AKAP12 Upregulation Associates With PDE8A to Accelerate Cardiac Dysfunction.

BACKGROUND: In heart failure, signaling downstream the ß2-adrenergic receptor is critical. Sympathetic stimulation of ß2-adrenergic receptor alters cAMP (cyclic adenosine 3',5'-monophosphate) and triggers PKA (protein kinase A)-dependent phosphorylation of proteins that regulate cardiac function. cAMP levels are regulated in part by PDEs (phosphodiesterases). Several AKAPs (A kinase anchoring proteins) regulate cardiac function and are proposed as targets for precise pharmacology. AKAP12 is expressed in the heart and has been reported to directly bind ß2-adrenergic receptor, PKA, and PDE4D. However, its roles in cardiac function are unclear. METHODS: cAMP accumulation in real time downstream of the ß2-adrenergic receptor was detected for 60 minutes in live cells using the luciferase-based biosensor (GloSensor) in AC16 human-derived cardiomyocyte cell lines overexpressing AKAP12 versus controls. Cardiomyocyte intracellular calcium and contractility were studied in adult primary cardiomyocytes from male and female mice overexpressing cardiac AKAP12 (AKAP12OX) and wild-type littermates post acute treatment with 100-nM isoproterenol (ISO). Systolic cardiac function was assessed in mice after 14 days of subcutaneous ISO administration (60 mg/kg per day). AKAP12 gene and protein expression levels were evaluated in left ventricular samples from patients with end-stage heart failure. RESULTS: AKAP12 upregulation significantly reduced total intracellular cAMP levels in AC16 cells through PDE8. Adult primary cardiomyocytes from AKAP12OX mice had significantly reduced contractility and impaired calcium handling in response to ISO, which was reversed in the presence of the selective PDE8 inhibitor (PF-04957325). AKAP12OX mice had deteriorated systolic cardiac function and enlarged left ventricles. Patients with end-stage heart failure had upregulated gene and protein levels of AKAP12. CONCLUSIONS: AKAP12 upregulation in cardiac tissue is associated with accelerated cardiac dysfunction through the AKAP12-PDE8 axis.

Functional LTCC-ß2AR Complex Needs Caveolin-3 and Is Disrupted in Heart Failure.

BACKGROUND: Beta-2 adrenergic receptors (ß2ARs) but not beta-2 adrenergic receptors (ß1ARs) form a functional complex with L-type Ca2+ channels (LTCCs) on the cardiomyocyte membrane. However, how microdomain localization in the plasma membrane affects the function of these complexes is unknown. We aim to study the coupling between LTCC and ß adrenergic receptors in different cardiomyocyte microdomains, the distinct involvement of PKA and CAMKII (Ca2+/calmodulin-dependent protein kinase II) and explore how this functional complex is disrupted in heart failure. METHODS: Global signaling between LTCCs and ß adrenergic receptors was assessed with whole-cell current recordings and western blot analysis. Super-resolution scanning patch-clamp was used to explore the local coupling between single LTCCs and ß1AR or ß2AR in different membrane microdomains in control and failing cardiomyocytes. RESULTS: LTCC open probability (Po) showed an increase from 0.054±0.003 to 0.092±0.008 when ß2AR was locally stimulated in the proximity of the channel (<350 nm) in the transverse tubule microdomain. In failing cardiomyocytes, from both rodents and humans, this transverse tubule coupling between LTCC and ß2AR was lost. Interestingly, local stimulation of ß1AR did not elicit any change in the Po of LTCCs, indicating a lack of proximal functional interaction between the two, but we confirmed a general activation of LTCC via ß1AR. By using blockers of PKA and CaMKII and a Caveolin-3-knockout mouse model, we conclude that the ß2AR-LTCC regulation requires the presence of caveolin-3 and the activation of the CaMKII pathway. By contrast, at a cellular "global" level PKA plays a major role downstream ß1AR and results in an increase in LTCC current. CONCLUSIONS: Regulation of the LTCC activity by proximity coupling mechanisms occurs only via ß2AR, but not ß1AR. This may explain how ß2ARs tune the response of LTCCs to adrenergic stimulation in healthy conditions. This coupling is lost in heart failure; restoring it could improve the adrenergic response of failing cardiomyocytes.

Norepinephrine/ß2-Adrenergic Receptor Pathway Promotes the Cell Proliferation and Nerve Growth Factor Production in Triple-Negative Breast Cancer.

