Molecular characterization of Clostridioides difficile ribotype 027 in a major Chinese hospital.

BACKGROUND: The rapid spread of C. difficile 027 has become one of the leading threats of healthcare-associated infections wordwild. However, C. difficile 027 infections have rarely been reported in China. The objective of this study was to strengthen the understanding of the molecular characterizations of C. difficile 027 in China. METHODS: In this study, stool specimens from 176 suspected CDI cases were collected from 1 Jan 2018 to 30 Jun 2019. These specimens were measured by GeneXpert test and C.difficile colonies were identified and analyzed. RESULTS: There were five samples positive for tcdA, tcdB, binary toxin genes and had deletions in tcdC gene. These five Clostridioides difficile isolates belonged to ST1 and confirmed as Clostridioides difficile 027 strains by PCR ribotyping. Through using whole genome sequencing, , we found that these five strains were closely clustered into the same predominant evolutionary branch and were highly similar to C. difficile 027 strain R20291. Antimicrobial susceptibility testing result showed they were highly resistant to fluoroquinolones. CONCLUSIONS: In Our study, five C. difficile 027 isolates were identified and characterized using MLST, PCR ribotyping and whole genome sequencing. We proposed that C. difficile 027 infections are probably neglected in China. Further epidemiological studies across the country together with the introduction of routine diagnostic testing and multi-center or national level surveillance are needed to ascertain the size of this potentially significant problem.

Smooth muscle-generated methylglyoxal impairs endothelial cell-mediated vasodilatation of cerebral microvessels in type 1 diabetic rats.

BACKGROUND AND PURPOSE: Endothelial cell-mediated vasodilatation of cerebral arterioles is impaired in individuals with Type 1 diabetes (T1D). This defect compromises haemodynamics and can lead to hypoxia, microbleeds, inflammation and exaggerated ischaemia-reperfusion injuries. The molecular causes for dysregulation of cerebral microvascular endothelial cells (cECs) in T1D remains poorly defined. This study tests the hypothesis that cECs dysregulation in T1D is triggered by increased generation of the mitochondrial toxin, methylglyoxal, by smooth muscle cells in cerebral arterioles (cSMCs). EXPERIMENTAL APPROACH: Endothelial cell-mediated vasodilatation, vascular transcytosis inflammation, hypoxia and ischaemia-reperfusion injury were assessed in brains of male Sprague-Dawley rats with streptozotocin-induced diabetes and compared with those in diabetic rats with increased expression of methylglyoxal-degrading enzyme glyoxalase-I (Glo-I) in cSMCs. KEY RESULTS: After 7-8 weeks of T1D, endothelial cell-mediated vasodilatation of cerebral arterioles was impaired. Microvascular leakage, gliosis, macrophage/neutrophil infiltration, NF-κB activity and TNF-α levels were increased, and density of perfused microvessels was reduced. Transient occlusion of a mid-cerebral artery exacerbated ischaemia-reperfusion injury. In cSMCs, Glo-I protein was decreased, and the methylglyoxal-synthesizing enzyme, vascular adhesion protein 1 (VAP-1) and methylglyoxal were increased. Restoring Glo-I protein in cSMCs of diabetic rats to control levels via gene transfer, blunted VAP-1 and methylglyoxal increases, cECs dysfunction, microvascular leakage, inflammation, ischaemia-reperfusion injury and increased microvessel perfusion. CONCLUSIONS AND IMPLICATIONS: Methylglyoxal generated by cSMCs induced cECs dysfunction, inflammation, hypoxia and exaggerated ischaemia-reperfusion injury in diabetic rats. Lowering methylglyoxal produced by cSMCs may be a viable therapeutic strategy to preserve cECs function and blunt deleterious downstream consequences in T1D.

CCDI: a new ligand that modulates mammalian type 1 ryanodine receptor (RyR1).

