Brown K.,New York Medical College |
Doss M.X.,New York Medical College |
Doss M.X.,Masonic Medical Research Laboratories |
Legros S.,New York Medical College |
And 3 more authors.
PLoS ONE | Year: 2010
Background: Initial specification of cardiomyocytes in the mouse results from interactions between the extraembryonic anterior visceral endoderm (AVE) and the nascent mesoderm. However the mechanism by which AVE activates cardiogenesis is not well understood, and the identity of specific cardiogenic factors in the endoderm remains elusive. Most mammalian studies of the cardiogenic potential of the endoderm have relied on the use of cell lines that are similar to the heart-inducing AVE. These include the embryonal-carcinoma-derived cell lines, END2 and PYS2. The recent development of protocols to isolate eXtraembryonic ENdoderm (XEN) stem cells, representing the extraembryonic endoderm lineage, from blastocyst stage mouse embryos offers new tools for the genetic dissection of cardiogenesis. Methodology/Principal Findings: Here, we demonstrate that XEN cell-conditioned media (CM) enhances cardiogenesis during Embryoid Body (EB) differentiation of mouse embryonic stem (ES) cells in a manner comparable to PYS2-CM and END2-CM. Addition of CM from each of these three cell lines enhanced the percentage of EBs that formed beating areas, but ultimately, only XEN-CM and PYS2-CM increased the total number of cardiomyocytes that formed. Furthermore, our observations revealed that both contact-independent and contact-dependent factors are required to mediate the full cardiogenic potential of the endoderm. Finally, we used gene array comparison to identify factors in these cell lines that could mediate their cardiogenic potential. Conclusions/Significance: These studies represent the first step in the use of XEN cells as a molecular genetic tool to study cardiomyocyte differentiation. Not only are XEN cells functionally similar to the heart-inducing AVE, but also can be used for the genetic dissection of the cardiogenic potential of AVE, since they can be isolated from both wild type and mutant blastocysts. These studies further demonstrate the importance of both contact-dependent and contact-independent factors in cardiogenesis and identify potential heart-inducing proteins in the endoderm. © 2010 Brown et al. Source
Jesty S.A.,Cornell University |
Jung S.W.,Cornell University |
Cordeiro J.M.,Masonic Medical Research Laboratories |
Gunn T.M.,Cornell University |
And 6 more authors.
Journal of Veterinary Cardiology | Year: 2013
Objective: To further characterize arrhythmic mechanisms in German shepherd dogs (GSDs) affected with inherited ventricular arrhythmias by evaluating intracellular calcium cycling and expression of calcium handling genes. Animals: Twenty five GSDs, 9 backcross dogs, and 6 normal mongrel dogs (controls) were studied. The GSDs and backcross dogs were from a research colony of inherited ventricular arrhythmias. The control research dogs were purchased. Methods: Action potentials (APs) and pseudo-electrocardiograms (ECG) were recorded from left ventricular (LV) wedge preparations of GSDs and normal dogs. Midmyocardial (Mid) LV cells from GSDs and normal mongrels were isolated by enzymatic digestion. Cells were either field stimulated or voltage clamped and calcium transients were measured by confocal microscopy using the indicator Fluo-3AM. Expression of calcium handling genes was measured by quantitative RT-PCR. Results: Mean calcium transient decay (tau) was not different between affected GSDs and control dogs, but striking cell-to-cell variability for tau was observed within affected GSDs and between affected GSDs and controls (P < 0.0001 each); within-dog variability accounted for 75% of total variability. Calcium sparks and afterdepolarizations occurred in GSD but not control cells. ATP2A2/SERCA2a expression was significantly reduced (P = 0.0063) in affected GSDs and inversely correlated (P = 0.0006) with severity of ventricular arrhythmias. Conclusions: German shepherd dogs with inherited ventricular arrhythmias have electrophysiologic abnormalities in calcium cycling associated with reduced ATP2A2/SERCA2a expression. These animals provide a unique opportunity to study calcium remodeling at the genetic and molecular level in familial ventricular arrhythmias. © 2013 Elsevier B.V. All rights reserved. Source