Asunto(s)
Insuficiencia Cardíaca , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/enzimología , Humanos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Animales , Etiocolanolona/análogos & derivados , Etiocolanolona/farmacología , Etiocolanolona/uso terapéutico , Enfermedad Crónica , Activadores de Enzimas/farmacología , Activadores de Enzimas/uso terapéuticoRESUMEN
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.
Asunto(s)
Etiocolanolona/análogos & derivados , Insuficiencia Cardíaca , Ratas , Ratones , Animales , Cobayas , Insuficiencia Cardíaca/metabolismo , Enfermedad Crónica , Inhibidores Enzimáticos , Cardiotónicos/uso terapéutico , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Miocitos Cardíacos/metabolismo , Calcio/metabolismoRESUMEN
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 Ca2+ 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: Ca2+ 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 Ca2+ compartments. Transcripts and proteins of relevant signaling pathways were evaluated. RESULTS: The mutation was characterized by hyperdynamic Ca2+ 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 Ca2+ buffering or SERCA2a blockade reduced O2 consumption with larger effect in the mutant. Signaling changes suggest cellular adaptation to perturbed Ca2+ 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 Ca2+ and activation of signaling responses point to an early involvement of metabolic stress in the pathogenesis of this ACM model.
Asunto(s)
Cardiomiopatías , Animales , Ratones , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Cardiomiopatías/genética , Ratones Transgénicos , Mutación , Miocitos Cardíacos/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
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.
Asunto(s)
Insuficiencia Cardíaca , Corazón , Animales , Humanos , Ratas , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/farmacología , Adenosina Trifosfatasas/uso terapéutico , Etiocolanolona/farmacología , Etiocolanolona/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Miocitos Cardíacos/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/uso terapéuticoRESUMEN
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.
Asunto(s)
Insuficiencia Cardíaca , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Animales , Arritmias Cardíacas , Calcio/metabolismo , Cobayas , Insuficiencia Cardíaca/metabolismo , Miocitos Cardíacos/metabolismo , RatasRESUMEN
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.
Asunto(s)
Diabetes Mellitus Experimental , Cardiomiopatías Diabéticas , Animales , Calcio/metabolismo , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Cardiomiopatías Diabéticas/tratamiento farmacológico , Cardiomiopatías Diabéticas/etiología , Cardiomiopatías Diabéticas/prevención & control , Etiocolanolona/análogos & derivados , Etiocolanolona/metabolismo , Etiocolanolona/farmacología , Etiocolanolona/uso terapéutico , Ratas , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
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 Ca2+ 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 Ca2+ transient decay. Although APD, DADs, BVR and Ca2+ transient decay recovered over time after the cessation of Dox treatment, subsequent Trz administration exacerbated these abnormalities. Trz, but not Dox, reduced Ca2+ transient amplitude and SR Ca2+ content, although only Dox treatment was associated with SERCA downregulation. Finally, Dox treatment increased Ca2+ spark frequency, resting Ca2+ waves, sarcoplasmic reticulum (SR) Ca2+ leak, and long-lasting Ca2+ release events (so-called Ca2+ "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 Ca2+ 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.
RESUMEN
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.
Asunto(s)
Androstanoles/uso terapéutico , Antihipertensivos/uso terapéutico , Hipertensión/tratamiento farmacológico , Hipertensión/genética , Losartán/uso terapéutico , Adulto , Pueblo Asiatico , Presión Sanguínea , Método Doble Ciego , Femenino , Perfilación de la Expresión Génica , Pruebas Genéticas , Humanos , Italia , Masculino , Persona de Mediana Edad , Ouabaína/metabolismo , Farmacogenética , Taiwán , Resultado del Tratamiento , Población BlancaRESUMEN
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.
Asunto(s)
Androstanoles/uso terapéutico , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/metabolismo , Riñón/efectos de los fármacos , Ouabaína/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Animales , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/sangre , Enfermedades Renales/patología , Nefrectomía , Ouabaína/sangre , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/sangre , Daño por Reperfusión/patologíaRESUMEN
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.