BACKGROUND AND PURPOSE: Ryanodine receptors (RyRs) are Ca(2+)-release channels on the sarco(endo)plasmic reticulum that modulate a wide array of physiological functions. Three RyR isoforms are present in cells: RyR1, RyR2 and RyR3. To date, there are no reports on ligands that modulate RyR in an isoform-selective manner. Such ligands are not only valuable research tools, but could serve as intermediates for development of therapeutics. EXPERIMENTAL APPROACH: Pyrrole-2-carboxylic acid and 1,3-dicyclohexylcarbodiimide were allowed to react in carbon tetrachloride for 24 h at low temperatures and pressures. The chemical structures of the two products isolated were elucidated using NMR spectrometry, mass spectrometry and elemental analyses. [(3) H]-ryanodine binding, lipid bilayer and time-lapsed confocal imaging were used to determine their effects on RyR isoforms. KEY RESULTS: The major product, 2-cyclohexyl-3-cyclohexylimino-2, 3, dihydro-pyrrolo[1,2-c]imidazol-1-one (CCDI) dose-dependently potentiated Ca(2+)-dependent binding of [(3)H]-ryanodine to RyR1, with no significant effects on [(3)H]-ryanodine binding to RyR2 or RyR3. CCDI also reversibly increased the open probability (P(o)) of RyR1 with minimal effects on RyR2 and RyR3. CCDI induced Ca(2+) transients in C2C12 skeletal myotubes, but not in rat ventricular myocytes. This effect was blocked by pretreating cells with ryanodine. The minor product 2-cyclohexyl-pyrrolo[1,2-c]imidazole-1,3-dione had no effect on either [(3)H]-ryanodine binding or P(o) of RyR1, RyR2 and RyR3. CONCLUSIONS AND IMPLICATIONS: A new ligand that preferentially modulates RyR1 was identified. In addition to being an important research tool, the pharmacophore of this small molecule could serve as a template for the synthesis of other isoform-selective modulators of RyRs.

Reactive carbonyl species and their roles in sarcoplasmic reticulum Ca2+ cycling defect in the diabetic heart.

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.

Malondialdehyde and 4-hydroxynonenal adducts are not formed on cardiac ryanodine receptor (RyR2) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) in diabetes.

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.

Dust from hog confinement facilities impairs Ca2+ mobilization from sarco(endo)plasmic reticulum by inhibiting ryanodine receptors.

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.

Carbonylation induces heterogeneity in cardiac ryanodine receptor function in diabetes mellitus.

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.

Gain of function of cardiac ryanodine receptor in a rat model of type 1 diabetes.

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.

Carbonylation contributes to SERCA2a activity loss and diastolic dysfunction in a rat model of type 1 diabetes.

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 Ca(2+)-ATPase (SERCA2a), the ATP-driven pump that translocates Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 MGO's 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.

Chloroform extract of hog barn dust modulates skeletal muscle ryanodine receptor calcium-release channel (RyR1).

Skeletal muscle weakness is a reported ailment in individuals working in commercial hog confinement facilities. To date, specific mechanisms responsible for this symptom remain undefined. The purpose of this study was to assess whether hog barn dust (HBD) contains components that are capable of binding to and modulating the activity of type 1 ryanodine receptor Ca2+-release channel (RyR1), a key regulator of skeletal muscle function. HBD collected from confinement facilities in Nebraska were extracted with chloroform, filtered, and rotary evaporated to dryness. Residues were resuspended in hexane-chloroform (20:1) and precipitates, referred to as HBDorg, were air-dried and studied further. In competition assays, HBDorg dose-dependently displaced [3H]ryanodine from binding sites on RyR1 with an IC50 of 1.5±0.1 microg/ml (Ki=0.4±0.0 microg/ml). In single-channel assays using RyR1 reconstituted into a lipid bilayer, HBDorg exhibited three distinct dose-dependent effects: first it increased the open probability of RyR1 by increasing its gating frequency and dwell time in the open state, then it induced a state of reduced conductance (55% of maximum) that was more likely to occur and persist at positive holding potentials, and finally it irreversibly closed RyR1. In differentiated C2C12 myotubes, addition of HBD triggered a rise in intracellular Ca2+ that was blocked by pretreatment with ryanodine. Since persistent activation and/or closure of RyR1 results in skeletal muscle weakness, these new data suggest that HBD is responsible, at least in part, for the muscle ailment reported by hog confinement workers.

Carbonylation of myosin heavy chains in rat heart during diabetes.

Cardiac inotropy progressively declines during diabetes mellitus. To date, the molecular mechanisms underlying this defect remain incompletely characterized. This study tests the hypothesis that ventricular myosin heavy chains (MHC) undergo carbonylation by reactive carbonyl species (RCS) during diabetes and these modifications contribute to the inotropic decline. Male Sprague-Dawley rats were injected with streptozotocin (STZ). Fourteen days later the animals were divided into two groups: one group was treated with the RCS blocker aminoguanidine for 6 weeks, while the other group received no treatment. After 8 weeks of diabetes, cardiac ejection fraction, fractional shortening, left ventricular pressure development (+dP/dt) and myocyte shortening were decreased by 9%, 16%, 34% and 18%, respectively. Ca(2+)- and Mg(2+)-actomyosin ATPase activities and peak actomyosin syneresis were also reduced by 35%, 28%, and 72%. MHC-alpha to MHC-beta ratio was 12:88. Mass spectrometry and Western blots revealed the presence of carbonyl adducts on MHC-alpha and MHC-beta. Aminoguanidine treatment did not alter MHC composition, but it blunted formation of carbonyl adducts and decreases in actomyosin Ca(2+)-sensitive ATPase activity, syneresis, myocyte shortening, cardiac ejection fraction, fractional shortening and +dP/dt induced by diabetes. From these new data it can be concluded that in addition to isozyme switching, modification of MHC by RCS also contributes to the inotropic decline seen during diabetes.

