The RSK Inhibitor BIX02565 Limits Cardiac Ischemia/Reperfusion Injury

Abstract

Aims: During ischemia/reperfusion (I/R), ribosomal S6 kinase (RSK) activates Na(+)/H(+) exchanger 1 (NHE1) by phosphorylating NHE1 at serine 703 (pS703-NHE1), which promotes cardiomyocyte death and injury. Pharmacologic inhibition of NHE1 effectively protects animal hearts from I/R. However, clinical trials using NHE1 inhibitors failed to show benefit in patients with acute myocardial infarction (MI). One possible explanation is those inhibitors block both agonist-stimulated activity (increasing I/R injury) and basal NHE1 activity (necessary for cell survival). We previously showed that dominant-negative RSK (DN-RSK) selectively blocked agonist-stimulated NHE1 activity. Therefore, we hypothesized that a novel RSK inhibitor (BIX02565) would blunt agonist-stimulated NHE1 and protect hearts from I/R.

Methods and results: Serum/angiotensin II-stimulated pS703-NHE1 was significantly decreased by BIX02565 in cultured cells. Intracellular pH recovery assay showed that BIX02565 selectively inhibited serum-stimulated NHE1 activity. Ischemia/reperfusion decreased left ventricular-developed pressure (LVDP; inhibited) to 8.7% of the basal level in non-transgenic littermate control (NLC) mouse hearts, which was significantly improved (44.6%) by BIX02565. Similar protection was observed in vehicle-treated, cardiac-specific DN-RSK-Tg mice (43%). No additional protective effect was seen in BIX02565-treated DN-RSK-Tg hearts. BIX02565 also improved LVDP in cardiac-specific wild-type (WT)-RSK-Tg mouse hearts (7.4%-40.9%, P < .01). Finally, Western Blotting results confirmed DN-RSK and BIX02565 significantly decreased I/R-induced pS703-NHE1.

Conclusion: The RSK plays a crucial role in I/R-induced activation of NHE1 and cardiac injury. The RSK inhibition may provide an alternative target for patients with MI.

Keywords: I/R; NHE1; RSK inhibitor; cardioprotection.

Figure 1

Serum-stimulated phosphorylation of Na⁺/H⁺ exchanger 1 (NHE1) at S703 is inhibited by ribosomal S6 Kinase (RSK) inhibitor BIX02565 in a dose-dependent manner. A, H9C2 cells were serum starved (0% fetal bovine serum [FBS]) overnight and stimulated with 20% FBS for 5 minutes. BIX02565 (100 nmol/L and 1 μmol/L) was added 1.5 hours before the serum stimulation. Protein lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with anti-phospho S703-NHE1 antibody. The same membrane was blotted with anti-phospho-ERK1/2 antibody to confirm the serum activation. Anti-NHE1 and antitubulin antibodies were used for the loading control. B, Quantified result of phospho S703-NHE1 normalized to total NHE1 protein levels (shown as mean ± SD, n = 3, *P < .01).

Figure 2

BIX02565 inhibits serum enhanced but not the basal Na⁺/H⁺ exchanger 1 (NHE1) activity. H9C2 cells were serum starved (0% fetal bovine serum [FBS]) for 24 hours and kept in the presence of 10% FBS (——) or 0% FBS (— —) for recording the enhanced and basal NHE1 activity (measured by the rate of intracellular pH [pHi] recovery). BIX 02565 was titrated to the cells with (—▲—) and without FBS (—●—). The 10% FBS-stimulated NHE1 activity was calibrated as 100%. The NHE1 activity under all the other treatments was calibrated as a ratio to the 10% FBS treatment. BIX 02565 inhibited serum-enhanced NHE1 activity in a dose-dependent manner while had no effect on basal NHE1 activity.

Figure 3

BIX02565 improves cardiac functional recovery in non-transgenic littermate control (NLC) mouse hearts in a dose-dependent manner. A, Diagram of ischemia/reperfusion (I/R) experimental protocol. In brief, hearts isolated from NLC mice were perfused with KH buffer for 20 minutes for stabilization. After this, hearts were perfused without or with BIX02565 (30, 100, and 300 nmol/L) for 10 minutes, followed by global ischemia for 40 minutes and reperfusion for 60 minutes. *Indicates the first data point in Figure 3B and D. B, Measurements of left ventricular–developed pressure (LVDP). C, dp/dt max. D, Rate pressure product (RPP) before and during I/R with vehicle or BIX02565 treatment in NLC mouse hearts. The RPP was calculated based on the equation: RPP = LVDP (mm Hg) × heart rate (bpm). Values are shown as mean ± standard error of the mean [SEM], n — 6, *P < .01.

Figure 4

The effect of BIX02565 is mediated by inhibition of ribosomal S6 Kinase (RSK). A, Hearts isolated from dominant-negative (DN)-RSK transgenic mice (DN-RSK-Tg) and non-transgenic littermate control (NLC) mice were perfused with KH buffer for 20 minutes for stabilization. Hearts were perfused with KH buffer or BIX02565 (100 nmol/L) for 10 minutes, followed by global ischemia for 40 minutes and reperfusion for 60 minutes. Both BIX-treated NLC hearts and untreated DN-RSK-Tg have significant increase in left ventricular–developed pressure (LVDP) after ischemia/reperfusion (I/R). A, Measurements of LVDP. B, dp/dt max. C, Rate pressure product (RPP) before and during I/R in NLC and DN-RSK-Tg mouse hearts (shown as mean ± standard error of the mean [SEM], n = 6, *P < .01).

Figure 5

Both BIX02565 and dominant-negative (DN)-RSK transgenic mice (DN-RSK-Tg) reduce infarction in mouse hearts after ischemia/reperfusion (I/R) injury. Quantified infarct area from 3,5-triphenyltetrazolium chloride (TTC)-stained heart sections collected from non-transgenic littermate control (NLC) and DN-RSK-Tg mouse hearts treated without or with 100 nmol/L BIX02565. Both BIX-treated NLC hearts and untreated DN-RSK-Tg have significantly decreased infarct area after I/R. However, no significant change is seen in BIX untreated and treated DN-RSK-Tg mouse hearts. Values are shown as mean ± standard error of the mean [SEM], n = 6, *P < .01.

Figure 6

Inhibiting ribosomal S6 kinase (RSK) blunted ischemia/reperfusion (I/R)-induced Na⁺/H⁺ exchanger 1 (NHE1) S703 phosphorylation. A, Mouse heart protein samples were collected from (1) non-transgenic littermate control (NLC) + Vehicle harvested at the end of 40 minutes ischemia (I), (2) NLC + BIX02565 (100 nmol/L), and (3) dominant-negative (DN)-RSK-Tg + vehicle groups harvested at 10 minutes of reperfusion (I/R). Protein lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrphoresis (SDS-PAGE) and immunoblotted with anti-phospho S703-NHE1 antibody. Anti-NHE1 and anti-tubulin antibodies were used for the loading control. B, Quantified result of pS703-NHE1 normalized to total NHE1 protein levels. Reperfusion significantly increases NHE1 phosphorylation at S703 (*P < .01). The phosphorylation of NHE1 is significantly reduced in NLC mouse heart treated with BIX and DN-RSK-Tg mouse hearts (#P < .01). Values are shown as mean ± standard deviation [SD], n = 4.