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Options for the treatment of multivessel coronary artery disease include medical therapy and revascularization with either coronary artery bypass grafting (CABG) or percutaneous transluminal coronary angioplasty (PTCA). CABG has been shown to prolong survival in patients with left main coronary disease or when at least two of the following factors are present: extensive coronary artery disease, significant ischemia as shown on stress testing and left ventricular dysfunction. In patients with extensive coronary artery disease but normal left ventricular function, either PTCA or CABG may be used initially without adversely affecting rates of survival. However, patients undergoing angioplasty have a higher rate of repeat revascularization procedures than those treated with bypass. The complex interplay of the various risks and benefits of medical therapy, angioplasty or bypass in the treatment of coronary artery disease requires an individualized approach to therapy and careful patient education. [ABSTRACT FROM AUTHOR]

Background: Transthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting TTR ., Methods: After conducting preclinical in vitro and in vivo studies, we evaluated the safety and pharmacodynamic effects of single escalating doses of NTLA-2001 in six patients with hereditary ATTR amyloidosis with polyneuropathy, three in each of the two initial dose groups (0.1 mg per kilogram and 0.3 mg per kilogram), within an ongoing phase 1 clinical study., Results: Preclinical studies showed durable knockout of TTR after a single dose. Serial assessments of safety during the first 28 days after infusion in patients revealed few adverse events, and those that did occur were mild in grade. Dose-dependent pharmacodynamic effects were observed. At day 28, the mean reduction from baseline in serum TTR protein concentration was 52% (range, 47 to 56) in the group that received a dose of 0.1 mg per kilogram and was 87% (range, 80 to 96) in the group that received a dose of 0.3 mg per kilogram., Conclusions: In a small group of patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of TTR . (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.)., (Copyright © 2021 Massachusetts Medical Society.)

The momentum of cardiovascular drug development has slowed dramatically. Use of validated cardiac biomarkers in clinical trials could accelerate development of much-needed therapies, but biomarkers have been used less for cardiovascular drug development than in therapeutic areas such as oncology. Moreover, there are inconsistences in biomarker use in clinical trials, such as sample type, collection times, analytical methods, and storage for future research. With these needs in mind, participants in a Cardiac Safety Research Consortium Think Tank proposed the development of international guidance in this area, together with improved quality assurance and analytical methods, to determine what biomarkers can reliably show. Participants recommended the development of systematic methods for sample collection, and the archiving of samples in all cardiovascular clinical trials (including creation of a biobank or repository). The academic and regulatory communities also agreed to work together to ensure that published information is fully and clearly expressed., Competing Interests: Funding Support and Author Disclosures Dr. Januzzi is supported in part by the Hutter Family Professorship; has served as a Trustee of the American College of Cardiology; is a board member of Imbria Pharmaceuticals; has received grant support from Applied Therapeutics, Innolife, Novartis Pharmaceuticals, and Abbott Diagnostics; has received consulting income from Abbott, Janssen, Novartis, and Roche Diagnostics; and has participated in clinical endpoint committees/data safety monitoring boards for Abbott, AbbVie, Amgen, Bayer, CVRx, Janssen, MyoKardia, and Takeda. Dr. Gintant is an employee of AbbVie, Inc. Dr. Gutstein is an employee of Janssen Pharmaceuticals. Dr. Jaffe has consulted or presently consults for most of the major diagnostics companies, including Beckman, Abbott, Siemens, ET Healthcare, Roche, Quidel, Sphingotec, Brava, Blade, and Novartis. Dr. Kaushik is an employee of Takeda Pharmaceuticals. Drs. Rambaran and Salas are employees of Daiichi-Sankyo, Inc. Ms. Rhyne is an employee of Prevencio, Inc. Dr. Shi is an employee of Novartis Pharmaceuticals, Inc. Dr. Udell has received grant support to his institutions from Amgen, AstraZeneca, Bayer, Boehringer Ingelheim, Janssen, Novartis, and Sanofi; and has received speaker/consulting honoraria from Amgen, Boehringer Ingelheim, Novartis, and Sanofi. Dr. Zabka is an employee of Genentech, Inc. Dr. Seltzer is an employee of WCG Clinical. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. The opinions expressed in this publication reflect those of the authors and do not necessarily reflect the positions or thinking of the U.S. Food and Drug Administration., (Copyright © 2021 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Most new chemical entities with systemic availability are required to be tested in a study specifically designed to exclude drug-induced corrected QT interval (QTc) effects, the so-called thorough QT/QTc study. Mirtazapine (Remeron™) is an antidepressant indicated for the treatment of episodes of major depression, which was originally approved in 1994 without a thorough QT study. To evaluate the proarrhythmic potential of mirtazapine, we performed a QT/QTc study with a novel design including implementation of an analysis of the relationship between drug concentration and the QTc interval as the primary assessment of proarrhythmic potential of mirtazapine. The least squares mean differences of the corrected QT interval between mirtazapine and placebo at the geometric mean maximum concentration of drug in blood plasma (90% confidence interval) were 2.39 milliseconds (0.70, 4.07) at the 45-mg dose and 4.00 milliseconds (1.18, 6.83) at the 75-mg dose level of mirtazapine. Modeling of the concentration/QTc relationship for moxifloxacin confirmed that the assay method was adequately sensitive. This trial showed a positive relationship between mirtazapine concentrations and prolongation of the QTc interval. However, the degree of QT prolongation observed with both 45-mg and 75-mg doses of mirtazapine was not at a level generally considered to be clinically meaningful. This study further demonstrates that analysis of the relationship between drug concentration and the QTc interval may be a reasonable alternative to traditional TQT studies to assess risk of QT prolongation., (© 2018, The American College of Clinical Pharmacology.)

Anacetrapib is a cholesteryl ester transfer protein inhibitor intended for the treatment of dyslipidemia. A phase 1 study was conducted to examine the pharmacokinetics and pharmacodynamics of multiple doses of anacetrapib in black compared to white healthy subjects. Although there was no apparent race-related pharmacokinetic effect, attenuation of the lipid response was observed in black subjects. Specifically, high-density lipoprotein cholesterol percentage increased 18.1% (absolute percentage points) less in black subjects (89.9%) when compared to increases in white subjects (108.0%). Similarly, the decrease in low-density lipoprotein cholesterol was 17.8% (absolute percentage points) less in blacks (-21.2%) relative to whites (-39.0%). In contrast, there were no apparent race-related differences in cholesteryl ester transfer protein mass or activity. Anacetrapib was generally well tolerated in this study. The results of this study suggest that there may be race-related differences in pharmacodynamics of anacetrapib independent of pharmacokinetics., (© 2018, The American College of Clinical Pharmacology.)

Background: We evaluated the incidence of hypersensitivity or anaphylaxis after repeated single-dose sugammadex administration in non-anaesthetised adults., Methods: In this multicentre, double-blind study (NCT02028065), healthy volunteer subjects were randomised (2:2:1 ratio) to one of three groups to receive three repeated intravenous injections of sugammadex 4 or 16 mg kg -1 , or placebo, separated by a ∼5 week intervals. Targeted hypersensitivity assessments were performed 0.5, 4, and 24 h post-dosing, and hypersensitivity signs/symptoms were referred to a blinded independent Adjudication Committee. Anaphylaxis was determined per Sampson (Criterion 1). The primary endpoint was the proportion with confirmed hypersensitivity., Results: Of 375 evaluable subjects, 25 had confirmed hypersensitivity [sugammadex 4 mg kg -1 : 10/151 (6.6%); sugammadex 16 mg kg -1 : 14/148 (9.5%); placebo: 1/76 (1.3%)]. The differences in incidence rates vs placebo were 5.3% (95% confidence interval: -0.9, 10.7) for sugammadex 4 mg kg -1 and 8.1% (1.7, 14.2) for 16 mg kg -1 . Incidence was similar across sugammadex doses and dosing occasions, including in subjects with reactions to previous doses. Three subjects (16 mg kg -1 group) required antihistamines/corticosteroids and discontinued the study, per protocol; symptoms resolved and no subject required epinephrine. One subject with anaphylaxis after the first 16 mg kg -1 dose recovered completely post-treatment. There were no clinically relevant anti-sugammadex antibody or tryptase findings., Conclusions: Hypersensitivity in response to sugammadex administration can occur in healthy subjects without history of previous sugammadex exposure. Hypersensitivity incidence was similar across sugammadex doses and numerically higher than placebo, with no evidence of sensitisation with repeated administration. Hypersensitivity is unlikely to be mediated through sugammadex-specific immunoglobulin G- or E-mediated mast cell stimulation in healthy volunteers., Clinical Trial Registration: NCT02028065., (Copyright © 2018 British Journal of Anaesthesia. All rights reserved.)

