Sophoridine manifests as a leading compound for anti-arrhythmia with multiple ion-channel blocking effects.

BACKGROUND: Sophoridine (SR) has shown the potential to be an antiarrhythmic agent. However, SR's electrophysiological properties and druggability research are relatively inadequate, which limits the development of SR as an antiarrhythmic candidate. PURPOSE: To facilitate the development process of SR as an antiarrhythmic candidate, we performed integrated studies on the electrophysiological properties of SR in vitro and ex vivo to gain more comprehensive insights into the multi-ion channel blocking effects of SR, which provided the foundation for the further drugability studies in antiarrhythmic and safety studies. Firstly, SR's electrophysiological properties and antiarrhythmic potentials were recorded and assessed at the cell and tissue levels by comprehensively integrating the patch clamp with the Electrical and Optical Mapping systems. Subsequently, the antiarrhythmic effects of SR were validated by aconitine and ouabain-induced arrhythmia in vivo. Finally, the safety of SR as an antiarrhythmic candidate compound was evaluated based on the guidelines of the Comprehensive in Vitro Proarrhythmia Assay (CiPA). STUDY DESIGN: The antiarrhythmic effect of SR was evaluated at the in vitro, ex vivo, and in vivo levels. METHODS: Isolated primary cardiomyocytes and stable cell lines were prepared to explore the electrophysiologic properties of being a multiple ion-channel blocker in vitro by whole-cell patch clamp. Using electrical and optical mapping, the negative chronotropic effect of SR was determined in langendorff-perfused rat or guinea-pig hearts.The antiarrhythmic activity of SR was assessed by the ex vivo tachyarrhythmia models induced by left coronary artery ligation (LCAL) and isoproterenol (ISO). Canonical models of aconitine and ouabain-induced arrhythmia were used to verify the antiarrhythmic effects in vivo. Finally, the pro-arrhythmic risk of SR was detected in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hSCCMs) using a Microelectrode array (MEA). RESULTS: Single-cell patch assay validated the multiple ion-channel blockers of SR in transient outward current potassium currents (Ito), l-type calcium currents (ICa-l), and rapid activation delayed rectifier potassium currents (IKr). SR ex vivo depressed heart rates (HR) and ventricular conduction velocity (CV) and prolonged Q-T intervals in a concentration-dependent manner. Consistent with the changes in HRs, SR extended the active time of hearts and increased the action potential duration measured at 90% repolarization (APD90). SR could also significantly lengthen the onset time and curtail the duration of spontaneous ventricular tachycardia (VT) in the ex vivo arrhythmic model induced by LCAL. Meanwhile, SR could also significantly upregulate the programmed electrical stimulation (PES) frequency after the ISO challenge in forming electrical alternans and re-entrant excitation. Furthermore, SR exerted antiarrhythmic effects in the tachyarrhythmia models induced by aconitine and ouabain in vivo. Notably, the pro-arrhythmic risk of SR was shallow for a moderate inhibition of the human ether-à-go-go-related gene (hERG) channel. Moreover, SR prolonged field potential duration (FPDc) of hSCCMs in a concentration-dependent manner without early after depolarization (EAD) and arrhythmia occurrence. CONCLUSION: Our results indicated that SR manifested as a multiple ion-channel blocker in the electrophysiological properties and exerts antiarrhythmic effects ex vivo and in vivo. Meanwhile, due to the low pro-arrhythmic risk in the hERG inhibition assay and the induction of EAD, SR has great potential as a leading candidate in the treatment of ventricular tachyarrhythmia.

A genome-wide positioning systems network algorithm for in silico drug repurposing.

Recent advances in DNA/RNA sequencing have made it possible to identify new targets rapidly and to repurpose approved drugs for treating heterogeneous diseases by the 'precise' targeting of individualized disease modules. In this study, we develop a Genome-wide Positioning Systems network (GPSnet) algorithm for drug repurposing by specifically targeting disease modules derived from individual patient's DNA and RNA sequencing profiles mapped to the human protein-protein interactome network. We investigate whole-exome sequencing and transcriptome profiles from ~5,000 patients across 15 cancer types from The Cancer Genome Atlas. We show that GPSnet-predicted disease modules can predict drug responses and prioritize new indications for 140 approved drugs. Importantly, we experimentally validate that an approved cardiac arrhythmia and heart failure drug, ouabain, shows potential antitumor activities in lung adenocarcinoma by uniquely targeting a HIF1α/LEO1-mediated cell metabolism pathway. In summary, GPSnet offers a network-based, in silico drug repurposing framework for more efficacious therapeutic selections.

