Calmodulin kinases II and IV and calcineurin are involved in leukemia inhibitory factor-induced cardiac hypertrophy in rats

We recently reported that leukemia inhibitory factor (LIF) enhances Ca²⁺](i) through an increase in L-type Ca²⁺ current (I(Ca,L)) in adult cardiomyocytes. The aim of this study was to investigate whether LIF activates Ca²⁺-dependent signaling molecules, such as calcineurin and calmodulin kinases II and IV (CaMKII and CaMKIV), and, if so, whether these Ca²⁺-mediated signaling events contribute to LIF-mediated cardiac hypertrophy. We first confirmed that LIF increased I(Ca,L) and [Ca²⁺](i) in primary cultured rat neonatal cardiomyocytes. Calcineurin, CaMKII, and CaMKIV activities increased at 2 minutes and peaked by 1.6-, 2.2-, and 2.2-fold, respectively, at 15 minutes. Nicardipine or verapamil fully inhibited these activities. Autophosphorylation of CaMKII was also observed to parallel the timing of CaMKII activity, and this phosphorylation was blocked by nicardipine, verapamil, or EGTA. LIF treatment led to a 3-fold increase in nuclear factor of activated T cell-luciferase activity. To confirm that inositol triphosphate (IP₃)-induced Ca²⁺ release from sarcoplasmic reticulum was not involved in this process, IP₃ content and phosphorylation of phospholipase Cγ were investigated. LIF did not increase IP₃ content or phosphorylate phospholipase Cγ. KN62 (an inhibitor of CaMKII and CaMKIV) attenuated c-fos, brain natriuretic peptide, α-skeletal actin, and atrial natriuretic peptide expression. KN62 suppressed the LIF-induced increase in [³H]phenylalanine uptake and cell size. Cyclosporin A and FK506 slightly attenuated brain natriuretic peptide but did not affect c-fos or atrial natriuretic peptide expression. Cyclosporin A significantly reduced the LIF-induced increase in [³H]phenylalanine uptake. These findings indicated that LIF activated CaMKII, CaMKIV, and calcineurin through an increase in I(Ca,L) and [Ca²⁺](i) and that CaMKII, CaMKIV, and calcineurin are critically involved in LIF-induced cardiac hypertrophy.