, fatigue, gastrointestinal disorders, and cardiovascular diseases, and the molecular mechanisms for these nonrespiratory ailments remain incompletely undefined. A common element underlying these diverse pathophysiologies is perturbation of intracellular Ca(2+) homeostasis. This study assessed whether the dust generated inside hog confinement facilities contains compounds that alter Ca(2+) mobilization via ryanodine receptors (RyRs), key intracellular channels responsible for mobilizing Ca(2+) from internal stores to elicit an array of physiologic functions. Hog barn dust (HBD) was extracted with phosphate-buffered saline, sterile-filtered (0.22 µm), and size-separated using Sephadex G-100 resin. Fractions (F) 1 through 9 (Mw >10,000 Da) had no measurable effects on RyR isoforms. However, F10 through F17, which contained compounds of Mw ≤2,000 Da, modulated the [(3)H]ryanodine binding to RyR1, RyR2, and RyR3 in a biphasic (Gaussian) manner. The Ki values for F13, the most potent fraction, were 3.8 ± 0.2 µg/ml for RyR1, 0.2 ± 0.01 µg/ml and 19.1 ± 2.8 µg/ml for RyR2 (two binding sites), and 44.9 ± 2.8 µg/ml and 501.6 ± 9.2 µg/ml for RyR3 (two binding sites). In lipid bilayer assays, F13 dose-dependently decreased the open probabilities of RyR1, RyR2, and RyR3. Pretreating differentiated mouse skeletal myotubes (C2C12 cells) with F13 blunted the amplitudes of ryanodine- and K(+)-induced Ca(2+) transients. Because RyRs are present in many cell types, impairment in Ca(2+) mobilization from internal stores via these channels is a possible mechanism by which HBD may trigger these seemingly unrelated pathophysiologies.


Subject(s)
Air Pollutants/metabolism , Calcium Channel Blockers/metabolism , Calcium/metabolism , Dust , Endoplasmic Reticulum/metabolism , Housing, Animal , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum/metabolism , Animals , Binding Sites , Male , Mice , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/metabolism , Potassium/metabolism , Rabbits , Rats , Rats, Sprague-Dawley , Swine
4.
Mol Pharmacol ; 82(3): 383-99, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22648972

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 Ca(2+)-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 [(3)H]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 Ca(2+) release in ventricular myocytes increased ~5-fold. Evoked Ca(2+) release from the SR was nonuniform (dyssynchronous). Total RyR2 protein levels remained unchanged, but the ability to bind the Ca(2+)-dependent ligand [(3)H]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 Ca(2+) responsiveness. The prototype RCS methylglyoxal increased and then decreased the RyR2 open probability. Methylglyoxal also increased spontaneous Ca(2+) release and induced Ca(2+) waves in healthy myocytes. Treatment of diabetic rats with RCS scavengers normalized spontaneous and evoked Ca(2+) release from the SR, reduced carbonylation of RyR2s, and increased binding of [(3)H]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.


Subject(s)
Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 1/metabolism , Myocytes, Cardiac/physiology , Protein Carbonylation/physiology , Ryanodine Receptor Calcium Release Channel/metabolism , Animals , Arrhythmias, Cardiac/genetics , Arrhythmias, Cardiac/metabolism , Arrhythmias, Cardiac/physiopathology , Calcium/metabolism , Calcium Channels/genetics , Calcium Channels/metabolism , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Type 1/genetics , Echocardiography/methods , HEK293 Cells , Heart Failure/genetics , Heart Failure/metabolism , Heart Failure/physiopathology , Humans , Mitochondria/genetics , Mitochondria/metabolism , Mitochondria/physiology , Myocytes, Cardiac/metabolism , Protein Processing, Post-Translational/genetics , Protein Processing, Post-Translational/physiology , Rats , Reactive Oxygen Species/metabolism , Ryanodine Receptor Calcium Release Channel/genetics , Sarcoplasmic Reticulum/genetics , Sarcoplasmic Reticulum/metabolism , Superoxides/metabolism
5.
Cardiovasc Res ; 91(2): 300-9, 2011 Jul 15.
Article in English | MEDLINE | ID: mdl-21421556

