Study of Cardiotonic Effect of Venom of the Green Toad Bufo viridis

In experiments on the isolated heart of frogs, cats, and rats, cardiotonic effect of the green toad Bufo viridis Laur. venom was studied. It has been shown that both the venom and the fraction of bufodienolides isolated from it produce an increase of the strength of cardiac contractions and, to a lesser extent, of the heart rate in cold-blooded and warm-blooded animals. A high selectivity of the venom inotropic effect was seen as an increase of the rate characteristics of elevation or reduction of the pressure in the rat heart left ventricle. The venom and bufadienolides increase the frog atrial trabecula contraction without a rise of the slow incoming (calcium) current. A similarity of mechanisms of cardiotonic effects of the venom and of the plant cardiac glycosides is discussed.     

The aqueous extract, not organic extracts, of Terminalia arjuna bark exerts cardiotonic effect on adult ventricular myocytes

The bark of Terminalia arjuna (TA) has been used for centuries in ayurvedic medicine as cardiotonics for treatment of cardiac disorders. It became recently available as over-the-counter supplements marketed for maintaining a healthy heart. However, the cellular mechanism of its cardiotonic effect remains undefined. The present study was designed to investigate the physicochemical property and inotropic effect of the aqueous extract of TA bark (TA_AqE) on adult rat ventricular myocytes in comparison with extracts prepared sequentially with organic solvents (organic extracts). The kinetics of myocyte contraction and caffeine-induced contraction were analyzed to assess the effect of TA_AqE on sarcoplasmic reticular (SR) function. The inotropic effect of TA_AqE was also compared with that of known cardiotonics, isoproterenol (ISO) and ouabain (Ouab). We found that TA_AqE decoctions exerted positive inotropy, accelerated myocyte relaxation and increased caffeine-induced contraction concentration-dependently. In contrast, TA organic extracts caused interruption of excitability and arrhythmias without consistent inotropic action. In conclusion, TA_AqE-induced cardiotonic action via enhancing SR function, a unique action minimizing the occurrence of arrhythmias, makes TA_AqE a promising and relatively safe cardiotonic beneficial to the healthy heart and the treatment for chronic heart disease. The cardiotonic effect of TA_AqE is consistent with the therapeutic property of TA bark used in ayurvedic medicine. The method of administration and/or selective omission of hydrophobic components from bark powder could be crucial to the efficacy and safety of TA bark in cardiac therapy and uses as over-the-counter supplements.     

ACTH-induced hypertension is dependent on the ouabain-binding site of the alpha2-Na+-K+-ATPase subunit

ACTH-induced-hypertension is commonly employed as a model of stress-related hypertension, and despite extensive investigation, the mechanisms underlying elevated blood pressure (BP) are not well understood. We have reported that ACTH treatment increases tail-cuff systolic pressure in wild-type mice but not in mutant mice expressing ouabain-resistant alpha(2)-Na(+)-K(+)-ATPase subunits (alpha2(R/R) mice). Since tail-cuff measurements involve restraint stress, the present study used telemetry to distinguish between an effect of ACTH on resting BP vs. an ACTH-enhanced stress response. We also sought to explore the mechanisms underlying ACTH-induced BP changes in mutant alpha2(R/R) mice vs. wild-type mice (ouabain-sensitive alpha(2)-Na(+)-K(+)-ATPase, alpha2(S/S) mice). Baseline BP was not different between the two genotypes, but after 5 days of ACTH treatment, BP increased in alpha2(S/S) (104.0 +/- 2.6 to 117.7 +/- 3.0 mmHg) but not in alpha2(R/R) mice (108.2 +/- 3.2 to 111.5 +/- 4.0 mmHg). To test the hypothesis that ACTH hypertension is related to inhibition of alpha(2)-Na(+)-K(+)-ATPase on vascular smooth muscle by endogenous cardiotonic steroids, we measured BP and regional blood flow. Results suggest a differential sensitivity of renal, mesenteric, and cerebral circulations to ACTH and that the response depends on the ouabain sensitivity of the alpha(2)-Na(+)-K(+)-ATPase. Baseline cardiac performance was elevated in alpha2(S/S) but not alpha2(R/R) mice. Overall, the data establish that the alpha(2)-Na(+)-K(+)-ATPase ouabain-binding site is of central importance in the development of ACTH-induced hypertension. The mechanism appears to be related to alterations in cardiac performance, and perhaps vascular tone in specific circulations, presumably caused by elevated levels of circulating cardiotonic steroids.     

