How to assess nonresponsiveness to vasodilator stress.

Myocardial perfusion imaging (MPI) is a powerful tool for the functional assessment of ischemia in patients with suspected or known coronary artery disease (CAD). Given that the diagnostic accuracy and prognostic value of MPI and post-test management are highly dependent on achieving an adequate stress vasodilatory response, it is critical to identify those who may not have adequately responded to vasodilator pharmacological stress agents such as adenosine, dipyridamole, and regadenoson. Caffeine, a potent inhibitor of the adenosine receptor, is a compound that can affect vasodilatory hemodynamics, result in false negative studies, and potentially alter management in cases of inaccurate test results. Vasodilator non-responsiveness can be suspected by examining hemodynamics, quantitative positron emission tomography (PET) metrics such as myocardial flow reserve (MFR), and splenic response to stress. Quantitative MFR values of 1-1.2 should raise suspicion for nonresponsiveness in the setting of normal perfusion, along with the absence of a splenic switch off. Newer metrics, such as splenic response ratio, can be used to aid in the identification of potential nonresponders to pharmacologic vasodilators.

The Medicare Appropriate Use Criteria Program: A Review of Recommendations for Testing in Coronary Artery Disease.

BACKGROUND: Congress established the Appropriate Use Criteria (AUC) Program to reduce unnecessary advanced imaging studies. Organizations that wish to develop AUC can apply to the Centers for Medicare & Medicaid Services (CMS) to qualify as provider-led entities (PLEs) under this program. Variable methods, content, and formatting of PLE-generated AUC could lead to clinician uncertainty about whether an advanced imaging test is appropriate or not. PURPOSE: To review AUC published by CMS-qualified PLEs focused on advanced imaging tests for coronary artery disease (CAD), a "priority clinical area" identified by CMS. DATA SOURCES: Publicly available data from the worldwide web searched on 29 August 2022. STUDY SELECTION: Approved AUC with recommendations related to testing for CAD. DATA EXTRACTION: Manual review of published AUC by all authors. DATA SYNTHESIS: Among the 17 CMS-qualified PLEs, only 7 had published AUC related to CAD. Substantial variation in the methods and formatting of these AUCs was observed. The number of clinical scenarios covered ranged from 6 to 210, and the number of advanced imaging methods covered ranged from 1 to 25. When specifically applied to clinical scenarios, many AUC offered no guidance on appropriateness; those that did conflicted with respect to appropriateness. LIMITATION: Other CMS-identified priority clinical areas were not evaluated. CONCLUSION: CMS-qualified AUC for imaging of CAD are heterogeneous and sometimes discrepant, creating substantial potential for uncertainty among clinicians seeking to provide their patients with appropriate imaging tests. PRIMARY FUNDING SOURCE: No funding was received for this study.

Accurate and efficient rapid acquisition early post-injection stress-first CZT SPECT myocardial perfusion imaging with tetrofosmin and attenuation correction.

INTRODUCTION: Myocardial perfusion imaging (MPI) protocols have not changed significantly despite advances in instrumentation and software. We compared an early post-injection, stress-first SPECT protocol to standard delayed imaging. METHODS: 95 patients referred for SPECT MPI were imaged upright and supine on a Spectrum Dynamics D-SPECT CZT system with CT attenuation correction. Patients received injection of 99mTc tetrofosmin at peak of regadenoson stress and were imaged. Early post-stress (mean 17 ± 2 minutes) and Standard 1-h delay (mean 61 ± 13 min). Three blinded readers evaluated images for overall interpretation, perceived need for rest imaging, image quality, and reader confidence. Laboratory efficiency was also evaluated. RESULTS: Blinded readers had the same response for the need for rest in 77.9% of studies. Studies also had the same interpretation in 89.5% of studies. Reader confidence was high (86.0% (Early) and 90.3% (Standard p = 0.52. Image quality was good or excellent in 87.4% Early vs 96.8% Standard (p = 0.09). Time between patient check-in and end of stress imaging was 104 ± (Standard) to 60 ± 18 minutes (Early) (p < 0.001). CONCLUSION: Early post-injection stress-only imaging using CZT SPECT/CT appears promising with Tc-99m tetrofosmin with similar image quality, reader confidence, diagnosis, and need for a rest scan.