Aims: Sugammadex rapidly reverses moderate and deep rocuronium- or vecuronium-induced neuromuscular blockade at doses of 4 mg/kg and 2 mg/kg, respectively. Sugammadex is renally eliminated. This study evaluated the pharmacokinetics of sugammadex in subjects with renal impairment versus those with normal renal function., Methods: This open-label, two-part, phase 1 study included adults with moderate (creatinine clearance (CL cr ) 30 - < 50 mL/min) and severe (CL cr < 30 mL/min) renal impairment and healthy controls (CL cr ≥ 80 mL/min). A single intravenous (IV) bolus injection of sugammadex 4 mg/kg was administered into a peripheral vein over 10 seconds directly by straight needle in part 1 (n = 24; 8/group), and via an IV catheter followed by a saline flush in part 2 (n = 18; 6/group). Plasma concentrations of sugammadex were collected after drug administration. Due to dosing issues in part 1, pharmacokinetic parameters were determined for part 2 only. Safety was assessed throughout the study., Results: Pharmacokinetic data were obtained from 18 subjects. Mean sugammadex exposure (AUC 0-∞ ) in subjects with moderate and severe renal impairment was 2.42- and 5.42-times, respectively, that of healthy controls. Clearance decreased and apparent terminal half-life was prolonged with increasing renal dysfunction. Similar C max values were observed in subjects with renal impairment and healthy controls. There were no serious adverse events., Conclusions: Sugammadex exposure is increased in subjects with moderate and severe renal insufficiency due to progressively decreased clearance as a function of worsening renal function. Sugammadex 4 mg/kg was well tolerated in subjects with renal impairment, with a safety profile similar to that of healthy subjects. These results indicate that dose adjustment of sugammadex is not required in patients with moderate renal impairment; however, current safety experience is insufficient to support the use of sugammadex in patients with CL cr < 30 mL/min.
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Objective: Lp(a) [lipoprotein (a)] is composed of apoB (apolipoprotein B) and apo(a) [apolipoprotein (a)] and is an independent risk factor for cardiovascular disease and aortic stenosis. In clinical trials, anacetrapib, a CETP (cholesteryl ester transfer protein) inhibitor, causes significant reductions in plasma Lp(a) levels. We conducted an exploratory study to examine the mechanism for Lp(a) lowering by anacetrapib., Approach and Results: We enrolled 39 participants in a fixed-sequence, double-blind study of the effects of anacetrapib on the metabolism of apoB and high-density lipoproteins. Twenty-nine patients were randomized to atorvastatin 20 mg/d, plus placebo for 4 weeks, and then atorvastatin plus anacetrapib (100 mg/d) for 8 weeks. The other 10 subjects were randomized to double placebo for 4 weeks followed by placebo plus anacetrapib for 8 weeks. We examined the mechanisms of Lp(a) lowering in a subset of 12 subjects having both Lp(a) levels >20 nmol/L and more than a 15% reduction in Lp(a) by the end of anacetrapib treatment. We performed stable isotope kinetic studies using 2 H 3 -leucine at the end of each treatment to measure apo(a) fractional catabolic rate and production rate. Median baseline Lp(a) levels were 21.5 nmol/L (interquartile range, 9.9-108.1 nmol/L) in the complete cohort (39 subjects) and 52.9 nmol/L (interquartile range, 38.4-121.3 nmol/L) in the subset selected for kinetic studies. Anacetrapib treatment lowered Lp(a) by 34.1% ( P ≤0.001) and 39.6% in the complete and subset cohort, respectively. The decreases in Lp(a) levels were because of a 41% reduction in the apo(a) production rate, with no effects on apo(a) fractional catabolic rate., Conclusions: Anacetrapib reduces Lp(a) levels by decreasing its production., Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00990808., (© 2017 The Authors.)

Cholesteryl ester transfer protein (CETP) mediates the transfer of HDL cholesteryl esters for triglyceride (TG) in VLDL/LDL. CETP inhibition, with anacetrapib, increases HDL-cholesterol, reduces LDL-cholesterol, and lowers TG levels. This study describes the mechanisms responsible for TG lowering by examining the kinetics of VLDL-TG, apoC-II, apoC-III, and apoE. Mildly hypercholesterolemic subjects were randomized to either placebo (N = 10) or atorvastatin 20 mg/qd (N = 29) for 4 weeks (period 1) followed by 8 weeks of anacetrapib, 100 mg/qd (period 2). Following each period, subjects underwent stable isotope metabolic studies to determine the fractional catabolic rates (FCRs) and production rates (PRs) of VLDL-TG and plasma apoC-II, apoC-III, and apoE. Anacetrapib reduced the VLDL-TG pool on a statin background due to an increased VLDL-TG FCR (29%; P = 0.002). Despite an increased VLDL-TG FCR following anacetrapib monotherapy (41%; P = 0.11), the VLDL-TG pool was unchanged due to an increase in the VLDL-TG PR (39%; P = 0.014). apoC-II, apoC-III, and apoE pool sizes increased following anacetrapib; however, the mechanisms responsible for these changes differed by treatment group. Anacetrapib increased the VLDL-TG FCR by enhancing the lipolytic potential of VLDL, which lowered the VLDL-TG pool on atorvastatin background. There was no change in the VLDL-TG pool in subjects treated with anacetrapib monotherapy due to an accompanying increase in the VLDL-TG PR., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Background: This study determined whether relaxin or matrix metalloproteinase (MMP)-9 influences angiotensin II (AngII)-induced abdominal aortic aneurysms (AAA).Methods and Results:Male C57BL/6 or apolipoprotein E -/- mice were infused with AngII with or without relaxin. Relaxin did not influence AngII-induced AAA in either mouse strain. Infusion of AngII reduced, but relaxin increased, MMP-9 mRNA in macrophages. We then determined the effects of MMP-9 deficiency on AAA in apolipoprotein E -/- mice. MMP-9 deficiency led to AAA formation in the absence of AngII, and augmented AngII-induced aortic rupture and AAA incidence., Conclusions: MMP-9 deficiency augmented AngII-induced AAA.

