Atrial-like cardiomyocytes from human pluripotent stem cells are a robust preclinical model for assessing atrial-selective pharmacology.

Drugs targeting atrial-specific ion channels, Kv1.5 or Kir3.1/3.4, are being developed as new therapeutic strategies for atrial fibrillation. However, current preclinical studies carried out in non-cardiac cell lines or animal models may not accurately represent the physiology of a human cardiomyocyte (CM). In the current study, we tested whether human embryonic stem cell (hESC)-derived atrial CMs could predict atrial selectivity of pharmacological compounds. By modulating retinoic acid signaling during hESC differentiation, we generated atrial-like (hESC-atrial) and ventricular-like (hESC-ventricular) CMs. We found the expression of atrial-specific ion channel genes, KCNA5 (encoding Kv1.5) and KCNJ3 (encoding Kir 3.1), in hESC-atrial CMs and further demonstrated that these ion channel genes are regulated by COUP-TF transcription factors. Moreover, in response to multiple ion channel blocker, vernakalant, and Kv1.5 blocker, XEN-D0101, hESC-atrial but not hESC-ventricular CMs showed action potential (AP) prolongation due to a reduction in early repolarization. In hESC-atrial CMs, XEN-R0703, a novel Kir3.1/3.4 blocker restored the AP shortening caused by CCh. Neither CCh nor XEN-R0703 had an effect on hESC-ventricular CMs. In summary, we demonstrate that hESC-atrial CMs are a robust model for pre-clinical testing to assess atrial selectivity of novel antiarrhythmic drugs.

Mutagenesis and functional analysis of ion channels heterologously expressed in mammalian cells.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4)--we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

Identification of dopaminergic neurons of nigral and ventral tegmental area subtypes in grafts of fetal ventral mesencephalon based on cell morphology, protein expression, and efferent projections.

Transplants of fetal ventral mesencephalic tissue are known to contain a mixture of two major dopamine (DA) neuron types: the A9 neurons of the substantia nigra pars compacta (SNpc) and the A10 neurons of the ventral tegmental area (VTA). Previous studies have suggested that these two DA neuron types may differ in their growth characteristics, but, because of technical limitations, it has so far been difficult to identify the two subtypes in fetal ventral mesencephalon (VM) grafts and trace their axonal projections. Here, we have made use of a transgenic mouse expressing green fluorescent protein (GFP) under the tyrosine hydroxylase promoter. The expression of the GFP reporter allowed for visualization of the grafted DA neurons and their axonal projections within the host brain. We show that the SNpc and VTA neuron subtypes in VM grafts can be identified on the basis of their morphology and location within the graft, and their expression of a G-protein-gated inwardly rectifying K+ channel subunit (Girk2) and calbindin, respectively, and also that the axonal projections of the two DA neuron types are markedly different. By retrograde axonal tracing, we show that dopaminergic innervation of the striatum is derived almost exclusively from the Girk2-positive SNpc cells, whereas the calbindin-positive VTA neurons project to the frontal cortex and probably also other forebrain areas. The results suggest the presence of axon guidance and target recognition mechanisms in the DA-denervated forebrain that can guide the growing axons to their appropriate targets and indicate that cell preparations used for cell replacement in Parkinson's disease will be therapeutically useful only if they contain cells capable of generating the correct nigral DA neuron phenotype.