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Heart disease is one of the leading causes of death in the United States and throughout the world. While there are different techniques for reducing or preventing the impact of heart disease, nitric oxide (NO) is administered as nitroglycerin for reversing angina or chest pain. Unfortunately, due to its gaseous and short-lived half-life, NO can be difficult to study or even administer. Therefore, controlled delivery of NO is desirable for therapeutic use. In the current study, the goal was to fabricate NO-releasing microspheres (MSs) using a donor molecule, S-Nitroso-N-Acetyl penicillamine, (SNAP), and encapsulating it in poly(ε-caprolactone) (PCL) using a single-emulsion technique that can provide sustained delivery of NO to cells over time without posing any toxicity risks. Optimization of the fabrication process was performed by varying the duration of homogenization (5, 10, and 20 min) and its effect on entrapment efficiency and size. The optimized SNAP-MS had an entrapment efficiency of ˃50%. Furthermore, we developed a modified method for NO detection by using NO microsensors to detect the NO release from SNAP-MSs in real time, showing sustained release behavior. The fabricated SNAP-MSs were tested for biocompatibility with HUVECs (human umbilical vein endothelial cells), which were found to be biocompatible. Lastly, we tested the effect of controlled NO delivery to human induced pluripotent stem-derived cardiomyocytes (hiPSC-CMs) via SNAP-MSs, which showed a significant improvement in the electrophysiological parameters and alleviated anoxic stress. [ABSTRACT FROM AUTHOR]

Cardiovascular diseases (CVDs) are one of the leading causes of mortality worldwide and a number one killer in the USA. Cell-based approaches to treat CVDs have only shown modest improvement due to poor survival, retention, and engraftment of the transplanted cells in the ischemic myocardium. Recently, tissue engineering and the use of 3D scaffolds for culturing and delivering stem cells for ischemic heart disease are gaining rapid potential. Here, we describe a protocol for the fabrication of aligned coaxial nanofibrous scaffold comprising of a polycaprolactone (PCL) core and gelatin shell. Furthermore, we describe a detailed protocol for the efficient seeding and maintenance of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on these nanofibrous scaffolds, which could have a potential application in the generation of functional "cardiac patch" for myocardial repair applications as well as an in vitro 3D cardiac tissue model to evaluate the efficacy of cardiovascular drugs and cardiac toxicities.

We earlier established the mouse embryonic stem (ES) cell "GS-2" line expressing enhanced green fluorescent protein (EGFP) and have been routinely using it to understand the molecular regulation of differentiation into cardiomyocytes. During such studies, we made a serendipitous discovery that functional cardiomyocytes derived from ES cells stopped beating when exposed to blue light. We observed a gradual cessation of contractility within a few minutes, regardless of wavelength (nm) ranges tested: blue (~420-495), green (~510-575), and red (~600-700), with green light manifesting the strongest impact. Following shifting of cultures back into the incubator (darkness), cardiac clusters regained beatings within a few hours. The observed light-induced contractility-inhibition effect was intrinsic to cardiomyocytes and not due to interference from other cell types. Also, this was not influenced by any physicochemical parameters or intracellular EGFP expression. Interestingly, the light-induced cardiomyocyte contractility inhibition was accompanied by increased intracellular reactive oxygen species (ROS), which could be abolished in the presence of N -acetylcysteine (ROS quencher). Besides, the increased intracardiomyocyte ROS levels were incidental to the inhibition of calcium transients and suppression of mitochondrial activity, both being essential for sarcomere function. To the best of our knowledge, ours is the first report to demonstrate the monochromatic light-mediated inhibition of contractions of cardiomyocytes with no apparent loss of cell viability and contractility. Our findings have implications in cardiac cell biology context in terms of 1 ) mechanistic insights into light impact on cardiomyocyte contraction, 2 ) potential use in laser beam-guided (cardiac) microsurgery, photo-optics-dependent medical diagnostics, 3 ) transient cessation of hearts during coronary artery bypass grafting, and 4 ) functional preservation of hearts for transplantation.

Drosophila CG10915 is an uncharacterized protein coding gene with sequence similarity to human Cortactin-binding protein 2 (CTTNBP2) and Cortactin-binding protein 2 N-terminal-like (CTTNBP2NL). Here, we have named this gene Nausicaa ( naus ) and characterize it through a combination of quantitative live-cell total internal reflection fluorescence microscopy, electron microscopy, RNAi depletion and genetics. We found that Naus co-localizes with F-actin and Cortactin in the lamellipodia of Drosophila S2R+ and D25c2 cells and this localization is lost following Cortactin or Arp2/3 depletion or by mutations that disrupt a conserved proline patch found in its mammalian homologs. Using permeabilization activated reduction in fluorescence and fluorescence recovery after photobleaching, we find that depletion of Cortactin alters Naus dynamics leading to a decrease in its half-life. Furthermore, we discovered that Naus depletion in S2R+ cells led to a decrease in actin retrograde flow and a lamellipodia characterized by long, unbranched filaments. We demonstrate that these alterations to the dynamics and underlying actin architecture also affect D25c2 cell migration and decrease arborization in Drosophila neurons. We present the hypothesis that Naus functions to slow Cortactin's disassociation from Arp2/3 nucleated branch junctions, thereby increasing both branch nucleation and junction stability., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)