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J Am Heart Assoc ; 10(19): e021985, 2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34583520


Background PKARIα (protein kinase A type I-α regulatory subunit) is redox-active independent of its physiologic agonist cAMP. However, it is unknown whether this alternative mechanism of PKARIα activation may be of relevance to cardiac excitation-contraction coupling. Methods and Results We used a redox-dead transgenic mouse model with homozygous knock-in replacement of redox-sensitive cysteine 17 with serine within the regulatory subunits of PKARIα (KI). Reactive oxygen species were acutely evoked by exposure of isolated cardiac myocytes to AngII (angiotensin II, 1 µmol/L). The long-term relevance of oxidized PKARIα was investigated in KI mice and their wild-type (WT) littermates following transverse aortic constriction (TAC). AngII increased reactive oxygen species in both groups but with RIα dimer formation in WT only. AngII induced translocation of PKARI to the cell membrane and resulted in protein kinase A-dependent stimulation of ICa (L-type Ca current) in WT with no effect in KI myocytes. Consequently, Ca transients were reduced in KI myocytes as compared with WT cells following acute AngII exposure. Transverse aortic constriction-related reactive oxygen species formation resulted in RIα oxidation in WT but not in KI mice. Within 6 weeks after TAC, KI mice showed an enhanced deterioration of contractile function and impaired survival compared with WT. In accordance, compared with WT, ventricular myocytes from failing KI mice displayed significantly reduced Ca transient amplitudes and lack of ICa stimulation. Conversely, direct pharmacological stimulation of ICa using Bay K8644 rescued Ca transients in AngII-treated KI myocytes and contractile function in failing KI mice in vivo. Conclusions Oxidative activation of PKARIα with subsequent stimulation of ICa preserves cardiac function in the setting of acute and chronic oxidative stress.

Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360742


Sodium-glucose co-transporter 2 inhibitors (SGLT2i) are emerging as a new treatment strategy for heart failure with reduced ejection fraction (HFrEF) and-depending on the wistfully awaited results of two clinical trials (DELIVER and EMPEROR-Preserved)-may be the first drug class to improve cardiovascular outcomes in patients suffering from heart failure with preserved ejection fraction (HFpEF). Proposed mechanisms of action of this class of drugs are diverse and include metabolic and hemodynamic effects as well as effects on inflammation, neurohumoral activation, and intracellular ion homeostasis. In this review we focus on the growing body of evidence for SGLT2i-mediated effects on cardiac intracellular Na+ as an upstream mechanism. Therefore, we will first give a short overview of physiological cardiomyocyte Na+ handling and its deterioration in heart failure. On this basis we discuss the salutary effects of SGLT2i on Na+ homeostasis by influencing NHE1 activity, late INa as well as CaMKII activity. Finally, we highlight the potential relevance of these effects for systolic and diastolic dysfunction as well as arrhythmogenesis.

Arritmias Cardíacas/tratamento farmacológico , Cardiotônicos/uso terapêutico , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Transportador 2 de Glucose-Sódio/metabolismo , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Humanos , Miocárdio/patologia , Miócitos Cardíacos/patologia
ESC Heart Fail ; 2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-32946200


AIMS: Recent clinical trials have proven gliflozins to be cardioprotective in diabetic and non-diabetic patients. However, the underlying mechanisms are incompletely understood. A potential inhibition of cardiac Na+ /H+ exchanger 1 (NHE1) has been suggested in animal models. We investigated the effect of empagliflozin on NHE1 activity in human atrial cardiomyocytes. METHODS AND RESULTS: Expression of NHE1 was assessed in human atrial and ventricular tissue via western blotting. NHE activity was measured as the maximal slope of pH recovery after NH4 + pulse in isolated carboxy-seminaphtarhodafluor 1 (SNARF1)-acetoxymethylester-loaded murine ventricular and human atrial cardiomyocytes. NHE1 is abundantly expressed in human atrial and ventricular tissue. Interestingly, compared with patients without heart failure (HF), atrial NHE1 expression was significantly increased in patients with HF with preserved ejection fraction and atrial fibrillation. The largest increase in atrial and ventricular NHE1 expression, however, was observed in patients with end-stage HF undergoing heart transplantation. Importantly, acute exposure to empagliflozin (1 µmol/L, 10 min) significantly inhibited NHE activity to a similar extent in human atrial myocytes and mouse ventricular myocytes. This inhibition was also achieved by incubation with the well-described selective NHE inhibitor cariporide (10 µmol/L, 10 min). CONCLUSIONS: This is the first study systematically analysing NHE1 expression in human atrial and ventricular myocardium of HF patients. We show that empagliflozin inhibits NHE in human cardiomyocytes. The extent of NHE inhibition was comparable with cariporide and may potentially contribute to the improved outcome of patients in clinical trials.

PLoS Biol ; 18(6): e3000722, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32569301


Inflammation and infection can trigger local tissue Na+ accumulation. This Na+-rich environment boosts proinflammatory activation of monocyte/macrophage-like cells (MΦs) and their antimicrobial activity. Enhanced Na+-driven MΦ function requires the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5), which augments nitric oxide (NO) production and contributes to increased autophagy. However, the mechanism of Na+ sensing in MΦs remained unclear. High extracellular Na+ levels (high salt [HS]) trigger a substantial Na+ influx and Ca2+ loss. Here, we show that the Na+/Ca2+ exchanger 1 (NCX1, also known as solute carrier family 8 member A1 [SLC8A1]) plays a critical role in HS-triggered Na+ influx, concomitant Ca2+ efflux, and subsequent augmented NFAT5 accumulation. Moreover, interfering with NCX1 activity impairs HS-boosted inflammatory signaling, infection-triggered autolysosome formation, and subsequent antibacterial activity. Taken together, this demonstrates that NCX1 is able to sense Na+ and is required for amplifying inflammatory and antimicrobial MΦ responses upon HS exposure. Manipulating NCX1 offers a new strategy to regulate MΦ function.

