Genome-wide association studies have problems due to confounding: Are family-based designs the answer?

Genome-wide association studies (GWASs) can be affected by confounding. Family-based GWAS uses random, within-family genetic variation to avoid this. A study in PLOS Biology details how different sources of confounding affect GWAS and whether family-based designs offer a solution.

Discovering genes that affect cognitive ability.

Twin and genomic studies indicate that genes play an important role in the development of cognitive ability. However, data limitations have made it difficult to pinpoint specific genes with a large impact. By examining the full gene sequences of >300 000 individuals, Chen et al. find eight such genes.

Cardiomyocyte PANX1 Controls Glycolysis and Neutrophil Recruitment in Hypertrophy.

BACKGROUND: PANX1 (pannexin 1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation, blood pressure regulation, and myocardial infarction. However, the possible role of PANX1 in cardiomyocytes in the progression of heart failure has not yet been investigated. METHOD: We generated a novel mouse line with constitutive deletion of PANX1 in cardiomyocytes (Panx1MyHC6). RESULTS: PANX1 deletion in cardiomyocytes had no effect on unstressed heart function but increased the glycolytic metabolism and resulting glycolytic ATP production, with a concurrent decrease in oxidative phosphorylation, both in vivo and in vitro. In vitro, treatment of H9c2 cardiomyocytes with isoproterenol led to PANX1-dependent release of ATP and Yo-Pro-1 uptake, as assessed by pharmacological blockade with spironolactone and siRNA-mediated knockdown of PANX1. To investigate nonischemic heart failure and the preceding cardiac hypertrophy, we administered isoproterenol, and we demonstrated that Panx1MyHC6 mice were protected from systolic and diastolic left ventricle volume increases as a result of cardiomyocyte hypertrophy. Moreover, we found that Panx1MyHC6 mice showed decreased isoproterenol-induced recruitment of immune cells (CD45+), particularly neutrophils (CD11b+, Ly6g+), to the myocardium. CONCLUSIONS: Together, these data demonstrate that PANX1 deficiency in cardiomyocytes increases glycolytic metabolism and protects against cardiac hypertrophy in nonischemic heart failure at least in part by reducing immune cell recruitment. Our study implies PANX1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in patients with heart failure.

A long noncoding RNA influences the choice of the X chromosome to be inactivated.

X chromosome inactivation (XCI) is the process of silencing one of the X chromosomes in cells of the female mammal which ensures dosage compensation between the sexes. Although theoretically random in somatic tissues, the choice of which X chromosome is chosen to be inactivated can be biased in mice by genetic element(s) associated with the so-called X-controlling element (Xce). Although the Xce was first described and genetically localized nearly 40 y ago, its mode of action remains elusive. In the approach presented here, we identify a single long noncoding RNA (lncRNA) within the Xce locus, Lppnx, which may be the driving factor in the choice of which X chromosome will be inactivated in the developing female mouse embryo. Comparing weak and strong Xce alleles we show that Lppnx modulates the expression of Xist lncRNA, one of the key factors in XCI, by controlling the occupancy of pluripotency factors at Intron1 of Xist. This effect is counteracted by enhanced binding of Rex1 in DxPas34, another key element in XCI regulating the activity of Tsix lncRNA, the main antagonist of Xist, in the strong but not in the weak Xce allele. These results suggest that the different susceptibility for XCI observed in weak and strong Xce alleles results from differential transcription factor binding of Xist Intron 1 and DxPas34, and that Lppnx represents a decisive factor in explaining the action of the Xce.

Inhibition of DYRK1a Enhances Cardiomyocyte Cycling After Myocardial Infarction.

