A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome.

Several diseases are associated with perturbations in redox signaling and aberrant hydrogen sulfide metabolism, and numerous analytical methods exist for the measurement of the sulfur-containing species affected. However, uncertainty remains about their concentrations and speciation in cells/biofluids, perhaps in part due to differences in sample processing and detection principles. Using ultrahigh-performance liquid chromatography in combination with electrospray-ionization tandem mass spectrometry we here outline a specific and sensitive platform for the simultaneous measurement of 12 analytes, including total and free thiols, their disulfides and sulfide in complex biological matrices such as blood, saliva and urine. Total assay run time is < 10 min, enabling high-throughput analysis. Enhanced sensitivity and avoidance of artifactual thiol oxidation is achieved by taking advantage of the rapid reaction of sulfhydryl groups with N-ethylmaleimide. We optimized the analytical procedure for detection and separation conditions, linearity and precision including three stable isotope labelled standards. Its versatility for future more comprehensive coverage of the thiol redox metabolome was demonstrated by implementing additional analytes such as methanethiol, N-acetylcysteine, and coenzyme A. Apparent plasma sulfide concentrations were found to vary substantially with sample pretreatment and nature of the alkylating agent. In addition to protein binding in the form of mixed disulfides (S-thiolation) a significant fraction of aminothiols and sulfide appears to be also non-covalently associated with proteins. Methodological accuracy was tested by comparing the plasma redox status of 10 healthy human volunteers to a well-established protocol optimized for reduced/oxidized glutathione. In a proof-of-principle study a deeper analysis of the thiol redox metabolome including free reduced/oxidized as well as bound thiols and sulfide was performed. Additional determination of acid-labile sulfide/thiols was demonstrated in human blood cells, urine and saliva. Using this simplified mass spectrometry-based workflow the thiol redox metabolome can be determined in samples from clinical and translational studies, providing a novel prognostic/diagnostic platform for patient stratification, drug monitoring, and identification of new therapeutic approaches in redox diseases.

Assessment of the Physiological Adaptations to Chronic Hypoxemia in Eisenmenger Syndrome.

OBJECTIVE: Eisenmenger syndrome is characterized by severe and lifelong hypoxemia and pulmonary hypertension. Despite this, patients do surprisingly well and report a reasonable quality of life. The aim of this study was to investigate whether these patients undergo adaptation of their skeletal and cardiac muscle energy metabolism which would help explain this paradox. DESIGN AND SETTING: Ten patients with Eisenmenger syndrome and eight age- and sex-matched healthy volunteers underwent symptom-limited treadmill cardiopulmonary exercise testing, transthoracic echocardiography and (31) P magnetic resonance spectroscopy of cardiac and skeletal muscle. Five subjects from each group also underwent near infrared spectroscopy to assess muscle oxygenation. RESULTS: Despite having a significantly lower peak VO2 , patients with Eisenmenger syndrome have a similar skeletal muscle phosphocreatine (PCr) recovery, a measure of oxidative capacity, when compared to healthy controls (34.9 s ± 2.9 s vs. 35.2 s ± 1.7 s, P = .9). Furthermore their intracellular pH falls to similar levels during exercise suggesting they are not reliant on early anaerobic metabolism (0.3 ± 0.06 vs. 0.28 ± 0.04, P = .7). While their right ventricular systolic function remained good, the Eisenmenger group had a lower cardiac PCr/ATP ratio compared to the control group (1.55 ± 0.10 vs. 2.17 ± 0.15, P < .05). CONCLUSIONS: These results show that adult patients with Eisenmenger syndrome have undergone beneficial physiological adaptations of both skeletal and cardiac muscle. This may, in part, explain their surprisingly good survival despite a lifetime of severe hypoxemia and adverse cardiopulmonary hemodynamics.

Pharmacology and therapeutic role of inorganic nitrite and nitrate in vasodilatation.