PURPOSE: Invasive ductal carcinoma (IDC) accounts for 90% of triple-negative breast cancer (TNBC). IDC is mainly derived from the breast ductal epithelium which is innervated by the 4th to 6th thoracic sympathetic nerves. However, little is known about the contribution of the interactions between sympathetic nerves and breast cancer cells to the malignant progression of TNBC. METHODS: The expression levels of the ß2-adrenergic receptor (ß2-AR, encoded by ADRB2 gene), nerve growth factor (NGF), and tropomyosin receptor kinase A (TrkA) were determined using immunohistochemistry (IHC). NGF expression levels in the serum were compared by enzyme-linked immunosorbent assay (ELISA). Cell proliferation was assessed using the Cell Counting Kit-8 assay. The ß2-AR, NGF, p-ERK, and p-CERB expression levels were determined using western blotting. TNBC cells and neuronal cells of the dorsal root ganglion (DRG) in 2-day-old Sprague Dawley rats were co-cultured. Using norepinephrine (NE), NGF, and ß2-AR, NGF/TrkA blocker pretreatments, the axon growth of each group of DRG neuron cells was detected by immunofluorescence analysis. RESULTS: The sympathetic adrenergic neurotransmitter NE activated the ERK signaling pathway in TNBC cells. NE/ß2-AR signaling promotes NGF secretion. NGF further facilitates the malignant progression of TNBC by increasing sympathetic neurogenesis. In the co-culture assay, the sympathetic adrenergic NE/ß2-AR signal pathway also enhanced NGF secretion. NGF binds TrkA in DRG neurons and promotes axonal growth. CONCLUSION: These results suggest that NE/ß2-AR pathway promotes cell proliferation and NGF production in triple-negative breast cancer.

Silymarin Administration Attenuates Cirrhotic-induced Cardiac Abnormality in the Rats: A Possible Role of ß1-adrenergic Receptors and L-type Voltage-Dependent Calcium Channels.

Background: Cirrhotic cardiomyopathy is a well-recognized cardiac dysfunction in cirrhotic patients. Studies have confirmed the protective effects of silymarin in different types of cardiac injury. This study aimed to examine the effectiveness and molecular mechanism of silymarin against myocardial dysfunction and hypertrophy in a rat model of cirrhosis. Methods: The experiment was performed at Alborz University of Medical Sciences (Karaj, Iran) during 2020-2021. Thirty-two male Wistar rats were randomly divided into four groups of Sham-operated (control group for surgical procedures), Bile Duct Ligated (BDL), and two Silymarin extract (SE)-treated groups of 300 and 600 mg/Kg/day. After 28 days, serum levels of AST, ALT, GGT, and ALP, liver histopathological status, as well as cardiac mechanical function, were assessed. Cardiac ß1-adrenergic receptors (ß1-AR), L-type voltage-dependent calcium channels (L-VDCC), and GATA4 mRNA expression were also determined using real-time RT-PCR. Data analysis was performed using the one-way ANOVA followed by Duncan's multiple range test. Histological data has been analyzed with Kruskal-Wallis nonparametric test. The analysis was performed at P≤0.05. Results: BDL was associated with a significant elevation in serum AST, ALT, GGT, and ALP, development of necrosis and fibrosis of the liver texture, increased Heart Weight and Heart Weight to Body Weight ratio, enhanced cardiac mechanical function as well as a significant up-regulation of ventricular ß1-AR and L-VDCC. Administration of SE600, but not SE300, significantly reduced the serum levels of the enzymes and alleviated signs of liver necrosis and fibrosis. Cirrhotic-induced cardiac dysfunction was also restored by SE600, but not by the lower dose. In addition, cardiac expression of the ß1-AR and L-VDCC was down-regulated toward normal values by either higher or lower doses of the SE. Conclusion: Silymarin treatment in higher dose attenuated cirrhosis-associated cardiac remodeling and reduced cardiac mechanical dysfunctions.

Exercise Causes Arrhythmogenic Remodeling of Intracellular Calcium Dynamics in Plakophilin-2-Deficient Hearts.

