Metabolic profiling of aortic stenosis and hypertrophic cardiomyopathy identifies mechanistic contrasts in substrate utilization.

Aortic stenosis (AS) and hypertrophic cardiomyopathy (HCM) are distinct disorders leading to left ventricular hypertrophy (LVH), but whether cardiac metabolism substantially differs between these in humans remains to be elucidated. We undertook an invasive (aortic root, coronary sinus) metabolic profiling in patients with severe AS and HCM in comparison with non-LVH controls to investigate cardiac fuel selection and metabolic remodeling. These patients were assessed under different physiological states (at rest, during stress induced by pacing). The identified changes in the metabolome were further validated by metabolomic and orthogonal transcriptomic analysis, in separately recruited patient cohorts. We identified a highly discriminant metabolomic signature in severe AS in all samples, regardless of sampling site, characterized by striking accumulation of long-chain acylcarnitines, intermediates of fatty acid transport across the inner mitochondrial membrane, and validated this in a separate cohort. Mechanistically, we identify a downregulation in the PPAR-α transcriptional network, including expression of genes regulating fatty acid oxidation (FAO). In silico modeling of ß-oxidation demonstrated that flux could be inhibited by both the accumulation of fatty acids as a substrate for mitochondria and the accumulation of medium-chain carnitines which induce competitive inhibition of the acyl-CoA dehydrogenases. We present a comprehensive analysis of changes in the metabolic pathways (transcriptome to metabolome) in severe AS, and its comparison to HCM. Our results demonstrate a progressive impairment of ß-oxidation from HCM to AS, particularly for FAO of long-chain fatty acids, and that the PPAR-α signaling network may be a specific metabolic therapeutic target in AS.

Fine-Scale Genetic Structure in the United Arab Emirates Reflects Endogamous and Consanguineous Culture, Population History, and Geography.

The indigenous population of the United Arab Emirates (UAE) has a unique demographic and cultural history. Its tradition of endogamy and consanguinity is expected to produce genetic homogeneity and partitioning of gene pools while population movements and intercontinental trade are likely to have contributed to genetic diversity. Emiratis and neighboring populations of the Middle East have been underrepresented in the population genetics literature with few studies covering the broader genetic history of the Arabian Peninsula. Here, we genotyped 1,198 individuals from the seven Emirates using 1.7 million markers and by employing haplotype-based algorithms and admixture analyses, we reveal the fine-scale genetic structure of the Emirati population. Shared ancestry and gene flow with neighboring populations display their unique geographic position while increased intra- versus inter-Emirati kinship and sharing of uniparental haplogroups, reflect the endogamous and consanguineous cultural traditions of the Emirates and their tribes.

Early childhood epilepsies: epidemiology, classification, aetiology, and socio-economic determinants.

Epilepsies of early childhood are frequently resistant to therapy and often associated with cognitive and behavioural comorbidity. Aetiology focused precision medicine, notably gene-based therapies, may prevent seizures and comorbidities. Epidemiological data utilizing modern diagnostic techniques including whole genome sequencing and neuroimaging can inform diagnostic strategies and therapeutic trials. We present a 3-year, multicentre prospective cohort study, involving all children under 3 years of age in Scotland presenting with epilepsies. We used two independent sources for case identification: clinical reporting and EEG record review. Capture-recapture methodology was then used to improve the accuracy of incidence estimates. Socio-demographic and clinical details were obtained at presentation, and 24 months later. Children were extensively investigated for aetiology. Whole genome sequencing was offered for all patients with drug-resistant epilepsy for whom no aetiology could yet be identified. Multivariate logistic regression modelling was used to determine associations between clinical features, aetiology, and outcome. Three hundred and ninety children were recruited over 3 years. The adjusted incidence of epilepsies presenting in the first 3 years of life was 239 per 100 000 live births [95% confidence interval (CI) 216-263]. There was a socio-economic gradient to incidence, with a significantly higher incidence in the most deprived quintile (301 per 100 000 live births, 95% CI 251-357) compared with the least deprived quintile (182 per 100 000 live births, 95% CI 139-233), χ2 odds ratio = 1.7 (95% CI 1.3-2.2). The relationship between deprivation and incidence was only observed in the group without identified aetiology, suggesting that populations living in higher deprivation areas have greater multifactorial risk for epilepsy. Aetiology was determined in 54% of children, and epilepsy syndrome was classified in 54%. Thirty-one per cent had an identified genetic cause for their epilepsy. We present novel data on the aetiological spectrum of the most commonly presenting epilepsies of early childhood. Twenty-four months after presentation, 36% of children had drug-resistant epilepsy (DRE), and 49% had global developmental delay (GDD). Identification of an aetiology was the strongest determinant of both DRE and GDD. Aetiology was determined in 82% of those with DRE, and 75% of those with GDD. In young children with epilepsy, genetic testing should be prioritized as it has the highest yield of any investigation and is most likely to inform precision therapy and prognosis. Epilepsies in early childhood are 30% more common than previously reported. Epilepsies of undetermined aetiology present more frequently in deprived communities. This likely reflects increased multifactorial risk within these populations.

