Alejandra M. Yeves

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.

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.

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.

Decreased Activity of the Na+/H+ Exchanger by Phosphodiesterase 5A Inhibition Is Attributed to an Increase in Protein Phosphatase Activity

The beneficial effect of phosphodiesterase 5A inhibition in ischemia/reperfusion injury and cardiac hypertrophy is well established. Inhibition of the cardiac Na+/H+ exchanger (NHE-1) exerts beneficial effects on these same conditions, and a possible link between these therapeutic strategies was suggested. Experiments were performed in isolated cat cardiomyocytes to gain insight into the intracellular pathway involved in the reduction of NHE-1 activity by phosphodiesterase 5A inhibition. NHE-1 activity was assessed by the rate of intracellular pH recovery from a sustained acidic load in the absence of bicarbonate. Phosphodiesterase 5A inhibition with sildenafil (1 &mgr;mol/L) did not affect basal intracellular pH; yet, it did decrease proton efflux (JH; in millimoles per liter per minute) after the acidic load (proton efflux: 6.97±0.43 in control versus 3.31±0.58 with sildenafil; P<0.05). The blockade of both protein phosphatase 1 and 2A with 100 nmol/L of okadaic acid reverted the sildenafil effect (proton efflux: 6.77±0.82). In contrast, selective inhibition of protein phosphatase 2A (1 nmol/L of okadaic acid or 100 &mgr;mol/L of endothall) did not (3.86±1.0 and 2.61±1.2), suggesting that only protein phosphatase 1 was involved in sildenafil-induced NHE-1 inhibition. Moreover, sildenafil prevented the acidosis-induced increase in NHE-1 phosphorylation without affecting activation of the extracellular signal–regulated kinase 1/2-p90RSK pathway. Our results suggest that phosphodiesterase 5A inhibition decreases NHE-1 activity, during intracellular pH recovery after an acidic load, by a protein phosphatase 1–dependent reduction in NHE-1 phosphorylation.

Early Hypertrophic Signals After Myocardial Stretch. Role of Reactive Oxygen Species and the Sodium/Hydrogen Exchanger

In this chapter 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 which in turn induce the release/formation of endothelin. The actions of these hormones trigger the production of mitochondrial reactive oxygen species that enhances NHE-1 activity, causing an increment in the intracellular Na+ concentration which promotes the increase in intracellular Ca2+ concentration ([Ca2+]i) through the Na+/Ca2+ exchanger. This [Ca2+]i increase would trigger cardiac hypertrophy by activation of widely recognized Ca2+-dependent intracellular signaling pathways.