Impact of telemedicine on glycemic control in type 2 diabetes mellitus during the COVID-19 lockdown period.

Background: The lockdown at the start of coronavirus disease 2019 (COVID-19) pandemic in Saudi Arabia (March 2020 to June 2020) shifted routine in-person care for patients with type 2 diabetes mellitus (T2DM) to telemedicine. The aim of this study was to investigate the impact telemedicine had during this period on glycemic control (HbA1c) in patients with T2DM. Methods: 4,266 patients with T2DM were screened from five Ministry of National Guard Health Affairs hospitals in the Kingdom of Saudi Arabia. Age, gender, body mass index (BMI), HbA1c (before and after the COVID-19 lockdown), duration of T2DM, comorbidities and antidiabetic medications data were obtained. Mean and standard deviation of differences in HbA1c were calculated to assess the impact of telemedicine intervention. Correlations between clinically significant variances (when change in the level is ≥0.5%) in HbA1c with demographics and clinical characteristic data were determined using chi square test. Results: Most of the participants were Saudis (97.7%) with 59.7% female and 56.4% ≥60 years of age. Obesity was 63.8%, dyslipidemia 91%, and hypertension 70%. Mean HbA1c of all patients slightly rose from 8.52% ± 1.5% before lockdown to 8.68% ± 1.6% after lockdown. There were n=1,064 patients (24.9%) whose HbA1c decreased by ≥0.5%, n =1,574 patients whose HbA1c increased by ≥0.5% (36.9%), and n =1,628 patients whose HbA1c changed by <0.5% in either direction (38.2%). More males had significant improvements in glycemia compared to females (28.1% vs 22.8%, p<0.0001), as were individuals below the age of 60 years (28.1% vs 22.5%, p<0.0001). Hypertensive individuals were less likely than non-hypertensive to have glycemic improvement (23.7% vs 27.9%, p=0.015). More patients on sulfonylureas had improvements in HbA1c (42.3% vs 37.9%, p=0.032), whereas patients on insulin had higher HbA1c (62.7% vs 56.2%, p=0.001). HbA1c changes were independent of BMI, duration of disease, hyperlipidemia, heart and kidney diseases. Conclusion: Telemedicine was helpful in delivering care to T2DM patients during COVID-19 lockdown, with 63.1% of patients maintaining HbA1c and improving glycemia. More males than females showed improvements. However, the HbA1c levels in this cohort of patients pre- and post-lockdown were unsatisfactorily high, and may be due to in part lifestyle, age, education, and hypertension.

Elevated plasma level of the glycolysis byproduct methylglyoxal on admission is an independent biomarker of mortality in ICU COVID-19 patients.

Biomarkers to identify ICU COVID-19 patients at high risk for mortality are urgently needed for therapeutic care and management. Here we found plasma levels of the glycolysis byproduct methylglyoxal (MG) were 4.4-fold higher in ICU patients upon admission that later died (n = 33), and 1.7-fold higher in ICU patients that survived (n = 32),compared to uninfected controls (n = 30). The increased MG in patients that died correlated inversely with the levels of the MG-degrading enzyme glyoxalase-1 (r2 = - 0.50), and its co-factor glutathione (r2 = - 0.63), and positively with monocytes (r2 = 0.29). The inflammation markers, SSAO (r2 = 0.52), TNF-α (r2 = 0.41), IL-1ß (r2 = 0.25), CRP (r2 = 0.26) also correlated positively with MG. Logistic regression analysis provides evidence of a significant relationship between the elevated MG upon admission into ICU and death (P < 0.0001), with 42% of the death variability explained. From these data we conclude that elevated plasma MG on admission is a novel independent biomarker that predicts mortality in ICU COVID-19 patients.

HIV-Tat Exacerbates the Actions of Atazanavir, Efavirenz, and Ritonavir on Cardiac Ryanodine Receptor (RyR2).

