Intra- and Inter-Molecular Signaling in a Cardiac Connexin: Role of Cytoplasmic Domain Dimerization and Phosphorylation

As critical mediators of cell-to-cell communication, gap junctions (GJs) are comprised of membrane channels that directly link the cytoplasm of adjacent coupled cells thereby allowing for the passage of ions, small metabolites, and secondary messengers. Each channel is formed by the apposition of two connexons from adjacent cells, each composed of six connexin (Cx) proteins. Each GJ channel functions to promote signal propagation and synchronization of cells and tissues in organs. Furthermore, GJs are essential for proper propagation of cardiac action potentials from one cell to the next, leading to the coordinated contraction and relaxation of heart muscle powering circulation. In diseased human hearts, the organization and expression of Cxs in the working myocardium is remodeled leading to impaired impulse propagation and risk of lethal arrhythmias. Diverse post-translational modifications (e.g., phosphorylation) and protein partner interactions, the majority of which involve the carboxyl-terminal (CT) domain, serve to regulate Cx function. The CxCT domain serves a critical role in the regulation of all aspects of the Cx life cycle including trafficking, assembly, channel open states, as well as internalization and degradation. Importantly, the CT domain is the site at which the greatest degree of sequence divergence is observed between family members. A number of studies have highlighted that little conservation of regulatory mechanisms contained within the CT domain exists between family members. Furthermore, a clear understanding of the molecular determinants that influence different Cx channel properties between family members is lacking. Thus, investigation into the specific mechanisms that regulate each family member is essential to develop a complete picture of Cx biology and to develop novel therapeutics with which to modulate Cx function. Previous work from our laboratory identified a novel feature of the Cx45CT domain, high-affinity (KD ~ 100 nM) dimerization. Here using a combination of biophysical, biochemical, and cell/molecular biology approaches we demonstrate that dimerization is essential for proper Cx45 turnover, localization, phosphorylation, and function. Additionally, we demonstrate that phosphorylation by three kinases that are dysregulated in heart disease alter the function of Cx45 to promote cell coupling and consequently may contribute to the pathogenic phenotype

Selective surface fabrication using instability patterning

For the first time, a room temperature-curing liquid resin was patterned using electrohydrodynamic instability (EHD) processing. The resulting patterns, on the micrometre scale, became permanent after the resin had hardened. The factors affecting the scale, symmetry and morphology of the patterns were investigated

Analyzing Mitochondrial Bioenergetics to Improve Organ Hibernation Methods.

β-hydroxybutyrate is a molecule synthesized in the liver that can be used as an alternative to glucose in cellular metabolism. BHB metabolism does not produce lactic acidosis which makes it an optimal source of energy for tissues in an ischemic state. Melatonin is a hormone produced in the pineal gland that aids in slowing the metabolic rate. Both BHB and Melatonin have antioxidant properties that assist in relieving oxidative stress due to an increased metabolic rate during post-ischemic reperfusion. Testing consisted of infusing donor pigs with either BHB/M or a vehicle control. The pigs were then made brain-dead and their hearts were harvested under standard conditions and placed in a UW solution or a UW+BHB/M solution under static cold conditions. Tissue samples were then analyzed immediately after harvest, 2 days, and 8 days post-harvest using a Seahorse XFe96 Flux Analyzer. This test analyzed the oxygen consumption rate in order to show mitochondrial function. We hypothesize that the BHB/M solution will aid in mitochondrial function while the tissues are in an ischemic state. We believe this will prolong the viability of harvested organs in order to increase the amount of available organs for transplantation. We also believe that future testing will show that the BHB/M will relieve the effects of oxidative stress during reperfusion of the tissues which will decrease the risk or organ failure post-transplantation.https://digitalcommons.unmc.edu/surp2023/1005/thumbnail.jp

Development of helical, fish-inspired cross-step filter for collecting harmful algae

A new filter was developed to collect harmful algae colonies by adapting the cross-step filtration structures and mechanisms discovered recently in filter-feeding fish. Extending beyond previously published models that closely emulated the basic morphology of the fish, the new cross-step filter\u27s major innovations are helical slots, radial symmetry, and rotation as an active anti-clogging mechanism. These innovations enable the transport of concentrated particles to the downstream end of the filter. This advance was made possible by recognizing that biologically imposed constraints such as bilateral symmetry do not apply to human-made filters. The use of helical slots was developed in a series of iterative tests that used water-tracing dye and algae-sized microspheres. The major products of the iterative tests were refinements in the helical design and an understanding of how varying the major structural parameters qualitatively influenced fluid flow and filter performance. Following the iterative tests, the clogging behavior of select filters was quantified at high particle concentrations. Vortices in the helical filter were effective at reducing clogging in the center of the slots. By considering the design space that is free of the biological constraints on the system and exploring the effects of variations in major structural parameters, our work has identified promising new directions for cross-step filtration and provided key insights into the biological system