Report on the current status of the use of real-world data (RWD) and real-world evidence (RWE) in drug development and regulation.

Radically expanding use of real-world data (RWD) and real-world evidence (RWE) holds the potential to substantially impact drug development, pharmaceutical regulation, and payment within health care systems. Central to this is the reconfiguration of data gathering and transformation of data to information, which can be used as evidence for decision making. We discuss applications of this paradigm in the light of recent developments in both the United States and Europe on RWD and RWE.

Improving Interpretation of Cardiac Phenotypes and Enhancing Discovery With Expanded Knowledge in the Gene Ontology.

BACKGROUND: A systems biology approach to cardiac physiology requires a comprehensive representation of how coordinated processes operate in the heart, as well as the ability to interpret relevant transcriptomic and proteomic experiments. The Gene Ontology (GO) Consortium provides structured, controlled vocabularies of biological terms that can be used to summarize and analyze functional knowledge for gene products. METHODS AND RESULTS: In this study, we created a computational resource to facilitate genetic studies of cardiac physiology by integrating literature curation with attention to an improved and expanded ontological representation of heart processes in the Gene Ontology. As a result, the Gene Ontology now contains terms that comprehensively describe the roles of proteins in cardiac muscle cell action potential, electrical coupling, and the transmission of the electrical impulse from the sinoatrial node to the ventricles. Evaluating the effectiveness of this approach to inform data analysis demonstrated that Gene Ontology annotations, analyzed within an expanded ontological context of heart processes, can help to identify candidate genes associated with arrhythmic disease risk loci. CONCLUSIONS: We determined that a combination of curation and ontology development for heart-specific genes and processes supports the identification and downstream analysis of genes responsible for the spread of the cardiac action potential through the heart. Annotating these genes and processes in a structured format facilitates data analysis and supports effective retrieval of gene-centric information about cardiac defects.

Hepatocyte Growth Factor Receptor c-Met Instructs T Cell Cardiotropism and Promotes T Cell Migration to the Heart via Autocrine Chemokine Release.

Effector-T-cell-mediated immunity depends on the efficient localization of antigen-primed lymphocytes to antigen-rich non-lymphoid tissue, which is facilitated by the expression of a unique set of "homing" receptors acquired by memory T cells. We report that engagement of the hepatocyte growth factor (HGF) receptor c-Met by heart-produced HGF during priming in the lymph nodes instructs T cell cardiotropism, which was associated with a specialized homing "signature" (c-Met(+)CCR4(+)CXCR3(+)). c-Met signals facilitated T cell recruitment to the heart via the chemokine receptor CCR5 by inducing autocrine CCR5 ligand release. c-Met triggering was sufficient to support cardiotropic T cell recirculation, while CCR4 and CXCR3 sustained recruitment during heart inflammation. Transient pharmacological blockade of c-Met during T cell priming led to enhanced survival of heart, but not skin, allografts associated with impaired localization of alloreactive T cells to heart grafts. These findings suggest c-Met as a target for development of organ-selective immunosuppressive therapies.

Absence of the Regulator of G-protein Signaling, RGS4, Predisposes to Atrial Fibrillation and Is Associated with Abnormal Calcium Handling.

The description of potential molecular substrates for predisposition to atrial fibrillation (AF) is incomplete, and it is unknown what role regulators of G-protein signaling might play. We address whether the attenuation of RGS4 function may promote AF and the mechanism through which this occurs. For this purpose, we studied a mouse with global genetic deletion of RGS4 (RGS4(-/-)) and the normal littermate controls (RGS4(+/+)). In vivo electrophysiology using atrial burst pacing revealed that mice with global RGS4 deletion developed AF more frequently than control littermates. Isolated atrial cells from RGS4(-/-) mice show an increase in Ca(2+) spark frequency under basal conditions and after the addition of endothelin-1 and abnormal spontaneous Ca(2+) release events after field stimulation. Isolated left atria studied on a multielectrode array revealed modest changes in path length for re-entry but abnormal electrical events after a pacing train in RGS4(-/-) mice. RGS4 deletion results in a predisposition to atrial fibrillation from enhanced activity in the Gαq/11-IP3 pathway, resulting in abnormal Ca(2+) release and corresponding electrical events.

Hypoxia signaling controls postnatal changes in cardiac mitochondrial morphology and function.

Fetal cardiomyocyte adaptation to low levels of oxygen in utero is incompletely understood, and is of interest as hypoxia tolerance is lost after birth, leading to vulnerability of adult cardiomyocytes. It is known that cardiac mitochondrial morphology, number and function change significantly following birth, although the underlying molecular mechanisms and physiological stimuli are undefined. Here we show that the decrease in cardiomyocyte HIF-signaling in cardiomyocytes immediately after birth acts as a physiological switch driving mitochondrial fusion and increased postnatal mitochondrial biogenesis. We also investigated mechanisms of ATP generation in embryonic cardiac mitochondria. We found that embryonic cardiac cardiomyocytes rely on both glycolysis and the tricarboxylic acid cycle to generate ATP, and that the balance between these two metabolic pathways in the heart is controlled around birth by the reduction in HIF signaling. We therefore propose that the increase in ambient oxygen encountered by the neonate at birth acts as a key physiological stimulus to cardiac mitochondrial adaptation.

Induction of the calcineurin variant CnAß1 after myocardial infarction reduces post-infarction ventricular remodelling by promoting infarct vascularization.

AIMS: Ventricular remodelling following myocardial infarction progressively leads to loss of contractile capacity and heart failure. Although calcineurin promotes maladaptive cardiac hypertrophy, we recently showed that the calcineurin splicing variant, CnAß1, has beneficial effects on the infarcted heart. However, whether this variant limits necrosis or improves remodelling is still unknown, precluding translation to the clinical arena. Here, we explored the effects and therapeutic potential of CnAß1 overexpression post-infarction. METHODS AND RESULTS: Double transgenic mice with inducible cardiomyocyte-specific overexpression of CnAß1 underwent left coronary artery ligation followed by reperfusion. Echocardiographic analysis showed depressed cardiac function in all infarcted mice 3 days post-infarction. Induction of CnAß1 overexpression 1 week after infarction improved function and reduced ventricular dilatation. CnAß1-overexpressing mice showed shorter, thicker scars, and reduced infarct expansion, accompanied by reduced myocardial remodelling. CnAß1 induced vascular endothelial growth factor (VEGF) expression in cardiomyocytes, which resulted in increased infarct vascularization. This paracrine angiogenic effect of CnAß1 was mediated by activation of the Akt/mammalian target of rapamycin pathway and VEGF. CONCLUSIONS: Our results indicate that CnAß1 exerts beneficial effects on the infarcted heart by promoting infarct vascularization and preventing infarct expansion. These findings emphasize the translational potential of CnAß1 for gene-based therapies.

Myosin heavy chain 15 is associated with bovine pulmonary arterial pressure.

Bovine pulmonary hypertension, brisket disease, causes significant morbidity and mortality at elevations above 2,000 m. Mean pulmonary arterial pressure (mPAP) is moderately heritable, with inheritance estimated to lie within a few major genes. Invasive mPAP measurement is currently the only tool available to identify cattle at risk of hypoxia-induced pulmonary hypertension. A genetic test could allow selection of cattle suitable for high altitude without the need for invasive testing. In this study we evaluated three candidate genes (myosin heavy chain 15 [MYH15], NADH dehydrogenase flavoprotein 2, and FK binding protein 1A) for association with mPAP in 166 yearling Angus bulls grazing at 2,182 m. The T allele (rs29016420) of MYH15 was linked to lower mPAP in a dominant manner (CC 47.2 ± 1.6 mmHg [mean ± standard error of the mean]; CT/TT 42.8 ± 0.7 mmHg; P = 0.02). The proportions of cattle with MYH15 CC, CT, and TT genotypes were 55%, 41%, and 4%, respectively. Given the high frequency of the deleterious allele, it is likely that the relative contribution of MYH15 polymorphisms to pulmonary hypertension is small, supporting previous predictions that the disease is polygenic. We evaluated allelic frequency of MYH15 in the Himalayan yak (Bos grunniens), a closely related species adapted to high altitude, and found 100% prevalence of T allele homozygosity. In summary, we identified a polymorphism in MYH15 significantly associated with mPAP. This finding may aid selection of cattle suitable for high altitude and contribute to understanding human hypoxia-induced pulmonary hypertension.

Hypoxic regulation of hand1 controls the fetal-neonatal switch in cardiac metabolism.

