Boston Biomedical Research Institute, Watertown, MA 02472, USA.
Biochem Biophys Res Commun. 2010 May 28;396(2):515-21. doi: 10.1016/j.bbrc.2010.04.129. Epub 2010 Apr 28.
We have recently shown that stimulation of cultured neonatal cardiomyocytes with endothelin-1 (ET-1) first produces conformational disorder within the ryanodine receptor (RyR2) and diastolic Ca(2+) leak from the sarcoplasmic reticulum (SR), then develops hypertrophy (HT) in the cardiomyocytes (Hamada et al., 2009 [3]). The present paper addresses the following question. By what mechanism does crosstalk between defective operation of RyR2 and activation of the HT gene program occur? Here we show that the immuno-stain of calmodulin (CaM) is localized chiefly in the cytoplasmic area in the control cells; whereas, in the ET-1-treated/hypertrophied cells, major immuno-staining is localized in the nuclear region. In addition, fluorescently labeled CaM that has been introduced into the cardiomyocytes using the BioPORTER system moves from the cytoplasm to the nucleus with the development of HT. The immuno-confocal imaging of Ca(2+)/CaM-dependent protein kinase II (CaMKII) also shows cytoplasm-to-nucleus shift of the immuno-staining pattern in the hypertrophied cells. In an early phase of hypertrophic growth, the frequency of spontaneous Ca(2+) transients increases, which accompanies with cytoplasm-to-nucleus translocation of CaM. In a later phase of hypertrophic growth, further increase in the frequency of spontaneous Ca(2+) transients results in the appearance of trains of Ca(2+) spikes, which accompanies with nuclear translocation of CaMKII. The cardio-protective reagent dantrolene (the reagent that corrects the de-stabilized inter-domain interaction within the RyR2 to a normal mode) ameliorates aberrant intracellular Ca(2+) events and prevents nuclear translocation of both CaM and CaMKII, then prevents the development of HT. These results suggest that translocation of CaM and CaMKII from the cytoplasm to the nucleus serves as messengers to transmit the pathogenic signal elicited in the surface membrane and in the RyR2 to the nuclear transcriptional sites to activate HT program.
我们最近的研究表明，内皮素-1（ET-1）刺激培养的新生大鼠心肌细胞，首先导致肌浆网（SR）内ryanodine 受体（RyR2）构象紊乱和舒张期 Ca2+渗漏，随后心肌细胞发生肥大（HT）（Hamada 等，2009 [3]）。本文探讨以下问题：RyR2 功能障碍和 HT 基因程序激活之间的串扰是通过什么机制发生的？我们发现，在对照组细胞中，钙调蛋白（CaM）的免疫染色主要定位于细胞质区域；而在 ET-1 处理/肥大的细胞中，主要的免疫染色定位于核区域。此外，使用 BioPORTER 系统将荧光标记的 CaM 导入心肌细胞后，随着 HT 的发展，CaM 从细胞质转移到细胞核。Ca2+/钙调蛋白依赖性蛋白激酶 II（CaMKII）的免疫共聚焦成像也显示了肥大细胞中免疫染色模式从细胞质到细胞核的转移。在肥大的早期阶段，自发性 Ca2+瞬变的频率增加，伴随着 CaM 的细胞质到细胞核的易位。在肥大的后期阶段，自发性 Ca2+瞬变频率的进一步增加导致 Ca2+峰的串出现，伴随着 CaMKII 的核易位。心脏保护试剂丹曲林（纠正 RyR2 内结构域间不稳定相互作用至正常模式的试剂）改善了异常的细胞内 Ca2+事件，并防止了 CaM 和 CaMKII 的核易位，从而防止了 HT 的发展。这些结果表明，CaM 和 CaMKII 从细胞质向细胞核的易位作为信使，将细胞膜表面和 RyR2 中引发的致病信号传递到核转录位点，激活 HT 程序。