Macrófagos/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Sódio/metabolismo , Processamento Alternativo/genética , Animais , Cálcio/metabolismo , Espaço Extracelular/metabolismo , Inativação Gênica/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Íons , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Óxido Nítrico/biossíntese , Células RAW 264.7 , Cloreto de Sódio/farmacologia
J Mol Cell Cardiol ; 144: 35-46, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32418916


OBJECTIVE: In myocardial pathology such as heart failure a late sodium current (INaL) augmentation is known to be involved in conditions of arrhythmogenesis. However, the underlying mechanisms of the INaL generation are not entirely understood. By now evidence is growing that non-cardiac sodium channel isoforms could also be involved in the INaL generation. The present study investigates the contribution of the neuronal sodium channel isoform NaV1.8 to arrhythmogenesis in a clearly-defined setting of enhanced INaL by using anemone toxin II (ATX-II) in the absence of structural heart disease. METHODS: Electrophysiological experiments were performed in order to measure INaL, action potential duration (APD), SR-Ca2+-leak and cellular proarrhythmic triggers in ATX-II exposed wild-type (WT) and SCN10A-/- mice cardiomyocytes. In addition, WT cardiomyocytes were stimulated with ATX-II in the presence or absence of NaV1.8 inhibitors. INCX was measured by using the whole cell patch clamp method. RESULTS: In WT cardiomyocytes exposure to ATX-II augmented INaL, prolonged APD, increased SR-Ca2+-leak and induced proarrhythmic triggers such as early afterdepolarizations (EADs) and Ca2+-waves. All of them could be significantly reduced by applying NaV1.8 blockers PF-01247324 and A-803467. Both blockers had no relevant effects on cellular electrophysiology of SCN10A-/- cardiomyocytes. Moreover, in SCN10A-/--cardiomyocytes, the ATX-II-dependent increase in INaL, SR-Ca2+-leak and APD prolongation was less than in WT and comparable to the results which were obtained with WT cardiomyocytes being exposed to ATX-II and NaV1.8 inhibitors in parallel. Moreover, we found a decrease in reverse mode NCX current and reduced CaMKII-dependent RyR2-phosphorylation after application of PF-01247324 as an underlying explanation for the Na+-mediated Ca2+-dependent proarrhythmic triggers. CONCLUSION: The current findings demonstrate that NaV1.8 is a significant contributor for INaL-induced arrhythmic triggers. Therefore, NaV1.8 inhibition under conditions of an enhanced INaL constitutes a promising antiarrhythmic strategy which merits further investigation.

ESC Heart Fail ; 5(4): 642-648, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30117720


AIMS: The EMPA-REG OUTCOME study showed reduced mortality and hospitalization due to heart failure (HF) in diabetic patients treated with empagliflozin. Overexpression and Ca2+ -dependent activation of Ca2+ /calmodulin-dependent kinase II (CaMKII) are hallmarks of HF, leading to contractile dysfunction and arrhythmias. We tested whether empagliflozin reduces CaMKII- activity and improves Ca2+ -handling in human and murine ventricular myocytes. METHODS AND RESULTS: Myocytes from wild-type mice, mice with transverse aortic constriction (TAC) as a model of HF, and human failing ventricular myocytes were exposed to empagliflozin (1 µmol/L) or vehicle. CaMKII activity was assessed by CaMKII-histone deacetylase pulldown assay. Ca2+ spark frequency (CaSpF) as a measure of sarcoplasmic reticulum (SR) Ca2+ leak was investigated by confocal microscopy. [Na+ ]i was measured using Na+ /Ca2+ -exchanger (NCX) currents (whole-cell patch clamp). Compared with vehicle, 24 h empagliflozin exposure of murine myocytes reduced CaMKII activity (1.6 ± 0.7 vs. 4.2 ± 0.9, P < 0.05, n = 10 mice), and also CaMKII-dependent ryanodine receptor phosphorylation (0.8 ± 0.1 vs. 1.0 ± 0.1, P < 0.05, n = 11 mice), with similar results upon TAC. In murine myocytes, empagliflozin reduced CaSpF (TAC: 1.7 ± 0.3 vs. 2.5 ± 0.4 1/100 µm-1  s-1 , P < 0.05, n = 4 mice) but increased SR Ca2+ load and Ca2+ transient amplitude. Importantly, empagliflozin also significantly reduced CaSpF in human failing ventricular myocytes (1 ± 0.2 vs. 3.3 ± 0.9, P < 0.05, n = 4 patients), while Ca2+ transient amplitude was increased (F/F0 : 0.53 ± 0.05 vs. 0.36 ± 0.02, P < 0.05, n = 3 patients). In contrast, 30 min exposure with empagliflozin did not affect CaMKII activity nor Ca2+ -handling but significantly reduced [Na+ ]i . CONCLUSIONS: We show for the first time that empagliflozin reduces CaMKII activity and CaMKII-dependent SR Ca2+ leak. Reduced Ca2+ leak and improved Ca2+ transients may contribute to the beneficial effects of empagliflozin in HF.

Compostos Benzidrílicos/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Glucosídeos/farmacologia , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Western Blotting , Sinalização do Cálcio , Células Cultivadas , Modelos Animais de Doenças , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia