Sudden cardiac death exhibits diurnal variation in both acquired and hereditary forms of heart disease, but the molecular basis of this variation is unknown. A common mechanism that underlies susceptibility to ventricular arrhythmias is abnormalities in the duration (for example, short or long QT syndromes and heart failure) or pattern (for example, Brugada’s syndrome) of myocardial repolarization. Here we provide molecular evidence that links circadian rhythms to vulnerability in ventricular arrhythmias in mice. Specifically, we show that cardiac ion-channel expression and QT-interval duration (an index of myocardial repolarization) exhibit endogenous circadian rhythmicity under the control of a clock-dependent oscillator, krüppel-like factor 15 (Klf15). Klf15 transcriptionally controls rhythmic expression of Kv channel-interacting protein 2 (KChIP2), a critical subunit required for generating the transient outward potassium current. Deficiency or excess of Klf15 causes loss of rhythmic QT variation, abnormal repolarization and enhanced susceptibility to ventricular arrhythmias. These findings identify circadian transcription of ion channels as a mechanism for cardiac arrhythmogenesis.
Although it is not known when or where life on Earth began, some of the earliest habitable environments may have been submarine-hydrothermal vents. Here we describe putative fossilized microorganisms that are at least 3,770 million and possibly 4,280 million years old in ferruginous sedimentary rocks, interpreted as seafloor-hydrothermal vent-related precipitates, from the Nuvvuagittuq belt in Quebec, Canada. These structures occur as micrometre-scale haematite tubes and filaments with morphologies and mineral assemblages similar to those of filamentous microorganisms from modern hydrothermal vent precipitates and analogous microfossils in younger rocks. The Nuvvuagittuq rocks contain isotopically light carbon in carbonate and carbonaceous material, which occurs as graphitic inclusions in diagenetic carbonate rosettes, apatite blades intergrown among carbonate rosettes and magnetite-haematite granules, and is associated with carbonate in direct contact with the putative microfossils. Collectively, these observations are consistent with an oxidized biomass and provide evidence for biological activity in submarine-hydrothermal environments more than 3,770 million years ago.
Here we report a unique cellular reprogramming phenomenon, called stimulus-triggered acquisition of pluripotency (STAP), which requires neither nuclear transfer nor the introduction of transcription factors. In STAP, strong external stimuli such as a transient low-pH stressor reprogrammed mammalian somatic cells, resulting in the generation of pluripotent cells. Through real-time imaging of STAP cells derived from purified lymphocytes, as well as gene rearrangement analysis, we found that committed somatic cells give rise to STAP cells by reprogramming rather than selection. STAP cells showed a substantial decrease in DNA methylation in the regulatory regions of pluripotency marker genes. Blastocyst injection showed that STAP cells efficiently contribute to chimaeric embryos and to offspring via germline transmission. We also demonstrate the derivation of robustly expandable pluripotent cell lines from STAP cells. Thus, our findings indicate that epigenetic fate determination of mammalian cells can be markedly converted in a context-dependent manner by strong environmental cues.
Transplantation studies in mice and rats have shown that human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) can improve the function of infarcted hearts, but two critical issues related to their electrophysiological behaviour in vivo remain unresolved. First, the risk of arrhythmias following hESC-CM transplantation in injured hearts has not been determined. Second, the electromechanical integration of hESC-CMs in injured hearts has not been demonstrated, so it is unclear whether these cells improve contractile function directly through addition of new force-generating units. Here we use a guinea-pig model to show that hESC-CM grafts in injured hearts protect against arrhythmias and can contract synchronously with host muscle. Injured hearts with hESC-CM grafts show improved mechanical function and a significantly reduced incidence of both spontaneous and induced ventricular tachycardia. To assess the activity of hESC-CM grafts in vivo, we transplanted hESC-CMs expressing the genetically encoded calcium sensor, GCaMP3 (refs 4, 5). By correlating the GCaMP3 fluorescent signal with the host ECG, we found that grafts in uninjured hearts have consistent 1:1 host–graft coupling. Grafts in injured hearts are more heterogeneous and typically include both coupled and uncoupled regions. Thus, human myocardial grafts meet physiological criteria for true heart regeneration, providing support for the continued development of hESC-based cardiac therapies for both mechanical and electrical repair.
