Characteristics of sublingual microcirculatory changes during the early postoperative period following cardiopulmonary bypass-assisted cardiac surgery-a prospective cohort study.

Background: Persistent microcirculatory dysfunction associated with increased morbidity and mortality. Interventions in the early resuscitation can be tailored to the changes of microcirculation and patient's need. However, there is usually an uncoupling of macrocirculatory and microcirculatory hemodynamics during resuscitation. Current research on the patterns of microcirculatory changes and recovery after cardiopulmonary bypass (CPB)-assisted cardiac surgery is limited. This study aimed to analyze changes in the microcirculatory parameters after CPB and their correlation with macrocirculation and to explore the characteristics of microcirculatory changes following CPB-assisted cardiac surgery. Methods: Between December 2018 and January 2019, 24 adult patients with indwelling pulmonary artery catheters after elective cardiac surgery using CPB were enrolled in this study. Both microcirculatory and macrocirculatory parameters were collected at 0, 6, 16, and 24 hours after admission to the intensive care unit (ICU). Video images of sublingual microcirculation were analyzed to obtain the microcirculatory parameters, including total vascular density (TVD), perfused small vessel density (PSVD), the proportion of perfused small vessels (PPV), microvascular flow index (MFI), and flow heterogeneity index (HI). The characteristics of microcirculatory parameter change following cardiac surgery and the correlation between microcirculatory parameters and macroscopic hemodynamic indicators, oxygen metabolic indicators, and carbon dioxide partial pressure difference (PCO2gap) were analyzed. Results: There were significant differences in the changes of TVD (P=0.012) and PSVD (P=0.005) during the first 24 hours postoperatively in patients who underwent CPB-assisted cardiac surgery. The microcirculatory density parameters (TVD: r=-0.5059, P=0.0456; PVD: r=-0.5499, P=0.0273) were correlated with oxygen delivery index (DO2I) at 24 hours after surgery. The microcirculatory flow parameters (PPV: r=0.4370, P=0.0327; MFI: r=0.6496, P=0.0006; and HI: r=-0.5350, P=0.0071) had a strong correlation with PCO2gap at 0 hour after surgery. Conclusions: TVD and PSVD might be two most sensitive indicators affected by CPB-assisted cardiac surgery. There was no consistency between microcirculation and macrocirculation until 24 hours following cardiac surgery, meaning the improvement of systemic hemodynamic indicators does not guarantee correspondently improvement in microcirculation. Early controlled oxygen supply after CPB-assisted cardiac surgery may be conducive to the resuscitation of patients to a certain extent.

Wumei Pill Ameliorates AOM/DSS-Induced Colitis-Associated Colon Cancer through Inhibition of Inflammation and Oxidative Stress by Regulating S-Adenosylhomocysteine Hydrolase- (AHCY-) Mediated Hedgehog Signaling in Mice.

Wumei Pill (WMP) is a traditional Chinese herbal formulation and widely used to treat digestive system diseases in clinical. S-Adenosylhomocysteine hydrolase (AHCY) can catalyze the hydrolysis of S-adenosylhomocysteine to adenosine and homocysteine in living organisms, and its abnormal expression is linked to the pathogenesis of many diseases including colorectal cancer (CRC). A previous study reported that WMP could prevent CRC in mice; however, the underlying mechanisms especially the roles of AHCY in WMP-induced anti-CRC remain largely unknown. Here, we investigated the regulatory roles and potential mechanisms of AHCY in WMP-induced anti-CRC. WMP notably alleviated the azoxymethane/dextran sulfate sodium- (AOM/DSS-) induced colitis-associated colon cancer (CAC) in mice. Besides, WMP inhibited the inflammation and oxidative stress in AOM/DSS-induced CAC mice. AHCY was high expression in clinical samples of colon cancer compared to the adjacent tissues. WMP inhibited the AHCY expression in AOM/DSS-induced CAC mice. An in vitro study found that AHCY overexpression induced cell proliferation, colony formation, invasion, and tumor angiogenesis, whereas its knockdown impaired its oncogenic function. AHCY overexpression enhanced, while its knockdown weakened the inflammation and oxidative stress in colon cancer cells. Interestingly, WMP potently suppressed the hedgehog (Hh) signaling in AOM/DSS-induced CAC mice. A further study showed that AHCY overexpression activated the Hh signaling while AHCY knockdown inactivated the Hh signaling. Moreover, activation of the Hh signaling reversed the effect of AHCY silencing on inflammation and oxidative stress in vitro. In conclusion, WMP alleviated the AOM/DSS-induced CAC through inhibition of inflammation and oxidative stress by regulating AHCY-mediated hedgehog signaling in mice. These findings uncovered a potential molecular mechanism underlying the anti-CAC effect of WMP and suggested WMP as a promising therapeutic candidate for CRC.