Asunto(s)
Antihipertensivos/uso terapéutico , Hipertensión/tratamiento farmacológico , Medicina de Precisión , Animales , Pruebas Genéticas , Humanos , Hipertensión/genética , Sodio/metabolismoRESUMEN
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.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Sodio/metabolismo , Canales de Sodio Activados por Voltaje/metabolismo , Animales , Línea Celular , Glucosa/farmacología , Humanos , Hipoglucemiantes/farmacología , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Potenciales de la Membrana , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/efectos de los fármacos , Canales de Potasio/genética , Canales de Potasio/metabolismo , Canales de potasio activados por Sodio , ARN Mensajero/metabolismo , Ratas , Factores de Tiempo , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Canales de Sodio Activados por Voltaje/efectos de los fármacos , Canales de Sodio Activados por Voltaje/genéticaRESUMEN
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.
Asunto(s)
Androstanoles/farmacología , Proteínas de Unión a Calmodulina/metabolismo , Variación Genética/genética , Ouabaína/efectos adversos , Podocitos/efectos de los fármacos , Proteinuria/tratamiento farmacológico , Animales , Antihipertensivos/farmacología , Proteínas de Unión a Calmodulina/genética , Modelos Animales de Enfermedad , Femenino , Hipertensión/inducido químicamente , Hipertensión/tratamiento farmacológico , Hipertensión/genética , Hipertensión/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Ratones , Ratones Noqueados , Podocitos/metabolismo , Proteinuria/inducido químicamente , Proteinuria/genética , Proteinuria/metabolismo , Ratas , Ratas Sprague-Dawley , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
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.
Asunto(s)
Cardiomegalia/prevención & control , Hipertrofia Ventricular Izquierda/prevención & control , Proteínas Serina-Treonina Quinasas/metabolismo , Cloruro de Sodio Dietético/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Cardiomegalia/enzimología , Humanos , Hipertrofia Ventricular Izquierda/enzimología , Inmunohistoquímica , Técnicas In Vitro , Masculino , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , RatasRESUMEN
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.
RESUMEN
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.
Asunto(s)
Proteína Quinasa Dependiente de GMP Cíclico Tipo I/genética , Hipertensión/genética , Riñón/metabolismo , Natriuresis/genética , Polimorfismo Genético , Sodio/metabolismo , Adulto , Anciano , Alelos , Presión Sanguínea/genética , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Hipertensión/metabolismo , Hipertensión/fisiopatología , Riñón/fisiopatología , Masculino , Persona de Mediana Edad , Sodio en la Dieta/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Unión al Calcio/antagonistas & inhibidores , Calcio/metabolismo , Etiocolanolona/análogos & derivados , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/farmacocinética , Retículo Sarcoplasmático/metabolismo , Animales , Calcio/farmacocinética , Perros , Etiocolanolona/farmacología , Cobayas , Humanos , Técnicas In Vitro , Masculino , Microsomas/metabolismo , Conejos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/efectos de los fármacos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , SpodopteraRESUMEN
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.
Asunto(s)
Lesión Renal Aguda/etiología , Puente de Arteria Coronaria , Válvulas Cardíacas/cirugía , Ouabaína/sangre , Lesión Renal Aguda/diagnóstico , Adulto , Anciano , Animales , Biomarcadores/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos Animales , Complicaciones Posoperatorias/diagnóstico , Valor Predictivo de las Pruebas , Periodo Preoperatorio , Estudios Prospectivos , Ratas , Ratas Sprague-DawleyRESUMEN
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.
Asunto(s)
Gasto Cardíaco Bajo/tratamiento farmacológico , Gasto Cardíaco Bajo/enzimología , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Animales , Gasto Cardíaco Bajo/metabolismo , Etiocolanolona/análogos & derivados , Etiocolanolona/uso terapéutico , Insuficiencia Cardíaca/metabolismo , Humanos , Modelos Biológicos , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
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.
Asunto(s)
Hipertensión/fisiopatología , Cloruro de Sodio/metabolismo , Sodio en la Dieta/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Aldosterona/sangre , Aldosterona/orina , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Diuréticos/farmacología , Furosemida/farmacología , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Endogámicas , Simportadores de Cloruro de Sodio-Potasio/genética , Miembro 1 de la Familia de Transportadores de Soluto 12 , Orina/químicaRESUMEN
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.