Nitroso-redox balance: a key mechanism in the regulation of the myocardial function in health and disease

Given the close interaction between nitric oxide (NO) and reactive oxygen species (ROS) in biological systems and their especial relevance in regulating aspects of the cardiovascular physiology, the concept of “nitroso-redox balance” has arisen as a more comprehensive manner of interpreting the intracellular redox state. Nitroso-redox signaling pathways participate in numerous cardiovascular mechanisms, including myocardial contractility and relaxation, mitochondrial respiration, and endothelial function. Alterations of this balance are involved in numerous aspects of cardiovascular pathophysiology. NO and ROS generating mechanisms play a major role in both regulating and responding to the redox state of the cell, which targets calcium handling, contractile and vasoactive mechanisms. Thus, the nitroso-redox signaling pathway is critically important in cardiac physiology and pathophysiology, and consequently a fundamental therapeutic target. This article briefly addresses the cardiovascular implications of the biological balance between NO and ROS, and their relevance in the development of heart diseases.Dada la íntima interacción entre el óxido nítrico (NO) y las especies reactivas de oxigeno (ROS) en los sistemas biológicos, y su especial relevancia en la regulación de la fisiología cardiovascular, ha surgido el concepto de “balance nitroso-redox” como una forma más inclusiva para interpretar el estado redox intracelular. Las vías de señalización nitroso-redox participan en numerosos mecanismos cardiovasculares, tales como contractilidad y relajación miocárdica, respiración mitocondrial y función endotelial. Las alteraciones de este balance están involucradas en muchos aspectos de la fisiopatología cardiovascular. Los sistemas que producen NO y ROS juegan un rol clave tanto en la regulación como en la respuesta al estado redox de la célula, el cual afecta el manejo de calcio y mecanismos contráctiles y vasoactivos. Así, las vías de señalización nitroso-redox son claves para la fisiología y fisiopatología cardiaca, y consecuentemente son un blanco terapéutico fundamental. Este artículo aborda brevemente las implicaciones cardiovasculares del balance biológico entre NO y ROS, y su relevancia en el desarrollo de enfermedades cardiacas.Sociedad Argentina de Fisiologí

Endothelin-1 stimulates the Na+/Ca2+ exchanger reverse mode through intracellular Na+ (Na+i) - Dependent and Na+i-independent pathways

This study aimed to explore the signaling pathways involved in the positive inotropic effect (PIE) of low doses of endothelin-1 (ET-1). Cat papillary muscles were used for force and intracellular Na+ concentration (Na+i) measurements, and isolated cat ventricular myocytes for patch-clamp experiments. ET-1 (5 nmol/L) induced a PIE and an associated increase in Na+i that were abolished by Na +/H+ exchanger (NHE) inhibition with HOE642. Reverse-mode Na+/Ca2+ exchanger (NCX) blockade with KB-R7943 reversed the ET-1-induced PIE. These results suggest that the ET-1-induced PIE is totally attributable to the NHE-mediated Na+i increase. However, an additional direct stimulating effect of ET-1 on NCX after the necessary increase in Na+i could occur. Thus, the ET-1-induced increase in Na+i and contractility was compared with that induced by partial inhibition of the Na+/K+ ATPase by lowering extracellular K+ (K+o) For a given Na+i, ET-1 induced a greater PIE than low K+o. In the presence of HOE642 and after increasing contractility and Na+i by low K+o, ET-1 induced an additional PIE that was reversed by KB-R7943 or the protein kinase C (PKC) inhibitor chelerythrine. ET-1 increased the NCX current and negatively shifted the NCX reversal potential (ENCX). HOE642 attenuated the increase in NCX outward current and abolished the ENCX shift. These results indicate that whereas the NHE-mediated ET-1-induced increase in Na+i seems to be mandatory to drive NCX in reverse and enhance contractility, Na+i-independent and PKC-dependent NCX stimulation appears to additionally contribute to the PIE. However, it is important to stress that the latter can only occur after the primary participation of the former.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

A low dose of angiotensin II increases inotropism through activation of reverse Na+/Ca2+ exchange by endothelin release