Cardiac Protection of a Novel Lupane-Type Triterpenoid from Injuries Induced by Hypoxia-Reperfusion.

Myocardial ischemia-reperfusion injury (MIRI) leads to cardiac remodeling and heart failure associated with acute myocardial infarction, which is one of the leading causes of death worldwide. Betulinic acid (BA), a widely distributed lupane-type triterpenoid, has been reported to possess antioxidative activity and inhibit apoptosis in MIRI. Due to the low bioavailability and water insolubility of BA, a previous study found a series of BA-derivative compounds by microbial transformation. In this study, we observe whether there are anti-MIRI effects of BTA07, a BA derivative, on cardiac injuries induced by hypoxia/reoxygenation (H/R) in adult rat cardiomyocytes in vitro and in Langendorff-perfused hearts ex vivo, and further explore its mechanism of cardioprotection to find more efficient BA derivatives. The hemodynamic parameters of isolated hearts were monitored and recorded by a Lab Chart system. The markers of oxidative stress and apoptosis in isolated hearts and adult rat cardiomyocytes (ARCMs) were evaluated. The expression levels of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax), protein kinase B (Akt) and phospho-Akt (pAkt, Ser473) induced by H/R were detected via Western blot. The Langendorff experiments showed that BTA07 improves hemodynamic parameters, reduces myocardium damage and infarct size, inhibits levels of myocardial tissue enzymes lactate dehydrogenase (LDH) and creatine kinase (CK) in the coronary outflow and reduces oxidative stress and the activation of caspase-3 in the myocardium. In vitro, BTA07 reduced cell death and caspase-3 activation and inhibited reactive oxygen species (ROS) generation. Furthermore, the protective effects of BTA07 were attenuated by inhibition of the PI3K/Akt signaling pathway with LY294002 in ARCMs. BTA07 protects ARCMs and isolated hearts from hypoxia-reperfusion partly by inhibiting oxidative stress and cardiomyocyte apoptosis.

Overview of piperlongumine analogues and their therapeutic potential.

Natural products have long been an important source for discovery of new drugs to treat human diseases. Piperlongumine (PL) is an amide alkaloid isolated from Piper longum L. (long piper) and other piper plants and has received widespread attention because of its diverse biological activities. A large number of PL derivatives have been designed, synthesized and assessed in many pharmacological functions, including antiplatelet aggregation, neuroprotective activities, anti-diabetic activities, anti-inflammatory activities, anti-senolytic activities, immune activities, and antitumor activities. Among them, the anti-tumor effects and application of PL and its derivatives are most extensively studied. We herein summarize the development of PL derivatives, the structure and activity relationships (SARs), and their therapeutic potential on the treatments of various diseases, especially against cancer. We also discussed the challenges and future directions associated with PL and its derivatives in these indications.

Efficacy of novel methylenecyclohexenone derivatives as TrxR inhibitors in suppressing the proliferation and metastasis of human cancer cells.

A series of mono- and di-methylenecyclohexenone derivatives, 3a-f and 4a-f, respectively, were designed and synthesized from piperlongumine (PL) and their in vitro and in vivo pharmacological properties were evaluated. A majority of the compounds exhibited a potent antiproliferative effect on five human cancer cell lines, especially those causing breast cancer. Compound 4f showed the highest antiproliferative potency among all of the compounds, almost a 10-fold higher inhibitory potency against thioredoxin reductase (TrxR) compared with PL in cells causing breast cancer. In addition, 4f was found to increase the levels of reactive oxygen species (ROS), thus leading to more potent antiproliferative effects. More importantly, the suppression assays of migration and invasion revealed that compound 4f could reverse the epithelial-mesenchymal transition induced by the transforming growth factor ß1, and exhibit prominent anti-metastasis effects. Compound 4f also showed strong inhibition potency toward solid tumors of breast cancer in vivo. Our findings show that compound 4f is a promising therapeutic candidate in the treatment of breast cancer, which, however, needs further research to be proved.