Nitrite has emerged as an important bioactive molecule that can be biotransformed to nitric oxide (NO) related metabolites in normoxia and reduced to NO under hypoxic and acidic conditions to exert vasodilatory effects and confer a variety of other benefits to the cardiovascular system. Abundant research is currently underway to understand the mechanisms involved and define the role of nitrite in health and disease. In this review we discuss the impact of nitrite and dietary nitrate on vascular function and the potential therapeutic role of nitrite in acute heart failure.

Left ventricular filling patterns and its relation to left ventricular untwist in patients with type 1 diabetes and normal ejection fraction.

BACKGROUND: We evaluated young patients with type 1 diabetes (T1DM) who had normal left ventricular (LV) ejection fraction and used speckle tracking echocardiography to assess changes in LV untwisting. We used cardiac magnetic resonance imaging (MRI) to assess the LV filling patterns in these subjects. METHODS: We recruited 33 T1DM patients and 32 age-matched healthy controls (HC) into the study. Study participants underwent echocardiography, cardiac MRI and metabolic exercise testing. RESULTS: The early peak LV untwisting rate (E) was similar in T1DM and HC (-11.9 ± 4.6 0/cm/s vs -11.3 ± 4.7 0/cm/s, P=0.29) but the late peak LV untwisting rate (A) was significantly increased in T1DM (-6.2 ± 3 0/cm/s vs -4.9 ± 3.9 0/cm/s, P<0.05). The time to early peak untwisting rate was not different (50.9 ± 9.6% vs 48.4 ± 7.3%, P=0.12) but the time to late peak untwisting rate was significantly delayed in T1DM patients (80.4 ± 12.5% vs 72.7 ± 14.6%, P<0.05). The LV filling patterns demonstrated a significantly increased left atrial (LA) contribution to LV filling in T1DM. On linear regression peak late filling rate (r=0.60, P<0.000), trans-mitral A wave (r=0.25, P<0.05) and A' (r=0.30, P<0.01) were predictors of LA contribution to LV filling. CONCLUSION: We demonstrate for the first time using speckle tracking that LV untwisting rate E is preserved and untwisting rate A is increased and delayed in young patients with uncomplicated T1DM. The LA contribution to LV filling is increased in these patients and is directly related to increases in other indices of LA function like peak late filling rate, trans-mitral A wave and A'.

Cardiac metabolism in hypertrophy and heart failure: implications for therapy.

The heart consumes huge amounts of energy to fulfil its function as a relentless pump. A highly sophisticated system of energy generation based on flexibility of substrate use and efficient energy production, effective energy sensing and energy transfer ensures function of the healthy heart across a range of physiological situations. In left ventricular hypertrophy and heart failure, these processes become disturbed, leading as will be discussed to impaired cardiac energetic status and to further impairment of cardiac function. These metabolic disturbances form a potential target for therapy.

The endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) predicts LV mass independent of afterload.

BACKGROUND: Nitric oxide (NO) is a modulator of left ventricular hypertrophy (LVH) and myocardial relaxation. The impact of NO availability on development of LVH has never been demonstrated in humans. We tested the hypotheses that elevation of asymmetric dimethylarginine (ADMA) concentrations (biochemical marker of decreased NO generation), and impairment of vascular responsiveness to NO donor GTN, would each predict the presence of LVH and associated LV diastolic dysfunction in a normal aging population. METHODS AND RESULTS: In 74 subjects aged 68±6 years, LV volumes and mass indexed to height(2.7) (LVMI) were calculated from cardiac MRI. Despite the absence of clinically-defined LVH, there was a relationship (r=0.29; p=0.01) between systolic BP and LVMI. Both elevation of ADMA levels to the highest quartile or impairment of GTN responsiveness (determined by applanation tonometry) to the lowest quartile were determinants of LVMI independent of systolic BP (p=0.01 and p=0.03, respectively). Filling pressure (E/E' ratio from echocardiography) was increased in patients with impaired vascular NO responsiveness (p<0.05) irrespective of LVMI. ADMA remained a significant determinant of LVMI on multivariate analysis. CONCLUSIONS: These data imply that NO bioavailability within the myocardium modulates earliest stages of LVH development and facilitates development of diastolic dysfunction at a given LV mass.

