Routine Coronary Angiography in Adults Undergoing Percutaneous Atrial Septal Defect Closure: Implications for Practice Guidelines.

OBJECTIVES: Secundum atrial septal defect (ASD) is a common adult congenital heart lesion for which percutaneous closure offers treatment in patients with suitable anatomy. We sought to determine the outcomes of coronary angiography in a population of adults >40 years of age who were undergoing percutaneous ASD closure. METHODS: Patients >40 years of age who underwent ASD closure between 2009-2016 were included in this retrospective chart review. Coronary angiograms were reviewed by 2 independent reviewers to evaluate the presence and degree of coronary artery disease (CAD) and the resulting clinical sequelae. RESULTS: A total of 398 patients underwent ASD closure, and 300 had coronary angiography at the time of closure. Mild CAD (10%-39% vessel stenosis) was found in 33 patients (11.0%), moderate CAD (40%-69% vessel stenosis) in 23 patients (7.7%), and severe CAD (≥70% vessel stenosis) in 25 patients (8.3%). Of the 48 patients with moderate to severe CAD, 24 had proximal vessel disease and 21 had multivessel disease. Four patients (8.3%) with moderate to severe CAD subsequently underwent percutaneous interventions, 16 patients (33.3%) had medication changes, 16 patients (33.3%) had perfusion testing followed by medication changes, and 12 patients (25%) had no changes in their medical management. CONCLUSIONS: Given the prevalence of CAD in this population, routine coronary angiography at the time of ASD closure should be reserved for patients with an unfavorable cardiovascular risk profile, who have a higher likelihood of CAD. While patients with ASDs suffer from chest pain and dyspnea both before and after percutaneous closure, few have established CAD.

Atrioventricular Node Dysfunction and Ion Channel Transcriptome in Pulmonary Hypertension.

BACKGROUND: Heart block is associated with pulmonary hypertension, and the aim of the study was to test the hypothesis that the heart block is the result of a change in the ion channel transcriptome of the atrioventricular (AV) node. METHODS AND RESULTS: The most commonly used animal model of pulmonary hypertension, the monocrotaline-injected rat, was used. The functional consequences of monocrotaline injection were determined by echocardiography, ECG recording, and electrophysiological experiments on the Langendorff-perfused heart and isolated AV node. The ion channel transcriptome was measured by quantitative PCR, and biophysically detailed computer modeling was used to explore the changes observed. After monocrotaline injection, echocardiography revealed the pattern of pulmonary artery blood flow characteristic of pulmonary hypertension and right-sided hypertrophy and failure; the Langendorff-perfused heart and isolated AV node revealed dysfunction of the AV node (eg, 50% incidence of heart block in isolated AV node); and quantitative PCR revealed a widespread downregulation of ion channel and related genes in the AV node (eg, >50% downregulation of Cav1.2/3 and HCN1/2/4 channels). Computer modeling predicted that the changes in the transcriptome if translated into protein and function would result in heart block. CONCLUSIONS: Pulmonary hypertension results in a derangement of the ion channel transcriptome in the AV node, and this is the likely cause of AV node dysfunction in this disease.

Modern management of adult coarctation: transcatheter and surgical options.

Coarctation of the aorta (CoA), a juxtaductal obstructive lesion in the descending aorta and commonly associated with hypoplasia of the aortic arch occurs in 5-8% of patients with congenital heart disease. Since the initial surgical corrections in the 1950, surgical and transcatheter options have constantly evolved. Nowadays, transcatheter options are widely accepted as the initial treatment of choice in adults presenting with native or recurrent CoA. Surgical techniques are mainly reserved for patients with complex aortic arch anatomy such as extended arch hypoplasia or stenosis or para-CoA aneurysm formation. Extended aneurysms can be covered by conformable stents but stent implantation may require preparative vascular surgery. Complex re-CoA my best be treated by an ascending to descending bypass conduit. The following review aims to describe current endovascular and surgical practice pointing out modern developments and their limitations.

Benefit of endovascular stenting for aortic coarctation on systemic hypertension in adults.