Objective: This work was aimed to prove that release/formation of endogenous endothelin acting in an autocrine/paracrine fashion contributes to the increase in contractility promoted by a low dose of angiotensin II. Methods: Isolated cat papillary muscles were used for force, pHi, [Na+]i and [Ca2+]i measurements and isolated cat myocytes for patch-clamp experiments. Results: In papillary muscles, 1.0 nmol/l angiotensin II increased force by 23±2% (n=4, P+]i by 2.2±0.2 mmol/l (n=4, P2+ from 0.674±0.11 to 0.768±0.13 μmol/l (n=4, P+]i increase were abolished by inhibition of the Na+/H+ exchanger (NHE) with the inhibitor HOE642, blockade of endothelin receptors with the nonselective antagonist TAK044 and by inhibition of the endothelin-converting enzyme with phosphoramidon. Force but not [Na+]i increase was abolished by inhibition of reverse Na+/Ca2+ exchange (NCX) with the inhibitor KB-R7943. Similar increase in force (21±2%, n=4, P+]i (2.4±0.4 mmol/l, n=4, P+]i. In isolated myocytes, exogenous endothelin-1 dose-dependently increased the NCX current and shifted the NCX reversal potential (ENCX) to a more negative value (ΔENCX: -10±3 and -17±5 mV, with 1 and 10 nmol/l endothelin-1, respectively, n=12). The latter effect was prevented by HOE642. Conclusion: Taken together, the results indicate that a low dose of angiotensin II induces release of endothelin, which, in autocrine/paracrine fashion activates the Na+/H+ exchanger, increases [Na+]i and changes ENCX, promoting the influx of Ca2+ that leads to a positive inotropic effect (PIE).Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

A low dose of angiotensin II increases inotropism through activation of reverse Na+/Ca2+ exchange by endothelin release

Objective: This work was aimed to prove that release/formation of endogenous endothelin acting in an autocrine/paracrine fashion contributes to the increase in contractility promoted by a low dose of angiotensin II. Methods: Isolated cat papillary muscles were used for force, pHi, [Na+]i and [Ca2+]i measurements and isolated cat myocytes for patch-clamp experiments. Results: In papillary muscles, 1.0 nmol/l angiotensin II increased force by 23±2% (n=4, P+]i by 2.2±0.2 mmol/l (n=4, P2+ from 0.674±0.11 to 0.768±0.13 μmol/l (n=4, P+]i increase were abolished by inhibition of the Na+/H+ exchanger (NHE) with the inhibitor HOE642, blockade of endothelin receptors with the nonselective antagonist TAK044 and by inhibition of the endothelin-converting enzyme with phosphoramidon. Force but not [Na+]i increase was abolished by inhibition of reverse Na+/Ca2+ exchange (NCX) with the inhibitor KB-R7943. Similar increase in force (21±2%, n=4, P+]i (2.4±0.4 mmol/l, n=4, P+]i. In isolated myocytes, exogenous endothelin-1 dose-dependently increased the NCX current and shifted the NCX reversal potential (ENCX) to a more negative value (ΔENCX: -10±3 and -17±5 mV, with 1 and 10 nmol/l endothelin-1, respectively, n=12). The latter effect was prevented by HOE642. Conclusion: Taken together, the results indicate that a low dose of angiotensin II induces release of endothelin, which, in autocrine/paracrine fashion activates the Na+/H+ exchanger, increases [Na+]i and changes ENCX, promoting the influx of Ca2+ that leads to a positive inotropic effect (PIE).Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

Endothelin-1 stimulates the Na+/Ca2+ exchanger reverse mode through intracellular Na+ (Na+i) - Dependent and Na+i-independent pathways

This study aimed to explore the signaling pathways involved in the positive inotropic effect (PIE) of low doses of endothelin-1 (ET-1). Cat papillary muscles were used for force and intracellular Na+ concentration (Na+i) measurements, and isolated cat ventricular myocytes for patch-clamp experiments. ET-1 (5 nmol/L) induced a PIE and an associated increase in Na+i that were abolished by Na +/H+ exchanger (NHE) inhibition with HOE642. Reverse-mode Na+/Ca2+ exchanger (NCX) blockade with KB-R7943 reversed the ET-1-induced PIE. These results suggest that the ET-1-induced PIE is totally attributable to the NHE-mediated Na+i increase. However, an additional direct stimulating effect of ET-1 on NCX after the necessary increase in Na+i could occur. Thus, the ET-1-induced increase in Na+i and contractility was compared with that induced by partial inhibition of the Na+/K+ ATPase by lowering extracellular K+ (K+o) For a given Na+i, ET-1 induced a greater PIE than low K+o. In the presence of HOE642 and after increasing contractility and Na+i by low K+o, ET-1 induced an additional PIE that was reversed by KB-R7943 or the protein kinase C (PKC) inhibitor chelerythrine. ET-1 increased the NCX current and negatively shifted the NCX reversal potential (ENCX). HOE642 attenuated the increase in NCX outward current and abolished the ENCX shift. These results indicate that whereas the NHE-mediated ET-1-induced increase in Na+i seems to be mandatory to drive NCX in reverse and enhance contractility, Na+i-independent and PKC-dependent NCX stimulation appears to additionally contribute to the PIE. However, it is important to stress that the latter can only occur after the primary participation of the former.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