Contribution of nitric oxide to the blood pressure and arterial responses to exercise in humans.

An exaggerated blood pressure (BP) response to exercise predicts future cardiovascular risk. The mechanisms underlying exercise-induced hypertension remain unclear, although endothelial dysfunction and elevated arterial stiffness may contribute. Given the association between reductions in nitric oxide (NO) and vascular dysfunction, we sought to determine whether acute inhibition of NO synthase with N(G)-monomethyl-L-arginine (L-NMMA) would lead to exaggerated BP responses to maximal exercise and attenuate exercise-induced reductions in arterial stiffness. In 10 healthy subjects (31±5 years), BP and heart rate (HR) were measured before, during and after an incremental cycling exercise test to determine maximal oxygen consumption (VO(2)max). Trials were performed with placebo (saline) or intravenous infusion of L-NMMA on separate days in a randomized, double-blind, crossover design. Central (aortic) and peripheral (femoral) arterial stiffness were assessed using pulse wave velocity (PWV). BP was increased with L-NMMA at rest and during sub-maximal exercise, but not at maximal exercise (mean BP 117±5 vs 118±8 mm Hg, saline vs L-NMMA, P>0.05). Furthermore, L-NMMA had no influence on exercising HR or VO(2)max (P<0.05). Notably, aortic PWV was similarly increased after exercise with either saline or L-NMMA (P<0.05), whereas postexercise decreases in femoral PWV were attenuated with L-NMMA (P<0.05). Our findings suggest that NO is an important contributor to reductions in femoral artery stiffness after maximal exercise in healthy individuals. Furthermore, acute pharmacological inhibition of NO synthase causes augmented BP responses to sub-maximal exercise, but does not lead to exaggerated BP responses to maximal exercise or reduce maximal oxygen consumption.

Relationship between coronary microvascular dysfunction and cardiac energetics impairment in type 1 diabetes mellitus.

BACKGROUND: Asymptomatic subjects with diabetes mellitus have an impaired cardiac energetics status that may play a significant role in the development of heart failure. In the present study, we assessed the role of microvascular dysfunction in the development of impaired cardiac energetics in subjects with type 1 diabetes mellitus. METHODS AND RESULTS: Twenty-five asymptomatic subjects with type 1 diabetes mellitus (mean age +/-1 SD 33+/-8 years) and 26 age-, sex-, and body mass index-matched healthy control subjects (32+/-8 years old) were recruited into the study. The type 1 diabetes mellitus subjects were divided into 2 age-matched groups (newly diagnosed [<5 years] and longer-duration [>10 years] diabetes) to assess the impact of microvascular disease. All subjects had an echocardiogram and an exercise ECG performed, followed by magnetic resonance spectroscopy and stress magnetic resonance imaging. Compared with healthy control subjects, the phosphocreatine/gamma-ATP ratio was reduced significantly both in subjects with longer-term (2.1+/-0.5 versus 1.5+/-0.4, P<0.000) and newly diagnosed (2.1+/-0.5 versus 1.6+/-0.2, P<0.000) diabetes. The phosphocreatine/gamma-ATP ratio was similar in newly diagnosed diabetes subjects and those with longer-term disease (1.6+/-0.2 versus 1.5+/-0.4, P=0.32). The mean myocardial perfusion reserve index in the longer-term type 1 diabetes mellitus subjects was significantly lower than in healthy control subjects (1.7+/-0.6 versus 2.3+/-0.4, P=0.005). On univariate analysis, there was no significant correlation of phosphocreatine/gamma-ATP ratio with myocardial perfusion reserve index (r=0.21, P=0.26). CONCLUSIONS: We demonstrate that young subjects with uncomplicated type 1 diabetes mellitus have impaired myocardial energetics irrespective of the duration of diabetes and that the impaired cardiac energetics status is independent of coronary microvascular function. We postulate that impairment of cardiac energetics in these subjects primarily results from metabolic dysfunction rather than microvascular impairment.

Echocardiography in hypertrophic cardiomyopathy diagnosis, prognosis, and role in management.