BACKGROUND: Endovascular stenting is a recognised treatment strategy for aortic coarctation (CoA) in adults. We assessed systemic hypertension control and the need for antihypertensive therapy after CoA stenting in adults. METHODS: Data were collected prospectively on 54 patients (36 men; mean age: 34 ± 16 years) who underwent endovascular stenting for CoA over a 7-year period. Five patients were excluded as they did not attend follow-up appointments. Patients underwent clinical examination, including right arm systolic blood pressure (SBP) and 24-hour ambulatory blood pressure monitoring at baseline, 6-12 weeks and 9-12 months. RESULTS: There was a significant fall in mean peak-to-peak systolic gradient (PG) across the CoA after stenting (26 ± 11 mmHg vs. 5 ± 4 mmHg; P<0.01). There were successive reductions in right arm SBP and ambulatory SBP at baseline, 6-12 weeks and 9-12 months post-procedure (right arm: 155 ± 18 mmHg vs. 137 ± 17 mmHg vs. 142 ± 16 mmHg, respectively; all P-values <0.01; ambulatory: 142 ± 14 mmHg vs. 132 ± 16 mmHg vs. 131 ± 15 mmHg, respectively; all P-values <0.01). Twenty-four patients had severe CoA (PG >25 mmHg before stenting); baseline SBP was significantly higher in severe versus non-severe patients (160 mmHg vs. 148 mmHg; P=0.02). The absolute reduction in PG after stenting was significantly higher in the severe group (31 ± 7 mmHg vs. 14 ± 5 mmHg; P<0.0001), but there was no significant difference in SBP between groups at 6-12 weeks (141 mmHg vs. 135 mmHg; P=0.21) or 9-12 months (139 mmHg vs. 139 mmHg; P=0.96). CONCLUSION: Endovascular stenting of CoA results in a significant reduction in SBP at 6-12 weeks, which is sustained at 9-12 months, with similar outcomes in severe and non-severe CoA groups.

Regulatory toxicology in the twenty-first century: challenges, perspectives and possible solutions.

The advent of new testing systems and "omics"-technologies has left regulatory toxicology facing one of the biggest challenges for decades. That is the question whether and how these methods can be used for regulatory purposes. The new methods undoubtedly enable regulators to address important open questions of toxicology such as species-specific toxicity, mixture toxicity, low-dose effects, endocrine effects or nanotoxicology, while promising faster and more efficient toxicity testing with the use of less animals. Consequently, the respective assays, methods and testing strategies are subject of several research programs worldwide. On the other hand, the practical application of such tests for regulatory purposes is a matter of ongoing debate. This document summarizes key aspects of this debate in the light of the European "regulatory status quo", while elucidating new perspectives for regulatory toxicity testing.

Biophysical characterization of the underappreciated and important relationship between heart rate variability and heart rate.

Heart rate (HR) variability (HRV; beat-to-beat changes in the R-wave to R-wave interval) has attracted considerable attention during the past 30+ years (PubMed currently lists >17 000 publications). Clinically, a decrease in HRV is correlated to higher morbidity and mortality in diverse conditions, from heart disease to fetal distress. It is usually attributed to fluctuation in cardiac autonomic nerve activity. We calculated HRV parameters from a variety of cardiac preparations (including humans, living animals, Langendorff-perfused heart, and single sinoatrial nodal cell) in diverse species, combining this with data from previously published articles. We show that regardless of conditions, there is a universal exponential decay-like relationship between HRV and HR. Using 2 biophysical models, we develop a theory for this and confirm that HRV is primarily dependent on HR and cannot be used in any simple way to assess autonomic nerve activity to the heart. We suggest that the correlation between a change in HRV and altered morbidity and mortality is substantially attributable to the concurrent change in HR. This calls for re-evaluation of the findings from many articles that have not adjusted properly or at all for HR differences when comparing HRV in multiple circumstances.

Structure, function and clinical relevance of the cardiac conduction system, including the atrioventricular ring and outflow tract tissues.

