Role of Protein Kinase C in Metabolic Regulation of Coronary Endothelial Small Conductance Calcium-Activated Potassium Channels.

BACKGROUND: Small conductance calcium-activated potassium (SK) channels are largely responsible for endothelium-dependent coronary arteriolar relaxation. Endothelial SK channels are downregulated by the reduced form of nicotinamide adenine dinucleotide (NADH), which is increased in the setting of diabetes, yet the mechanisms of these changes are unclear. PKC (protein kinase C) is an important mediator of diabetes-induced coronary endothelial dysfunction. Thus, we aimed to determine whether NADH signaling downregulates endothelial SK channel function via PKC. METHODS AND RESULTS: SK channel currents of human coronary artery endothelial cells were measured by whole cell patch clamp method in the presence/absence of NADH, PKC activator phorbol 12-myristate 13-acetate, PKC inhibitors, or endothelial PKCα/PKCß knockdown by using small interfering RNA. Human coronary arteriolar reactivity in response to the selective SK activator NS309 was measured by vessel myography in the presence of NADH and PKCß inhibitor LY333531. NADH (30-300 µmol/L) or PKC activator phorbol 12-myristate 13-acetate (30-300 nmol/L) reduced endothelial SK current density, whereas the selective PKCᵦ inhibitor LY333531 significantly reversed the NADH-induced SK channel inhibition. PKCß small interfering RNA, but not PKCα small interfering RNA, significantly prevented the NADH- and phorbol 12-myristate 13-acetate-induced SK inhibition. Incubation of human coronary artery endothelial cells with NADH significantly increased endothelial PKC activity and PKCß expression and activation. Treating vessels with NADH decreased coronary arteriolar relaxation in response to the selective SK activator NS309, and this inhibitive effect was blocked by coadministration with PKCß inhibitor LY333531. CONCLUSIONS: NADH-induced inhibition of endothelial SK channel function is mediated via PKCß. These findings may provide insight into novel therapeutic strategies to preserve coronary microvascular function in patients with metabolic syndrome and coronary disease.

Inflammatory Macrophage Interleukin-1ß Mediates High-Fat Diet-Induced Heart Failure With Preserved Ejection Fraction.

Diabetes mellitus (DM) is a main risk factor for diastolic dysfunction (DD) and heart failure with preserved ejection fraction. High-fat diet (HFD) mice presented with diabetes mellitus, DD, higher cardiac interleukin (IL)-1ß levels, and proinflammatory cardiac macrophage accumulation. DD was significantly ameliorated by suppressing IL-1ß signaling or depleting macrophages. Mice with macrophages unable to adopt a proinflammatory phenotype were low in cardiac IL-1ß levels and were resistant to HFD-induced DD. IL-1ß enhanced mitochondrial reactive oxygen species (mitoROS) in cardiomyocytes, and scavenging mitoROS improved HFD-induced DD. In conclusion, macrophage-mediated inflammation contributed to HFD-associated DD through IL-1ß and mitoROS production.

Circulating S-Glutathionylated cMyBP-C as a Biomarker for Cardiac Diastolic Dysfunction.

Background cMyBP-C (Cardiac myosin binding protein-C) regulates cardiac contraction and relaxation. Previously, we demonstrated that elevated myocardial S-glutathionylation of cMyBP-C correlates with diastolic dysfunction (DD) in animal models. In this study, we tested whether circulating S-glutathionylated cMyBP-C would be a biomarker for DD. Methods and Results Humans, African Green monkeys, and mice had DD determined by echocardiography. Blood samples were acquired and analyzed for S-glutathionylated cMyBP-C by immunoprecipitation. Circulating S-glutathionylated cMyBP-C in human participants with DD (n=24) was elevated (1.46±0.13-fold, P=0.014) when compared with the non-DD controls (n=13). Similarly, circulating S-glutathionylated cMyBP-C was upregulated by 2.13±0.47-fold (P=0.047) in DD monkeys (n=6), and by 1.49 (1.22-2.06)-fold (P=0.031) in DD mice (n=5) compared with the respective non-DD controls. Circulating S-glutathionylated cMyBP-C was positively correlated with DD in humans. Conclusions Circulating S-glutathionylated cMyBP-C was elevated in humans, monkeys, and mice with DD. S-glutathionylated cMyBP-C may represent a novel biomarker for the presence of DD.

Hsa_circ_0010729 is Involved in Oxygen-Glucose Deprivation/Reoxygenation-Induced Human Microvascular Endothelial Cell Deprivation by Targeting miR-665/ING5.

Ischemic stroke is a disease with high mortality. Circular RNA_0010729 (hsa_circ_0010729) has been reported to be involved in ischemic heart disease. However, it is not clear whether hsa_circ_0010729 is involved in the regulation of ischemic stroke. In this study, we used oxygen-glucose deprivation/reoxygenation (OGD/R) to stimulate human brain microvascular endothelial cells (HBMECs) model to investigate the potential role of hsa_circ_0010729 in stroke in vitro. The expression levels of hsa_circ_0010729, miR-665, and ING5 in ischemic stroke were detected by quantitative real-time polymerase chain reaction (qRT-PCR). HBMECs proliferation was detected by CCK-8. Cell apoptosis was detected by flow cytometry. The levels of inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA). Western blot was used to detect the related protein expression. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) were used to examine the target relationship between miR-665 and hsa_circ_0010729 or ING5. Compared with the control group, hsa_circ_0010729 and ING5 were highly expressed in OGD/R-induced HBMECs, while miR-665 was lowly expressed. Hsa_circ_0010729 silencing promoted OGD/R-induced cell proliferation and inhibited apoptosis. However, the effect of hsa_circ_0010729 down-regulation on OGD/R-induced cell was partially restored after co-transfection with miR-665 inhibitor. Overexpression of miR-665 can promote the proliferation and inhibit apoptosis of OGD/R-induced HBMECs by inhibiting ING5 expression. In OGD/R-induced HBMECs, hsa_circ_0010729 silencing decreased ING5 expression by upregulating miR-665. Hsa_circ_0010729 regulated miR-665/ING5 axis in OGD/R-induced HBMECs. Therefore, hsa_circ_0010729 may be a new therapeutic target for ischemic stroke.

Glycemic control is not associated with neurocognitive decline after cardiac surgery.

BACKGROUND: Whether perioperative glycemic control is associated with neurocognitive decline (NCD) after cardiac surgery was examined. METHODS: Thirty patients undergoing cardiac surgery utilizing cardiopulmonary bypass (CPB) were screened for NCD preoperatively and on postoperative day 4 (POD4). Indices of glucose control were examined. Serum cytokine levels were measured and human transcriptome analysis was performed on blood samples. Neurocognitive data are presented as a change from baseline to POD4 in a score standardized with respect to age and gender. RESULTS: A decline in neurocognitive function was identified in 73% (22/30) of patients on POD4. There was no difference in neurocognitive function between patients with elevated HbA1c levels preoperatively (p = .973) or elevated fasting blood glucose levels the morning of surgery (>126 mg/dl, p = .910), or a higher maximum blood glucose levels during CPB (>180 mg/dl, p = .252), or higher average glucose levels during CPB (>160 mg/dl, p = .639). Patients with postoperative leukocytosis (WBC ≥ 10.5) had more NCD when compared to their baseline function (p = .03). Patients with elevated IL-8 levels at 6 h postoperatively had a significant decline in NCD at POD4 (p = .04). Human transcriptome analysis demonstrated unique and differential patterns of gene expression in patients depending on the presence of DM and NCD. CONCLUSIONS: Perioperative glycemic control does not have an effect on NCD soon after cardiac surgery. The profile of gene expression was altered in patients with NCD with or without diabetes.

Inhibition of mitochondrial reactive oxygen species improves coronary endothelial function after cardioplegic hypoxia/reoxygenation.

