Quo vadis Cardiac Glycoside Research?

Cardiac glycosides (CGs), toxins well-known for numerous human and cattle poisoning, are natural compounds, the biosynthesis of which occurs in various plants and animals as a self-protective mechanism to prevent grazing and predation. Interestingly, some insect species can take advantage of the CG's toxicity and by absorbing them, they are also protected from predation. The mechanism of action of CG's toxicity is inhibition of Na+/K+-ATPase (the sodium-potassium pump, NKA), which disrupts the ionic homeostasis leading to elevated Ca2+ concentration resulting in cell death. Thus, NKA serves as a molecular target for CGs (although it is not the only one) and even though CGs are toxic for humans and some animals, they can also be used as remedies for various diseases, such as cardiovascular ones, and possibly cancer. Although the anticancer mechanism of CGs has not been fully elucidated, yet, it is thought to be connected with the second role of NKA being a receptor that can induce several cell signaling cascades and even serve as a growth factor and, thus, inhibit cancer cell proliferation at low nontoxic concentrations. These growth inhibitory effects are often observed only in cancer cells, thereby, offering a possibility for CGs to be repositioned for cancer treatment serving not only as chemotherapeutic agents but also as immunogenic cell death triggers. Therefore, here, we report on CG's chemical structures, production optimization, and biological activity with possible use in cancer therapy, as well as, discuss their antiviral potential which was discovered quite recently. Special attention has been devoted to digitoxin, digoxin, and ouabain.

Ouabain worsens diastolic sarcomere length in myocytes from a cardiomyopathy mouse model.

Diastolic dysfunction is a major feature of hypertrophic cardiomyopathy (HCM). Data from patient tissue and animal models associate increased Ca2+ sensitivity of myofilaments with altered Na+ and Ca2+ ion homeostasis in cardiomyocytes with diastolic dysfunction. In this study, we tested the acute effects of ouabain on ventricular myocytes of an HCM mouse model. The effects of ouabain on contractility and Ca2+ transients were tested in intact adult mouse ventricular myocytes (AMVMs) of Mybpc3-targeted knock-in (KI) and wild-type (WT) mice. Concentration-response assessment of contractile function revealed low sensitivity of AMVMs to ouabain (10 µM) compared to literature data on human cardiomyocytes (100 nM). Three hundred µM ouabain increased contraction amplitude (WT ~1.8-fold; KI ~1.5-fold) and diastolic intracellular Ca2+ in both WT and KI (+12-18%), but further decreased diastolic sarcomere length in KI cardiomyocytes (-5%). Western Blot analysis of whole heart protein extracts revealed 50% lower amounts of Na+/K+ ATPase (NKA) in KI than in WT. Ouabain worsened the diastolic phenotype of KI cardiomyocytes at concentrations which did not impair WT diastolic function. Ouabain led to an elevation of intracellular Ca2+, which was poorly tolerated in KI showing already high cytosolic Ca2+ at baseline due to increased myofilament Ca2+ sensitivity. Lower amounts of NKA in KI could amplify the need to exchange excessive intracellular Na+ for Ca2+ and thereby explain the general tendency to higher diastolic Ca2+ in KI.

Comparative Cardiotoxicity of Low Doses of Digoxin, Ouabain, and Oleandrin.