Interaction of cardiac troponin with cardiotonic drugs: a structural perspective

Over the 40 years since its discovery, many studies have focused on understanding the role of troponin as a myofilament based molecular switch in regulating the Ca²⁺-dependent activation of striated muscle contraction. Recently, studies have explored the role of cardiac troponin as a target for cardiotonic agents. These drugs are clinically useful for treating heart failure, a condition in which the heart is no longer able to pump enough blood to other organs. These agents act via a mechanism that modulates the Ca²⁺-sensitivity of troponin; such a mode of action is therapeutically desirable because intracellular Ca²⁺ concentration is not perturbed, preserving the regulation of other Ca²⁺-based signaling pathways. This review describes molecular details of the interaction of cardiac troponin with a variety of cardiotonic drugs. We present recent structural work that has identified the docking sites of several cardiotonic drugs in the troponin C-troponin I interface and discuss their relevance in the design of troponin based drugs for the treatment of heart disease.     

Effects of cardiotonic steroids on dermal collagen synthesis and wound healing.

We previously reported that cardiotonic steroids stimulate collagen synthesis by cardiac fibroblasts in a process that involves signaling through the Na-K-ATPase pathway (Elkareh et al. Hypertension 49: 215-224, 2007). In this study, we examined the effect of cardiotonic steroids on dermal fibroblasts collagen synthesis and on wound healing. Increased collagen expression by human dermal fibroblasts was noted in response to the cardiotonic steroid marinobufagenin in a dose- and time-dependent fashion. An eightfold increase in collagen synthesis was noted when cells were exposed to 10 nM marinobufagenin for 24 h (P < 0.01). Similar increases in proline incorporation were seen following treatment with digoxin, ouabain, and marinobufagenin (10 nM x 24 h, all results P < 0.01 vs. control). The coadministration of the Src inhibitor PP2 or N-acetylcysteine completely prevented collagen stimulation by marinobufagenin. Next, we examined the effect of digoxin, ouabain, and marinobufagenin on the rate of wound closure in an in vitro model where human dermal fibroblasts cultures were wounded with a pipette tip and monitored by digital microscopy. Finally, we administered digoxin in an in vivo wound healing model. Olive oil was chosen as the digoxin carrier because of a favorable partition coefficient observed for labeled digoxin with saline. This application significantly accelerated in vivo wound healing in rats wounded with an 8-mm biopsy cut. Increased collagen accumulation was noted 9 days after wounding (both P < 0.01). The data suggest that cardiotonic steroids induce increases in collagen synthesis by dermal fibroblasts, as could potentially be exploited to accelerate wound healing.     

The effect of FK664, a new cardiovascular drug, on systemic capacitance vessels in anesthetized dogs.

To characterize the cardiovascular effect of FK664, a compound developed for the treatment of heart failure, the mean circulatory pressure (MCP), cardiac output and other parameters were measured in open-chest anesthetized dogs. Milrinone, a cardiotonic agent, and nifedipine, a calcium channel blocker were used as reference substances. Nifedipine (10 micrograms/kg), FK664 (0.1 mg/kg) or milrinone (0.1 mg/kg) given intravenously reduced the total peripheral resistance in a similar extent (35-40%). Whereas nifedipine had no effect on MCP, FK664 produced a significant decrease in MCP. Milrinone caused a minimal decrease in MCP, but not significantly. These results indicate that FK664 dilates the systemic capacitance vessels. This action to reduce the pre-load would be beneficial in the treatment of heart failure.     

Improved heart failure protection by FK664, a novel positive inotropic agent, in dog heart-lung preparations.

The effects of FK664, a novel positive inotropic agent, and enoximone on pentobarbital-induced heart failure were compared in dog heart-lung preparations. Both FK664 and enoximone improved the cardiac function curve in a dose-dependent manner and restored it to the control level at drug concentrations of 1 microgram/ml and 10 micrograms/ml, respectively. Therefore, the cardiotonic potency of FK664 appears to be 10 times that of enoximone. These agents were almost equal in force-rate separation of cardiac effect. Neither of the agents produced arrhythmia at any dose tested. These results suggest that FK664 may be a potent cardiotonic agent for the treatment of heart failure.     