BACKGROUND: Exercise training, and catecholaminergic stimulation, increase the incidence of arrhythmic events in patients affected with arrhythmogenic right ventricular cardiomyopathy correlated with plakophilin-2 (PKP2) mutations. Separate data show that reduced abundance of PKP2 leads to dysregulation of intracellular Ca2+ (Ca2+i) homeostasis. Here, we study the relation between excercise, catecholaminergic stimulation, Ca2+i homeostasis, and arrhythmogenesis in PKP2-deficient murine hearts. METHODS: Experiments were performed in myocytes from a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout murine line (PKP2cKO). For training, mice underwent 75 minutes of treadmill running once per day, 5 days each week for 6 weeks. We used multiple approaches including imaging, high-resolution mass spectrometry, electrocardiography, and pharmacological challenges to study the functional properties of cells/hearts in vitro and in vivo. RESULTS: In myocytes from PKP2cKO animals, training increased sarcoplasmic reticulum Ca2+ load, increased the frequency and amplitude of spontaneous ryanodine receptor (ryanodine receptor 2)-mediated Ca2+ release events (sparks), and changed the time course of sarcomeric shortening. Phosphoproteomics analysis revealed that training led to hyperphosphorylation of phospholamban in residues 16 and 17, suggesting a catecholaminergic component. Isoproterenol-induced increase in Ca2+i transient amplitude showed a differential response to ß-adrenergic blockade that depended on the purported ability of the blockers to reach intracellular receptors. Additional experiments showed significant reduction of isoproterenol-induced Ca2+i sparks and ventricular arrhythmias in PKP2cKO hearts exposed to an experimental blocker of ryanodine receptor 2 channels. CONCLUSIONS: Exercise disproportionately affects Ca2+i homeostasis in PKP2-deficient hearts in a manner facilitated by stimulation of intracellular ß-adrenergic receptors and hyperphosphorylation of phospholamban. These cellular changes create a proarrhythmogenic state that can be mitigated by ryanodine receptor 2 blockade. Our data unveil an arrhythmogenic mechanism for exercise-induced or catecholaminergic life-threatening arrhythmias in the setting of PKP2 deficit. We suggest that membrane-permeable ß-blockers are potentially more efficient for patients with arrhythmogenic right ventricular cardiomyopathy, highlight the potential for ryanodine receptor 2 channel blockers as treatment for the control of heart rhythm in the population at risk, and propose that PKP2-dependent and phospholamban-dependent arrhythmogenic right ventricular cardiomyopathy-related arrhythmias have a common mechanism.

Effects of gene polymorphisms of β1 adrenergic receptor G1165C and A145G locus on myocardial hypertrophy and the efficacy in patients with hypertension / 中国医师进修杂志

Objective:To investigate the effect of gene polymorphism of β1 adrenergic receptor (β1-AR) G1165C and A145G locus on myocardial hypertrophy and the efficacy in patients with hypertension.Methods:Two hundred and twenty-seven cases of patients with hypertension admitted to Binhai County People′s Hospital from January to December 2019 were enrolled. Among them, there were 113 cases of hypertension with myocardial hypertrophy and 114 cases of hypertension without myocardial hypertrophy. In addition, 115 patients with normal physical examination during the same period were selected as the control group. DNA in the peripheral blood leukocytes was extracted, polymerase chain reaction-restriction fragment length polymorphism method was used to detect β1-AR G1165C and A145G locus gene polymorphism, and the differences in the efficacy of β blockers in hypertensive patients with different genotypes were compared.Results:There was no statistically significant differences in the distribution of β1-AR A145G genotypes among the three groups ( P>0.05). Compared with the healthy control group, the frequency of Gly/Gly genotype carrying β1-AR G1165C locus was higher in hypertension with myocardial hypertrophy group, and the frequency of Gly/Arg and Arg/Arg gene were lower; compared with hypertension without myocardial hypertrophy group, the frequency of Gly/Arg+Gly/Gly gene in hypertension with myocardial hypertrophy group was higher; taking Arg/Arg genotype as the control group, carrying Gly/Gly genotype could increase the risk of cardiac hypertrophy in hypertensive patients by 3.159 times ( OR = 3.159, 95% CI 1.240 - 7.412, P<0.05).The frequency of G1165C allele Arg in the hypertension with myocardial hypertrophy group was significantly lower than that in the control group and the hypertension without myocardial hypertrophy group ( P<0.05); the frequency of G1165C allele Gly was significantly higher than that in the control group and the hypertension without myocardial hypertrophy group ( P<0.05); taking Arg/Arg genotype as the control, carrying Gly/Gly genotype could increase the risk of cardiac hypertrophy in hypertensive ( OR = 3.417, 95% CI 1.357 - 7.965, P<0.05). The left ventricular mass index of Gly/Gly genotype patients was (120.38 ± 28.41) g/m 2, which was significantly higher than (99.76 ± 25.16) g/m 2 and (90.30 ± 19.54) g/m 2 of Gly/Arg and Arg/Arg, with statistically significant differences ( F = 10.89, P<0.01). After the treatment, the resting heart rate, systolic blood pressure, diastolic blood pressure and mean arterial blood pressure of patients with G1165C allele Arg hypertension with myocardial hypertrophy were lower than those with G1165C allele Gly, with statistically significant differences ( P<0.05). Conclusions:β1-AR G1165C gene polymorphism is related to the risk of myocardial hypertrophy in hypertensive patients. Carrying the G1165C allele Gly may increase the risk of susceptibility to cardiac hypertrophy, and β-blockers are more effective in hypertensive patients with myocardial hypertrophy who carry the G1165C allele Arg.