The aim of this study was to evaluate the comparative effects of CGs on heart physiology. Twenty-eight Wistar rats were distributed into four groups (n = 7), control group received NaCl 0.9% every 24 h for 21 days; treated groups received respectively 50 µg/kg of digoxin (DIG), ouabain (OUA) and oleandrin (OLE) every 24 h for 21 days. Serial ECGs were performed, as well as serum levels of creatinine kinase (CK), its MB fraction, troponin I (cTnI), calcium (Ca2+) and lactic dehydrogenase (LDH). Heart tissue was processed for histology, scanning electron microscopy and Western blot analysis for cTnI, brain natriuretic peptide (BNP), sodium potassium pump alpha-1 and alpha-2. Ventricle samples were also analyzed for thiobarbituric acid reactive substances and antioxidant enzymes (SOD, GPX, and CAT). ECGs showed decrease in QT and progressive shortening of QRS. No arrhythmias were observed. No significant differences were associated with CGs treatment and serum levels of CK, CK-MB, and cTnI. Only oleandrin increased LDH levels. Histological analysis showed degenerative changes and only oleandrin promoted moderate focal necrosis of cardiomyocytes. Scanning microscopy also confirmed the greatest effect of oleandrin, with rupture and shortening of cardiac fibers. The expression of troponin I and alpha-1 isoform were not altered, however, the protein levels of BNP and alpha-2 were higher in the groups that received oleandrin and ouabain in relation to the digoxin group. All GCs affected the production of ROS, without causing lipid peroxidation, through the activation of different antioxidant pathways. It is concluded that the administration of digoxin, ouabain, and oleandrin at 50 µg/kg for 21 days caused cardiovascular damage that represent an important limitation into its future use in heart failure and antineoplastic therapy.

The cardenolides strophanthidin, digoxigenin and dihydroouabain act as activators of the human RORγ/RORγT receptors.

Two isoforms of a ligand-activated nuclear receptor, RORγ and RORγT, have been implicated in various physiological functions, including energy metabolism, circadian rhythm and immune system development. Using a stably transfected reporter cell line, we screened two chemical libraries and identified three cardenolides (natural, plant-derived pesticides) as activators of RORγ-dependent transcription. These compounds increased G6PC and NPAS2 expression in HepG2 cells, accompanied by increased occupancy of RORγ within the promoters of these genes. Further, strophanthidin, digoxigenin and dihydroouabain upregulated IL17A and IL17F expression and enhanced IL17 secretion in Th17 human lymphocytes. Molecular docking analyses of these compounds to the RORγ LBD showed favorable docking scores, suggesting that cardenolides may act as agonists of the receptor. Thus, our results provide new chemical structures for further development of RORγ-selective modulators with virtual therapeutic potential.

Dynamic changes in microglial and macrophage characteristics during degeneration and regeneration of the zebrafish retina.

BACKGROUND: In contrast to mammals, zebrafish have the capacity to regenerate retinal neurons following a variety of injuries. Two types of glial cells, Müller glia (MG) and microglia, are known to exist in the zebrafish retina. Recent work has shown that MG give rise to regenerated retinal neurons, but the role of resident microglia, and the innate immune system more generally, during retinal regeneration is not well defined. Specifically, characteristics of the immune system and microglia following substantial neuron death and a successful regenerative response have not been documented. METHODS: The neurotoxin ouabain was used to induce a substantial retinal lesion of the inner retina in zebrafish. This lesion results in a regenerative response that largely restores retinal architecture, neuronal morphologies, and connectivities, as well as recovery of visual function. We analyzed cryosections from damaged eyes following immunofluorescence and H&E staining to characterize the initial immune response to the lesion. Whole retinas were analyzed by confocal microscopy to characterize microglia morphology and distribution. Statistical analysis was performed using a two-tailed Student's t test comparing damaged to control samples. RESULTS: We find evidence of early leukocyte infiltration to the retina in response to ouabain injection followed by a period of immune cell proliferation that likely includes both resident microglia and substantial numbers of proliferating, extra-retinally derived macrophages, leading to rapid accumulation upon retinal damage. Following immune cell proliferation, Müller glia re-enter the cell cycle. In retinas that have regenerated the layers lost to the initial injury (histologically regenerated), microglia retain morphological features of activation, suggesting ongoing functions that are likely essential to restoration of retinal function. CONCLUSIONS: Collectively, these results indicate that microglia and the immune system are dynamic during a successful regenerative response in the retina. This study provides an important framework to probe inflammation in the initiation of, and functional roles of microglia during retinal regeneration.

Comparative Action of Cardiotonic Steroids on Intracellular Processes in Rat Cortical Neurons.

Binding to Na+,K+-ATPase, cardiotonic steroids (CTS) activate intracellular signaling cascades that affect gene expression and regulation of proliferation and apoptosis in cells. Ouabain is the main CTS used for studying these processes. The effects of other CTS on nervous tissue are practically uncharacterized. Previously, we have shown that ouabain affects the activation of mitogen-activated protein kinases (MAP kinases) ERK1/2, p38, and JNK. In this study, we compared the effects of digoxin and bufalin, which belong to different subclasses of CTS, on primary culture of rat cortical cells. We found that CTS toxicity is not directly related to the degree of Na+,K+-ATPase inhibition, and that bufalin and digoxin, like ouabain, are capable of activating ERK1/2 and p38, but with different concentration and time profiles. Unlike bufalin and ouabain, digoxin did not decrease JNK activation after long-term incubation. We concluded that the toxic effect of CTS in concentrations that inhibit less than 80% of Na+,K+-ATPase activity is related to ERK1/2 activation as well as the complex profile of MAP kinase activation. A direct correlation between Na+,K+-ATPase inhibition and the degree of MAP kinase activation is only observed for ERK1/2. The different action of the three CTS on JNK and p38 activation may indicate that it is associated with intracellular signaling cascades triggered by protein-protein interactions between Na+,K+-ATPase and various partner proteins. Activation of MAP kinase pathways by these CTS occurs at concentrations that inhibit Na+,K+-ATPase containing the α1 subunit, suggesting that these signaling cascades are realized via α1. The results show that the signaling processes in neurons caused by CTS can differ not only because of different inhibitory constants for Na+,K+-ATPase.

Lithium and Tamoxifen Modulate Behavior and Protein Kinase C Activity in the Animal Model of Mania Induced by Ouabain.

Background: The intracerebroventricular injection of ouabain, a specific inhibitor of the Na+/K+-adenosine-triphosphatase (Na+/K+-ATPase) enzyme, induces hyperactivity in rats in a putative animal model of mania. Several evidences have suggested that the protein kinase C signaling pathway is involved in bipolar disorder. In addition, it is known that protein kinase C inhibitors, such as lithium and tamoxifen, are effective in treating acute mania. Methods: In the present study, we investigated the effects of lithium and tamoxifen on the protein kinase C signaling pathway in the frontal cortex and hippocampus of rats submitted to the animal model of mania induced by ouabain. We showed that ouabain induced hyperlocomotion in the rats. Results: Ouabain increased the protein kinase C activity and the protein kinase C and MARCKS phosphorylation in frontal cortex and hippocampus of rats. Lithium and tamoxifen reversed the behavioral and protein kinase C pathway changes induced by ouabain. These findings indicate that the Na+/K+-ATPase inhibition can lead to protein kinase C alteration. Conclusions: The present study showed that lithium and tamoxifen modulate changes in the behavior and protein kinase C signalling pathway alterations induced by ouabain, underlining the need for more studies of protein kinase C as a possible target for treatment of bipolar disorder.

Cardiotonic Steroids Stimulate Macrophage Inflammatory Responses Through a Pathway Involving CD36, TLR4, and Na/K-ATPase.