Cardiomyocytes are vulnerable to hypoxia in the adult, but adapted to hypoxia in utero. Current understanding of endogenous cardiac oxygen sensing pathways is limited. Myocardial oxygen consumption is determined by regulation of energy metabolism, which shifts from glycolysis to lipid oxidation soon after birth, and is reversed in failing adult hearts, accompanying re-expression of several "fetal" genes whose role in disease phenotypes remains unknown. Here we show that hypoxia-controlled expression of the transcription factor Hand1 determines oxygen consumption by inhibition of lipid metabolism in the fetal and adult cardiomyocyte, leading to downregulation of mitochondrial energy generation. Hand1 is under direct transcriptional control by HIF1α. Transgenic mice prolonging cardiac Hand1 expression die immediately following birth, failing to activate the neonatal lipid metabolising gene expression programme. Deletion of Hand1 in embryonic cardiomyocytes results in premature expression of these genes. Using metabolic flux analysis, we show that Hand1 expression controls cardiomyocyte oxygen consumption by direct transcriptional repression of lipid metabolising genes. This leads, in turn, to increased production of lactate from glucose, decreased lipid oxidation, reduced inner mitochondrial membrane potential, and mitochondrial ATP generation. We found that this pathway is active in adult cardiomyocytes. Up-regulation of Hand1 is protective in a mouse model of myocardial ischaemia. We propose that Hand1 is part of a novel regulatory pathway linking cardiac oxygen levels with oxygen consumption. Understanding hypoxia adaptation in the fetal heart may allow development of strategies to protect cardiomyocytes vulnerable to ischaemia, for example during cardiac ischaemia or surgery.

Hypoxia at the heart of sudden infant death syndrome?

Sudden infant death syndrome (SIDS) is a significant clinical problem without an accepted pathological mechanism, but with multiple conflicting models. Mutations in a growing number of genes have been found postmortem in SIDS cases, notably genes encoding ion channels. This can only account for a minority of cases, however. Our recent work on a novel mouse model of SIDS suggests a potentially more widespread role for cardiac arrhythmia in SIDS without needing to invoke the inheritance of abnormal ion-channel genes. We propose a model for SIDS pathogenesis whereby postnatal hypoxia leads to delayed maturation of the cardiac conduction system and an increased risk of cardiac arrhythmia. Our model may integrate several epidemiological findings related to risks factors for SIDS, and agrees with previous work suggesting a common final pathological pathway in SIDS.

Abundance, distribution, mobility and oligomeric state of M2 muscarinic acetylcholine receptors in live cardiac muscle.

M2 muscarinic acetylcholine receptors modulate cardiac rhythm via regulation of the inward potassium current. To increase our understanding of M2 receptor physiology we used Total Internal Reflection Fluorescence Microscopy to visualize individual receptors at the plasma membrane of transformed CHO(M2) cells, a cardiac cell line (HL-1), primary cardiomyocytes and tissue slices from pre- and post-natal mice. Receptor expression levels between individual cells in dissociated cardiomyocytes and heart slices were highly variable and only 10% of murine cardiomyocytes expressed muscarinic receptors. M2 receptors were evenly distributed across individual cells and their density in freshly isolated embryonic cardiomyocytes was ~1µm(-2), increasing at birth (to ~3µm(-2)) and decreasing back to ~1µm(-2) after birth. M2 receptors were primarily monomeric but formed reversible dimers. They diffused freely at the plasma membrane, moving approximately 4-times faster in heart slices than in cultured cardiomyocytes. Knowledge of receptor density and mobility has allowed receptor collision rate to be modeled by Monte Carlo simulations. Our estimated encounter rate of 5-10 collisions per second, may explain the latency between acetylcholine application and GIRK channel opening.

A mouse model to study the link between hypoxia, long QT interval and sudden infant death syndrome.

The pathology of sudden infant death syndrome (SIDS) is poorly understood. Many risk factors, including hypoxia, have been identified. Prolongation of the ECG QTc interval is associated with elevated risk of SIDS but its aetiology in most cases remains unknown. We have characterised ECG changes in the newborn mouse in the hours and days following birth. There was a steady increase in heart rate alongside significant decreases in QTc interval, QRS duration and QTc dispersion over the first 10 postnatal days. Birth into hypoxia (10% FiO2) prevented electrocardiac maturation, downregulated cardiac ion-channel expression and led to neonatal death. We found that risk of death decreased with increasing age of exposure to hypoxia. Genetic elevation of cardiac hypoxia-signalling after birth in αMHC-Cre::VHL(fl/fl) mice also prevented electrocardiographic maturation, leading to arrhythmia and death before weaning. Immunohistochemistry and western blotting revealed internalisation and dephosphorylation of Connexin43. We conclude that increased ambient oxygen concentration after birth drives maturation of the cardiac electrical conduction system, failure of which leads to aberrant ion channel and Connexin43 expression and predisposes to arrhythmia and sudden death. This is consistent with known risk factors of SIDS and provides a link between neonatal hypoxia, ECG abnormalities and sudden death.