OBJECTIVE: Cardioplegic ischemia-reperfusion and diabetes mellitus are correlated with coronary endothelial dysfunction and inactivation of small conductance calcium-activated potassium channels. Increased reactive oxidative species, such as mitochondrial reactive oxidative species, may contribute to oxidative injury. Thus, we hypothesized that inhibition of mitochondrial reactive oxidative species may protect coronary small conductance calcium-activated potassium channels and endothelial function against cardioplegic ischemia-reperfusion-induced injury. METHODS: Small coronary arteries and endothelial cells from the hearts of mice with and without diabetes mellitus were isolated and examined by using a cardioplegic hypoxia and reoxygenation model to determine whether the mitochondria-targeted antioxidant Mito-Tempo could protect against coronary endothelial and small conductance calcium-activated potassium channel dysfunction. The microvessels or mouse heart endothelial cells were treated with or without Mito-Tempo (0-10 µM) 5 minutes before and during cardioplegic hypoxia and reoxygenation. Microvascular function was assessed in vitro by vessel myography. K+ currents of mouse heart endothelial cells were measured by whole-cell patch clamp. The levels of intracellular cytosolic free calcium (Ca2+) concentration, mitochondrial reactive oxidative species, and small conductance calcium-activated potassium protein expression of mouse heart endothelial cells were measured by Rhod-2 fluorescence staining, MitoSox, and Western blotting, respectively. RESULTS: Cardioplegic hypoxia and reoxygenation significantly attenuated endothelial small conductance calcium-activated potassium channel activity, caused calcium overload, and increased mitochondrial reactive oxidative species of mouse heart endothelial cells in both the nondiabetic and diabetes mellitus groups. In addition, treating mouse heart endothelial cells with Mito-Tempo (10 µM) reduced cardioplegic hypoxia and reoxygenation-induced Ca2+ and mitochondrial reactive oxidative species overload in both the nondiabetic and diabetes mellitus groups, respectively (P < .05). Treatment with Mito-Tempo (10 µM) significantly enhanced coronary relaxation responses to adenosine 5'-diphosphate and NS309 (P < .05), and endothelial small conductance calcium-activated potassium channel currents in both the nondiabetic and diabetes mellitus groups (P < .05). CONCLUSIONS: Administration of Mito-Tempo improves endothelial function and small conductance calcium-activated potassium channel activity, which may contribute to its enhancement of endothelium-dependent vasorelaxation after cardioplegic hypoxia and reoxygenation.

Lactobacillus plantarum probiotic induces Nrf2-mediated antioxidant signaling and eNOS expression resulting in improvement of myocardial diastolic function.

Yorkshire swine were fed standard diet (n = 7) or standard diet containing applesauce rich in caffeic acid with Lactobacillus plantarum (n = 7) for 3 wk. An ameroid constrictor was next placed around the left coronary circumflex artery, and the dietary regimens were continued. At 14 wk, cardiac function, myocardial perfusion, vascular density, and molecular signaling in ischemic myocardium were evaluated. The L. plantarum-applesauce augmented NF-E2-related factor 2 (Nrf2) in the ischemic myocardium and induced Nrf2-regulated antioxidant enzymes heme oxygenase-1 (HO-1), NADPH dehydrogenase quinone 1 (NQO-1), and thioredoxin reductase (TRXR-1). Improved left ventricular diastolic function and decreased myocardial collagen expression were seen in animals receiving the L. plantarum-applesauce supplements. The expression of endothelial nitric oxide synthase (eNOS) was increased in ischemic myocardial tissue of the treatment group, whereas levels of asymmetric dimethyl arginine (ADMA), hypoxia inducible factor 1α (HIF-1α), and phosphorylated MAPK (pMAPK) were decreased. Collateral-dependent myocardial perfusion was unaffected, whereas arteriolar and capillary densities were reduced as determined by α-smooth muscle cell actin and CD31 immunofluorescence in ischemic myocardial tissue. Dietary supplementation with L. plantarum-applesauce is a safe and effective method of enhancing Nrf2-mediated antioxidant signaling cascade in ischemic myocardium. Although this experimental diet was associated with a reduction in hypoxic stimuli, decreased vascular density, and without any change in collateral-dependent perfusion, the net effect of an increase in antioxidant activity and eNOS expression resulted in improvement in diastolic function.NEW & NOTEWORTHY Colonization of the gut microbiome with certain strains of L. Plantarum has been shown to convert caffeic acid readily available in applesauce to 4-vinyl-catechol, a potent activator of the Nrf2 antioxidant defense pathway. In this exciting study, we show that simple dietary supplementation with L. Plantarum-applesauce-mediated Nrf2 activation supports vascular function, ameliorates myocardial ischemic diastolic dysfunction, and upregulates expression of eNOS.