Piperlonguminine and Piperine Analogues as TrxR Inhibitors that Promote ROS and Autophagy and Regulate p38 and Akt/mTOR Signaling.

The natural products piperlongumine and piperine have been shown to inhibit cancer cell proliferation through elevation of reactive oxidative species (ROS) and eventually cell death, but only have modest cytotoxic potencies. A series of 14 novel phenylallylidenecyclohexenone analogues based on piperlongumine and piperine therefore were designed and synthesized, and their pharmacological properties were evaluated. Most of the compounds produced antiproliferative activities against five human cancer cells with IC50 values lower than those of piperlongumine and piperine. Among these, compound 9m exerted the most potent antiproliferative activity against drug-resistant Bel-7402/5-FU human liver cancer 5-FU resistant cells (IC50 = 0.8 µM), which was approximately 10-fold lower than piperlongumine (IC50 = 8.4 µM). Further, 9m showed considerably lower cytotoxicity against LO2 human normal liver epithelial cells compared to Bel-7402/5-FU. Mechanistically, compound 9m inhibited thioredoxin reductase (TrxR) activity, increased ROS levels, reduced mitochondrial transmembrane potential (MTP), and induced autophagy in Bel-7402/5-FU cells via regulation of autophagy-related proteins LC3, p62, and beclin-1. Finally, 9m activated significantly the p38 signaling pathways and suppressed the Akt/mTOR signaling pathways. In conclusion, 9m could be a promising candidate for the treatment of drug-resistant cancer cells and, as such, warrants further investigation.

Optical mapping of human embryonic stem cell-derived cardiomyocyte graft electrical activity in injured hearts.

BACKGROUND: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) show tremendous promise for cardiac regeneration, but the successful development of hESC-CM-based therapies requires improved tools to investigate their electrical behavior in recipient hearts. While optical voltage mapping is a powerful technique for studying myocardial electrical activity ex vivo, we have previously shown that intra-cardiac hESC-CM grafts are not labeled by conventional voltage-sensitive fluorescent dyes. We hypothesized that the water-soluble voltage-sensitive dye di-2-ANEPEQ would label engrafted hESC-CMs and thereby facilitate characterization of graft electrical function and integration. METHODS: We developed and validated a novel optical voltage mapping strategy based on the simultaneous imaging of the calcium-sensitive fluorescent protein GCaMP3, a graft-autonomous reporter of graft activation, and optical action potentials (oAPs) derived from di-2-ANEPEQ, which labels both graft and host myocardium. Cardiomyocytes from three different GCaMP3+ hESC lines (H7, RUES2, or ESI-17) were transplanted into guinea pig models of subacute and chronic infarction, followed by optical mapping at 2 weeks post-transplantation. RESULTS: Use of a water-soluble voltage-sensitive dye revealed pro-arrhythmic properties of GCaMP3+ hESC-CM grafts from all three lines including slow conduction velocity, incomplete host-graft coupling, and spatially heterogeneous patterns of activation that varied beat-to-beat. GCaMP3+ hESC-CMs from the RUES2 and ESI-17 lines both showed prolonged oAP durations both in vitro and in vivo. Although hESC-CMs partially remuscularize the injured hearts, histological evaluation revealed immature graft structure and impaired gap junction expression at this early timepoint. CONCLUSION: Simultaneous imaging of GCaMP3 and di-2-ANEPEQ allowed us to acquire the first unambiguously graft-derived oAPs from hESC-CM-engrafted hearts and yielded critical insights into their arrhythmogenic potential and line-to-line variation.

Development of pH/Glutathione-Responsive Theranostic Agents Activated by Glutathione S-Transferase π for Human Colon Cancer.