It is now over 100years since the discovery of the cardiac conduction system, consisting of three main parts, the sinus node, the atrioventricular node and the His-Purkinje system. The system is vital for the initiation and coordination of the heartbeat. Over the last decade, immense strides have been made in our understanding of the cardiac conduction system and these recent developments are reviewed here. It has been shown that the system has a unique embryological origin, distinct from that of the working myocardium, and is more extensive than originally thought with additional structures: atrioventricular rings, a third node (so called retroaortic node) and pulmonary and aortic sleeves. It has been shown that the expression of ion channels, intracellular Ca(2+)-handling proteins and gap junction channels in the system is specialised (different from that in the ordinary working myocardium), but appropriate to explain the functioning of the system, although there is continued debate concerning the ionic basis of pacemaking. We are beginning to understand the mechanisms (fibrosis and remodelling of ion channels and related proteins) responsible for dysfunction of the system (bradycardia, heart block and bundle branch block) associated with atrial fibrillation and heart failure and even athletic training. Equally, we are beginning to appreciate how naturally occurring mutations in ion channels cause congenital cardiac conduction system dysfunction. Finally, current therapies, the status of a new therapeutic strategy (use of a specific heart rate lowering drug) and a potential new therapeutic strategy (biopacemaking) are reviewed.

Standalone balloon aortic valvuloplasty: indications and outcomes from the UK in the transcatheter valve era.

OBJECTIVES: We sought to characterize UK-wide balloon aortic valvuloplasty (BAV) experience in the TAVI era. BACKGROUND: BAV for acquired calcific aortic stenosis is in a phase of renaissance, largely due to the development of transcatheter aortic valve implantation (TAVI). METHODS: Data from 423 patients at 14 centers across the UK were analyzed. RESULTS: Patients were aged 80.9 ± 9.5 years; 52.5% were male. Mean logistic EuroScore was 27.3% ± 16.8%. Mean peak transaortic gradient fell from 62.0 ± 26.3 to 28.3 ± 16.2 mm Hg. Aortic valve area increased from 0.58 ± 0.19 to 0.80 ± 0.25 cm(2) echocardiographically. Procedural complication rate was 6.3%, comprising death (2.4%), blood transfusion ≥ 2 U (1.2%), cardiac tamponade (1.0%), stroke (1.0%), vascular surgical repair (1.0%), coronary embolism (0.5%), and permanent pacemaker (0.2%). Mortality was 13.8% at 30 days and 36.3% at 12 months. Subsequently, 18.3% of patients underwent TAVI and 7.0% sAVR, with improved survival compared to those who had no further intervention (logrank < 0.0001). Multivariate Cox proportional hazard analysis demonstrated that survival was adversely effected by the presence of coronary artery disease (HR 1.53, 95%CI 1.08-2.17, P = 0.018), poor LV function (HR 1.54, 95%CI 1.09-2.16, P = 0.014), and either urgent (HR 1.70, 95%CI 1.18-2.45; P = 0.004) or emergent presentation (HR 3.72, 95%CI 2.27-6.08; P < 0.0001). CONCLUSION: Balloon aortic valvuloplasty offers good immediate hemodynamic efficacy at an acceptable risk of major complications. Medium-term prognosis is poor in the absence of definitive therapy.

Hydroxymethyl-substituted furans: mutagenicity in Salmonella typhimurium strains engineered for expression of various human and rodent sulphotransferases.