Endogenous cardiotonic steroids.

The search for endogenous digitalis led to the isolation of ouabain from blood adrenals and hypothalamus. Additional cardiotonic steroids of the cardenolid and bufadienolide type seem to circulate in blood. Adrenal cortical cells in tissue culture release ouabain upon addition of angiotensin 11. Ouabain in blood is increased in 50% of Caucasians with low renin hypertension. Analogous to other steroid hormones, cardiotonic steroid hormones in blood are bound to a specific cardiac glycoside binding globulin. Since ouabain induced growth of myocytes in tissue culture, this effect probably mediates by partial inhibition of the sodium pump and consecutive rise of intracellular Ca2+ the thickening of the wall of arteries and myocardium. PST 2238, an antagonist of cardiac glycoside function at the sodium pump, leads in rats under prolonged therapy to a decrease of hypertension. The finding of ouabain as a new adrenal hormone of the Na+ metabolism and of ouabain antagonists opens new possibilities of therapy of hypertension and congestive heart failure.     

Endogenous cardiac glycosides, a new class of steroid hormones.

The search for endogenous digitalis has led to the isolation of ouabain as well as several additional cardiotonic steroids of the cardenolide and bufadienolide type from blood, adrenals, and hypothalamus. The concentration of endogenous ouabain is elevated in blood upon increased Na(+) uptake, hypoxia, and physical exercise. Changes in blood levels of ouabain upon physical exercise occur rapidly. Adrenal cortical cells in tissue culture release ouabain upon addition of angiotensin II and epinephrine, and it is thought that ouabain is released from adrenal cortex in vivo. Ouabain levels in blood are elevated in 50% of Caucasians with low-renin hypertension. Infusion over several weeks of low concentrations of ouabain, but not of digoxin, induces hypertension in rats. A digoxin-like compound, which has been isolated from human urine and adrenals, as well various other endogenous cardiac glycosides may counterbalance their actions within a regulatory framework of water and salt metabolism. Marinobufagenin, for instance, whose concentration is increased after cardiac infarction, may show natriuretic properties because it inhibits the alpha1 isoform of Na(+)/K(+)-ATPase, the main sodium pump isoform of the kidney, much better than other sodium pump isoforms. In analogy to other steroid hormones, cardiotonic steroid hormones in blood are bound to a specific cardiac glycoside binding globulin. The discovery of ouabain as a new adrenal hormone affecting Na(+) metabolism and the development of the new ouabain antagonist PST 2238 allows for new possibilities for the therapy of hypertension and congestive heart failure. This will lead in turn to a better understanding of the disease on a physiological and endocrinological level and of the action of ouabain on the cellular level as a signal that is transduced to the plasma membrane as well as to the cell nucleus.     

Mice expressing ouabain-sensitive α1-Na,K-ATPase have increased susceptibility to pressure overload-induced cardiac hypertrophy

The Na,K-ATPase is a ubiquitous transmembrane pump and a specific receptor for cardiac glycosides such as ouabain and digoxin, which are used in the management of congestive heart failure (CHF). A potential role for these so-called endogenous cardiotonic steroids (CS) has been explored, and it has become apparent that such compounds are elevated and may play an important role in a variety of physiological and pathophysiological conditions such as hypertension and CHF. Recent evidence suggests that the Na,K-ATPase may act as a signal transducer upon CS binding and induce nonproliferative cardiac growth, implicating a role for endogenous CS in the development of cardiac hypertrophy and progressive failure of the heart. In the present study, we tested whether hypertrophic responses to pressure overload would be altered in mutant mice that specifically express ouabain-sensitive or ouabain-resistant α1- and α2-Na,K-ATPase subunits, as follows: α1-resistant, α2-resistant (α1(R/R)α2(R/R)); α1-sensitive, α2-resistant (α1(S/S)α2(R/R)); and α1-resistant, α2-sensitive (α1(R/R)α2(S/S), wild-type). In α1(S/S)α2(R/R) mice, pressure overload by transverse aortic coarctation induced severe left ventricular (LV) hypertrophy with extensive perivascular and replacement fibrosis at only 4 wk. Responses in α1(R/R)α2(S/S) and α1(R/R)α2(R/R) mice were comparatively mild. Mutant α1(S/S)α2(R/R) mice also had LV dilatation and depressed LV systolic contractile function by 4 wk of pressure overload. In separate experiments, chronic Digibind treatment prevented the rapid progression of cardiac hypertrophy and fibrosis in α1(S/S)α2(R/R) mice. These data demonstrate that mice with a ouabain-sensitive α1-Na,K-ATPase subunit have a dramatic susceptibility to the development of cardiac hypertrophy, and failure from LV pressure overload and provide evidence for the involvement of endogenous CS in this process.     

