Selective SERCA2a activator as a candidate for chronic heart failure therapy.

BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+ 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 Ca2+ 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.

Istaroxime Metabolite PST3093 Selectively Stimulates SERCA2a and Reverses Disease-Induced Changes in Cardiac Function.

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 Ca2+ 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, Ca2+ 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 Ca2+ 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.

Antihypertensive treatment guided by genetics: PEARL-HT, the randomized proof-of-concept trial comparing rostafuroxin with losartan.

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.

High-Fidelity Simulation Training for Helicopter Emergency Medical Services Flight Nurses: A Report From the First Italian Experience.

OBJECTIVE: Simulation-based training has a significant effect in enhancing professionals' skills in the helicopter emergency medical service (HEMS) setting and is reported to be more effective than other learning strategies. The main objective of this study was to assess the efficacy of a specific high-fidelity simulation (HFS) training course for HEMS nurses before entering into operational service. METHODS: This report describes the first Italian experience of a specifically designed HFS training program for HEMS nurses held in Bologna, Italy, in a dedicated scenario with a mock-up helicopter and an advanced mannequin. RESULTS: A total of 14 nurses participated in the SAFE2019 (Simulazione ad Alta Fedeltà per l'Elisoccorso-High-Fidelity Simulation for HEMS) courses. The aspects considered most useful and effective were related to team working, communication issues, and nontechnical skills (NTS) development. Moreover, participants suggested implementing a series of scheduled HFS retraining sessions. CONCLUSION: HFS in a dedicated HEMS scenario during the orientation period has contributed to enhanced technical competencies and NTS, increasing comfort and situational awareness for new entrant flight nurses. Simulation may represent a valuable resource to assess and maintain competencies through periodic retraining sessions for full HEMS teams or when new devices are adopted.

Nursing perspectives from an Italian ICU.

ABSTRACT: In Europe, the novel coronavirus outbreak started in the northern regions of Italy at the end of February 2020. Initially, resources were insufficient to deal with the abrupt influx of critically ill patients requiring respiratory support. This article recounts the authors' experiences caring for patients with COVID-19 in an Italian ICU during this period in order to help colleagues in the international critical care nursing community successfully manage similar circumstances in the ongoing pandemic.

Personalized Therapy of Hypertension: the Past and the Future.

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.

Rostafuroxin protects from podocyte injury and proteinuria induced by adducin genetic variants and ouabain.

Glomerulopathies are important causes of morbidity and mortality. Selective therapies that address the underlying mechanisms are still lacking. Recently, two mechanisms, mutant ß-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 ß-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 ß-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 ß-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 ß-adducin- and ouabain-induced effects on nephrin protein expression and proteinuria. We conclude that rostafuroxin prevented podocyte lesions and proteinuria due to mutant ß-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.

Preoperative endogenous ouabain predicts acute kidney injury in cardiac surgery patients.

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.

Cross talk between plasma membrane Na(+)/Ca (2+) exchanger-1 and TRPC/Orai-containing channels: key players in arterial hypertension.

Arterial smooth muscle (ASM) Na(+)/Ca(2+) exchanger type 1 (NCX1) and TRPC/Orai-containing receptor/store-operated cation channels (ROC/SOC) are clustered with α2 Na(+) pumps in plasma membrane microdomains adjacent to the underlying junctional sarcoplasmic reticulum. This arrangement enables these transport proteins to function as integrated units to help regulate local Na(+) metabolism, Ca(2+) signaling, and arterial tone. They thus influence vascular resistance and blood pressure (BP). For instance, upregulation of NCX1 and TRPC6 has been implicated in the pathogenesis of high BP in several models of essential hypertension. The models include ouabain-induced hypertensive rats, Milan hypertensive rats, and Dahl salt-sensitive hypertensive rats, all of which exhibit elevated plasma ouabain levels. We suggest that these molecular mechanisms are key contributors to the increased vascular resistance ("whole body autoregulation") that elevates BP in essential hypertension. Enhanced expression and function of ASM NCX1 and TRPC/Orai1-containing channels in hypertension implies that these proteins are potential targets for pharmacological intervention.