Coronary vascular dysfunction is associated with increased risk of death in patients with peripheral artery disease.

BACKGROUND: Peripheral artery disease (PAD) and coronary vascular dysfunction are common in patients with cardiometabolic disease. Neither the prevalence of coronary vascular dysfunction among patients with PAD nor the prognostic impact with these two conditions present together has been well studied. METHODS: Consecutive patients who underwent PET MPI were analyzed for presence of coronary vascular dysfunction [myocardial blood flow reserve (MBFR) < 2]. Cox regression was used to examine the association of reduced MBFR with mortality in patients with PAD, as well as the association of comorbid MBFR < 2 and PAD with all-cause death. RESULTS: Among 13,940 patients, 1936 (14%) had PAD, 7782 (56%) had MBFR < 2 and 1346 (10%) had both PAD and MBFR < 2. Reduced MBFR was very common (69.5%) and was associated with increased risk of all-cause death (HR 1.69, 95%CI 1.32, 2.16, p < 0.01) in patients with PAD. Patients with both PAD and MBFR < 2, and those with either PAD or reduced MBFR had increased risk of death compared to those with neither condition: PAD + MBFR < 2 [(HR 95%CI), 2.30; 1.97-2.68], PAD + MBFR ≥ 2 (1.37; (1.08-1.72), PAD - MBFR < 2 (1.98; 1.75-2.25), p < 0.001 for all). CONCLUSION: Coronary vascular dysfunction was common in patients with PAD and was associated with increased risk of death.

Improved Performance of PET Myocardial Perfusion Imaging Compared to SPECT in the Evaluation of Suspected CAD.

PURPOSE OF REVIEW: Myocardial perfusion imaging (MPI) with single photon emission computed tomography (SPECT) has played a central role in the non-invasive evaluation of patients with obstructive coronary artery disease (CAD) for decades. In this review, we discuss the key differences and advantages of positron emission tomography (PET) MPI over SPECT MPI as it relates to the diagnosis, prognosis, as well as clinical decision-making in patients with suspected CAD. RECENT FINDINGS: Stress-induced perfusion abnormalities on SPECT help estimate presence, extent, and location of ischemia and flow-limiting obstructive CAD, help with risk stratification, and serve as a gatekeeper to identify patients who will benefit from downstream revascularization versus medical management. Some of the major limitations of SPECT include soft-tissue attenuation artifacts, underestimation of ischemia due to reliance on relative perfusion assessment, and longer protocols with higher radiation dose when performed with traditional equipment. PET MPI addresses most of these limitations and offers better quality images, higher diagnostic accuracy along with shorter protocols and lower radiation dose to the patient. A special advantage of PET scanning lies in the ability to quantify absolute myocardial blood flow and assess true extent of epicardial involvement along with identifying non-obstructive phenotypes of CAD such as diffuse atherosclerosis and microvascular dysfunction. In addition, stress acquisition at/near peak stress with PET allows us to measure left ventricular ejection fraction reserve and myocardial blood flow reserve, which help with identifying patients at a higher risk of future cardiac events and optimally select candidates for revascularization. The several technical advantages of PET MPI position as a superior method to diagnose obstructive and non-obstructive phenotypes of ischemic heart disease affecting the entirety of the coronary circulation offer incremental value for risk stratification and guide post-test management strategy for patients with suspected CAD.

Reasons for discordance between positron emission tomography (PET) myocardial perfusion imaging (MPI) results and subsequent management.

