Reproducibility of results in biomedical research is an area of concern that should be paramount for all researchers. Importantly, this issue has been examined for experiments concerning cardiac electrophysiology. Specifically, multiple labs have found differences in results when comparing cardiac conduction velocity (CV) between healthy mice and mice that were heterozygous null for the gap junction (GJ) forming protein, Connexin 43. While the results of the comparison study showed differing extracellular ionic concentrations of the perfusates, specifically sodium, potassium, and calcium ([Na+]o, [K+]o, and [Ca2+]o), there was a lack of understanding why certain combinations of the aforementioned ions led to specific CV changes. However, more research from our lab indicates that these changes can predict modifications to a secondary form of cardiac coupling known as ephaptic coupling (EpC). Therefore the work in this dissertation was twofold, 1) to examine the effects of modulating EpC through perfusate ionic concentrations while also modulating GJC and 2) to investigate the effects of modulating all three of the main ions contributed with cardiac conduction (Na+, K+, Ca2+) and the interplay between them.
Firstly I designed and tested changes from the use of 3D printed bath for optical mapping procedures. After verification that the bath did not modify electrophysiological or contrile parameters, I studied the effects of physiologic changes to EpC determinants ([Na+]o and [K+]o) on CV during various states of GJ inhibition using the non-specific GJ uncoupler carbenoxolone (CBX). Multiple pacing rates were used to further modify EpC, as an increased pacing rate leads to a decrease in sodium channel availability through modification of the resting membrane potential. with no to low (0 and 15 µM CBX) GJ inhibition, physiologic changes in [Na+]o and [K+]o did not affect CV, however increasing pacing rate decreased CV as expected. When CBX was increased to 30 µM, a combination of decreasing [Na+]o and increasing [K+]o significantly decreased cardiac CV, specifically when pacing rate was increased.
Next, the combinatory effects of cations associated with EpC (Na+, K+, and Ca2+) were tested in to examine how cardiac CV reacts to changes in perfusate solution and how this may explain differences in experimental outcomes between laboratories. Briefly, experiments were run where [K+]o was varied throughout an experiment and the values for [Na+]o and [Ca2+]o were at one of two specific values during an experiment. 30 µM CBX was added to half of the experiments to see the changes in the CV-[K+]o relationship with GJ inhibition. With unaltered GJ coupling, elevated [Na+]o maintains CV during hyperkalemia. Interestingly, both [Na+]o and [Ca2+]o must be increased to maintain normal CV during hyperkalemia with reduced GJ coupling. These data suggest that optimized fluids can sustain normal conduction under pathophysiologic conditions like hyperkalemia and GJ uncoupling. Taken as a whole, this dissertation attempts to shed light on the importance of ionic concentration balance in perfusate solutions on cardiac conduction. / Ph. D. / The use of fluid replacement therapy was first used during the outbreak of Blue Cholera in the 1830s. However, after the development of basic fluids for intravenous fluid therapy, there have been very few changes in the fluid recipes. This same principle can be applied to cardiac research, where blood substitute perfusates are used during experimentation. However, there have been disagreements in experimental outcomes between various labs running matching studies which only varied in choice of perfusate solution. Therefore, one of the goals of this dissertation was to explore how changing ionic concentrations in cardiac perfusate solutions affected cardiac electrophysiological parameters. To fully appreciate changes in cardiac conduction, we also had to investigate changes to gap junctional coupling (GJC), which is the canonical determinant of cardiac conduction. Gap junctions are low resistance pathways which allow direct cell-to-cell coupling, which leads to synchronized cardiac conduction and contraction. However, there have been recent studies that have found a secondary form of cardiac coupling, known as ephaptic coupling (EpC), which is controlled through extracellular ionic concentrations, especially sodium, potassium, and calcium ([Na⁺]₀, [K⁺]₀, and [Ca²⁺]₀ respectively) and extracellular nano-domains known as the perinexus. We first investigate making small physiologic changes to [Na⁺]₀ and [K⁺]₀, while also inhibiting GJs to find the relationship between EpC and GJC. The results indicated that these EpC modulators could indeed modulate conduction, but only after GJs were sufficiently inhibited. However, results from this study disagreed with historical work indicating that [K⁺]₀ had a biphasic relationship with CV. Therefore, we then examined the effects of [Na⁺]₀ and [Ca²⁺]₀ on the CV-[K⁺]₀ relationship. Interestingly, it was found that inclusion of [Na⁺]₀ and [Ca²⁺]₀ had varying effects, depending on the level of GJ in the hearts. Specifically, hyperkalemia (high levels of potassium) v is associated with decreases cardiac CV. With a full complement of GJs it was found that increased [Na⁺]₀ was able to maintain cardiac CV at control levels. However, with inhibited GJ coupling, both increased [Na⁺]₀ and [Ca²⁺]₀ were needed to maintain conduction. This indicated that increasing EpC during GJ inhibition could be a possible safety mechanism for cardiac CV. The data in this dissertation aim to provide information to the importance of perfusate composition when regarding scientific data.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/81823 |
| Date | 16 January 2018 |
| Creators | Entz, Michael William II |
| Contributors | Biomedical Engineering and Mechanics, Poelzing, Steven, Gourdie, Robert G., Smyth, James W., Huckle, William R., Lee, Yong Woo |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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