Hypotonic swelling promotes nitric oxide release in cardiac ventricular myocytes: Impact on swelling-induced negative inotropic effect

Aims Cardiomyocyte swelling occurs in multiple pathological situations and has been associated with contractile dysfunction, cell death, and enhanced propensity to arrhythmias.We investigate whether hypotonic swelling promotes nitric oxide (NO) release in cardiomyocytes, and whether it impacts on swelling-induced contractile dysfunction. Methods and results Superfusing rat cardiomyocytes with a hypotonic solution (HS; 217 mOsm), increased cell volume, reduced myocyte contraction and Ca2+ transient, and increased NO-sensitive 4-amino-5-methylamino-2', 7'-difluorofluorescein diacetate (DAF-FM) fluorescence. When cells were exposed to HS + 2.5 mMof theNOsynthase inhibitor L-NAME, cell swelling occurred in the absence of NOrelease. Swelling-induced NO release was also prevented by the nitric oxide synthase 1 (NOS1) inhibitor, nitroguanidine, and significantly reduced in NOS1 knockout mice. Additionally, colchicine (inhibitor of microtubule polymerization) prevented the increase in DAF-FM fluorescence induced by HS, indicating that microtubule integrity is necessary for swelling-inducedNOrelease. The swelling-induced negative inotropic effectwas exacerbated in the presence of either L-NAME, nitroguandine, the guanylate cyclase inhibitor, ODQ, or the PKG inhibitor, KT5823, suggesting that NOS1-derived NO provides contractile support via a cGMP/PKG-dependent mechanism. Indeed, ODQ reduced Ca2+ wave velocity and both ODQ and KT5823 reduced the HS-induced increment in ryanodine receptor (RyR2, Ser2808) phosphorylation, suggesting that in this context, cGMP/PKG may contribute to preserve contractile function by enhancing sarcoplasmic reticulum Ca2+ release. Conclusions Our findings suggest a novel mechanism for NO release in cardiomyocytes with putative pathophysiological relevance determined, at least in part, by its capability to reduce the extent of contractile dysfunction associated with hypotonic swelling.Centro de Investigaciones Cardiovasculare

Mitochondrial reactive oxygen species activate the slow force response to stretch in feline myocardium

When the length of the myocardium is increased, a biphasic response to stretch occurs involving an initial rapid increase in force followed by a delayed slow increase called the slow force response (SFR). Confirming previous findings involving angiotensin II in the SFR, it was blunted by AT1 receptor blockade (losartan). The SFR was accompanied by an increase in reactive oxygen species (ROS) of ∼30% and in intracellular Na+ concentration ([Na⁺]i) of ∼2.5 mmol l⁻¹ over basal detected by H2DCFDA and SBFI fluorescence, respectively. Abolition of ROS by 2-mercapto-propionyl-glycine (MPG) and EUK8 suppressed the increase in [Na⁺]i and the SFR, which were also blunted by Na⁺/H⁺ exchanger (NHE-1) inhibition (HOE642). NADPH oxidase inhibition (apocynin or DPI) or blockade of the ATP-sensitive mitochondrial potassium channels (5HD or glybenclamide) suppressed both the SFR and the increase in [Na⁺]i after stretch, suggesting that endogenous angiotensin II activated NADPH oxidase leading to ROS release by the ATP-sensitive mitochondrial potassium channels, which promoted NHE-1 activation. Supporting the notion of ROS-mediated NHE-1 activation, stretch increased the ERK1/2 and p90rsk kinases phosphorylation, effect that was cancelled by losartan. In agreement, the SFR was cancelled by inhibiting the ERK1/2 signalling pathway with PD98059. Angiotensin II at a dose that mimics the SFR (1 nmol l⁻¹) induced an increase in ·O₂− production of ∼30–40% detected by lucigenin in cardiac slices, an effect that was blunted by losartan, MPG, apocynin, 5HD and glybenclamide. Taken together the data suggest a pivotal role of mitochondrial ROS in the genesis of the SFR to stretch.Centro de Investigaciones Cardiovasculare

Influence of Na+-Independent Cl¯-HCO3¯ Exchange on the Slow Force Response to Myocardial Stretch

