Department of Medicine, SUNY Upstate Medical University, Syracuse, New York, USA.
Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, New York, USA.
Cytoskeleton (Hoboken). 2024 Sep;81(9-10):448-472. doi: 10.1002/cm.21850. Epub 2024 Mar 12.
Tropomyosin (TPM) is an essential sarcomeric component, stabilizing the thin filament and facilitating actin's interaction with myosin. In mammals, including humans, there are four TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which generates a multitude of TPM isoforms via alternative splicing and using different promoters. In this study, we have examined the expression of transcripts as well as proteins of various sarcomeric TPM isoforms during human inducible pluripotent stem cell differentiation into cardiomyocytes. During the differentiation time course, we harvested cells on Days 0, 5, 10, 15, and 20 to analyze for various sarcomeric TPM transcripts by qRT-PCR and for sarcomeric TPM proteins using two-dimensional Western blot with sarcomeric TPM-specific CH1 monoclonal antibody followed by mass spectra analyses. Our results show increasing levels of total TPM transcripts and proteins during the period of differentiation, but varying levels of specific TPM isoforms during the same period. By Day 20, the rank order of TPM transcripts was TPM1α > TPM1κ > TPM2α > TPM1μ > TPM3α > TPM4α. TPM1α was the dominant protein produced with some TPM2 and much less TPM1κ and μ. Interestingly, small amounts of two lower molecular weight TPM3 isoforms were detected on Day 15. To the best of our knowledge this is the first demonstration of TPM1μ non-muscle isoform protein expression before and during cardiac differentiation.
原肌球蛋白（TPM）是一种重要的肌节成分，可稳定细肌丝并促进肌动蛋白与肌球蛋白的相互作用。在哺乳动物（包括人类）中，有四个 TPM 基因（TPM1、TPM2、TPM3 和 TPM4），每个基因通过选择性剪接产生多种 TPM 异构体，并使用不同的启动子。在这项研究中，我们检查了人诱导多能干细胞分化为心肌细胞过程中各种肌节 TPM 异构体的转录本和蛋白质的表达。在分化过程中，我们在第 0、5、10、15 和 20 天收获细胞，通过 qRT-PCR 分析各种肌节 TPM 转录本，并使用二维 Western blot 结合肌节 TPM 特异性 CH1 单克隆抗体进行分析，然后进行质谱分析分析肌节 TPM 蛋白。我们的结果表明，在分化过程中总 TPM 转录本和蛋白质水平逐渐增加，但在同一时期特定 TPM 异构体水平不同。到第 20 天，TPM 转录本的等级顺序为 TPM1α>TPM1κ>TPM2α>TPM1μ>TPM3α>TPM4α。TPM1α 是产生的主要蛋白，带有一些 TPM2 和较少的 TPM1κ 和 μ。有趣的是，在第 15 天检测到两种较低分子量的 TPM3 异构体的少量存在。据我们所知，这是在心脏分化之前和期间首次证明 TPM1μ 非肌肉同工型蛋白表达。
