Mariela B. Nolly

Endurance Training in the Spontaneously Hypertensive Rat: Conversion of Pathological into Physiological Cardiac Hypertrophy

The effect of endurance training (swimming 90 min/d for 5 days a week for 60 days) on cardiac hypertrophy was investigated in the spontaneously hypertensive rat (SHR). Sedentary SHRs (SHR-Cs) and normotensive Wistar rats were used as controls. Exercise training enhanced myocardial hypertrophy assessed by left ventricular weight/tibial length (228±7 versus 251±5 mg/cm in SHR-Cs and exercised SHRs [SHR-Es], respectively). Myocyte cross-sectional area increased ≈40%, collagen volume fraction decreased ≈50%, and capillary density increased ≈45% in SHR-Es compared with SHR-Cs. The mRNA abundance of atrial natriuretic factor and myosin light chain 2 was decreased by the swimming routine (100±19% versus 41±10% and 100±8% versus 61±9% for atrial natriuretic factor and myosin light chain 2 in SHR-Cs and SHR-Es, respectively). The expression of sarcoplasmic reticulum Ca2+ pump was significantly augmented, whereas that of Na+/Ca2+ exchanger was unchanged (93±7% versus 167±8% and 158±13% versus 157±7%, sarcoplasmic reticulum Ca2+ pump and Na+/Ca2+ exchanger in SHR-Cs and SHR-Es, respectively; P<0.05). Endurance training inhibited apoptosis, as reflected by a decrease in caspase 3 activation and poly(ADP-ribose) polymerase-1 cleavage, and normalized calcineurin activity without inducing significant changes in the phosphatidylinositol 3-kinase/Akt pathway. The swimming routine improved midventricular shortening determined by echocardiography (32.4±0.9% versus 36.9±1.1% in SHR-Cs and SHR-Es, respectively; P<0.05) and decreased the left ventricular free wall thickness/left ventricular cavity radius toward an eccentric model of cardiac hypertrophy (0.59±0.02 versus 0.53±0.01 in SHR-Cs and SHR-Es, respectively; P<0.05). In conclusion, we present data demonstrating the effectiveness of endurance training to convert pathological into physiological hypertrophy improving cardiac performance. The reduction of myocardial fibrosis and calcineurin activity plus the increase in capillary density represent factors to be considered in determining this beneficial effect.

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.

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 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.

Mineralocorticoid receptor activation is crucial in the signalling pathway leading to the Anrep effect

Non‐technical summary  Myocardial stretch increases force in two phases. The first one is immediate and attributed to an increase in myofilament Ca2+ responsiveness (Frank–Starling mechanism). The second phase gradually develops and is known as slow force response (SFR) or Anrep effect due to an increase in intracellular Ca2+ transient. We previously showed that Ca2+ entry through reverse Na+/Ca2+ exchange underlies the SFR, as the final step of an autocrine/paracrine loop involving release of angiotensin II/endothelin, transactivation of the epidermal growth factor receptor, increased mitochondrial oxidative stress and a Na+/H+ exchanger (NHE‐1) activation‐mediated rise in Na+. In the present study we show that mineralocorticoid receptor activation is a necessary step between endothelin and epidermal growth factor receptor activation in the stretch‐triggered reactive oxygen species‐mediated NHE‐1 activation leading to the SFR.

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.

Cardiac hypertrophy reduction in SHR by specific silencing of myocardial Na+/H+ exchanger

We examined the effect of specific and local silencing of sodium/hydrogen exchanger isoform 1 (NHE1) with a small hairpin RNA delivered by lentivirus (L-shNHE1) in the cardiac left ventricle (LV) wall of spontaneously hypertensive rats, to reduce cardiac hypertrophy. Thirty days after the lentivirus was injected, NHE1 protein expression was reduced 53.3 ± 3% in the LV of the L-shNHE1 compared with the control group injected with L-shSCR (NHE1 scrambled sequence), without affecting its expression in other organs, such as liver and lung. Hypertrophic parameters as LV weight-to-body weight and LV weight-to-tibia length ratio were significantly reduced in animals injected with L-shNHE1 (2.32 ± 0.5 and 19.30 ± 0.42 mg/mm, respectively) compared with L-shSCR-injected rats (2.68 ± 0.06 and 21.53 ± 0.64 mg/mm, respectively). Histochemical analysis demonstrated a reduction of cardiomyocytes cross-sectional area in animals treated with L-shNHE1 compared with L-shSCR (309,81 ± 20,86 vs. 424,52 ± 21 μm(2), P < 0.05). Echocardiography at the beginning and at the end of the treatment showed that shNHE1 expression for 30 days induced 9% reduction of LV mass. Also, animals treated with L-shNHE1 exhibited a reduced LV wall thickness without changing LV diastolic dimension and arterial pressure, indicating an increased parietal stress. In addition, midwall shortening was not modified, despite the increased wall tension, suggesting an improvement of cardiac function. Chronic shNHE1 expression in the heart emerges as a possible methodology to reduce pathological cardiac hypertrophy, avoiding potentially undesired effects caused from a body-wide inhibition of NHE1.