BACKGROUND: DYRK1a (dual-specificity tyrosine phosphorylation-regulated kinase 1a) contributes to the control of cycling cells, including cardiomyocytes. However, the effects of inhibition of DYRK1a on cardiac function and cycling cardiomyocytes after myocardial infarction (MI) remain unknown. METHODS: We investigated the impacts of pharmacological inhibition and conditional genetic ablation of DYRK1a on endogenous cardiomyocyte cycling and left ventricular systolic function in ischemia-reperfusion (I/R) MI using αMHC-MerDreMer-Ki67p-RoxedCre::Rox-Lox-tdTomato-eGFP (RLTG) (denoted αDKRC::RLTG) and αMHC-Cre::Fucci2aR::DYRK1aflox/flox mice. RESULTS: We observed that harmine, an inhibitor of DYRK1a, improved left ventricular ejection fraction (39.5±1.6% and 29.1±1.6%, harmine versus placebo, respectively), 2 weeks after I/R MI. Harmine also increased cardiomyocyte cycling after I/R MI in αDKRC::RLTG mice, 10.8±1.5 versus 24.3±2.6 enhanced Green Fluorescent Protein (eGFP)+ cardiomyocytes, placebo versus harmine, respectively, P=1.0×10-3. The effects of harmine on left ventricular ejection fraction were attenuated in αDKRC::DTA mice that expressed an inducible diphtheria toxin in adult cycling cardiomyocytes. The conditional cardiomyocyte-specific genetic ablation of DYRK1a in αMHC-Cre::Fucci2aR::DYRK1aflox/flox (denoted DYRK1a k/o) mice caused cardiomyocyte hyperplasia at baseline (210±28 versus 126±5 cardiomyocytes per 40× field, DYRK1a k/o versus controls, respectively, P=1.7×10-2) without changes in cardiac function compared with controls, or compensatory changes in the expression of other DYRK isoforms. After I/R MI, DYRK1a k/o mice had improved left ventricular function (left ventricular ejection fraction 41.8±2.2% and 26.4±0.8%, DYRK1a k/o versus control, respectively, P=3.7×10-2). RNAseq of cardiomyocytes isolated from αMHC-Cre::Fucci2aR::DYRK1aflox/flox and αMHC-Cre::Fucci2aR mice after I/R MI or Sham surgeries identified enrichment in mitotic cell cycle genes in αMHC-Cre::Fucci2aR::DYRK1aflox/flox compared with αMHC-Cre::Fucci2aR. CONCLUSIONS: The pharmacological inhibition or cardiomyocyte-specific ablation of DYRK1a caused baseline hyperplasia and improved cardiac function after I/R MI, with an increase in cell cycle gene expression, suggesting the inhibition of DYRK1a may serve as a therapeutic target to treat MI.

Mitochondria-localized AMPK responds to local energetics and contributes to exercise and energetic stress-induced mitophagy.

Mitochondria form a complex, interconnected reticulum that is maintained through coordination among biogenesis, dynamic fission, and fusion and mitophagy, which are initiated in response to various cues to maintain energetic homeostasis. These cellular events, which make up mitochondrial quality control, act with remarkable spatial precision, but what governs such spatial specificity is poorly understood. Herein, we demonstrate that specific isoforms of the cellular bioenergetic sensor, 5' AMP-activated protein kinase (AMPKα1/α2/ß2/γ1), are localized on the outer mitochondrial membrane, referred to as mitoAMPK, in various tissues in mice and humans. Activation of mitoAMPK varies across the reticulum in response to energetic stress, and inhibition of mitoAMPK activity attenuates exercise-induced mitophagy in skeletal muscle in vivo. Discovery of a mitochondrial pool of AMPK and its local importance for mitochondrial quality control underscores the complexity of sensing cellular energetics in vivo that has implications for targeting mitochondrial energetics for disease treatment.

The Characteristics of People with Serious Mental Illness Who are at High Risk for Hospitalization or Death.

Many individuals with serious mental illness are at high risk for hospitalization or death due to inadequate treatment of medical conditions or unhealthy behaviors. The authors describe demographic and clinical characteristics associated with increased risk in this population. Electronic data were obtained for individuals in treatment at a large Veterans' healthcare system who were at high risk according to a validated model. A random sample of these individuals was assessed in person. Multivariable regressions estimated the effect of numerous demographic, health, and clinical characteristics on risk. Emergency visits and hospitalizations were common. Greater risk was associated with being male, not married, and having more diagnoses. While risk varied by race, this effect was no longer significant after controlling for other factors. Health-related quality of life worsened with increasing risk. Routine data identify a large population of high-risk individuals who may benefit from outreach to provide healthcare services.

Loss of Endogenously Cycling Adult Cardiomyocytes Worsens Myocardial Function.