The incidence of sudden cardiac death (SCD) in people living with HIV infection (PLWH), especially those with inadequate viral suppression, is high and the reasons for this remain incompletely characterized. The timely opening and closing of type 2 ryanodine receptor (RyR2) is critical for ensuring rhythmic cardiac contraction-relaxation cycles, and the disruption of these processes can elicit Ca2+ waves, ventricular arrhythmias, and SCD. Herein, we show that the HIV protein Tat (HIV-Tat: 0-52 ng/mL) and therapeutic levels of the antiretroviral drugs atazanavir (ATV: 0-25,344 ng/mL), efavirenz (EFV: 0-11,376 ng/mL), and ritonavir (RTV: 0-25,956 ng/mL) bind to and modulate the opening and closing of RyR2. Abacavir (0-14,315 ng/mL), bictegravir (0-22,469 ng/mL), Rilpivirine (0-14,360 ng/mL), and tenofovir disoproxil fumarate (0-18,321 ng/mL) did not alter [3H]ryanodine binding to RyR2. Pretreating RyR2 with low HIV-Tat (14 ng/mL) potentiated the abilities of ATV and RTV to bind to open RyR2 and enhanced their ability to bind to EFV to close RyR2. In silico molecular docking using a Schrodinger Prime protein-protein docking algorithm identified three thermodynamically favored interacting sites for HIV-Tat on RyR2. The most favored site resides between amino acids (AA) 1702-1963; the second favored site resides between AA 467-1465, and the third site resides between AA 201-1816. Collectively, these new data show that HIV-Tat, ATV, EFV, and RTV can bind to and modulate the activity of RyR2 and that HIV-Tat can exacerbate the actions of ATV, EFV, and RTV on RyR2. Whether the modulation of RyR2 by these agents increases the risk of arrhythmias and SCD remains to be explored.

A Link Between Methylglyoxal and Heart Failure During HIV-1 Infection.

Early-onset heart failure (HF) continues to be a major cause of morbidity and mortality in people living with human immunodeficiency virus type one (HIV-1) infection (PLWH), yet the molecular causes for this remain poorly understood. Herein NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ humanized mice (Hu-mice), plasma from PLWH, and autopsied cardiac tissues from deceased HIV seropositive individuals were used to assess if there is a link between the glycolysis byproduct methylglyoxal (MG) and HF in the setting of HIV-1 infection. At five weeks post HIV infection, Hu-mice developed grade III-IV diastolic dysfunction (DD) with an associated two-fold increase in plasma MG. At sixteen-seventeen weeks post infection, cardiac ejection fraction and fractional shortening also declined by 26 and 35%, and plasma MG increased to four-fold higher than uninfected controls. Histopathological and biochemical analyses of cardiac tissues from Hu-mice 17 weeks post-infection affirmed MG increase with a concomitant decrease in expression of the MG-degrading enzyme glyoxalase-1 (Glo1). The endothelial cell marker CD31 was found to be lower, and coronary microvascular leakage and myocardial fibrosis were prominent. Increasing expression of Glo1 in Hu-mice five weeks post-infection using a single dose of an engineered AAV2/9 (1.7 × 1012 virion particles/kg), attenuated the increases in plasma and cardiac MG levels. Increasing Glo1 also blunted microvascular leakage, fibrosis, and HF seen at sixteen weeks post-infection, without changes in plasma viral loads. In plasma from virally suppressed PLWH, MG was also 3.7-fold higher. In autopsied cardiac tissues from seropositive, HIV individuals with low viral log, MG was 4.2-fold higher and Glo1 was 50% lower compared to uninfected controls. These data show for the first time a causal link between accumulation of MG and HF in the setting of HIV infection.

Efavirenz, atazanavir, and ritonavir disrupt sarcoplasmic reticulum Ca2+ homeostasis in skeletal muscles.

While muscle fatigue, pain and weakness are common co-morbidities in HIV-1 infected people, their underlying cause remain poorly defined. To this end, we evaluated whether the common antiretroviral drugs efavirenz (EFV), atazanavir (ATV) and ritonavir (RTV) could be a contributing factor by pertubating sarcoplasmic reticulum (SR) Ca2+ cycling. In live-cell imaging, EFV (6.0 µM), ATV (6.0 µM), and RTV (3.0 µM) elicited Ca2+ transients and blebbing of the plasma membranes of C2C12 skeletal muscle myotubes. Pretreating C2C12 skeletal muscle myotubes with the SR Ca2+ release channel blocker ryanodine (50 µM), slowed the rate and amplitude of Ca2+ release from and reuptake of Ca2+ into the SR. EFV, ATV and RTV (1 nM - 20 µM) potentiated and then displaced [3H] ryanodine binding to rabbit skeletal muscle ryanodine receptor Ca2+ release channel (RyR1). These drugs at concentrations 0.25-31.2 µM also increased and or decreased the open probability of RyR1 by altering its gating and conductance. ATV (≤5 µM) potentiated and >5µM inhibited the ability of sarco (endo)plasmic reticulum Ca2+-ATPase (SERCA1) to hydrolyze ATP and transport Ca2+. RTV (2.5-31.5 µM) dose-dependently inhibited SERCA1-mediated, ATP-dependent Ca2+ transport. EFV (0.25-31.5 µM) had no measurable effect on SERCA1's ability to hydrolyze ATP and transport Ca2+. These data support the notion that EFV, ATV and RTV could be contributing to skeletal muscle co-morbidities in PLWH by modulating SR Ca2+ homeostasis.