Factor XI (FXI) is an integral component of the intrinsic pathway of the coagulation cascade and plays a critical role in thrombus formation. Because its role in the pathogenesis of cerebral microembolic signals (MES) is unclear, this study used a potent and selective small molecule inhibitor of FXIa, compound 1, to assess the effect of FXI blockade in our recently established preclinical model of cerebral MES induced by FeCl 3 injury of the carotid artery in male New Zealand White rabbits. Ascending doses of compound 1 were evaluated simultaneously for both carotid arterial thrombosis by a Doppler flowmeter and MES in the middle cerebral artery by a transcranial Doppler. Plasma drug exposure and pharmacodynamic responses to compound 1 treatment were also assessed. The effective dose for 50% inhibition (ED 50 ) of thrombus formation was 0.003 mg/kg/h compound 1, i.v. for the integrated blood flow, 0.004 mg/kg/h for reduction in thrombus weight, and 0.106 mg/kg/h for prevention of MES. The highest dose, 3 mg/kg/h compound 1, achieved complete inhibition in both thrombus formation and MES. In addition, we assessed the potential bleeding liability of compound 1 (5 mg/kg/h, i.v., >1250-fold ED 50 levels in arterial thrombosis) in rabbits using a cuticle bleeding model, and observed about 2-fold (not statistically significant) prolongation in bleeding time. Our study demonstrates that compound 1 produced a robust and dose-dependent inhibition of both arterial thrombosis and MES, suggesting that FXIa blockade may represent a novel therapeutic strategy for the reduction in MES in patients at risk for ischemic stroke., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Coagulation Factor XII (FXII) plays a critical role in thrombosis. What is unclear is the level of enzyme occupancy of FXIIa that is needed for efficacy and the impact of FXIIa inhibition on cerebral embolism. A selective activated FXII (FXIIa) inhibitor, recombinant human albumin-tagged mutant Infestin-4 (rHA-Mut-inf), was generated to address these questions. rHA-Mut-inf displayed potency comparable to the original wild-type HA-Infestin-4 (human FXIIa inhibition constant = 0.07 and 0.12 nM, respectively), with markedly improved selectivity against Factor Xa (FXa) and plasmin. rHA-Mut-inf binds FXIIa, but not FXII zymogen, and competitively inhibits FXIIa protease activity. Its mode of action is hence akin to typical small-molecule inhibitors. Plasma shift and aPTT studies with rHA-Mut-inf demonstrated that calculated enzyme occupancy for FXIIa in achieving a putative aPTT doubling target in human, nonhuman primate, and rabbit is more than 99.0%. The effects of rHA-Mut-inf in carotid arterial thrombosis and microembolic signal (MES) in middle cerebral artery were assessed simultaneously in rabbits. Dose-dependent inhibition was observed for both arterial thrombosis and MES. The ED 50 of thrombus formation was 0.17 mg/kg i.v. rHA-Mut-inf for the integrated blood flow and 0.16 mg/kg for thrombus weight; the ED 50 for MES was 0.06 mg/kg. Ex vivo aPTT tracked with efficacy. In summary, our findings demonstrated that very high enzyme occupancy will be required for FXIIa active site inhibitors, highlighting the high potency and exquisite selectivity necessary for achieving efficacy in humans. Our MES studies suggest that targeting FXIIa may offer a promising strategy for stroke prevention associated with thromboembolic events., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Cerebral microembolic signal (MES) is an independent predictor of stroke risk and prognosis. The objective of this study is to assess the effects of apixaban, as a representative of the novel oral anticoagulant class, on a rabbit model of cerebral MES. A clinical transcranial Doppler ultrasound instrument was used to assess MESs in the middle cerebral artery in a 30% FeCl3-induced carotid arterial thrombosis model in male New Zealand White rabbits. Ascending doses of apixaban were evaluated as monotherapy and in combination with aspirin on both arterial thrombosis and MES. Pharmacokinetic and pharmacodynamic responses were also evaluated. The effective dose for 50% inhibition (ED50) of thrombus formation for monotherapy was 0.04 mg/kg per hour apixaban, i.v. (0.03 μM plasma exposure) for the integrated blood flow, 0.13 mg/kg per hour apixaban (0.10 μM plasma exposure) for thrombus weight, and 0.03 mg/kg per hour apixaban (0.02 μM plasma exposure) for MES. Dual treatment with aspirin (5 mg/kg, PO) and apixaban (0.015 mg/kg per hour, i.v.) resulted in a significant reduction in cerebral MES (P < 0.05) compared with monotherapy with either agent. Pharmacokinetic analysis of apixaban and pharmacodynamic assays using activated partial thromboplastin time (aPTT) and prothrombin time (PT) for apixaban- and arachidonic acid-induced platelet aggregation for aspirin were used to confirm the exposure-response relationships. In summary, our study demonstrates that apixaban in a concentration-dependent manner inhibits both arterial thrombosis and MES, suggesting a potential association between factor Xa (FXa) blockade and the reduction in MES in patients at risk of ischemic stroke., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Unlabelled: Essentials Microembolic signal (MES) is an independent predictor of stroke risk in patients. A rabbit model of cerebral microembolic signals was established. Therapeutic efficacy was demonstrated for aspirin and clopidogrel on microembolic signals. Potential translational value of this preclinical model of MES was demonstrated., Summary: Objectives Cerebral microembolic signals (MESs) detected by transcranial Doppler (TCD) ultrasound constitute an independent predictor of stroke risk and prognosis. The aim of this study was to develop a novel preclinical model of MESs to facilitate translational research. Methods A clinical TCD ultrasound machine was used to detect MESs in the cerebral circulation of New Zealand White rabbits. Technical feasibility was assessed for the measurement of MESs in the middle cerebral artery (MCA) by TCD. FeCl3 -induced carotid arterial thrombosis was optimized for the generation of endogenous microemboli. Ascending doses of two antithrombotic agents (aspirin and clopidogrel) were evaluated individually and in combination for their effects on both arterial thrombosis and MESs in a 30% FeCl3 -induced carotid arterial thrombosis model, along with ex vivo functional assays. Results Dose-dependent FeCl3 -induced arterial thrombosis studies showed that 30% FeCl3 resulted in the most consistent and reproducible MESs in the MCA (3.3 ± 0.7 MESs h(-1) ). Ascending-dose studies showed that the effective doses for 50% inhibition (ED50 ) of thrombus formation, based on integrated blood flow and thrombus weight, respectively, were 3.1 mg kg(-1) and 4.2 mg kg(-1) orally for aspirin, and 0.3 mg kg(-1) and 0.28 mg kg(-1) orally for clopidogrel. The ED50 values for MES incidence were 12.7 mg kg(-1) orally for aspirin, and 0.25 mg kg(-1) orally for clopidogrel. Dual treatment with aspirin (5 mg kg(-1) ) and clopidogel (0.3 mg kg(-1) ) resulted in significant reductions in cerebral MESs (P < 0.05) as compared with monotherapy with either agent. Conclusions Our study demonstrated the successful establishment of the MES model in rabbits, and it may provide translational value for MESs and ischemic stroke research., (© 2016 International Society on Thrombosis and Haemostasis.)

Background: Nitric oxide donors are widely used to treat cardiovascular disease, but their major limitation is the development of tolerance, a multifactorial process to which the in vivo release of nitric oxide is thought to contribute. Here we describe the preclinical and clinical results of a translational drug development effort to create a next-generation nitric oxide donor with improved pharmacokinetic properties and a unique mechanism of nitric oxide release through CYP3A4 metabolism that was designed to circumvent the development of tolerance., Methods and Results: Single- and multiple-dose studies in telemetered dogs showed that MK-8150 induced robust blood-pressure lowering that was sustained over 14 days. The molecule was safe and well tolerated in humans, and single doses reduced systolic blood pressure by 5 to 20 mm Hg in hypertensive patients. Multiple-dose studies in hypertensive patients showed that the blood-pressure-lowering effect diminished after 10 days, and 28-day studies showed that the hemodynamic effects were completely lost by day 28, even when the dose of MK-8150 was increased during the dosing period., Conclusions: The novel nitric oxide donor MK-8150 induced significant blood-pressure lowering in dogs and humans for up to 14 days. However, despite a unique mechanism of nitric oxide release mediated by CYP3A4 metabolism, tolerance developed over 28 days, suggesting that tolerance to nitric oxide donors is multifactorial and cannot be overcome solely through altered in vivo release of nitric oxide., Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01590810 and NCT01656408., (© 2016 The Authors and Merck & Co., Inc. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Objective: Anacetrapib (ANA), an inhibitor of cholesteryl ester transfer protein (CETP) activity, increases plasma concentrations of high-density lipoprotein cholesterol (HDL-C), apolipoprotein A-I (apoA)-I, apoA-II, and CETP. The mechanisms responsible for these treatment-related increases in apolipoproteins and plasma CETP are unknown. We performed a randomized, placebo (PBO)-controlled, double-blind, fixed-sequence study to examine the effects of ANA on the metabolism of HDL apoA-I and apoA-II and plasma CETP., Approach and Results: Twenty-nine participants received atorvastatin (ATV) 20 mg/d plus PBO for 4 weeks, followed by ATV plus ANA 100 mg/d for 8 weeks (ATV-ANA). Ten participants received double PBO for 4 weeks followed by PBO plus ANA for 8 weeks (PBO-ANA). At the end of each treatment, we examined the kinetics of HDL apoA-I, HDL apoA-II, and plasma CETP after D3-leucine administration as well as 2D gel analysis of HDL subspecies. In the combined ATV-ANA and PBO-ANA groups, ANA treatment increased plasma HDL-C (63.0%; P<0.001) and apoA-I levels (29.5%; P<0.001). These increases were associated with reductions in HDL apoA-I fractional clearance rate (18.2%; P=0.002) without changes in production rate. Although the apoA-II levels increased by 12.6% (P<0.001), we could not discern significant changes in either apoA-II fractional clearance rate or production rate. CETP levels increased 102% (P<0.001) on ANA because of a significant reduction in the fractional clearance rate of CETP (57.6%, P<0.001) with no change in CETP production rate., Conclusions: ANA treatment increases HDL apoA-I and CETP levels by decreasing the fractional clearance rate of each protein., (© 2016 American Heart Association, Inc.)

