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The clinical monitoring of therapy with cardiac glycosides such as digoxin may at times be difficult. The presence of a therapeutic response is not always easy to determine and most of the signs and symptoms of digoxin toxicity may also occur because of the condition being treated. For these reasons attempts have been made to monitor therapy by biochemical pharmacological means. Most previous efforts have examined the usefulness of the measurement of plasma or serum digoxin concentrations (an index of the pharmacokinetic behaviour of the drug) while only a few have considered measurements which reflect the pharmacological or pharmacodynamic effects of the drug. Plasma digoxin concentration measurement has been shown by many workers to be of value in helping to distinguish between patients who are clinically toxic and those who are not although doubt has recently been cast on its true usefulness. Such measurements, however, have not been shown conclusively to be of value in reflecting the therapeutic status of the individual patient. The studies reported here have been carried out in order to examine further the usefulness of plasma digoxin concentration measurements in the diagnosis of digoxin toxicity and to determine if any such usefulness might be extended; to develop a technique for monitoring the pharmacodynamic effects of digoxin (the measurement of the ability of a patient's own erythrocytes to transport 86rubidium - termed 86Rb uptake for short), to determine the usefulness of that technique in monitoring digoxin therapy and to compare its value with that of plasma digoxin concentration measurements; and to analyse critically current practice in terms of recommended dosage regimens and existing aids (nomograms and equations) to determining suitable dosages of digoxin clinically. The last set of aims has led in addition to an evaluation of a proposed set of guidelines for digoxin therapy.

Triple-negative breast cancer (TNBC) remains clinically challenging as effective targeted therapies are still lacking. In addition, patient mortality mainly results from the metastasized lesions. Therefore, there is an unmet need to develop novel therapies against metastatic TNBC (mTNBC). CXCR4 has been identified to be one of the major chemokine receptors involved in breast cancer metastasis. Previously, our lab had identified LIM and SH3 Protein 1 (LASP1) to be a key mediator in CXCR4-driven invasion. To further investigate the role of LASP1 in this process, a proteomic screen was employed and identified a novel protein-protein interaction between LASP1 and components of the eukaryotic initiation 4F complex (eIF4F). We hypothesized that activation of the CXCR4-LASP1-eIF4F axis may contribute to the preferential translation of oncogenic mRNAs leading to an altered pro-oncogenic proteome that facilitates breast cancer progression and metastasis. To test this hypothesis, we first confirmed that the gene expression of CXCR4, LASP1, and eIF4A are upregulated in invasive breast cancer. Moreover, we demonstrate that LASP1 specifically associated with eIF4A, a mRNA helicase, in a CXCL12-dependent manner via a proximity ligation assay. We validated this finding through many approaches including co-immunoprecipitation and GST-pulldown assays. Furthermore, we showed an association with eIF4B, an ancillary protein that enhances the helicase activity of eIF4A. Activation of CXCR4 signaling by its ligand, CXCL12, increased the translation of oncoproteins downstream of eIF4A. Interestingly, genetic silencing of LASP1 interrupted the ability of eIF4A to translate oncogenic mRNAs into oncoproteins implicating a role for LASP1 in mediating the signaling from CXCR4. This impaired ability of eIF4A was confirmed by a previously established luciferase reporter assay which harbors a 5'-leader dependent on eIF4F. Finally, depletion of LASP1 sensitizes 231S cells to the pharmacological inhibition of eIF4A by Rocaglamide A as evident through reduced expression of BIRC5, ROCK1, Cyclin D1, and Mdm2 which are downstream effectors of eIF4A and serves as an endogenous measure of the level of its activity. Importantly, the viability of 231 cells is compromised with the concomitant depletion of LASP1 and the pharmacological inhibition of eIF4A. Overall, our work identified that the CXCR4-LASP1 axis is a novel mediator in oncogenic protein translation through activation of the helicase activity of eIF4A. Next, we attempted to identify novel small molecule inhibitors against the helicase activity of eIF4A which is part of the CXCR4-LASP1-eIF4F axis. To accomplish this goal, a (CGG)4-tdTomato-luciferase reporter system was established to determine the helicase activity of eIF4A in three distinct TNBC cell lines. The Prestwick Chemical Library, consisting of mostly FDA-approved and off-patent drugs, was then screened. This led to identification of cardiac glycosides as potential inhibitors of eIF4A-mediated translation. We hypothesize that cardiac glycosides inhibit the expression of eIF4A through decreases in c-Myc. The combination of rocaglamide A and a standard cardiac glycoside such as digoxin exhibited synergistic anti-cancer activity against TNBC cells in vitro. Thus, digoxin and other related cardiac glycosides could be potentially harnessed to target the helicase activity of eIF4A in order to disrupt the CXCR4-LASP1-eIF4F axis in mTNBC.