Digitaloid pregnanes promote potassium-sparing diuresis in the guinea pig.

The synthesis of 17 alpha-acetoxy-3 beta-[(beta-D-glucopyranosyl)oxy]- 6 alpha-methylpregn-4-en-20-one, the glucoside of medroxyprogesterone acetate (MPA-glu), is described. MPA-glu and 14-amino-20 beta-hydroxy-3 beta-[(alpha-L-rhamnopyranosyl)oxy]-5 beta, 14 beta-pregnane (LND 623), pregnane glycosides that bind to the digitalis receptor, and digoxin, a cardiac glycoside, were infused intravenously into the anesthetized guinea pig. Each of the three steroids significantly enhanced urinary volume and sodium excretion without affecting blood pressure and creatinine clearance. Potassium excretion was markedly enhanced by digoxin but unaffected by MPA-glu or LND 623. These observations conform to previous work that demonstrated, in the rat, potassium-sparing diuresis by the glucoside of 14 beta-hydroxyprogesterone, a cardiotonic pregnane. There is a dissociation between potency to inhibit [3H]ouabain binding and the extra ATPase actions of the digitaloid pregnanes.     

Cellular basis for the species differences in sensitivity to cardiac glycosides (digitalis)

The relative toxicity of numerous cardiotonic steroids (viz. ouabain, digitoxin, digoxin, convallatoxin, SC4453, bufalin, gitaloxin, digoxigenin, actodigin, oleandrin, digitoxigenin, gitoxin, strophanthidin, gitoxigenin, lanatosides A, B and C, alpha- and beta-acetyl digoxin, alpha- and beta-methyl digoxin) and related compounds towards a number of independent cell lines established from human, monkey, mouse, Syrian hamster, and Chinese hamster have been determined. All cardiac glycosides and their genins, as well as the cardiotonic alkaloid cassaine, exhibited greater than 100-fold higher toxicity towards cultured human and monkey cells in comparison to the cell lines of mouse, Syrian hamster, and Chinese hamster origins. These differences are species-related as all cell lines (both normal as well as transformed) from any one species, as well as cells from the closely related species (e.g., man and monkey or mouse, Chinese hamster, and Syrian hamster), showed similar sensitivity towards these drugs. The failure to see any significant differences in cellular toxicity for a larger number of other compounds which either bear limited structural resemblance to cardiac glycosides (viz. estradiol 17-beta-acetate, testosterone propionate, 21-acetoxy pregnenolone, beta-estradiol, digitonin, tigogenin, and tomatine) or interact with the Na+/K+ ATPase in a different manner (viz. veratridine, sanguinarine nitrate, penicillic acid, vanadium pentoxide, harmaline-HCI,5,5'-diphenyl hydantoin, quindonium bromide, and methyl quinolizinum bromide) provides strong evidence that the observed species-related differences are highly specific for cardiotonic steroids. Studies on the binding of [3H]ouabain show that, in comparison to human and monkey cell lines, no significant binding of the drug is observed in cells derived from the resistant species (i.e., mouse and Chinese hamster). The Na+/K+ ATPase from cells of the resistant species is inhibited at much higher concentrations of ouabain and digitoxin in comparison to the enzyme from human cells, and a good correlation is observed between these concentrations and those reported for inhibition of the enzyme from isolated heart muscles of the same species. These results provide strong evidence that the species-related differences in sensitivity to digitalis have a cellular basis and that the cultured cells from various mammalian species provide a useful model system for investigating the mechanism of action of cardiac glycosides.     

