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Aims: The heterozygous phospholamban (PLN) mutation R14del (PLN R14del +/- ) is associated with a severe arrhythmogenic cardiomyopathy (ACM) developing in the adult. "Superinhibition" of SERCA2a by PLN R14del is widely assumed to underlie the pathogenesis, but alternative mechanisms such abnormal energy metabolism have also been reported. This work aims to (1) to evaluate Ca 2+ dynamics and energy metabolism in a transgenic (TG) mouse model of the mutation prior to cardiomyopathy development; (2) to test whether they are causally connected., Methods: Ca 2+ dynamics, energy metabolism parameters, reporters of mitochondrial integrity, energy, and redox homeostasis were measured in ventricular myocytes of 8-12 weeks-old, phenotypically silent, TG mice. Mutation effects were compared to pharmacological PLN antagonism and analyzed during modulation of sarcoplasmic reticulum (SR) and cytosolic Ca 2+ compartments. Transcripts and proteins of relevant signaling pathways were evaluated., Results: The mutation was characterized by hyperdynamic Ca 2+ handling, compatible with a loss of SERCA2a inhibition by PLN. All components of energy metabolism were depressed; myocyte energy charge was preserved under quiescence but reduced during stimulation. Cytosolic Ca 2+ buffering or SERCA2a blockade reduced O 2 consumption with larger effect in the mutant. Signaling changes suggest cellular adaptation to perturbed Ca 2+ dynamics and response to stress., Conclusions: (1) PLN R14del +/- loses its ability to inhibit SERCA2a, which argues against SERCA2a superinhibition as a pathogenetic mechanism; (2) depressed energy metabolism, its enhanced dependency on Ca 2+ and activation of signaling responses point to an early involvement of metabolic stress in the pathogenesis of this ACM model., (© 2024 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Background: The sarcoplasmic reticulum (SR) Ca 2+ ATPase (SERCA2a) depression substantially contributes to diastolic dysfunction in heart failure (HF), suggesting that SERCA2a stimulation may be a mechanism-based HF therapy. Istaroxime is a drug endowed with both a SERCA2a stimulatory activity and a Na + /K + pump inhibitory activity for acute HF treatment. Its main metabolite PST3093 shows a more favorable therapeutic profile as compared to the parent drug, but it is still unsuitable for chronic usage. Novel PST3093 derivatives have been recently developed for oral (chronic) HF treatment; compound 8 was selected among them and here characterized., Methods: Effects of compound 8 were evaluated in a context of SERCA2a depression, by using streptozotocin-treated rats, a well-known model of diastolic dysfunction. The impact of SERCA2a stimulation by compound 8 was assessed at the cellular level ad in vivo, following i.v. infusion (acute effects) or oral administration (chronic effects)., Results: As expected from SERCA2a stimulation, compound 8 induced SR Ca 2+ compartmentalization in STZ myocytes. In-vivo echocardiographic analysis during i.v. infusion and after repeated oral administration of compound 8, detected a significant improvement of diastolic function. Moreover, compound 8 did not affect electrical activity of healthy guinea-pig myocytes, in line with the absence of off-target effects. Finally, compound 8 was well tolerated in mice with no evidence of acute toxicity., Conclusions: The pharmacological evaluation of compound 8 indicates that it may be a safe and selective drug for a mechanism-based treatment of chronic HF by restoring SERCA2a activity., (© 2024. The Author(s).)

Heart failure (HF) therapeutic toolkit would strongly benefit from the availability of ino-lusitropic agents with a favorable pharmacodynamics and safety profile. Istaroxime is a promising agent, which combines Na + /K + pump inhibition with sarcoplasmic reticulum Ca 2+ ATPase (SERCA2a) stimulation; however, it has a very short half-life and extensive metabolism to a molecule named PST3093. The present work aims to investigate whether PST3093 still retains the pharmacodynamic and pharmacokinetic properties of its parent compound. We studied PST3093 for its effects on SERCA2a and Na + /K + ATPase activities, Ca 2+ dynamics in isolated myocytes, and hemodynamic effects in an in vivo rat model of diabetic [streptozotocin (STZ)-induced] cardiomyopathy. Istaroxime infusion in HF patients led to accumulation of PST3093 in the plasma; clearance was substantially slower for PST3093 than for istaroxime. In cardiac rat preparations, PST3093 did not inhibit the Na + /K + ATPase activity but retained SERCA2a stimulatory activity. In in vivo echocardiographic assessment, PST3093 improved overall cardiac performance and reversed most STZ-induced abnormalities. PST3093 intravenous toxicity was considerably lower than that of istaroxime, and it failed to significantly interact with 50 off-targets. Overall, PST3093 is a "selective" SERCA2a activator, the prototype of a novel pharmacodynamic category with a potential in the ino-lusitropic approach to HF with prevailing diastolic dysfunction. Its pharmacodynamics are peculiar, and its pharmacokinetics are suitable to prolong the cardiac beneficial effect of istaroxime infusion. SIGNIFICANCE STATEMENT: Heart failure (HF) treatment would benefit from the availability of ino-lusitropic agents with favourable profiles. PST3093 is the main metabolite of istaroxime, a promising agent combining Na + /K + pump inhibition and sarcoplasmic reticulum Ca 2+ ATPase2a (SERCA2a) stimulation. PST3093 shows a longer half-life in human circulation compared to istaroxime, selectively activates SERCA2a, and improves cardiac performance in a model of diabetic cardiomyopathy. Overall, PST3093 as a selective SERCA2a activator can be considered the prototype of a novel pharmacodynamic category for HF treatment., (Copyright © 2022 by The Author(s).)

The stimulation of sarcoplasmic reticulum calcium ATPase SERCA2a emerged as a novel therapeutic strategy to efficiently improve overall cardiac function in heart failure (HF) with reduced arrhythmogenic risk. Istaroxime is a clinical-phase IIb compound with a double mechanism of action, Na + /K + ATPase inhibition and SERCA2a stimulation. Starting from the observation that istaroxime metabolite PST3093 does not inhibit Na + /K + ATPase while stimulates SERCA2a, we synthesized a series of bioisosteric PST3093 analogues devoid of Na + /K + ATPase inhibitory activity. Most of them retained SERCA2a stimulatory action with nanomolar potency in cardiac preparations from healthy guinea pigs and streptozotocin (STZ)-treated rats. One compound was further characterized in isolated cardiomyocytes, confirming SERCA2a stimulation and in vivo showing a safety profile and improvement of cardiac performance following acute infusion in STZ rats. We identified a new class of selective SERCA2a activators as first-in-class drug candidates for HF treatment.

Aims: Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the dual properties to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). This project aims to characterize istaroxime effects at a concentration (100 nmol/L) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in an animal model of mild diabetes., Methods and Results: Streptozotocin (STZ) treated diabetic rats were studied at 9  weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed (i) marked DD not associated to cardiac fibrosis, (ii) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, (iii) reduced LV SERCA2 protein level and activity and (iv) slower SR Ca2+ uptake rate, (v) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, (vi) increased diastolic Ca2+, and (vii) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nmol/L) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent., Conclusions: SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

We compared a standard antihypertensive losartan treatment with a pharmacogenomics-guided rostafuroxin treatment in never-treated Caucasian and Chinese patients with primary hypertension. Rostafuroxin is a digitoxigenin derivative that selectively disrupts the binding to the cSrc-SH2 domain of mutant α-adducin and of the ouabain-activated Na-K pump at 10 -11  M. Of 902 patients screened, 172 were enrolled in Italy and 107 in Taiwan. After stratification for country and genetic background, patients were randomized to rostafuroxin or losartan, being the difference in the fall in office systolic blood pressure (OSBP) after 2-month treatment the primary endpoint. Three pharmacogenomic profiles (P) were examined, considering: P1, adding to the gene variants included in the subsequent P2, the variants detected by post-hoc analysis of a previous trial; P2, variants of genes encoding enzymes for endogenous ouabain (EO) synthesis (LSS and HSD3B1), EO transport (MDR1/ABCB1), adducin (ADD1 and ADD3); P3, variants of the LSS gene only. In Caucasians, the group differences (rostafuroxin 50 μg minus losartan 50 mg in OSBP mmHg) were significant both in P2 adjusted for genetic heterogeneity (P2a) and P3 LSS rs2254524 AA [9.8 (0.6-19.0), P = 0.038 and 13.4 (25.4-2.5), P = 0.031, respectively]. In human H295R cells transfected with LSS A and LSS C variants, the EO production was greater in the former (P = 0.038); this difference was abolished by rostafuroxin at 10 -11  M. Chinese patients had a similar drop in OSBP to Caucasians with losartan but no change in OSBP with rostafuroxin. These results show that genetics may guide drug treatment for primary hypertension in Caucasians.