Acute myocardial infarction, the clinical manifestation of ischemia-reperfusion (IR) injury, is a leading cause of death worldwide. Although percutaneous coronary interventions and thrombolytic therapies are effective in limiting the duration of ischemia, the re-introduction of blood flow to previously ischemic area causes additional damage, collectively known as reperfusion injury. One of the most effective ways to reduce reperfusion injury is ischemic preconditioning (IPC), which is induced by several cycles of brief ischemia and reperfusion bouts prior to the prolonged ischemia. IPC was found to be mediated through signaling pathways (including activation of Src, PI3K-IB, and PKCe), and mimicked by a number of pharmacological or mechanical interventions. However, 25 years after the first report of IPC, preconditioning research has not translated into clinical application against cardiac reperfusion injury. Contributing to this somewhat surprising and disappointing failure to translate preconditioning into the clinic, the applicability and efficacy of preconditioning treatments in the setting of acute myocardial infarction (MI) have not always been carefully considered in the research setting. In particular, the impact of comorbidities on cardioprotective signaling or the unpredictable nature of MI has limited the impact of IPC. Against this background, the overall objective of this work was to investigate the potential benefit of using cardiac glycosides (CG) drugs to trigger cardioprotection in conditions relevant to acute myocardial infarction. Indeed, treatment with low doses of the CG ouabain before ischemia has been shown to induce cardioprotective effects against IR injury through a mechanism known as ouabain preconditioning (OPC). Rather than the classic specific inhibition of Na/K-ATPase-mediated ion transport, the mechanism underlying OPC is the activation of the more recently recognized signaling function of Na/K-ATPase, which includes Src-PKCe, ROS and the mKATP channel. Accordingly, a first study was undertaken to test whether digoxin, which is the only FDA-approved CG, could mimic OPC. Further, we tested whether CG could improve cardiac recovery when administered at the onset of reperfusion rather than before ischemia (i.e. postconditioning rather than preconditioning), which is highly suitable in the setting of acute MI. In Langendorff-perfused mouse heart preparations, ouabain and digoxin 10 µM similarly inhibited Na/K-ATPase activity by about 30% and activated PKCe translocation by about 50%. Four min of perfusion with 10 µM ouabain (OPC protocol) or digoxin (DigPC protocol), followed by 8 min washout period prior to 40 min ischemia and 30 min reperfusion significantly improved the recovery of left ventricle developed pressure (LVDP), end-diastolic pressure (EDP), and protected Na/K-ATPase activity. Ouabain and digoxin treatments also significantly decreased LDH release and reduced infarct size, in a similar manner, further suggesting protection against IR-induced cell death. These findings were consistent with previous reports of OPC-induced protection against IR in the rat and rabbit cardiac tissue, and revealed that OPC and DigPC were equally effective. Postconditioning protocols consisting of a single bolus injection of 100 nmoles of ouabain or digoxin in the coronary tree at the beginning of reperfusion revealed that both ouabain and digoxin postconditioning improved the recovery of LVDP and decreased LDH release, suggesting a functional and structural protection as efficient as the one provided by OPC. This series of experiments therefore led to the conclusion that both CG ouabain and FDA-approved digoxin are effective preconditioners and postconditioners. Digoxin postconditioning protocols may be of particular interest in clinical settings. Our second study explored mechanistic differences between IPC and OPC, which could make the CG-based interventions more suitable in diseases where IPC has reduced efficiency. Specifically, Class I PI3K activation is required for IPC, but its role in OPC has not been investigated. While PI3K-IB is critical to IPC, studies have suggested that ouabain signaling is PI3K-IA-specific. A pharmacological approach was used to test the hypothesis that OPC and IPC rely on distinct PI3K-I isoforms. In Langendorff-perfused mouse hearts, OPC was initiated by 4 min of ouabain 10µM and IPC by 4 cycles of 5 min ischemia and reperfusion prior to 40 min of global ischemia and 30 min of reperfusion. Without affecting PI3K-IB, ouabain doubled PI3K-IA activity and Akt Ser473 phosphorylation. The PI3K-IA inhibitor PI-103 (100 nM) blocked ouabain-induced Akt activation. IPC and OPC significantly preserved cardiac contractile function and tissue viability as evidenced by left ventricular developing pressure and end-diastolic pressure recovery, reduced lactate dehydrogenase release, and decreased infarct size. OPC protection was blunted by PI-103 but not by the PI3K-IB inhibitor AS-604850 (1 µM). In contrast, IPC-mediated protection was not affected by PI-103 but was blocked by AS-604850 (15 µM). These data suggested that PI3K-IA activation is required for OPC while PI3K-IB activation is needed for IPC. Ouabain-induced PKCe translocation, previously shown to be critical for OPC, was unaffected by PI-103 co-treatment and further studies shall reveal the identity of the downstream targets of this new PI3K IA-dependent branch of OPC. In summary, cardiac glycosides can work in both preconditioning and postconditioning manners. In addition, cardiac glycosides preconditioning is mediated through PI3K-IA while ischemic preconditioning (IPC) relies on PI3K-IB activation. These findings may be of clinical relevance in the setting of AMI in patients presenting with diseases and medications that could differentially affect the integrity of cardiac PI3K-IA and IB pathways.

