María C. Villa-Abrille

The Positive Inotropic Effect of Angiotensin II: Role of Endothelin-1 and Reactive Oxygen Species

Many effects believed to be because of angiotensin II (Ang II) are attributable to the action of endothelin (ET)-1, which is released/produced by Ang II. We investigated whether Ang II elicits its positive inotropic effect (PIE) by the action of endogenous ET-1, in addition to the role played by reactive oxygen species (ROS) in this mechanism. Cat cardiomyocytes were used for: (1) sarcomere shortening measurements; (2) ROS measurements by epifluorescence; (3) immunohistochemical staining for preproET-1, BigET-1, and ET-1; and (4) measurement of preproET-1 mRNA by RT-PCR. Cells were exposed to 1 nmol/L Ang II for 15 minutes. This low concentration of Ang II increases sarcomere shortening by 29.2±3.7% (P<0.05). This PIE was abrogated by Na+/H+ exchanger or Na+/Ca2+ exchanger reverse mode inhibition. The production of ROS increased in response to Ang II treatment (&Dgr;ROS respect to control: 68±15 fluorescence units; P<0.05). The Ang II–induced PIE and ROS production were blocked by the Ang II type 1 receptor blocker losartan, the nonselective ET-1 receptor blocker TAK044, the selective ETA receptor blocker BQ-123, or the ROS scavenger N-(2-mercapto-propionyl)glycine. Exogenous ET-1 (0.4 nmol/L) induced a similar PIE and increase in ROS production to those caused by Ang II. Immunostaining for preproET-1, BigET-1, and ET-1 was positive in cardiomyocytes. The preproET-1 mRNA abundance increased from 100±4.6% in control to 241.9±39.9% in Ang II–treated cells (P<0.05). We conclude that the PIE after exposure to 1 nmol/L Ang II is due to endogenous ET-1 acting through the ETA receptor and triggering ROS production, Na+/H+ exchanger stimulation, and Na+/Ca2+ exchanger reverse mode activation.

The positive inotropic effect of endothelin-1 is mediated by mitochondrial reactive oxygen species.

We have previously demonstrated the participation of reactive oxygen species (ROS) in the positive inotropic effect of a physiological concentration of Angiotensin II (Ang II, 1 nM). The objective of the present work was to evaluate the role and source of ROS generation in the positive inotropic effect produced by an equipotent concentration of endothelin-1 (ET-1, 0.4 nM). Isolated cat ventricular myocytes were used to measure sarcomere shortening with a video-camera, superoxide anion (()O(2)(-)) with chemiluminescence, and ROS production and intracellular pH (pH(i)) with epifluorescence. The ET-1-induced positive inotropic effect (40.4+/-3.1%, n=10, p<0.05) was associated to an increase in ROS production (105+/-29 fluorescence units above control, n=6, p<0.05). ET-1 also induced an increase in ()O(2)(-) production that was inhibited by the NADPH oxidase blocker, apocynin, and by the blockers of mitochondrial ATP-sensitive K(+) channels (mK(ATP)), glibenclamide and 5 hydroxydecanoic acid. The ET-1-induced positive inotropic effect was inhibited by apocynin (0.3 mM; 6.3+/-6.6%, n=13), glibenclamide (50 microM; 8.8+/-3.5%, n=6), 5 hydroxydecanoic acid (500 microM; 14.1+/-8.1, n=9), and by scavenging ROS with MPG (2 mM; 0.92+/-5.6%, n=8). ET-1 enhanced proton efflux (J(H)) carried by the Na(+)/H(+) exchanger (NHE) after an acid load, effect that was blocked by MPG. Consistently, the ET-induced positive inotropic effect was also inhibited by the NHE selective blocker HOE642 (5 microM; 9.37+/-6.07%, n=7). The data show that the effect of a concentration of ET-1 that induces an increase in contractility of about 40% is totally mediated by an intracellular pathway triggered by mitochondrial ROS formation and stimulation of the NHE.

Aldosterone Stimulates the Cardiac Na+/H+ Exchanger via Transactivation of the Epidermal Growth Factor Receptor

The use of antagonists of the mineralocorticoid receptor in the treatment of myocardial hypertrophy and heart failure has gained increasing importance in the last years. The cardiac Na+/H+ exchanger (NHE-1) upregulation induced by aldosterone could account for the genesis of these pathologies. We tested whether aldosterone-induced NHE-1 stimulation involves the transactivation of the epidermal growth factor receptor (EGFR). Rat ventricular myocytes were used to measure intracellular pH with epifluorescence. Aldosterone enhanced the NHE-1 activity. This effect was canceled by spironolactone or eplerenone (mineralocorticoid receptor antagonists), but not by mifepristone (glucocorticoid receptor antagonist) or cycloheximide (protein synthesis inhibitor), indicating that the mechanism is mediated by the mineralocorticoid receptor triggering nongenomic pathways. Aldosterone-induced NHE-1 stimulation was abolished by the EGFR kinase inhibitor AG1478, suggesting that is mediated by transactivation of EGFR. The increase in the phosphorylation level of the kinase p90RSK and NHE-1 serine703 induced by aldosterone was also blocked by AG1478. Exogenous epidermal growth factor mimicked the effects of aldosterone on NHE-1 activity. Epidermal growth factor was also able to increase reactive oxygen species production, and the epidermal growth factor–induced activation of the NHE-1 was abrogated by the reactive oxygen species scavenger N-2-mercaptopropionyl glycine, indicating that reactive oxygen species are participating as signaling molecules in this mechanism. Aldosterone enhances the NHE-1 activity via transactivation of the EGFR, formation of reactive oxygen species, and phosphorylation of the exchanger. These results call attention to the consideration of the EGFR as a new potential therapeutic target of the cardiovascular pathologies involving the participation of aldosterone.