Changes in cardiac rhythm in humans during breathing compressed air under pressure up to 1.1 MPa (results of the rhythmo-cardiographic study)]

Dynamics of cardiac rhythm has been considered according to rhythmocardiographic characteristics of heart rate under orthostatic test and one-stage step-test in four altitude chamber experiments where air under pressure of 0.4-1.1 MPa is used as a breathing mixture. It is shown that these characteristics linearly depend on the partial nitrogen and oxygen pressure and hyperbaric bradycardia essentially decreases in the final period of isopression due to toxic oxygen effect. Cytochrome C decreases hyperbaric bradycardia. Under hyperbaric conditions the regulation of cardiac rhythm proceeds with altered central vegetative effects provided a direct effect of higher nitrogen and oxygen pressure on the sinusal node cells.     

Membranotropic action of cardiotonic agents and ubiquinone

Interaction of cardiotonic drugs (strophantidine acetate, suphan, para-oxybenzoic acid) and ubiquinone with phospholipid bilayers has been studied. Exothermic effect of the reaction followed by an increase in microviscosity and hydrophobicity of the bilayer from cardiolipin, but by a decrease of the microviscosity of the bilayer from lecithin has been estimated. A correlation is observed between changes in the lecithin bilayer fluidity and the heat effect of the interaction at the initial period of time after mixing of reagents.     

Effect of the new anti-arrhythmia preparation Bonnecor on the hemodynamics and activity of the ischemic heart

The effect of newly-developed anti-arrhythmia drug Bonnecor compared to that of ethacizin and lidocaine has been studied on anesthetized rats under acute myocardial ischemia. The drugs injected intravenously prior to coronary artery occlusion have been shown to prevent to a great degree the appearance of hemodynamic and functional disturbances of the heart. When compared to control, less reduction in systemic arterial pressure, systolic discharge cardiac output, contractility and heart power were observed. In addition, Bonnecor has been found to have certain advantages over reference drugs.     

Sugar moiety of cardiac glycosides is essential for the inhibitory action on the palytoxin-induced K+ release from red blood cells

Palytoxin (PTX), a highly toxic and sugar-containing substance isolated from Palythoa tuberculosa, caused K+ release from rabbit red blood cells. Cardiac glycosides, such as ouabain, convallatoxin, cymarin, digoxin and digitoxin, inhibited the PTX-induced K+ release. Their corresponding aglycones did not inhibit the K+ release, but antagonized the inhibitory effect of the glycosides. All these cardiotonic steroids equally inhibited the activity of (Na+ + K+)-ATPase prepared from hog cerebral cortex. These results suggest that the sugar moiety of the cardiac glycosides is important for the inhibitory effect on the K+ release induced by PTX and that the inhibition is not related to their inhibitory potency on the (Na+ + K+)-ATPase activity.     

Cardiotonic and antidysrhythmic effects of oleanolic and ursolic acids, methyl maslinate and uvaol.

The cardiotonic and antidysrhythmic effects of four triterpenoid derivatives, namely oleanolic acid (OA), ursolic acid (UA), and uvaol (UV), isolated from the leaves of African wild olive (Olea europaea, subsp. africana) as well as methyl maslinate (MM) isolated from the leaves of Olea europaea (Cape cultivar) were examined. The derivatives showed low toxicity on brine shrimp test. They displayed significant, dose-response vasodepressor effect and sinus bradicardia, most prominent for OA and MM. The derivatives acted as beta-adrenergic antagonists, blocking the effect of adrenaline and isoprenaline. The established positive inotropic and dromotropic effects were most distinctive for OA and MM. The antidysrhythmic effects were evaluated on CaCl2- and adrenaline-induced chemical arrhythmias, and on ischemia-reperfusion arrhythmia. OA and UA displayed antidysrhythmic effects on both types of chemical arrhythmia; OA and UV in dose 40 mg/kg conferred significant antidysrhythmic activity on ischemia and reperfusion arrhythmias. The effect was comparable to that of propranolol and suggestive of beta-adrenergic antagonistic activity. On the basis of the vasodepressor, cardiotonic and antidysrhythmic effects of these compounds, it was concluded that OA and UV isolated from wild African olive leaves, or crude extract containing all components, can provide a cheap and accessible source of additive to conventional treatment of hypertension, complicated by stenocardia and cardiac failure.     

Endogenous cardiotonic steroids and cardiovascular disease,where to next?