Our laboratory previously reported that ouabain decreases the transcellular sodium transport in LLCPK1 cell line by mechanisms other than affecting intracellular sodium concentration (Liu J 2002). We utilized for our experiments LLCPK1 cell line, which is an epithelial cells derived from pig renal proximal tubules. When cell were treated for 12 hours with low dose of ouabain (50nM and 100 nM), the intracellular sodium concentration was not affected. Moreover, NHE3, the major apical sodium transporter in proximal tubules, was significantly downregulated on mRNA level, as measured by StaRT-PCR. NHE3 protein was also downregulated when measured by westernblot. Total NHE activity was also decreased when measured by intracellular PH recovery after acidification. Ouabain-induced downregulation of NHE3 was blocked after inhibiting c-src or PI3K with PP2 or wortmannin respectively. Both c-src and PI3K are essential part of Na/K ATpase signaling cascade. We utilized transiently transfected cells with NHE3 promoter constructs in luceferase reporter vector to map the response elements. We mapped the ouabain response elements on NHE3 promoter to be (-450 to -1194). Furthermore, Ouabain-induced downregulation of NHE3 promoter activity was blocked in C2-9 cell line (siRNA-mediated caveolin-1 depleted cell line). We used C2-9 cell line to confirm the role of pump endocytosis in the ouabian signaling cascade to regulate NHE3 expression. Our laboratory identified that ouabain decreases the binding of transcription factors TR and Sp1 to their cognate cis-elements on the promoter. Our group utilized protein/DNA array analysis, chromatin immunoprecipitation and electrophoretic mobility shift assay (EMSA) to confirm the decrease of the transcription factors binding induced by ouabain. We concluded that ouabain chronically downregulates NHE3 expression and acitivty in proximal tubule cells by endocytosis-mediated signaling cascade from Na/K ATPase. The signaling pathway involves c-src and PI3K to atenuate TR and Sp1 binding to NHE3 promoter.

In skeletal muscle, the Na+,K+-ATPase enzyme regulates trans-membrane Na+ and K+ fluxes during contractions, and therefore also affects muscle excitability and plays an important role in delaying muscle fatigue. Consequently, any modulation of Na+,K+-ATPase content or activity has the potential to affect muscle fatiguability. Thus, this thesis investigated three factors thought to impair or down-regulate the skeletal muscle Na+,K+-ATPase – acute exercise, renal disease and digoxin. The related effects on plasma [K+] during exercise and on muscle performance were also examined. This thesis firstly investigated the acute effects of brief intense exercise on muscle Na+,K+-ATPase content and maximal activity (Study 1). Study 2 investigated the effects of end-stage renal disease on plasma [K+] regulation during exercise; examined the relationship between impaired [K+] regulation and muscle performance, and investigated the effects of endurance training in these patients. Study 3 investigated the impacts of end-stage renal disease and renal transplantation on skeletal muscle Na+,K+-ATPase and its relationship with muscle performance. Finally, Study 4 investigated the effects of chronic digoxin administration on skeletal muscle Na+,K+-ATPase content and maximal activity and on muscle performance in healthy humans.