Exercise training during diabetes attenuates cardiac ryanodine receptor dysregulation.

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 Ca(2+) spark frequency increased threefold, and evoked Ca(2+) release was dyssynchronous with diastolic Ca(2+) releases. Steady-state type 2 ryanodine receptor (RyR2) protein did not change, but its response to Ca(2+) was altered. RyR2 also exhibited 1.8- and 1.5-fold increases in phosphorylation at Ser(2808) and Ser(2814). 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 Ca(2+) spark frequency, and minimized dyssynchronous and diastolic Ca(2+) releases. ExT also normalized the responsiveness of RyR2 to Ca(2+) activation, attenuated increases in RyR2 phosphorylation at Ser(2808) and Ser(2814), and normalized CaMKII and PKA activities. These data are the first to show that ExT during diabetes normalizes RyR2 function and Ca(2+) release from the sarcoplasmic reticulum, providing insights into mechanisms by which ExT during diabetes improves cardiac function.

Exercise training initiated after the onset of diabetes preserves myocardial function: effects on expression of beta-adrenoceptors.

The present study was undertaken to assess cardiac function and characterize beta-adrenoceptor subtypes in hearts of diabetic rats that underwent exercise training (ExT) after the onset of diabetes. Type 1 diabetes was induced in male Sprague-Dawley rats using streptozotocin. Four weeks after induction, rats were randomly divided into two groups. One group was exercised trained for 3 wk while the other group remained sedentary. At the end of the protocol, cardiac parameters were assessed using M-mode echocardiography. A Millar catheter was also used to assess left ventricular hemodynamics with and without isoproterenol stimulation. beta-Adrenoceptors were assessed using Western blots and [(3)H]dihydroalprenolol binding. After 7 wk of diabetes, heart rate decreased by 21%, fractional shortening by 20%, ejection fraction by 9%, and basal and isoproterenol-induced dP/dt by 35%. beta(1)- and beta(2)-adrenoceptor proteins were reduced by 60% and 40%, respectively, while beta(3)-adrenoceptor protein increased by 125%. Ventricular homogenates from diabetic rats bound 52% less [(3)H]dihydroalprenolol, consistent with reductions in beta(1)- and beta(2)-adrenoceptors. Three weeks of ExT initiated 4 wk after the onset of diabetes minimized cardiac function loss. ExT also blunted loss of beta(1)-adrenoceptor expression. Interestingly, ExT did not prevent diabetes-induced reduction in beta(2)-adrenoceptor or the increase of beta(3)-adrenoceptor expression. ExT also increased [(3)H]dihydroalprenolol binding, consistent with increased beta(1)-adrenoceptor expression. These findings demonstrate for the first time that ExT initiated after the onset of diabetes blunts primarily beta(1)-adrenoceptor expression loss, providing mechanistic insights for exercise-induced improvements in cardiac function.

Dyssynchronous (non-uniform) Ca2+ release in myocytes from streptozotocin-induced diabetic rats.

Using biochemical/pharmacological approaches, we previously showed that type 2 ryanodine receptors (RyR2) become dysfunctional in hearts of streptozotocin-induced type 1 diabetic rats. However, the functional consequence of this observation remains incompletely understood. Here we use laser confocal microscopy to investigate whether RyR2 dysfunction during diabetes alters evoked and spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR). After 7-8 weeks of diabetes, steady-state levels of RyR2 remain unchanged in hearts of male Sprague-Dawley rats, but the number of functional receptors decreased by >37%. Interestingly, residual functional RyR2 from diabetic rat hearts exhibited increased sensitivity to Ca(2+) activation (EC(50activation) decreased from 80 microM to 40 microM, peak Ca(2+) activation decreased from 425 microM to 160 microM). When field stimulated, intracellular Ca(2+) release in diabetic ventricular myocytes was dyssynchronous (non-uniform) and this was independent of L-type Ca(2+) currents. Time to peak Ca(2+) increased 3.7-fold. Diabetic myocytes also exhibited diastolic Ca(2+) release and 2-fold higher frequency of spontaneous Ca(2+) sparks, albeit at a lower amplitude. The amplitude of caffeine-releasable Ca(2+) was also lower in diabetic myocytes. RyR2 from diabetic rat hearts exhibited increased phosphorylation at Ser2809 and contained reduced levels of FKBP12.6 (calstablin2). Collectively, these data suggest that RyR2 becomes leaky during diabetes and this defect may be responsible to the reduced SR Ca(2+) load. Diastolic Ca(2+) release could also serve as a substrate for delayed after-depolarizations, contributing to the increased incidence of arrhythmias and sudden cardiac death in type 1 diabetes.