The polar oxy-metabolome reveals the 4-hydroxymandelate CoQ10 synthesis pathway.

Oxygen is critical for a multitude of metabolic processes that are essential for human life. Biological processes can be identified by treating cells with 18O2 or other isotopically labelled gases and systematically identifying biomolecules incorporating labeled atoms. Here we labelled cell lines of distinct tissue origins with 18O2 to identify the polar oxy-metabolome, defined as polar metabolites labelled with 18O under different physiological O2 tensions. The most highly 18O-labelled feature was 4-hydroxymandelate (4-HMA). We demonstrate that 4-HMA is produced by hydroxyphenylpyruvate dioxygenase-like (HPDL), a protein of previously unknown function in human cells. We identify 4-HMA as an intermediate involved in the biosynthesis of the coenzyme Q10 (CoQ10) headgroup in human cells. The connection of HPDL to CoQ10 biosynthesis provides crucial insights into the mechanisms underlying recently described neurological diseases related to HPDL deficiencies1-4 and cancers with HPDL overexpression5.

Cardiac Resynchronization and Circulating Markers of Sarcoplasmic Reticulum Calcium Handling and Sudden Death Risk.

Cardiac resynchronization therapy (CRT) can improve heart function and decrease arrhythmic events. We tested whether CRT altered circulating markers of calcium handling and sudden death risk. Circulating cardiac sodium channel messenger RNA (mRNA) splicing variants indicate arrhythmic risk, and a reduction in sarco/endoplasmic reticulum calcium adenosine triphosphatase 2a (SERCA2a) is thought to diminish contractility in heart failure. CRT was associated with a decreased proportion of circulating, nonfunctional sodium channels and improved SERCA2a mRNA expression. Patients without CRT did not have improvement in the biomarkers. These changes might explain the lower arrhythmic risk and improved contractility associated with CRT.

Inhibition of the unfolded protein response reduces arrhythmia risk after myocardial infarction.

Ischemic cardiomyopathy is associated with an increased risk of sudden death, activation of the unfolded protein response (UPR), and reductions in multiple cardiac ion channels. When activated, the protein kinase-like ER kinase (PERK) branch of the UPR reduces protein translation and abundance. We hypothesized that PERK inhibition could prevent ion channel downregulation and reduce arrhythmia risk after myocardial infarct (MI). MI induced in mice by coronary artery ligation resulted in reduced ion channel levels, ventricular tachycardia (VT), and prolonged corrected intervals between the Q and T waves on the ECGs (QTc). Protein levels of major cardiac ion channels were decreased. MI cardiomyocytes showed significantly prolonged action potential duration and decreased maximum upstroke velocity. Cardiac-specific PERK KO reduced electrical remodeling in response to MI, with shortened QTc intervals, fewer VT episodes, and higher survival rates. Pharmacological PERK inhibition had similar effects. In conclusion, we found that activated PERK during MI contributed to arrhythmia risk by the downregulation of select cardiac ion channels. PERK inhibition prevented these changes and reduced arrhythmia risk. These results suggest that ion channel downregulation during MI is a fundamental arrhythmia mechanism and that maintenance of ion channel levels is antiarrhythmic.

Chronic Inhibition of mROS Protects Against Coronary Endothelial Dysfunction in Mice With Diabetes.

