Expression of Nav1.5 was quantified using ImageJ software (National Institute of Health, NIH). 2.12. heterologous models. Our objective was to determine the properties of the sodium current in iPS-CM with a mutation in associated with Brugada syndrome. Dermal fibroblasts from a Brugada syndrome patient with a mutation in (c.1100G?>?A, leading to Nav1.5_p.R367H) were reprogrammed to iPS cells. Clones were characterized and differentiated to form beating clusters and sheets. Patient and control iPS-CM were structurally indistinguishable. Sodium current properties of patient and control iPS-CM were compared. These results were contrasted with those obtained in tsA201 cells heterologously expressing sodium channels with the same mutation. Patient-derived iPS-CM showed a 33.1C45.5% Alosetron (Hydrochloride(1:X)) reduction in recapitulate the loss of function of sodium channel current associated with this syndrome; including pro-arrhythmic changes in channel function not detected using conventional heterologous expression systems. oocytes) deviate considerably from human cardiomyocytes in many relevant aspects. These cells do not reflect the modulatory effects of accessory channel subunits or the influence of potential compensatory pathways, both of which could take place in native cardiomyocytes. Thus, studies of mutant channels using Rabbit polyclonal to NFKBIZ such expression systems might be missing important characteristics of native cardiomyocytes relevant to pathophysiology. The differentiation of induced pluripotent stem (iPS) cells from patients with cardiac diseases into cardiomyocytes (iPS-CM) provides a cell model highly homologous to native human cardiomyocytes. The use of these surrogate cells allows investigators to study mutant ion channels in their native patient-specific cell environment. This includes all their regulatory proteins, and importantly, a physiologically controlled level of protein expression. To date, several cardiac channelopathies including long QT syndrome (LQT), catecholaminergic polymorphic ventricular tachycardia and Timothy syndrome have been modeled using the iPS cell approach . Likewise, Davis et al. used iPS-CM to model an overlap LQT/Brugada syndrome . Recently, BrS was modeled using patient-specific iPS-CM . However, to date no reports exist that provide a complete characterization of the sodium current properties in Brugada syndrome patient-specific iPS-CM. We generated iPS-CM from a patient diagnosed with Brugada Syndrome who carries a heterozygous missense mutation in (c.1100G?>?A, leading to Nav1.5_p.R367H). This mutation had been previously found in Brugada Syndrome patients , . Moreover, recombinant channels with this mutation were previously studied in homozygosis in both HEK293 cells and oocytes , , , , . These studies showed a total loss of function of the sodium current. Thus, we expected that, in iPS-CM, the presence of the Nav1.5_p.R367H mutation in heterozygosis would cause a decrease near 50% of the total current due to the expression of non-functional channels translated from the mutant allele. To assess this assumption, we analyzed and compared sodium current properties of iPS-CM derived from the patient and from a healthy individual without this mutation. 2.?Materials and methods Detailed experimental procedures are available in the Online Data Supplement. 2.1. Isolation and reprogramming of fibroblasts to induced pluripotent stem (iPS) cells This study was approved by the South East Scotland Research Ethics Committee REC reference 11-SS-0095 and written informed consent was obtained from the two subjects included in the study. Dermal biopsies were dissected into 1?mm3 pieces, which were transferred to culture plastic, covered with a glass coverslip and cultured for 2?weeks before harvest. 5??105 fibroblasts were reprogrammed Alosetron (Hydrochloride(1:X)) with the Addgene episomal vectors pCXLE-hOCT3/4-shp53-F (encoding for Oct4 and shp53, Addgene plasmid # Alosetron (Hydrochloride(1:X)) 27077), pCXLE-hSK (Sox2 and Klf, Addgene plasmid # 27078) and pCXLE-hUL (LMyc and Lin28, Addgene plasmid # 27080). Reprogrammed fibroblasts were replated onto 0.1% gelatin and medium was changed to stem cell selection medium (TeSR-E8, STEMCELL Technologies SARL, Grenoble, France) 7?days post electroporation. Colonies appeared 15C20?days later. Individual clones were picked into Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) coated tissue culture plates and maintained in Alosetron (Hydrochloride(1:X)) TeSR-E8. 2.2. Sequencing For all iPS.