Background: Combined treatment with anthracyclines (e.g., doxorubicin; Dox) and trastuzumab (Trz), a humanized anti-human epidermal growth factor receptor 2 (HER2; ErbB2) antibody, in patients with HER2-positive cancer is limited by cardiotoxicity, as manifested by contractile dysfunction and arrhythmia. The respective roles of the two agents in the cardiotoxicity of the combined therapy are incompletely understood., Objective: To assess cardiac performance, T-tubule organization, electrophysiological changes and intracellular Ca 2+ handling in cardiac myocytes (CMs) using an in vivo rat model of Dox/Trz-related cardiotoxicity., Methods and Results: Adult rats received 6 doses of either Dox or Trz, or the two agents sequentially. Dox-mediated left ventricular (LV) dysfunction was aggravated by Trz administration. Dox treatment, but not Trz, induced T-tubule disarray. Moreover, Dox, but not Trz monotherapy, induced prolonged action potential duration (APD), increased incidence of delayed afterdepolarizations (DADs) and beat-to-beat variability of repolarization (BVR), and slower Ca 2+ transient decay. Although APD, DADs, BVR and Ca 2+ transient decay recovered over time after the cessation of Dox treatment, subsequent Trz administration exacerbated these abnormalities. Trz, but not Dox, reduced Ca 2+ transient amplitude and SR Ca 2+ content, although only Dox treatment was associated with SERCA downregulation. Finally, Dox treatment increased Ca 2+ spark frequency, resting Ca 2+ waves, sarcoplasmic reticulum (SR) Ca 2+ leak, and long-lasting Ca 2+ release events (so-called Ca 2+ "embers"), partially reproduced by Trz treatment., Conclusion: These results suggest that in vivo Dox but not Trz administration causes T-tubule disarray and pronounced changes in electrical activity of CMs. While adaptive changes may account for normal AP shape and reduced DADs late after Dox administration, subsequent Trz administration interferes with such adaptive changes. Intracellular Ca 2+ handling was differently affected by Dox and Trz treatment, leading to SR instability in both cases. These findings illustrate the specific roles of Dox and Trz, and their interactions in cardiotoxicity and arrhythmogenicity., Competing Interests: MF was employed by company Windtree Therapeutics Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Altomare, Lodrini, Milano, Biemmi, Lazzarini, Bolis, Pernigoni, Torre, Arici, Ferrandi, Barile, Rocchetti and Vassalli.)

Warm renal ischemia performed during partial nephrectomy has been found to be associated with kidney disease. Since endogenous ouabain (EO) is a neuro-endocrine hormone involved in renal damage, we evaluated the role of EO in renal ischemia-reperfusion injury (IRI). We measured plasma and renal EO variations and markers of glomerular and tubular damage (nephrin, KIM-1, Kidney-Injury-Molecule-1, α1 Na-K ATPase) and the protective effect of the ouabain inhibitor, rostafuroxin. We studied five groups of rats: (1) normal; (2) infused for eight weeks with ouabain (30 µg/kg/day, OHR) or (3) saline; (4) ouabain; or (5) saline-infused rats orally treated with 100 µg/kg/day rostafuroxin for four weeks. In group 1, 2-3 h after IRI, EO increased in ischemic kidneys while decreased in plasma. Nephrin progressively decreased and KIM-1 mRNA increased starting from 24 h. Ouabain infusion (group 2) increased blood pressure (from 111.7 to 153.4 mmHg) and ouabain levels in plasma and kidneys. In OHR ischemic kidneys at 120 h from IRI, nephrin, and KIM-1 changes were greater than those detected in the controls infused with saline (group 3). All these changes were blunted by rostafuroxin treatment (groups 4 and 5). These findings support the role of EO in IRI and suggest that rostafuroxin pre-treatment of patients before partial nephrectomy with warm ischemia may reduce IRI, particularly in those with high EO., Competing Interests: The authors declare no conflict of interest.

During the past 20 years, the studies on genetics or pharmacogenomics of primary hypertension provided interesting results supporting the role of genetics, but no actionable finding ready to be translated into personalized medicine. Two types of approaches have been applied: a "hypothesis-driven" approach on the candidate genes, coding for proteins involved in the biochemical machinery underlying the regulation of BP, and an "unbiased hypothesis-free" approach with GWAS, based on the randomness principles of frequentist statistics. During the past 10-15 years, the application of the latter has overtaken the application of the former leading to an enlargement of the number of previously unknown candidate loci or genes but without any actionable result for the therapy of hypertension. In the present review, we summarize the results of our hypothesis-driven approach based on studies carried out in rats with genetic hypertension and in humans with essential hypertension at the pre-hypertensive and early hypertensive stages. These studies led to the identification of mutant adducin and endogenous ouabain as candidate genetic-molecular mechanisms in both species. Rostafuroxin has been developed for its ability to selectively correct Na(+) pump abnormalities sustained by the two abovementioned mechanisms and to selectively reduce BP in rats and in humans carrying the gene variants underlying the mutant adducin and endogenous ouabain (EO) effects. A clinical trial is ongoing to substantiate these findings. Future studies should apply both the candidate gene and GWAS approaches to fully exploit the potential of genetics in optimizing the personalized therapy.

Recent evidence of beneficial effects of ranolazine (RAN) in type II diabetes motivates interest in the role of the late sodium current (INaL) in glucose-stimulated insulin secretion. In the present work, we characterize INaL and its function in rat INS-1E cells and human islets cells. INaL was identified as steady-state current blocked by 10 μM RAN (IRAN) or 0.5 μM tetrodotoxin (TTX) (ITTX). Veratridine (VERA, 40 μM) was used as INaL enhancer. Baseline INaL was similar between INS-1E and human islet cells. In INS-1E cells, activated by glucose or tolbutamide, TTX or RAN hyperpolarized membrane potential (V m). VERA-induced depolarization was countered by TTX or RAN. ITTX and IRAN reversal potentials were negative to Na(+) equilibrium one, but they approached it after Na(+) substitution with Li(+) or when K(+) channels were blocked. This revealed INaL coupling with Na(+)-activated K(+) current (IKNa); expression of IKNa channels (Slick/Slack) was confirmed by transcript analysis and Western blot. RAN or TTX blunted cytosolic Ca(2+) response to depolarization. Long-term incubation in high (33 mM) glucose (CHG) constitutively enhanced INaL. VERA immediately increased glucose-stimulated insulin secretion. CHG increased glucose-independent secretion instead and abolished the secretory response to glucose. RAN or TTX countered VERA- and CHG-induced changes in insulin secretion. Our study demonstrated that (1) INaL was expressed in insulin-secreting cells and coupled to IKNa; INaL affected cytosolic Ca(2+) but, unless enhanced, barely contributed to glucose-stimulated insulin secretion (GSIS); and (2) sustained hyperglycemic stress enhanced INaL, which contributed to the attending increase of glucose-independent insulin "leak" and GSIS impairment.