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.

The Anrep effect requires transactivation of the epidermal growth factor receptor

Myocardial stretch elicits a biphasic contractile response: the Frank–Starling mechanism followed by the slow force response (SFR) or Anrep effect. In this study we hypothesized that the SFR depends on epidermal growth factor receptor (EGFR) transactivation after the myocardial stretch‐induced angiotensin II (Ang II)/endothelin (ET) release. Experiments were performed in isolated cat papillary muscles stretched from 92 to 98% of the length at which maximal twitch force was developed (Lmax). The SFR was 123 ± 1% of the immediate rapid phase (n= 6, P < 0.05) and was blunted by preventing EGFR transactivation with the Src‐kinase inhibitor PP1 (99 ± 2%, n= 4), matrix metalloproteinase inhibitor MMPI (108 ± 4%, n= 11), the EGFR blocker AG1478 (98 ± 2%, n= 6) or the mitochondrial transition pore blocker clyclosporine (99 ± 3%, n= 6). Stretch increased ERK1/2 phosphorylation by 196 ± 17% of control (n= 7, P < 0.05), an effect that was prevented by PP1 (124 ± 22%, n= 7) and AG1478 (131 ± 17%, n= 4). In myocardial slices, Ang II (which enhances ET mRNA) or endothelin‐1 (ET‐1)‐induced increase in O2− production (146 ± 14%, n= 9, and 191 ± 17%, n= 13, of control, respectively, P < 0.05) was cancelled by AG1478 (94 ± 5%, n= 12, and 98 ± 15%, n= 8, respectively) or PP1 (100 ± 4%, n= 6, and 99 ± 8%, n= 3, respectively). EGF increased O2− production by 149 ± 4% of control (n= 9, P < 0.05), an effect cancelled by inhibiting NADPH oxidase with apocynin (110 ± 6%n= 7), mKATP channels with 5‐hydroxydecanoic acid (5‐HD; 105 ± 5%, n= 8), the respiratory chain with rotenone (110 ± 7%, n= 7) or the mitochondrial permeability transition pore with cyclosporine (111 ± 10%, n= 6). EGF increased ERK1/2 phosphorylation (136 ± 8% of control, n= 9, P < 0.05), which was blunted by 5‐HD (97 ± 5%, n= 4), suggesting that ERK1/2 activation is downstream of mitochondrial oxidative stress. Finally, stretch increased Ser703 Na+/H+ exchanger‐1 (NHE‐1) phosphorylation by 172 ± 24% of control (n= 4, P < 0.05), an effect that was cancelled by AG1478 (94 ± 17%, n= 4). In conclusion, our data show for the first time that EGFR transactivation is crucial in the chain of events leading to the Anrep effect.

Silencing of cardiac mitochondrial NHE1 prevents mitochondrial permeability transition pore opening.

Inhibition of Na(+)/H(+) exchanger 1 (NHE1) reduces cardiac ischemia-reperfusion (I/R) injury and also cardiac hypertrophy and failure. Although the mechanisms underlying these NHE1-mediated effects suggest delay of mitochondrial permeability transition pore (MPTP) opening, and reduction of mitochondrial-derived superoxide production, the possibility of NHE1 blockade targeting mitochondria has been incompletely explored. A short-hairpin RNA sequence mediating specific knock down of NHE1 expression was incorporated into a lentiviral vector (shRNA-NHE1) and transduced in the rat myocardium. NHE1 expression of mitochondrial lysates revealed that shRNA-NHE1 transductions reduced mitochondrial NHE1 (mNHE1) by ∼60%, supporting the expression of NHE1 in mitochondria membranes. Electron microscopy studies corroborate the presence of NHE1 in heart mitochondria. Immunostaining of rat cardiomyocytes also suggests colocalization of NHE1 with the mitochondrial marker cytochrome c oxidase. To examine the functional role of mNHE1, mitochondrial suspensions were exposed to increasing concentrations of CaCl(2) to induce MPTP opening and consequently mitochondrial swelling. shRNA-NHE1 transduction reduced CaCl(2)-induced mitochondrial swelling by 64 ± 4%. Whereas the NHE1 inhibitor HOE-642 (10 μM) decreased mitochondrial Ca(2+)-induced swelling in rats transduced with nonsilencing RNAi (37 ± 6%), no additional HOE-642 effects were detected in mitochondria from rats transduced with shRNA-NHE1. We have characterized the expression and function of NHE1 in rat heart mitochondria. Because mitochondria from rats injected with shRNA-NHE1 present a high threshold for MPTP formation, the beneficial effects of NHE1 inhibition in I/R resulting from mitochondrial targeting should be considered.