OBJECTIVE: Circulating levels of cardiotonic steroids (CTS) are elevated in various chronic inflammatory conditions, but the role of CTS in inflammation remains largely unknown. We have previously shown that the CTS ouabain stimulates proinflammatory responses in murine macrophages. In this study, we aim to explore the mechanism how CTS induce proinflammatory responses in primary murine and human macrophages. APPROACH AND RESULTS: Using both murine peritoneal macrophages and human monocyte-derived macrophages, we demonstrated that ouabain activated NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), leading to proinflammatory cytokine (eg, MCP-1 [monocyte chemotactic protein 1], TNF-α [tumor necrosis factor-α], IL-1ß [interleukin-1ß], and IL-6) production. By applying siRNA techniques and murine peritoneal macrophages isolated from genetically modified mice, we showed that macrophages partially deficient in Na/K-ATPase, the receptor for CTS, or fully deficient in the scavenger receptor CD36 or TLR4 (Toll-like receptor) were resistant to ouabain-induced NF-κB activation, suggesting an indispensable role of these 3 receptors in this pathway. Mechanistically, this effect of ouabain was independent of the ion transport function of the Na/K-ATPase. Instead, ouabain stimulated a signaling complex, including Na/K-ATPase, CD36, and TLR4. Subsequently, TLR4 recruited MyD88 adaptor protein for NF-κB activation. Furthermore, intraperitoneal injection of ouabain into mice specifically recruited Ly6C+CCR2+ monocyte subtypes to the peritoneal cavities, indicating that the CTS ouabain triggers inflammation in vivo. CONCLUSIONS: CTS activate NF-κB leading to proinflammatory cytokine production in primary macrophages through a signaling complex, including CD36, TLR4, and Na/K-ATPase. These findings warrant further studies on endogenous CTS in chronic inflammatory diseases, such as atherosclerosis.

Human neuronal cell based assay: A new in vitro model for toxicity evaluation of ciguatoxin.

Ciguatoxins (CTXs) are emerging marine neurotoxins representing the main cause of ciguatera fish poisoning, an intoxication syndrome which configures a health emergency and constitutes an evolving issue constantly changing due to new vectors and derivatives of CTXs, as well as their presence in new non-endemic areas. The study applied the neuroblastoma cell model of human origin (SH-SY5Y) to evaluate species-specific mechanistic information on CTX toxicity. Metabolic functionality, cell morphology, cytosolic Ca2+i responses, neuronal cell growth and proliferation were assessed after short- (4-24h) and long-term exposure (10days) to P-CTX-3C. In SH-SY5Y, P-CTX-3C displayed a powerful cytotoxicity requiring the presence of both Veratridine and Ouabain. SH-SY5Y were very sensitive to Ouabain: 10 and 0.25nM appeared the optimal concentrations, for short- and long-term toxicity studies, respectively, to be used in co-incubation with Veratridine (25µM), simulating the physiological and pathological endogenous Ouabain levels in humans. P-CTX-3C cytotoxic effect, on human neurons co-incubated with OV (Ouabain+Veratridine) mix, was expressed starting from 100pM after short- and 25pM after long-term exposure. Notably, P-CTX-3C alone at 25nM induced cytotoxicity after 24h and prolonged exposure. This human brain-derived cell line appears a suitable cell-based-model to evaluate cytotoxicity of CTX present in marine food contaminated at low toxic levels and to characterize the toxicological profile of other/new congeners.

Screening, verification, and analysis of biomarkers for drug-induced cardiac toxicity in vitro based on RTCA coupled with PCR Array technology.

Cardiotoxicity is one of the most serious side effects of new drugs. Early detection of the drug induced cardiotoxicity based on the biomarkers provides an important preventative strategy for detecting potential cardiotoxicity of candidate drugs. In this study, we aim to identify the predictive genomics biomarkers for drug-induced cardiac toxicity based on the RTCA coupled with PCR Array technology in primary cells. Three prototypical cardiotoxic compounds (doxorubicin, isoproterenol, ouabain) with different mechanisms were firstly real-time monitored to diagnose the cytotoxicity by using the RTCA, while the functional alterations of cardiomyocytes were also monitored by analyzing the beating frequency of cardiomyocytes. Then cardiac specific toxicity gene expression changes were studied by using the technology of PCR Array, which can detect the changes of 84 cardiac functions related genes. Rps6kb1 was identified to be the common cardiac biomarkers by using multivariate statistical and integration analyses. The biomarker was further verified by selecting other drugs with or without cardiotoxicity, and the results showed that the gene exhibited specific changes in cardiac toxicity. Moreover, IPA was applied to combine relevant pathways of Rps6kb1, and identify the main types of cardiac toxicity. These results would further enrich the evaluating strategy of drug-induced cardiotoxicity in vitro, and Rps6kb1 could be used as the specific biomarker of cardiotoxcity during safety assessment of the novel drug candidates.