Glomerulopathies are important causes of morbidity and mortality. Selective therapies that address the underlying mechanisms are still lacking. Recently, two mechanisms, mutant β-adducin and ouabain, have been found to be involved in glomerular podocytopathies and proteinuria through nephrin downregulation. The main purpose of the present study was to investigate whether rostafuroxin, a novel antihypertensive agent developed as a selective inhibitor of Src-SH2 interaction with mutant adducin- and ouabain-activated Na,K-ATPase, may protect podocytes from adducin- and ouabain-induced effects, thus representing a novel pharmacologic approach for the therapy of podocytopathies and proteinuria caused by the aforementioned mechanisms. To study the effect of rostafuroxin on podocyte protein changes and proteinuria, mice carrying mutant β-adducin and ouabain hypertensive rats were orally treated with 100 μg/kg per day rostafuroxin. Primary podocytes from congenic rats carrying mutant α-adducin or β-adducin (NB) from Milan hypertensive rats and normal rat podocytes incubated with 10(-9) M ouabain were cultured with 10(-9) M rostafuroxin. The results indicated that mutant β-adducin and ouabain caused podocyte nephrin loss and proteinuria in animal models. These alterations were reproduced in primary podocytes from NB rats and normal rats incubated with ouabain. Treatment of animals, or incubation of cultured podocytes with rostafuroxin, reverted mutant β-adducin- and ouabain-induced effects on nephrin protein expression and proteinuria. We conclude that rostafuroxin prevented podocyte lesions and proteinuria due to mutant β-adducin and ouabain in animal models. This suggests a potential therapeutic effect of rostafuroxin in patients with glomerular disease progression associated with these two mechanisms., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Hypertension is a prevalent disorder in the world representing one of the major risk factors for heart attack and stroke. These risks are increased in salt sensitive individuals. Hypertension and salt sensitivity are complex phenotypes whose pathophysiology remains poorly understood and, remarkably, salt sensitivity is still laborious to diagnose. Here we present a urinary proteomic study specifically designed to identify urinary proteins relevant for the pathogenesis of hypertension and salt sensitivity. Despite previous studies that underlined the association of UMOD gene variants with hypertension, this work provides novel evidence showing different uromodulin protein level in the urine of hypertensive patients compared to healthy individuals. Notably, we also show that patients with higher level of uromodulin are homozygous for UMOD risk variant and display a decreased level of salt excretion, highlighting the essential role of UMOD in the regulation of salt reabsorption in hypertension. Additionally, we found that urinary nephrin 1, a marker of glomerular slit diaphragm, may predict a salt sensitive phenotype and positively correlate with increased albuminuria associated with this type of hypertension.

Mutant α-adducin and endogenous ouabain levels exert a causal role in hypertension by affecting renal Na-K ATPase. In addition, mutant β-adducin is involved in glomerular damage through nephrin down-regulation. Recently, the salt-inducible kinase 1 (SIK1) has been shown to exert a permissive role on mutant α-adducin effects on renal Na-K ATPase activity involved in blood pressure (BP) regulation and a SIK1 rs3746951 polymorphism has been associated with changes in vascular Na-K ATPase activity and BP. Here, we addressed the role of SIK1 on nephrin and glomerular functional modifications induced by mutant β-adducin and ouabain, by using congenic substrains of the Milan rats expressing either mutant α- or β-adducin, alone or in combination, ouabain hypertensive rats (OHR) and hypertensive patients. SIK1 co-localized and co-immunoprecipitated with nephrin from glomerular podocytes and associated with caveolar nephrin signaling. In cultured podocytes, nephrin-gene silencing decreased SIK1 expression. In mutant β-adducin congenic rats and in OHR, the podocyte damage was associated with decreased nephrin and SIK1 expression. Conversely, when the effects of β-adducin on podocytes were blocked by the presence of mutant α-adducin, nephrin and SIK1 expressions were restored. Ouabain effects were also reproduced in cultured podocytes. In hypertensive patients, nephrinuria, but not albuminuria, was higher in carriers of mutant SIK1 rs3746951 than in wild-type, implying a more direct effect of SIK1 on glomerular damage. These results demonstrate that, through nephrin, SIK1 is involved in the glomerular effects of mutant adducin and ouabain and a direct effect of SIK1 is also likely to occur in humans., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Cardiac left ventricle hypertrophy (LVH) constitutes a major risk factor for heart failure. Although LVH is most commonly caused by chronic elevation in arterial blood pressure, reduction of blood pressure to normal levels does not always result in regression of LVH, suggesting that additional factors contribute to the development of this pathology. We tested whether genetic preconditions associated with the imbalance in sodium homeostasis could trigger the development of LVH without concomitant increases in blood pressure. The results showed that the presence of a hypertensive variant of α-adducin gene in Milan rats (before they become hypertensive) resulted in elevated expression of genes associated with LVH, and of salt-inducible kinase 2 (SIK2) in the left ventricle (LV). Moreover, the mRNA expression levels of SIK2, α-adducin, and several markers of cardiac hypertrophy were positively correlated in tissue biopsies obtained from human hearts. In addition, we found in cardiac myocytes that α-adducin regulates the expression of SIK2, which in turn mediates the effects of adducin on hypertrophy markers gene activation. Furthermore, evidence that SIK2 is critical for the development of LVH in response to chronic high salt diet (HS) was obtained in mice with ablation of the sik2 gene. Increases in the expression of genes associated with LVH, as well as increases in LV wall thickness upon HS, occurred only in sik2+/+ but not in sik2-/- mice. Thus LVH triggered by HS or the presence of a genetic variant of α-adducin requires SIK2 and is independent of elevated blood pressure. Inhibitors of SIK2 may constitute part of a novel therapeutic regimen aimed at prevention/regression of LVH.

Defective pressure-natriuresis related to abnormalities in the natriuretic response has been associated with hypertension development. A major signaling pathway mediating pressure natriuresis involves the cGMP-dependent protein kinase 1 (PRKG1) that, once activated by Src kinase, inhibits renal Na(+) reabsorption via a direct action on basolateral Na-K ATPase and luminal Na-H exchanger type 3, as shown in renal tubuli of animals. Because a clear implication of PRKG1 in humans is still lacking, here we addressed whether PRKG1 polymorphisms affect pressure-natriuresis in patients. Naive hypertensive patients (n = 574), genotyped for PRKG1 rs1904694, rs7897633, and rs7905063 single nucleotide polymorphisms (SNPs), underwent an acute Na(+) loading, and the slope of the pressure-natriuresis relationship between blood pressure and Na(+) excretion was calculated. The underlying molecular mechanism was investigated by immunoblotting protein quantifications in human kidneys. The results demonstrate that the PRKG1 risk haplotype GAT (rs1904694, rs7897633, rs7905063, respectively) associates with a rightward shift of the pressure-natriuresis curve (0.017 ± 0.004 μEq/mm Hg per minute) compared with the ACC (0.0013 ± 0.003 μEq/mm Hg per minute; P = 0.001). In human kidneys, a positive correlation of protein expression levels between PRKG1 and Src (r = 0.83; P<0.001) or α1 Na-K ATPase (r = 0.557; P<0.01) and between α1 Na-K ATPase and Na-H exchanger type 3 (r = 0.584; P<0.01) or Src (r = 0.691; P<0.001) was observed in patients carrying PRKG1 risk GAT (n = 23) but not ACC (n = 14) variants. A functional signaling complex among PRKG1, α1 Na-K ATPase, and Src was shown by immunoprecipitation from human renal caveolae. These findings indicate that PRKG1 risk alleles associate with salt-sensitivity related to a loss of the inhibitory control of renal Na(+) reabsorption, suggestive of a blunt pressure-natriuresis response.

Background and Purpose: Calcium handling is known to be deranged in heart failure. Interventions aimed at improving cell Ca(2) (+) cycling may represent a promising approach to heart failure therapy. Istaroxime is a new luso-inotropic compound that stimulates cardiac contractility and relaxation in healthy and failing animal models and in patients with acute heart failure (AHF) syndrome. Istaroxime is a Na-K ATPase inhibitor with the unique property of increasing sarcoplasmic reticulum (SR) SERCA2a activity as shown in heart microsomes from humans and guinea pigs. The present study addressed the molecular mechanism by which istaroxime increases SERCA2a activity., Experimental Approach: To study the effect of istaroxime on SERCA2a-phospholamban (PLB) complex, we applied different methodologies in native dog healthy and failing heart preparations and heterologous canine SERCA2a/PLB co-expressed in Spodoptera frugiperda (Sf21) insect cells., Key Results: We showed that istaroxime enhances SERCA2a activity, Ca(2) (+) uptake and the Ca(2) (+) -dependent charge movements into dog healthy and failing cardiac SR vesicles. Although not directly demonstrated, the most probable explanation of these activities is the displacement of PLB from SERCA2a.E2 conformation, independently from cAMP/PKA. We propose that this displacement may favour the SERCA2a conformational transition from E2 to E1, thus resulting in the acceleration of Ca(2) (+) cycling., Conclusions and Implications: Istaroxime represents the first example of a small molecule that exerts a luso-inotropic effect in the failing human heart through the stimulation of SERCA2a ATPase activity and the enhancement of Ca(2) (+) uptake into the SR by relieving the PLB inhibitory effect on SERCA2a in a cAMP/PKA independent way., (© 2013 The British Pharmacological Society.)