RATIONALE: Endogenously cycling adult cardiomyocytes increase after myocardial infarction (MI) but remain scarce and are generally thought not to contribute to myocardial function. However, this broadly held assumption has not been tested, mainly because of the lack of transgenic reporters that restrict Cre expression to adult cardiomyocytes that reenter the cell cycle. OBJECTIVE: We created and validated a new transgenic mouse, αMHC (alpha myosin heavy chain)-MerDreMer-Ki67p-RoxedCre (denoted αDKRC [cardiomyocyte-specific αMHC-MerDreMer-Ki67p-RoxedCre]) that restricts Cre expression to cycling adult cardiomyocytes and uniquely integrates spatial and temporal adult cardiomyocyte cycling events based on the DNA specificities of orthologous Dre and Cre recombinases. We then created αDKRC::DTA mice that expressed an inducible diphtheria toxin in adult cycling cardiomyocytes and examined the effects of ablating these endogenously cycling cardiomyocytes on myocardial function after ischemic-reperfusion (I/R) MI. METHODS AND RESULTS: A tandem αDKRC transgene was designed, validated in cultured cells, and used to make transgenic mice. The αDKRC transgene integrated between MYH6 and MYH7 and did not disrupt expression of the surrounding genes. Compared with controls, αDKRC::RLTG (Rox-Lox-tdTomato-eGFP) mice treated with Tamoxifen expressed tdTomato+ in cardiomyocytes with rare Bromodeoxyuridine+, eGFP+ cardiomyocytes, consistent with reentry of the cell cycle. We then pretreated αDKRC::RLTG mice with Tamoxifen to activate the reporter before sham or reperfusion (I/R) MI surgeries. Compared with Sham surgery, the I/R MI group had increased single and paired eGFP+ (enhanced green fluorescent protein)+ cardiomyocytes predominantly in the border zones (5.8±0.5 versus 3.3±0.3 cardiomyocytes per 10-micron section, N=8-9 mice per group, n=16-24 sections per mouse), indicative of cycled cardiomyocytes. The single to paired eGFP+ cardiomyocyte ratio was ≈9 to 1 (5.2±0.4 single versus 0.6±0.2 paired cardiomyocytes) in the I/R MI group after MI, suggesting that cycling cardiomyocytes were more likely to undergo polyploidy than replication. The ablation of endogenously cycling adult cardiomyocytes in αDKRC::DTA (diphtheria) mice caused progressive worsening left ventricular chamber size and function after I/R MI, compared with controls. CONCLUSIONS: Although scarce, endogenously cycling adult cardiomyocytes contribute to myocardial function after injury, suggesting that these cells may be physiologically relevant.

Predictors of Functioning and Recovery Among Men and Women Veterans with Schizophrenia.

Gender differences may play a role in functional outcomes for individuals with schizophrenia. To better understand differences, an exploratory secondary analysis was conducted using data from a large, multi-site study of individuals with schizophrenia in treatment at Veterans Affairs medical centers. Participants completed surveys at baseline (n = 801; 734 men, 67 women) to assess demographics, symptoms, social supports, and recovery; and one year (n = 662; 604 men, 58 women) to assess quality of life and functioning. Hierarchical linear regressions examined interactions of baseline factors with functioning and quality of life. Women and men did not differ significantly in baseline social support, psychiatric symptoms, or recovery. Female gender predicted higher occupational functioning, while social functioning in men was inversely related to baseline symptom severity. Being married predicted higher quality of life for women, but not men. These findings may inform gender tailoring of services for schizophrenia.

Tractable limitations of current polygenic scores do not excuse genetically confounded social science.

Burt's critique of using polygenic scores in social science conflates the "scientific costs" of sociogenomics with "sociopolitical and ethical" concerns. Furthermore, she paradoxically enlists recent advances in controlling for environmental confounding to argue such confounding is scientifically "intractable." Disinterested social scientists should support ongoing efforts to improve this technology rather than obstructing progress and excusing genetically confounded research.

The Effectiveness of a Specialized Primary Care Medical Home for Patients with Serious Mental Illness.

