BackgroundHuman induced pluripotent stem cells (hiPSC) harbor the potential to differentiate into diverse cardiac cell types. Previous experimental efforts were primarily directed at the generation of hiPSC-derived cells with ventricular cardiomyocyte characteristics. Aiming at a straightforward approach for pacemaker cell modeling and replacement, we sought to selectively differentiate cells with nodal-type properties.MethodshiPSC were differentiated into spontaneously beating clusters by co-culturing with visceral endoderm-like cells in a serum-free medium. Subsequent culturing in a specified fetal bovine serum (FBS)-enriched cell medium produced a pacemaker-type phenotype that was studied in detail using quantitative real-time polymerase chain reaction (qRT-PCR), immunocytochemistry, and patch-clamp electrophysiology. Further investigations comprised pharmacological stimulations and co-culturing with neonatal cardiomyocytes.ResultshiPSC co-cultured in a serum-free medium with the visceral endoderm-like cell line END-2 produced spontaneously beating clusters after 10–12 days of culture. The pacemaker-specific genes HCN4, TBX3, and TBX18 were abundantly expressed at this early developmental stage, while levels of sarcomeric gene products remained low. We observed that working-type cardiomyogenic differentiation can be suppressed by transfer of early clusters into a FBS-enriched cell medium immediately after beating onset. After 6 weeks under these conditions, sinoatrial node (SAN) hallmark genes remained at high levels, while working-type myocardial transcripts (NKX2.5, TBX5) were low. Clusters were characterized by regular activity and robust beating rates (70–90 beats/min) and were triggered by spontaneous Ca2+ transients recapitulating calcium clock properties of genuine pacemaker cells. They were responsive to adrenergic/cholinergic stimulation and able to pace neonatal rat ventricular myocytes in co-culture experiments. Action potential (AP) measurements of cells individualized from clusters exhibited nodal-type (63.4%) and atrial-type (36.6%) AP morphologies, while ventricular AP configurations were not observed.ConclusionWe provide a novel culture media-based, transgene-free approach for targeted generation of hiPSC-derived pacemaker-type cells that grow in clusters and offer the potential for disease modeling, drug testing, and individualized cell-based replacement therapy of the SAN.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0681-4) contains supplementary material, which is available to authorized users.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Concomitant heart failure (HF) poses a particular therapeutic challenge and is associated with prolonged atrial electrical refractoriness compared with non-failing hearts. We hypothesized that downregulation of atrial repolarizing TREK-1 (K2.1) K channels contributes to electrical remodeling during AF with HF, and that TREK-1 gene transfer would provide rhythm control via normalization of atrial effective refractory periods in this AF subset. In patients with chronic AF and HF, atrial TREK-1 mRNA levels were reduced by 82% (left atrium) and 81% (right atrium) compared with sinus rhythm (SR) subjects. Human findings were recapitulated in a porcine model of atrial tachypacing-induced AF and reduced left ventricular function. TREK-1 mRNA (-66%) and protein (-61%) was suppressed in AF animals at 14-day follow-up compared with SR controls. Downregulation of repolarizing TREK-1 channels was associated with prolongation of atrial effective refractory periods versus baseline conditions, consistent with prior observations in humans with HF. In a preclinical therapeutic approach, pigs were randomized to either atrial Ad-TREK-1 gene therapy or sham treatment. Gene transfer effectively increased TREK-1 protein levels and attenuated atrial effective refractory period prolongation in the porcine AF model. Ad-TREK-1 increased the SR prevalence to 62% during follow-up in AF animals, compared to 35% in the untreated AF group. In conclusion, TREK-1 downregulation and rhythm control by Ad-TREK-1 transfer suggest mechanistic and potential therapeutic significance of TREK-1 channels in a subgroup of AF patients with HF and prolonged atrial effective refractory periods. Functional correction of ionic remodeling through TREK-1 gene therapy represents a novel paradigm to optimize and specify AF management.

HCN channels underlie the depolarizing funny current (I f ) that contributes importantly to cardiac pacemaking. I f is upregulated in failing and infarcted hearts, but its implication in disease mechanisms remained unresolved. We generated transgenic mice ( HCN4 tg/wt ) to assess functional consequences of HCN4 overexpression-mediated I f increase in cardiomyocytes to levels observed in human heart failure. HCN4 tg/wt animals exhibit a dilated cardiomyopathy phenotype with increased cellular arrhythmogenicity but unchanged heart rate and conduction parameters. I f augmentation induces a diastolic Na + influx shifting the Na + /Ca 2+ exchanger equilibrium towards ‘reverse mode’ leading to increased [Ca 2+ ] i . Changed Ca 2+ homeostasis results in significantly higher systolic [Ca 2+ ] i transients and stimulates apoptosis. Pharmacological inhibition of I f prevents the rise of [Ca 2+ ] i and protects from ventricular remodeling. Here we report that augmented myocardial I f alters intracellular Ca 2+ homeostasis leading to structural cardiac changes and increased arrhythmogenicity. Inhibition of myocardial I f per se may constitute a therapeutic mechanism to prevent cardiomyopathy.

The rodent hippocampus expresses a variety of neuronal network oscillations depending on the behavioral state of the animal. Locomotion and active exploration are accompanied by theta‐nested gamma oscillations while resting states and slow‐wave sleep are dominated by intermittent sharp wave‐ripple complexes. It is believed that gamma rhythms create a framework for efficient acquisition of information whereas sharp wave‐ripples are thought to be involved in consolidation and retrieval of memory. While not strictly mutually exclusive, one of the two patterns usually dominates in a given behavioral state. Here we explore how different input patterns induce either of the two network states, using an optogenetic stimulation approach in hippocampal brain slices of mice. We report that the pattern of the evoked oscillation depends strongly on the initial synchrony of activation of excitatory cells within CA3. Short, synchronous activation favors the emergence of sharp wave‐ripple complexes while persistent but less synchronous activity—as typical for sensory input during exploratory behavior—supports the generation of gamma oscillations. This dichotomy is reflected by different degrees of synchrony of excitatory and inhibitory synaptic currents within these two states. Importantly, the induction of these two fundamental network patterns does not depend on the presence of any neuromodulatory transmitter like acetylcholine, but is merely based on a different synchrony in the initial activation pattern.

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