Insulin regulation of glutathione and contractile phenotype in diabetic rat ventricular myocytes.

Cardiovascular complications of diabetes mellitus involve oxidative stress and profound changes in reduced glutathione (GSH), an essential tripeptide that controls many redox-sensitive cell functions. This study examined regulation of GSH by insulin to identify mechanisms controlling cardiac redox state and to define the functional impact of GSH depletion. GSH was measured by fluorescence microscopy in ventricular myocytes isolated from Sprague-Dawley rats made diabetic by streptozotocin, and video and confocal microscopy were used to measure mechanical properties and Ca(2+) transients, respectively. Spectrophotometric assays of tissue extracts were also done to measure the activities of enzymes that control GSH levels. Four weeks after injection of streptozotocin, mean GSH concentration ([GSH]) in isolated diabetic rat myocytes was approximately 36% less than in control, correlating with decreased activities of two major enzymes regulating GSH levels: glutathione reductase and gamma-glutamylcysteine synthetase. Treatment of diabetic rat myocytes with insulin normalized [GSH] after a delay of 3-4 h. A more rapid but transient upregulation of [GSH] occurred in myocytes treated with dichloroacetate, an activator of pyruvate dehydrogenase. Inhibitor experiments indicated that insulin normalized [GSH] via the pentose pathway and gamma-glutamylcysteine synthetase, although the basal activity of glucose-6-phosphate dehydrogenase was not different between diabetic and control hearts. Diabetic rat myocytes were characterized by significant mechanical dysfunction that correlated with diminished and prolonged Ca(2+) transients. This phenotype was reversed by in vitro treatment with insulin and also by exogenous GSH or N-acetylcysteine, a precursor of GSH. Our data suggest that insulin regulates GSH through pathways involving de novo GSH synthesis and reduction of its oxidized form. It is proposed that a key function of glucose metabolism in heart is to supply reducing equivalents required to maintain adequate GSH levels for the redox control of Ca(2+) handling proteins and contraction.

Dysfunction of cardiac ryanodine receptors in the metabolic syndrome.

This study examined the hypothesis that the prediabetic metabolic syndrome alters expression, phosphorylation state and binding affinity of cardiac RyR2. Real-time PCR and Western blot analysis were used to assess mRNA and protein expression in the left ventricle, right ventricle and right atrium from control (n=5) and chronically high-fat-fed (n=5) dogs with the metabolic syndrome. Functional integrity of RyR2 was assessed by RyR2-Ser2809 phosphorylation and the receptor's ability to bind [3H]ryanodine. We found that RyR2 phosphorylation at Ser2809 was significantly elevated in right and left ventricle from high-fat-fed dogs compared to normal control dogs. This hyperphosphorylation was associated with a decrease in RyR2 binding affinity in right and left ventricle (high-fat diet=80.2 and 90.5 fmol/mg protein vs. control=243.6 and 200.9 fmol/mg protein, respectively) and a decrease in cardiac index in exercising dogs. RyR2 phosphorylation at Ser2809 and RyR2 binding affinity were not altered in the right atria of high-fat-fed dogs. In addition, no significant differences in cardiac RyR2 mRNA or protein expression were noted between groups. These data suggest that alterations in RyR2 could be an important mechanism of early cardiac dysfunction in obesity and insulin resistance.

Simultaneous phosphorus and nitrogen removal in a continuous-flow two-sludge system.

The ability of simultaneous biological phosphorus and nitrogen removal was investigated in a lab-scale continuous-flow two-sludge system. Alternating anaerobic and anoxic conditions were combined with contact oxidation stage for treating raw municipal wastewater. Long-term experiments showed that the contradiction of competing for the organic substrate between denitrifying bacteria and PAOs (phosphorus accumulating organisms) in traditional phosphorus and nitrogen removal system has been resolved. The system can adapt to low influent COD/TN ratio (C/N). Furthermore the SRT (sludge retention time) of nitrifying sludge and denitrifying phosphorus removal sludge can be controlled at optimal conditions respectively. The removal efficiency of COD, TP, TN, and NH4-N was 81.78%, 92.51%,75.75%, and 84.47% respectively. It was also found that the appropriate influent C/N should be controlled at the range of 3.8-6, while the optimal C/N to the system ranged between 4-5, and the BFR (bypass sludge flow rate) should be controlled at 0.35 around.

Diabetes increases formation of advanced glycation end products on Sarco(endo)plasmic reticulum Ca2+-ATPase.

Prolongation of relaxation is a hallmark of diabetic cardiomyopathy. Most studies attribute this defect to decreases in sarco(endo)plasmic reticulum Ca(2+)-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.