Adeno-Associated Viral Transfer of Glyoxalase-1 Blunts Carbonyl and Oxidative Stresses in Hearts of Type 1 Diabetic Rats.

Accumulation of methylglyoxal (MG) arising from downregulation of its primary degrading enzyme glyoxalase-1 (Glo1) is an underlying cause of diabetic cardiomyopathy (DC). This study investigated if expressing Glo1 in rat hearts shortly after the onset of Type 1 diabetes mellitus (T1DM) would blunt the development of DC employing the streptozotocin-induced T1DM rat model, an adeno-associated virus containing Glo1 driven by the endothelin-1 promoter (AAV2/9-Endo-Glo1), echocardiography, video edge, confocal imaging, and biochemical/histopathological assays. After eight weeks of T1DM, rats developed DC characterized by a decreased E:A ratio, fractional shortening, and ejection fraction, and increased isovolumetric relaxation time, E: e' ratio, and circumferential and longitudinal strains. Evoked Ca2+ transients and contractile kinetics were also impaired in ventricular myocytes. Hearts from eight weeks T1DM rats had lower Glo1 and GSH levels, elevated carbonyl/oxidative stress, microvascular leakage, inflammation, and fibrosis. A single injection of AAV2/9 Endo-Glo1 (1.7 × 1012 viron particles/kg) one week after onset of T1DM, potentiated GSH, and blunted MG accumulation, carbonyl/oxidative stress, microvascular leakage, inflammation, fibrosis, and impairments in cardiac and myocyte functions that develop after eight weeks of T1DM. These new data indicate that preventing Glo1 downregulation by administering AAV2/9-Endo-Glo1 to rats one week after the onset of T1DM, blunted the DC that develops after eight weeks of diabetes by attenuating carbonyl/oxidative stresses, microvascular leakage, inflammation, and fibrosis.

HIV-1-Associated Left Ventricular Cardiac Dysfunction in Humanized Mice.

The molecular cause(s) for early onset heart failure in people living with HIV-1 infection (PLWH) remains poorly defined. Herein, longitudinal echocardiography was used to assess whether NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice reconstituted with human hematopoietic stem cells (Hu-NSG mice) and infected with HIV-1ADA can recapitulate the salient features of this progressive human disease. Four weeks post infection, Hu-NSG mice of both sexes developed left ventricular (LV) diastolic dysfunction (DD), with 25% exhibiting grade III/IV restrictive DD with mitral regurgitation. Increases in global longitudinal and circumferential strains and declines in LV ejection fraction and fractional shortening were observed eight weeks post infection. After twelve weeks of infection, 33% of Hu-NSG mice exhibited LV dyskinesia and dyssynchrony. Histopathological analyses of hearts seventeen weeks post infection revealed coronary microvascular leakage, fibrosis and immune cell infiltration into the myocardium. These data show for the first time that HIV-1ADA-infected Hu-NSG mice can recapitulate key left ventricular cardiac deficits and pathophysiological changes reported in humans with progressive HIV-1 infection. The results also suggest that HIV-1 infected Hu-NSG mice may be a useful model to screen for pharmacological agents to blunt LV dysfunction and associated pathophysiologic causes reported in PLWH.

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.

Evidence of contribution of iPLA2ß-mediated events during islet ß-cell apoptosis due to proinflammatory cytokines suggests a role for iPLA2ß in T1D development.