Hypertrophic cardiomyopathy (HCM) is diagnosed on the basis of left ventricular (LV) hypertrophy for which there is insufficient explanation (e.g. mild hypertension or mild aortic stenosis with marked hypertrophy). Echocardiography is an invaluable tool in the diagnosis and follow-up of patients with HCM. Echocardiographic assessment requires a comprehensive assessment in several imaging planes with careful attention to correct beam alignment in order to minimize errors in the measurement of LV wall thickness and appropriate identification of hypertrophy with an unusual distribution.

Development and validation of a clinical index to predict survival after cardiac resynchronisation therapy.

OBJECTIVE: To develop and validate a prognostic risk index of cardiovascular mortality after cardiac resynchronisation therapy (CRT). DESIGN: Prospective cohort study. SETTING: District general hospital. PATIENTS: 148 patients with heart failure (mean age 66.7 (SD 10.4) years), New York Heart Association class III or IV, LVEF <35%) who underwent CRT. INTERVENTIONS: CRT device implantation. MAIN OUTCOME MEASURES: Value of a composite index in predicting cardiovascular mortality, validated internally by bootstrapping. The predictive value of the index was compared to factors that are known to predict mortality in patients with heart failure. RESULTS: All patients underwent assessment of 16 prognostic risk factors, including cardiovascular magnetic resonance (CMR) measures of myocardial scarring (gadolinium-hyperenhancement) and dyssynchrony, before implantation. Clinical events were assessed after a median follow-up of 913 (interquartile range 967) days. At follow-up, 37/148 (25%) of patients died from cardiovascular causes. In Cox proportional hazards analyses, (DSC) Dyssynchrony, posterolateral Scar location (both p<0.0001) and Creatinine (p = 0.0046) emerged as independent predictors of cardiovascular mortality. The DSC index, derived from these variables combined, emerged as a powerful predictor of cardiovascular mortality. Compared to patients with a DSC <3, cardiovascular mortality in patients in the intermediate DSC index (3-5; HR: 11.1 (95% confidence interval (CI) 3.00 to 41.1), p = 0.0003) and high DSC index (> or =5; HR: 30.5 (95% CI 9.15 to 101.8), p<0.0001) were higher. Bootstrap validation confirmed excellent calibration and internal validity of the prediction model. CONCLUSION: The DSC index, derived from a standard CMR scan and plasma creatinine before implantation, is a powerful predictor of cardiovascular mortality after CRT.

The heart metabolism: pathophysiological aspects in ischaemia and heart failure.

The morbidity and mortality of coronary heart disease and of heart failure remain unacceptably high despite major advances in their management. The main focus of treatment has been revascularisation for ischaemic heart disease and neuro-humoral modification for heart failure. There is an urgent need for new modalities of treatment to improve mortality and morbidity. Recently, there has been a great deal of interest in the role of disturbances in cardiac energetics and myocardial metabolism in the pathophysiology of both ischaemic heart disease and heart failure and of therapeutic potential of metabolic modulation. The myocardium is a metabolic omnivore, but mainly uses fatty acids and glucose for generation of Adenosine-5'-triphosphate (ATP). This review focuses on the key changes that occur to the metabolism of the heart in ischaemia and in heart failure and its effects on cardiac energetics.

Antioxidant treatment for heart failure: friend or foe?

Increasing studies demonstrate a pivotal role for oxidant stress in the pathophysiology of heart failure (HF). Recent meta-analyses also reveal the potential pitfall of a mono-dimensional antioxidant approach. This review article summarizes the main biological pathways involved in oxidant stress and HF, the possible deleterious nature of certain antioxidant monotherapy and proposes potential antioxidant strategies necessary to challenge specific HF aetiology and progression.

Episodic syncope in hypertrophic cardiomyopathy: evidence for inappropriate vasodilation.