Background: Individuals treated with the cholesteryl ester transfer protein (CETP) inhibitor anacetrapib exhibit a reduction in both LDL cholesterol and apolipoprotein B (ApoB) in response to monotherapy or combination therapy with a statin. It is not clear how anacetrapib exerts these effects; therefore, the goal of this study was to determine the kinetic mechanism responsible for the reduction in LDL and ApoB in response to anacetrapib., Methods: We performed a trial of the effects of anacetrapib on ApoB kinetics. Mildly hypercholesterolemic subjects were randomized to background treatment of either placebo (n = 10) or 20 mg atorvastatin (ATV) (n = 29) for 4 weeks. All subjects then added 100 mg anacetrapib to background treatment for 8 weeks. Following each study period, subjects underwent a metabolic study to determine the LDL-ApoB-100 and proprotein convertase subtilisin/kexin type 9 (PCSK9) production rate (PR) and fractional catabolic rate (FCR)., Results: Anacetrapib markedly reduced the LDL-ApoB-100 pool size (PS) in both the placebo and ATV groups. These changes in PS resulted from substantial increases in LDL-ApoB-100 FCRs in both groups. Anacetrapib had no effect on LDL-ApoB-100 PRs in either treatment group. Moreover, there were no changes in the PCSK9 PS, FCR, or PR in either group. Anacetrapib treatment was associated with considerable increases in the LDL triglyceride/cholesterol ratio and LDL size by NMR., Conclusion: These data indicate that anacetrapib, given alone or in combination with a statin, reduces LDL-ApoB-100 levels by increasing the rate of ApoB-100 fractional clearance., Trial Registration: ClinicalTrials.gov NCT00990808., Funding: Merck & Co. Inc., Kenilworth, New Jersey, USA. Additional support for instrumentation was obtained from the National Center for Advancing Translational Sciences (UL1TR000003 and UL1TR000040).

Background: For a more complete understanding of pharmacodynamic, metabolic, and pathophysiologic effects, protein kinetics, such as production rate and fractional catabolic rate, can offer substantially more information than protein concentration alone. Kinetic experiments with stable isotope tracers typically require laborious sample preparation and are most often used for studying abundant proteins. Here we describe a practical methodology for measuring isotope enrichment into low-abundance proteins that uses an automated procedure and immunoaffinity enrichment (IA) with LC-MS. Low-abundance plasma proteins cholesteryl ester transfer protein (CETP) and proprotein convertase subtilisin/kexin type 9 (PCSK9) were studied as examples., Methods: Human participants (n = 39) were infused with [(2)H(3)]leucine, and blood samples were collected at multiple time points. Sample preparation and analysis were automated and multiplexed to increase throughput. Proteins were concentrated from plasma by use of IA and digested with trypsin to yield proteotypic peptides that were analyzed by microflow chromatography-mass spectrometry to measure isotope enrichment., Results: The IA procedure was optimized to provide the greatest signal intensity. Use of a gel-free method increased throughput while increasing the signal. The intra- and interassay CVs were <15% at all isotope enrichment levels studied. More than 1400 samples were analyzed in <3 weeks without the need for instrument stoppages or user interventions., Conclusions: The use of automated gel-free methods to multiplex the measurement of isotope enrichment was applied to the low-abundance proteins CETP and PCSK9., (© 2014 American Association for Clinical Chemistry.)

Objective: To investigate the potential effect of sugammadex on anti-Xa anticoagulantactivity of enoxaparin and the activated partial thromboplastin time (APTT) of unfractionated heparin (UFH)., Methods: This two-part, randomized, double-blind, placebocontrolled, four-period cross-over study was performed in healthy males (18 - 45 years). In each period, subjects received 40 mg enoxaparin (in part 1), 5,000 units UFH (in part 2), or placebo followed by 4 or 16 mg/kg sugammadex, or placebo. Treatments were separated by ≥ 4 days. Primary endpoints were anti-Xa activity and APTT both time-averaged from 3 to 30 minutes post-dose. Geometric mean ratios (GMRs) and their two-sided 90% confidence limits were calculated for anticoagulant plus sugammadex (4 or 16 mg/kg) vs. anticoagulant plus placebo. The pre-specified threshold for a potential effect of clinical relevance was a 90% upper confidence limit (UCL) > 1.50., Results: In part 1 (n = 13), the 90% UCLs were 1.07 and 1.08 for GMRs of anti-Xa activity after dosing with 4 and 16 mg/kg sugammadex, respectively. In part 2 (n = 43), the 90% UCLs for GMRs of APTT were 1.06 and 1.15. Neither sugammadex dose produced a treatment effect that met the pre-specified criterion for potential clinical relevance. Treatments were generally well tolerated., Conclusions: In healthy subjects, treatment with 4 mg/kg and 16 mg/kg sugammadex did not change either anti-Xa activity or APTT to a clinically meaningful extent following pretreatments with enoxaparin or UFH.

LC/MS quantification of multiple plasma proteins that differ by several orders of magnitude in concentration from a single sample is challenging. We present a strategy that allows the simultaneous determination of the concentration and turnover kinetics of higher and lower abundant proteins from a single digestion mixture. Our attention was directed at a cluster of proteins that interact to affect the absorption and interorgan lipid trafficking. We demonstrate that apos involved in TG metabolism such as apoC2, C3, E, and A4 (micromolar concentration), and apoB48 and apoA5 (single-digit nanomolar concentration) can be quantified from a single digestion mixture. A high degree of correlation between LC/MS and immunobased measurements for apoC2, C3, E, and B48 was observed. Moreover, apoA5 fractional synthesis rate was measured in humans for the first time. Finally, the method can be directly applied to studies involving nonhuman primates because peptide sequences used in the method are conserved between humans and nonhuman primates., (Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.)

Anacetrapib is a novel cholesteryl ester transfer protein (CETP) inhibitor in development for treatment of dyslipidemia. This open-label, fixed-sequence, 3-period study was intended to evaluate the potential of anacetrapib to be a victim of OATP1B1/3 inhibition and strong CYP3A induction using acute and chronic dosing of rifampin, respectively, as a probe. In this study, 16 healthy subjects received 100 mg anacetrapib administered without rifampin (Day 1, Period 1), with single-dose (SD) 600 mg rifampin (Day 1, Period 2), and with multiple-dose (MD) 600 mg rifampin for 20 days (Day 14, Period 3). Log-transformed anacetrapib AUC0-∞ and Cmax were analyzed by a linear mixed effects model. The GMRs and 90% CIs for anacetrapib AUC0-∞ and Cmax were 1.25 (1.04, 1.51) and 1.43 (1.13, 1.82) for SD rifampin (Period 2/Period 1) and 0.35 (0.29, 0.42) and 0.26 (0.21, 0.32) for MD rifampin (Period 3/Period 1), respectively. Anacetrapib was generally well tolerated in both the absence/presence of SD and MD rifampin. In conclusion, treatment with SD rifampin, which inhibits the OATP1B1/3 transporter system, did not substantially influence the SD pharmacokinetics of anacetrapib, while chronic (20 days) administration of rifampin, which strongly induces CYP3A isozymes, reduced mean systemic exposure to SD anacetrapib by 65%., (© The Author(s) 2013.)