Objectives: Acute kidney injury is a frequent complication of cardiac surgery and increases morbidity and mortality. As preoperative biomarkers predicting the development of acute kidney injury are not available, we have tested the hypothesis that preoperative plasma levels of endogenous ouabain may function as this type of biomarker., Rationale and Design: Endogenous ouabain is an adrenal stress hormone associated with adverse cardiovascular outcomes. Its involvement in acute kidney injury is unknown. With studies in patients and animal settings, including isolated podocytes, we tested the above mentioned hypothesis., Patients: Preoperative endogenous ouabain was measured in 407 patients admitted for elective cardiac surgery and in a validation population of 219 other patients. We also studied the effect of prolonged elevations of circulating exogenous ouabain on renal parameters in rats and the influence of ouabain on podocyte proteins both "in vivo" and "in vitro.", Main Results: In the first group of patients, acute kidney injury (2.8%, 8.3%, 20.3%, p < 0.001) and ICU stay (1.4±0.38, 1.7±0.41, 2.4±0.59 days, p = 0.014) increased with each incremental preoperative endogenous ouabain tertile. In a linear regression analysis, the circulating endogenous ouabain value before surgery was the strongest predictor of acute kidney injury. In the validation cohort, acute kidney injury (0%, 5.9%, 8.2%, p < 0.0001) and ICU stay (1.2±0.09, 1.4±0.23, 2.2±0.77 days, p = 0.003) increased with the preoperative endogenous ouabain tertile. Values for preoperative endogenous ouabain significantly improved (area under curve: 0.85) risk prediction over the clinical score alone as measured by integrate discrimination improvement and net reclassification improvement. Finally, in the rat model, elevated circulating ouabain reduced creatinine clearance (-18%, p < 0.05), increased urinary protein excretion (+ 54%, p < 0.05), and reduced expression of podocyte nephrin (-29%, p < 0.01). This last finding was replicated ex vivo by incubating podocyte primary cell cultures with low-dose ouabain., Conclusions: Preoperative plasma endogenous ouabain levels are powerful biomarkers of acute kidney injury and postoperative complications and may be a direct cause of podocyte damage.

The Milan hypertensive strain of rats (MHS) develops hypertension as a consequence of the increased tubular Na(+) reabsorption sustained by enhanced expression and activity of the renal tubular Na-K-ATPase. To verify whether the Na-K-2Cl cotransporter (NKCC2) is involved in the maintenance of hypertension in MHS rats, we have analysed the phosphorylation state and the activation of NKCC2 in Milan rats. Western blotting and immunofluorescence experiments were performed using specific antibodies against the regulatory phospho-threonines in the NKCC2 N terminus (R5 antibody). The phosphorylation levels of NKCC2 were significantly increased in the kidney of MHS rats. Moreover, the administration of furosemide in vivo decreased the blood pressure and increased the urine output and natriuresis in MHS rats demonstrating the actual involvement of NKCC2 activity in the pathogenesis of hypertension in this strain of rats. The up-regulation of NKCC2 activity is most probably mediated by a STE20/SPS1-related proline/alanine-rich kinase (SPAK) phosphorylation at serine-325 since it was significantly increased in MHS rats. Interestingly, aldosterone treatment caused an increase in NKCC2 phosphorylation in NKCC2-expressing MDCK cells. In conclusion, we demonstrated an increase in the activity of NKCC2 along the TAL that significantly contributes to the increase in systemic blood pressure in MHS rats. The elevated plasma levels of aldosterone, found in MHS rats, may influence Na(+) balance through a SPAK-dependent regulation of NKCC2 accounting for the maintenance of the hypertensive state in MHS rats.

Heart failure (HF) patients are a medically complex and heterogeneous population with multiple cardiac and non-cardiac comorbidities. Although there are a multitude of etiologic substrates and initiating and amplifying mechanisms contributing to disease progression, these pathophysiologic processes ultimately all lead to impaired myocardial function. The myocardium must both pump oxygenated, nutrient-rich blood throughout the body (systolic function) and receive deoxygenated, nutrient-poor blood returning from the periphery (diastolic function). At the molecular level, it is well-established that Ca2+ plays a central role in excitation-contracting coupling with action potentials stimulating the opening of L-type Ca2+ in the plasma membrane and ryanodine receptor 2 (RyR2) in the sarcoplasmic reticulum (SR) membrane during systole and the Na-Ca2+ exchanger and SERCA2a returning Ca2+ to the extracellular space and SR, respectively, during diastole. However, there is increasing recognition that impaired Ca2+ cycling may contribute to myocardial dysfunction. Preclinical studies and clinical trials indicate that combining SERCA2a activation and Na-K ATPase inhibition may increase contractility (inotropy) and facilitate active relaxation (lusitropy), improving both systolic and diastolic functions. Istaroxime, a novel luso-inotrope that activates SERCA2a and inhibits the Na-K ATPase, is currently in phase II clinical development and has been shown to improve systolic and diastolic functions and central hemodynamics, increase systolic but not diastolic blood pressure, and decrease substantially heart rate. Irrespective of its clinical utility, the development of istaroxime has evolved our understanding of the clinical importance of inhibiting the Na-K ATPase in order to obtain a clinically significant effect from SERCA2a activation in the setting of myocardial failure.

Background: Damage to renal artery myogenic tone is universally associated with progressive kidney damage. Recently, we have observed that mutations in the beta adducin subunit are associated with proteinuria in the Milan rat. Because of the role of adducin as a component of the cytoskeleton we hypothesized that this mutation may be associated with changes in myogenic tone., Methods and Results: Congenic rats were generated with beta adducin subunit mutation (NB rats) and compared with a previously studied rat model with alpha adducin subunit mutation (NAs rats). Blood pressure and urinary protein excretion were studied at two time points: 6 weeks and 4 months of age, and at these time points, small renal, middle cerebral and skeletal (cremaster) arteries were isolated and studied using pressure myography. Agonist-induced vasoconstriction was not different between the two groups at any age. However, myogenic tone in renal arteries was significantly damaged in the NB rat compared to its NAs counterpart and this was associated with a decrease in vascular distensibility. There was a smaller reduction in myogenic tone in the middle cerebral arteries from the NB rat, whereas in the skeletal arteries there was no difference between the two strains. In the NB rat, this tissue-specific damage to myogenic tone was associated with progressive proteinuria despite lower blood pressure than the NAs rat., Conclusions: Mutations in the beta subunit of the adducin protein result in damage to renal artery myogenic tone and this is associated with renal damage as manifest by proteinuria.

Essential hypertension is a complex, multifactorial disease associated with a high cardiovascular risk and whose genetic-molecular basis is heterogeneous and largely unknown. Although multiple antihypertensive therapies are available, the large individual variability in drug response results in only a modest reduction of the cardiovascular risk and unsatisfactory control of blood pressure in the hypertensive population as a whole. Two mechanisms, among others, are associated with essential hypertension and related organ damage: mutant α-adducin variants and high concentrations of endogenous ouabain. An antihypertensive agent, rostafuroxin, selectively inhibits these mechanisms in rodents. We investigated the molecular and functional effects of mutant α-adducin, ouabain, and rostafuroxin in hypertensive rats, human cells, and cell-free systems and demonstrated that both mutant α-adducin variants and the ouabain-Na,K-ATPase (Na(+)- and K(+)-dependent adenosine triphosphatase) complex can interact with the Src-SH2 (Src homology 2) domain, increasing Src activity and the Src-dependent Na,K-ATPase phosphorylation and activity. Wild-type α-adducin or Na,K-ATPase in the absence of ouabain showed no interaction with the Src-SH2 domain. Rostafuroxin disrupted the interactions between the Src-SH2 domain and mutant α-adducin or the ouabain-Na,K-ATPase complex and blunted Src activation and Na,K-ATPase phosphorylation, resulting in blood pressure normalization in the hypertensive rats. We have also shown the translatability of these data to humans in a pharmacogenomic clinical trial, as described in the companion paper.

Twenty years of genetic studies have not contributed to improvement in the clinical management of primary arterial hypertension. Genetic heterogeneity, epistatic-environmental-biological interactions, and the pathophysiological complexity of hypertension have hampered the clinical application of genetic findings. In the companion article, we furnished data from rodents and human cells demonstrating two hypertension-triggering mechanisms--variants of adducin and elevated concentrations of endogenous ouabain (within a particular range)--and their selective inhibition by the drug rostafuroxin. Here, we have investigated the relationship between variants of genes encoding enzymes for ouabain synthesis [LSS (lanosterol synthase) and HSD3B1 (hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 1)], ouabain transport {MDR1/ABCB1 [ATP-binding cassette, sub-family B (MDR/TAP), member 1]}, and adducin activity [ADD1 (adducin 1) and ADD3], and the responses to antihypertensive medications. We determined the presence of these variants in newly recruited, never-treated patients. The genetic profile defined by these variants predicted the antihypertensive effect of rostafuroxin (a mean placebo-corrected systolic blood pressure fall of 14 millimeters of mercury) but not that of losartan or hydrochlorothiazide. The magnitude of the rostafuroxin antihypertensive effect was twice that of antihypertensive drugs recently tested in phase 2 clinical trials. One-quarter of patients with primary hypertension display these variants of adducin or concentrations of endogenous ouabain and would be expected to respond to therapy with rostafuroxin. Because the mechanisms that are inhibited by rostafuroxin also underlie hypertension-related organ damage, this drug may also reduce the cardiovascular risk in these patients beyond that expected by the reduction in systolic blood pressure alone.