Antibodies against the cardiac sodium/bicarbonate co-transporter (NBCe1) as pharmacological tools

BACKGROUND AND PURPOSE Na+/HCO3‐ co‐transport (NBC) regulates intracellular pH (pHi) in the heart. We have studied the electrogenic NBC isoform NBCe1 by examining the effect of functional antibodies to this protein.

The electrogenic Na+/HCO3− cotransport modulates resting membrane potential and action potential duration in cat ventricular myocytes

Perforated whole‐cell configuration of patch clamp was used to determine the contribution of the electrogenic Na+/HCO3− cotransport (NBC) on the shape of the action potential in cat ventricular myocytes. Switching from Hepes to HCO3− buffer at constant extracellular pH (pHo) hyperpolarized resting membrane potential (RMP) by 2.67 ± 0.42 mV (n= 9, P < 0.05). The duration of action potential measured at 50% of repolarization time (APD50) was 35.8 ± 6.8% shorter in the presence of HCO3− than in its absence (n= 9, P < 0.05). The anion blocker SITS prevented and reversed the HCO3−‐induced hyperpolarization and shortening of APD. In addition, no HCO3−‐induced hyperpolarization and APD shortening was observed in the absence of extracellular Na+. Quasi‐steady‐state currents were evoked by 8 s duration voltage‐clamped ramps ranging from −130 to +30 mV. A novel component of SITS‐sensitive current was observed in the presence of HCO3−. The HCO3−‐sensitive current reversed at −87 ± 5 mV (n= 7), a value close to the expected reversal potential of an electrogenic Na+/HCO3− cotransport with a HCO3−:Na+ stoichiometry ratio of 2: 1. The above results allow us to conclude that the cardiac electrogenic Na+/HCO3− cotransport has a relevant influence on RMP and APD of cat ventricular cells.

Silencing of NHE-1 blunts the slow force response to myocardial stretch

Myocardial stretch induces a biphasic force response: a first abrupt increase followed by a slow force response (SFR), believed to be the in vitro manifestation of the Anrep effect. The SFR is due to an increase in Ca²⁺ transient of unclear mechanism. We proposed that Na⁺/H⁺ exchanger (NHE-1) activation is a key factor in determining the contractile response, but recent reports challenged our findings. We aimed to specifically test the role of the NHE-1 in the SFR. To this purpose small hairpin interference RNA capable of mediating specific NHE-1 knockdown was incorporated into a lentiviral vector (l-shNHE1) and injected into the left ventricular wall of Wistar rats. Injection of a lentiviral vector expressing a nonsilencing sequence (scramble) served as control. Myocardial NHE-1 protein expression and function (the latter evaluated by the recovery of pH(i) after an acidic load and the SFR) were evaluated. Animals transduced with l-shNHE1 showed reduced NHE-1 expression (45 ± 8% of controls; P < 0.05), and the presence of the lentivirus in the left ventricular myocardium, far from the site of injection, was evidenced by confocal microscopy. These findings correlated with depressed basal pH(i) recovery after acidosis [(max)dpH(i)/dt 0.055 ± 0.008 (scramble) vs. 0.009 ± 0.004 (l-shNHE1) pH units/min, P < 0.05], leftward shift of the relationship between J(H⁺) (H⁺ efflux corrected by the intrinsic buffer capacity), and abolishment of SFR (124 ± 2 vs. 101 ± 2% of rapid phase; P < 0.05) despite preserved ERK1/2 phosphorylation [247 ± 12 (stretch) and 263 ± 23 (stretch l-shNHE1) % of control; P < 0.05 vs. nonstretched control], well-known NHE-1 activators. Our results provide strong evidence to propose NHE-1 activation as key factor in determining the SFR to stretch.

Early signals after stretch leading to cardiac hypertrophy. Key role of NHE-1.

The enhanced activity of the cardiac Na+/H+ exchanger (NHE-1) after myocardial stretch is considered a key step of the intracellular signaling pathway leading to the slow force response to stretch as well as an early signal for the development of cardiac hypertrophy. We propose that the chain of events triggered by stretch begins with the release of small amounts of Angiotensin II (Ang II)/endothelin (ET) and ends with the increase in intracellular Ca2+ concentration ([Ca2+]i) through the Na+/Ca2+ exchanger in reverse mode (NCX(rev)), which triggers cardiac hypertrophy by activation of widely recognized Ca2+-dependent intracellular signaling pathways.