Romina G. Díaz

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.

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.

Inhibition of carbonic anhydrase prevents the Na(+)/H(+) exchanger 1-dependent slow force response to rat myocardial stretch.

Myocardial stretch is an established signal that leads to hypertrophy. Myocardial stretch induces a first immediate force increase followed by a slow force response (SFR), which is a consequence of an increased Ca(2+) transient that follows the NHE1 Na(+)/H(+) exchanger activation. Carbonic anhydrase II (CAII) binds to the extreme COOH terminus of NHE1 and regulates its transport activity. We aimed to test the role of CAII bound to NHE1 in the SFR. The SFR and changes in intracellular pH (pHi) were evaluated in rat papillary muscle bathed with CO2/HCO3(-) buffer and stretched from 92% to 98% of the muscle maximal force development length for 10 min in the presence of the CA inhibitor 6-ethoxzolamide (ETZ, 100 μM). SFR control was 120 ± 3% (n = 8) of the rapid initial phase and was fully blocked by ETZ (99 ± 4%, n = 6). The SFR corresponded to a maximal increase in pHi of 0.18 ± 0.02 pH units (n = 4), and pHi changes were blocked by ETZ (0.04 ± 0.04, n = 6), as monitored by epifluorescence. NHE1/CAII physical association was examined in the SFR by coimmunoprecipitation, using muscle lysates. CAII immunoprecipitated with an anti-NHE1 antibody and the CAII immunoprecipitated protein levels increased 58 ± 9% (n = 6) upon stretch of muscles, assessed by immunoblots. The p90(RSK) kinase inhibitor SL0101-1 (10 μM) blocked the SFR of heart muscles after stretch 102 ± 2% (n = 4) and reduced the binding of CAII to NHE1, suggesting that the stretch-induced phosphorylation of NHE1 increases its binding to CAII. CAII/NHE1 interaction constitutes a component of the SFR to heart muscle stretch, which potentiates NHE1-mediated H(+) transport in the myocardium.

Epidermal Growth Factor Receptor Silencing Blunts the Slow Force Response to Myocardial Stretch

Background Myocardial stretch increases force biphasically: the Frank‐Starling mechanism followed by the slow force response (SFR). Based on pharmacological strategies, we proposed that epidermal growth factor (EGF) receptor (EGFR or ErbB1) activation is crucial for SFR development. Pharmacological inhibitors could block ErbB4, a member of the ErbB family present in the adult heart. We aimed to specifically test the role of EGFR activation after stretch, with an interference RNA incorporated into a lentiviral vector (small hairpin RNA [shRNA]‐EGFR). Methods and Results Silencing capability of p‐shEGFR was assessed in EGFR‐GFP transiently transfected HEK293T cells. Four weeks after lentivirus injection into the left ventricular wall of Wistar rats, shRNA‐EGFR–injected hearts showed ≈60% reduction of EGFR protein expression compared with shRNA‐SCR–injected hearts. ErbB2 and ErbB4 expression did not change. The SFR to stretch evaluated in isolated papillary muscles was ≈130% of initial rapid phase in the shRNA‐SCR group, while it was blunted in shRNA‐EGFR–expressing muscles. Angiotensin II (Ang II)‐dependent Na+/H+ exchanger 1 activation was indirectly evaluated by intracellular pH measurements in bicarbonate‐free medium, demonstrating an increase in shRNA‐SCR–injected myocardium, an effect not observed in the silenced group. Ang II‐ or EGF‐triggered reactive oxygen species production was significantly reduced in shRNA‐EGFR–injected hearts compared with that in the shRNA‐SCR group. Chronic lentivirus treatment affected neither the myocardial basal redox state (thiobarbituric acid reactive substances) nor NADPH oxidase activity or expression. Finally, Ang II or EGF triggered a redox‐sensitive pathway, leading to p90RSK activation in shRNA‐SCR‐injected myocardium, an effect that was absent in the shRNA‐EGFR group. Conclusions Our results provide evidence that specific EGFR activation after myocardial stretch is a key factor in promoting the redox‐sensitive kinase activation pathway, leading to SFR development.

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.

Carbonic anhydrase inhibitors reduce cardiac dysfunction after sustained coronary artery ligation in rats

BACKGROUND Two potent carbonic anhydrase (CA) inhibitors with widely differing membrane permeability, poorly diffusible benzolamide (BZ), and highly diffusible ethoxzolamide (ETZ) were assessed to determine whether they can reduce cardiac dysfunction in rats subjected to coronary artery ligation (CAL)-induced myocardial infarction. METHODS AND RESULTS Rats with evidence of heart failure (HF) at 32 weeks following a permanent left anterior coronary artery occlusion were treated with placebo, BZ, or ETZ (4 mg kgday-1) for 4 weeks at which time left ventricular function and structure were evaluated. Lung weight/body weight (LW/BW) ratio increased in CAL rats by 17±1% vs. control, suggesting pulmonary edema. There was a trend for BZ and ETZ to ameliorate the increase in LW/BW by almost 50% (9±5% and 9±8%, respectively, versus CAL) (P=.16, NS). Echocardiographic assessment showed decreased left ventricular midwall shortening in HF rats, 21±1% vs. control 32±1%, which was improved by BZ to 29±1% and ETZ to 27±1%, and reduced endocardial shortening in HF rats 38±3% vs. control 62±1%, partially restored by BZ and ETZ to ~50%. Expression of the hypoxia-inducible membrane-associated CAIX isoform increased by ~60% in HF rat hearts, and this effect was blocked by ETZ. CONCLUSIONS We conclude that CAL-induced myocardial interstitial fibrosis and associated decline in left ventricular function were diminished with BZ or ETZ treatment. The reductions in cardiac remodeling in HF with both ETZ and BZ CA inhibitors suggest that inhibition of a membrane-bound CA appears to be the critical site for this protection.

Benzolamide perpetuates acidic conditions during reperfusion and reduces myocardial ischemia-reperfusion injury

During ischemia, increased anaerobic glycolysis results in intracellular acidosis. Activation of alkalinizing transport mechanisms associated with carbonic anhydrases (CAs) leads to myocardial intracellular Ca2+ increase. We characterize the effects of inhibition of CA with benzolamide (BZ) during cardiac ischemia-reperfusion (I/R). Langendorff-perfused isolated rat hearts were subjected to 30 min of global ischemia and 60 min of reperfusion. Other hearts were treated with BZ (5 μM) during the initial 10 min of reperfusion or perfused with acid solution (AR, pH 6.4) during the first 3 min of reperfusion. p38MAPK, a kinase linked to membrane transporters and involved in cardioprotection, was examined in hearts treated with BZ in presence of the p38MAPK inhibitor SB202190 (10 μM). Infarct size (IZ) and myocardial function were assessed, and phosphorylated forms of p38MAPK, Akt, and PKCε were evaluated by immunoblotting. We determined the rate of intracellular pH (pHi) normalization after transient acid loading in the absence and presence of BZ or BZ + SB202190 in heart papillary muscles (HPMs). Mitochondrial membrane potential (ΔΨm), Ca2+ retention capacity and Ca2+-mediated swelling after I/R were also measured. BZ, similarly to AR, reduced IZ, improved postischemic recovery of myocardial contractility, increased phosphorylation of Akt, PKCε, and p38MAPK, and normalized ΔΨm and Ca2+ homeostasis, effects abolished after p38MAPK inhibition. In HPMs, BZ slowed pHi recovery, an effect that was restored after p38MAPK inhibition. We conclude that prolongation of acidic conditions during reperfusion by BZ could be responsible for the cardioprotective benefits of reduced infarction and better myocontractile function, through p38MAPK-dependent pathways. NEW & NOTEWORTHY Carbonic anhydrase inhibition by benzolamide (BZ) maintains acidity, decreases infarct size, and improves postischemic myocardial dysfunction in ischemia-reperfusion (I/R) hearts. Protection afforded by BZ mimicked the beneficial effects elicited by an acidic solution (AR). Increased phosphorylation of p38MAPK occurs in I/R hearts reperfused with BZ or with AR. Mitochondria from I/R hearts possess abnormal Ca2+ handling and a more depolarized membrane potential compared with control hearts, and these changes were restored by treatment with BZ or AR.