Rationale: Apolipoprotein(a) [apo(a)] is the defining protein component of lipoprotein(a) [Lp(a)], an independent risk factor for cardiovascular disease. The regulation of Lp(a) levels in blood is poorly understood in part due to technical challenges in measuring Lp(a) kinetics. Improvements in the ability to readily and reliably measure the kinetics of apo(a) using a stable isotope labeled tracer is expected to facilitate studies of the role of Lp(a) in cardiovascular disease. Since investigators typically determine the isotopic labeling of protein-bound amino acids following acid-catalyzed hydrolysis of a protein of interest [e.g., apo(a)], studies of protein synthesis require extensive protein purification which limits throughput and often requires large sample volumes. We aimed to develop a rapid and efficient method for studying apo(a) kinetics that is suitable for use in studies involving human subjects., Methods: Microfluidic device and tandem mass spectrometry were used to quantify the incorporation of [(2)H3]-leucine tracer into protein-derived peptides., Results: We demonstrated that it is feasible to quantify the incorporation of [(2)H3]-leucine tracer into a proteolytic peptide from the non-kringle repeat region of apo(a) in human subjects. Specific attention was directed toward optimizing the multiple reaction monitoring (MRM) transitions, mass spectrometer settings, and chromatography (i.e., critical parameters that affect the sensitivity and reproducibility of isotopic enrichment measurements). The results demonstrated significant advantages with the use of a microfluidic device technology for studying apo(a) kinetics, including enhanced sensitivity relative to conventional micro-flow chromatography, a virtually drift-free elution profile, and a stable and robust electrospray., Conclusions: The technological advances described herein enabled the implementation of a novel method for studying the kinetics of apo(a) in human subjects infused with [(2)H3]-leucine., (Copyright © 2013 John Wiley & Sons, Ltd.)

Recent advances in electrocardiographic monitoring and waveform analysis have significantly improved the ability to detect drug-induced changes in cardiac repolarization manifested as changes in the QT/corrected QT interval. These advances have also improved the ability to detect drug-induced changes in cardiac conduction. This White Paper summarizes current opinion, reached by consensus among experts at the Cardiac Safety Research Consortium, on the assessment of electrocardiogram-based safety measurements of the PR and QRS intervals, representing atrioventricular and ventricular conduction, respectively, during drug development., (Copyright © 2013 Mosby, Inc. All rights reserved.)

The ability to make informed benefit-risk assessments for potentially cardiotoxic new compounds is of considerable interest and importance at the public health, drug development, and individual patient levels. Cardiac imaging approaches in the evaluation of drug-induced myocardial dysfunction will likely play an increasing role. However, the optimal choice of myocardial imaging modality and the recommended frequency of monitoring are undefined. These decisions are complicated by the array of imaging techniques, which have varying sensitivities, specificities, availabilities, local expertise, safety, and costs, and by the variable time-course of tissue damage, functional myocardial depression, or recovery of function. This White Paper summarizes scientific discussions of members of the Cardiac Safety Research Consortium on the main factors to consider when selecting nonclinical and clinical cardiac function imaging techniques in drug development. We focus on 3 commonly used imaging modalities in the evaluation of cardiac function: echocardiography, magnetic resonance imaging, and radionuclide (nuclear) imaging and highlight areas for future research., (Copyright © 2012 Mosby, Inc. All rights reserved.)

D-dimer is a product of the coagulation cascade and is associated with venous thromboembolism, disseminated intravascular coagulation, and additional clinical conditions. Despite its importance, D-dimer measurement has limited clinical utility due in part to the lack of reliable assays. The difficulty in developing an immunoassay that is specific for D-dimer arises from the inherent heterogeneity in its structure. In this report, we describe a highly specific method for the quantification of D-dimer level in human plasma. In our method, the reciprocally cross-linked peptide resulting from factor XIIIa-catalyzed dimerization of fibrin γ chains was selected to represent the D-dimer antigen. Using an antipeptide antibody, we enriched the cross-linked peptide from trypsin-digested plasma prior to quantitative analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The assay has a quantitative range of 500 pmol/L to 100 nmol/L in human plasma. In further characterization of the assay, we found that it exhibited good correlation with fibrinolytic activity in human donors and with thrombin generation and clot strength in an in vitro thromboelastography assay. These observations thus establish the biological relevance of the assay and suggest it may be a valuable biomarker in characterization and treatment of blood coagulation disorders.

Aims: To characterize monocyte response in a delayed-type hypersensitivity reaction to intradermal tetanus toxoid (TT) injection., Materials & Methods: Men with positive serum anti-tetanus titers were stratified by last TT vaccination. Subjects were administered three intradermal injections of TT and one saline control on the same side of the back. Skin biopsies were taken post-injection. After 2 weeks, the procedure was repeated on the contralateral side., Results: Men who received TT booster vaccination 1 month before the study showed greater reproducibility and lower variability in monocyte responses than those who were not revaccinated. Monocyte concentration in subjects re-vaccinated within 1 month of study start appeared maximal at 48 h post-injection., Conclusion: This assay represents a novel approach that allows for quantification of dermal monocyte/macrophage influx. This clinical methodology has potential utility in the pharmacodynamic evaluation of therapies targeting inflammatory disorders, which involve monocyte tissue recruitment, like the delayed-type hypersensitivity response.

Hematopoietic stem cell (HSC) aging has become a concern in chemotherapy of older patients. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment (HM) control HSC activity during regenerative hematopoiesis. Connexin-43 (Cx43), a connexin constituent of gap junctions (GJs) is expressed in HSCs, down-regulated during differentiation, and postulated to be a self-renewal gene. Our studies, however, reveal that hematopoietic-specific Cx43 deficiency does not result in significant long-term competitive repopulation deficiency. Instead, hematopoietic Cx43 (H-Cx43) deficiency delays hematopoietic recovery after myeloablation with 5-fluorouracil (5-FU). 5-FU-treated H-Cx43-deficient HSC and progenitors (HSC/P) cells display decreased survival and fail to enter the cell cycle to proliferate. Cell cycle quiescence is associated with down-regulation of cyclin D1, up-regulation of the cyclin-dependent kinase inhibitors, p21(cip1.) and p16(INK4a), and Forkhead transcriptional factor 1 (Foxo1), and activation of p38 mitogen-activated protein kinase (MAPK), indicating that H-Cx43-deficient HSCs are prone to senescence. The mechanism of increased senescence in H-Cx43-deficient HSC/P cells depends on their inability to transfer reactive oxygen species (ROS) to the HM, leading to accumulation of ROS within HSCs. In vivo antioxidant administration prevents the defective hematopoietic regeneration, as well as exogenous expression of Cx43 in HSC/P cells. Furthermore, ROS transfer from HSC/P cells to BM stromal cells is also rescued by reexpression of Cx43 in HSC/P. Finally, the deficiency of Cx43 in the HM phenocopies the hematopoietic defect in vivo. These results indicate that Cx43 exerts a protective role and regulates the HSC/P ROS content through ROS transfer to the HM, resulting in HSC protection during stress hematopoietic regeneration.

Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animals.

Cholesteryl ester transfer protein (CETP) inhibition is a promising experimental strategy to raise high-density lipoprotein cholesterol (HDL-C) and reduce cardiovascular risk. This review focuses on the highly selective and potent CE TP inhibitor anacetrapib and discusses the available preclinical and clinical information pertaining to it. We also describe strategies to target HDL-C, discuss the mechanism underlying CETP inhibition and its effects on lipid biology, and give an overview of other CETP inhibitors that are currently in development.

Connexin43 (Cx43), a component of gap junctions, has a relatively large carboxy-terminal region with multiple proteomic interactions. Proteomic interactions with its cytoplasmic loop, however, are poorly defined. The goal of this study is to examine proteomic interactions involving the cytoplasmic loop (CL) of Cx43. The authors utilized various techniques, including glutathione-S-transferase (GST) pull-down, immunoblot analysis, two-dimensional (2D) gel electrophoresis, and mass spectrometry, to elucidate binding partners for Cx43-CL. The authors identified novel interactions with Cx43-CL involving α- and β-tubulin, myelin basic protein, and Purα. Because tubulin interacts with the C-terminus of Cx43 (Cx43-CT), the authors further investigated the nature of the interaction between β-tubulin and Cx43-CL. β-Tubulin binds with the full length of Cx43-CL with approximately one-fifth the affinity of the interaction between Cx43-CT and β-tubulin. This study demonstrates novel proteomic interactions involving Cx43-CL that may lead to a more complete understanding of trafficking and gating of gap junction channels.