Early-onset autoimmunity associated with SOCS1 haploinsufficiency.

Autoimmunity can occur when a checkpoint of self-tolerance fails. The study of familial autoimmune diseases can reveal pathophysiological mechanisms involved in more common autoimmune diseases. Here, by whole-exome/genome sequencing we identify heterozygous, autosomal-dominant, germline loss-of-function mutations in the SOCS1 gene in ten patients from five unrelated families with early onset autoimmune manifestations. The intracellular protein SOCS1 is known to downregulate cytokine signaling by inhibiting the JAK-STAT pathway. Accordingly, patient-derived lymphocytes exhibit increased STAT activation in vitro in response to interferon-γ, IL-2 and IL-4 that is reverted by the JAK1/JAK2 inhibitor ruxolitinib. This effect is associated with a series of in vitro and in vivo immune abnormalities consistent with lymphocyte hyperactivity. Hence, SOCS1 haploinsufficiency causes a dominantly inherited predisposition to early onset autoimmune diseases related to cytokine hypersensitivity of immune cells.

Elevated risk of invasive group A streptococcal disease and host genetic variation in the human leucocyte antigen locus.

Invasive group A streptococcal (GAS) disease is uncommon but carries a high case-fatality rate relative to other infectious diseases. Given the ubiquity of mild GAS infections, it remains unclear why healthy individuals will occasionally develop life-threatening infections, raising the possibility of host genetic predisposition. Here, we present the results of a case-control study including 43 invasive GAS cases and 1540 controls. Using HLA imputation and linear mixed models, we find each copy of the HLA-DQA1*01:03 allele associates with a twofold increased risk of disease (odds ratio 2.3, 95% confidence interval 1.3-4.4, P = 0.009), an association which persists with classical HLA typing of a subset of cases and analysis with an alternative large control dataset with validated HLA data. Moreover, we propose the association is driven by the allele itself rather than the background haplotype. Overall this finding provides impetus for further investigation of the immunogenetic basis of this devastating bacterial disease.

The interplay between metabolic alterations, diastolic strain rate and exercise capacity in mild heart failure with preserved ejection fraction: a cardiovascular magnetic resonance study.

BACKGROUND: Heart failure (HF) is characterized by altered myocardial substrate metabolism which can lead to myocardial triglyceride accumulation (steatosis) and lipotoxicity. However its role in mild HF with preserved ejection fraction (HFpEF) is uncertain. We measured myocardial triglyceride content (MTG) in HFpEF and assessed its relationships with diastolic function and exercise capacity. METHODS: Twenty seven HFpEF (clinical features of HF, left ventricular EF >50%, evidence of mild diastolic dysfunction and evidence of exercise limitation as assessed by cardiopulmonary exercise test) and 14 controls underwent 1H-cardiovascular magnetic resonance spectroscopy (1H-CMRS) to measure MTG (lipid/water, %), 31P-CMRS to measure myocardial energetics (phosphocreatine-to-adenosine triphosphate - PCr/ATP) and feature-tracking cardiovascular magnetic resonance (CMR) imaging for diastolic strain rate. RESULTS: When compared to controls, HFpEF had 2.3 fold higher in MTG (1.45 ± 0.25% vs. 0.64 ± 0.16%, p = 0.009) and reduced PCr/ATP (1.60 ± 0.09 vs. 2.00 ± 0.10, p = 0.005). HFpEF had significantly reduced diastolic strain rate and maximal oxygen consumption (VO2 max), which both correlated significantly with elevated MTG and reduced PCr/ATP. On multivariate analyses, MTG was independently associated with diastolic strain rate while diastolic strain rate was independently associated with VO2 max. CONCLUSIONS: Myocardial steatosis is pronounced in mild HFpEF, and is independently associated with impaired diastolic strain rate which is itself related to exercise capacity. Steatosis may adversely affect exercise capacity by indirect effect occurring via impairment in diastolic function. As such, myocardial triglyceride may become a potential therapeutic target to treat the increasing number of patients with HFpEF.