BACKGROUND: Referral patterns to coronary angiography following positron emission tomography (PET) myocardial perfusion imaging (MPI) and reasons for non-referral following abnormal PET MPI are largely unknown. METHODS: Referral rates to coronary angiography within 90 days post PET MPI were determined. A random subset of 100 patients with severe (≥ 10%) ischemia on MPI between 2014-16 who were not referred for angiography were examined to better understand reasons as to why patients with high-risk MPI findings did not undergo coronary angiography. RESULTS: Among 19,282 unique patients, overall rate of 90-day coronary angiography was 18.5% (3574/19282). Among patients with severe ischemia, 64.1% (1930/3011) underwent angiography within 90 days; the rate was lower in those with mild-moderate (20.6% [1010/4898]) and no ischemia (5.6% [634/11373]). In the random sample of 100 patients, the most common physician reasons for non-referral were uncertainty regarding whether the test results were responsible for the patient's presenting symptoms, renal failure, and patient age, frailty, or cognitive status, while patient preference for medical management was by far the most common patient reason. CONCLUSION: Referral rates for coronary angiography after PET correlate with severity of ischemia. However, there appear to be opportunities to reconsider testing for instances when results will not change clinical management.

The Potential of F-18 Flurpiridaz PET/CT Myocardial Perfusion Imaging for Precision Imaging.

PURPOSE OF THE REVIEW: Myocardial perfusion imaging (MPI) with single photon emission computed tomography (SPECT) has been the main method for assessing patients with known or suspected coronary artery disease (CAD) for decades. Based on a strong and growing evidence base, positron emission tomography (PET) MPI is increasingly favored when it is available. However, currently available PET perfusion tracers have limitations that have hampered broad utilization. RECENT FINDINGS: F-18 flurpiridaz is a novel PET MPI agent that is nearing completion of studies necessary to obtain regulatory approval. It has unique capabilities that will facilitate further expansion of PET MPI utilization. In addition, it has characteristics that may define it as the best MPI agent to date, in terms of the potential to equalize accuracy independent of patient size, gender, complexity, or ability to perform exercise stress. The combination of excellent image quality and accurate absolute blood flow quantification hold the potential of its being an ideal precision tool for non-invasive assessment of myocardial blood flow and entire spectrum of ischemic heart disease.

Global patterns of comprehensive cardiovascular risk factor control in patients with type 2 diabetes mellitus: Insights from the DISCOVER study.

AIM: To investigate global patterns of cardiovascular risk factor control in patients with type 2 diabetes mellitus (T2D). METHODS: DISCOVER is an international, observational cohort study of patients with T2D beginning second-line glucose-lowering therapy. Risk factor management was examined among eligible patients (ie, those with the risk factor) at study baseline. Inter-country variability was estimated using median odds ratios (MORs). RESULTS: Among 14 343 patients with T2D from 34 countries, the mean age was 57.4 ± 12.0 years and the median (interquartile range) duration of T2D was 4.2 (2.0-8.0) years; 11.8% had documented atherosclerotic cardiovascular disease (ASCVD). Among eligible patients, blood pressure was controlled in 67.5% (9284/13756), statins were prescribed in 43.7% (5775/13208), angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers were prescribed in 55.6% (5292/9512), aspirin was prescribed in 53.3% of those with established ASCVD (876/1645), and 84.4% (12 102/14343) were non-smoking. Only 21.5% of patients (3088/14343) had optimal risk factor management (defined as control of all eligible measures), with wide inter-country variability (10%-44%), even after adjusting for patient and site differences (MOR 1.47, 95% confidence interval 1.24-1.66). CONCLUSION: Globally, comprehensive control of ASCVD risk factors is not being achieved in most patients, with wide variability among countries unaccounted for by patient and site differences. Better country-specific strategies are needed to implement comprehensive cardiovascular risk factor control consistently in patients with T2D to improve long-term outcomes.

Stress myocardial perfusion imaging in patients presenting with syncope: Comparison of PET vs. SPECT.