Myocardial Mineralocorticoid Receptor Activation by Stretching and Its Functional Consequences

Myocardial stretch triggers an angiotensin II–dependent autocrine/paracrine loop of intracellular signals, leading to reactive oxygen species–mediated activation of redox-sensitive kinases. Based on pharmacological strategies, we previously proposed that mineralocorticoid receptor (MR) is necessary for this stretch-triggered mechanism. Now, we aimed to test the role of MR after stretch by using a molecular approach to avoid secondary effects of pharmacological MR blockers. Small hairpin interference RNA capable of specifically knocking down the MR was incorporated into a lentiviral vector (l-shMR) and injected into the left ventricular wall of Wistar rats. The same vector but expressing a nonsilencing sequence (scramble) was used as control. Lentivirus propagation through the left ventricle was evidenced by confocal microscopy. Myocardial MR expression, stretch-triggered activation of redox-sensitive kinases (ERK1/2-p90RSK), the consequent Na+/H+ exchanger–mediated changes in pHi (HEPES-buffer), and its mechanical counterpart, the slow force response, were evaluated. Furthermore, reactive oxygen species production in response to a low concentration of angiotensin II (1.0 nmol/L) or an equipotent concentration of epidermal growth factor (0.1 &mgr;g/mL) was compared in myocardial tissue slices from both groups. Compared with scramble, animals transduced with l-shMR showed (1) reduced cardiac MR expression, (2) cancellation of angiotensin II–induced reactive oxygen species production but preservation of epidermal growth factor–induced reactive oxygen species production, (3) cancellation of stretch-triggered increase in ERK1/2-p90RSK phosphorylation, (4) lack of stretch-induced Na+/H+ exchanger activation, and (5) abolishment of the slow force response. Our results provide strong evidence that MR activation occurs after myocardial stretch and is a key factor to promote redox-sensitive kinase activation and their downstream consequences.

Phosphodiesterase 5A Inhibition Decreases NHE-1 Activity Without Altering Steady State pHi: Role of Phosphatases

Background/Aims: This study aimed to identify the signaling pathway for the proposed link between phosphodiesterase-5A (PDE5A) inhibition and decreased cardiac Na+/H+ exchanger (NHE-1) activity. Methods: NHE-1 activity was assessed in rat isolated papillary muscles by the Na+-dependent initial pHi recovery from a sustained acidosis (ammonium prepulse). ERK1/2, p90RSK and NHE-1 phosphorylation state during acidosis was determined. Results: PDE5A inhibition (1 µmol/L sildenafil, SIL) did not modify basal pHi but significantly blunted pHi recovery after sustained acidosis. Although preventing ERK1/2- p90RSK signaling pathway (10 µmol/L U0126) mimicked SIL effect, SIL did not blunt the acidosis-mediated increase in kinases activation. SIL+U0126 did not show additive effect on NHE-1 activity. Then, we hypothesized that SIL could be activating phophasatases (PP1 and/or PP2A) to directly dephosphorylate NHE-1 despite preserved ERK1/2-p90RSK activation. Non-specific phosphatases inhibition (1 µmol/L okadaic acid) canceled SIL effect on pHi recovery from acidosis. Same result was observed by inhibiting PP2A either with a lower dose of okadaic acid (1 nmol/L) or, more specifically, with 100 µmol/L endothall. Consistently, NHE-1 phosphorylation at Ser703 increased after acidosis, SIL prevented this effect and PP2A inhibition (endothall) reverted SIL effect. Conclusion: We suggest that PDE5A inhibitors decrease NHE-1 phosphorylation and activity through a mechanism that involves PP2A activation.