Association between beta-2 adrenergic receptor variants and clinical outcomes in children and adolescents with acute asthma

ABSTRACT Objective To investigate whether different genotypes of p.Arg16Gly, p.Gln27Glu, p.Arg19Cys and p.Thr164Ile variants interfere in response to treatment in children and adolescents with moderate to severe acute asthma. Methods This sample comprised patients aged 2 to 17 years with a history of at least two wheezing episodes and current moderate to severe asthma exacerbation. All patients received multiple doses of albuterol and ipratropium bromide delivered via pressurized metered-dose inhaler with holding chamber and systemic corticosteroids. Hospital admission was defined as the primary outcome. Secondary outcomes were changes in forced expiratory volume in the first second after 1 hour of treatment, and for outpatients, length of stay in the emergency room. Variants were genotyped by sequencing. Results A total of 60 patients were evaluated. Hospital admission rates were significantly higher in carriers of the genotype AA relative to those with genotype AG or GG, within the p.Arg16Gly variant (p=0.03, test χ2, alpha=0.05). Secondary outcomes did not differ between genotypes. Conclusion Hospital admission rates were significantly higher among carriers of the genotype AA within the p.Arg16Gly variant. Trial registration: ClinicalTrials.gov: NCT01323010

Heterodimerization With 5-HT2BR Is Indispensable for ß2AR-Mediated Cardioprotection.

RATIONALE: The ß2-adrenoceptor (ß2-AR), a prototypical GPCR (G protein-coupled receptor), couples to both Gs and Gi proteins. Stimulation of the ß2-AR is beneficial to humans and animals with heart failure presumably because it activates the downstream Gi-PI3K-Akt cell survival pathway. Cardiac ß2-AR signaling can be regulated by crosstalk or heterodimerization with other GPCRs, but the physiological and pathophysiological significance of this type of regulation has not been sufficiently demonstrated. OBJECTIVE: Here, we aim to investigate the potential cardioprotective effect of ß2-adrenergic stimulation with a subtype-selective agonist, (R,R')-4-methoxy-1-naphthylfenoterol (MNF), and to decipher the underlying mechanism with a particular emphasis on the role of heterodimerization of ß2-ARs with another GPCR, 5-hydroxytryptamine receptors 2B (5-HT2BRs). METHODS AND RESULTS: Using pharmacological, genetic and biophysical protein-protein interaction approaches, we studied the cardioprotective effect of the ß2-agonist, MNF, and explored the underlying mechanism in both in vivo in mice and cultured rodent cardiomyocytes insulted with doxorubicin, hydrogen peroxide (H2O2) or ischemia/reperfusion. In doxorubicin (Dox)-treated mice, MNF reduced mortality and body weight loss, while improving cardiac function and cardiomyocyte viability. MNF also alleviated myocardial ischemia/reperfusion injury. In cultured rodent cardiomyocytes, MNF inhibited DNA damage and cell death caused by Dox, H2O2 or hypoxia/reoxygenation. Mechanistically, we found that MNF or another ß2-agonist zinterol markedly promoted heterodimerization of ß2-ARs with 5-HT2BRs. Upregulation of the heterodimerized 5-HT2BRs and ß2-ARs enhanced ß2-AR-stimulated Gi-Akt signaling and cardioprotection while knockdown or pharmacological inhibition of the 5-HT2BR attenuated ß2-AR-stimulated Gi signaling and cardioprotection. CONCLUSIONS: These data demonstrate that the ß2-AR-stimulated cardioprotective Gi signaling depends on the heterodimerization of ß2-ARs and 5-HT2BRs.

Beta-adrenergic pathway activation in head and neck cancer: a comprehensive analysis of the clinical and genomic features using integrated bioinformatics / Ativação da via beta-adrenérgica no câncer de cabeça e pescoço: uma análise abrangente das características clínicas e gênomicas utilizando a bioinformática