Previous work demonstrated that the slow force response (SFR) to stretch is due to the increase in calcium transients (Ca2+T) produced by an autocrine-paracrine mechanism of locally produced angiotensin II/endothelin activating Na+-H+ exchange. Although a rise in pHi is presumed to follow stretch, it was observed only in the absence of extracellular bicarbonate, suggesting pHi compensation through the Na+-independent Cl¯-HCO3¯ exchange (AE) mechanism. Because available AE inhibitors do not distinguish between different bicarbonate-dependent mechanisms or even between AE isoforms, we developed a functional inhibitory antibody against both the AE3c and AE3fl isoforms (anti-AE3Loop III) that was used to explore if pHi would rise in stretched cat papillary muscles superfused with bicarbonate after AE3 inhibition. In addition, the influence of this potential increase in pHi on the SFR was analyzed. In this study, we present evidence that cancellation of AE3 isoforms activity (either by superfusion with bicarbonate-free buffer or with anti-AE3Loop III) results in pHi increase after stretch and the magnitude of the SFR was larger than when AE was operative, despite of similar increases in [Na+]i and Ca2+T under both conditions. Inhibition of reverse mode Na+-Ca2+ exchange reduced the SFR to the half when the AE was inactive and totally suppressed it when AE3 was active. The difference in the SFR magnitude and response to inhibition of reverse mode Na+-Ca2+ exchange can be ascribed to a pHi-induced increase in myofilament Ca2+ responsiveness.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

Influence of Na+-Independent Cl¯-HCO3¯ Exchange on the Slow Force Response to Myocardial Stretch

Previous work demonstrated that the slow force response (SFR) to stretch is due to the increase in calcium transients (Ca2+T) produced by an autocrine-paracrine mechanism of locally produced angiotensin II/endothelin activating Na+-H+ exchange. Although a rise in pHi is presumed to follow stretch, it was observed only in the absence of extracellular bicarbonate, suggesting pHi compensation through the Na+-independent Cl¯-HCO3¯ exchange (AE) mechanism. Because available AE inhibitors do not distinguish between different bicarbonate-dependent mechanisms or even between AE isoforms, we developed a functional inhibitory antibody against both the AE3c and AE3fl isoforms (anti-AE3Loop III) that was used to explore if pHi would rise in stretched cat papillary muscles superfused with bicarbonate after AE3 inhibition. In addition, the influence of this potential increase in pHi on the SFR was analyzed. In this study, we present evidence that cancellation of AE3 isoforms activity (either by superfusion with bicarbonate-free buffer or with anti-AE3Loop III) results in pHi increase after stretch and the magnitude of the SFR was larger than when AE was operative, despite of similar increases in [Na+]i and Ca2+T under both conditions. Inhibition of reverse mode Na+-Ca2+ exchange reduced the SFR to the half when the AE was inactive and totally suppressed it when AE3 was active. The difference in the SFR magnitude and response to inhibition of reverse mode Na+-Ca2+ exchange can be ascribed to a pHi-induced increase in myofilament Ca2+ responsiveness.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

A low dose of angiotensin II increases inotropism through activation of reverse Na+/Ca2+ exchange by endothelin release

Objective: This work was aimed to prove that release/formation of endogenous endothelin acting in an autocrine/paracrine fashion contributes to the increase in contractility promoted by a low dose of angiotensin II. Methods: Isolated cat papillary muscles were used for force, pHi, [Na+]i and [Ca2+]i measurements and isolated cat myocytes for patch-clamp experiments. Results: In papillary muscles, 1.0 nmol/l angiotensin II increased force by 23±2% (n=4, P+]i by 2.2±0.2 mmol/l (n=4, P2+ from 0.674±0.11 to 0.768±0.13 μmol/l (n=4, P+]i increase were abolished by inhibition of the Na+/H+ exchanger (NHE) with the inhibitor HOE642, blockade of endothelin receptors with the nonselective antagonist TAK044 and by inhibition of the endothelin-converting enzyme with phosphoramidon. Force but not [Na+]i increase was abolished by inhibition of reverse Na+/Ca2+ exchange (NCX) with the inhibitor KB-R7943. Similar increase in force (21±2%, n=4, P+]i (2.4±0.4 mmol/l, n=4, P+]i. In isolated myocytes, exogenous endothelin-1 dose-dependently increased the NCX current and shifted the NCX reversal potential (ENCX) to a more negative value (ΔENCX: -10±3 and -17±5 mV, with 1 and 10 nmol/l endothelin-1, respectively, n=12). The latter effect was prevented by HOE642. Conclusion: Taken together, the results indicate that a low dose of angiotensin II induces release of endothelin, which, in autocrine/paracrine fashion activates the Na+/H+ exchanger, increases [Na+]i and changes ENCX, promoting the influx of Ca2+ that leads to a positive inotropic effect (PIE).Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare