Development, validation and utilisation of experimental assays for detection and quantification of endogenous cardiotonic steroids

Digoxin is commonly used for the treatment of patients with atrial fibrillation (AF) with/without heart failure (HF). However, in clinical practice the response to digoxin therapy is variable. The primary cellular target of digoxin, the Na+/K+ ATPase pump, is shared with a group of hormones called cardiotonic steroids (CTS). CTS are elevated in patients with cardiovascular disease. Differing levels of endogenous CTS in individual patients may explain the varied response to digoxin and susceptibility to adverse drug reactions. Thus validated methodologies to measurements of levels and effects of CTS are necessary. In this thesis two methodologies were developed 1) a cell-based assay for assessment of effects of CTS and 2) an ouabain competitive ELISA with aim to use for patients from the RATE control Therapy Evaluation in permanent Atrial Fibrillation (RATE-AF) trial. HEK293t cell-based assay developed were able to measure consistently changes in sodium sensitive fluorescents using SBFI sodium fluorescent dye. Changes were observed between different CTS were observed in IC50 values and maximal effects on sodium sensitive fluorescents and when calibrated to sodium levels. Extraction methodology was able to be optimised to extract potential cardiotonic steroids out of spiked human plasma. Competitive ELISA methodology developed and validated for detecting of ouabain levels from spiked and extracted samples. Some cross-reactivity with the primary antibody was observed from digoxin. Significant lowered intracellular Na+ levels was observed in HEK293t cells when applied with serum from patients with type 2 diabetes, heart failure, myocardial infarction and with a number of unexpected hospitalisations due to AF or HF. Lastly, increased levels of endogenous ouabain were detected in more severe HF (NYHA IV vs NYHA II) and in unexpected hospitalisations caused by AF or HF. Furthermore, endogenous ouabain concentrations correlated with AF (mEHRA) and HF (NYHA class) disease severity. The results presented in this thesis suggests endogenous CTS to have an important role in HF and potentially also AF. Thus, further work with CTS and the potential of these assays to be used to measure biomarkers could help better patient stratification

Comparative investigation of copper tolerance and identification of putative tolerance related genes in tardigrades

Tardigrades are microscopic aquatic animals renowned for their tolerance toward extreme environmental conditions. The current study is the first to investigate their tolerance toward heavy metals and we present a novel tardigrade toxicant tolerance assay based on activity assessments as a measure of survival. Specifically, we compare tolerance toward copper in four species representing different evolutionary lineages, habitats and adaptation strategies, i.e., a marine heterotardigrade, Echiniscoides sigismundi, a limno-terrestrial heterotardigrade, Echiniscus testudo, a limno-terrestrial eutardigrade, Ramazzottius oberhaeuseri, and a marine eutardigrade, Halobiotus crispae. The latter was sampled at a time of year, when the population is predominantly represented by aberrant P1 cysts, while the other species were in normal active states prior to exposure. Based on volume measurements and a general relation between body mass and copper tolerance, expected tardigrade EC50 values were estimated at 0.5–2 μg l(−1). Following 24 h of exposure, tolerance was high with no apparent link to lineage or habitat. EC50s (95% CI), 24 h after exposure, were estimated at 178 (168–186) and 310 (295–328) μg l(−1), respectively, for E. sigismundi and R. oberhaeuseri, whereas E. testudo and H. crispae were less affected. Highest tolerance was observed in H. crispae with a mean ± s.e.m. activity of 77 ± 2% (n = 3) 24 h after removal from ~3 mg l(−1) copper, suggesting that tardigrade cysts have increased tolerance toward toxicants. In order to identify putative tolerance related genes, an E. sigismundi transcriptome was searched for key enzymes involved in osmoregulation, antioxidant defense and copper metabolism. We found high expression of Na/K ATPase and carbonic anhydrase, known targets for copper. Our transcriptome, furthermore, revealed high expression of antioxidant enzymes, copper transporters, ATOX1, and a Cu-ATPase. In summary, our results indicate that tardigrades express well-known key osmoregulatory enzymes, supporting the hypothesis that copper inhibits sodium turnover as demonstrated for other aquatic organisms. Tardigrades, nevertheless, have high tolerance toward the toxicant, which is likely linked to high expression of antioxidant enzymes and an ability to enter dormant states. Tardigrades, furthermore, seem to have a well-developed battery of cuproproteins involved in copper homeostasis, providing basis for active copper sequestering and excretion