Increased arterial smooth muscle Ca2+ signaling, vasoconstriction, and myogenic reactivity in Milan hypertensive rats.

The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na(+) reabsorption. Recently we demonstrated that Ca(2+) signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na(+)/Ca(2+) exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca(2+) signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 ± 1 mmHg) than in MNS (112 ± 1 mmHg; P < 0.001; n = 16 each) rats. Pressurized mesenteric resistance arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1-100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca(2+) signals in response to 5 µM PE or ATP in the absence and presence of extracellular Ca(2+). These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca(2+) release and increased Ca(2+) entry, respectively. The increased SR Ca(2+) release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a ∼70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca(2+) signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca(2+) signaling. These molecular and functional changes provide a mechanism for the increased peripheral vascular resistance (whole body autoregulation) that underlies the sustained hypertension.

In-Situ Simulation for Intensive Care Nurses During the COVID-19 Pandemic in Italy: Advantages and Challenges.

The COVID-19 pandemic required a global increase in intensive care unit (ICU) resources and the recruitment of a great number of nurses without any tprior critical care experience. The opportunities for traditional education and supervised clinical training were limited to prevent infections. The massive increase of nursing staff resources required a rethinking of the usual educational strategies for newly acquired nurses. This short communication describes our experience of an "in-situ" simulation training course in an Italian tertiary level hospital. A series of two-part classes were structured with short lectures on fundamental principles of intensive care nursing and brief hands-on sessions, and a set of simulated scenarios, based upon the most common situations to be faced in the ICU. In-situ simulation offers greater realism and transferability and represents a cost-effective strategy, avoiding the costs and the maintenance of a dedicated simulation center. The simulated multidisciplinary teamwork in the real ICU setting contributes to an effective experiential learning, improving staff familiarity with devices, equipment, and environment, and allows trainees to improve both technical and nontechnical skills.

Optimizing nursing workload in the intensive care unit during the COVID-19 pandemic: Planning prone positioning.

Background: Prone positioning is a complex, time-consuming task, involving significant intensive care unit staff. The increased workload during the COVID-19 pandemic and the reduced staffing boosted the burden of intensive care unit nurses, which might have a negative impact on patients' safety and outcomes. Methods: Retrospective chart review, analysing the hourly distribution of pronation and supination procedures in mechanically ventilated critically ill patients during the first and the second wave of the COVID-19 pandemic (March 2020-May 2021). Results: 303 procedures were analysed: 77 pronation manoeuvres out of 156 (49.3%) and 82 supination out of 147 (55.8%) were performed in dedicated time slots in the afternoon (15.30-19.00) and in the morning (9.30-12.30) shifts, when the nursing staff was increased. At least five healthcare providers performed pronation manoeuvres. Six device displacements were registered. Conclusions: Planning complex activities such as prone positioning needs an effective strategy to optimize nursing staff workload in the intensive care unit. This organization allowed to perform pronation cycles with a duration of at least 16 h, according to current clinical recommendations.

Highly Selective SERCA2a Activators: Preclinical Development of a Congeneric Group of First-in-Class Drug Leads against Heart Failure.

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.

SERCA2a stimulation by istaroxime improves intracellular Ca2+ handling and diastolic dysfunction in a model of diabetic cardiomyopathy.

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.

Rostafuroxin: an ouabain-inhibitor counteracting specific forms of hypertension.