Low dose ouabain stimulates NaK ATPase α1 subunit association with angiotensin II type 1 receptor in renal proximal tubule cells.

Our laboratory has recently demonstrated that low concentrations of ouabain increase blood pressure in rats associated with stimulation of NaK ATPase activity and activation of the Src signaling cascade in NHE1-dependent manner. Proteomic analysis of human kidney proximal tubule cells (HKC11) suggested that the Angiotensin II type 1 receptor (AT1R) as an ouabain-associating protein. We hypothesize that ouabain-induced stimulation of NaK ATPase activity is mediated through AT1R. To test this hypothesis, we examined the effect of ouabain on renal cell angiotensin II production, the effect of AT1R inhibition on ouabain-stimulated NKA activity, and the effect of ouabain on NKA-AT1R association. Ouabain increased plasma angiotensin II levels in rats treated with ouabain (1µg/kg body wt./day) for 9days and increased angiotensin II levels in cell culture media after 24h treatment with ouabain in human (HKC11), mouse (MRPT), and human adrenal cells. Ouabain 10pM stimulated NKA-mediated 86Rb uptake and phosphorylation of EGFR, Src, and ERK1/2. These effects were prevented by the AT1R receptor blocker candesartan. FRET and TIRF microscopy using Bodipy-labeled ouabain and mCherry-NKA or mCherry-AT1R demonstrated association of ouabain with AT1R and NKA. Further our FRET and TIRF studies demonstrated increased association between AT1R and NKA upon treatment with low dose ouabain. We conclude that ouabain stimulates NKA in renal proximal tubule cells through an angiotensin/AT1R-dependent mechanism and that this pathway contributes to cardiac glycoside associated hypertension.

Inversions and adaptation to the plant toxin ouabain shape DNA sequence variation within and between chromosomal inversions of Drosophila subobscura.

Adaptation is defined as an evolutionary process allowing organisms to succeed in certain habitats or conditions. Chromosomal inversions have the potential to be key in the adaptation processes, since they can contribute to the maintenance of favoured combinations of adaptive alleles through reduced recombination between individuals carrying different inversions. We have analysed six genes (Pif1A, Abi, Sqd, Yrt, Atpα and Fmr1), located inside and outside three inversions of the O chromosome in European populations of Drosophila subobscura. Genetic differentiation was significant between inversions despite extensive recombination inside inverted regions, irrespective of gene distance to the inversion breakpoints. Surprisingly, the highest level of genetic differentiation between arrangements was found for the Atpα gene, which is located outside the O1 and O7 inversions. Two derived unrelated arrangements (O3+4+1 and O3+4+7) are nearly fixed for several amino acid substitutions at the Atpα gene that have been described to confer resistance in other species to the cardenolide ouabain, a plant toxin capable of blocking ATPases. Similarities in the Atpα variants, conferring ouabain resistance in both arrangements, may be the result of convergent substitution and be favoured in response to selective pressures presumably related to the presence of plants containing ouabain in the geographic locations where both inversions are present.

Critical role of the α1-Na(+), K(+)-ATPase subunit in insensitivity of rodent cells to cytotoxic action of ouabain.