Clinical Use of Digitalis: A State of the Art Review.

The history of digitalis is rich and interesting, with the first use usually attributed to William Withering and his study on the foxglove published in 1785. However, some knowledge of plants with digitalis-like effects used for congestive heart failure (CHF) was in evidence as early as Roman times. The active components of the foxglove (Digitalis purpurea and Digitalis lanata) are classified as cardiac glycosides or cardiotonic steroids and include the well-known digitalis leaf, digitoxin, and digoxin; ouabain is a rapid-acting glycoside usually obtained from Strophanthus gratus. These drugs are potent inhibitors of cellular membrane sodium-potassium adenosine triphosphatase (Na+/K+-ATPase). For most of the twentieth century, digitalis and its derivatives, especially digoxin, were the available standard of care for CHF. However, as the century closed, many doubts, especially regarding safety, were raised about their use as other treatments for CHF, such as decreasing the preload of the left ventricle, were developed. Careful attention is needed to maintain the serum digoxin level at ≤ 1.0 ng/ml because of the very narrow therapeutic window of the medication. Evidence for benefit exists for CHF with reduced ejection fraction (EF), also referred to as heart failure with reduced EF (HFrEF), especially when considering the combination of mortality, morbidity, and decreased hospitalizations. However, the major support for using digoxin is in atrial fibrillation (AF) with a rapid ventricular response when a rate control approach is planned. The strongest support of all for digoxin is for its use in rate control in AF in the presence of a marginal blood pressure, since all other rate control medications contribute to additional hypotension. In summary, these days, digoxin appears to be of most use in HFrEF and in AF with rapid ventricular response for rate control, especially when associated with hypotension. The valuable history of the foxglove continues; it has been modified but not relegated to the garden or the medical history book, as some would advocate.

Prevention of apoptosis averts glomerular tubular disconnection and podocyte loss in proteinuric kidney disease.

There is a great need for treatment that arrests progression of chronic kidney disease. Increased albumin in urine leads to apoptosis and fibrosis of podocytes and tubular cells and is a major cause of functional deterioration. There have been many attempts to target fibrosis, but because of the lack of appropriate agents, few have targeted apoptosis. Our group has described an ouabain-activated Na,K-ATPase/IP3R signalosome, which protects from apoptosis. Here we show that albumin uptake in primary rat renal epithelial cells is accompanied by a time- and dose-dependent mitochondrial accumulation of the apoptotic factor Bax, down-regulation of the antiapoptotic factor Bcl-xL and mitochondrial membrane depolarization. Ouabain opposes these effects and protects from apoptosis in albumin-exposed proximal tubule cells and podocytes. The efficacy of ouabain as an antiapoptotic and kidney-protective therapeutic tool was then tested in rats with passive Heymann nephritis, a model of proteinuric chronic kidney disease. Chronic ouabain treatment preserved renal function, protected from renal cortical apoptosis, up-regulated Bax, down-regulated Bcl-xL, and rescued from glomerular tubular disconnection and podocyte loss. Thus we have identified a novel clinically feasible therapeutic tool, which has the potential to protect from apoptosis and rescue from loss of functional tissue in chronic proteinuric kidney disease.

Central Gain Restores Auditory Processing following Near-Complete Cochlear Denervation.

Sensory organ damage induces a host of cellular and physiological changes in the periphery and the brain. Here, we show that some aspects of auditory processing recover after profound cochlear denervation due to a progressive, compensatory plasticity at higher stages of the central auditory pathway. Lesioning >95% of cochlear nerve afferent synapses, while sparing hair cells, in adult mice virtually eliminated the auditory brainstem response and acoustic startle reflex, yet tone detection behavior was nearly normal. As sound-evoked responses from the auditory nerve grew progressively weaker following denervation, sound-evoked activity in the cortex-and, to a lesser extent, the midbrain-rebounded or surpassed control levels. Increased central gain supported the recovery of rudimentary sound features encoded by firing rate, but not features encoded by precise spike timing such as modulated noise or speech. These findings underscore the importance of central plasticity in the perceptual sequelae of cochlear hearing impairment.