BACKGROUND: There are unmet primary care needs among people with serious mental illness that might be improved with integrated care and medical care management. Many healthcare organizations have attempted to address this problem, but few interventions have been rigorously studied and found to be effective. OBJECTIVE: Study the implementation and effectiveness of a novel, specialized primary care medical home designed to improve the healthcare of patients with serious mental illness. DESIGN, SETTING, AND PARTICIPANTS: Clustered controlled trial for a median of 401 days. One Veterans Health Administration medical center was assigned to intervention and two were assigned to usual care (control). Thirty-nine clinicians and managers were included in the study, as well as 331 patients who met eligibility criteria. INTERVENTION: A specialized medical home with systematic patient engagement, proactive nurse panel management, a collaborative care psychiatrist, and a primary care physician providing care that included psychiatric treatment. MAIN MEASURES: Quality of care, chronic illness care and care experience, symptoms, and quality of life. KEY RESULTS: Sixty-five intervention patients (40%) moved all psychiatric care to the primary care team. No adverse events were attributable to the intervention. Compared with control, intervention patients had greater improvement over time in appropriate screening for body mass index, lipids, and glucose (χ2 = 6.9, 14.3, and 3.9; P's < .05); greater improvement in all domains of chronic illness care (activation, decision support, goal-setting, counseling, coordination) and care experience (doctor-patient interaction, shared decision-making, care coordination, access; F for each 10-24, P's < .05); and greater improvement in mental health-related quality of life (F = 3.9, P = .05) and psychotic symptoms (F = 3.9, P = .05). CONCLUSION: A primary care medical home for serious mental illness can be feasible to implement, safe, and more effective than usual care. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01668355.

Synthetic cannabinoids reduce the inflammatory activity of microglia and subsequently improve neuronal survival in vitro.

Microglia are resident immune cells of the brain that survey the microenvironment, provide trophic support to neurons, and clear debris to maintain homeostasis and healthy brain function. Microglia are also drivers of neuroinflammation in several neurodegenerative diseases. Microglia produce endocannabinoids and express both cannabinoid receptor subtypes suggesting that this system is a target to suppress neuroinflammation. We tested whether cannabinoid type 1 (CB1) or type 2 (CB2) receptors could be targeted selectively or in combination to dampen the pro-inflammatory behavior of microglia, and whether this would have functional relevance to decrease secondary neuronal damage. We determined that components of the endocannabinoid system were altered when microglia are treated with lipopolysaccharide and interferon-gamma and shift to a pro-inflammatory phenotype. Furthermore, pro-inflammatory microglia released cytotoxic factors that induced cell death in cultured STHdhQ7/Q7 neurons. Treatment with synthetic cannabinoids that were selective for CB1 receptors (ACEA) or CB2 receptors (HU-308) dampened the release of nitric oxide (NO) and pro-inflammatory cytokines and decreased levels of mRNA for several pro-inflammatory markers. A nonselective agonist (CP 55,940) exhibited similar influence over NO release but to a lesser extent relative to ACEA or HU-308. All three classes of synthetic cannabinoids ultimately reduced the secondary damage to the cultured neurons. The mechanism for the observed neuroprotective effects appeared to be related to cannabinoid-mediated suppression of MAPK signaling in microglia. Taken together, the data indicate that activation of CB1 or CB2 receptors interfered with the pro-inflammatory activity of microglia in a manner that also reduced secondary damage to neurons.

Limits of epidemic prediction using SIR models.

The Susceptible-Infectious-Recovered (SIR) equations and their extensions comprise a commonly utilized set of models for understanding and predicting the course of an epidemic. In practice, it is of substantial interest to estimate the model parameters based on noisy observations early in the outbreak, well before the epidemic reaches its peak. This allows prediction of the subsequent course of the epidemic and design of appropriate interventions. However, accurately inferring SIR model parameters in such scenarios is problematic. This article provides novel, theoretical insight on this issue of practical identifiability of the SIR model. Our theory provides new understanding of the inferential limits of routinely used epidemic models and provides a valuable addition to current simulate-and-check methods. We illustrate some practical implications through application to a real-world epidemic data set.

Factors associated with recovery from homelessness among veterans in permanent supportive housing.

AIMS: We sought to identify person- and program-level factors distinguishing permanent supportive housing (PSH) residents with higher versus lower social integration; and higher versus lower instrumental functioning. METHODS: Among 60 PSH residents at Los Angeles' VA, surveys and medical records captured person-level factors. Using a median split, we dichotomized participants with higher versus lower social integration; and higher versus lower instrumental functioning. Recursive partitioning (RP) identified variables that best-differentiated these subgroups. Interviews with 26 participants captured their perceptions on social integration and instrumental functioning. RESULTS: Using RP, health-related quality of life, psychiatric symptoms and case management frequency best-differentiated the social integration subgroups. Few perceived that PSH affected social integration. RP did not yield a stable model to differentiate the instrumental functioning subgroups; participants perceived that PSH addressed most functional deficits. CONCLUSIONS: Services that enhance social integration may benefit PSH residents with poor health; existing services may adequately address instrumental functioning.