O estresse crônico leva à ativação da via de sinalização beta-adrenérgica. Sua ativação tem sido implicada na progressão de diferentes tipos de câncer, mas seu papel nos carcinomas espinocelulares de cabeça e pescoço (CECPs) permanece indefinido. O objetivo deste estudo foi investigar o papel da ativação da via betaadrenérgica na progressão dos CECPs, avaliar seu impacto na sobrevida dos pacientes e buscar possíveis terapias para pacientes que encontravam-se com a via beta-adrenérgica ativa. Quinhentos e vinte pacientes do The Cancer Genome Atlas com CECPs primários foram divididos em dois grupos: ADRB2baixa / SLC6A2baixa e ADRB2alta / SLC6A2alta. A associação de características clinicopatológicas e genômicas entre os grupos foram analisadas utilizando bioinformática. Os genes diferencialmente expressos (DEGs) foram identificados através da análise da expressão diferencial. A análise de sobrevida também foi realizada com base nas expressões ADRB2 e SLC6A2. Foram identificados medicamentos em potencial para tratamento de CECPs com base nos DEGs. Houve associação entre as expressões ADRB2 e SLC6A2 com idade, raça, localização do tumor, grau histológico, invasão perineural e status do HPV p16. Foram identificados 898 DEGs entre os grupos. Foi demonstrado que a expressão ADRB2alta / SLC6A2alta influenciou a proliferação, adesão e invasão de células CECPs além da angiogênese. Pacientes com carcinomas espinocelular de laringe e faringe apresentando expressão ADRB2alta / SLC6A2alta tiveram menor sobrevida. Por fim, 56 drogas antineoplásicas e imunoterápicas aprovadas pelo Food Drugs Administration foram identificadas como potenciais alvos para o tratamento personalizado. Significância: Estes achados sugerem fortemente um papel proeminente da sinalização beta-adrenérgica no CECPs ao estimular um fenótipo tumoral mais agressivo. Estas alterações tiveram um impacto negativo no prognóstico dos pacientes com CECP em região de faringe e laringe(AU)

Chronic stress leads to the activation of the beta-adrenergic pathway. Its activation has been implicated in the progression of different types of cancer but its role on head and neck squamous cell carcinomas (HNSCCs) remains undefined. The aim of this study was to investigate the influence of the beta-adrenergic pathway activation in the progression of HNSCCs, assess its impact in the survival of the patients, and explore the potential targets. Five hundred and twenty The Cancer Genome Altas patients with primary HNSCCs were divided in two groups: ADRB2low / SLC6A2low and ADRB2high / SLC6A2high. The association of clinicopathological and genomic features between the groups was analyzed using a bioinformatic approach. Differentially expressed genes (DEGs) were identified through differential expression analysis. Survival analysis was also performed based on ADRB2 and SLC6A2 expressions. Potential drugs for treatment of HNSCC were identified based on the DEGs. There was association between ADRB2 and SLC6A2 expressions with age, race, tumor site, histologic grade, perineural invasion, and HPV p16 status. It was identified 898 DEGs between the groups. It was demonstrated that ADRB2high / SLC6A2high expression influenced HNSCC cells proliferation, adhesion, invasion, and angiogenesis. Patients with larynx and pharynx squamous cell carcinomas presenting ADRB2high / SLC6A2high expression showed had lower survival rates. Finally, 56 Food Drugs Administration-approved antineoplastic and immunotherapeutic drugs were identified as potential targets for the personalized treatment. Significance: These findings strongly suggest a prominent role of beta-adrenergic pathway in HNSCC by stimulating a more aggressive tumoral phenotype. These alterations were shown to negatively impact the prognosis of patients with larynx and pharynx squamous cell carcinomas(AU)

Labetalol Use Is Associated With Increased In-Hospital Infection Compared With Nicardipine Use in Intracerebral Hemorrhage.

BACKGROUND AND PURPOSE: Increased sympathetic tone causes hypertension after intracerebral hemorrhage, and blood pressure reduction has been studied as a way to decrease hemorrhage growth and improve outcomes. It is unknown if the antihypertensive used to achieve blood pressure goals influences either. Because sympatholytic drugs reduce death and infection in animal models, we hypothesized that labetalol would improve outcomes compared with nicardipine. METHODS: Prospective data from a single center were retrospectively reviewed. Patients receiving labetalol, nicardipine, or both during their first 3 days of hospitalization were included. Outcomes included in-hospital death; discharge modified Rankin Score >2; and in-hospital urinary tract infection, pneumonia, or bacteremia. Patients were compared with propensity scoring and analyzed with linear models adjusted for significant confounders. RESULTS: Of 1066 admissions, 525 were treated with labetalol or nicardipine and are included; 229 (43.6%) received labetalol, 107 (20.4%) received nicardipine, and 189 (36.0%) received both. Mortality and infection rates were 40.2% and 15.8%, respectively, 77.2% had a modified Rankin Score >2. After adjustment, compared with nicardipine alone, labetalol alone was associated with infection (odds ratio, 3.12; confidence interval, 1.27-7.64; P=0.013) but not when combined with nicardipine (odds ratio, 2.44; confidence interval, 0.98-6.07; P=0.055). Labetalol, with or without nicardipine, was not associated with death or discharge modified Rankin Score >2. CONCLUSIONS: Compared with nicardipine, labetalol was associated with increased in-hospital infections, but not mortality or modified Rankin Score >2. These findings do not support our hypothesis that labetalol use improves outcomes relative to nicardipine in intracerebral hemorrhage.