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate lectrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology

Comparative Investigation of Copper Tolerance and Identification of Putative Tolerance Related Genes in Tardigrades

Tardigrades are microscopic aquatic animals renowned for their tolerance toward extreme environmental conditions. The current study is the first to investigate their tolerance toward heavy metals and we present a novel tardigrade toxicant tolerance assay based on activity assessments as a measure of survival. Specifically, we compare tolerance toward copper in four species representing different evolutionary lineages, habitats and adaptation strategies, i.e., a marine heterotardigrade, Echiniscoides sigismundi , a limno-terrestrial heterotardigrade, Echiniscus testudo , a limno-terrestrial eutardigrade, Ramazzottius oberhaeuseri , and a marine eutardigrade, Halobiotus crispae . The latter was sampled at a time of year, when the population is predominantly represented by aberrant P1 cysts, while the other species were in normal active states prior to exposure. Based on volume measurements and a general relation between body mass and copper tolerance, expected tardigrade EC50 values were estimated at 0.5–2 μg l −1 . Following 24 h of exposure, tolerance was high with no apparent link to lineage or habitat. EC50s (95% CI), 24 h after exposure, were estimated at 178 (168–186) and 310 (295–328) μg l −1 , respectively, for E. sigismundi and R. oberhaeuseri , whereas E. testudo and H. crispae were less affected. Highest tolerance was observed in H. crispae with a mean ± s.e.m . activity of 77 ± 2% ( n = 3) 24 h after removal from ~3 mg l −1 copper, suggesting that tardigrade cysts have increased tolerance toward toxicants. In order to identify putative tolerance related genes, an E. sigismundi transcriptome was searched for key enzymes involved in osmoregulation, antioxidant defense and copper metabolism. We found high expression of Na/K ATPase and carbonic anhydrase, known targets for copper. Our transcriptome, furthermore, revealed high expression of antioxidant enzymes, copper transporters, ATOX1, and a Cu-ATPase. In summary, our results indicate that tardigrades express well-known key osmoregulatory enzymes, supporting the hypothesis that copper inhibits sodium turnover as demonstrated for other aquatic organisms. Tardigrades, nevertheless, have high tolerance toward the toxicant, which is likely linked to high expression of antioxidant enzymes and an ability to enter dormant states. Tardigrades, furthermore, seem to have a well-developed battery of cuproproteins involved in copper homeostasis, providing basis for active copper sequestering and excretion

The atrial resting membrane potential confers sodium current sensitivity to propafenone, flecainide and dronedarone

Background: Although atrial fibrillation (AF) ablation is increasingly used for rhythm control therapy, antiarrhythmic drugs (AADs) are commonly used, either alone or in combination with ablation. The effectiveness of AADs is highly variable. Prior work from our group suggests that alterations in the atrial resting membrane potential (RMP) induced by low Pitx2 expression could explain the variable effect of flecainide. Objective: This study assessed whether alterations in the atrial/cardiac RMP modify the effectiveness of multiple clinically used AADs. Methods: The sodium channel blocking effects of propafenone (300nM, 1μM), flecainide (1μM) and dronedarone (5μM, 10μM) were measured in human stem cell derived cardiac myocytes, HEK293 expressing human Nav1.5, primary murine atrial cardiac myocytes and murine hearts with reduced Pitx2c. Results: A more positive atrial RMP delayed INa recovery, slowed channel inactivation and decreased the peak AP upstroke velocity. All three AADs displayed enhanced sodium channel block at more positive atrial RMPs. Dronedarone was the most sensitive to changes in the atrial RMP. Dronedarone caused greater reductions in AP amplitude and peak AP upstroke velocity at more positive RMPs. Dronedarone evoked greater prolongation of the atrial effective refractory period and post-repolarisation refractoriness in murine Langendorff-perfused Pitx2c+/- hearts, which have a more positive RMP compared to wild-type. Conclusion: The atrial RMP modifies the effectiveness of several clinically used AADs. Dronedarone is more sensitive to changes in atrial RMP than flecainide or propafenone. Identifying and modifying the atrial RMP may offer a novel approach to enhancing the effectiveness of AADs or personalizing AAD selection