Diabetes is associated with coronary endothelial dysfunction. Persistent oxidative stress during diabetes contributes to coronary endothelial dysfunction. The mitochondria are main sources of reactive oxygen species (ROS) in diabetes, and mitochondria-targeted antioxidant mito-Tempo can prevent mitochondrial reactive oxygen species (mROS) generation in a variety of disorders. Inhibition/inactivation of small-conductance Ca2+-activated K+ (SK) channels contribute to diabetic downregulation of coronary endothelial function/relaxation. However, few investigated the role of mROS on endothelial dysfunction/vasodilation and endothelial SK channel downregulation in diabetes. The aim of present study was to investigate the chronic administration of mito-Tempo, on coronary vasodilation, and endothelial SK channel activity of mice with or without diabetes. Mito-Tempo (1 mg/kg/day) was applied to the mice with or without diabetes (n = 10/group) for 4 weeks. In vitro relaxation response of pre-contracted arteries was examined in the presence or absence of the vasodilatory agents. SK channel currents of the isolated mouse heart endothelial cells were measured using whole-cell patch clamp methods. At baseline, coronary endothelium-dependent relaxation responses to ADP and the selective SK channel activator NS309 and endothelial SK channel currents were decreased in diabetic mice compared with that in non-diabetic (ND) mice (p < 0.05). After a 4-week treatment with mito-Tempo, coronary endothelium-dependent relaxation response to ADP or NS309 and endothelial SK channel currents in the diabetic mice was significantly improved when compared with that in untreated diabetic mice (p < 0.05). Interestingly, coronary relaxation responses to ADP and NS309 and endothelial SK channel currents were not significantly changed in ND mice after mito-Tempo treatment, as compared to that of untreated control group. Chronic inhibition of endothelial mROS appears to improve coronary endothelial function/dilation and SK channel activity in diabetes, and mROS inhibitors may be a novel strategy to treat vascular complications in diabetes.

Interleukin-1ß, Oxidative Stress, and Abnormal Calcium Handling Mediate Diabetic Arrhythmic Risk.

Diabetes mellitus (DM) is associated with increased arrhythmia. Type 2 DM (T2DM) mice showed prolonged QT interval and increased ventricular arrhythmic inducibility, accompanied by elevated cardiac interleukin (IL)-1ß, increased mitochondrial reactive oxygen species (mitoROS), and oxidation of the sarcoplasmic reticulum (SR) Ca2+ release channel (ryanodine receptor 2 [RyR2]). Inhibiting IL-1ß and mitoROS reduced RyR2 oxidation and the ventricular arrhythmia in DM. Inhibiting SR Ca2+ leak by stabilizing the oxidized RyR2 channel reversed the diabetic arrhythmic risk. In conclusion, cardiac IL-1ß mediated the DM-associated arrhythmia through mitoROS generation that enhances SR Ca2+ leak. The mechanistic link between inflammation and arrhythmias provides new therapeutic options.

Effects of High Fat Versus Normal Diet on Extracellular Vesicle-Induced Angiogenesis in a Swine Model of Chronic Myocardial Ischemia.

Background Mesenchymal stem cell-derived extracellular vesicles (EVs) promote angiogenesis in the ischemic myocardium. This study examines the difference in vascular density, myocardial perfusion, molecular signaling, and gene expression between normal diet (ND) and high fat diet (HFD) groups at baseline and following intramyocardial injection of EVs. Methods and Results Intact male Yorkshire swine fed either an ND (n=17) or HFD (n=14) underwent placement of an ameroid constrictor on the left circumflex coronary artery. Subsequently, animals received either intramyocardial injection of vehicle-saline as controls; (ND-controls n=7, HFD-controls, n=6) or EVs; (ND-EVs n=10, HFD-EVs n=8) into the ischemic territory. Five weeks later, myocardial function, perfusion, vascular density, cell signaling, and gene expression were examined. EVs improved indices of myocardial contractile function, myocardial perfusion, and arteriogenesis in both dietary cohorts. Interestingly, quantification of alpha smooth muscle actin demonstrated higher basal arteriolar density in HFD swine compared with their ND counterparts; whereas EVs were associated with increased CD31-labeled endothelial cell density only in the ND tissue, which approached significance. Levels of total endothelial nitric oxide synthase, FOXO1 (forkhead box protein O1) , transforming growth factor-ß, phosphorylated VEGFR2 (vascular endothelial growth factor receptor 2), and phosphorylated MAPK ERK1/ERK2 (mitogen-activated protein kinase) were higher in ischemic myocardial lysates from ND-controls compared with HFD-controls. Conversely, HFD-control tissue showed increased expression of phosphorylated endothelial nitric oxide synthase, phosphorylated FOXO1, VEGFR2, and MAPK ERK1/ERK2 with respect to ND-controls. Preliminary gene expression studies indicate differential modulation of transcriptional activity by EVs between the 2 dietary cohorts. Conclusions HFD produces a profound metabolic disorder that dysregulates the molecular mechanisms of collateral vessel formation in the ischemic myocardium, which may hinder the therapeutic angiogenic effects of EVs.