BACKGROUND: The role of myocardial perfusion imaging (MPI) in patients with suspected coronary artery disease (CAD) presenting with syncope is controversial. We aimed to determine diagnostic yield of MPI for evaluation of syncope in patients without known CAD, as a function of pre-test patient risk and test modality (PET vs SPECT). METHODS: Between 1/2010 and 12/2016, 1324 consecutive patients presenting with syncope without known CAD underwent MPI with PET (n = 640) or SPECT (n = 684). Rates of abnormal MPI (summed difference score (SDS) > 2 or left ventricular ejection fraction (LVEF) reserve ≤ 0 for PET and SDS > 2 or post-stress LVEF ≤ 45% for SPECT) were determined among patients stratified by pre-test risk. In patients who were referred for coronary angiography, diagnostic yield of obstructive CAD was calculated in the overall cohort as well as in a propensity-matched cohort compared to patients without syncope. RESULTS: Abnormal MPI was noted in 36.5% (201/551) of patients who had PET compared with 13.0% (87/671) who had SPECT (P < 0.001), which is largely related to higher comorbidity burden and greater pre-test CAD risk in the PET population. Among patients who had an abnormal MPI, 8.5% (47/551) with PET and 0.7% (5/671) with SPECT were found to have obstructive CAD if referred for coronary angiography. Patients at intermediate-high pre-test risk had a higher proportion of abnormal MPIs and obstructive CAD as compared to those at low risk in both the PET and SPECT cohorts. The rate of abnormal testing and diagnostic yield of PET MPI was similar and proportionate to pre-test likelihood among matched patients with and without syncope. CONCLUSIONS: Among patients referred for PET MPI with syncope at an intermediate-high pre-test CAD risk, 1 in 3 had an abnormal MPI and 1 in 10 had obstructive CAD. The value of MPI was related to pre-test risk as opposed to the presence of syncope, and MPI testing with PET or SPECT in the low-risk population was low value.

Myocardial blood flow reserve assessed by positron emission tomography myocardial perfusion imaging identifies patients with a survival benefit from early revascularization.

AIMS: Positron emission tomography (PET) myocardial perfusion imaging (MPI) can non-invasively measure myocardial blood flow reserve (MBFR). We aimed to examine whether MBFR identifies patients with a survival benefit after revascularization, helping to guide post-test management. METHODS AND RESULTS: We examined all-cause mortality in 12 594 consecutive patients undergoing Rb82 rest/stress PET MPI from January 2010 to December 2016, after excluding those with cardiomyopathy, prior coronary artery bypass surgery (CABG), and missing MBFR. Myocardial blood flow reserve was calculated as the ratio of stress to rest absolute myocardial blood flow. A Cox model adjusted for patient and test characteristics, early revascularization (percutaneous coronary intervention or CABG ≤90 days of MPI), and the interaction between MBFR and early revascularization was developed to identify predictors of all-cause mortality. After a median follow-up of 3.2 years, 897 patients (7.1%) underwent early revascularization and 1699 patients (13.5%) died. Ischaemia was present in 4051 (32.3%) patients, with 1413 (11.2%) having ≥10% ischaemia. Mean MBFR was 2.0 ± 1.3, with MBFR <1.8 in 4836 (38.5%). After multivariable adjustment, every 0.1 unit decrease in MBFR was associated with 9% greater hazard of all-cause death (hazard ratio 1.09, 95% confidence interval 1.08-1.10; P < 0.001). There was a significant interaction between MBFR and early revascularization (P < 0.001); such that patients with MBFR ≤1.8 had a survival benefit with early revascularization, regardless of type of revascularization or level of ischaemia. CONCLUSION: Myocardial blood flow reserve on PET MPI is associated with all-cause mortality and can identify patients who receive a survival benefit with early revascularization compared to medical therapy. This may be used to guide revascularization, and prospective validation is needed.