Type 1 diabetes (T1D) results from autoimmune destruction of islet ß-cells, but the underlying mechanisms that contribute to this process are incompletely understood, especially the role of lipid signals generated by ß-cells. Proinflammatory cytokines induce ER stress in ß-cells and we previously found that the Ca(2+)-independent phospholipase A2ß (iPLA2ß) participates in ER stress-induced ß-cell apoptosis. In view of reports of elevated iPLA2ß in T1D, we examined if iPLA2ß participates in cytokine-mediated islet ß-cell apoptosis. We find that the proinflammatory cytokine combination IL-1ß+IFNγ, induces: a) ER stress, mSREBP-1, and iPLA2ß, b) lysophosphatidylcholine (LPC) generation, c) neutral sphingomyelinase-2 (NSMase2), d) ceramide accumulation, e) mitochondrial membrane decompensation, f) caspase-3 activation, and g) ß-cell apoptosis. The presence of a sterol regulatory element in the iPLA2ß gene raises the possibility that activation of SREBP-1 after proinflammatory cytokine exposure contributes to iPLA2ß induction. The IL-1ß+IFNγ-induced outcomes (b-g) are all inhibited by iPLA2ß inactivation, suggesting that iPLA2ß-derived lipid signals contribute to consequential islet ß-cell death. Consistent with this possibility, ER stress and ß-cell apoptosis induced by proinflammatory cytokines are exacerbated in islets from RIP-iPLA2ß-Tg mice and blunted in islets from iPLA2ß-KO mice. These observations suggest that iPLA2ß-mediated events participate in amplifying ß-cell apoptosis due to proinflammatory cytokines and also that iPLA2ß activation may have a reciprocal impact on ER stress development. They raise the possibility that iPLA2ß inhibition, leading to ameliorations in ER stress, apoptosis, and immune responses resulting from LPC-stimulated immune cell chemotaxis, may be beneficial in preserving ß-cell mass and delaying/preventing T1D evolution.

Selenite cataracts: activation of endoplasmic reticulum stress and loss of Nrf2/Keap1-dependent stress protection.

Cataract-induced by sodium selenite in suckling rats is one of the suitable animal models to study the basic mechanism of human cataract formation. The aim of this present investigation is to study the endoplasmic reticulum (ER) stress-mediated activation of unfolded protein response (UPR), overproduction of reactive oxygen species (ROS), and suppression of Nrf2/Keap1-dependent antioxidant protection through endoplasmic reticulum-associated degradation (ERAD) pathway and Keap1 promoter DNA demethylation in human lens epithelial cells (HLECs) treated with sodium selenite. Lenses enucleated from sodium selenite injected rats generated overproduction of ROS in lens epithelial cells and newly formed lens fiber cells resulting in massive lens epithelial cells death after 1-5days. All these lenses developed nuclear cataracts after 4-5days. Sodium selenite treated HLECs induced ER stress and activated the UPR leading to release of Ca(2+) from ER, ROS overproduction and finally HLECs death. Sodium selenite also activated the mRNA expressions of passive DNA demethylation pathway enzymes such as Dnmt1, Dnmt3a, and Dnmt3b, and active DNA demethylation pathway enzyme, Tet1 leading to DNA demethylation in the Keap1 promoter of HLECs. This demethylated Keap1 promoter results in overexpression of Keap1 mRNA and protein. Overexpression Keap1 protein suppresses the Nrf2 protein through ERAD leading to suppression of Nrf2/Keap1 dependent antioxidant protection in the HLECs treated with sodium selenite. As an outcome, the cellular redox status is altered towards lens oxidation and results in cataract formation.

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.

Methylglyoxal induces endoplasmic reticulum stress and DNA demethylation in the Keap1 promoter of human lens epithelial cells and age-related cataracts.

Age-related cataracts are a leading cause of blindness. Previously, we have demonstrated the association of the unfolded protein response with various cataractogenic stressors. However, DNA methylation alterations leading to suppression of lenticular antioxidant protection remains unclear. Here, we report the methylglyoxal-mediated sequential events responsible for Keap1 promoter DNA demethylation in human lens epithelial cells, because Keap1 is a negative regulatory protein that regulates the Nrf2 antioxidant protein. Methylglyoxal induces endoplasmic reticulum stress and activates the unfolded protein response leading to overproduction of reactive oxygen species before human lens epithelial cell death. Methylglyoxal also suppresses Nrf2 and DNA methyltransferases but activates the DNA demethylation pathway enzyme TET1. Bisulfite genomic DNA sequencing confirms the methylglyoxal-mediated Keap1 promoter DNA demethylation leading to overexpression of Keap1 mRNA and protein. Similarly, bisulfite genomic DNA sequencing shows that human clear lenses (n = 15) slowly lose 5-methylcytosine in the Keap1 promoter throughout life, at a rate of 1% per year. By contrast, diabetic cataractous lenses (n = 21) lose an average of 90% of the 5-methylcytosine regardless of age. Overexpressed Keap1 protein is responsible for decreasing Nrf2 by proteasomal degradation, thereby suppressing Nrf2-dependent stress protection. This study demonstrates for the first time the associations of unfolded protein response activation, Nrf2-dependent antioxidant system failure, and loss of Keap1 promoter methylation because of altered active and passive DNA demethylation pathway enzymes in human lens epithelial cells by methylglyoxal. As an outcome, the cellular redox balance is altered toward lens oxidation and cataract formation.