Symptoms of impaired consciousness (syncope and pre-syncope) occur in 15-25% of patients with hypertrophic cardiomyopathy (HCM).1 In young patients a history of recurrent syncope is associated with an increased risk of sudden death.2(-)5 Syncope usually occurs without warning or symptoms suggestive of the cause. Detailed investigations identify a probable mechanism in a minority, usually paroxysmal atrial fibrillation or ventricular tachycardia. In the majority however no likely mechanism is found despite repeated 24-hour ambulatory echocardiography (ECG) or patient-activated monitoring, exercise testing and invasive electrophysiological studies.1 6 Empirical treatment with amiodarone, a pacemaker or an implantable cardioverter-defibrillator is commonly employed, but is often unsuccessful in relieving the symptoms. We have previously observed that approximately 30% of patients with HCM have abnormal blood pressure response during maximal upright exercise.7 8 This was due in the majority of patients to an exaggerated fall in systemic vascular resistance, possibly arising from abnormal activation of stretch-sensitive left ventricular mechanoreceptors,9 10 by a mechanism similar to that described in aortic stenosis.11 However, in some patients an inadequate cardiac output response to exercise may be responsible.12 We hypothesised that abnormal vasodepressor-mediated hypotension may also occur during daily life in patients with HCM, and that this may be an important mechanism of syncope when conventional investigations fail to reveal a cause.

Heart failure is not a diagnosis.

Heart failure (HF) is a syndrome and not a diagnosis. Aetiology and precipitants for decompensation are often not sought. Care is also often based upon protocols, with widespread prescription of drugs validated in systolic HF, for patients with other forms of HF for example HF with preserved ejection fraction which can account for almost half of patients with HF in the UK. Therefore, service design and configuration by healthcare providers should based upon quality and not only feasibility, as protocol-based treatment will inevitably diminish the quality of care for patients with HF and result in both inappropriate care in many cases as well as reduced access to advanced evidence based and NICE approved therapies. Expertise is therefore of paramount importance in managing patients with HF.

Long-term effects of cardiac resynchronisation therapy in patients with atrial fibrillation.

OBJECTIVE: To compare the effects of cardiac resynchronisation therapy (CRT) in patients with heart failure (HF) in either atrial fibrillation (AF) or sinus rhythm (SR). DESIGN: Prospective observational study. PATIENTS: 295 consecutive patients with HF (permanent AF in 66, paroxysmal AF in 20, SR in 209; New York Heart Association (NYHA) class III or IV; left ventricular ejection fraction (LVEF) or=120 ms). INTERVENTIONS: All patients underwent CRT without atrioventricular junction ablation. MAIN OUTCOME MEASURES: The primary end point was the composite of cardiovascular death or unplanned hospitalisation for major cardiovascular events. Secondary end points included the composite of cardiovascular death or hospitalisation for worsening HF. Cardiovascular mortality, total mortality and changes in NYHA class, 6-minute walking distance, quality of life (Minnesota Living with Heart Failure questionnaire) and echocardiographic variables were also considered. RESULTS: Over a follow-up period of up to 6.8 years, no differences emerged between patients in AF or SR in any of the mortality or morbidity end points. The AF and SR groups derived similar improvements in mean NYHA class (-1.3 vs -1.2), 6-minute walking distance (92.3 vs 78.4 m) and quality of life scores (-25.2 vs -18.7) (all p<0.001). In both the AF and the SR groups, reductions were seen in left ventricular end-systolic (-25.9 vs -34.5 ml, both p<0.001) and end-diastolic (-20.2 ml, p = 0.001 vs 26.2 ml, p<0.001) volumes and improvements in LVEF (4.69% vs 7.86%, both p<0.001). CONCLUSIONS: Cardiac resynchronisation therapy leads to similar prognostic and symptomatic benefits in patients in AF and SR, even without atrioventricular junction ablation. Echocardiographic improvements are also comparable.

Prolonged deterioration of endothelial dysfunction in response to postprandial lipaemia is attenuated by vitamin C in Type 2 diabetes.

BACKGROUND: Endothelial dysfunction (ED) has been described in Type 2 diabetes (T2DM). We have described previously a diminution of flow-mediated arterial dilatation and, by implication, further ED in T2DM in response to postprandial lipaemia (PPL) at 4 h. This is possibly mediated by oxidative stress/alteration of the nitric oxide (NO) pathway. T2DM subjects tend to exhibit both exaggerated and prolonged PPL. We therefore studied the relationship of PPL to the duration of ED in T2DM subjects and oxidative stress with or without the antioxidant, vitamin C. METHODS: Twenty subjects with T2DM with moderate glycaemic control (mean HbA1c 8.4%) were studied. After an overnight fast, all subjects consumed a standard fat meal. Endothelial function (EF), lipid profiles, and venous free radicals were measured in the fasting, peak lipaemic phase (4 h) and postprandially to 8 h. The study was repeated in a double-blinded manner with placebo, vitamin C (1 g) therapy for 2 days prior to re-testing and with the fat meal. Oxidative stress was assessed by lipid-derived free radicals in plasma, ex vivo by electron paramagnetic resonance spectroscopy (EPR) and by markers of lipid peroxidation (TBARS). Endothelial function was assessed by flow-mediated vasodilatation (FMD) of the brachial artery. RESULTS: There was a significant decrease in endothelial function in response to PPL from baseline (B) 1.3 +/- 1.3% to 4 h 0.22 +/- 1.1% (P < 0.05) and 8 h 0.7 +/- 0.9% (P < 0.05) (mean +/- sem). The endothelial dysfunction seen was attenuated at each time point with vitamin C. Baseline EF with vitamin C changed from (fasting) 3.8 +/- 0.9-2.8 +/- 0.8 (at 4 h) and 2.9 +/- 1.3 (at 8 h) in response to PPL. Vitamin C attenuated postprandial (PP) oxidative stress significantly only at the 4-h time point [301.1 +/- 118 (B) to 224.7 +/- 72 P < 0.05] and not at 8 h 301.1 +/- 118 (B) to 260 +/- 183 (P = NS). There were no changes with placebo treatment in any variable. PPL was associated with a PP rise in TG levels (in mmol/l) from (B) 1.8 +/- 1 to 2.7 +/- 1 at 4 h and 1.95 +/- 1.2 at 8 h (P = 0.0002 and 0.33, respectively). CONCLUSION: PPL is associated with prolonged endothelial dysfunction for at least 8 h after a fatty meal. Vitamin C treatment improves endothelial dysfunction at all time points and attenuates PPL-induced oxidative stress. This highlights the importance of low-fat meals in T2DM and suggests a role for vitamin C therapy to improve endothelial function during meal ingestion.

Role of resynchronisation therapy and implantable cardioverter defibrillators in heart failure.

The worldwide prevalence of heart failure is increasing in part because of an aging population. In the developed world, heart failure affects 1%-2% of the general population, accounting for 5% of adult hospital admissions. There is now convincing evidence supporting the beneficial effects of cardiac resynchronisation therapy for the treatment of heart failure. Numerous observational studies, as well as a series of randomised controlled trials, have shown the safety, efficacy, and long term benefits for patients with chronic systolic heart failure who have broad QRS complexes and refractory symptoms despite optimal medical therapy. These studies have consistently found statistically significant improvements in quality of life, New York Heart Association functional class, exercise tolerance, and left ventricular reverse remodelling. Recent evidence suggests that the benefit may at least in part be because of a reduction in mechanical dysynchrony.

The role of cardiac resynchronization therapy in heart failure.

The worldwide prevalence of heart failure is increasing in part due to an ageing population. In the developed world, heart failure affects 1-2% of the general population, accounting for 5% of adult hospital admissions. There is now convincing evidence supporting the beneficial effects of cardiac resynchronization therapy for the treatment of heart failure. Numerous observational studies, as well as a series of randomised controlled trials, have demonstrated the safety, efficacy, and long-term benefits for patients with chronic systolic heart failure who have broad QRS complexes and refractory symptoms despite optimal medical therapy. These studies have consistently demonstrated statistically significant improvements in quality of life, NYHA functional class, exercise tolerance, and left ventricular reverse remodeling. Recent evidence suggests that the benefit may at least in part be due to a reduction in mechanical dyssynchrony.