Two novel theranostic agents HJTA and HJTB have been designed and synthesized by covalently linking a ß-carboline derivative, with antitumor activities and pH-responsive fluorescence, with a 2-exomethylenecyclohexanone moiety, which can be activated by the tumor-targeting glutathione (GSH)/glutathione S-transferase π (GSTπ). These agents showed pH- and GSH-dual-responsive fluorescence in tumor cells but not in normal cells. Importantly, HJTA selectively illuminated tumor tissue for up to 7 h and generated precise visualization of orthotopic colonic tumors through the blood circulation system in intraoperative mice. Furthermore, HJTA exhibited potent and selective antiproliferative activities and colonic tumor inhibition in mice. Finally, HJTA induced great cancer cell apoptosis and autophagy by regulating the expression of apoptotic and autophagic proteins. Therefore, this pH/GSH-dual-responsive fluorescent probe with cancer-targeting therapeutic activity provides a novel tool for precise diagnosis and tumor treatment, therefore broadening the impact of multifunctional agents as theranostic precision medicines.

Design and synthesis of benzylidenecyclohexenones as TrxR inhibitors displaying high anticancer activity and inducing ROS, apoptosis, and autophagy.

Oxidative therapy, a strategy that specifically increases reactive oxygen species (ROS) levels in tumor cells by disrupting the redox homeostasis has gained increasing interest. The antitumor effects of the natural product piperlongumine (PL) appear to result from its ability to increase intracellular ROS levels via inhibition of antioxidative thioredoxin reductase (TrxR). Twenty-seven benzylidenecyclohexenone-based PL analogues (2a-v and 15a-e) were designed, synthesized and evaluated for their pharmacological properties. Most of the compounds exhibited potent antiproliferative activities against five human cancer cell lines, especially against breast tumor cells. One of the most promising analogueues 2c showed 12-fold higher inhibitory activity against the thioredoxin reductase (TrxR) than PL and surpressed the expression of TrxR1 protein in breast cancer cells and inhibited TrxR enzymatic activity. In addition, 2c increased ROS levels and resulted in marked apoptosis by regulating apoptosis-related proteins expressed in the breast cancer cells. Compound 2c also triggered the formation of autophagosomes and autolysosomes by promoting the expression of LC3-II and Beclin-1 and diminishing the expression of LC3-I and p62 proteins. Finally, 2c displayed low acute toxicity and good inhibitory potency to tumors in mice. Overall, 2c is a promising anti-breast cancer candidate warranting further investigation.

O-GlcNAc transferase affects the signal transduction of ß1 adrenoceptor in adult rat cardiomyocytes by increasing the O-GlcNAcylation of ß1 adrenoceptor.

O-GlcNAcylation was first found by Torres and Hart in monocytes. It is a dynamic and reversible post-translational modification catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation is increased in diabetic cardiomyopathy (DCM) patients and it has been reported that OGT plays an important role in the regulation of cardiac gene transcription, cell cycle and calcium homeostasis. The purpose of this study is to investigate the effects of OGT on signal transduction and function of ß1-adrenoceptor (ß1AR) in adult rat cardiomyocytes. We found that after overexpressing OGT by adenovirus vector in adult rat cardiomyocytes, cAMP formation and phosphorylation of phospholamban (PLB) at Ser16 (p16-PLB) were decreased under isoprenaline (ISO) stimulation. Over expression of OGT increased the intracellular [Ca2+]i and deteriorated the death of cardiomyocytes induced by prolonged stimulation with ISO. ß1-adrenoceptor was overexpressed using a plasmid vector and then co-immunoprecipitation (co-IP) followed by Western blot was employed to define the O-GlcNAcylation of ß1-adrenoceptor. The results showed that O-GlcNAcylation of ß1-adrenoceptor was increased in OGT overexpressed cells, and there was no significant change in the formation of cAMP and phosphorylation of PLB after ß1-adrenoceptor was blocked by CGP20712A. Given that OGT affects the signal transduction of ß1-adrenoceptor in adult rat cardiomyocytes by increasing the O-GlcNAcylation of ß1-adrenoceptor, the mechanism revealed in this study indicates that OGT and ß1AR may be therapeutic targets in patients undergoing diabetic cardiomyopathy.

ß-Arrestin 2 mediates arginine vasopressin-induced IL-6 induction via the ERK1/2-NF-κB signal pathway in murine hearts.