Gap junction redistribution and reduced expression, a phenomenon termed gap junction remodeling (GJR), is often seen in diseased hearts and may predispose toward arrhythmias. We have recently shown that short-term pacing in the mouse is associated with changes in connexin43 (Cx43) expression and localization but not with increased inducibility into sustained arrhythmias. We hypothesized that short-term pacing, if imposed on murine hearts with decreased Cx43 abundance, could serve as a model for evaluating the electrophysiological effects of GJR. We paced wild-type (normal Cx43 abundance) and heterozygous Cx43 knockout (Cx43+/-; 66% mean reduction in Cx43) mice for 6 h at 10-15% above their average sinus rate. We investigated the electrophysiological effects of pacing on the whole animal using programmed electrical stimulation and in isolated ventricular myocytes with patch-clamp studies. Cx43+/- myocytes had significantly shorter action potential durations (APD) and increased steady-state (Iss) and inward rectifier (I(K1)) potassium currents compared with those of wild-type littermate cells. In Cx43+/- hearts, pacing resulted in a significant prolongation of ventricular effective refractory period and APD and significant diminution of Iss compared with unpaced Cx43+/- hearts. However, these changes were not seen in paced wild-type mice. These data suggest that Cx43 abundance plays a critical role in regulating currents involved in myocardial repolarization and their response to pacing. Our study may aid in understanding how dyssynchronous activation of diseased, Cx43-deficient myocardial tissue can lead to electrophysiological changes, which may contribute to the worsened prognosis often associated with pacing in the failing heart.

Impulse propagation in cardiac tissue is a complex process in which intercellular coupling through gap junction channels is a critical component. Connexin40 (Cx40) is an abundant gap junction protein that is expressed in atrial myocytes. Alterations in the expression of Cx40 have been implicated in atrial arrhythmogenesis. The purpose of the current study was to assess the role of Cx40 in atrial impulse propagation. High-resolution optical mapping was used to study conduction in the right and left atrial appendages of isolated Langendorff-perfused murine hearts. Wild-type (Cx40(+/+)), heterozygous (Cx40(+/-)), and knockout (Cx40(-/-)) mice, both adult and embryonic, were studied to assess the effects of reduced Cx40 expression on sinus node function and conduction velocity at different pacing cycle lengths (100 and 60 ms). In both adult and late-stage embryonic Cx40(+/+) mice, heterogeneity in CV was found between the right and left atrial appendages. Either partial (Cx40(+/-)) or complete (Cx40(-/-)) deletion of Cx40 was associated with the loss of conduction heterogeneity in both adult and embryonic mice. Additionally, sinus node impulse initiation was found to be ectopic in Cx40(-/-) mice at 15.5 days postcoitus, whereas Cx40(+/+) mice showed normal activation occurring near the crista terminalis. Our findings suggest that Cx40 plays an essential role in establishing interatrial conduction velocity heterogeneity in the murine model. Additionally, we describe for the first time a developmental requirement for Cx40 in normal sinus node impulse initiation at 15.5 days postcoitus.

Mutations in human KCNE2, which encodes the MiRP1 potassium channel ancillary subunit, associate with long QT syndrome (LQTS), a defect in ventricular repolarization. The precise cardiac role of MiRP1 remains controversial, in part, because it has marked functional promiscuity in vitro. Here, we disrupted the murine kcne2 gene to define the role of MiRP1 in murine ventricles. kcne2 disruption prolonged ventricular action potential duration (APD), suggestive of reduced repolarization capacity. Accordingly, kcne2 (-/-) ventricles exhibited a 50% reduction in I(K,slow1), generated by Kv1.5--a previously unknown partner for MiRP1. I(to,f), generated by Kv4 alpha subunits, was also diminished, by approximately 25%. Ventricular MiRP1 protein coimmunoprecipitated with native Kv1.5 and Kv4.2 but not Kv1.4 or Kv4.3. Unexpectedly, kcne2 (-/-) ventricular membrane fractions exhibited 50% less mature Kv1.5 protein than wild type, and disruption of Kv1.5 trafficking to the intercalated discs. Consistent with the reduction in ventricular K(+) currents and prolonged ventricular APD, kcne2 deletion lengthened the QT(c) under sevoflurane anesthesia. Thus, targeted disruption of kcne2 has revealed a novel cardiac partner for MiRP1, a novel role for MiRPs in alpha subunit targeting in vivo, and a role for MiRP1 in murine ventricular repolarization with parallels to that proposed for the human heart.

In the uterus, the formation of new maternal blood vessels in the stromal compartment at the time of embryonic implantation is critical for the establishment and maintenance of pregnancy. Although uterine angiogenesis is known to be influenced by the steroid hormones estrogen (E) and progesterone (P), the underlying molecular pathways remain poorly understood. Here, we report that the expression of connexin 43 (Cx43), a major gap junction protein, is markedly enhanced in response to E in uterine stromal cells surrounding the implanted embryo during the early phases of pregnancy. Conditional deletion of the Cx43 gene in these stromal cells and the consequent disruption of their gap junctions led to a striking impairment in the development of new blood vessels within the stromal compartment, resulting in the arrest of embryo growth and early pregnancy loss. Further analysis of this phenotypical defect revealed that loss of Cx43 expression resulted in aberrant differentiation of uterine stromal cells and impaired production of several key angiogenic factors, including the vascular endothelial growth factor (Vegf). Ablation of CX43 expression in human endometrial stromal cells in vitro led to similar findings. Collectively, these results uncovered a unique link between steroid hormone-regulated cell-cell communication within the pregnant uterus and the development of an elaborate vascular network that supports embryonic growth. Our study presents the first evidence that Cx43-type gap junctions play a critical and conserved role in modulating stromal differentiation, and regulate the consequent production of crucial paracrine signals that control uterine neovascularization during implantation.

Background: Cardiac insults such as ischemia, infarction, hypertrophy and dilatation are often accompanied by altered abundance and/or localization of the connexin43 gap junction protein, which may predispose towards arrhythmic complications. Models of chronic dyssynchronous cardiac activation have also been shown to result in redistribution of connexin43 in cardiomyocytes. We hypothesized that alterations in connexin43 expression and localization in the mouse heart might be induced by ventricular pacing over a short period of time., Results: The subdiaphragmatic approach was used to pace a series of wild type mice for six hours before the hearts were removed for analysis. Mice were paced at 10-15% above their average anesthetized sinus rate and monitored to ensure 1:1 capture. Short-term pacing resulted in a significant reduction in connexin43 mRNA abundance, a partial redistribution of connexin43 from the sarcolemma to a non-sarcolemmal fraction, and accumulation of ubiquitinated connexin43 without a significant change in overall connexin43 protein levels. These early pacing-induced changes in connexin43 expression were not accompanied by decreased cardiac function, prolonged refractoriness or increased inducibility into sustained arrhythmias., Conclusion: Our data suggest that short-term pacing is associated with incipient changes in the expression of the connexin43 gap junction, possibly including decreased production and a slowed rate of degradation. This murine model may facilitate the study of early molecular changes induced by pacing and may ultimately assist in the development of strategies to prevent gap junction remodeling and the associated arrhythmic complications of cardiac disease.

Loss of connexin43 (Cx43) gap junction channels in the heart results in a marked increase in the incidence of spontaneous and inducible polymorphic ventricular tachyarrhythmias (PVTs). The mechanisms resulting in this phenotype remain unclear. We hypothesized that uncoupling promotes regional ion channel remodeling, thereby increasing electrical heterogeneity and facilitating the development of PVT. In isolated-perfused control hearts, programmed electrical stimulation elicited infrequent monomorphic ventricular tachyarrhythmias (MVT), and dominant frequencies (DFs) during MVT were similar in the right ventricle (RV) and left ventricle (LV). Moreover, conduction properties, action potential durations (APDs), and repolarizing current densities were similar in RV and LV myocytes. In contrast, PVT was common in Cx43 conditional knockout (OCKO) hearts, and arrhythmias were characterized by significantly higher DFs in the RV compared to the LV. APDs in OCKO myocytes were significantly shorter than those from chamber-matched controls, with RV OCKO myocytes being most affected. APD shortening was associated with higher levels of sustained current in myocytes from both chambers as well as higher levels of the inward rectifier current only in RV myocytes. Thus, alterations in cell-cell coupling lead to regional changes in potassium current expression, which in this case facilitates the development of reentrant arrhythmias. We propose a new mechanistic link between electrical uncoupling and ion channel remodeling. These findings may be relevant not only in cardiac tissue but also to other organ systems where gap junction remodeling is known to occur.

Latent TGF-beta binding protein 1 (LTBP1) is a member of the LTBP/fibrillin family of extracellular proteins. Due to the usage of different promoters, LTBP1 exists in two major forms, long (L) and short (S), each expressed in a temporally and spatially unique fashion. Both LTBP1 molecules covalently interact with latent TGF-beta and regulate its function, presumably via interaction with the extracellular matrix (ECM). To explore the in vivo role of Ltbp1 in mouse development, at the time when only the L isoform is expressed, we mutated the Ltbp1L locus by gene targeting. Ltbp1L-null animals die shortly after birth from defects in heart development, consisting of the improper septation of the cardiac outflow tract (OFT) and remodeling of the associated vessels. These cardiac anomalies present as persistent truncus arteriosus (PTA) and interrupted aortic arch (IAA), which are associated with the faulty function of cardiac neural crest cells (CNCCs). The lack of Ltbp1L in the ECM of the septating OFT and associated vessels results in altered gene expression and function of CNCCs and decreased Tgf-beta activity in the OFT. This phenotype reveals a crucial role for Ltbp1L and matrix as extracellular regulators of Tgf-beta activity in heart organogenesis.

K(ATP) channels are involved in regulating coronary function, but the contribution of endothelial K(ATP) channels remains largely uncharacterized. We generated a transgenic mouse model to specifically target endothelial K(ATP) channels by expressing a dominant negative Kir6.1 subunit only in the endothelium. These animals had no obvious overt phenotype and no early mortality. Histologically, the coronary endothelium in these animals was preserved. There was no evidence of increased susceptibility to ergonovine-induced coronary vasospasm. However, isolated hearts from these animals had a substantially elevated basal coronary perfusion pressure. The K(ATP) channel openers, adenosine and levcromakalim, decreased the perfusion pressure whereas the K(ATP) channel blocker glibenclamide failed to produce a vasoconstrictive response. The inducible endothelial nitric oxide pathway was intact, as evidenced by vasodilation caused by bradykinin. In contrast, basal endothelin-1 release was significantly elevated in the coronary effluent from these hearts. Treatment of mice with bosentan (endothelin-1 receptor antagonist) normalized the coronary perfusion pressure, demonstrating that the elevated endothelin-1 release was sufficient to account for the increased coronary perfusion pressure. Pharmacological blockade of K(ATP) channels led to elevated endothelin-1 levels in the coronary effluent of isolated mouse and rat hearts as well as enhanced endothelin-1 secretion from isolated human coronary endothelial cells. These data are consistent with a role for endothelial K(ATP) channels to control the coronary blood flow by modulating the release of the vasoconstrictor, endothelin-1.

Imaging of the mammalian cardiac right ventricle (RV) is particularly challenging, especially when a two-dimensional method such as conventional histology is used to evaluate the morphology of this asymmetric, crescent-shaped chamber. MRI may improve the characterization of mutants with RV phenotypes by allowing analysis of the samples in any plane and by facilitating three-dimensional image reconstruction. MRI was used to examine the conditional knockout Cx43-PCKO mouse line known to have RV malformations. To help delineate the cardiovascular system and facilitate identification of the right ventricular outflow tract (RVOT), embryonic day (E) 17.5 embryos were perfusion fixed through the umbilical vein followed by a gadolinium-based contrast agent mixed in 7% gelatin. Micro-MRI experiments were performed at 7 T and followed by paraffin embedding of specimens, histological sectioning and hematoxylin and eosin (H&E) staining. Imaging of up to four embryos simultaneously allowed for higher throughput than traditional individual imaging techniques, while intravascular contrast afforded excellent signal-to-noise characteristics. All control embryos (n = 4) and heterozygous Cx43 knockout embryos (n = 4) had normal-appearing right ventricular outflow tract contours by MRI. Obvious abnormalities in the RVOT, including abnormal bulging and infiltration of contrast into the wall of the RV, were seen in three out of four Cx43-PCKO mutants with MRI. Furthermore, three-dimensional reconstruction of MR images with orthogonal projections as well as maximum-intensity projection allowed for visualization of the relationship of infundibular bulging segments to the pulmonary trunk in Cx43-PCKO mutant hearts. The addition of MRI to standard histology in the characterization of RV malformations in mutant mouse embryos aids in the assessment and understanding of morphologic abnormalities. Flexibility in the viewing of MR images, which can be retrospectively sectioned in any desired orientation, is particularly useful in the investigation of the RV, an asymmetric chamber that is difficult to analyze with two-dimensional techniques., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

GJA1 (also known and referred to here as connexin 43 and abbreviated CX43) is the predominant testicular gap junction protein, and CX43 may regulate Sertoli cell maturation and spermatogenesis. We hypothesized that lack of CX43 would inhibit Sertoli cell differentiation and extend proliferation. To test this, a Sertoli cell-specific Cx43 knockout (SC-Cx43 KO) mouse was generated using Cre-lox technology. Immunohistochemistry indicated that CX43 was not expressed in the Sertoli cells of SC-Cx43 KO mice, but was normal in organs such as the heart. Testicular weight was reduced by 41% and 76% in SC-Cx43 KO mice at 20 and 60 days, respectively, vs. wild-type (wt) mice. Seminiferous tubules of SC-Cx43 KO mice contained only Sertoli cells and actively proliferating early spermatogonia. Sertoli cells normally cease proliferation at 2 wk of age in mice and become terminally differentiated. However, proliferating Sertoli cells were present in SC-Cx43 KO but not wt mice at 20 and 60 days of age. Thyroid hormone receptor alpha (THRA) is high in proliferating Sertoli cells, then declines sharply in adulthood. Thra mRNA expression was increased in 20-day SC-Cx43 KO vs. wt mice, and it showed a trend toward an increase in 60-day mice, indicating that loss of CX43 in Sertoli cells inhibited their maturation. In conclusion, we have generated mice lacking CX43 in Sertoli cells but not other tissues. Our data indicate that CX43 in Sertoli cells is essential for spermatogenesis but not spermatogonial maintenance/proliferation. SC-Cx43 KO mice showed continued Sertoli cell proliferation and delayed maturation in adulthood, indicating that CX43 plays key roles in Sertoli cell development.

We report on the use of centerline analysis of cardiac-gated magnetic resonance images to measure wall motion abnormalities in mice infected with Trypanosoma cruzi. To our knowledge, this is the first report of segmental wall motion abnormalities in an animal model of Chagas' disease. Chagas' disease patients with severe cardiac involvement exhibit mild hypokinesis in an extensive region of the left ventricle and dyskinesis in the apical region. We observed dyskinetic segments in a similar region of the hearts of infected wild-type mice. Dyskinesis was not observed in infected mice lacking macrophage inflammatory protein-1alpha, a chemokine that may play an important role in the cardiac remodeling that is normally observed in mouse models of Chagas' disease and in human patients. This study aimed to demonstrate the utility of cardiac-gated magnetic resonance imaging and centerline analysis as a straightforward method for monitoring regional left ventricular wall motion in transgenic and/or diseased mice.