Valproic acid suppresses Nrf2/Keap1 dependent antioxidant protection through induction of endoplasmic reticulum stress and Keap1 promoter DNA demethylation in human lens epithelial cells.

Recent epidemiological studies confirm the prevalence of cataract in epileptic patients. Similarly, the drugs used to treat epilepsy also show the connection with increased cataract formation. In this present study, we investigated the suppression of Nrf2/Keap1 dependent antioxidant protection through induction of endoplasmic (ER) stress and Keap1 promoter DNA demethylation in human lens epithelial cells (HLECs) treated with valproic acid (VPA), an antiepileptic drug. 20 mM VPA induces ER stress and activates the unfolded protein response (UPR) within 4 h by activating the ER stress sensor proteins, such as PERK, IRE1α, and ATF6 in HLECs. Consequently, the integrated ER stress signals, such as eIF2α, ATF4, BiP, and CHOP are altered accordingly to induce ER-Ca2+ release, reactive oxygen species (ROS) overproduction, and cell death in HLECs treated with VPA. VPA also suppresses the Nrf2, catalase, and glutathione reductase expressions with significant increases in Keap1 protein. Bisulphite genomic DNA sequencing reveals the promoter DNA demethylation in the Keap1 promoter, which results in the overexpression of Keap1 mRNA and protein in HLECs treated with 20 mM VPA. VPA also alters the expression profiles of passive DNA demethylation pathway enzymes such Dnmt1, Dnmt3a, Dnmt3b, and active DNA demethylation pathway enzyme, TET1 leading to DNA demethylation in the Keap1 promoter of HLECs. Overexpressed Keap1 decreases the Nrf2 level, thereby abolishing the Nrf2 dependent antioxidant protection. This might be responsible for lenticular proteins oxidation and cataract formation.

FMLP-, thapsigargin-, and H2O2-evoked changes in intracellular free calcium concentration in lymphocytes and neutrophils of type 2 diabetic patients.

Type 2 diabetic (T2DM) patients are immune-compromised having a higher susceptibility to infections and long-term complications in different parts of the body contributing to increased morbidity and mortality. A derangement in the homeostasis of intracellular free calcium concentration [Ca²âº](i) is known to be associated with several diseases in the body including T2DM. Both neutrophils and lymphocytes play active protective roles in host immune response to infection showing impairment in microbicidal functions including phagocytosis and chemotaxis which are calcium-dependent processes. This study evaluated the process of [Ca²âº]i mobilization from both neutrophils and lymphocytes taken from blood of both T2DM patients and healthy age-matched control subjects investigating the effect of N-formyl-methionyl-leucyl-phenylalanine (fMLP), thapsigargin (TG), and hydrogen peroxide (H2O2) on [Ca²âº](i) homeostasis. This study employed isolated peripheral blood neutrophils and lymphocytes from 24 T2DM patients and 24 healthy volunteers. Either neutrophils or lymphocytes were stimulated separately with fMLP, TG, or H2O2. Induced changes in [Ca²âº] in both neutrophils and lymphocytes were evaluated using spectrofluorometric methods. Stimulation of human neutrophils and lymphocytes with fMLP, TG, or H2O2 in the presence of [Ca²âº]o resulted in significant decreases in [Ca²âº](i) mobilization from T2DM patients compared with healthy controls. These data indicate that neutrophils and lymphocytes from T2DM patients are less responsive to calcium mobilizing agents compared with granulocytes from healthy controls and this is possibly due to the hyperglycemia. The results suggest that agonist-evoked decrease in [Ca²âº](i) in immune cells might be one of the possible mechanisms of impaired immunity in diabetic patients.

A glutathione-independent glyoxalase of the DJ-1 superfamily plays an important role in managing metabolically generated methylglyoxal in Candida albicans.

