Department of Biomedical Science, College of Medicine, Florida Atlantic University, Boca Raton, FL, 33431, USA.
Cardiovasc Toxicol. 2015 Jan;15(1):29-40. doi: 10.1007/s12012-014-9265-z.
We evaluated the effect of shz-1, a cardiogenic molecule, on the expression of various tropomyosin (TM) isoforms in the Mexican axolotl (Ambystoma mexicanum) hearts. qRT-PCR data show a ~1.5-fold increase in cardiac transcripts of the Nkx2.5 gene, which plays a crucial role in cardiogenesis in vertebrates. Shz-1 augments the expression of transcripts of the total sarcomeric TPM1 (both TPM1α & TPM1κ) and sarcomeric TPM4α. In order to understand the mechanism by which shz-1 augments the expression of sarcomeric TPM transcription in axolotl hearts, we transfected C2C12 cells with pGL3.axolotl. We transfected C2C12 cells with pGL3-axolotl TPM4 promoter constructs containing the firefly luciferase reporter gene. The transfected C2C12 cells were grown in the absence or presence of shz-1 (5 μM). Subsequently, we determined the firefly luciferase activity in the extracts of transfected cells. The results suggest that shz-1 activates the axolotl TPM4 promoter-driven ectopic expression in C2C12 cells. Also, we transfected C2C12 cells with a pGL3.1 vector containing the promoter of the mouse skeletal muscle troponin-I and observed a similar increase in the luciferase activity in shz-1-treated cells. We conclude that shz-1 activates the promoters of a variety of genes including axolotl TPM4. We have quantified the expression of the total sarcomeric TPM1 and observed a 1.5-fold increase in treated cells. Western blot analyses with CH1 monoclonal antibody specific for sarcomeric isoforms show that shz-1 does not increase the expression of TM protein in axolotl hearts, whereas it does in C2C12 cells. These findings support our hypothesis that cardiac TM expression in axolotl undergoes translational control.
我们评估了心脏生成分子 shz-1 对墨西哥蝾螈（Ambystoma mexicanum）心脏中各种原肌球蛋白（TM）同工型表达的影响。qRT-PCR 数据显示，Nkx2.5 基因的心脏转录物增加了约 1.5 倍，该基因在脊椎动物的心脏发生中起着至关重要的作用。Shz-1 增强了总肌节 TPM1（TPM1α 和 TPM1κ）和肌节 TPM4α 的转录物的表达。为了了解 shz-1 增强蝾螈心脏肌节 TPM 转录表达的机制，我们将 pGL3.axolotl 转染到 C2C12 细胞中。我们将包含萤火虫荧光素酶报告基因的 pGL3-axolotl TPM4 启动子构建体转染到 C2C12 细胞中。将转染的 C2C12 细胞在存在或不存在 shz-1（5 μM）的情况下培养。随后，我们测定了转染细胞提取物中的萤火虫荧光酶活性。结果表明，shz-1 激活了在 C2C12 细胞中驱动的异位表达 axolotl TPM4 启动子。此外，我们将 pGL3.1 载体转染到 C2C12 细胞中，该载体包含小鼠骨骼肌肌钙蛋白 I 的启动子，并观察到 shz-1 处理的细胞中荧光素酶活性的类似增加。我们得出结论，shz-1 激活了包括 axolotl TPM4 在内的多种基因的启动子。我们已经定量了总肌节 TPM1 的表达，并在处理的细胞中观察到 1.5 倍的增加。用针对肌节同工型的 CH1 单克隆抗体进行的 Western blot 分析表明，shz-1 不会增加 axolotl 心脏中 TM 蛋白的表达，但在 C2C12 细胞中会增加。这些发现支持我们的假设，即 axolotl 心脏中的 TM 表达经历翻译控制。