Intravenous injection of extracellular vesicles to treat chronic myocardial ischemia.

BACKGROUND: Mesenchymal stem cell-derived extracellular vesicles (EVs) appear to be a very exciting treatment option for heart disease. Here, we used a swine model of chronic myocardial ischemia to evaluate the efficacy of a less-invasive method of injection of EVs via a peripheral intravenous route. METHODS: Sixteen Yorkshire swine underwent placement of an ameroid constrictor on the left circumflex (LCx) artery at age 11 weeks to induce chronic myocardial ischemia. Two weeks later, they were divided into two groups: control (CON; n = 8), and intravenous injection of EVs (EVIV; n = 8). At 18 weeks of age, animals underwent final analysis and euthanasia. The chronically ischemic myocardium (LCx territory) was harvested for analysis. RESULTS: Intravenous injection (IV) of EVs induced several pro-angiogenic markers such as MAPK, JNK but not Akt. Whereas IV injections of EVs decreased VEGFR2 expression and inhibited apoptotic signaling (caspase 3), they increased expression of VEGFR1 that is believed to be anti-angiogenic. Injection of EVs did not result in an increase in vessel density and blood flow when compared to the control group. CONCLUSIONS: Although IV injection of EVs upregulated several pro-angiogenic signaling pathways, it failed to induce changes in vascular density in the chronically ischemic myocardium. Thus, a lack of increase in vascular density at the doses tested failed to elicit a functional response in ischemic myocardium.

Effects of Inorganic Arsenic on Human Prostate Stem-Progenitor Cell Transformation, Autophagic Flux Blockade, and NRF2 Pathway Activation.

BACKGROUND: Inorganic arsenic (iAs) is an environmental toxicant associated with an increased risk of prostate cancer in chronically exposed populations worldwide. However, the biological mechanisms underlying iAs-induced prostate carcinogenesis remain unclear. OBJECTIVES: We studied how iAs affects normal human prostate stem-progenitor cells (PrSPCs) and drives transformation and interrogated the molecular mechanisms involved. METHODS: PrSPCs were enriched by spheroid culture from normal human primary or immortalized prostate epithelial cells, and their differentiation capability was evaluated by organoid culture. Microarray analysis was conducted to identify iAs-dysregulated genes, and lentiviral infection was used for stable manipulation of identified genes. Soft agar colony growth assays were applied to examine iAs-induced transformation. For in vivo study, PrSPCs mixed with rat urogenital sinus mesenchyme were grafted under the renal capsule of nude mice to generate prostatelike tissues, and mice were exposed to 5 ppm (∼65µM) iAs in drinking water for 3 months. RESULTS: Low-dose iAs (1µM) disturbed PrSPC homeostasis in vitro, leading to increased self-renewal and suppressed differentiation. Transcriptomic analysis indicated that iAs activated oncogenic pathways in PrSPCs, including the KEAP1-NRF2 pathway. Further, iAs-exposed proliferative progenitor cells exhibited NRF2 pathway activation that was sustained in their progeny cells. Knockdown of NRF2 inhibited spheroid formation by driving PrSPC differentiation, whereas its activation enhanced spheroid growth. Importantly, iAs-induced transformation was suppressed by NRF2 knockdown. Mechanistically, iAs suppressed Vacuolar ATPase subunit VMA5 expression, impairing lysosome acidification and inhibiting autophagic protein degradation including p62, which further activated NRF2. In vivo, chronic iAs exposure activated NRF2 in both epithelial and stroma cells of chimeric human prostate grafts and induced premalignant events. CONCLUSIONS: Low-dose iAs increased self-renewal and decreased differentiation of human PrSPCs by activating the p62-NRF2 axis, resulting in epithelial cell transformation. NRF2 is activated by iAs through specific autophagic flux blockade in progenitor cells, which may have potential therapeutic implications. https://doi.org/10.1289/EHP6471.

Coronary endothelial dysfunction prevented by small-conductance calcium-activated potassium channel activator in mice and patients with diabetes.