Mutant Muscle LIM Protein C58G causes cardiomyopathy through protein depletion.

Cysteine and glycine rich protein 3 (CSRP3) encodes Muscle LIM Protein (MLP), a well-established disease gene for Hypertrophic Cardiomyopathy (HCM). MLP, in contrast to the proteins encoded by the other recognised HCM disease genes, is non-sarcomeric, and has important signalling functions in cardiomyocytes. To gain insight into the disease mechanisms involved, we generated a knock-in mouse (KI) model, carrying the well documented HCM-causing CSRP3 mutation C58G. In vivo phenotyping of homozygous KI/KI mice revealed a robust cardiomyopathy phenotype with diastolic and systolic left ventricular dysfunction, which was supported by increased heart weight measurements. Transcriptome analysis by RNA-seq identified activation of pro-fibrotic signalling, induction of the fetal gene programme and activation of markers of hypertrophic signalling in these hearts. Further ex vivo analyses validated the activation of these pathways at transcript and protein level. Intriguingly, the abundance of MLP decreased in KI/KI mice by 80% and in KI/+ mice by 50%. Protein depletion was also observed in cellular studies for two further HCM-causing CSRP3 mutations (L44P and S54R/E55G). We show that MLP depletion is caused by proteasome action. Moreover, MLP C58G interacts with Bag3 and results in a proteotoxic response in the homozygous knock-in mice, as shown by induction of Bag3 and associated heat shock proteins. In conclusion, the newly generated mouse model provides insights into the underlying disease mechanisms of cardiomyopathy caused by mutations in the non-sarcomeric protein MLP. Furthermore, our cellular experiments suggest that protein depletion and proteasomal overload also play a role in other HCM-causing CSPR3 mutations that we investigated, indicating that reduced levels of functional MLP may be a common mechanism for HCM-causing CSPR3 mutations.

MiR-184 expression is regulated by AMPK in pancreatic islets.

AMPK is a critical energy sensor and target for widely used antidiabetic drugs. In ß cells, elevated glucose concentrations lower AMPK activity, and the ablation of both catalytic subunits [ß-cell-specific AMPK double-knockout (ßAMPKdKO) mice] impairs insulin secretion in vivo and ß-cell identity. MicroRNAs (miRNAs) are small RNAs that silence gene expression that are essential for pancreatic ß-cell function and identity and altered in diabetes. Here, we have explored the miRNAs acting downstream of AMPK in mouse and human ß cells. We identified 14 down-regulated and 9 up-regulated miRNAs in ßAMPKdKO vs. control islets. Gene ontology analysis of targeted transcripts revealed enrichment in pathways important for ß-cell function and identity. The most down-regulated miRNA was miR-184 (miR-184-3p), an important regulator of ß-cell function and compensatory expansion that is controlled by glucose and reduced in diabetes. We demonstrate that AMPK is a potent regulator and an important mediator of the negative effects of glucose on miR-184 expression. Additionally, we reveal sexual dimorphism in miR-184 expression in mouse and human islets. Collectively, these data demonstrate that glucose-mediated changes in AMPK activity are central for the regulation of miR-184 and other miRNAs in islets and provide a link between energy status and gene expression in ß cells.-Martinez-Sanchez, A., Nguyen-Tu, M.-S., Cebola, I., Yavari, A., Marchetti, P., Piemonti, L., de Koning, E., Shapiro, A. M. J., Johnson, P., Sakamoto, K., Smith, D. M., Leclerc, I., Ashrafian, H., Ferrer, J., Rutter, G. A. MiR-184 expression is regulated by AMPK in pancreatic islets.