5-Hydroxymethylfurfural (HMF) and furfuryl alcohol (FFA) are present in numerous foodstuffs at high levels. FFA is also used for the production of polymers. Both compounds had demonstrated some evidence of carcinogenic activity in 2-year bioassays. We tested these compounds and four congeners for mutagenicity in Salmonella typhimurium TA100 and TA100-derived strains expressing human or rodent sulphotransferases (SULTs). 5-Hydroxymethylfuroic acid, a metabolite of HMF, was not mutagenic in any strain. 3-Hydroxymethylfuran was weakly mutagenic in all strains independently of SULT expression. HMF, 2,5-(bishydroxymethyl)furan (metabolite of HMF), FFA and 5-methyl-FFA were inactive in TA100 but strongly mutagenic when human SULT1C2 was expressed. This form has been detected in ovary, kidney and foetal tissues. Human SULT1A1, SULT1A2 and SULT1A3 as well as murine Sult1a1 and Sult1d1 also activated some hydroxymethyl-substituted furans to varying degrees. Whereas chemically synthesised 5-sulphooxymethylfurfural was mutagenic in TA100, furfuryl sulphate was bacteriotoxic, only leading to marginal increases in the number of revertants. Furfuryl acetate, an uncharged ester of FFA, used as fragrance and food flavouring, was clearly mutagenic. We determined half-life times of 120 min, 20 s and 10 h, respectively, for 5-sulphooxymethylfurfural, furfuryl sulphate and furfuryl acetate at 37°C in water. It is likely that the short lifespan of furfuryl sulphate, together with its charge, led to insufficient penetration of the bacteria when added externally, although it was mutagenic when generated by appropriate SULTs from FFA within the cell.

Efficient oxidation of promutagenic hydroxymethylpyrenes by cDNA-expressed human alcohol dehydrogenase ADH2 and its inhibition by various agents.

Alkylated polycyclic aromatic hydrocarbons can be metabolically activated via benzylic hydroxylation and sulphation to electrophilically reactive esters. However, we previously found that the predominant biotransformation route for the hepatocarcinogen 1-hydroxymethylpyrene (1-HMP) in the rat in vivo is the oxidation of the side chain by alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases to the carboxylic acid. Inhibition of this pathway by ethanol (competing ADH substrate) or 4-methylpyrazole (ADH inhibitor) led to a dramatic increase in the 1-HMP-induced DNA adduct formation in rat tissues in the preceding study. In order to elucidate the role of individual ADHs in the metabolism of alkylated polycyclic aromatic hydrocarbons, we expressed the various members of the human ADH family in bacteria. Cytosolic preparations from bacteria expressing ADH2 clearly oxidized hydroxymethylpyrene isomers (1-, 2- and 4-HMP) with the highest rate. This form was purified to near homogeneity to perform detailed kinetic analyses. High catalytic efficiencies (V(max)/K(m)) were observed with HMPs. Thus, this value was 10,000-fold higher for 2-HMP than for the reference substrate, ethanol. The corresponding aldehydes were also efficiently reduced by ADH2. 4-Methylpyrazole inhibited the oxidation of the HMP isomers as well as the reverse reaction. Daidzein, cimetidine and the competing substrate ethanol were further compounds that inhibited the ADH2-mediated oxidative detoxification of 1-HMP.

SULT1C3, an orphan sequence of the human genome, encodes an enzyme activating various promutagens.

The human genome contains a sequence that is homologous to genes encoding soluble sulphotransferases (SULTs) based on the nucleotide sequence and possible intron/exon splice sites. The putative coding sequence (termed SULT1C3) was synthesized and integrated into a bacterial expression vector. We used the cDNA-expressed protein for raising an antiserum and studying enzyme activities. No activity was detected with 4-nitrophenol and 1-naphthol, known substrates of all other members of the human SULT1 subfamily. The activity was also negligible with paracetamol, ethanol, 5-hydroxymethylfurfural, 2-hydroxymethylpyrene, 2-(alpha-hydroxy)ethylpyrene, and corticosterone, compounds for which we have developed sensitive enzyme assays with direct determination of the product by HPLC-UV, HPLC-fluorescence or HPLC-MS/MS. Since diverse sulpho conjugates are chemically reactive - often short-lived and mutagenic - we expressed SULT1C3 in Ames'Salmonella typhimurium strains TA1538 and TA100, as we had done with many other SULTs previously. The expression level of SULT1C3 protein amounted to 2% of the total cytosolic proteins, which is in the middle range of other SULTs expressed in this model. Using recombinant bacterial tester strains in mutagenicity assays, we observed SULT1C3-mediated activation of several large benzylic alcohols derived from alkylated polycyclic hydrocarbons: 1-hydroxymethylpyrene, both enantiomers of 1-(alpha-hydroxy)ethylpyrene, 6-hydroxymethylbenzo[a]pyrene and 6-hydroxymethylanthanthrene. 1'-Hydroxysafrole was the smallest molecule activated by SULT1C3 up to date. Our study demonstrates that SULT1C3 has sulphotransferase activity and that it prefers relatively large substrates. The substrates detected were activated to mutagens, which cannot be the regular function of the enzyme. The physiological substrates remain to be identified. Probably, they are relatively large, endogenous or common exogenous, molecules.