Ever since British Physician William Withering first described the use of foxglove extract for treatment of patients with congestive heart failure in 1785, cardiotonic steroids have been used clinically to treat heart failure and more recently atrial fibrillation. Due to their ability to bind and inhibit the ubiquitous transport enzyme sodium potassium pump, thus regulating intracellular Na⁺ concentration in every living cell, they are also an essential tool for research into the sodium potassium pump structure and function. Exogenous CTS have been clearly demonstrated to affect cardiovascular system through modulation of vagal tone, cardiac contraction (via ionic changes) and altered natriuresis. Reports of a number of endogenous CTS, since the 1980s, have intensified research into their physiologic and pathophysiologic roles and opened up novel therapeutic targets. Substantive evidence pointing to the role of endogenous ouabain and marinobufagenin, the two most prominent CTS, in development of cardiovascular disease has accumulated. Nevertheless, their presence, structure, biosynthesis pathways and even mechanism of action remain unclear or controversial. In this review the current state-of-the-art, the controversies and the remaining questions surrounding the role of endogenous cardiotonic steroids in health and disease are discussed.     

A Structural View on the Functional Importance of the Sugar Moiety and Steroid Hydroxyls of Cardiotonic Steroids in Binding to Na,K-ATPase

The Na,K-ATPase is specifically inhibited by cardiotonic steroids (CTSs) like digoxin and is of significant therapeutic value in the treatment of congestive heart failure and arrhythmia. Recently, new interest has arisen in developing Na,K-ATPase inhibitors as anticancer agents. In the present study, we compare the potency and rate of inhibition as well as the reactivation of enzyme activity following inhibition by various cardiac glycosides and their aglycones at different pH values using shark Na,K-ATPase stabilized in the E2MgP_i or in the E2BeF_x conformations. The effects of the number and nature of various sugar residues as well as changes in the positions of hydroxyl groups on the β-side of the steroid core of cardiotonic steroids were investigated by comparing various cardiac glycoside compounds like ouabain, digoxin, digitoxin, and gitoxin with their aglycones. The results confirm our previous hypothesis that CTS binds primarily to the E2-P ground state through an extracellular access channel and that binding of extracellular Na⁺ ions to K⁺ binding sites relieved the CTS inhibition. This reactivation depended on the presence or absence of the sugar moiety on the CTS, and a single sugar is enough to impede reactivation. Finally, increasing the number of hydroxyl groups of the steroid was sterically unfavorable and was found to decrease the inhibitory potency and to confer high pH sensitivity, depending on their position on the steroid β-face. The results are discussed with reference to the recent crystal structures of Na,K-ATPase in the unbound and ouabain-bound states.     

Two classes of ouabain receptors in chick ventricular cardiac cells and their relation to (Na+,K+)-ATPase inhibition, intracellular Na+ accumulation, Ca2+ influx, and cardiotonic effect

The biochemical and pharmacological properties of the (Na+,K+)-ATPase have been studied at different stages of chick embryonic heart development in ovo and under cell culture conditions. The results show the existence of two families of ouabain binding sites: a low affinity binding site with a dissociation constant (Kd) of 2-6 microM for the ouabain-receptor complex and a high affinity binding site with a Kd of 26-48 nM. Levels of high affinity sites gradually decrease during cardiac ontogenesis to reach a plateau near 14 days of development. Conversely the number of low affinity binding sites is essentially invariant between 5 days and hatching. Cultured cardiac cells display the same binding characteristics as those found in intact ventricles. Inhibition of 86Rb+ uptake in cultured cardiac cells and an increase in intracellular Na+ concentration, due to (Na+,K+)-ATPase blockade, occur in a ouabain concentration range corresponding to the saturation of the low affinity ouabain site. Ouabain-stimulated 45Ca2+ uptake increases in parallel with the increase in the intracellular Na+ concentration. It is suppressed in Na+-free medium or when Na+ is replaced by Li+ suggesting that the increase is due to the indirect activation of the Na+/Ca2+ exchange system in the plasma membrane. Dose-response curves for the inotropic effects of ouabain on papillary muscle and on ventricular cells in culture indicate that the development of the cardiotonic properties is parallel to the saturation of the low affinity binding site for ouabain. Therefore, inhibition of the cardiac (Na+,K+)-ATPase corresponding to low affinity ouabain binding sites seems to be responsible for both the cardiotonic and cardiotoxic effects of the drug.