Background and purpose:

The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have an important role in several diseases like heart failure and cancer. Phosphorylation of ERK1/2 can cause dimerization that subsequently triggers an autophosphorylation at threonine 188 (pERK^T188), which leads to nuclear ERK1/2 signaling, and was shown to induce pathological hypertrophy. However, activation of ERK1/2 is also essential for cardiomyocyte survival and inhibition of these kinases in cancer therapy can thus cause cardiac side-effects. In this study we investigated a potential strategy to selectively inhibit pERK^T188 by interference with ERK dimerization to prevent ERK1/2 mediated hypertrophy while preserving its anti-apoptotic effects.

Methods and results:

To assess the function of monomeric ERK in the heart, we generated mice with cardiac overexpression of a ERK2 mutant that is unable to dimerize (ERK2^Δ174-177) under the control of the aMHC promoter and induced chronic left ventricular pressure overload by transverse aortic constriction (TAC). This mutant showed a reduction in pERK^T188 in ERK2^Δ174-177 compared to wildtype mice in response to TAC whereas ERK1/2 activation was unaffected as confirmed by western blot analysis. We determined cardiac function by echocardiography and cardiac catheterization as well as cardiac fibrosis by Sirius red staining, cardiomyocyte size by H&E staining and apoptosis by TUNEL assay. ERK2^Δ174-177 expression had no adverse effects on cell survival, remodeling and heart function and even reduced pathological cardiac hypertrophy after TAC.

To interfere with endogenous ERK dimerization, we generated the ERK dimerization inhibitor peptide (EDI) and assessed its effects in neonatal rat cardiomyocytes (NRCM). In line with our hypothesis, EDI reduced phenylephrine (PE) induced pERK^T188 while ERK1/2 activation was not influenced by this peptide. Furthermore, EDI prevented nuclear translocation of YFP-ERK2^wt as assessed by confocal microscopy and subsequently reduced nuclear ERK1/2 target activation (pELK1) as well as cardiomyocyte size after PE stimulation. In accordance with an unaltered ERK1/2 activation, cell survival was not impaired by EDI. As cardiotoxic side-effects often arise in cancer therapy with inhibitors of the Raf/MEK/ERK1/2 cascade, we compared the cardio-safe strategy of inhibiting pERK^T188 with clinically used drugs (e.g. trametinib, selumetinib). These substances also abolished pERK^T188 and the hypertrophic response but increased apoptosis in contrast to EDI. Of note, EDI reduced colon cancer cell proliferation at least as effective as the used MEK-inhibitors as assessed by [3H]-thymidine incorporation. Furthermore, application of an adeno-associated virus serotype 9 encoding for EDI protected mice from pERK^T188, pathological cardiac hypertrophy and impaired cardiac function after TAC.

Conclusion:

These findings suggest that the ERK dimerization interface is a promising target to selectively inhibit pERK^T188 mediated maladaptive cardiac hypertrophy and cancer cell proliferation without impairing the anti-apoptotic ERK1/2 effects. Thus, the peptide-based inhibition of ERK dimerization represents a cardio-safe principle to help people with heart failure as well as patients suffering from cardiac side-effects of anti-cancer drugs.