Ouabain rescues rat nephrogenesis during intrauterine growth restriction by regulating the complement and coagulation cascades and calcium signaling pathway.

Intrauterine growth restriction (IUGR) is associated with a reduction in the numbers of nephrons in neonates, which increases the risk of hypertension. Our previous study showed that ouabain protects the development of the embryonic kidney during IUGR. To explore this molecular mechanism, IUGR rats were induced by protein and calorie restriction throughout pregnancy, and ouabain was delivered using a mini osmotic pump. RNA sequencing technology was used to identify the differentially expressed genes (DEGs) of the embryonic kidneys. DEGs were submitted to the Database for Annotation and Visualization and Integrated Discovery, and gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted. Maternal malnutrition significantly reduced fetal weight, but ouabain treatment had no significant effect on body weight. A total of 322 (177 upregulated and 145 downregulated) DEGs were detected between control and the IUGR group. Meanwhile, 318 DEGs were found to be differentially expressed (180 increased and 138 decreased) between the IUGR group and the ouabain-treated group. KEGG pathway analysis indicated that maternal undernutrition mainly disrupts the complement and coagulation cascades and the calcium signaling pathway, which could be protected by ouabain treatment. Taken together, these two biological pathways may play an important role in nephrogenesis, indicating potential novel therapeutic targets against the unfavorable effects of IUGR.

Modulation of signal-transducing function of neuronal membrane Na+,K+-ATPase by endogenous ouabain and low-power infrared radiation leads to pain relief.

Effects of infrared (IR) radiation generated by a low-power Co2-laser on sensory neurons of chick embryos were investigated by organotypic culture method. Low-power IR radiation firstly results in marked neurite suppressing action, probably induced by activation of Na+,K+-ATPase signal-transducing function. A further increase in energy of radiation leads to stimulation of neurite growth. We suggest that this effect is triggered by activation of Na+,K+-ATPase pumping function. Involvement of Na+,K+-ATPase in the control of the transduction process was proved by results obtained after application of ouabain at very low concentrations. Physiological significance of low-power IR radiation and effects of ouabain at nanomolar level was investigated in behavioral experiments (formalin test). It is shown that inflammatory pain induced by injection of formalin is relieved both due to ouabain action and after IR irradiation.

Response of sodium pump to ouabain challenge in human glioblastoma cells in culture.

Bipolar disorder is a severe psychiatric condition that manifests with abnormalities in ion regulation. Previous studies have suggested that glia may be specifically involved in the pathophysiology of this condition. Since the potent sodium pump inhibitor, ouabain, has been used previously to model the ionic changes of bipolar illness, we investigated its effect of on sodium pump expression and activity in a human glioblastoma cell line. LN229 cells were grown with or without ouabain 10(-7) M for 3 days, and the effect of a therapeutic concentration of lithium was also examined. The mRNA transcription of sodium pump isoforms was determined by reverse transcriptase polymerase chain reaction (RT-PCR), and the protein expression of phosphorylated and non-phosphorylated pump isoforms was semi-quantified utilizing Western blot. Ouabain treatment caused an increase of some 6-fold in alpha1 protein expression and a doubling of alpha1 mRNA. alpha3 protein and alpha2 and alpha3 mRNA more than doubled. Lithium treatment alone had no effect, but lithium co-administered with ouabain normalized Na pump protein and mRNA expression for alpha1 and 2, but not alpha3. These results suggest that disturbance of ion regulation induces changes in glial cell sodium regulatory systems which are normalized by lithium treatment.

[Ouabain: from an arrow poison to a new steroidal hormone]. / Ouabaín--od sípového jedu k novému steroidnému hormónu.