Dopamine has a central role in motivation and reward. Dopaminergic neurons in the ventral tegmental area (VTA) signal the discrepancy between expected and actual rewards (that is, reward prediction error), but how they compute such signals is unknown. We recorded the activity of VTA neurons while mice associated different odour cues with appetitive and aversive outcomes. We found three types of neuron based on responses to odours and outcomes: approximately half of the neurons (type I, 52%) showed phasic excitation after reward-predicting odours and rewards in a manner consistent with reward prediction error coding; the other half of neurons showed persistent activity during the delay between odour and outcome that was modulated positively (type II, 31%) or negatively (type III, 18%) by the value of outcomes. Whereas the activity of type I neurons was sensitive to actual outcomes (that is, when the reward was delivered as expected compared to when it was unexpectedly omitted), the activity of type II and type III neurons was determined predominantly by reward-predicting odours. We ‘tagged’ dopaminergic and GABAergic neurons with the light-sensitive protein channelrhodopsin-2 and identified them based on their responses to optical stimulation while recording. All identified dopaminergic neurons were of type I and all GABAergic neurons were of type II. These results show that VTA GABAergic neurons signal expected reward, a key variable for dopaminergic neurons to calculate reward prediction error.
To correlate the variable clinical features of oestrogen-receptor-positive breast cancer with somatic alterations, we studied pretreatment tumour biopsies accrued from patients in two studies of neoadjuvant aromatase inhibitor therapy by massively parallel sequencing and analysis. Eighteen significantly mutated genes were identified, including five genes (RUNX1, CBFB, MYH9, MLL3 and SF3B1) previously linked to haematopoietic disorders. Mutant MAP3K1 was associated with luminal A status, low-grade histology and low proliferation rates, whereas mutant TP53 was associated with the opposite pattern. Moreover, mutant GATA3 correlated with suppression of proliferation upon aromatase inhibitor treatment. Pathway analysis demonstrated that mutations in MAP2K4, a MAP3K1 substrate, produced similar perturbations as MAP3K1 loss. Distinct phenotypes in oestrogen-receptor-positive breast cancer are associated with specific patterns of somatic mutations that map into cellular pathways linked to tumour biology, but most recurrent mutations are relatively infrequent. Prospective clinical trials based on these findings will require comprehensive genome sequencing.
Discovering the unintended ‘off-targets’ that predict adverse drug reactions is daunting by empirical methods alone. Drugs can act on several protein targets, some of which can be unrelated by conventional molecular metrics, and hundreds of proteins have been implicated in side effects. Here we use a computational strategy to predict the activity of 656 marketed drugs on 73 unintended ‘side-effect’ targets. Approximately half of the predictions were confirmed, either from proprietary databases unknown to the method or by new experimental assays. Affinities for these new off-targets ranged from 1 nM to 30 μM. To explore relevance, we developed an association metric to prioritize those new off-targets that explained side effects better than any known target of a given drug, creating a drug-target-adverse drug reaction network. Among these new associations was the prediction that the abdominal pain side effect of the synthetic oestrogen chlorotrianisene was mediated through its newly discovered inhibition of the enzyme cyclooxygenase-1. The clinical relevance of this inhibition was borne out in whole human blood platelet aggregation assays. This approach may have wide application to de-risking toxicological liabilities in drug discovery.
The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.
The posterior parietal cortex (PPC) has an important role in many cognitive behaviours; however, the neural circuit dynamics underlying PPC function are not well understood. Here we optically imaged the spatial and temporal activity patterns of neuronal populations in mice performing a PPC-dependent task that combined a perceptual decision and memory-guided navigation in a virtual environment. Individual neurons had transient activation staggered relative to one another in time, forming a sequence of neuronal activation spanning the entire length of a task trial. Distinct sequences of neurons were triggered on trials with opposite behavioural choices and defined divergent, choice-specific trajectories through a state space of neuronal population activity. Cells participating in the different sequences and at distinct time points in the task were anatomically intermixed over microcircuit length scales (<100 micrometres). During working memory decision tasks, the PPC may therefore perform computations through sequence-based circuit dynamics, rather than long-lived stable states, implemented using anatomically intermingled microcircuits.
As vertebrate embryos develop to adulthood, their organs undergo marked changes in size and tissue architecture. The heart acquires muscle mass and matures structurally to fulfil increasing circulatory needs, a process that is incompletely understood. Here we used multicolour clonal analysis to define the contributions of individual cardiomyocytes as the zebrafish heart undergoes morphogenesis from a primitive embryonic structure into its complex adult form. We find that the single-cardiomyocyte-thick wall of the juvenile ventricle forms by lateral expansion of several dozen cardiomyocytes into muscle patches of variable sizes and shapes. As juvenile zebrafish mature into adults, this structure becomes fully enveloped by a new lineage of cortical muscle. Adult cortical muscle originates from a small number of cardiomyocytes–an average of approximately eight per animal–that display clonal dominance reminiscent of stem cell populations. Cortical cardiomyocytes initially emerge from internal myofibres that in rare events breach the juvenile ventricular wall, and then expand over the surface. Our results illuminate the dynamic proliferative behaviours that generate adult cardiac structure, revealing clonal dominance as a key mechanism that shapes a vertebrate organ.