Evidence to date suggests that ß-arrestins act beyond their role as adapter proteins. Arginine vasopressin (AVP) may be a factor in inflammation and fibrosis in the pathogenesis of heart failure. In the present study we investigated the effect of AVP on inflammatory cytokine IL-6 production in murine hearts and the impact of ß-arrestin 2-dependent signaling on AVP-induced IL-6 production. We found that administration of AVP (0.5 U/kg, iv) markedly increased the levels of IL-6 mRNA in rat hearts with the maximum level occurred at 6 h. In ß-arrestin 2 KO mouse hearts, deletion of ß-arrestin 2 decreased AVP-induced IL-6 mRNA expression. We then performed in vitro experiments in adult rat cardiac fibroblasts (ARCFs). We found that AVP (10-9-10-6 M) dose-dependently increased the expression of IL-6 mRNA and protein, activation of NF-κB signaling and ERK1/2 phosphorylation, whereas knockdown of ß-arrestin 2 blocked AVP-induced IL-6 increase, NF-κB activation and ERK1/2 phosphorylation. Pharmacological blockade of ERK1/2 using PD98059 diminished AVP-induced NF-κB activation and IL-6 production. The selective V1A receptor antagonist SR49059 effectively blocked AVP-induced NF-κB phosphorylation and activation as well as IL-6 expression in ARCFs. In AVP-treated mice, pre-injection of SR49059 (2 mg/kg, iv) abolished AVP-induced NF-κB activation and IL-6 production in hearts. The above results suggest that AVP induces IL-6 induction in murine hearts via the V1A receptor-mediated ß-arrestin2/ERK1/2/NF-κB pathway, thus reveal a novel mechanism of myocardial inflammation in heart failure involving the V1A/ß-arrestin 2/ERK1/2/NF-κB signaling pathway.

O-GlcNAc Transferase Promotes Compensated Cardiac Function and Protein Kinase A O-GlcNAcylation During Early and Established Pathological Hypertrophy From Pressure Overload.

Background Protein posttranslational modifications by O-linked ß-N-acetylglucosamine (O-GlcNAc) increase with cardiac hypertrophy, yet the functional effects of these changes are incompletely understood. In other organs, O-GlcNAc promotes adaptation to acute physiological stressors; however, prolonged O-GlcNAc elevations are believed to be detrimental. We hypothesize that early O-GlcNAcylation improves cardiac function during initial response to pressure overload hypertrophy, but that sustained elevations during established pathological hypertrophy negatively impact cardiac function by adversely affecting calcium handling proteins. Methods and Results Transverse aortic constriction or sham surgeries were performed on littermate controls or cardiac-specific, inducible O-GlcNAc transferase knockout (OGTKO) mice to reduce O-GlcNAc levels. O-GlcNAc transferase deficiency was induced at different times. To evaluate the initial response to pressure overload, OGTKO was completed preoperatively and mice were followed for 2 weeks post-surgery. To assess prolonged O-GlcNAcylation during established hypertrophy, OGTKO was performed starting 18 days after surgery and mice were followed until 6 weeks post-surgery. In both groups, OGTKO with transverse aortic constriction caused significant left ventricular dysfunction. OGTKO did not affect levels of the calcium handling protein SERCA2a. OGTKO reduced phosphorylation of phospholamban and cardiac troponin I, which would negatively impact cardiac function. O-GlcNAcylation of protein kinase A catalytic subunit, a kinase for phospholamban, decreased with OGTKO. Conclusions O-GlcNAcylation promotes compensated cardiac function in both early and established pathological hypertrophy. We identified a novel O-GlcNAcylation of protein kinase A catalytic subunit, which may regulate calcium handling and cardiac function.

Cardiomyocyte PKA Ablation Enhances Basal Contractility While Eliminates Cardiac ß-Adrenergic Response Without Adverse Effects on the Heart.

