Kato T, Sano M, Miyoshi S, Sato T, Hakuno D, Ishida H, Kinoshita-Nakazawa H, Fukuda K, Ogawa S
Cardiopulmonary Division, Department of Internal Medicine, Keio University, Tokyo, Japan.
Circ Res. 2000 Nov 10;87(10):937-45. doi: 10.1161/01.res.87.10.937.
We recently reported that leukemia inhibitory factor (LIF) enhances Ca(2+)](i) through an increase in L-type Ca(2+) current (I(Ca,L)) in adult cardiomyocytes. The aim of this study was to investigate whether LIF activates Ca(2+)-dependent signaling molecules, such as calcineurin and calmodulin kinases II and IV (CaMKII and CaMKIV), and, if so, whether these Ca(2+)-mediated signaling events contribute to LIF-mediated cardiac hypertrophy. We first confirmed that LIF increased I(Ca,L) and Ca(2+) 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 (IP(3))-induced Ca(2+) release from sarcoplasmic reticulum was not involved in this process, IP(3) content and phosphorylation of phospholipase Cgamma were investigated. LIF did not increase IP(3) content or phosphorylate phospholipase Cgamma. KN62 (an inhibitor of CaMKII and CaMKIV) attenuated c-fos, brain natriuretic peptide, alpha-skeletal actin, and atrial natriuretic peptide expression. KN62 suppressed the LIF-induced increase in [(3)H]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 [(3)H]phenylalanine uptake. These findings indicated that LIF activated CaMKII, CaMKIV, and calcineurin through an increase in I:(Ca,L) and Ca(2+) and that CaMKII, CaMKIV, and calcineurin are critically involved in LIF-induced cardiac hypertrophy.
我们最近报道，白血病抑制因子（LIF）通过增加成年心肌细胞中的L型钙电流（I(Ca,L)）来增强细胞内钙离子浓度（Ca(2+)）。本研究的目的是探讨LIF是否激活钙依赖性信号分子，如钙调神经磷酸酶、钙调蛋白激酶II和IV（CaMKII和CaMKIV），如果是，这些钙介导的信号事件是否促成LIF介导的心肌肥大。我们首先证实LIF增加了原代培养的新生大鼠心肌细胞中的I(Ca,L)和Ca(2+)。钙调神经磷酸酶、CaMKII和CaMKIV的活性在2分钟时增加，并分别在15分钟时达到峰值，增幅分别为1.6倍、2.2倍和2.2倍。尼卡地平或维拉帕米完全抑制了这些活性。还观察到CaMKII的自磷酸化与CaMKII活性的时间变化平行，并且这种磷酸化被尼卡地平、维拉帕米或乙二醇双乙胺醚（EGTA）阻断。LIF处理导致活化T细胞核因子荧光素酶活性增加3倍。为了证实肌醇三磷酸（IP(3)）诱导的肌浆网钙离子释放不参与此过程，研究了IP(3)含量和磷脂酶Cγ的磷酸化情况。LIF没有增加IP(3)含量或使磷脂酶Cγ磷酸化。KN62（CaMKII和CaMKIV的抑制剂）减弱了c-fos、脑钠肽、α-骨骼肌肌动蛋白和心钠肽的表达。KN62抑制了LIF诱导的[3H]苯丙氨酸摄取增加和细胞大小增加。环孢素A和FK506略微减弱了脑钠肽的表达，但不影响c-fos或心钠肽的表达。环孢素A显著降低了LIF诱导的[3H]苯丙氨酸摄取增加。这些发现表明，LIF通过增加I:(Ca,L)和Ca(2+)激活了CaMKII、CaMKIV和钙调神经磷酸酶，并且CaMKII、CaMKIV和钙调神经磷酸酶在LIF诱导的心肌肥大中起关键作用。