Adrenergic Receptors in Individual Ventricular Myocytes: The Beta-1 and Alpha-1B Are in All Cells, the Alpha-1A Is in a Subpopulation, and the Beta-2 and Beta-3 Are Mostly Absent.

RATIONALE: It is unknown whether every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), ß1, ß2, ß3, α1A, and α1B. The ß1 and ß2 are thought to be the dominant myocyte ARs. OBJECTIVE: Quantify the 5 cardiac ARs in individual ventricular myocytes. METHODS AND RESULTS: We studied ventricular myocytes from wild-type mice, mice with α1A and α1B knockin reporters, and ß1 and ß2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of extracellular signal-regulated kinase and phospholamban), and contraction. We found that the ß1 and α1B were present in all myocytes. The α1A was present in 60%, with high levels in 20%. The ß2 and ß3 were detected in only ≈5% of myocytes, mostly in different cells. In intact heart, 30% of total ß-ARs were ß2 and 20% were ß3, both mainly in nonmyocytes. CONCLUSION: The dominant ventricular myocyte ARs present in all cells are the ß1 and α1B. The ß2 and ß3 are mostly absent in myocytes but are abundant in nonmyocytes. The α1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with ß1 and α1B only; 60% that also have the α1A; and 5% each that also have the ß2 or ß3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms.

Nucleoside Diphosphate Kinase-C Suppresses cAMP Formation in Human Heart Failure.

BACKGROUND: Chronic heart failure (HF) is associated with altered signal transduction via ß-adrenoceptors and G proteins and with reduced cAMP formation. Nucleoside diphosphate kinases (NDPKs) are enriched at the plasma membrane of patients with end-stage HF, but the functional consequences of this are largely unknown, particularly for NDPK-C. Here, we investigated the potential role of NDPK-C in cardiac cAMP formation and contractility. METHODS: Real-time polymerase chain reaction, (far) Western blot, immunoprecipitation, and immunocytochemistry were used to study the expression, interaction with G proteins, and localization of NDPKs. cAMP levels were determined with immunoassays or fluorescent resonance energy transfer, and contractility was determined in cardiomyocytes (cell shortening) and in vivo (fractional shortening). RESULTS: NDPK-C was essential for the formation of an NDPK-B/G protein complex. Protein and mRNA levels of NDPK-C were upregulated in end-stage human HF, in rats after long-term isoprenaline stimulation through osmotic minipumps, and after incubation of rat neonatal cardiomyocytes with isoprenaline. Isoprenaline also promoted translocation of NDPK-C to the plasma membrane. Overexpression of NDPK-C in cardiomyocytes increased cAMP levels and sensitized cardiomyocytes to isoprenaline-induced augmentation of contractility, whereas NDPK-C knockdown decreased cAMP levels. In vivo, depletion of NDPK-C in zebrafish embryos caused cardiac edema and ventricular dysfunction. NDPK-B knockout mice had unaltered NDPK-C expression but showed contractile dysfunction and exacerbated cardiac remodeling during long-term isoprenaline stimulation. In human end-stage HF, the complex formation between NDPK-C and Gαi2 was increased whereas the NDPK-C/Gαs interaction was decreased, producing a switch that may contribute to an NDPK-C-dependent cAMP reduction in HF. CONCLUSIONS: Our findings identify NDPK-C as an essential requirement for both the interaction between NDPK isoforms and between NDPK isoforms and G proteins. NDPK-C is a novel critical regulator of ß-adrenoceptor/cAMP signaling and cardiac contractility. By switching from Gαs to Gαi2 activation, NDPK-C may contribute to lower cAMP levels and the related contractile dysfunction in HF.

β3-adrenoceptor activation promotes cholesterol efflux from foam cells by upregulating apoA-Ⅰ expression / 中华老年心脑血管病杂志

Objective To study the mechanism of β3 adrenoceptor (β3-AR) activation underlying cholesterol efflux by activating or inhibiting the β3-AR of HepG2 cells.Methods Cultured HepG2 cells were randomly divided into control group,β3-AR agonist group and β3-AR antagonist group.Serum levels of apoA-Ⅰ,apoA-Ⅱ,and β3-AR in supernatant fluid,and cholesterol,free cholesterol,cholesterol ester in HepG2 cells were measured by ELISA.Cholesterol efflux from macrophages was tested by 3H-labled cholesterol.Expressions of ABCA1 and LXRα mRNA and protein were detected by RT-PCR and Western blot respectively.Results The efflux rate of apoA-Ⅰ,cholesterol and cholesterol ester was significantly higher while the serum levels of cholesterol and cholesterol ester were significantly lower and the expression levels of ABCA1 and LXRα mRNA and protein were significantly higher in β3 AR agonist group than in control group.The serum levels of cholesterol and cholesterol ester were significantly higher while the efflux rate of cholesterol and cholesterol ester and the expression levels of ABCA1 and LXRα mRNA and protein were significantly lower in β3-AR antagonist groupt than in β3-AR agonist group (0.49±0.10 vs 1.24±0.02,0.85±0.05 vs 1.32±0.05,0.38±0.01 vs 1.45±0.20,0.08±0.01 vs 0.76±0.02,P＜0.01).Conclusion β3 AR promotes cholesterol efflux by upregulating the expression of apoA-Ⅰin HepG2 cells.