OBJECTIVE: To investigate coronary endothelial protection of a small-conductance calcium-activated potassium (SK) channel activator against a period of cardioplegic-hypoxia and reoxygenation (CP-H/R) injury in mice and patients with diabetes (DM) and those without diabetes (nondiabetic [ND]). METHODS: Mouse small coronary arteries/heart endothelial cells (MHECs) and human coronary arterial endothelial cells (HCAECs) were dissected from the harvested hearts of mice (n = 16/group) and from discarded right atrial tissue samples of patients with DM and without DM (n = 8/group). The SK current density of MHECs was measured. The in vitro small arteries/arterioles, MHECs, and HCAECs were subjected to 60 minutes of CP hypoxia, followed by 60 minutes of oxygenation. Vessels were treated with or without the selective SK activator NS309 for 5 minutes before and during CP hypoxia. RESULTS: DM and/or CP-H/R significantly inhibited the total SK currents of MHECs and HCAECs and significantly diminished the mouse coronary relaxation response to NS309. Administration of NS309 immediately before and during CP hypoxia significantly improved the recovery of coronary endothelial function, as demonstrated by increased relaxation responses to adenosine 5'-diphosphate and substance P compared with those seen in controls (P < .05). This protective effect was more pronounced in vessels from ND mice and patients compared with DM mice and patients (P < .05). Cell surface membrane SK3 expression was significantly reduced after hypoxia, whereas cytosolic SK3 expression was greater than that of the sham control group (P < .05). CONCLUSIONS: Application of NS309 immediately before and during CP hypoxia protects mouse and human coronary microvasculature against CP-H/R injury, but this effect is diminished in the diabetic coronary microvasculature. SK inhibition/inactivation and/or internalization/redistribution may contribute to CP-H/R-induced coronary endothelial and vascular relaxation dysfunction.

Metabolic regulation of endothelial SK channels and human coronary microvascular function.

BACKGROUND: Diabetic (DM) inactivation of small conductance calcium-activated potassium (SK) channels contributes to coronary endothelial dysfunction. However, the mechanisms responsible for this down-regulation of endothelial SK channels are poorly understood. Thus, we hypothesized that the altered metabolic signaling in diabetes regulates endothelial SK channels and human coronary microvascular function. METHODS: Human atrial tissue, coronary arterioles and coronary artery endothelial cells (HCAECs) obtained from DM and non-diabetic (ND) patients (n = 12/group) undergoing cardiac surgery were used to analyze metabolic alterations, endothelial SK channel function, coronary microvascular reactivity and SK gene/protein expression/localization. RESULTS: The relaxation response of DM coronary arterioles to the selective SK channel activator SKA-31 and calcium ionophore A23187 was significantly decreased compared to that of ND arterioles (p < 0.05). Diabetes increases the level of NADH and the NADH/NAD+ ratio in human myocardium and HCAECs (p < 0.05). Increase in intracellular NADH (100 µM) in the HCAECs caused a significant decrease in endothelial SK channel currents (p < 0.05), whereas, intracellular application of NAD+ (500 µM) increased the endothelial SK channel currents (p < 0.05). Mitochondrial reactive oxygen species (mROS) of HCAECs and NADPH oxidase (NOX) and PKC protein expression in the human myocardium and coronary microvasculature were increased respectively (p < 0.05). CONCLUSIONS: Diabetes is associated with metabolic changes in the human myocardium, coronary microvasculature and HCAECs. Endothelial SK channel function is regulated by the metabolite pyridine nucleotides, NADH and NAD+, suggesting that metabolic regulation of endothelial SK channels may contribute to coronary endothelial dysfunction in the DM patients with diabetes.

Younger age is associated with greater early neurocognitive decline postcardiopulmonary bypass.

