Myocardial Mineralocorticoid Receptor Activation by Stretching and Its Functional Consequences

Abstract

Abstract-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 redoxsensitive kinases (ERK1/2-p90 RSK ), the consequent Na + /H + exchanger-mediated changes in pH i (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 μg/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-p90 RSK phosphorylation, (4)

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

“…It is possible that some amount of the lentivirus injected in the heart could have leaked into the bloodstream, reducing the NHE1 expression and activity in other organs. NHE1 protein expression in the lung or liver remains unaltered, confirming a local silencing effect restricted to the heart, similarly to a previous report using the same technique (11). The present study also shows that 1 mo of treatment with L-shNHE1 did not significantly affect the increased myocardial interstitial fibrosis of SHR.…”

“…In consequence, cardiac shNHE1 expression alters the normal course of the LV growth in the SHR, preventing an excessive mass development. Similar results were obtained specifically silencing in vivo other genes by chronic and local expression of shRNA in the heart (11,22,35,37,39). Expression of shRNA-NF-B/p65 (22) or shRNA-thyrotropin releasing hormone (39), delivered with a lentivirus into the LV wall of a hypertrophic heart, rendered a 49.6 and 53% reduction of mRNA expression, respectively.…”

“…This reduction suggests that the silencing effect was not restricted to the single injection area, but extended into the LV mass. It was demonstrated that lentivirus transduction takes place with a nonuniform pattern, and that shRNA molecules can have a limited spread throughout the LV (11,25,31). In consequence, NHE1 protein determination in the whole LV homogenate included regions that could have not received the shNHE1.…”

The Multifaceted Mineralocorticoid Receptor

“…Rapid nongenomic MR activation of the N + /H + exchanger NHE-1 results in rapid changes in ion transport in the tubular epithelial cells of the kidney and in intracellular Ca ++ transients necessary for vasoconstriction of mesenteric resistance vessels (303). Stretch-triggered reactive oxygen species generation in the cardiomyocytes was found to activate redox-sensitive kinases upstream of the NHE-1, resulting in mobilization of intracellular Ca ++ (59, 102). …”

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

“…After reaching deep anesthesia, a left thoracotomy was performed via the fourth intercostal space and the lungs were retracted to expose the heart. Following this, the shRNA‐EGFR or the shRNA‐SCR lentivirus (≈2×10 7 transducing units in 200 μL, multiplicity of infection ≈80) were injected at two sites in the free wall of the left ventricle, close to the cardiac apex, using a 30‐gauge needle . Immediately after surgery, rats were returned to their cages and carried to a recovery room and subsequently returned to the animal facility until sacrifice (1 month later).…”