Adducins are cytoskeletal actin-binding proteins (alpha, beta, gamma) that function as heterodimers and heterotetramers and are encoded by distinct genes. Experimental and clinical evidence implicates alpha- and beta-adducin variants in hypertension and renal dysfunction. Here, we have addressed the role of alpha- and beta-adducin on glomerular function and disease using beta-adducin null mice, congenic substrains for alpha- and beta-adducin from the Milan hypertensive (MHS) and Milan normotensive (MNS) rats and patients with IgA nephropathy. Targeted deletion of beta-adducin in mice reduced urinary protein excretion, preceded by an increase of podocyte protein expression (phospho-nephrin, synaptopodin, alpha-actinin, ZO-1, Fyn). The introgression of polymorphic MHS beta-adducin locus into MNS (Add2, 529R) rats was associated with an early reduction of podocyte protein expression (nephrin, synaptopodin, alpha-actinin, ZO-1, podocin, Fyn), followed by severe glomerular and interstitial lesions and increased urinary protein excretion. These alterations were markedly attenuated when the polymorphic MHS alpha-adducin locus was also present (Add1, 316Y). In patients with IgA nephropathy, the rate of decline of renal function over time was associated to polymorphic beta-adducin (ADD2, 1797T, rs4984) with a significant interaction with alpha-adducin (ADD1, 460W, rs4961). These findings suggest that adducin genetic variants participate in the development of glomerular lesions by modulating the expression of specific podocyte proteins.

An endogenous ouabain has been isolated and conclusively identified from several mammalian tissues, including human plasma, by a number of independent laboratories. Substantial evidence from independent laboratories in several continents is consistent with an adrenal source for most if not all of the circulating endogenous ouabain. Accumulating evidence suggests that circulating levels of endogenous ouabain in humans are modulated by dietary salt and chronic volume status. Endogenous ouabain is linked significantly with vascular function in hypertension and likely impacts the pathogenesis of heart and renal failure. Moreover, the molecular mechanism of endogenous ouabain-linked hypertension involves the sodium pump/sodium-calcium exchange duet. The outstanding analytical issues include the elucidation of distal events in the biosynthetic pathway for endogenous ouabain and identification of molecular mechanisms that regulate its secretion and clearance.

Rats of the Milan hypertensive strain (MHS) are resistant to both hypertensive and diabetic renal disease. Genetically determined hypertrophy of intrarenal arteries has been suggested as the putative mechanism preventing transmission of systemic hypertension to the glomerular microcirculation or diabetes-induced loss of autoregulation, which lead to glomerular hypertension and consequent podocyte injury and proteinuria. This study aimed to investigate glomerular barrier function and structure in ageing and diabetic MHS rats under basal conditions and after injection of 2.5 g of bovine serum albumin (BSA) causing increased workload and possibly removing haemodynamic protection by inducing renal cortical vasodilatation. Genetically related rats of the Milan normotensive strain (MNS) served as a proteinuric counterpart. No change in renal function or structure was detected in diabetic MHS rats, whereas MNS rats developed diabetic nephropathy superimposed on that occurring spontaneously in this strain. Diabetic, but not non-diabetic, MHS rats showed significantly reduced synaptopodin and nephrin expression, though to a lesser extent than non-diabetic and diabetic MNS rats, together with unchanged podocyte number, density and structure and no proteinuria. Agrin expression was significantly altered in diabetic versus non-diabetic MHS animals, whereas collagen I was expressed only in diabetic MHS rats and collagen IV content did not change significantly between the two groups. Upon BSA injection, proteinuria increased markedly and abundant BSA was detected only in kidneys from diabetic MHS rats. BSA injection was associated with changes in intrarenal arteries suggesting vasodilatation, without any influx of inflammatory cells. These data indicate that while MNS rats show marked changes in the glomerular filtration barrier with either age or diabetes, glomerulosclerosis-resistant MHS rats develop only minor diabetes-induced podocyte (and extracellular matrix) alterations, which are not associated with proteinuria unless they are unmasked by an increased workload or removal of the haemodynamic protection.

Hypertension is an important public health problem affecting more than 50 million individuals in the USA alone. The most common form, essential hypertension, results from the complex interplay between genetic predisposition and environmental influences. Epidemiological, migration, intervention and genetic studies in humans and animals provide very strong evidence of a causal link between high salt intake and high blood pressure. One of the candidate genes for salt-sensitive hypertension is adducin. Adducin is a heterodimeric cytoskeleton protein, the three subunits of which are encoded by genes (ADD1, ADD2 and ADD3) that map to three different chromosomes. A long series of parallel studies in the Milan hypertensive rat strain model of hypertension and humans indicated that an altered adducin function might cause hypertension through enhanced constitutive tubular sodium reabsorption. An example of a prospective efficacy of pharmacogenetics and pharmacogenomics is the detection and impact of adducin polymorphisms on hypertension. In particular, the selective advantages of diuretics in preventing myocardial infarction and stroke over other antihypertensive therapies that produce a similar blood pressure reduction in carriers of the mutated adducin may support new strategies aimed at optimizing the use of new antihypertensive agents for the prevention of hypertension-associated organ damage.

Interventions involving calcium cycling may represent a promising approach to heart failure (HF) therapy because calcium handling is known to be deranged in human and experimental HF. Istaroxime is a sodium-potassium adenosine triphosphatase (ATPase) inhibitor with the unique property of increasing sarcoplasmic reticulum calcium ATPase (SERCA) isoform 2a (SERCA2a) activity. Because this was demonstrated in normal experimental models, we investigated whether istaroxime is able to improve global cardiac function and stimulate SERCA in failing hearts. In guinea pigs with 3-month aortic banding (AoB), echocardiographic results showed that istaroxime intravenous infusion (0.11 mg/kg per min) significantly increased both indices of contraction and relaxation (fractional shortening, +18+/-3.7%; aortic flow rate, +19+/-2.9%; peak myocardial systolic velocity, +36+/-7%; circumferential fiber shortening, +24+/-4.1%; peak atrial flow velocity, +69+/-8.6%; isovolumic relaxation time, +19+/-6.9%; and peak myocardial early diastolic velocity, +42+/-12%). In left ventricular sarcoplasmic reticulum microsomes from AoB animals, 100 nmol/L istaroxime normalized the depressed (-32%) SERCA2a maximum velocity and increased SERCA activity (+17%). In muscle strips from hearts from patients undergoing cardiac transplantation, istaroxime (0.1-1.0 micromol/L) increased (in a concentration-dependent manner) developed tension, the maximum and minimum first derivative of tension, and absolute velocity of contraction, while stimulating SERCA activity in sarcoplasmic reticulum microsomes at physiologic free calcium concentrations. In conclusion, istaroxime is presently the only available compound that stimulates SERCA2a activity and produces a luso-inotropic effect in HF.

