Department of Internal Medicine (Cardiology), University of Texas Southwestern Medical Center, Dallas, TX, USA.
Mol Cell Biol. 2012 Oct;32(19):4025-34. doi: 10.1128/MCB.05948-11. Epub 2012 Jul 30.
Stress-induced hypertrophic growth of the heart predisposes the heart to arrhythmia, contractile dysfunction, and clinical heart failure. FHL2 (four-and-a-half LIM domain protein 2) is expressed predominantly in the heart, and inactivation of the gene coding for FHL2 leads to exaggerated responsiveness to adrenergic stress. Activation of calcineurin occurs downstream of β-adrenergic signaling and is required for isoproterenol-induced myocardial hypertrophy. Based on these facts, we hypothesized that FHL2 suppresses stress-induced activation of calcineurin. FHL2 is upregulated in mouse hearts exposed to isoproterenol, a β-adrenergic agonist, and isoproterenol-induced increases in the NFAT target genes RCAN1.4 and BNP were amplified significantly in FHL2 knockout (FHL2(-/-)) mice compared with levels in wild-type (WT) mice. To determine whether the effect of FHL2 on NFAT target gene transcript levels occurred at the level of transcription, HEK 293 cells and neonatal rat ventricular myocytes (NRVMs) were transfected with a luciferase reporter construct harboring the NFAT-dependent promoters of either RCAN1 or interleukin 2 (IL-2). Consistent with the in vivo data, small interfering RNA (siRNA) knockdown of FHL2 led to increased activation of these promoters by constitutively active calcineurin or the calcium ionophore ionomycin. Importantly, activation of the RCAN1 promoter by ionomycin, in control and FHL2 knockdown cells, was abolished by the calcineurin inhibitor cyclosporine, confirming the calcineurin dependence of the response. Overexpression of FHL2 inhibited activation of both NFAT reporter constructs. Furthermore, NRVMs overexpressing FHL2 exhibited reduced hypertrophic growth in response to constitutively active calcineurin, as measured by cell cross-sectional area and fetal gene expression. Finally, immunostaining in isolated adult cardiomyocytes revealed colocalization of FHL2 and calcineurin predominantly at the sarcomere and activation of calcineurin by endothelin-1-facilitated interaction between FHL2 and calcineurin. FHL2 is an endogenous, agonist-dependent suppressor of calcineurin.
应激诱导的心脏肥厚使心脏容易发生心律失常、收缩功能障碍和临床心力衰竭。FHL2（四个半 LIM 结构域蛋白 2）主要在心脏中表达，编码 FHL2 的基因失活会导致对肾上腺素能应激的反应过度。钙调神经磷酸酶的激活发生在β-肾上腺素能信号下游，是异丙肾上腺素诱导心肌肥厚所必需的。基于这些事实，我们假设 FHL2 抑制应激诱导的钙调神经磷酸酶激活。在暴露于异丙肾上腺素（β-肾上腺素能激动剂）的小鼠心脏中，FHL2 上调，与野生型（WT）小鼠相比，FHL2 基因敲除（FHL2(-/-)) 小鼠的 NFAT 靶基因 RCAN1.4 和 BNP 的异丙肾上腺素诱导增加显著放大。为了确定 FHL2 对 NFAT 靶基因转录水平的影响是否发生在转录水平，用含有 NFAT 依赖性启动子的荧光素酶报告基因构建体转染 HEK 293 细胞和新生大鼠心室肌细胞（NRVMs）。RCAN1 或白细胞介素 2（IL-2）。与体内数据一致，FHL2 的小干扰 RNA（siRNA）敲低导致组成型活性钙调神经磷酸酶或钙离子载体离子霉素对这些启动子的激活增加。重要的是，在对照和 FHL2 敲低细胞中，离子霉素对 RCAN1 启动子的激活被钙调神经磷酸酶抑制剂环孢菌素消除，证实了反应的钙调神经磷酸酶依赖性。FHL2 的过表达抑制了两个 NFAT 报告基因构建体的激活。此外，过表达 FHL2 的 NRVMs 对组成型活性钙调神经磷酸酶的反应表现出减少的肥厚生长，如细胞横截面积和胎儿基因表达所测量的。最后，在分离的成年心肌细胞中的免疫染色显示 FHL2 和钙调神经磷酸酶主要在肌节处共定位，并且内皮素-1 促进 FHL2 和钙调神经磷酸酶之间的相互作用激活钙调神经磷酸酶。FHL2 是一种内源性、激动剂依赖性钙调神经磷酸酶抑制剂。