RATIONALE: PKA (Protein Kinase A) is a major mediator of ß-AR (ß-adrenergic) regulation of cardiac function, but other mediators have also been suggested. Reduced PKA basal activity and activation are linked to cardiac diseases. However, how complete loss of PKA activity impacts on cardiac physiology and if it causes cardiac dysfunction have never been determined. OBJECTIVES: We set to determine how the heart adapts to the loss of cardiomyocyte PKA activity and if it elicits cardiac abnormalities. METHODS AND RESULTS: (1) Cardiac PKA activity was almost completely inhibited by expressing a PKA inhibitor peptide in cardiomyocytes (cPKAi) in mice; (2) cPKAi reduced basal phosphorylation of 2 myofilament proteins (TnI [troponin I] and cardiac myosin binding protein C), and one longitudinal SR (sarcoplasmic reticulum) protein (PLB [phospholamban]) but not of the sarcolemmal proteins (Cav1.2 α1c and PLM [phospholemman]), dyadic protein RyR2, and nuclear protein CREB (cAMP response element binding protein) at their PKA phosphorylation sites; (3) cPKAi increased the expression of CaMKII (Ca2+/calmodulin-dependent kinase II), the Cav1.2 ß subunits and current, but decreased CaMKII phosphorylation and CaMKII-mediated phosphorylation of PLB and RyR2; (4) These changes resulted in significantly enhanced myofilament Ca2+ sensitivity, prolonged contraction, slowed relaxation but increased myocyte Ca2+ transient and contraction amplitudes; (5) Isoproterenol-induced PKA and CaMKII activation and their phosphorylation of proteins were prevented by cPKAi; (6) cPKAi abolished the increases of heart rate, and cardiac and myocyte contractility by a ß-AR agonist (isoproterenol), showing an important role of PKA and a minimal role of PKA-independent ß-AR signaling in acute cardiac regulation; (7) cPKAi mice have partial exercise capability probably by enhancing vascular constriction and ventricular filling during ß-AR stimulation; and (8) cPKAi mice did not show any cardiac functional or structural abnormalities during the 1-year study period. CONCLUSIONS: PKA activity suppression induces a unique Ca2+ handling phenotype, eliminates ß-AR regulation of heart rates and cardiac contractility but does not cause cardiac abnormalities.

The PXXP domain is critical for the protective effect of BAG3 in cardiomyocytes.

Bcl-2-associated athanogene3(BAG3) protects the heart and cardiomyocytes from ischaemia/reperfusion (I/R) injury. Although the anti-apoptosis effect of BAG3 has been demonstrated in multiple cell types, the structural domain of BAG3, which is responsible for its anti-apoptosis effect, is not well understood. BAG3 protein consists of various characteristic amino acid motifs/regions that permit the interaction of BAG3 with numerous proteins involved in many cellular key pathways. The purpose of this study is to determine whether the proline-rich (PXXP) domain of BAG3 is necessary for its cellular protection against hypoxia-reoxygenation (H/R) stress by binding to its chaperone, heat shock cognate 71 kDa protein (HSC70). Cell apoptosis induced by H/R was evaluated using propidium iodide (PI) staining, caspase 3/7 activation and TUNEL staining in cultured H9C2 cells. The expression levels of BAG3 and HSC70 were manipulated, where BAG3 or its mutant, which lacked the PXXP domain, was overexpressed using a plasmid and adenovirus vector, and HSC70 expression was silenced using siRNA. Co-immunoprecipitation (co-IP) followed by western blot was employed to define the complex of BAG3 binding to its chaperones. The PXXP domain of BAG3 was determined to be critical for BAG3-mediated attenuation of H9C2 cell apoptosis induced by H/R through the binding of PXXP with HSC70. The abolished cellular protection of BAG3 induced by the knockdown of HSC70 is associated with reduced binding to HSC70. Given that the structural domain PXXP of BAG3 is necessary for the cellular protection of BAG3 from I/R injury, the mechanism revealed in this study indicates that BAG3 may be a therapeutic target in patients undergoing reperfusion after myocardial infarction.

SNPs, linkage disequilibrium, and chronic mountain sickness in Tibetan Chinese.