Methylglyoxal is a cytotoxic reactive carbonyl compound produced by central metabolism. Dedicated glyoxalases convert methylglyoxal to d-lactate using multiple catalytic strategies. In this study, the DJ-1 superfamily member ORF 19.251/GLX3 from Candida albicans is shown to possess glyoxalase activity, making this the first demonstrated glutathione-independent glyoxalase in fungi. The crystal structure of Glx3p indicates that the protein is a monomer containing the catalytic triad Cys(136)-His(137)-Glu(168). Purified Glx3p has an in vitro methylglyoxalase activity (Km = 5.5 mM and kcat = 7.8 s(-1)) that is significantly greater than that of more distantly related members of the DJ-1 superfamily. A close Glx3p homolog from Saccharomyces cerevisiae (YDR533C/Hsp31) also has glyoxalase activity, suggesting that fungal members of the Hsp31 clade of the DJ-1 superfamily are all probable glutathione-independent glyoxalases. A homozygous glx3 null mutant in C. albicans strain SC5314 displays greater sensitivity to millimolar levels of exogenous methylglyoxal, elevated levels of intracellular methylglyoxal, and carbon source-dependent growth defects, especially when grown on glycerol. These phenotypic defects are complemented by restoration of the wild-type GLX3 locus. The growth defect of Glx3-deficient cells in glycerol is also partially complemented by added inorganic phosphate, which is not observed for wild-type or glucose-grown cells. Therefore, C. albicans Glx3 and its fungal homologs are physiologically relevant glutathione-independent glyoxalases that are not redundant with the previously characterized glutathione-dependent GLO1/GLO2 system. In addition to its role in detoxifying glyoxals, Glx3 and its close homologs may have other important roles in stress response.

Chronic effects of mild hyperglycaemia on left ventricle transcriptional profile and structural remodelling in the spontaneously type 2 diabetic Goto-Kakizaki rat.

Heart failure in chronic type 2 diabetes mellitus is partly attributable to adverse structural remodelling of the left ventricle (LV), but the contribution of hyperglycaemia (HG) per se in remodelling processes is debated. In this study, we examined the molecular signature of LV remodelling in 18-month-old spontaneously diabetic male Goto-Kakizaki (GK) rats that represent a long-term mildly diabetic phenotype, using histological, immunoblotting and quantitative gene expression approaches. Relative to age-matched Wistar controls, mildly diabetic GK rats presented with LV hypertrophy, increased expression of natriuretic peptides and phosphorylation of pro-hypertrophic Akt. Fibrosis proliferation in the GK LV paralleled increased transcriptional and biologically active pro-fibrogenic transforming growth factor-ß1 (TGFß1) in the LV with upregulated mRNA abundance for key extracellular matrix (ECM) components such as fibronectin, collagen type(s) 1 and 3α and regulators including matrix metalloproteinases 2 and 9, and their tissue inhibitor (TIMP) 4, connexin 43 and α5-integrin. GK rats also presented with altered mRNA expression for cardiac sarcoplasmic reticulum Ca(2+)ATPase, Na(+)/Ca(2+) exchanger and the L-type Ca(2+) channels which may contribute to the altered Ca(2+) transient kinetics previously observed in this model at 18 months of age (t test, p < 0.05 vs. age-matched Wistar control for all parameters). The results indicate that chronic mild HG can produce the molecular and structural correlates of a hypertrophic myopathy. Diffuse ECM proliferation in this model is possibly a product of HG-induced TGFß1 upregulation and altered transcriptional profile of the ECM.

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.

Combination of real time three-dimensional echocardiography with diagnostic catheterization to derive left ventricular pressure-volume relations.

AIMS: The aim of this study was to investigate the left ventricular (LV) myocardial contractility index-Emax using transesophageal real time three-dimensional echocardiography (RT3DE) combined with catheterization. METHODS: Transesophageal RT3DE (single beat, X7-2 × matrix, iE33, Philips) was used to obtain real time LV volumes in pigs. Volumes were integrated with LV pressures from conductance catheterization (CC) to create RT3DE pressure-volume relations. At the same time, CC was used for measuring conventional pressure-volume relations that served as reference. The slope Emax was determined from RT3DE and CC end-systolic pressure-volume relations. All measurements were made at rest and during dobutamine infusion. RESULTS: In six pigs, the mean ± SD (mmHg/mL) values were Emax-CC 1.86 ± 1.1 and Emax-RT3DE 1.78 ± 1.2 (P = 0.502) at baseline. On dobutamine, mean Emax-CC was 3.43 ± 1.5 and Emax-RT3DE 3.60 ± 1.23 (P = 0.171). Bland-Altman analysis showed good agreements between the RT3DE- and CC-derived Emax for measurements performed at baseline and on dobutamine. CONCLUSIONS: Emax can be determined from RT3DE integrated with catheterization-derived pressures. RT3DE is a promising method for enhancing clinical applicability of pressure-volume relations for assessment of myocardial contractility.

