To bridge the gap between two-dimensional cell culture and tissue, various three-dimensional (3-D) cell culture approaches have been developed for the investigation of cardiac myocytes (CMs) and cardiac fibroblasts (CFs). However, several limitations still exist. This study was designed to develop a cardiac 3-D culture model with a scaffold-free technology that can easily and inexpensively generate large numbers of microtissues with cellular distribution and functional behavior similar to cardiac tissue. Using micromolded nonadhesive agarose hydrogels containing 822 concave recesses (800 μm deep × 400 μm wide), we demonstrated that neonatal rat ventricular CMs and CFs alone or in combination self-assembled into viable (Live/Dead stain) spherical-shaped microtissues. Importantly, when seeded simultaneously or sequentially, CMs and CFs self-sorted to be interspersed, reminiscent of their myocardial distribution, as shown by cell type-specific CellTracker or antibody labeling. Microelectrode recordings and optical mapping revealed characteristic triangular action potentials (APs) with a resting membrane potential of -66 ± 7 mV (n = 4) in spontaneously contracting CM microtissues. Under pacing, optically mapped AP duration at 90% repolarization and conduction velocity were 100 ± 30 ms and 18.0 ± 1.9 cm/s, respectively (n = 5 each). The presence of CFs led to a twofold AP prolongation in heterogenous microtissues (CM-to-CF ratio of 1:1). Importantly, Ba(2+)-sensitive inward rectifier K(+) currents and Ca(2+)-handling proteins, including sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, were detected in CM-containing microtissues. Furthermore, cell type-specific adenoviral gene transfer was achieved, with no impact on microtissue formation or cell viability. In conclusion, we developed a novel scaffold-free cardiac 3-D culture model with several advancements for the investigation of CM and CF function and cross-regulation.

Drug discovery in epilepsy has historically been biased towards transmembrane channels and receptors. Ibhazehiebo et al. describe a novel, metabolism-based drug discovery platform that assays for bioenergetic changes in zebrafish models, and demonstrate that histone deacetylases 1 and 3 decrease mitochondrial hyperexcitability and represent a combined target for anti-seizure drug development.

Mice expressing a germline mutation in the PLC-γ1 binding site of LAT (linker for activation of T cells) show progressive lymphoproliferation and ultimately die at 4–6 months of age. The hyper-activated T cells in these mice show defective TCR-induced calcium flux, but enhanced Ras/ERK activation that is critical for disease progression. Despite the loss of LAT-dependent PLC-γ1 binding and activation, genetic analysis revealed RasGRP1, and not Sos1 or Sos2, to be the major RasGEF responsible for ERK activation and the lymphoproliferative phenotype in these mice. Analysis of isolated CD4+ T cells from LAT-Y136F mice showed altered proximal TCR-dependent kinase signaling, which activated a Zap70- and LAT-independent pathway. Moreover, LAT-Y136F T cells showed ERK activation that was dependent on Lck and/or Fyn, PKCθ, and RasGRP1. These data demonstrate a novel route to Ras activation in vivo in a pathological setting.