Aggressive restenosis after percutaneous intervention in two coronary loci in a patient with human immunodeficiency virus infection.

A 54-year-old black African woman, 22 years human immunodeficiency virus (HIV)-positive, presented with an acute coronary syndrome. She was taking two nucleoside reverse transcriptase inhibitors and two protease inhibitors. Viral load and CD4 count were stable. Angiography revealed a right coronary artery lesion, which was treated with everolimus eluting stent. She also underwent balloon angioplasty to the first diagonal. She re-presented on three different occasions and technically successful coronary intervention was performed. The patient has reported satisfactory compliance with dual anti platelet therapy throughout. She was successfully treated with surgical revascularisation. The patient did not experience any clinical recurrence on follow up. This case demonstrates exceptionally aggressive multifocal and recurrent instent restenosis in a patient treated for HIV infection, raising the possibility of an association with HIV infection or potentially components of retro viral therapy.

Human Second Window Pre-Conditioning and Post-Conditioning by Nitrite Is Influenced by a Common Polymorphism in Mitochondrial Aldehyde Dehydrogenase.

Pre-conditioning is an exciting physiological phenomenon that, despite great efforts, has so far resisted translation to mainstream clinical medicine. Many potential triggers (e.g., ischemia of the organ in question or a remote organ, many different drugs) have been investigated, but recent work has implicated activation of mitochondrial aldehyde dehydrogenase (ALDH2) as central to the process. A genetic polymorphism, known as ALDH2*2, is common worldwide (present in up to 40% of Han Chinese people) and produces a functionally different enzyme. The authors used a variety of protocols in the human ischemic forearm model, in participants with both enzyme types, to assess cytoprotection with low-dose sodium nitrite and attempt to further elucidate the role of ALDH2.

Benzimidazole derivative small-molecule 991 enhances AMPK activity and glucose uptake induced by AICAR or contraction in skeletal muscle.

AMP-activated protein kinase (AMPK) plays diverse roles and coordinates complex metabolic pathways for maintenance of energy homeostasis. This could be explained by the fact that AMPK exists as multiple heterotrimer complexes comprising a catalytic α-subunit (α1 and α2) and regulatory ß (ß1 and ß2)- and γ (γ1, γ2, γ3)-subunits, which are uniquely distributed across different cell types. There has been keen interest in developing specific and isoform-selective AMPK-activating drugs for therapeutic use and also as research tools. Moreover, establishing ways of enhancing cellular AMPK activity would be beneficial for both purposes. Here, we investigated if a recently described potent AMPK activator called 991, in combination with the commonly used activator 5-aminoimidazole-4-carboxamide riboside or contraction, further enhances AMPK activity and glucose transport in mouse skeletal muscle ex vivo. Given that the γ3-subunit is exclusively expressed in skeletal muscle and has been implicated in contraction-induced glucose transport, we measured the activity of AMPKγ3 as well as ubiquitously expressed γ1-containing complexes. We initially validated the specificity of the antibodies for the assessment of isoform-specific AMPK activity using AMPK-deficient mouse models. We observed that a low dose of 991 (5 µM) stimulated a modest or negligible activity of both γ1- and γ3-containing AMPK complexes. Strikingly, dual treatment with 991 and 5-aminoimidazole-4-carboxamide riboside or 991 and contraction profoundly enhanced AMPKγ1/γ3 complex activation and glucose transport compared with any of the single treatments. The study demonstrates the utility of a dual activator approach to achieve a greater activation of AMPK and downstream physiological responses in various cell types, including skeletal muscle.

Mutation of Fnip1 is associated with B-cell deficiency, cardiomyopathy, and elevated AMPK activity.