In rodents, ubiquitous α1-Na(+), K(+)-ATPase is inhibited by ouabain and other cardiotonic steroids (CTS) at ~10(3)-fold higher concentrations than those effective in other mammals. To examine the specific roles of the CTS-sensitive α1S- and CTS-resistant α1R-Na(+), K(+)-ATPase isoforms, we compared the effects of ouabain on intracellular Na(+) and K(+) content, cell survival, and mitogen-activated protein kinases (MAPK) in human and rat vascular smooth muscle cells (HASMC and RASMC), human and rat endothelial cells (HUVEC and RAEC), and human and rat brain astrocytes. 6-h exposure of HASMC and HUVEC to 3 µM ouabain dramatically increased the intracellular [Na(+)]/[K(+)] ratio to the same extend as in RASMC and RAEC treated with 3000 µM ouabain. In 24, 3 µM ouabain triggered the death of all types of human cells used in this study. Unlike human cells, we did not detect any effect of 3000-5000 µM ouabain on the survival of rat cells, or smooth muscle cells from mouse aorta (MASMC). Unlike in the wild-type α1(R/R) mouse, ouabain triggered death of MASMC from α1(S/S) mouse expressing human α1-Na(+), K(+)-ATPase. Furthermore, transfection of HUVEC with rat α1R-Na(+), K(+)-ATPase protected them from the ouabain-induced death. In HUVEC, ouabain led to phosphorylation of p38 MAPK, whereas in RAEC it stimulated phosphorylation of ERK1/2. Overall, our results, demonstrate that the drastic differences in cytotoxic action of ouabain on human and rodent cells are caused by unique features of α1S/α1R-Na(+), K(+)-ATPase, rather than by any downstream CTS-sensitive/resistant components of the cell death machinery.

Potassium aspartate inhibits SH-SY5Y cell damage and apoptosis induced by ouabain and H2O2.

The present study aimed to investigate the effects of L-aspartic acid potassium salt (potassium aspartate, K-asp) on SH-SY5Y cells treated with ouabain and H2O2. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to investigate the effects of K-asp on SH-SY5Y cell death induced by ouabain. Nissl staining was used to demonstrate the morphological changes of the SH-SY5Y cells. Light microscopy and 4',6-diamidino-2-phenylindole (DAPI) staining were performed to visualize apoptosis in SH-SY5Y cells incubated with ouabain for 6, 24 and 48 h. Transmission electron microscopy was used to observe the effect of K-asp on ultrastructural changes of the SH-SY5Y cells following incubation with ouabain for 24 and 48 h. An annexin V-fluorescein isothiocyanate/propidium binding assay and flow cytometry were performed successively to investigate how K-asp affected the H2O2-induced cell apoptosis. The MTT assay demonstrated that K-asp attenuated the cytotoxicity of the SH-SY5Y cells following treatment with ouabain, in a dose-dependent manner. The cell survival rates following 48 h incubation in the K-asp (15 mM) and K-asp (25 mM) groups were higher compared with the KCl and MK801 groups. Nissl staining demonstrated that the severity of cell injury in the KCl and K-asp (25 mM) groups were alleviated. In the DAPI staining and transmission electron microscopy analyses, KCl and K-asp (25 mM) reduced the rate of ouabain-induced apoptosis. Flow cytometry revealed that K-asp (25 mM) reduced H2O2 -induced apoptosis. These results demonstrated that K-asp (25 mM) inhibited the ouabain and H2O2-induced SH-SY5Y cell damage and apoptosis, possibly by supplementing levels of intracellular K(+).

Ouabain mediates integrin switch in human lung cancer cells.

BACKGROUND: Physiological effects of ouabain, an endogenous human hormone, are being intensively investigated. However, its role in regulation of integrin pattern in lung cancer is largely unknown. The switching in the expression pattern of integrins is recognized as an important factor facilitating metastasis of several cancers. MATERIALS AND METHODS: Cytotoxicity and proliferative effects of ouabain on H460 lung cancer cells were evaluated by the MTT assay. The levels of integrin proteins in response to ouabain were determined by western blotting. Anchorage-independent growth and migration behaviors were performed by the wound healing assay and colony formation assay, respectively. RESULTS: Herein, the results suggested that exposure of the lung cancer cells to physiological concentrations of ouabain significantly altered the level of integrins. Ouabain suppressed integrin α4, α5, αv, ß3 and ß4, whereas it had no significant effect on integrin ß1 and ß4. According to the switch patterns of integrins, ouabain treatment resulted in a dramatic reduction of cell colony size and inhibition of cancer cell migration. However, ouabain-induced integrin switch had only a slight effect on chemotherapeutic drug susceptibility. CONCLUSION: Ouabain may have a role in suppressing cancer metastasis via integrin regulation.