For more than 200 years, cardiotonic glycosides have been used for the treatment of congestive heart failure. Ouabain is a well-known arrow poison obtained from different Acokanthera or Strophanthus species. Much information has now accumulated that this plant toxin and its congeners are mammalian steroid hormones involved in the pathophysiology of cardiovascular diseases. There is an interesting fact that 50% patients with essential hypertension have elevated levels of endogenous ouabain. A better knowledge of the interactions of these compounds with the hormones of salt and water metabolism might help to improve the diagnosis and therapy of hypertension.

Norepinephrine increases alveolar fluid reabsorption and Na,K-ATPase activity.

The purpose of this study was to determine whether alpha-adrenergic receptor agonists have a role in alveolar fluid reabsorption, via Na,K-ATPase, in the alveolar epithelium. Alveolar fluid reabsorption increased approximately twofold with increasing concentrations of norepinephrine (NE) as compared with control rats. Treatment with the nonselective alpha-adrenergic receptor agonist, octopamine, and the specific alpha(1) agonist, phenylephrine, increased alveolar fluid reabsorption by 54 and 40%, respectively, as compared with control. The specific alpha(1)-adrenergic receptor antagonist, prazosin, inhibited the stimulatory effects of NE by approximately 30%, whereas alpha(2)-adrenergic antagonist, yohimbine, did not prevent the stimulatory effects of NE. The administration of ouabain, Na,K-ATPase inhibitor, prevented the NE-mediated increase in alveolar fluid reabsorption. In parallel with these changes, NE increased Na,K-ATPase activity and protein abundance in alveolar epithelial type II cells via the alpha(1)- and beta-adrenergic receptor. We report here that NE increased alveolar fluid reabsorption via the activation of both alpha(1)- and beta-adrenergic receptors, but not alpha(2)-adrenergic receptors. These effects are due to increased activity and abundance of the Na,K-ATPase in the basolateral membrane of ATII cells.

[The effect of ouabain on the cardiovascular system of rats with chronic heart failure]. / Vliianie uabaina na serdechno-sosudistuiu sistemu krys s khronicheskoi serdechnoi nedostatochnost'iu.

The response of the rat cardiovascular system to the cardiac glycoside ouabain was studied in an original model of heart failure developed on day 21 of coronary embolization with 15 microns radioactive microspheres introduced into the left ventricular cavity during ascending aortic occlusion. To examine systemic and regional hemodynamic parameters, the tracer technique with microspheres. Ouabain infused to rats with heart failure caused an increase in the index of left ventricular contractility, cardiac output, stroke volume, myocardial contractility, blood flow in most viscera and a decrease in end-diastolic pressure. The findings suggest that 21 days after coronary embolization the rats with heart failure proved more sensitive to the glycoside than did those sham-operated and thus, the presented model adequately reflects the status of the cardiovascular system when heart failure is evolving.

The in vitro effects of griffonin and ouabain on erythrocyte sodium content obtained from normal subjects and sickle cell patients.

The in vitro effects of griffonin and ouabain on erythrocyte sodium content have been investigated in 6 normal subjects and 6 sickle cell patients. Intracellular sodium contents of normal or sickle cells incubated for 8 h in tris buffer, griffonin/tris buffer, or ouabain/tris buffer were determined. Incubation of normal cells in tris buffer or 0.5 mmol/l griffonin had little effect on the cell sodium content. However, 1.0 mmol/l griffonin/tris buffer raised the cell sodium level (P less than 0.05) over the incubation period. Ouabain/tris buffer (0.5 mmol/l or 1.0 mmol/l) also raised the sodium content (P less than 0.05 to P less than 0.001). Incubation of sickle cells in tris buffer raised the cell sodium (P less than 0.05) as did 0.5 mmol/l or 1.0 mmol/l griffonin (P less than 0.05 to P less than 0.001). Ouabain/tris buffer (0.5 mmol/l or 1.0 mmol/l) raised the intra-erythrocyte sodium level (P less than 0.01 to P less than 0.001). These findings suggest that ouabain and griffonin both have similar actions on intra-erythrocyte sodium content although ouabain was more potent. It is suggested therefore that griffonin could be a useful anti-sickling drug for sickle cell disease crisis.