Folliculin (FLCN) is a tumor-suppressor protein mutated in the Birt-Hogg-Dubé (BHD) syndrome, which associates with two paralogous proteins, folliculin-interacting protein (FNIP)1 and FNIP2, forming a complex that interacts with the AMP-activated protein kinase (AMPK). Although it is clear that this complex influences AMPK and other metabolic regulators, reports of its effects have been inconsistent. To address this issue, we created a recessive loss-of-function variant of Fnip1 Homozygous FNIP1 deficiency resulted in profound B-cell deficiency, partially restored by overexpression of the antiapoptotic protein BCL2, whereas heterozygous deficiency caused a loss of marginal zone B cells. FNIP1-deficient mice developed cardiomyopathy characterized by left ventricular hypertrophy and glycogen accumulation, with close parallels to mice and humans bearing gain-of-function mutations in the γ2 subunit of AMPK. Concordantly, γ2-specific AMPK activity was elevated in neonatal FNIP1-deficient myocardium, whereas AMPK-dependent unc-51-like autophagy activating kinase 1 (ULK1) phosphorylation and autophagy were increased in FNIP1-deficient B-cell progenitors. These data support a role for FNIP1 as a negative regulator of AMPK.

Adenosine Receptor Activation in the "Trigger" Limb of Remote Pre-Conditioning Mediates Human Endothelial Conditioning and Release of Circulating Cardioprotective Factor(s).

Remote ischemic pre-conditioning (rIPC) has emerged as a potential mechanism to reduce ischemia-reperfusion injury. Clinical data, however, have been mixed, and its physiological basis remains unclear, although it appears to involve release of circulating factor(s) and/or neural pathways. Here, the authors demonstrate that adenosine receptor activation is an important step in initiating human pre-conditioning; that pre-conditioning liberates circulating cardioprotective factor(s); and that exogenous adenosine infusion is able to recapitulate release of this factor. However, blockade of adenosine receptors in ischemic tissue does not block the protection afforded by pre-conditioning. These data have important implications for defining the physiology of human pre-conditioning and its translation to future clinical trials.

No Evidence of Myocardial Oxygen Deprivation in Nonischemic Heart Failure.

BACKGROUND: Whether the myocardium in nonischemic heart failure experiences oxygen limitation remains a long-standing controversy. We addressed this question in patients with dilated cardiomyopathy (DCM) using a dual approach. First, we tested the changes in myocardial oxygenation between rest and stress states, using oxygenation-sensitive cardiovascular magnetic resonance. Second, we sought to assess the functional consequences of oxygen limitation at rest by measuring myocardial energetics before and after short-term oxygen supplementation. METHODS AND RESULTS: Twenty-six subjects (14 DCM and 12 normal) underwent cardiac magnetic resonance imaging at 3 Tesla to assess cardiac volumes, function, oxygenation, and first-pass perfusion (0.03 mmol/kg Gd-DTPA bolus) at stress and rest (4-6 minutes IV adenosine, 140 µg/kg per minute). Signal intensity change (SIΔ) and myocardial perfusion reserve index (MPRI) were measured from oxygenation and perfusion images, respectively. Furthermore, the effect of oxygen supplementation on resting myocardial energy metabolism was tested using (31)P MR spectroscopy, measuring PCr/ATP ratios in both groups at baseline and after 4 hours of oxygen via facemask in the DCM group. During stress, there were equivalent rises in rate pressure product in both groups (DCM, 76±15% and normal, 79±9%; P=0.84). MPRI was significantly reduced in DCM (1.51±0.11 versus normal 1.86±0.10; P=0.03). However, there was no difference in oxygenation between groups: SIΔ in DCM 17±3% versus normal 20±2% (P=0.38). Furthermore, at a left ventricular segmental level, there was no correlation between oxygenation-sensitive SIΔ and MPRI (R=0.06; P=0.43). Resting PCr/ATP was reduced in DCM (1.66±0.07 versus normal 2.12±0.06; P=0.002). With oxygen supplementation, there was no change in PCr/ATP (1.61±0.08; P=0.58; Δ=0.04±0.05). There was also no effect of oxygen on systolic function (ejection fraction pre oxygen, 34±1%; post oxygen, 36±2%; P=0.46; Δ 2±1%). CONCLUSIONS: Our results demonstrate dissociation between microvascular dysfunction and oxygenation in DCM, suggesting that the impairment of perfusion is not sufficient to cause deoxygenation during stress. Cardiac energetics are unaffected by oxygen supplementation, indicating the absence of relevant myocardial hypoxia at rest. Our study suggests that novel treatments for nonischemic heart failure should focus on efforts to directly target cardiomyocyte function and metabolism rather than oxygen delivery and microvascular function.