An innovative approach to the therapy of essential hypertension (EH) and the related complications has been pursued by our group with the aim of defining specific genetic-molecular mechanisms underlying the disease in sub-sets of patients. This approach is anticipated to have a major effect on the clinical practice, diagnostics and development of new drugs able to selectively target such mechanisms. The final achievement is the definition of biomarkers for identifying patients who more likely should benefit for a given therapy both in terms of efficacy and reduction of the adverse reactions. Among many, two mechanisms have been defined and addressed:Both alterations lead to hypertension, organ hypertrophy, negative vascular remodeling and increased cardiovascular risk by affecting the renal Na(+) handling, through the up-regulation of the Na(+)-K(+) pump and the activation of the Src-dependent signal transduction pathway. A novel antihypertensive agent, rostafuroxin (PST2238), has been selected and developed for its ability to correct the renal Na(+)-K(+) pump abnormalities sustained by the mutant adducin and EO-dependent mechanisms. It is endowed with high potency and efficacy in reducing blood pressure (BP) 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 mutant adducin and antagonizes the EO triggering of the Src-EGFr-dependent signaling pathway leading to renal Na(+)-K(+) pump and ERK phosphorylation and activation. In the vasculature, it normalizes the increased myogenic tone caused by ouabain. A very high safety ratio and the absence of interaction with other mechanisms involved in BP regulation, together with 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. A recently concluded Phase II clinical trial (OASIS) has provided the proof of concept that such a compound is effective in the subset of patients where these two mechanisms are at work.

NKCC2 is activated in Milan hypertensive rats contributing to the maintenance of salt-sensitive hypertension.

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.

Upregulation of Na+/Ca2+ exchanger and TRPC6 contributes to abnormal Ca2+ homeostasis in arterial smooth muscle cells from Milan hypertensive rats.

The Milan hypertensive strain (MHS) of rats is a model for hypertension in humans. Inherited defects in renal function have been well studied in MHS rats, but the mechanisms that underlie the elevated vascular resistance are unclear. Altered Ca(2+) signaling plays a key role in the vascular dysfunction associated with arterial hypertension. Here we compared Ca(2+) signaling in mesenteric artery smooth muscle cells from MHS rats and its normotensive counterpart (MNS). Systolic blood pressure was higher in MHS than in MNS rats (144 +/- 2 vs. 113 +/- 1 mmHg, P < 0.05). Resting cytosolic free Ca(2+) concentration (measured with fura-2) and ATP-induced Ca(2+) transients were augmented in freshly dissociated arterial myocytes from MHS rats. Ba(2+) entry activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (a measure of receptor-operated channel activity) was much greater in MHS than MNS arterial myocytes. This correlated with a threefold upregulation of transient receptor potential canonical 6 (TRPC6) protein. TRPC3, the other component of receptor-operated channels, was marginally, but not significantly, upregulated. The expression of TRPC1/5, components of store-operated channels, was not altered in MHS mesenteric artery smooth muscle. Immunoblots also revealed that the Na(+)/Ca(2+) exchanger-1 (NCX1) was greatly upregulated in MHS mesenteric artery (by approximately 13-fold), whereas the expression of plasma membrane Ca(2+)-ATPase was not altered. Ca(2+) entry via the reverse mode of NCX1 evoked by the removal of extracellular Na(+) induced a rapid increase in cytosolic free Ca(2+) concentration that was significantly larger in MHS arterial myocytes. The expression of alpha(1)/alpha(2) Na(+) pumps in MHS mesenteric arteries was not changed. Immunocytochemical observations showed that NCX1 and TRPC6 are clustered in plasma membrane microdomains adjacent to the underlying sarcoplasmic reticulum. In summary, MHS arteries exhibit upregulated TRPC6 and NCX1 and augmented Ca(2+) signaling. We suggest that the increased Ca(2+) signaling contributes to the enhanced vasoconstriction and elevated blood pressure in MHS rats.

Novel analogues of Istaroxime, a potent inhibitor of Na(+),K(+)-ATPase: Synthesis, structure-activity relationship and 3D-quantitative structure-activity relationship of derivatives at position 6 on the androstane scaffold.

We report the synthesis and biological properties of novel analogues of Istaroxime acting as positive inotropic compounds through the inhibition of the Na(+),K(+)-ATPase. We explored the chemical space around the position 6 of the steroidal scaffold by changing the functional groups at that position and maintaining a basic oximic chain in position 3. Some compounds showed inhibitory potencies of the Na(+),K(+)-ATPase higher than Istaroxime and many of the compounds tested in vivo were safer than digoxin, the classic digitalis compound currently used for the treatment of congestive heart failure as inotropic agent. The 3D-QSAR analyses using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods have been successfully applied to a set of 63 androstane derivatives as Na(+),K(+)-ATPase inhibitors. The contour plots provide many useful insights into relationships between structural features and inhibitory potency.