From zebrafish heart jogging genes to mouse and human orthologs: using Gene Ontology to investigate mammalian heart development.

For the majority of organs in developing vertebrate embryos, left-right asymmetry is controlled by a ciliated region; the left-right organizer node in the mouse and human, and the Kuppfer's vesicle in the zebrafish. In the zebrafish, laterality cues from the Kuppfer's vesicle determine asymmetry in the developing heart, the direction of 'heart jogging' and the direction of 'heart looping'.  'Heart jogging' is the term given to the process by which the symmetrical zebrafish heart tube is displaced relative to the dorsal midline, with a leftward 'jog'. Heart jogging is not considered to occur in mammals, although a leftward shift of the developing mouse caudal heart does occur prior to looping, which may be analogous to zebrafish heart jogging. Previous studies have characterized 30 genes involved in zebrafish heart jogging, the majority of which have well defined orthologs in mouse and human and many of these orthologs have been associated with early mammalian heart development.    We undertook manual curation of a specific set of genes associated with heart development and we describe the use of Gene Ontology term enrichment analyses to examine the cellular processes associated with heart jogging.  We found that the human, mouse and zebrafish 'heart jogging orthologs' are involved in similar organ developmental processes across the three species, such as heart, kidney and nervous system development, as well as more specific cellular processes such as cilium development and function. The results of these analyses are consistent with a role for cilia in the determination of left-right asymmetry of many internal organs, in addition to their known role in zebrafish heart jogging.    This study highlights the importance of model organisms in the study of human heart development, and emphasises both the conservation and divergence of developmental processes across vertebrates, as well as the limitations of this approach.

Chemical labelling of active serum thioester proteins for quantification.

The complement serum proteins C3 and C4 and the protease inhibitor α-2 macroglobulin are all members of the C3/α-2M thioester protein family, an evolutionarily ancient and conserved family that contains an intrachain thioester bond. The chemistry of the thioester bond is a key to the function of the thioester proteins. All these proteins function by covalently linking to their target by acyl transfer of the protein via the thioester moiety. We show that the signature thioester bond can be targeted with nucleophiles linked to a bioreporter molecule, site-specifically modifying the whole, intact thioester protein. Conditions were optimised to label selectively and efficiently pull-down unprocessed thioester-containing proteins from serum. We demonstrated pull-down of full-length C3, α-2M and C4 from sera in high salt, using a biotinylated nucleophile and streptavidin-coated resin, confirmed by MALDI-TOF MS identification of the gel bands. The potential for the development of a quantitative method for measuring active C3 in serum was investigated in patient sera pre and post operation. Quantifying active C3 in clinical assays using current methods is difficult. Methods based on antibody detection (e.g. nephelometry) do not distinguish between active C3 and inactive breakdown products. C3-specific haemolytic assays can be used, but these require use of relatively unstable reagents. The current work represents a promising robust, enzyme- and antibody-free chemical method for detecting active thioester proteins in blood, plasma or serum.

The representation of heart development in the gene ontology.

An understanding of heart development is critical in any systems biology approach to cardiovascular disease. The interpretation of data generated from high-throughput technologies (such as microarray and proteomics) is also essential to this approach. However, characterizing the role of genes in the processes underlying heart development and cardiovascular disease involves the non-trivial task of data analysis and integration of previous knowledge. The Gene Ontology (GO) Consortium provides structured controlled biological vocabularies that are used to summarize previous functional knowledge for gene products across all species. One aspect of GO describes biological processes, such as development and signaling. In order to support high-throughput cardiovascular research, we have initiated an effort to fully describe heart development in GO; expanding the number of GO terms describing heart development from 12 to over 280. This new ontology describes heart morphogenesis, the differentiation of specific cardiac cell types, and the involvement of signaling pathways in heart development. This work also aligns GO with the current views of the heart development research community and its representation in the literature. This extension of GO allows gene product annotators to comprehensively capture the genetic program leading to the developmental progression of the heart. This will enable users to integrate heart development data across species, resulting in the comprehensive retrieval of information about this subject. The revised GO structure, combined with gene product annotations, should improve the interpretation of data from high-throughput methods in a variety of cardiovascular research areas, including heart development, congenital cardiac disease, and cardiac stem cell research. Additionally, we invite the heart development community to contribute to the expansion of this important dataset for the benefit of future research in this area.