Effect of Selective Heart Rate Slowing in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is associated with significant morbidity and mortality but is currently refractory to therapy. Despite limited evidence, heart rate reduction has been advocated, on the basis of physiological considerations, as a therapeutic strategy in HFpEF. We tested the hypothesis that heart rate reduction improves exercise capacity in HFpEF. METHODS AND RESULTS: We conducted a randomized, crossover study comparing selective heart rate reduction with the If blocker ivabradine at 7.5 mg twice daily versus placebo for 2 weeks each in 22 symptomatic patients with HFpEF who had objective evidence of exercise limitation (peak oxygen consumption at maximal exercise [o2 peak] <80% predicted for age and sex). The result was compared with 22 similarly treated matched asymptomatic hypertensive volunteers. The primary end point was the change in o2 peak. Secondary outcomes included tissue Doppler-derived E/e' at echocardiography, plasma brain natriuretic peptide, and quality-of-life scores. Ivabradine significantly reduced peak heart rate compared with placebo in the HFpEF (107 versus 129 bpm; P<0.0001) and hypertensive (127 versus 145 bpm; P=0.003) cohorts. Ivabradine compared with placebo significantly worsened the change in o2 peak in the HFpEF cohort (-2.1 versus 0.9 mL·kg(-1)·min(-1); P=0.003) and significantly reduced submaximal exercise capacity, as determined by the oxygen uptake efficiency slope. No significant effects on the secondary end points were discernable. CONCLUSION: Our observations bring into question the value of heart rate reduction with ivabradine for improving symptoms in a HFpEF population characterized by exercise limitation. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02354573.

Resistance of Dynamin-related Protein 1 Oligomers to Disassembly Impairs Mitophagy, Resulting in Myocardial Inflammation and Heart Failure.

We have reported previously that a missense mutation in the mitochondrial fission gene Dynamin-related protein 1 (Drp1) underlies the Python mouse model of monogenic dilated cardiomyopathy. The aim of this study was to investigate the consequences of the C452F mutation on Drp1 protein function and to define the cellular sequelae leading to heart failure in the Python monogenic dilated cardiomyopathy model. We found that the C452F mutation increased Drp1 GTPase activity. The mutation also conferred resistance to oligomer disassembly by guanine nucleotides and high ionic strength solutions. In a mouse embryonic fibroblast model, Drp1 C452F cells exhibited abnormal mitochondrial morphology and defective mitophagy. Mitochondria in C452F mouse embryonic fibroblasts were depolarized and had reduced calcium uptake with impaired ATP production by oxidative phosphorylation. In the Python heart, we found a corresponding progressive decline in oxidative phosphorylation with age and activation of sterile inflammation. As a corollary, enhancing autophagy by exposure to a prolonged low-protein diet improved cardiac function in Python mice. In conclusion, failure of Drp1 disassembly impairs mitophagy, leading to a downstream cascade of mitochondrial depolarization, aberrant calcium handling, impaired ATP synthesis, and activation of sterile myocardial inflammation, resulting in heart failure.

Targets for therapy in sarcomeric cardiomyopathies.

To date, no compounds or interventions exist that treat or prevent sarcomeric cardiomyopathies. Established therapies currently improve the outcome, but novel therapies may be able to more fundamentally affect the disease process and course. Investigations of the pathomechanisms are generating molecular insights that can be useful for the design of novel specific drugs suitable for clinical use. As perturbations in the heart are stage-specific, proper timing of drug treatment is essential to prevent initiation and progression of cardiac disease in mutation carrier individuals. In this review, we emphasize potential novel therapies which may prevent, delay, or even reverse hypertrophic cardiomyopathy caused by sarcomeric gene mutations. These include corrections of genetic defects, altered sarcomere function, perturbations in intracellular ion homeostasis, and impaired myocardial energetics.