Modulation of sarcoplasmic reticulum function by PST2744 [istaroxime; (E,Z)-3-((2-aminoethoxy)imino) androstane-6,17-dione hydrochloride)] in a pressure-overload heart failure model.

PST2744 [Istaroxime; (E,Z)-3-((2-aminoethoxy)imino) androstane-6,17-dione hydrochloride)] is a novel inotropic agent that enhances sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA) 2 activity. We investigated the istaroxime effect on Ca(2+) handling abnormalities in myocardial hypertrophy/failure (HF). Guinea pig myocytes were studied 12 weeks after aortic banding (AoB) and compared with those of sham-operated animals (sham). The gain of calcium-induced Ca(2+) release (CICR), sarcoplasmic reticulum (SR) Ca(2+) content, Na(+)/Ca(2+) exchanger (NCX) function, and the rate of SR reloading after caffeine-induced depletion (SR Ca(2+) uptake, measured during NCX blockade) were evaluated by measurement of cytosolic Ca(2+) and membrane currents. HF characterization: AoB caused hypertrophy and failure in 100 and 25% of animals, respectively. Although CICR gain during constant pacing was preserved, SR Ca(2+) content and SR Ca(2+) uptake were strongly depressed. Resting Ca(2+) and the slope of the Na(+)/Ca(2+) exchanger current (I(NCX))/Ca(2+) relationship were unchanged by AoB. Istaroxime effects: CICR gain, SR Ca(2+) content, and SR Ca(2+) uptake rate were increased by istaroxime in sham myocytes and, to a significantly larger extent, in AoB myocytes; this led to almost complete recovery of SR Ca(2+) uptake in AoB myocytes. Istaroxime increased resting Ca(2+) and the slope of the I(NCX)/Ca(2+) relationship similarly in sham and AoB myocytes. Istaroxime failed to increase SERCA activity in skeletal muscle microsomes devoid of phospholamban. Thus, clear-cut abnormalities in Ca(2+) handling occurred in this model of hypertrophy, with mild decompensation. Istaroxime enhanced SR function more in HF myocytes than in normal ones; almost complete drug-induced recovery suggests a purely functional nature of SR dysfunction in this HF model.

Role of rat alpha adducin in angiogenesis: null effect of the F316Y polymorphism.

OBJECTIVE: Rat alpha adducin point mutation (F316Y) has been associated with primary systemic arterial hypertension. As microcirculatory abnormalities are present in most forms of hypertension, the aim of the present study was to investigate whether rat alpha adducin may regulate endothelial cell (EC) functions in vitro and in vivo. METHODS AND RESULTS: The overexpression of rat wild type alpha adducin (WT-Add1) in ECs induced capillary-like structure development in Matrigel in vitro and enhanced capillary formation in Matrigel implants in vivo in CD1 mice. In contrast, the overexpression of the mutated form (MUT-Add1) of rat alpha adducin had a Null effect in vitro and lacked any significant activity in vivo. Further, adenovirus-mediated rat WT-Add1 but not MUT-Add1 gene transfer to murine ischemic hindlimb enhanced capillary formation in skeletal muscles. Gene profiling of human umbilical vein endothelial cells overexpressing alpha adducin was performed in order to identify putative effector molecules of alpha adducin-mediated activities on ECs. Interestingly, among a number of genes involved in angiogenesis regulation, retinoic acid-induced protein (RAI17) was found to be upregulated in WT-Add1 vs MUT-Add1 overexpressing cells, possibly representing a key molecule/axis for the functional Add1-induced effect. CONCLUSIONS: Rat WT alpha adducin enhanced EC functions both in vitro and in vivo. The expression of the F316Y variant, associated with the hypertensive phenotype, had a Null effect and might contribute to endothelial rarefaction/dysfunction in hypertension. RAI17 was found to be a putative effector molecule differentially regulated by the overexpression of the two forms of Add1 in endothelial cells.