Cardiac ATP-sensitive K+ (K(ATP)) channels are formed by Kir6.2 and SUR2A subunits. We produced transgenic mice that express dominant negative Kir6.x pore-forming subunits (Kir6.1-AAA or Kir6.2-AAA) in cardiac myocytes by driving their expression with the alpha-myosin heavy chain promoter. Weight gain and development after birth of these mice were similar to nontransgenic mice, but an increased mortality was noted after the age of 4-5 mo. Transgenic mice lacked cardiac K(ATP) channel activity as assessed with patch clamp techniques. Consistent with a decreased current density observed at positive voltages, the action potential duration was increased in these mice. Some myocytes developed EADs after isoproterenol treatment. Hemodynamic measurements revealed no significant effects on ventricular function (apart from a slightly elevated heart rate), whereas in vivo electrophysiological recordings revealed a prolonged ventricular effective refractory period in transgenic mice. The transgenic mice tolerated stress less well as evident from treadmill stress tests. The proarrhythmogenic features and lack of adaptation to a stress response in transgenic mice suggest that these features are intrinsic to the myocardium and that K(ATP) channels in the myocardium have an important role in protecting the heart from lethal arrhythmias and adaptation to stress situations.

Connexin 43 (Cx43) is expressed in the embryonic heart, cardiac neural crest (CNC) and neural tube, and germline knockout (KO) of Cx43 results in aberrant cardiac outflow tract (OFT) formation and abnormal coronary deployment. Prior studies suggest a vital role for CNC expression of Cx43 in heart development. Surprisingly, we found that conditional knockout (CKO) of Cx43 in the dorsal neural tube and CNC mediated by Wnt1-Cre failed to recapitulate the Cx43-null OFT phenotype, although coronary vasculature was abnormal in this mutant line. A broader CKO mediated by P3pro (Pax3)-Cre, involving both ventral and dorsal aspects of the thoracic neural tube and CNC, resulted in infundibular bulging and coronary anomalies similar to those seen in germline Cx43-null hearts. P3pro-Cre-mediated loss of Cx43 in the neural tube was characterized by a late phase of cellular delamination from the dorsal and lateral neural tube, a markedly increased abundance of neuroepithelium-derived cells outside of the neural tube and an excess of such cells infiltrating the heart and infundibulum. Thus, expression of Cx43 in the CNC is crucial for normal coronary deployment, but Cx43 is not required in the CNC for normal OFT morphogenesis. Rather, this study suggests a novel function for Cx43 in which Cx43 acts through non-crest neuroepithelial cells to suppress cellular delamination from the neural tube and thereby preserve normal OFT development.

Genetic studies in the mouse have demonstrated that conditional cardiac-restricted loss of connexin43 (Cx43), the major ventricular gap junction protein, is highly arrhythmogenic. However, whether more focal gap junction remodeling, as is commonly seen in acquired cardiomyopathies, influences the propensity for arrhythmogenesis is not known. We examined electrophysiological properties and the frequency of spontaneous and inducible arrhythmias in genetically engineered chimeric mice derived from injection of Cx43-deficient embryonic stem cells into wild-type recipient blastocysts. Chimeric mice had numerous well-circumscribed microscopic Cx43-negative foci in their hearts, comprising approximately 15% of the total surface area as determined by immunohistochemical analysis. Systolic function in the chimeric mice was significantly depressed as measured echocardiographically (19.0% decline in fractional shortening compared with controls, P < 0.05) and by invasive hemodynamics (17.6% reduction in change of pressure over time, P < 0.01). Chimeras had significantly more spontaneous arrhythmic events than controls (P < 0.01), including frequent runs of nonsustained ventricular tachycardia in some of the chimeric mice. However, in contrast to mice with conditional cardiac-resricted loss of Cx43 in the heart, no sustained ventricular tachyarrhythmias were observed. We conclude that focal areas of uncoupling in the myocardium increase the likelihood of arrhythmic triggers, but more widespread uncoupling is required to support sustained arrhythmias.

Contact between bone marrow (BM) hematopoietic stem cells (HSC) and osteoblast/stromal (OS) cells has been shown to be crucial in the regulation of hematopoiesis. However, very little is known about the regulatory mechanisms of direct cell-to-cell communication in the hematopoietic microenvironment. Gap junction channels (connexons) are formed by polypeptides (connexins) arranged in hexamers and represent the best described intercellular communication system. Connexin-43 (Cx43) is expressed by BM OS cells and has been associated with the cadherin/beta -catenin signaling pathway, recently reported as relevant in the OS/HSC interaction at the stem cell niche. Here, we employed an inducible gene-targeted murine approach to study the role of Cx43 in HSC proliferation and differentiation in vivo. Mx-Cre/Cx43+/+ and Mx-Cre/Cx43flox/flox littermates have been analyzed after gene deletion induced in vivo by the interferon-inducer poly (I)-poly (C), generating control (Cx43+) and Cx43-deficient (Cx43-/-) mice. After one week, Cx43+ and Cx43-/- mice were treated with 5-fluorouracil (5-FU). Cx43 expression in Cx43-/- BM was markedly reduced (> 90%) as analyzed on day +14 post-5-FU treatment. Cx43 deficiency did not induce a significant change in peripheral blood counts before 5-FU treatment, but the hematopoiesis recovery after 5-FU treatment was severely impaired as demonstrated by absence of recovery of peripheral blood counts, including profound neutropenia, anemia with reticulocytopenia, thrombocytopenia and a 5- to 8-fold decrease of cellularity and hematopoietic progenitor content (granulomacrophagic colony-forming-units (CFU-GM-), erythroid burst forming units (BFU-E) and mixed colony forming units (CFU-mix-) in BM and spleen on day +14 post-5-FU treatment. However, the femoral content of Lin-/c-kit+/Sca1+ cells in Cx43-/- BM was maintained when compared to Cx43+ BM. Short-term competitive repopulation ability of Cx43-/- BM cells was diminished as compared to Cx43+ mice, specifically for myeloid and B lymphoid cells, but showed spared long-term competitive repopulation ability with roughly normal hematopoietic differentiation. These data suggest that hematopoietic regeneration after cycle-specific chemotherapy is blocked in Cx43-deficient mice at the long-term HSC repopulating level. Cx43 expression within the BM appears to be crucial in the development of an efficient response to hematopoietic stress.

Ventricular tachycardia is a common heart rhythm disorder and a frequent cause of sudden cardiac death. Aberrant cell-cell coupling through gap junction channels, a process termed gap junction remodeling, is observed in many of the major forms of human heart disease and is associated with increased arrhythmic risk in both humans and in animal models. Genetically engineered mice with cardiac-restricted knockout of Connexin43, the major cardiac gap junctional protein, uniformly develop sudden cardiac death, although a detailed electrophysiological understanding of their profound arrhythmic propensity is unclear. Using voltage-sensitive dyes and high resolution optical mapping techniques, we found that uncoupling of the ventricular myocardium results in ectopic sites of ventricular activation. Our data indicate that this behavior reflects alterations in source-sink relationships and paradoxical conduction across normally quiescent Purkinje-ventricular muscle junctions. The aberrant activation profiles are associated with wavefront collisions, which in the setting of slow conduction may account for the highly arrhythmogenic behavior of Connexin43-deficient hearts. Thus, the extent of gap junction remodeling in diseased myocardium is a critical determinant of cardiac excitation patterns and arrhythmia susceptibility.

Caveolin-1 and caveolin-3 are expressed in the mammalian heart. Mice deficient in caveolin 1 or 3 exhibit cardiac abnormalities including left ventricular hypertrophy and reduced fractional shortening. Cardiac imaging technologies such as transthoracic echocardiography and cardiac-gated magnetic resonance imaging (MRI) are effective tools for the study of left ventricular morphology and function in mice; however, there has not been widespread use of these technologies in studies of right ventricular morphology. In particular, right ventricular wall thickness has been difficult to assess using cardiac imaging technologies. We report here the use of centerline analysis of cardiac-gated MR images to more accurately determine right ventricular wall thickness in the mouse heart. Right ventricular wall thickness was evaluated in Cav-1 null, Cav-3 null and Cav-1/3 null mice, as well as wild-type control mice. Using this technique, we find that caveolin null mice exhibit significant thickening of the right ventricular wall as compared with age-matched wild-type controls. Interestingly, right ventricular wall thickening is greatest in the Cav-1/3 null mice. Furthermore, significant right ventricular wall thickening is also seen in the Cav-1 null mice. Histological analyses revealed right ventricular hypertrophy consistent with the imaging results. These studies demonstrate the utility of MRI in determining right ventricular wall thickness and underscore the severity of the right ventricular hypertrophy in caveolin null mice.