Drivers of radiation dose reduction with myocardial perfusion imaging: A large health system experience.

BACKGROUND: Despite increasing emphasis on reducing radiation exposure from myocardial perfusion imaging (MPI), the use of radiation-sparing practices (RSP) at nuclear laboratories remains limited. Defining real-world impact of RSPs on effective radiation dose (E) can potentially further motivate their adoption. METHODS: MPI studies performed between 1/2010 and 12/2016 within a single health system were included. Mean E was compared between sites with 'basic' RSP (defined as elimination of thallium-based protocols and use of stress-only (SO) imaging on conventional single photon emission computed tomography (SPECT) cameras) and those with 'advanced' capabilities (sites that additionally used solid-state detector (SSD) SPECT cameras, advanced post-processing software (APPS) or positron emission tomography (PET) imaging), after matching patients by age, gender, and weight. Contributions of individual RSP to E reduction were determined using multiple linear regression after adjusting for factors affecting tracer dose. RESULTS: Among 55,930 MPI studies performed, the use of advanced RSP was associated with significantly lower mean E compared to basic RSP (7 ± 5.6 mSv and 16 ± 5.4 mSv, respectively; P < 0.001), with a greater likelihood of achieving E < 9 mSv (65.7% vs. 10.8%, respectively; OR 15.8 [95% CI 14 to 17.8]; P < 0.0001). Main driver of E reduction was SO-SSD SPECT (mean reduction = 11.5 mSv), followed by use of SO-SPECT + APPS (mean reduction = 10.1 mSv), ;ET (mean reduction = 9.7 mSv); and elimination of thallium protocols (mean reduction = 9.1 mSv); P < 0.0001 for all comparisons. CONCLUSION: In a natural experiment with implementation of radiation-saving practices at a large health system, stress-only protocols used in conjunction with modern SPECT technologies, the use of PET and elimination of thallium-based protocols were associated with greatest reductions in radiation dose. Availability of several approaches to dose reduction within a health system can facilitate achievement of targeted radiation benchmarks in a greater number of performed studies.

Changes in Health and Well-Being during Residents' Training.

OBJECTIVES: Previous studies have characterized the negative effects of graduate medical education on physicians; however, there is limited longitudinal data on how physicians' well-being changes during their training. This study aimed to demonstrate and quantify changes to trainees' wellness and health habits during the course of their first 2 years of graduate medical education. METHODS: A longitudinal survey study of postgraduate year 1 trainees at the Cleveland Clinic was administered at 3 time points: the initial survey during orientation week, a second survey at 1 year, and a final survey at 2 years. RESULTS: Of the 170 trainees contacted, 59 (35%) completed the initial survey and 34 (58%) completed the first follow-up survey. Between the initial survey and the first follow-up survey, respondents reported that their health was worse than the prior year (P < 0.001). They also reported sleeping on average 1 hour less per night and exercising on average one fewer day per week. The number of individuals who reported not eating breakfast increased by 22%, whereas the number of individuals eating out at lunch more than doubled. Twenty-seven people completed the second follow-up survey. Between the first follow-up survey and the final survey, respondents gained on average 2.12 lb (P = 0.039). Breakfast, lunch, and sleeping habits persisted through the second follow-up survey. CONCLUSIONS: Residents' health and wellness habits deteriorated during internship and did not improve in the second year of residency. Efforts to promote healthy habits in this population should be a priority.

Association Between Prompt Defibrillation and Epinephrine Treatment With Long-Term Survival After In-Hospital Cardiac Arrest.

BACKGROUND: Prior studies have reported higher in-hospital survival with prompt defibrillation and epinephrine treatment in patients with in-hospital cardiac arrest (IHCA). Whether this survival benefit persists after discharge is unknown. METHODS: We linked data from a national IHCA registry with Medicare files and identified 36 961 patients ≥65 years of age with an IHCA at 517 hospitals between 2000 and 2011. Patients with IHCA caused by pulseless ventricular tachycardia or ventricular fibrillation were stratified by prompt (≤2 minutes) versus delayed (>2 minutes) defibrillation, whereas patients with IHCA caused by asystole or pulseless electric activity were stratified by prompt (≤5 minutes) versus delayed (>5 minutes) epinephrine treatment. The association between prompt treatment and long-term survival for each rhythm type was assessed with multivariable hierarchical modified Poisson regression models. RESULTS: Of 8119 patients with an IHCA caused by ventricular tachycardia or ventricular fibrillation, the rate of 1-year survival was higher in those treated with prompt defibrillation than with delayed defibrillation (25.7% [1466 of 5714] versus 15.5% [373 of 2405]; adjusted relative risk [RR], 1.49; 95% confidence interval [CI] 1.32-1.69; P<0.0001). This survival advantage persisted at 3 years (19.1% versus 11.0%; adjusted RR, 1.45; 95% CI, 1.23-1.69; P<0.0001) and at 5 years (14.7% versus 7.9%; adjusted RR, 1.50; 95% CI, 1.22-1.83; P<0.0001). Of 28 842 patients with an IHCA caused by asystole/pulseless electric activity, the rate of 1-year survival with prompt epinephrine treatment was higher than with delayed treatment (5.4% [1341 of 24 885] versus 4.3% [168 of 3957]; adjusted RR, 1.20; 95% CI, 1.02-1.41; P=0.02), but this survival benefit was no longer present at 3 years (3.5% versus 2.9%; adjusted RR, 1.17; 95% CI, 0.95-1.45; P=0.15) and at 5 years (2.3% versus 1.9%; adjusted RR, 1.18; 95% CI, 0.88-1.58; P=0.27). CONCLUSIONS: Prompt defibrillation for IHCA caused by ventricular tachycardia or ventricular fibrillation was associated with higher rates of long-term survival throughout 5 years of follow-up, whereas prompt epinephrine treatment for asystole/pulseless electric activity was associated with greater survival at 1 year but not at 3 or 5 years. By quantifying the greater survival associated with timely defibrillation and epinephrine administration, these findings provide important insights into the durability of survival benefits for 2 process-of-care measures in current resuscitation guidelines.

Validation of the Seattle angina questionnaire in women with ischemic heart disease.

BACKGROUND: Although the Seattle Angina Questionnaire (SAQ) has been widely used to assess disease-specific health status in patients with ischemic heart disease, it was originally developed in a predominantly male population and its validity in women has been questioned. METHODS: Using data from 8892 men and 4013 women across 2 multicenter trials and 5 registries, we assessed the construct validity, test-retest reliability, responsiveness to clinical change, and predictive validity of the SAQ Summary Score (SS) and its 5 subdomains (Physical Limitation (PL), Anginal Stability (AS), Angina Frequency (AF), Treatment Satisfaction (TS), and Quality of Life (QoL)) separately in men and women. RESULTS: Comparable correlations of the SAQ SS with Canadian Cardiovascular Society class was demonstrated in both men and women (-0.48 for men, -0.46 for women). Similar correlations between the SAQ PL scale with treadmill exercise duration and Short Form-12 (SF-12) Physical Component Summary were observed in women and men (0.34-0.63 and 0.40-0.63, respectively). SAQ AS scores were significantly lower for both men and women with acute syndromes compared with 1 month later. The SAQ AF scale was strongly correlated with daily angina diaries (0.62 for men and 0.66 for women). The SAQ QoL scores were moderately correlated with the EQ5D visual analog scale and SF-12 general health question in men (0.43-0.50) and women (0.33-0.39). All SAQ scales demonstrated excellent reliability (intraclass correlation ≥0.78) in both men and women with stable CAD and were very sensitive to change after percutaneous coronary intervention (≥15-point difference in scores, standardized response mean ≥ 0.67). The SAQ SS was similarly predictive of 1-year mortality and cardiac re-hospitalizations for both men and women. CONCLUSION: The SAQ demonstrates similar psychometric properties in men and women with CAD. These findings provide evidence for validity of the SAQ in assessing women with IHD.