In vitro effects of propranolol and isoproterenol on the expression of beta-2 adrenergic receptor on infantile hemangioma endothelial cells / 中华皮肤科杂志

Objective To explore the role of beta-2 adrenergic receptor in the pathogenesis of infantile hemangioma.Methods In vitro cultured infantile hemangioma endothelial cells were divided into propranolol and isoproterenol groups.The propranolol groups were further classified into 5 groups to be treated with propranolol solutions at concentrations of 10,15,20 μg/ml,EGM-2 medium (blank control group 1),and EGM-2 medium containing 0.16％ DMSO (DMSO group) respectively,while the isoproterenol groups were classified into 4 groups to be treated with isoproterenol solutions at concentrations of 5,10,20 μg/ml and EGM-2 medium (blank control group 2) respectively.After 24-and 48-hour treatment,enzyme-linked immunosorbent assay (ELISA) was performed to measure the expression of beta-2 adrenergic receptor in cell culture supernatants in the above groups.Results After 24-hour treatment,15-μg/ml and 20-μg/ml propranolol groups showed significantly decreased expression of beta-2 adrenergic receptor compared with blank control group 1 and DMSO group (all P ＜ 0.05).After 48-hour treatment,all the propranolol groups showed significantly decreased expression of beta-2 adrenergic receptor compared with the blank control group 1 (all P ＜ 0.05).However,the expression of beta-2 adrenergic receptor was significantly higher in the 10-and 20-μg/ml isoproterenol groups than in the blank control group 2 after 24-hour treatment (all P ＜ 0.05),and higher in the 20-μg/ml isoproterenol group than in the blank control group 2 after 48-hour treatment (P ＜ 0.05).Conclusion Propranolol can down-regulate the expression of beta-2 adrenergic receptor on the surface of vascular endothelial cells,while isoproterenol can up-regulate its expression.

β3-adrenoceptor in heart and lungs of elderly heart failure rats / 中华老年心脑血管病杂志

Objective To study the β3-adrenoceptor (β3-AR) in heart and lungs of elderly heart failure (HF) rats.Methods Forty-eight elderly HF Wistar rats were included in this study.A HF model of rats was established by ligating the aorta.The rats were divided into sham operation group (n=24) and HF group (n=24).The rats in each group were further divided into 4 subgroups at weeks 5,7,9 and 11 after operation (6 in each group).The hemodynamics,pathology and expression of β3-AR mRNA and protein in heart and lungs were detected at weeks 5,7,9 and 11 respectively after operation.Results The heart rate,LVESP,and dp/dtmax were significantly lower in HF group than in sham operation group at weeks 9 and 11 after operation while the LVEDP was significantly higher in HF group than in sham operation group at weeks 5,7,9 and 11 after operation (P＜0.01).Pulmonary edema occurred at week 7 after operation and myocardial necrosis was detected at week 9 after operation.The expression level of β3-AR mRNA in lungs was significantly lower in HF group at weeks 5,7,9 and 11 than at week 2 after operation (P＜0.05).The expression level of β3-AR mRNA in heart was significantly higher in HF group than in sham operation group at weeks 9 and 11 after operation (1.21±0.26 vs 0.98±0.22,1.26±0.23 vs 1.05±0.24,P＜0.01).Conclusion The β3-AR mRNA expression is downregulated in the lungs and upregulated in the heart.

Impact of the ß-1 adrenergic receptor polymorphism on tolerability and efficacy of bisoprolol therapy in Korean heart failure patients: association between ß adrenergic receptor polymorphism and bisoprolol therapy in heart failure (ABBA) study.

