PDE5 inhibition improves cardiac morphology and function in SHR by reducing NHE1 activity: Repurposing Sildenafil for the treatment of hypertensive cardiac hypertrophy.

Previously, we have shown that an increased cGMP-activated protein Kinase (PKG) activity after phosphodiesterase 5 (PDE5) inhibition by Sildenafil (SIL), leads to myocardial Na+/H+ exchanger (NHE1) inhibition preserving its basal homeostatic function. Since NHE1 is hyperactive in the hypertrophied myocardium of spontaneous hypertensive rats (SHR), while its inhibition was shown to prevent and revert this pathology, the current study was aimed to evaluate the potential antihypertrophic effect of SIL on adult SHR myocardium. We initially tested the inhibitory capability of SIL on NHE1 in isolated cardiomyocytes of SHR by comparing H+ efflux during the recovery from an acid load. After confirmed that effect, eight-month-old SHR were chronically treated for one month with SIL through drinking water. Compared to their littermate controls, SIL-treated rats presented a decreased NHE1 activity, which correlated with a reduction in its phosphorylation level assigned to activation of a PKG-p38 MAP kinase-PP2A signaling pathway. Moreover, treated animals showed a decreased oxidative stress that appears to be a consequence of a decreased mitochondrial NHE1 phosphorylation. Treated SHR showed a significant reduction in the pro-hypertrophic phosphatase calcineurin, despite slight tendency to decrease hypertrophy was detected. When SIL treatment was prolonged to three months, a significant decrease in myocardial hypertrophy and interstitial fibrosis that correlated with a lower myocardial stiffness was observed. In conclusion, the current study provides evidence concerning the ability of SIL to revert established cardiac hypertrophy in SHR, a clinically relevant animal model that resembles human essential hypertension.

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.