Essential hypertension is a heterogeneous multifactorial syndrome associated with a high cardiovascular risk. A multiple choice of antihypertensive drugs is available; however, a high individual variability to the antihypertensive therapy is still responsible for a modest reduction of the CV risk and not satisfactory control of blood pressure levels. The success of future hypertension treatment will depend upon the understanding of the genetic molecular mechanisms operating in subsets of patients, and the ability of new drugs to specifically correct such alterations. Two mechanisms, among others, are involved in determining the abnormalities of tubular Na(+) reabsorption observed in essential hypertension: the polymorphism of the cytoskeletal protein alpha-adducin and the increased circulating levels of endogenous ouabain (EO). Both lead to increased activity and expression of the renal Na-K pump, the driving force for tubular Na transport. Morphological and functional cardiovascular alterations have also been associated with adducin and EO. Rostafuroxin is a new oral antihypertensive agent able to selectively antagonize adducin and EO hypertensive and molecular effects. It is endowed with high potency and efficacy in reducing blood pressure and preventing organ hypertrophy in animal models representative of both adducin and EO mechanisms. At molecular level, in the kidney, Rostafuroxin normalizes the enhanced activity of the Na-K pump induced by adducin mutation and antagonizes the EO triggering of the Src-EGFr-dependent signaling pathway leading to renal Na-K pump, and ERK Tyrosin phosphorylation and activation. In the vasculature, it normalizes the increased myogenic tone caused by ouabain. A very high safety ratio and an absence of interaction with other mechanisms involved in blood pressure regulation, together with initial evidence of high tolerability and efficacy in hypertensive patients, indicate Rostafuroxin as the first example of a new class of antihypertensive agents designed to antagonize adducin and EO-hypertensive mechanisms. Currently, a phase II multicenter European clinical trial is ongoing for providing the proof of concept that such a compound is effective in the subset of patients where these two mechanisms are at work.

Experimental and clinical evidence indicates that Endogenous Ouabain (EO) and Adducin polymorphism play a pathogenetic role in hypertension and related organ complications. These effects occur through a complex interaction of genetic molecular mechanisms regulating renal sodium reabsorption and vascular function. The activation of a Na-K ATPase-Src-EGFr-ERK signaling pathway within the restricted membrane subdomains of caveolae by Ouabain has been associated to hypertension and cardiac remodeling. Rostafuroxin (PST 2238) is a novel anti-hypertensive compound able to selectively antagonize EO/Ouabain and Adducin hypertensive effect and Ouabain-induced cardiac hypertrophy in rats. Studies have been conducted in vivo and in a cell-free system to prove that Rostafuroxin exerts its antihypertensive and antihypertrophic effects by antagonizing the Src-dependent signaling triggered by Ouabain. At the vascular level, Rostafuroxin antagonizes the Ouabain-mediated increase of myogenic vascular tone. This peculiar and novel mechanism of action, together with a good tolerability and efficacy both in animal models and hypertensive patients, make Rostafuroxin the prototype of a new class of antihypertensive compounds able to antagonize EO/ Ouabain and Adducin molecular effects.

The genetic and environmental heterogeneity of essential hypertension is responsible for the individual variability of antihypertensive therapy. An understanding of the molecular mechanisms underlying hypertension and related organ complications is a key aspect for developing new, effective, and safe antihypertensive agents able to cure the cause of the disease. Two mechanisms, among others, are involved in determining the abnormalities of tubular Na+ reabsorption observed in essential hypertension: the polymorphism of the cytoskeletal protein alpha-adducin and the increased circulating levels of endogenous ouabain (EO). Both lead to increased activity and expression of the renal Na+-K+ pump, the driving force for tubular Na transport. Morphological and functional vascular alterations have also been associated with EO. Rostafuroxin (PST 2238) is a new oral antihypertensive agent able to selectively antagonize EO, adducin pressor, and molecular effects. It is endowed with high potency and efficacy in reducing blood pressure and preventing organ hypertrophy in animal models representative of both adducin and EO mechanisms. At molecular level, in the kidney, Rostafuroxin antagonizes EO triggering of the Src-epidermal growth factor receptor (EGFr)-dependent signaling pathway leading to renal Na+-K+ pump, and ERK tyrosine phosphorylation and activation. In the vasculature, it normalizes the increased myogenic tone caused by nanomolar ouabain. A very high safety ratio and an absence of interaction with other mechanisms involved in blood pressure regulation, together with initial evidence of high tolerability and efficacy in hypertensive patients, indicate Rostafuroxin as the first example of a new class of antihypertensive agents designed to antagonize adducin and EO-hypertensive mechanisms.

Endogenous Ouabain (EO) and Adducin enhance the Na-K pump function and play an important role in sodium homeostasis and blood pressure (BP) regulation. In the general population, plasma EO modulates BP either by inhibiting the prohypertensive effect of an excessive salt intake or counteracting the depressor action of normal-moderate salt intake. Almost 50% of hypertensive patients have increased circulating plasma levels of EO. EO has been associated both to left ventricular dysfunction and hypertrophy. A new antihypertensive agent, PST2238, (17beta-(3-furyl)-5beta-androstan-3beta, 14beta, 17alpha-triol a digitoxigenin derivative) able to selectively antagonize both the EO and adducin prohypertensive and molecular effects, has been developed. In hypertensive rats (MHS strain) carrying both adducin mutations and increased plasma EO and in ouabain-infused rats (OS), PST2238 lowers BP by normalizing the renal Na-K pump function. In OS rats, PST antagonized the cardiac and renal pro-hypertrophic ouabain effect associated to the activation of the Src-EGFr-ERK(1/2) signaling cascade. Phase 1 clinical studies demonstrated a high tolerability of PST2238. In a preliminary phase 2 study on 42 mild never-treated hypertensive patients, PST2238 given for 3 months at 0.5 mg/day, significantly reduced BP in subjects with moderate salt intake, implying that it may be selectively effective in conditions where EO plays a prohypertensive role. In conclusion, PST2238, because of its peculiar action mechanism, represents a new tool to disentangle the complex relationship between salt intake, genetic control of renal sodium handling and EO effect.

Elevated levels of the endogenous ouabain (EO), a closely related isomer of ouabain, are implicated in rat and human hypertension and in related cardiovascular complications. The pathogenetic mechanisms through which EO affects the cardiovascular system involve the modulation of the renal Na/K-ATPase, implicated in renal tubular sodium reabsorption, and the activation of signal transduction pathways, promoting the transcription of growth-related genes. Experimental and clinical evidence on rats and humans stimulated the pharmacological research for developing novel anti-hypertensive agents able to antagonize the cellular and molecular alterations mediated by EO. Among them, the digitoxigenin derivate, PST 2238, has been selected for its ability to antagonize the ouabain-induced effects on blood pressure and organ hypertrophy at oral doses of microgram/kg/day in vivo. The pharmacological selectivity and safety of PST 2238 suggests that the compound may be effective for the treatment of those forms of hypertension in which renal sodium handling alterations and cardiovascular complications are associated with increased production of EO.

The evidence that high levels of endogenous ouabain (EO), a closely related isomer of ouabain, are implicated in human hypertension and cardiac hypertrophy and failure stimulated the pharmacological research for developing novel anti-hypertensive agents active as ouabain antagonists. The pathogenetic mechanisms through which increased EO levels affect cardiovascular system involve the modulation of Na-K ATPase, the key enzyme responsible for renal tubular sodium reabsorption and the activation of signalling transduction pathways implicated in growth-related gene transcription. By studying both genetic and experimental rat models of hypertension and comparing them with humans, our group has demonstrated that elevated levels of circulating EO and the genetic polymorphism of the cytoskeletal protein adducin associate with hypertension and high renal Na-K pump activity. Ouabain itself induces hypertension and up-regulates renal Na-K pump when chronically infused at low doses into rats (OS). In renal cultured cells, either incubated for several days with nanomolar concentrations of ouabain or transfected with the hypertensive adducin genetic variant, the Na-K pump results enhanced. Moreover, both EO and adducin polymorphism affect cardiac complications associated to hypertension, the former through the activation of a signalling transduction pathway. As a consequence, a compound able to interact with the cellular and molecular alterations, sustained by EO or mutated adducin, may represent the suitable treatment for those patients in whom these mechanisms are at work. A new antihypertensive compound, PST 2238, that selectively antagonises the pressor effect and the alteration of renal Na-K pump, sustained both by ouabain and adducin polymorphism, is described. A selective ability of PST 2238 to antagonise the ouabain-induced organ hypertrophy is also documented. The specificity of PST 2238 mechanism of action is supported by the absence of interactions with receptors or hormones involved in blood pressure regulation and by the lack of diuretic activity and diuretic-associated side effects. It is concluded that this compound could be useful for the treatment of those forms of essential hypertension in which renal Na handling alterations and cardiac complications are associated with either increased EO levels and/or adducin polymorphism.