Bone marrow stromal cell transplantation enhances recovery of motor function after lacunar stroke in rats.

This study was aimed to clarify if the bone marrow stromal cells (BMSCs) significantly improve functional outcome after lacunar stroke when stereotactically transplanted into the brain. Ouabain, a Na/K ATPase pump inhibitor, was stereotactically injected into the right striatum of Wistar rats. One week later, the superparamagnetic iron oxide (SPIO)-labeled rat BMSCs (n=7) or vehicle (n=8) were stereotactically transplanted into the left striatum. Using rotarod test, motor function was serially evaluated through the experiment. A 7.0-T MR apparatus was employed to serially monitor the migration of BMSCs in the host brain. Histological analysis was performed at 7 weeks after ouabain injection, i.e., 6 weeks after BMSC transplantation. Ouabain injection yielded the reproducible, focal lesion in the right striatum, causing continuous motor dysfunction throughout the experiment. BMSC transplantation significantly enhanced the recovery of motor function after ouabain injection. MR imaging demonstrated that the BMSCs aggressively migrated towards the lesion through the corpus callosum. Histological analysis supported the findings on MRI. The BMSCs significantly enhanced the neurogenesis in the subventricular zone (SVZ) on both sides. Some of them also expressed neuronal or astrocytic phenotypes in the neocortex, SVZ, corpus callosum, and peri-lesion area. These findings strongly suggest that the BMSCs may serve therapeutic impacts on lacunar stroke when stereotactically transplanted at clinically relevant timing.

Stem cell transplantation via the cochlear lateral wall for replacement of degenerated spiral ganglion neurons.

Spiral ganglion neurons (SGNs) are poorly regenerated in the mammalian inner ear. Because of this, stem cell transplantation has been used to replace injured SGNs, and several studies have addressed this approach. However, the difficulty of delivering stem cells into the cochlea and encouraging their migration to Rosenthal's canal (RC), where the SGNs are located, severely restricts this therapeutic strategy. In this study, we attempted to establish a new stem cell transplantation route into the cochlea via the cochlear lateral wall (CLW). First, we tested the precision of this route by injecting Fluorogold into the CLW and next assessed its safety by mock surgeries. Then, using a degenerated SGN animal model, we transplanted neural stem cells (NSCs), derived from the olfactory bulb of C57BL/6-green fluorescent protein (GFP) mice, via the CLW route and examined the cells' distribution in the cochlea. We found the CLW transplantation route is precise and safe. In addition, NSCs migrated into RC with a high efficiency and differentiated into neurons in a degenerated SGN rat model after the CLW transplantation. This result revealed that the basilar membrane (BM) may have crevices permitting the migration of NSCs. The result of this study demonstrates a novel route for cell transplantation to the inner ear, which is important for the replacement of degenerated SGNs and may contribute to the treatment of sensorineural hearing loss.

Regenerative potential of the zebrafish corneal endothelium.

Corneal transparency, critical for clear vision, is maintained in part by the pump function of the corneal endothelial cells that are arrested in G(1) phase of the cell cycle in adult humans. Thus loss of endothelial cells leads to a decrease in endothelial cell density. A decrease below a critical threshold results in corneal edema and subsequent vision loss. Corneal edema due to endothelial dysfunction is a common indication for transplantation in developed countries. The zebrafish has emerged as a model for vertebrate regeneration due to its ease of genetic manipulation and remarkable regenerative capacity. The purpose of this study was to investigate the response and regenerative potential of the zebrafish corneal endothelium to pharmacological and mechanical injury. Similar to the human cornea, Na(+)/K(+) ATPase activity is necessary to maintain the pump function as intracameral injection of ouabain resulted in an increase in central corneal thickness. Surgical removal of the majority of the central corneal endothelium resulted in a similar increase in corneal thickness. Remarkably, by just one week post-injury the central corneal endothelium had largely re-formed. Immunofluorescence of phosphorylated histone H3 indicated that this recovery correlated with corneal endothelial cells re-entering the cell cycle. In conclusion, our results establish zebrafish as a useful model of corneal injury and repair that may offer insights into the mechanism of cell cycle arrest in human corneal endothelial cells.