Prevalence of Elevated Cardiovascular Risks in Young Adults: A Cross-sectional Analysis of National Health and Nutrition Examination Surveys.

BACKGROUND: The 2013 cholesterol management guidelines from the American College of Cardiology and American Heart Association (ACC/AHA) recommend lipid screening in all adults older than 20 years to identify those at increased risk for atherosclerotic cardiovascular disease (ASCVD). Statins may be considered for patients with elevated 10-year risk (>5%) or a low-density lipoprotein cholesterol (LDL-C) level of 4.92 mmol/L (190 mg/dL) or greater. OBJECTIVE: To describe the prevalence of elevated ASCVD risk among nondiabetic adults younger than 50 years. DESIGN: Cross-sectional. SETTING: NHANES (National Health and Nutrition Examination Survey), 1999 to 2000 through 2011 to 2012. PARTICIPANTS: Adults aged 30 to 49 years without known ASCVD or diabetes. MEASUREMENTS: 10-year ASCVD risk was estimated by using the 2013 ACC/AHA ASCVD risk calculator. Participants were subdivided by age, sex, and history of smoking and hypertension. The percentages of adults in each subgroup with a 10-year ASCVD risk greater than 5% and of those with an LDL-C level of 4.92 mmol/L (190 mg/dL) or greater were estimated. Low-prevalence subgroups were defined as those in which a greater than 1% prevalence of elevated cardiovascular risk could be ruled out (that is, the upper 95% confidence bound for prevalence was ≤1%). RESULTS: Overall, 9608 NHANES participants representing 67.9 million adults were included, with approximately half (47.12%, representing 32 million adults) in low-prevalence subgroups. In the absence of smoking or hypertension, 0.09% (95% CI, 0.02% to 0.35%) of adult men younger than 40 years and 0.04% (CI, 0.0% to 0.26%) of adult women younger than 50 years had an elevated risk. Among other subgroups, 0% to 75.9% of participants had an increased risk. Overall, 2.9% (CI, 2.3% to 3.5%) had an LDL-C level of 4.92 mmol/L (190 mg/dL) or greater. LIMITATION: No information was available regarding cardiovascular outcomes. CONCLUSION: In the absence of risk factors, the prevalence of increased ASCVD risk is low among women younger than 50 and men younger than 40 years. PRIMARY FUNDING SOURCE: None.

False responses of Renilla luciferase reporter control to nuclear receptor TR4.

Renilla luciferase reporter is a widely used internal control in dual luciferase reporter assay system, where its transcription is driven by a constitutively active promoter. However, the authenticity of the Renilla luciferase response in some experimental settings has recently been questioned. Testicular receptor 4 (TR4, also known as NR2C2) belongs to the subfamily 2 of nuclear receptors. TR4 binds to a direct repeat regulatory element in the promoter of a variety of target genes and plays a key role in tumorigenesis, lipoprotein regulation, and central nervous system development. In our experimental system using murine pituitary corticotroph tumor AtT20 cells to investigate TR4 actions on POMC transcription, we found that overexpression of TR4 resulted in reduced Renilla luciferase expression whereas knockdown TR4 increased Renilla luciferase expression. The TR4 inhibitory effect was mediated by the TR4 DNA-binding domain and behaved similarly to the GR and its agonist, Dexamethasone. We further demonstrated that the chimeric intron, commonly present in various Renilla plasmid backbones such as pRL-Null, pRL-SV40, and pRL-TK, was responsible for TR4's inhibitory effect. The results suggest that an intron-free Renilla luciferase reporter may provide a satisfactory internal control for TR4 at certain dose range. Our findings advocate caution on the use of Renilla luciferase as an internal control in TR4-directed studies to avoid misleading data interpretation.