A survey of personnel and services offered in 32 outpatient US clozapine clinics.

BACKGROUND: Clozapine clinics can facilitate greater access to clozapine, but there is a paucity of data on their structure in the US. METHODS: A 23-item survey was administered to participants recruited from the SMI Adviser Clozapine Center of Excellence listserv to understand characteristics of clozapine clinics. RESULTS: Clozapine clinics (N = 32) had a median caseload of 45 (IQR = 21-88) patients and utilized a median of 5 (IQR = 4-6) interdisciplinary roles. The most common roles included psychiatrists (100%), pharmacists (65.6%), nurses (65.6%), psychiatric nurse practitioners (53.1%), and case managers (53.1%). The majority of clinics outreached to patients who were overdue for labs (78.1%) and had access to on-site phlebotomy (62.5%). Less than half had on call services (46.9%). CONCLUSIONS: In this first systematic description of clozapine clinics in the US, there was variation in the size, staffing, and services offered. These findings may serve as a window into configurations of clozapine teams.

A novel "video-game" simulator for training fetoscopic laser coagulation of anastomoses in twin-to-twin transfusion syndrome.

 : We have developed a high-fidelity interactive "video-game" simulator in order to teach fetoscopic laser ablation of placental anastomoses for twin-twin transfusion syndrome This simulator may be used by teachers in order to provide metrics-based simulator education to multiple trainees, in both hands-on and distanced learning settings WHAT IS ALREADY KNOWN ABOUT THIS TOPIC?: The use of simulation improves training of the fetoscopic laser techniques utilized in the treatment of twin-twin transfusion syndrome A number of mannequins have been developed to aid this education WHAT DOES THIS STUDY ADD?: Two new simulators are described for twin-twin transfusion syndrome training-silicone and digital The digital simulator is a novel digital video game virtual format This new format has enhanced interactivity and has the potential to enable distance learning.

How important are parents in the development of child anxiety and depression? A genomic analysis of parent-offspring trios in the Norwegian Mother Father and Child Cohort Study (MoBa).

BACKGROUND: Many studies detect associations between parent behaviour and child symptoms of anxiety and depression. Despite knowledge that anxiety and depression are influenced by a complex interplay of genetic and environmental risk factors, most studies do not account for shared familial genetic risk. Quantitative genetic designs provide a means of controlling for shared genetics, but rely on observed putative exposure variables, and require data from highly specific family structures. METHODS: The intergenerational genomic method, Relatedness Disequilibrium Regression (RDR), indexes environmental effects of parents on child traits using measured genotypes. RDR estimates how much the parent genome influences the child indirectly via the environment, over and above effects of genetic factors acting directly in the child. This 'genetic nurture' effect is agnostic to parent phenotype and captures unmeasured heritable parent behaviours. We applied RDR in a sample of 11,598 parent-offspring trios from the Norwegian Mother, Father and Child Cohort Study (MoBa) to estimate parental genetic nurture separately from direct child genetic effects on anxiety and depression symptoms at age 8. We tested for mediation of genetic nurture via maternal anxiety and depression symptoms. Results were compared to a complementary non-genomic pedigree model. RESULTS: Parental genetic nurture explained 14% of the variance in depression symptoms at age 8. Subsequent analyses suggested that maternal anxiety and depression partially mediated this effect. The genetic nurture effect was mirrored by the finding of family environmental influence in our pedigree model. In contrast, variance in anxiety symptoms was not significantly influenced by common genetic variation in children or parents, despite a moderate pedigree heritability. CONCLUSIONS: Genomic methods like RDR represent new opportunities for genetically sensitive family research on complex human traits, which until now has been largely confined to adoption, twin and other pedigree designs. Our results are relevant to debates about the role of parents in the development of anxiety and depression in children, and possibly where to intervene to reduce problems.

Heteromer formation between cannabinoid type 1 and dopamine type 2 receptors is altered by combination cannabinoid and antipsychotic treatments.