Mutagenicity of arbutin in mammalian cells after activation by human intestinal bacteria.

Arbutin (hydroquinone-beta-D-glucopyranoside) is present in various food plants. Its aglycone, hydroquinone, is mutagenic and carcinogenic. We investigated whether hydroquinone may be released under conditions encountered in the human gastrointestinal tract. Arbutin was stable in artificial gastric juice. Fecal slurries from nine human subjects completely converted arbutin (2 mM) into hydroquinone. Four of nine representative human intestinal species investigated, namely Eubacterium ramulus, Enterococcus casseliflavus, Bacteroides distasonis, and Bifidobacterium adolescentis, deglycosylated arbutin at rates of 21.08, 16.62, 8.43 and 3.59 nmol x min(-1) x (mg protein)(-1), respectively. In contrast, homogenates from small intestinal mucosa and cytosolic fractions from colon mucosa deglycosylated arbutin at substantially lower rates: 0.50 and 0.09 nmol x min(-1) x (mg protein)(-1), respectively. Arbutin, unlike hydroquinone, did not induce gene mutations in Chinese hamster V79 cells in the absence of an activating system. However, in the presence of cytosolic fractions from E. ramulus or B. distasonis, arbutin was strongly mutagenic. Cytosolic fraction from Escherichia coli, showing no arbutin glycosidase activity, was not able to activate arbutin in this model system. The release of the proximate mutagen hydroquinone from arbutin by intestinal bacteria in the immediate vicinity of the colon mucosa may pose a potential risk.

V79-hCYP2E1-hSULT1A1, a cell line for the sensitive detection of genotoxic effects induced by carbohydrate pyrolysis products and other food-borne chemicals.

We recently constructed a Chinese hamster V79-derived cell line that stably expresses human cytochrome P450 (CYP) 2E1 and human sulphotransferase (SULT) 1A1. These enzymes are involved in the bioactivation of numerous promutagens/procarcinogens, but are not taken into account in standard in vitro mutagenicity assays. Various carbohydrate pyrolysis products and other food contaminants that induce tumours or preneoplastic lesions in laboratory animals are inactive or only weakly active in standard in vitro genotoxicity assays. This is the case for acrylamide, furan, 5-hydroxymethylfurfural, nitrofen and N-nitrosodimethylamine. These compounds were investigated for induction of sister chromatid exchange (SCE) in V79-hCYP2E1-hSULT1A1 cells. All test compounds showed positive results over a wide concentration range, starting at 0.01 microM for N-nitrosodimethylamine, 3 microM for furan, 12.5 microM for nitrofen, 20 microM for 5-hydroxymethylfurfural, and 200 microM for acrylamide. The concentration-response curve of furan was unusual, as this compound induced a statistically significant, but rather constant and weak increase in SCE over an extremely wide concentration range (3-16,000 microM). Furan was slightly less active, whereas the remaining compounds were much less active in the parental V79 cell line than in V79-hCYP2E1-hSULT1A1 cells. Compared to many other genotoxic effects, the study of SCE only requires small numbers of cells (and incubation volumes) and usually is detected even at low concentrations of the genotoxicant. Therefore, induction of SCE in V79-hCYP2E1-hSULT1A1 cells may be useful in the genotoxicity testing of preparations of heated food and in their bioassay-directed fractionation.

Sulpho-conjugation of ethanol in humans in vivo and by individual sulphotransferase forms in vitro.