Objective: To examine the effect of aging on postoperative neurocognitive decline (NCD) in cardiac surgery patients. Methods: Patients undergoing coronary artery bypass graft or open aortic valve replacement were administered the Repeatable Battery for the Assessment of Neuropsychological Status at preoperative, postoperative day (POD) 4, and 1 month. Blood samples were collected at preoperative, 6 hours postoperative, and POD 4. Plasma interleukin (IL)-6, tumor necrosis factor-α, and C-reactive protein (CRP) levels were quantified. Quality of life was measured with the 12-Item Short Form Health Survey. Data were analyzed using paired ratio and unpaired t tests with Welch's correction, and linear regression for cytokine levels. Results: NCD occurred in 15 patients (N = 33, 45.5%). Dichotomized at age extremes (<60 years; ≥75 years), youngest patients had greater preoperative scores (P = .02) with lower scores by POD 4 (P = .03). There was no NCD in the oldest patients, and scores were not different between age groups on POD 4 (P = .08). Regression at 1 month showed NCD scores again declined by age (n = 15), with younger scores returning toward baseline (P = .008). Regression analyses showed decline by age at 6 hours postoperative and POD 4 in plasma CRP levels (P = .05 6 hours, P = .02 POD 4). Dichotomizing IL-6 levels by age (<70 years, ≥70 years) demonstrated that levels were greater in younger versus older patients at 6 hours postoperative (P = .03), but not on POD 4. Conclusions: Younger patients tend to have better cognitive scores before surgery but scores at POD 4 are similar to those of older patients, with this trend disappearing at 1 month. IL-6 and CRP upregulation is greater in younger patients, suggesting that a robust perioperative inflammatory response may be associated with reduction in neurocognitive function, and this may be greater in younger versus older patients.

Laboratory-Based Investigation into Stress Corrosion Cracking of Cable Bolts.

Cable-bolt failures due to stress corrosion cracking (SCC) could significantly compromise the sustainability and long-term stability of underground constructions. To fully understand the SCC of cable bolts, a two-step methodology was implemented: (i) long-term cable-bolt coupon tests using mineralogical materials collected from underground mines; and (ii) accelerated full-scale cable-bolt tests using an acidified solution. In the long-term tests, a novel three-point bending coupon was designed. The effects of mineralogical materials on SCC were evaluated under the simulated underground bolting conditions through the application of "corrosion cells". For accelerated tests, SCC resistance of different type of cable bolts was examined using the new designed tensile-loading apparatus under the periodically increasing strain-rate loading mechanism. It was identified that mineralogical materials and applied stress intensity accelerated the corrosion process of the cable bolts. The number of wires and wire surface conditions in different types of cable bolt directly affected SCC susceptibility. The cable bolts with a greater number of wires provided higher resistance to SCC. The developed experimental methodologies can be applied to study SCC in other reinforcement materials and the results can be used to design optimal support systems in different environmental and geotechnical conditions.

Increased coronary arteriolar contraction to serotonin in juvenile pigs with metabolic syndrome.

Metabolic syndrome (MetS) is associated with alterations in coronary vascular smooth muscle and endothelial function. The current study examined the contractile response of the isolated coronary arterioles to serotonin in pigs with and without MetS and investigated the signaling pathways responsible for serotonin-induced vasomotor tone. The MetS pigs (8-weeks old) were fed with a hyper-caloric, fat/cholesterol diet and the control animals (lean) were fed with a regular diet for 12 weeks (n = 6/group). The coronary arterioles (90-180 µm in diameter) were dissected from the harvested pig myocardial tissues and the in vitro coronary arteriolar response to serotonin was measured in the presence of pharmacological inhibitors. The protein expressions of phospholipase A2 (PLA2), TXA2 synthase, and the thromboxane-prostanoid (TP) receptor in the pigs' left ventricular tissue samples were measured using Western blotting. Serotonin (10-9-10-5 M) induced dose-dependent contractions of coronary-resistant arterioles in both non-MetS control (lean) and MetS pigs. This effect was more pronounced in the MetS vessels compared with those of non-MetS controls (lean, P < 0.05]. Serotonin-induced contraction of the MetS vessels was significantly inhibited in the presence of the selective PLA2 inhibitor quinacrine (10-6 M), the COX inhibitor indomethacin (10-5 M), and the TP receptor antagonist SQ29548 (10-6 M), respectively (P < 0.05). MetS exhibited significant increases in tissue levels of TXA2 synthase and TP receptors (P < 0.05 vs. lean), respectively. MetS is associated with increased contractile response of porcine coronary arterioles to serotonin, which is in part via upregulation/activation of PLA2, COX, and subsequent TXA2, suggesting that alteration of vasomotor function may occur at an early stage of MetS and juvenile obesity.