Summary: Ouabain, also known as g-strophanthin, is a cardiac glycoside and a plant derived toxic substance that was traditionally used as an arrow poison in Africa. Ouabain is also found naturally within the human body in tiny quantities. In lower doses, it is used medically to treat hypotension and some arrhythmias. It acts by inhibiting the Na/K-ATPase, also known as the sodium–potassium ion pump. It is classified as an extremely hazardous substance in the United States. Interestingly, mild hypomorphic mutations in the sodium pump significantly improve longevity (by 25% in fruit flies). A very low dose of Ouabain was able to reproduce this longevity effect, while higher doses of Ouabain reduced longevity. Research from several papers shows that at low doses Ouabain (1) is a powerful senolytic and anti-cancer agent, (2) induces transcription changes that mimic those induced by heterochronic parabiosis, and (3) reduced the epigenetic age of of mouse livers by 40%. Ouabain induces activation of RhoA/ROCK signaling. It synergizes with anti-cancer drugs to kill tumor cells and eliminate the senescent cells that accumulate after irradiation. It also eliminates senescent preneoplastic cells.
Transcriptomic Analysis: The paper "Transcriptomic Hallmarks of Mortality Reveal Universal and Specific Mechanisms of Aging, Chronic Disease, and Rejuvenation" (see attached) integrated data from over 4,000 rodent tissues representing aging and responses to genetic, pharmacological, and dietary interventions with established survival data, to develop multi-tissue transcriptomic biomarkers of mortality that are capable of quantifying aging and change in lifespan in both short-lived and long-lived models. These tools were further extended to single-cell and human data, demonstrating common mechanisms of molecular aging across cell types and species. The authors then tested various substances for their effect on longevity. As predicted, the authors found effect from Rapamycin.
With respect to Ouabain, the authors found that:
According to both chronological and mortality multi-tissue transcriptomic clocks, 3-month treatment with ouabain was able to significantly decrease biological age of livers of 24-month-old female mice (p.adjusted < 4.10-18) (Fig. 7F; Extended Data Fig. 28B). Remarkably, this compound decreased predicted chronological tAge of mouse livers by ~10 months, suggesting that it did not just slow down the accumulation of aging-associated changes but also activated mechanisms of molecular rejuvenation. Indeed, top drivers of the decrease in tAge induced by ouabain included many biomarkers of mortality shared by other models of rejuvenation, such as upregulation of Nrep, and downregulation of Cdkn1a, Vcam1, Gpnmb, and Ccl5 (Fig. 7G). Overall, the mortality-associated gene expression signature of ouabain demonstrated a strong positive correlation with the rejuvenation signature of heterochronic parabiosis (Pearson’s r = 0.55) (Fig. 7H), indicating that this drug may be the first discovered pharmacological mimetic of HPB. As in the parabiosis model, made available under aCC-BY 4.0 International license. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is bioRxiv preprint doi: https://doi.org/10.1...4.07.04.601982; this version posted July 7, 2024. The copyright holder for this preprint 46 the top module contributing to ouabain-induced molecular rejuvenation was the one representing innate immune response and inflammation (Extended Data Fig. 28C). At the same time, most module-specific clocks demonstrated a statistically significant decrease of chronological and mortality tAge induced by this compound, suggesting that ouabain may produce systemic rejuvenation of multiple cellular components, including immune response, respiration, ECM organization, mRNA splicing, and others (Fig. 7I). In agreement with our observations, experimental data demonstrated the reversal of multiple age-related phenotypes produced by ouabain, including the reduction of senescence and local inflammation in the liver revealed by immunohistochemistry, restoration of albumin and phosphate levels in blood, and improvement of motor coordination and strength152. Therefore, ouabain appears to be a promising candidate for future studies that may reveal its effect on other organs and molecular modalities and shed light on specific mechanisms of tissue rejuvenation induced by this compound.
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Remarkably, the effect of ouabain was systemic, supported by most module-specific clocks, biochemical assays, and physiological tests, while its anti-mortality signature was strongly correlated with that of HPB, suggesting that ouabain mimics many effects of heterochronic parabiosis. Indeed, ouabain induced multiple rejuvenation hallmarks shared by HPB, such as downregulation of Cdkn1a and Vcam1, a vascular cell adhesion molecule shown to modulate neuroinflammation and impair the cognitive function of old mice129. Therefore, transcriptomic biomarkers developed in this study provide an opportunity to identify interventions promoting or counteracting molecular mechanisms of mortality, and characterize specific targets associated with their effects at the level of cell types, intracellular functional components, and individual genes.
Senolytic Properties: The paper "Cardiac glycosides are broad-spectrum senolytics" found that Ouabain is a senolytic agent with broad activity. The paper found that:
Here, we show that the cardiac glycoside, ouabain, is a senolytic agent with broad activity. Senescent cells are sensitized to ouabain-induced apoptosis, a process mediated in part by induction of the pro-apoptotic Bcl2-family protein NOXA. We show that cardiac glycosides synergize with anti-cancer drugs to kill tumor cells and eliminate senescent cells that accumulate after irradiation or in old mice. Ouabain also eliminates senescent preneoplastic cells. Our findings suggest that cardiac glycosides may be effective anti-cancer drugs by acting through multiple mechanism. Given the broad range of senescent cells targeted by cardiac glycosides their use against age-related diseases warrants further exploration.
Mutation Analysis: Studies of longevity have demonstrated that mild hypomorphic mutations in the sodium pump significantly improve longevity. The paper "Novel mutations affecting the Na, K ATPase alpha model complex neurological diseases and implicate the sodium pump in increased longevity" discusses these mutations and how administration of Ouabain reproduces the longevity effect of these mutations in fruit flies lacking the mutation. Interestingly, the lowest dose of Ouabain used had the greatest longevity effect (a 25% increase in maximum lifespan) while the highest dose of Ouabain used actually worsened lifespan relative to controls. The paper found that:
he Na+, K+ ATPase has numerous diverse functions including ion transport and non-pumping functions. Activity of the Na+, K+ ATPase is known to serve the essential roles of maintaining ion gradients in numerous tissues, including the neuromuscular system where high membrane potentials are required for signaling and in numerous other tissues where they are tied to various cellular homeostatic processes. To confirm that dose-dependent loss-of-function of the Na+, K+ ATPase is responsible for modulating longevity, we utilized the well-characterized pharmacological antagonist ouabain. These experiments allowed us to examine the effect of varied Na+, K+ ATPase impairment within a standardized control genotype (isogenic Canton S) to completely control for genetic background. These experiments revealed a dose-dependent increase in longevity, where low doses exhibited the most striking increase in lifespan (25%) and high doses exhibited toxicity, as predicted (Fig. 8). The finding that genetic and pharmacologic impairment of the Na+, K+ ATPase each exhibit a similar 22–25% increase in longevity suggests an important role for this protein in regulating organism lifespan. ATPalpha mutant longevity and other phenotypes are summarized in Table 2.T
See chart of lifespan here: https://www.ncbi.nlm...3_Fig8_HTML.jpg
The paper also determined that the mechanism of lifespan extension exercised by the sodium pump mutations and Ouabain was distinct from calorie restriction.
Although there was variability in consumption noted between strains, none of the mutants examined showed a marked reduction in feeding (Fig. 9). There was no correlation between feeding and animal longevity (Pearson r = 0.383) or between mass and longevity (Pearson r = −0.308). Although the feeding assay media is distinct from the standard Drosophila media used in longevity assay, the feeding assay results clearly demonstrate the animals are all capable of normal levels of nutrient consumption. Thus, the data argue that the increase in longevity observed from loss of Na+, K+ ATPase function in Drosophila is likely distinct from that reported for eat-6 mutants.
ROCK Activation: The paper "Ouabain Induces Transcript Changes and Activation of RhoA/ROCK Signaling in Cultured Epithelial Cells (MDCK)" found that Ouabain, at the concentrations found naturally in the body, activates RhoA and its downstream effector ROCK. This is an interesting result, since ROCK inhibition has been identified in other research as a mechanism of enhancing cellular dedifferentiation and improving longevity. Perhaps in elevated doses Ouabain has a different effect on ROCK? Or perhaps Ouabain's longevity enhancing effect occurs despite its ROCK activation.
Ouabain, an organic compound with the ability to strengthen the contraction of the heart muscle, was originally derived from plants. It has been observed that certain mammalian species, including humans, naturally produce ouabain, leading to its classification as a new type of hormone. When ouabain binds to Na+/K+-ATPase, it elicits various physiological effects, although these effects are not well characterized. Previous studies have demonstrated that ouabain, within the concentration range found naturally in the body (10 nmol/L), affects the polarity of epithelial cells and their intercellular contacts, such as tight junctions, adherens junctions, and gap junctional communication. This is achieved by activating signaling pathways involving cSrc and Erk1/2. To further investigate the effects of ouabain within the hormonally relevant concentration range (10 nmol/L), mRNA-seq, a high-throughput sequencing technique, was employed to identify differentially expressed transcripts. The discovery that the transcript encoding MYO9A was among the genes affected prompted an exploration of whether RhoA and its downstream effector ROCK were involved in the signaling pathways through which ouabain influences cell-to-cell contacts in epithelial cells. Supporting this hypothesis, this study reveals the following: (1) Ouabain increases the activation of RhoA. (2) Treatment with inhibitors of RhoA activation (Y27) and ROCK (C3) eliminates the enhancing effect of ouabain on the tight junction seal and intercellular communication via gap junctions. These findings further support the notion that ouabain acts as a hormone to emphasize the epithelial phenotype.
Conclusion: Ouabain is an interesting substance with much potential for longevity, although great hurdles remain with respect to its toxicity and sensitive dosing. Since Ouabain is already used as a prescription drug, there is sufficient information on its pharmacology and side effect profile to make an informed decision. Ouabain's mechanism of action is promising, but still mysterious. As a mimetic for hetetrochronic parabiosis at the transcriptional level, it may be unique among existing substances.
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