Mitogen-Activated Protein Kinases Are Differently Activated in Early Hypertrophic Response to Acute Pressure Overload in Rat Myocardium

Abstract

P55 Mitogen-activated protein kinases (MAPKs) have been reported as important signaling molecules mediating cardiomyocyte hypertrophic response to various stimuli. Four subfamilies of MAPKs have been described in mammalian cells: c-jun NH2 terminal kinases (JNKs), p38-MAPKs (p38), extracellular response kinases (ERKs) and big MAP-kinase (BMK). Our objective was to study the activation of these kinases in the early phases of hypertrophic response, which is currently obscure. Transverse aorta constriction (TAC) was performed in male Wistar rats (160-200g) and left ventricles were studied up to 3 h following the surgery. To study the activation of ERKs, JNKs and p38, heart homogenates were separated into cytosolic (C) and nuclear (N) fractions and their kinase activities were determined by Western Blot using anti p-ERK, p-JNK and p-p38 antibodies. The membranes were then stripped and blotted with anti-ERK, anti-JNK and anti-p38 to determine the protein contents. The amount of BMK was studied in total homogenates using anti-BMK antibody and its activity was studied by measuring its in vitro autophosphorylation. ERK1/2 were transiently activated in both subcellular fractions, reaching a maximum 30 min after TAC (ERK1 - 236% in C and 196% in N; ERK2 - 280% in C and 248% in N) and returning toward basal levels after 1 h. p38 showed a sustained increase in its activity only in C (to 259%) but not in N after 1 h of TAC. JNKs showed a biphasic response with an early and major increase in their activities after 10 min (to 313% for JNK1 and to 288% for JNK2 in C) and a second and minor one following 3 h of TAC. In parallel, TAC induced a significant nuclear translocation of JNK1. The total amount of BMK remained stable during the studied period. We observed a progressive increase in its kinase activity which was directly related to TAC time course (45% in 10 min; 159% in 1 h and 173 % in 2h). These results indicate that pressure overload induces an early activation of the four subfamilies of MAPKs. Differences in their time course activation suggest that they might play different roles in the early hypertrophic response in adult rat myocardium.