The cannabinoid type 1 (CB1 ) receptor and the dopamine type 2 (D2 ) receptor are co-localized on medium spiny neuron terminals in the globus pallidus where they modulate neural circuits involved in voluntary movement. Physical interactions between the two receptors have been shown to alter receptor signaling in cell culture. The objectives of the current study were to identify the presence of CB1 /D2 heteromers in the globus pallidus of C57BL/6J male mice, define how CB1 /D2 heteromer levels are altered following treatment with cannabinoids and/or antipsychotics, and determine if fluctuating levels of CB1 /D2 heteromers have functional consequences. Using in situ proximity ligation assays, we observed CB1 /D2 heteromers in the globus pallidus of C57BL/6J mice. The abundance of the heteromers increased following treatment with the nonselective cannabinoid receptor agonist, CP55,940. In contrast, treatment with the typical antipsychotic haloperidol reduced the number of CB1 /D2 heteromers, whereas the atypical antipsychotic olanzapine treatment had no effect. Co-treatment with CP55,940 and haloperidol had similar effects to haloperidol alone, whereas co-treatment with CP55,940 and olanzapine had similar effects to CP55,940. The observed changes were found to have functional consequences as the differential effects of haloperidol and olanzapine also applied to γ-aminobutyric acid release in STHdhQ7/Q7 cells and motor function in C57BL/6J male mice. This work highlights the clinical relevance of co-exposure to cannabinoids and different antipsychotics over acute and prolonged time periods.

Do Collaborative Care Managers and Technology Enhance Primary Care Satisfaction with Care from Embedded Mental Health Providers?

BACKGROUND: To improve mental health care access, the Veterans Health Administration (VA) implemented Primary Care-Mental Health Integration (PC-MHI) in clinics nationally. Primary care clinical leader satisfaction can inform model implementation and may be facilitated by collaborative care managers and technology supporting cross-specialty collaboration. OBJECTIVE: (1) To determine primary care clinical leaders' overall satisfaction with care from embedded mental health providers for a range of conditions and (2) to examine the association between overall satisfaction and two program features (care managers, technology). DESIGN: Cross-sectional organizational survey in one VA region (Southern California, Arizona, and New Mexico), 2018. PARTICIPANTS: Sixty-nine physicians or other designated clinical leaders in each VA primary care clinic (94% response rate). MAIN MEASURES: We assessed primary care clinical leader satisfaction with embedded mental health care on four groups of conditions: target, non-target mental health, behavioral health, suicide risk management. They additionally responded about the availability of mental health care managers and the sufficiency of information technology (telemental health, e-consult, instant messaging). We examined relationships between satisfaction and the two program features using χ2 tests and multivariable regressions. KEY RESULTS: Most primary care clinical leaders were "very satisfied" with care for targeted anxiety (71%) and depression (69%), but not for other common conditions (37% alcohol misuse, 19% pain). Care manager availability was significantly associated with "very satisfied" responses for depression (p = .02) and anxiety care by embedded mental health providers (p = .02). Highly rated sufficiency of communication technology (only 19%) was associated with "very satisfied" responses to suicide risk management (p = .002). CONCLUSIONS: Care from embedded mental health providers for depression and anxiety was highly satisfactory, which may guide improvement among less satisfactory conditions (alcohol misuse, pain). Observed associations between overall satisfaction and collaborative care features may inform clinics on how to optimize staffing and technology based on priority conditions.

Endothelin receptor heteromerization inhibits ß-arrestin function in HEK293 cells.

The endothelin receptor A (ETA) and endothelin receptor B (ETB) are G protein-coupled receptors that are co-expressed in vascular smooth muscle cells. Endothelin-1 (ET-1) activates endothelin receptors to cause microvascular vasoconstriction. Previous studies have shown that heteromerization between ETA and ETB prolongs Ca2+ transients, leading to prolongation of Gαq-dependent signaling and sustained vasoconstriction. We hypothesized that these effects are in part mediated by the resistance of ETA/ETB heteromers to ß-arrestin recruitment and subsequent desensitization. Using bioluminescence resonance energy transfer 2 (BRET2), we found that ETB has a relatively equal affinity to form either homomers or heteromers with ETA when co-expressed in the human embryonic kidney 293 (HEK293) cells. When co-expressed, activation of ETA and ETB by ET-1 caused a heteromer-specific reduction and delay in ß-arrestin-2 recruitment with a corresponding reduction and delay in ET-1-induced ETA/ETB co-internalization. Furthermore, the co-expression of ETA and ETB inhibited ET-1-induced ß-arrestin-1-dependent extracellular signal-regulated kinase (ERK) phosphorylation while prolonging ET-1-induced Gαq-dependent ERK phosphorylation. ETA/ETB heteromerization mediates the long-lasting vasoconstrictor response to ET-1 by the prolongation of Gαq-dependent signaling and inhibition of ß-arrestin function.