Impaired Ang II - mediated ERK1/2 activation in human failing ventricular myocytes

Background. With the progression to failure human ventricular cardiomyocytes lose the capability to produce ET-1 or IGF-I following Ang II stimulation notwithstanding the markedly increased Ang II cardiac formation and the normal expression of myocyte AT1 receptors. Therefore an altered Ang II activated intra-myocyte signaling can be hypothesized. Methods. The signaling pathway(s) responsible for Ang II (100 nmol/L)-stimulated IGF-I and ET-1 formation were investigated using ERK1/2 (PD98059, 30 µmol/L), JNK(SP600125, 10 µmol/L), p38(SB203580, 10 µmol/L), and JAK2(AG490, 10 µmol/L) inhibitors, in human ventricular non-failing (NF) and failing myocytes (105cell/mL) obtained from donor hearts (n=5) and from patients with dilated (n=19) or ischemic (n=14) cardiomyopathy. ERK1/2 and JNK activities of lysate-stimulated cells were investigated by measuring the phosphorylation of a synthetic ERK substrate, and of c-Jun protein beads, respectively. Intramyocyte amounts signaling proteins were investigated in Western blotting studies. The myocyte expression of MKP-1, MKP-2 and MKP-3 was also investigated. Results. ERK1/2 inhibition completely blocked AT-1 mediated IGF-I and Big ET-1 formation in Ang II stimulated freshly-isolated NF human ventricular myocytes, whereas JNK antagonism only mildly reduced Big ET-1 production. Neither IGF-I or Big ET-1 formation was affected by p38, or JAK2 inhibitors. In NF myocytes, Ang II significantly enhanced both phosphorylation (+100% at 10 min) and enzymatic activity of ERK1/2 (+117% vs controls after 10 min), via AT1 receptor stimulation. Conversely, Ang II stimulation failed to increase ERK1/2 phosphorylation and enzymatic activity at all experimental times in failing myocytes from both DCM and ICM hearts. In contrast with the ERK1/2 pattern, JNK phosphorylation by Ang II was preserved in the failing myocytes. Western blot analysis revealed higher levels (2.5 times) of MKP-1, MKP-2 and MKP-3 in failing than in NF unstimulated myocytes. Ang II stimulation in NF myocytes resulted in increased myocyte levels of MKP-1, MKP-2 and MKP-3 after 30-60 min, paralleling the return to baseline of MAPKs phosphorylation. Conclusions. The reduced IGF-I and ET-1 formation by Ang II stimulated human failing ventricular myocytes is associated with an important functional alteration of ERK1/2 signaling.