Chronic mountain sickness (CMS) is estimated at 1.2% in Tibetans living at the Qinghai-Tibetan Plateau. Eighteen single-nucleotide polymorphisms (SNPs) from nine nuclear genes that have an association with CMS in Tibetans have been analyzed by using pairwise linkage disequilibrium (LD). The SNPs included are the angiotensin-converting enzyme (rs4340), the angiotensinogen (rs699), and the angiotensin II type 1 receptor (AGTR1) (rs5186) from the renin-angiotensin system. A low-density lipoprotein apolipoprotein B (rs693) SNP was also included. From the hypoxia-inducible factor oxygen signaling pathway, the endothetal Per-Arnt-Sim domain protein 1 (EPAS1) and the egl nine homolog 1 (ENGL1) (rs480902) SNPs were included in the study. SNPs from the vascular endothelial growth factor (VEGF) signaling pathway included are the v-akt murine thymoma viral oncogene homolog 3 (rs4590656 and rs2291409), the endothelial cell nitric oxide synthase 3 (rs1007311 and rs1799983), and the (VEGFA) (rs699947, rs34357231, rs79469752, rs13207351, rs28357093, rs1570360, rs2010963, and rs3025039). An increase in LD occurred in 40 pairwise comparisons, whereas a decrease in LD was found in 55 pairwise comparisons between the controls and CMS patients. These changes were found to occur within and between signaling pathways, which suggests that there is an interaction between SNP alleles from different areas of the genome that affect CMS.

GRK2 Mediates Arginine Vasopressin-Induced Interleukin-6 Production via Nuclear Factor-κB Signaling Neonatal Rat Cardiac Fibroblast.

Interleukin 6 (IL-6), which is elevated in patients with congestive heart failure and acts as both a chronic marker of inflammation and an acute-phase reactant, is associated with myocardial damage. Circulating levels of arginine vasopressin (AVP) are elevated during cardiac stress and could be a factor for cardiac inflammation and fibrosis. Our previous study has shown that AVP promotes the proliferation of neonatal rat cardiac fibroblasts (NRCFs) throughV1A vasopressin receptor-mediated G protein-coupled receptor kinase 2 (GRK2) signaling. In the present study, we investigated the impact of the GRK2-dependent signaling. Using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, we measured the levels of interleukin-6 (IL-6) mRNA and protein in NRCFs, respectively. Manipulation of GRK2 activation either pharmacologically or through overexpression of GRK2-ct was used to determine the role of GRK2 in regulating the effects of AVP on IL-6 production. Phosphorylation and activation of nuclear factor κ-B (NF-κB) evoked by AVP stimulation were measured by immunoblot and NF-kB luciferase reporter gene transfected in NRCFs, respectively. Present studies have found that: 1) AVP increased the level of IL-6 protein and mRNA in a dose- and time-dependent manner in NRCFs; 2) inhibition of GRK2 abolished the AVP-induced IL-6 production and NF-κB activation; and 3) blocking NF-κB signaling using the pharmacologic approach diminished AVP-induced IL-6 production. In summary, AVP induces IL-6 production of NRCFs by activating V1A receptor signaling via a GRK2/NF-κB pathway. These findings provide a possible molecular mechanism for inflammation that occurs in heart failure and other types of cardiac stress.

Vasopressin V1A receptor mediates cell proliferation through GRK2-EGFR-ERK1/2 pathway in A7r5 cells.

Abnormal proliferation and hypertrophy of vascular smooth muscle (VSMC), as the main structural component of the vasculature, is an important pathological mechanism of hypertension. Recently, increased levels of arginine vasopressin (AVP) and copeptin, the C-terminal fragment of provasopressin, have been shown to correlate with the development of preeclampsia. AVP targets on the Gq-coupled vasopressin V1A receptor and the Gs-coupled V2 receptor in VSMC and the kidneys to regulate vascular tone and water homeostasis. However, the role of the vasopressin receptor on VSM cell proliferation during vascular remodeling is unclear. Here, we studied the effects of AVP on the proliferation of the rat VSMC-derived A7r5 cells. AVP, in a time- and concentration-dependent manner, promoted A7r5 cell proliferation as indicated by the induction of proliferating cell nuclear antigen expression, methylthiazolyldiphenyl-tetrazolium reduction and incorporation of 5'-bromodeoxyuridine into cellular DNA. These effects, coupled with the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), were blocked by a V1A receptor antagonist SR45059 but not by a V2 receptor antagonist lixivaptan. Although acute activation of V1A receptor induced ERK1/2 phosphorylation via a protein kinase C-dependent pathway, this effect was not involved in cell proliferation. Cell proliferation and ERK1/2 phosphorylation in response to prolonged stimulation with AVP were abolished by inhibition of G protein-coupled receptor kinase 2 (GRK2) and epidermal growth factor receptor (EGFR) using specific inhibitors or small hairpin RNA knock-down. These results suggest that activation of V1A, but not V2 receptor, produces a cell proliferative signal in A7r5 cells via a GRK2/EGFR/ERK1/2-dependent mechanism.