Pyruvate restores ß-adrenergic sensitivity of L-type Ca(2+) channels in failing rat heart: role of protein phosphatase.

Oxidative stress plays a major role in the pathogenesis of heart failure, where the contractile response to ß-adrenergic stimulation is profoundly depressed. This condition involves L-type Ca(2+) channels, but the mechanisms underlying their impaired adrenergic regulation are unclear. Thus the present study explored the basis for impaired adrenergic control of Ca(2+) channels in a rat infarction model of heart failure. Patch-clamp recordings of L-type Ca(2+) current (I(Ca,L)) from ventricular myocytes isolated from infarcted hearts showed a blunted response to intracellular cAMP that was reversed by treatment with exogenous pyruvate. Biochemical studies showed that basal and cAMP-stimulated protein kinase A activities were similar in infarcted and sham-operated hearts, whereas molecular analysis also found that binding of protein kinase A to the α(1C) subunit of voltage-gated Ca(2+) channel isoform 1.2 was not different between groups. By contrast, protein phosphatase 2A (PP2A) activity and binding to α(1C) were significantly less in infarcted hearts. The PP2A inhibitor okadaic acid markedly increased I(Ca,L) in sham-operated myocytes, but this response was significantly less in myocytes from infarcted hearts. However, pyruvate normalized I(Ca,L) stimulation by okadaic acid, and this effect was blocked by inhibitors of thioredoxin reductase, implicating a functional role for the redox-active thioredoxin system. Our data suggest that blunted ß-adrenergic stimulation of I(CaL) in failing hearts results from hyperphosphorylation of Ca(2+) channels secondary to oxidation-induced impairment of PP2A function. We propose that the redox state of Ca(2+) channels or PP2A is controlled by the thioredoxin system which plays a key role in Ca(2+) channel remodeling of the failing heart.

Validation of admittance computed left ventricular volumes against real-time three-dimensional echocardiography in the porcine heart.

The admittance and Wei's equation is a new technique for ventricular volumetry to determine pressure-volume relations that addresses traditional conductance-related issues of parallel conductance and field correction factor. These issues with conductance have prevented researchers from obtaining real-time absolute ventricular volumes. Moreover, the time-consuming steps involved in processing conductance catheter data warrant the need for a better catheter-based technique for ventricular volumetry. We aimed to compare the accuracy of left ventricular (LV) volumetry between the new admittance catheterization technique and transoesophageal real-time three-dimensional echocardiography (RT3DE) in a large-animal model. Eight anaesthetized pigs were used. A 7 French admittance catheter was positioned in the LV via the right carotid artery. The catheter was connected to an admittance control unit (ADVantage; Transonic Scisense Inc.), and data were recorded on a four-channel acquisition system (FA404; iWorx Systems). Admittance catheterization data and transoesophageal RT3DE (X7-2; Philips) data were simultaneously obtained with the animal ventilated, under neuromuscular blockade and monitored in baseline conditions and during dobutamine infusion. Left ventricular volumes measured from admittance catheterization (Labscribe; iWorx Systems) and RT3DE (Qlab; Philips) were compared. In a subset of four animals, admittance volumes were compared with those obtained from traditional conductance catheterization (MPVS Ultra; Millar Instruments). Of 37 sets of measurements compared, admittance- and RT3DE-derived LV volumes and ejection fractions at baseline and in the presence of dobutamine exhibited general agreement, with mean percentage intermethod differences of 10% for end-diastolic volumes, 14% for end-systolic volumes and 9% for ejection fraction; the respective intermethod differences between admittance and conductance in eight data sets compared were 11, 11 and 12%. Admittance volumes were generally higher than those obtained by RT3DE, especially among the larger ventricles. It is concluded that it is feasible to derive pressure-volume relations using admittance catheterization in large animals. This study demonstrated agreements between admittance and RT3DE to within 10-14% mean intermethod difference in the estimation of LV volumes. Further investigation will be required to examine the accuracy of volumes in largest ventricles, where intermethod divergence is greatest.