This laboratory has shown that exercise in humans impairs skeletal muscle Na+,K+-ATPase maximal in vitro activity, whilst in isolated rat muscles, Na+,K+-ATPase inhibition with ouabain leads to early muscle fatigue. Hence, Na+,K+-ATPase function is likely to be important for skeletal muscle performance. Digoxin is a specific inhibitor of the Na+,K+- ATPase and is used to treat patients with severe heart failure. This thesis investigated whether in-vivo inhibition of Na+,K+-ATPase by digoxin adversely effected muscle performance and Na+,K+-ATPase isoform expression and protein abundance in skeletal muscle of healthy individuals. Ten active, but not well-trained healthy volunteers (9 M, 1 F) gave written informed consent. Subjects performed incremental cycle ergometer exercise to measure VO2peak and to determine 33, 67 and 90% VO2peak workrates. Exercise tests were performed after taking digoxin (DIG, 0.25 mg.d-1) or a placebo (CON) for 13 day (Cybex) or 14 day (cycling), in a randomised, counterbalanced, cross-over, double-blind design, with trials separated by at least 6 weeks. On day 13 subjects performed tests of quadriceps muscle strength and endurance of the dominant leg, on a Cybex isokinetic dynamometer ( Cybex Norm 770, Henley Healthcare, USA). On day 14 subjects completed 10 min cycling at each of 33% and 67% VO2peak, then to fatigue at 90% VO2peak on cycle ergometer (Lode Excalibur, Groningen, the Netherlands), with arterial blood sampling for plasma [K+] determinations. A muscle biopsy was taken at rest, after exercise at 67% and 90%VO2peak and at 3 hr recovery. Muscle was analysed for Na+, K+-pump isoform (alpha1-alpha3, beta1-beta3) mRNA expression (real-time RT-PCR, GeneAmp 7500 Sequence Detection System) and whole homogenate protein abundance (immunoblotting, Kodak Digital Science Image Station 400CF, Eastman Kodak Company, CT, USA). Serum digoxin was 0.7±0.1 nM at day 13 and 0.8±0.1 nM at day 14 (Mean±SEM) and was less than the lowest detection limit of 0.4 nM in control trials. There were no differences in VO2 or time to fatigue (DIG 262±156 vs CON 254 ±125 s) between DIG and CON during exercise. Arterial plasma [K+] increased above rest at 67% VO2peak and increased further at fatigue (P

The fundamental aspects of the recently developed NEELA system were explored along with ways to improve and expand the versatility of the technique. The overall usefulness of NEELA was enhanced by demonstrating that NEELA can be employed for competitive binding assays of small molecules (e.g., biotin, digoxin) in addition to sandwich type assays for proteins developed previously. Competitive assays were subsequently used to determine the relationship between assay performance and reagent binding affinity and to develop novel detection schemes for the NEELA system. It was found through a series of competitive assays towards digoxin, utilizing monoclonal antibodies with different affinities towards digoxin, that the NEELA system requires antibodies with KA ≥ 1 x 1010 M-1 and becomes qualitative, at best, when KA ≤ 1 x 109 M-1. Additionally, it was demonstrated that labeling enzymes other than alkaline phosphatase (ALP) could be used in NEELA via competitive assays towards biotin employing beta-galactosidase (beta-GAL) and glucose oxidase (GOX) as labels. An enzyme channeling system with ALP as the label and immobilized tyrosinase completing the enzyme pair was also shown to be an effective detection scheme. The systems which used enzyme channeling and (beta-GAL) exhibit lower limits of detection (LODs) of approximately 10 nM towards biotin, while the GOX system had a LOD of 1 nM. Studies involving alternate enzyme labels combined with confocal microscopy experiments showed that it is unlikely that the pH gradient across the sensing membrane present in the NEELA system plays a crucial role in the spatial resolution of bound and unbound enzyme label. Indeed, the profile of pH vs. distance from the microporous nylon membrane surface with time shows that the gradient observed when using ALP as a label extends into the bulk of the solution. Results from competitive assays suggest that the specific activity of the enzyme label towards the substrate being employed is crucial for achieving low LODs with the NEELA system. It is also shown herein, that protein immobilized within the pores of the nylon membranes used for NEELA provides no significant contribution to the signal observed in a competitive assay for biotin. Additional studies comparing porous gold electrodes modified non-specifically to electrodes modified using activated thioctic acid in both competitive assays towards biotin and sandwich assays toward human chorionic gonadotropin, indicated that the use of thioctic: acid is not necessary to achieve good assay performance.

Five antibodies directed against digoxin were grown and characterized. Two separation techniques were tested to find the better method of separating bound the free fractions of ligand after competitive reactions with the antibodies. Charcoal separation was chosen. The antibodies were analyzed for affinity constant by the Scatchard Analysis. Cross reactivity data for two of the antibodies were collected. Four of the antibodies have satisfactory affinity constants for use in a digoxin assay. Cross reactivity studies showed varying amounts of cross reactivity with digoxin metabolites, but none with the steroids and lipid tested.

2031-01-01