The H1-H2 domain of the α1 isoform of Na+-K+-ATPase is involved in ouabain toxicity in rat ventricular myocytes.

The composition of different isoforms of Na+-K+-ATPase (NKA, Na/K pump) in ventricular myocytes is an important factor in determining the therapeutic effect and toxicity of cardiac glycosides (CGs) on heart failure. The mechanism whereby CGs cause these effects is still not completely clear. In the present study, we prepared two site-specific antibodies (SSA78 and WJS) against the H1-H2 domain of α1 and α2 isoforms of NKA in rat heart, respectively, and compared their influences on the effect of ouabain (OUA) in isolated rat ventricular myocytes. SSA78 or WJS, which can specifically bind with the α1 or α2 isoform, were assessed with enzyme linked immunosorbent assay (ELISA), Western blot and immunofluorescent staining methods. Preincubation of myocytes with SSA78 inhibited low OUA affinity pump current but not high OUA affinity pump current, reduced the rise in cytosolic calcium concentration ([Ca²âº](i)), attenuated mitochondrial Ca²âº overload, restored mitochondrial membrane potential reduction, and delayed the decrease of the myocardial contractile force as well as the occurrence of arrhythmic contraction induced by high concentrations (1 mM) but not low concentrations (1 µM) of OUA. Similarly, preincubation of myocytes with WJS inhibited high OUA affinity pump current, reduced the increase of [Ca²âº](i) and the contractility induced by 1 µM but not that induced by 1 mM OUA. These results indicate that the H1-H2 domain of the NKA α1 isoform mediates OUA-induced cardiac toxicity in rat ventricular myocytes, and inhibitors for this binding site may be used as an adjunct to CGs treatment for cardiovascular disease.

Insulin interacts directly with Na⁺/K⁺ATPase and protects from digoxin toxicity.

Insulin has shown to have cardioprotective effect in diabetic patient after digoxin intoxication. The latter, prompted us to study whether insulin interacts directly with Na⁺/K⁺-ATPase. The interaction of insulin with Na⁺/K⁺-ATPase was explored using enzyme activity, Biacore and Western blot. We also used, flow cytometry, immunohistochemistry and chronotropy on both neonatal and adult rats cardiomyocytes. Insulin at concentration 1.7e⁻7 M blunted the effect of digoxin on Na⁺/K⁺-ATPase activity. In Western blot, the same insulin concentration decreased enzyme α subunit immunoreactivity. Insulin and digoxin decreased both enzyme α subunit immunoreactivity but insulin/digoxin co-treatment did not. Biacore confirmed a direct interaction between insulin and Na⁺/K⁺-ATPase. In neonatal rat cardiomyocytes, insulin plus digoxin induced cell apoptosis but not alone. In adult rat cardiomyocytes, insulin at optimal dose did not induce apoptosis but prevented the one induced by digoxin. In immunocytochemsitry both insulin and digoxin altered Na⁺/K⁺-ATPase α subunit immunoreactivity while their association did not. Finally, insulin increased the beating rate of neonatal rat cardiomyocytes (45±7 beats/min); so did digoxin (36±13 beats/min). The effect of insulin was prevented after pre-treated with digoxin. These results demonstrate that insulin interacts directly with Na⁺/K⁺-ATPase pump and alters the effect of digoxin. This would have important clinical relevance in cardiac complications related to type I and II diabetes.