Genetic variants of alpha adducin (ADD1) taken alone or in interaction with those of beta (ADD2) and gamma (ADD3) subunits have been associated with primary hypertension in humans and in Milan hypertensive (MHS) rats. In this study, we report the dissection of the individual contribution of each rat Add gene to blood pressure, by congenic substitution mapping. Congenic strains were developed by introgressing Add1, Add2, and Add3 genes (and chr14, chr4, and chr1 associated segments) of MHS in the Milan normotensive rat (MNS) genetic background (MNS.H-Add1, MNS.H-Add2, and MNS.H-Add3) and vice versa (MHS.N-Add1, MHS.N-Add2, and MHS.N-Add3). Systolic blood pressure (SBP) of MNS.H-Add1 rats was significantly higher (+10 mmHg) than that of MNS, whereas SBP of MHS.N-Add1 was significantly lower (-10 mmHg) than that of MHS. The differences account for 43% of the blood pressure differences between MHS and MNS. In contrast, SBPs of Add2 and Add3 congenic strains were not different from those of the correspondent recipient parental strain. The fine mapping of chr14 congenic segment supports the identity of blood pressure QTL with Add1 gene.

In addition to inhibition of the Na-K ATPase, ouabain activates a signal transduction function, triggering growth and proliferation of cultured cells even at nanomolar concentrations. An isomer of ouabain (EO) circulates in mammalians at subnanomolar concentrations, and increased levels are associated with cardiac hypertrophy and hypertension. We present here a study of cardiac and renal hypertrophy induced by ouabain infused into rats for prolonged periods and relate this effect to the recently described ouabain-induced activation of the Src-EGFr-ERK signaling pathway. Ouabain infusion into rats (15 microg/kg/day for 18 weeks) doubled plasma ouabain levels from 0.3 to 0.7 nm and increased blood pressure by 20 mm Hg (p < 0.001), cardiac left ventricle (+11%, p < 0.05), and kidney weight (+9%, p < 0.01). These effects in vivo are associated with a significant enrichment of alpha1, beta1, gammaa Na-K ATPase subunits together with Src and EGFr in isolated renal caveolae membranes and activation of ERK1/2. In caveolae, direct Na-K ATPase/Src interactions can be demonstrated by co-immunoprecipitation. The interaction is amplified by ouabain, at a high affinity binding site, detectable in caveolae but not in total rat renal membranes. The high affinity site for ouabain is associated with Src-dependent tyrosine phosphorylation of rat alpha1 Na-K ATPase. The antihypertensive compound, PST 2238, antagonized all ouabain-induced effects at 10 microg/kg/day in vivo or 10(-10)-10(-8) m in vitro. These findings provide a molecular mechanism for the in vivo pro-hypertrophic and hypertensinogenic activity of ouabain, or by analogy those of EO in humans. They also explain the pharmacological basis for PST 2238 treatment.

A primary impairment of the kidney sodium excretion has been documented both in hypertensive patients (EH) and genetic animal models (Milan hypertensive rat [MHS]) carrying mutations of the cytoskeletal protein adducin and/or increased plasma levels of endogenous ouabain (EO). Ouabain (OU) itself induces hypertension in rats and both OU and mutated adducin activate the renal Na/K-ATPase function both in vivo and in cultured renal cells (NRK). A new antihypertensive agent, PST 2238, able to selectively interact with these alterations has been developed. PST lowers blood pressure (BP) by normalizing the expression and activity of the renal Na-K pump selectively in those rat models carrying the adducin mutation (MHS) and/or increased EO levels (OS) at oral doses of 0.1-10 micro g/kg. In NRK cells either transfected with mutated adducin or incubated with 10(-9) M OU, PST normalizes the Na-K pump activity. Recently, an association between EO and cardiac complications has been observed in both EH and rat models consistent with a prohypertrophic activity of OU. OS rats showed a 10% increase of left ventricle and kidney weights as compared with controls, and PST 2238 (1 micro g/kg OS) prevented both ventricle and renal hypertrophy. This effect was associated with the ability of PST to antagonize the OU-dependent activation of growth-related genes, in the membrane subdomains of caveolae. In conclusion, PST is a new antihypertensive agent that may prevent cardiovascular complications associated with hypertension through the selective modulation of the Na-K pump function.

PST 2238 is a new antihypertensive compound that is able to correct the molecular and functional alterations of the renal Na-K pump and the pressor effect associated with either alpha-adducin mutations or high circulating levels of endogenous ouabain (EO) in genetic and experimental rat models. Due to the close relationship between renal Na-K pump function and tubular Na reabsorption, PST 2238 was investigated to determine whether it is endowed with diuretic activity and consequently might trigger alterations of the renin-aldosterone system and the carbohydrate and lipid metabolism often associated with chronic diuretic therapy. In Milan hypertensive (MHS) rats, in which hypertension is genetically associated with alpha-adducin mutation, increased tubular Na reabsorption, and hyperactivation of the renal Na-K pump. PST 2238 reduced blood pressure and normalized the renal Na-K pump activity at oral doses of micro g/kg, but did not induce, either acutely or chronically, any diuretic activity or hormonal or metabolic alterations. In contrast, HCTZ, given to MHS rats orally at 40 mg/kg, although it displayed diuretic activity and reduced the renal Na-K pump activity, did not lower blood pressure and caused hyperactivation of the renin-aldosterone system, hypokaliemia, and hyperglycemia. The findings lead to the conclusion that PST 2238 is a new antihypertensive compound that normalizes the altered function of the renal Na-K pump associated with hypertension in rat models, but that it is devoid of diuretic activity and does not induce the diuretic-associated side effects.

An ouabain-like factor has been implicated repeatedly in salt-sensitive hypertension as a natriuretic agent. However, the response of plasma ouabain-like factor to acute and chronic variation of body sodium is unclear. We studied 138 patients with essential hypertension who underwent an acute volume expansion/contraction maneuver (2 days) and 20 patients who entered a blind randomized crossover design involving chronically controlled sodium intake and depletion (170 to 70 mmol/d; 2 weeks each period). In both studies, plasma levels of ouabain-like factor were higher during sodium depletion (acute: 338.8+/-17.4 and 402.7+/-22.8 pmol/L for baseline and low sodium, respectively, P<0.01; chronic: 320.4+/-32.0 versus 481.0+/-48.1 pmol/L, P=0.01). No significant change in plasma ouabain-like factor was observed after a 2-hour saline infusion (333.4+/-23.9 pmol/L) or controlled sodium (402.1+/-34.9 pmol/L). When patients were divided into salt-sensitive or salt-resistant groups, no differences in plasma ouabain-like factor were observed in the 2 groups at baseline or in response to the 2 protocols: salt resistant (n=69, 340.1+/-25.9 pmol/L) versus salt sensitive (n=69, 337.4+/-23.6 pmol/L) and chronic salt resistant (n=11, 336.0+/-53.2) versus salt sensitive (n=9, 301.1+/-331.4 pmol/L). However, circulating ouabain-like factor was increased by sodium depletion in both groups. These results demonstrate that circulating ouabain-like factor is raised specifically by maneuvers that promote the loss of body sodium. Acute expansion of body fluids with isotonic saline is not a stimulus to plasma ouabain-like factor. Moreover, basal levels of plasma ouabain-like factor do not differ among patients with salt-sensitive or salt-resistant hypertension. Taken together, these new results suggest that ouabain-like factor is involved in the adaptation of humans to sodium depletion and argue against the hypothesis that ouabain-like factor is a natriuretic hormone.

We sought to evaluate the relationships among circulating levels of an endogenous ouabain-like factor (EO) and systemic hemodynamics and left ventricular (LV) geometry in patients with recently diagnosed essential hypertension. We selected 92 never-treated patients with essential hypertension. Blood samples were drawn for estimation of plasma EO (radioimmunoassay) and subjects underwent echocardiographic examination to evaluate LV end-systolic and end-diastolic wall thickness and internal dimensions. LV volumes, stroke volume, cardiac output, total peripheral resistance, LV mass, and relative wall thickness were calculated, and all except the last parameter were indexed by body surface area. LV mass also was indexed by height. On the basis of the values of LV mass index (body surface area or height) and relative wall thickness, subjects were divided into groups with either normal geometry, concentric remodeling, concentric hypertrophy, or eccentric nondilated hypertrophy. In the study population as a whole, circulating EO levels were significantly and directly correlated with mean blood pressure (r = 0.21, P = .048), relative wall thickness (r = 0.34, P = .001), and total peripheral resistance index (r = 0.37, P = .0003). Plasma EO also was significantly and inversely correlated with LV end-diastolic volume index (r = -0.32, P = .002), stroke index (r = -0.34, P = .0009), and cardiac index (r = -0.35, P = .0007). In multiple regression analysis, plasma EO was an independent correlate of total peripheral resistance index, cardiac index, and relative wall thickness. Regardless of the indexation method used for LV mass, plasma EO was higher in patients with concentric remodeling than in those with either normal geometry or concentric hypertrophy. Plasma EO tended to be higher (indexation by body surface area) or was significantly higher (indexation by height) in subjects with concentric remodeling than in those with eccentric nondilated hypertrophy. Patients with concentric remodeling showed the highest total peripheral resistance index and the lowest cardiac index. Our data suggest that EO plays a role in regulating systemic hemodynamics and LV geometry in patients with essential hypertension.