BACKGROUND/AIMS: We evaluated the association between coding region variants of adrenergic receptor genes and therapeutic effect in patients with congestive heart failure (CHF). METHODS: One hundred patients with stable CHF (left ventricular ejection fraction [LVEF] < 45%) were enrolled. Enrolled patients started 1.25 mg bisoprolol treatment once daily, then up-titrated to the maximally tolerable dose, at which they were treated for 1 year. RESULTS: Genotypic analysis was carried out, but the results were blinded to the investigators throughout the study period. At position 389 of the ß-1 adrenergic receptor gene (ADRB1), the observed minor Gly allele frequency (Gly389Arg + Gly389Gly) was 0.21, and no deviation from Hardy-Weinberg equilibrium was observed in the genotypic distribution of Arg389Gly (p = 0.75). Heart rate was reduced from 80.8 ± 14.3 to 70.0 ± 15.0 beats per minute (p < 0.0001). There was no significant difference in final heart rate across genotypes. However, the Arg389Arg genotype group required significantly more bisoprolol compared to the Gly389X (Gly389Arg + Gly389Gly) group (5.26 ± 2.62 mg vs. 3.96 ± 2.05 mg, p = 0.022). There were no significant differences in LVEF changes or remodeling between two groups. Also, changes in exercise capacity and brain natriuretic peptide level were not significant. However, interestingly, there was a two-fold higher rate of readmission (21.2% vs. 10.0%, p = 0.162) and one CHF-related death in the Arg389Arg group. CONCLUSIONS: The ADRB1 Gly389X genotype showed greater response to bisoprolol than the Arg389Arg genotype, suggesting the potential of individually tailoring ß-blocker therapy according to genotype.

Isoproterenol Promotes Rapid Ryanodine Receptor Movement to Bridging Integrator 1 (BIN1)-Organized Dyads.

BACKGROUND: The key pathophysiology of human acquired heart failure is impaired calcium transient, which is initiated at dyads consisting of ryanodine receptors (RyRs) at sarcoplasmic reticulum apposing CaV1.2 channels at t-tubules. Sympathetic tone regulates myocardial calcium transients through ß-adrenergic receptor (ß-AR)-mediated phosphorylation of dyadic proteins. Phosphorylated RyRs (P-RyR) have increased calcium sensitivity and open probability, amplifying calcium transient at a cost of receptor instability. Given that bridging integrator 1 (BIN1) organizes t-tubule microfolds and facilitates CaV1.2 delivery, we explored whether ß-AR-regulated RyRs are also affected by BIN1. METHODS AND RESULTS: Isolated adult mouse hearts or cardiomyocytes were perfused for 5 minutes with the ß-AR agonist isoproterenol (1 µmol/L) or the blockers CGP+ICI (baseline). Using biochemistry and superresolution fluorescent imaging, we identified that BIN1 clusters P-RyR and CaV1.2. Acute ß-AR activation increases coimmunoprecipitation between P-RyR and cardiac spliced BIN1+13+17 (with exons 13 and 17). Isoproterenol redistributes BIN1 to t-tubules, recruiting P-RyRs and improving the calcium transient. In cardiac-specific Bin1 heterozygote mice, isoproterenol fails to concentrate BIN1 to t-tubules, impairing P-RyR recruitment. The resultant accumulation of uncoupled P-RyRs increases the incidence of spontaneous calcium release. In human hearts with end-stage ischemic cardiomyopathy, we find that BIN1 is also 50% reduced, with diminished P-RyR association with BIN1. CONCLUSIONS: On ß-AR activation, reorganization of BIN1-induced microdomains recruits P-RyR into dyads, increasing the calcium transient while preserving electric stability. When BIN1 is reduced as in human acquired heart failure, acute stress impairs microdomain formation, limiting contractility and promoting arrhythmias.

Comparative Efficacy of Nebivolol and Metoprolol to Prevent Tachycardia-Induced Cardiomyopathy in a Porcine Model.

Chronic tachycardia is a well-known cause of nonischemic cardiomyopathy. We hypothesized that nebivolol, a ß-blocker with nitric oxide activity, would be superior to a pure ß-blocker in preventing tachycardia-induced cardiomyopathy in a porcine model. Fifteen healthy Yucatan pigs were randomly assigned to receive nebivolol, metoprolol, or placebo once a day. All pigs underwent dual-chamber pacemaker implantation. The medication was started the day after the pacemaker implantation. On day 7 after implantation, each pacemaker was set at atrioventricular pace (rate, 170 beats/min), and the pigs were observed for another 7 weeks. Transthoracic echocardiograms, serum catecholamine levels, and blood chemistry data were obtained at baseline and at the end of the study. At the end of week 8, the pigs were euthanized, and complete histopathologic studies were performed. All the pigs developed left ventricular cardiomyopathy but remained hemodynamically stable and survived to the end of the study. The mean left ventricular ejection fraction decreased from baseline by 34%, 20%, and 20% in the nebivolol, metoprolol, and placebo groups, respectively. These changes did not differ significantly among the 3 groups (P =0.51). Histopathologic analysis revealed mild left ventricular perivascular fibrosis with cardiomyocyte hypertrophy in 14 of the 15 pigs. Both nebivolol and metoprolol failed to prevent cardiomyopathy in our animal model of persistent tachycardia and a high catecholamine state.