We studied whether ethanol is sulphonated in humans with the perspective of using the urinary excretion of ethyl sulphate after ethanol consumption as a biomarker for SULT (sulphotransferase) activity. We developed a sensitive and selective HPLC-MS/MS method for determining ethyl sulphate in urine. Ten volunteers received a low dose of ethanol (0.1 g/kg of body mass). In general, excretion of ethyl sulphate was maximal in the first or second hour after dosage. Within 8 h, 2.5-6.8 micromol of ethyl sulphate was excreted. A 5-fold increase in the dose of ethanol led to an increase in the amount of ethyl sulphate excreted within 8 h (28-95 micromol) and the presence of this metabolite in urine for at least 24 h. Since ethyl sulphate was still being excreted for a substantial period after the elimination of ethanol, it might be used as a medium-time biomarker for preceding ethanol consumption. We have expressed previously all human SULT forms identified in Salmonella typhimurium. Ethanol sulphonation was studied in cytosolic preparations of these strains. The highest activities were observed with SULT1A2, 1B1 and 1C2, followed by 1A3. Activities were markedly lower with SULT1E1, 1A1 and 2A1, and were negligible with SULT1C1, 2B1a, 2B1b and 4A1. If the expression levels in tissues are additionally taken into account, SULT1A3 might be the predominant form for the sulphonation of ethanol in vivo, although a robust estimate requires further studies. With this limitation, urinary ethyl sulphate excretion appears very promising as a biomarker for SULT activity in vivo.

Extractionless method for the determination of urinary caffeine metabolites using high-performance liquid chromatography coupled with tandem mass spectrometry.

Caffeine is metabolised in humans primarily by cytochromes P450 1A2 and 2A6, xanthine dehydrogenase/oxidase, and N-acetyltransferase 2. The activities of these enzymes show a large variation due to genetic polymorphisms and/or induction by xenobiotics. Ratios of different caffeine metabolites in urine or other body fluids are frequently used to characterise the individual/actual activity of these enzymes. The common analytical method involves extensive sample preparation, followed by HPLC-UV. The presence of numerous other UV-absorbing chemicals in body fluids affects the sensitivity and selectivity of this method. We have developed an HPLC-electrospray-MS-MS method for the determination of 11 caffeine metabolites and two internal standards after a simple, extractionless preparation. Blank urine, obtained after 5 days on a methylxanthine-free diet, contained small amounts of some caffeine metabolites, but no other components producing any confounding signals. Eleven metabolites and internal standards were recovered at 90 to 110% after addition to the blank urine (0.1 to 2.5 micro M in the final sample involving a 20-fold dilution of urine) in the 0.1-2.5 micro M concentration range. Other metabolites, 5-acetylamino-6-amino-3-methyluracil (AAMU) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU), were detected with similar recovery and precision, but required higher concentrations (3 to 30 micro M). AFMU was completely converted into AAMU by a short alkalisation of urine. The method was explored in six healthy individuals after consuming coffee (4 mg caffeine per kg body mass). These experiments demonstrated the simplicity, high sensitivity and selectivity of the method under conditions used for phenotyping.

In vivo pharmacokinetics of calreticulin S-domain, an inhibitor of the classical complement pathway.

Inhibition of the complement system is potentially therapeutic in diseases where uncontrolled or overshooting complement activation plays a significant role in the pathogenesis of the disorder. Calreticulin (CRT) is a multifunctional protein whose cell-surface form (ectocalreticulin) is reported to be a C1q receptor. A 124-residue domain within CRT, the S-domain, binds to C1q, prevents the formation of C1 and so inhibits activation of the classical pathway. To assess the usefulness of CRT S-domain as a complement inhibitor, recombinant S-domain was expressed, radiolabeled, and the fate of the radiolabeled peptide followed in vivo. In rats, CRT-S-domain shows a half-life of 1.21 +/- 0.34 and 40.5 +/- 2.7 min in the distribution and elimination phases from plasma, respectively. The peptide remains largely intact, and is cleared from the circulation by the kidneys, where it accumulates in the proximal convoluted tubules, but is not excreted. Much smaller amounts of the peptide accumulate in other tissues, and essentially none crosses the blood-brain barrier.