Department of Pediatrics, Osaka University Graduate School of Medicine, Osaka, Japan.
Am J Physiol Heart Circ Physiol. 2011 Oct;301(4):H1531-9. doi: 10.1152/ajpheart.00216.2011. Epub 2011 Jul 29.
LEOPARD syndrome (LS) is an autosomal dominant inherited multisystemic disorder. Most cases involve mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase Src homology 2-containing protein phosphatase 2 (SHP2). LS frequently causes severe hypertrophic cardiomyopathy (HCM), even from the fetal period. However, the molecular pathogenesis has not been clearly elucidated. Here, we analyzed the roles of the LS-type SHP2 mutant Gln510Glu (Q510E), which showed the most severe type of HCM in LS, in cardiomyocyte differentiation, and in morphological changes. We generated mutant P19CL6 cell lines, the most convenient cardiomyocyte differentiation model, which continuously expressed SHP2-Q510E, SHP2-D61N (Noonan-type mutant), wild-type SHP2, and green fluorescent protein (native SHP2 expression only). SHP2-Q510E mutant P19CL6 cells showed significant attenuation of myofibrillogenesis, with increased proliferative activity. Mature cardiomyocytes from the SHP2-Q510E mutant were significantly larger than those of controls and the other mutants. However, expression of cardiac-specific transcriptional factors (Gata4, Tbx5, and Nkx2.5) did not differ significantly between the LS-type SHP2-Q510E mutants and the other mutants and controls. Our results indicate that SHP2-Q510E mutants can differentiate into cardiac progenitors but are inhibited from undergoing terminal differentiation into mature cardiomyocytes. In contrast, Akt and glycogen synthase kinase (GSK)-3β phosphorylation were upregulated, and nuclear β-catenin at the late stage of differentiation was highly accumulated in SHP2-Q510E mutant P19CL6 cells. Supplementation with the phosphoinositide 3-kinase/Akt inhibitor LY-294002 during the late stage of differentiation was found to partially restore myofibrillogenesis while suppressing the increase in size of individual mature cardiomyocytes derived from the SHP2-Q510E mutants. Our findings suggest that dysregulation of the Akt/GSK-3β/β-catenin pathway can contribute to the pathogenesis of HCM in LS patients, not only through hypertrophic changes in individual cardiac cells but also via the expansion of cardiac progenitors.
莱伯先天性黑矇综合征（LS）是一种常染色体显性遗传性多系统疾病。大多数病例涉及 PTPN11 基因的突变，该基因编码蛋白酪氨酸磷酸酶Src 同源 2 结构域含有蛋白磷酸酶 2（SHP2）。LS 常导致严重的肥厚型心肌病（HCM），甚至在胎儿期就已如此。然而，其分子发病机制尚未明确。在此，我们分析了 LS 中最严重的 HCM 类型的 LS 型 SHP2 突变 Gln510Glu（Q510E）在心肌细胞分化和形态变化中的作用。我们生成了突变 P19CL6 细胞系，这是最方便的心肌细胞分化模型，持续表达 SHP2-Q510E、SHP2-D61N（Noonan 型突变）、野生型 SHP2 和绿色荧光蛋白（仅表达天然 SHP2）。SHP2-Q510E 突变 P19CL6 细胞的肌原纤维发生明显减弱，增殖活性增加。来自 SHP2-Q510E 突变体的成熟心肌细胞明显大于对照和其他突变体。然而，LS 型 SHP2-Q510E 突变体与其他突变体和对照之间的心脏特异性转录因子（Gata4、Tbx5 和 Nkx2.5）的表达没有显著差异。我们的结果表明，SHP2-Q510E 突变体可以分化为心脏祖细胞，但阻止其向成熟心肌细胞的终末分化。相比之下，Akt 和糖原合酶激酶（GSK）-3β 的磷酸化上调，并且在 SHP2-Q510E 突变 P19CL6 细胞分化的晚期，细胞核中的 β-连环蛋白高度积累。在分化的晚期添加磷酸肌醇 3-激酶/Akt 抑制剂 LY-294002 被发现部分恢复肌原纤维发生，同时抑制来自 SHP2-Q510E 突变体的单个成熟心肌细胞大小的增加。我们的研究结果表明，Akt/GSK-3β/β-连环蛋白通路的失调可能导致 LS 患者的 HCM 发病机制，不仅通过单个心肌细胞的肥大变化，还通过心脏祖细胞的扩增。
