Onduction. In distinct, we set to test the hypothesis that vulnerability to conduction failure across a cardiomyocytedonor cell interface is governed by an interplay of AP duration and strength of electrical coupling in donor cells.NIHPA Author Manuscript NIHPA Author Manuscript Techniques NIHPA Author ManuscriptMicropatterned fibronectin lines13 (Figure I in the onlineonly Information Supplement) plus a polydimethylsiloxane (PDMS) frame were applied to create 150 wide homocellular (“host” or “donor”) or heterocellular (“hostdonor”) strands (Figure 1). Host cells in the strands were represented by NRVMs even though donor cells were represented by among two genetically engineered excitable HEK293 monoclonal cell lines: 1) the poorlycoupled “Excitable Slow” or “ExS” engineered HEK293 cell line stably expressing human voltagegated cardiac sodium (Nav1.five) and inward rectifier potassium (Kir2.1) channels and two) the wellcoupled “Excitable Fast” or “ExF” engineered HEK293 cell line derived by the more steady expression of rat connexin43 (Cx43) gap junctions.18 Action potential propagation along the strands was optically mapped at 10magnification utilizing a voltagesensitive dye (ANNINE6plus).19 An S1S2 pacing protocol was applied towards the donor cells to study vulnerability to conduction block across the interface amongst host NRVMs and donor excitable HEK293 cells. An expanded Approaches section is supplied within the onlineonly Data Supplement.Circ Arrhythm Electrophysiol. Author manuscript; offered in PMC 2014 December 01.Kirkton et al.PageResultsOptical mapping in Heterocellular HostDonor StrandsNIHPA Author Manuscript NIHPA Author Manuscript NIHPA Author ManuscriptThe steady expression of fluorescent reporters in ExS (GFP, mCherry) and ExF (GFP, mCherry, mOrange) donor cells (Figure III within the onlineonly Information Supplement) allowed us to specifically localize the hostdonor interface in cocultured strands (Figure 2A) and, beneath 10magnification, align and spatially register strands with recording web pages from an optical fiber array (Figure 2B). Intense membrane staining from the cells using the voltage sensitive dye, ANNINE6plus,19 further revealed the differences in size (smaller sized vs. larger) and geometry (round vs. elongated) in donor cells vs. host NRVMs. (Figure 2B). Immunostaining showed the existence of a seamless interface in between the two cell forms with Cx43 gap junctions located involving NRVMs and ExF (but not ExS) cells (Figure IIID within the onlineonly Information Supplement). The difference (“mismatch”) involving the APD with the ExF or ExS donor cells (31.9.7 or 34.6.1 ms, respectively) and that from the host NRVMs (153.2.3 ms) yielded the formation of a monotonic APD profile (APD transform along the strand) that extended over a length of 1.Prussian blue insoluble Order 2 mm across the hostdonor interface (Figure 2B, and Figure IVA and IVB, Movie I in the onlineonly Data Supplement).Methyl 2,3-dihydroxypropanoate site Pacing from the donor finish of ExFNRVM strands resulted in unhindered conduction across the heterocellular interface (Figure 2C) as evidenced by equally spaced activation isochrones and linear boost in activation time (AT) indicative of your robust intercellular coupling and comparable CVs involving the host NRVM and donor ExF cells (22.PMID:35901518 three.3 and 22.1.4 cm/s, respectively). In contrast, the poorlycoupled ExS cells (ie, electrically connected by weak endogenous HEK293 gap junctions besides Cx43)18 displayed substantially slower CV (3.1.1 cm/s) as evidenced by dense activation isochrones as well as a steep AT slope in comparison with NRVMs, thereby crea.