Hydrocortisone cardioprotection in ischaemia/reperfusion injury involves antioxidant mechanisms.

BACKGROUND: Glucocorticoid (GR) and mineralocorticoid (MR) receptors are highly expressed in cardiac tissue, and both can be activated by corticosteroids. MR activation, in acute myocardial infarction (AMI), worsens cardiac function, and increase NHE activity contributing to the deleterious process. In contrast, effects of GR activation are not fully understood, probably because of the controversial scenario generated by using different doses or potencies of corticosteroids. AIMS: We tested the hypothesis that an acute dose of hydrocortisone (HC), a low-potency glucocorticoid, in a murine model of AMI could be cardioprotective by regulating NHE1 activity, leading to a decrease in oxidative stress. MATERIALS AND METHODS: Isolated hearts from Wistar rats were subjected to regional ischemic protocol. HC (10 nmol/L) was added to the perfusate during early reperfusion. Infarct size and oxidative stress were determined. Isolated papillary muscles from non-infarcted hearts were used to evaluate HC effect on sodium-proton exchanger 1 (NHE1) by analysing intracellular pH recovery from acute transient acidosis. RESULTS: HC treatment decreased infarct size, improved cardiac mechanics, reduced oxidative stress after AMI, while restoring the decreased level of the pro-fusion mitochondrial protein MFN-2. Co-treatment with the GR-blocker Mifepristone avoided these effects. HC reduced NHE1 activity by increasing the NHE1 pro-inhibiting Ser648 phosphorylation site and its upstream kinase AKT. HC restored the decreased AKT phosphorylation and anti-apoptotic BCL-2 protein expression detected after AMI. CONCLUSIONS: Our results provide the first evidence that acute HC treatment during early reperfusion induces cardioprotection against AMI, associated with a non-genomic HC-triggered NHE1 inhibition by AKT and antioxidant action that might involves mitochondrial dynamics improvement.

Silencing of epidermal growth factor receptor reduces Na+/H+ exchanger 1 activity and hypertensive cardiac hypertrophy.

Pathological cardiac hypertrophy (PCH) can be triggered by epidermal growth factor receptor (EGFR) transactivation. Progression of PCH can be prevented by inhibition of hyperactive Na+/H+ exchanger isoform 1 (NHE1). We first aimed, to limit PCH of spontaneously hypertensive rats (SHR) by specific and localized silencing of cardiac EGFR, and second to study the connection of its activation pathway with cardiac NHE1 activity. Short hairpin RNA (shRNA) against EGFR was delivered with a lentivirus (l-shEGFR) in the cardiac left ventricle (LV) wall. Protein expression was analyzed by immunoblots, and NHE1 activity was indirectly measured in isolated papillary muscles by rate of pHi recovery from transient acidification. EGFR protein expression in the LV was reduced compared to the group injected with l-shSCR (Scrambled sequence) without changes in ErbB2 or ErbB4. Hypertrophic parameters together with cardiomyocytes cross sectional area were reduced in animals injected with l-shEGFR. Echocardiographic analysis exhibited a reduced fractional shortening in the l-shSCR group 30 days following treatment that was not observed in l-shEGFR group. l-shEGFR treated rats presented a reduced basal production of reactive oxygen species and decreased lipid peroxidation. NHE1 activity was significantly diminished in hearts with a partial EGFR silencing, without modification of its protein expression. We conclude that specifically silencing cardiac EGFR expression prevents progression of PCH through a pathway that involves a decrease in the NHE1 activity. Lentiviral vectors prove to be a valuable tool for long term expression of shRNA, bringing the possibility to extend its use in clinical area.

p38 mitogen activated protein kinase mediates cardiac Na+/H+ exchanger inhibition induced by Sildenafil.

Since the original description as potent antianginal compounds, phosphodiesterase 5A inhibitors have continuously increased their possible therapeutic applications. In the heart, Sildenafil was shown to protect against an ischemic insult by decreasing cardiac Na+/H+ exchanger (NHE1) activity, action that was mediated by protein kinase G. p38 mitogen activated protein kinase (p38MAPK) activation was described in cardiac ischemia, but its precise role remains elusive. It has been shown that p38MAPK is activated by protein kinase G (PKG) in certain non-cardiac tissues, while in others modulates NHE1 activity. Current study was aimed to seek the role of p38MAPK in the Sildenafil-triggered pathway leading to NHE1 inhibition in myocardium. Rat isolated papillary muscles were used to evaluate NHE1 activity during intracellular pH recovery from an acidic load. Protein kinases phosphorylation (activation) was determined by western blot. Sustained acidosis promoted NHE1 hyperactivity by enhancing Ser703 phosphorylation, effect that was blunted by Sildenafil. p38MAPK inhibition reversed the effect of Sildenafil on NHE1. Activation of p38MAPK, by Sodium Arsenite or Anisomycin, mimicked the inhibitory effect of Sildenafil on the exchanger. Consistently, Sildenafil induced p38MAPK phosphorylation/activation during acidosis. Neither Sildenafil nor p38MAPK inhibition affected extracellular signal-regulated kinases 1/2 phosphorylation, kinases upstream NHE1. Furthermore, inhibition of NHE1 after p38MAPK activation was precluded by preventing the activation of protein phosphatase 2A with Okadaic Acid. Taken together, these results suggest that activation of p38MAPK is a necessary step to trigger the inhibitory effect of Sildenafil on cardiac NHE1 activity, thorough a mechanism that involves protein phosphatase 2A-mediated exchanger dephosphorylation.

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.