ABSTRACT

AIMS: Ventricular myocytes isolated from hearts of streptozotocin (STZ)-diabetic rats exhibit increased spontaneous Ca(2+) release. Studies attribute this defect to an enhancement in activity of type 2 ryanodine receptor (RyR2). To date, underlying reasons for RyR2 dysregulation remain undefined. This study assesses whether the responsiveness of RyR2 following stimulation by intrinsic ligands is being altered during experimental type 1 diabetes (T1D). METHODS AND RESULTS: M-mode echocardiography established a cardiomyopathy in 8 weeks STZ-diabetic rats. Confocal microscopy confirmed an increase in the spontaneous Ca(2+) release in isolated ventricular myocytes. Western blots revealed no significant change in steady-state levels of the RyR2 protein. When purified to homogeneity and incorporated into planar lipid bilayers, RyR2 from STZ-diabetic rats (dRyR2) exhibited reduced current amplitude at ±35 mV. dRyR2 was also more responsive to intrinsic cytoplasmic activators Ca(2+), adenosine triphosphate, and cyclic adenosine diphosphate ribose and less responsive to the cytoplasmic deactivator Mg(2+). Threshold for the activation of RyR2 by trans (luminal) Ca(2+) was also reduced. These changes were independent of phosphorylation at Ser2808 and Ser2814. Two weeks of insulin treatment starting after 6 weeks of diabetes blunted the phenotype change, indicating that the gain of function is specific to the diabetes and not the result of STZ interacting directly with RyR2. CONCLUSION: These data show, for the first time, that RyR2 is acquiring a gain-of-function phenotype independent of its phosphorylation status during T1D and provides new insights for the enhanced spontaneous Ca(2+) release in myocytes from T1D rats.


Subject(s)
Calcium Signaling , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 1/metabolism , Diabetic Cardiomyopathies/metabolism , Myocytes, Cardiac/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Adenosine Triphosphate/metabolism , Analysis of Variance , Animals , Blotting, Western , Cyclic ADP-Ribose/analogs & derivatives , Cyclic ADP-Ribose/metabolism , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/diagnostic imaging , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Type 1/complications , Diabetes Mellitus, Type 1/diagnostic imaging , Diabetes Mellitus, Type 1/drug therapy , Diabetic Cardiomyopathies/diagnostic imaging , Diabetic Cardiomyopathies/drug therapy , Diabetic Cardiomyopathies/etiology , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Ligands , Magnesium/metabolism , Membrane Potentials , Microscopy, Confocal , Phenotype , Phosphorylation , Rats , Ryanodine/metabolism , Ultrasonography
6.
Int J Environ Res Public Health ; 2(1): 51-7, 2005 Apr.
Article in English | MEDLINE | ID: mdl-16705801

ABSTRACT

Ultraviolet (UV)-induced cataracts are becoming a major environmental health concern because of the possible decrease in the stratospheric ozone layer. Experiments were designed to isolate gene(s) affected by UV irradiation in rabbit cornea tissues using fluorescent differential display-reverse transcription-polymerase chain reaction (FDDRT-PCR). The epithelial cells were grown in standard medium for 2 or 4 hours post treatment. Cornea epithelial cells were irradiated with UVB for 20 minutes. RNA was extracted and amplified by reverse transcriptase-polymerase chain reaction using poly A+ specific anchoring primers and random arbitrary primers. Polyacrylamide gel electrophoresis revealed several differentially expressed genes in untreated versus UV irradiated cells. Complimentary DNA (cDNA) fragments resulting from fluorescent differentially expressed mRNAs were eluted from the gel and re-amplified. The re-amplified PCR products were cloned directly into the PCR-TRAP cloning system. These data showed that FDDRT-PCR is a useful technique to elucidate UV-regulated gene expressions. Future experiments will involve sequence analysis of cloned inserts. The identification of these genes through sequence analysis could lead to a better understanding of cataract formation via DNA damage and mechanisms of prevention.


Subject(s)
Epithelial Cells/radiation effects , Epithelium, Corneal/radiation effects , Ultraviolet Rays , Animals , Cataract , Cells, Cultured , DNA Damage , Epithelial Cells/metabolism , Epithelium, Corneal/metabolism , Gene Expression Regulation/radiation effects , RNA, Messenger/metabolism , Rabbits , Reverse Transcriptase Polymerase Chain Reaction/methods
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