A primary renal alteration due to a genetic polymorphism of the cytoskeletal protein adducin associated with an up-regulation of the renal Na-K pump and increased levels of ouabainlike factor (OLF) has been identified as a possible causes of hypertension in Milan rats (MHS). This adducin polymorphism has also been found to be associated with hypertension and the blood pressure changes related to renal Na handling in humans and increased OLF levels have been found in a relevant portion of hypertensive patients. Increased activity and expression of the Na-K pump has also been observed under the following 'in vitro' and 'in vivo' conditions: rat renal cells transfected with the 'hypertensive' variant of adducin, as compared with normal cells; normal rat renal cells incubated for 5 days with 10(-9) M ouabain and normal rats made hypertensive by a chronic infusion of low doses of ouabain (OS rats). An up-regulation of the Na-K pump seems therefore to be a common biochemical alteration induced both by an adducin polymorphism and/or chronic exposure to low concentrations of ouabain (or OLF). A new antihypertensive compound, PST 2238, that selectively antagonizes the pressor effect and the alteration of the renal Na-K pump induced both by an adducin polymorphism and OLF, is described. The ability of PST 2238 to lower blood pressure and normalize the Na-K pump both in MHS and OS rats suggests that this compound could be useful in the treatment of those forms of essential hypertension in which renal Na-handling alterations are associated with either adducin polymorphisms and/or increased OLF levels.

This review focuses on the most recent data published in the field of the sodium-potassium pump inhibitors regarding the hypothetical structure, the secretory stimuli and the pathophysiological implications for particular diseases, such as hypertension. On the basis of the findings published so far, we reconsider and discuss the 'natriuretic hypothesis' for explaining the role of the endogenous sodium-potassium ATPase inhibitor. We propose the ouabain-like factor as a modulator of the renal sodium-potassium pump, that can be considered as a new pharmacological target for hypertension therapy.

Adducin point mutations are associated with genetic hypertension in Milan hypertensive strain (MHS) rats and in humans. In transfected cells, adducin affects actin cytoskeleton organization and increases the Na(+)-K(+)-pump rate. The present study has investigated whether rat and human adducin polymorphisms differently modulate rat renal Na(+)-K(+)-ATPase in vitro. We report the following. 1) Both rat and human adducins stimulate Na(+)-K(+)-ATPase activity, with apparent affinity in tens of nanomolar concentrations. 2) MHS and Milan normotensive strain (MNS) adducins raise the apparent ATP affinity for Na(+)-K(+)-ATPase. 3) The mechanism of action of adducin appears to involve a selective acceleration of the rate of the conformational change E(2) (K) --> E(1) (Na) or E(2)(K). ATP --> E(1)Na. ATP. 4) Apparent affinities for mutant rat and human adducins are significantly higher than those for wild types. 5) Recombinant human alpha- and beta-adducins stimulate Na(+)-K(+)-ATPase activity, as do the COOH-terminal tails, and the mutant proteins display higher affinities than the wild types. 6) The cytoskeletal protein ankyrin, which is known to bind to Na(+)-K(+)-ATPase, also stimulates enzyme activity, whereas BSA is without effect; the effects of adducin and ankyrin when acting together are not additive. 7) Pig kidney medulla microsomes appear to contain endogenous adducin; in contrast with purified pig kidney Na(+)-K(+)-ATPase, which does not contain adducin, added adducin stimulates the Na(+)-K(+)-ATPase activity of microsomes only about one-half as much as that of purified Na(+)-K(+)-ATPase. Our findings strongly imply the existence of a direct and specific interaction between adducin and Na(+)-K(+)-ATPase in vitro and also suggest the possibility of such an interaction in intact renal membranes.

Many patients with essential hypertension (EH) exhibit increased left ventricular mass. Similarly, elevated circulating levels of an endogenous ouabainlike factor (OLF) have been described in some but not all patients with EH. Moreover, ouabain has a hypertrophic influence on isolated cardiac myocytes. Accordingly, we investigated relationships among plasma OLF, left ventricular mass, and cardiac function in patients with EH. Plasma OLF was determined in 110 normotensive subjects and 128 patients with EH. Echocardiographic parameters and humoral determinants were measured in EH. Plasma OLF levels were increased (P<0.0001) in patients with EH (377+/-19 pmol/L) versus normotensive (253+/-53 pmol/L) subjects. The distribution of plasma OLF was unimodal in normotensives, whereas it was bimodal in EH. Twenty-four-hour diastolic ambulatory blood pressure was slighter higher in EH with high OLF compared with EH with normal OLF (93.2+/-1.14 versus 89.4+/-1.33 mm Hg, P=0.03). Left ventricular mass index and stroke volume in EH with high OLF were greater than in EH with normal OLF (101.9+/-3.3 versus 86.1+/-2.5 g/m(2), P=0.0003, and 57.10+/-1.48 versus 52.30+/-1.14 mL/m(2), P=0. 02, respectively), although heart rate was slower (74.2+/-1.3 versus 80.5+/-1.3 bpm, P=0.005). Multiple regression analysis that tested the influence of body mass index, age, gender, 24-hour blood pressure, and OLF on left ventricular mass revealed independent contributions of systolic (13.2%) and diastolic (12.4%) blood pressure and plasma OLF (11.6%) to left ventricular mass. We conclude that approximately 50% of patients with uncomplicated EH have elevated-high circulating OLF levels, higher diastolic blood pressure, greater left ventricular mass and stroke volume, and reduced heart rate. We propose that the OLF affects cardiovascular function and structure and should be considered as a factor that contributes to the risk of morbid events.

A genetic alteration in the adducin genes is associated with hypertension and up-regulation of the expression of renal Na, K-ATPase in Milan-hypertensive (MHS) rats, in which increased ouabain-like factor (OLF) levels are also observed. PST 2238, a new antihypertensive compound that antagonizes the pressor effect of ouabain in vivo and normalizes ouabain-dependent up-regulation of the renal Na-K pump, was evaluated for its ability to lower blood pressure and regulate renal Na,K-ATPase activity in MHS genetic hypertension. In this study, we show that PST 2238, given orally at very low doses (1 and 10 microg/kg for 5-6 weeks), reduced the development of hypertension in MHS rats and normalized the increased renal Na,K-ATPase activity and mRNA levels, whereas it did not affect either blood pressure or Na,K-ATPase in Milan-normotensive (MNS) rats. In addition, a similar antihypertensive effect was observed in adult MHS rats after a short-term treatment. In cultured rat renal cells with increased Na-K pump activity at Vmax due to overexpression of the hypertensive variant of adducin, 5 days of incubation with PST 2238 (10(-10-)-10(-9) M) lowered the pump rate to the level of normal wild-type cells, which in turn were not affected by the drug. In conclusion, PST 2238 is a very potent compound that in MHS rats reduces blood pressure and normalizes Na-K pump alterations caused by a genetic alteration of the cytoskeletal adducin. Because adducin gene mutations have been associated with human essential hypertension, it is suggested that PST 2238 may display greater antihypertensive activity in those patients carrying such a genetic alteration.

Endogenous ouabain-like factor (OLF) is present in mammal tissues and after standardized extraction procedure can be similarly quantified by two independent assays: RIA and Na-KATPase inhibition. OLF was quantified both from plasma and tissues obtained from MHS hypertensive and MNS normotensive rats, maintained under the same environmental and dietary conditions, and from plasma of healthy volunteers and essential hypertensive patients. OLF biochemical characterization shows that it behaves like ouabain except for a 1000-fold higher affinity for the ouabain low-affinity Na-KATPase isoforms than ouabain. Tissue and plasma levels of OLF are higher in MHS than in MNS rats and are not influenced by exogenous OLF sources. Plasma OLF is also increased in a subgroup of hypertensive patients. Both in rats and humans a primary cell membrane alteration affecting ion transports seems to be linked to the increased levels of OLF. An antihypertensive compound which selectively antagonizes the pressor effect of OLF and corrects the ion transport defect is under development and can represent a new pharmacological approach to the treatment of hypertension.