ß-adrenergic receptor-mediated cardiac contractility is inhibited via vasopressin type 1A-receptor-dependent signaling.

BACKGROUND: Enhanced arginine vasopressin levels are associated with increased mortality during end-stage human heart failure, and cardiac arginine vasopressin type 1A receptor (V1AR) expression becomes increased. Additionally, mice with cardiac-restricted V1AR overexpression develop cardiomyopathy and decreased ß-adrenergic receptor (ßAR) responsiveness. This led us to hypothesize that V1AR signaling regulates ßAR responsiveness and in doing so contributes to development of heart failure. METHODS AND RESULTS: Transaortic constriction resulted in decreased cardiac function and ßAR density and increased cardiac V1AR expression, effects reversed by a V1AR-selective antagonist. Molecularly, V1AR stimulation led to decreased ßAR ligand affinity, as well as ßAR-induced Ca(2+) mobilization and cAMP generation in isolated adult cardiomyocytes, effects recapitulated via ex vivo Langendorff analysis. V1AR-mediated regulation of ßAR responsiveness was demonstrated to occur in a previously unrecognized Gq protein-independent/G protein receptor kinase-dependent manner. CONCLUSIONS: This newly discovered relationship between cardiac V1AR and ßAR may be informative for the treatment of patients with acute decompensated heart failure and elevated arginine vasopressin.

Methods for assessing the electromechanical integration of human pluripotent stem cell-derived cardiomyocyte grafts.

Cardiomyocytes derived from human pluripotent stem cells show tremendous promise for the replacement of myocardium and contractile function lost to infarction. However, until recently, no methods were available to directly determine whether these stem cell-derived grafts actually couple with host myocardium and fire synchronously following transplantation in either intact or injured hearts. To resolve this uncertainty, our group has developed techniques for the intravital imaging of hearts engrafted with stem cell-derived cardiomyocytes that have been modified to express the genetically encoded protein calcium sensor, GCaMP. When combined with the simultaneously recorded electrocardiogram, this protocol allows one to make quantitative assessments as to the presence and extent of host-graft electrical coupling as well as the timing and pattern of graft activation. As described here, this system has been employed to investigate the electromechanical integration of human embryonic stem cell-derived cardiomyocytes in a guinea pig model of cardiac injury, but analogous approaches should be applicable to other human graft cell types and animal models.

VEGFA SNPs and transcriptional factor binding sites associated with high altitude sickness in Han and Tibetan Chinese at the Qinghai-Tibetan Plateau.

Mountain sickness (MS) occurs among humans visiting or inhabiting high altitude environments. We conducted genetic analyses of seven single nucleotide polymorphisms (SNPs) in the promoter region of VEGFA gene for lowland (Han) and highland (Tibetan) Chinese. The seven SNPs were evaluated in Han and Tibetan patients with acute (A) and chronic (C) MS. We compared 64 patients with AMS with 64 Han unaffected with MS, as well as 48 CMS patients with 32 unaffected Tibetans. The SNPs studied are rs699947, rs34357231, rs79469752, rs13207351, rs28357093, rs1570360, and rs2010963 which are found in the promoter ranging from -2,578 to -634 bp from the transcriptional start site (TSS), respectively. Direct sequencing was used to identify individual genotypes for these SNPs. Arterial oxygen saturation of hemoglobin (SaO2) was found to be significantly associated with the rs699947, rs34357231, rs13207351, and rs1570360 SNPs in Han patients with AMS, while the rs2010963 SNP was found to approach significance in the AMS study group, but found to be significantly associated in the normal Tibetan study group. The Han and Tibetan control groups were found to diverge significantly for the rs28357093 and rs2010963 SNPs, as measured by genetic distances of 0.073 and 0.054, respectively. All the SNPs are found in transcriptional factor binding sites (TFBS), and their possible role in gene regulation was evaluated with regard to MS. MS was found to be significantly associated with these SNPs compared with their Han and Tibetan control groups, indicating that these nucleotide substitutions result in TFBS changes which apparently have a physiological effect on the development of high altitude sickness.