Department of Developmental Biology, University of Pittsburgh, Pittsburgh, PA 15201, USA.
Peptide Synthesis Facility, University of Pittsburgh, Pittsburgh, PA 15201, USA.
Biomolecules. 2018 Nov 14;8(4):147. doi: 10.3390/biom8040147.
Our previous work identified a 12-amino acid peptide that targets the heart, termed cardiac targeting peptide (CTP). We now quantitatively assess the bio-distribution of CTP, show a clinical application with the imaging of the murine heart, and study its mechanisms of transduction. Bio-distribution studies of cyanine5.5--Hydroxysuccinimide (Cy5.5) labeled CTP were undertaken in wild-type mice. Cardiac targeting peptide was labeled with Technetium 99m (Tc) using the chelator hydrazino-nicotinamide (HYNIC), and imaging performed using micro-single photon emission computerized tomography/computerized tomography (SPECT/CT). Human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMCs) were incubated with dual-labeled CTP, and imaged using confocal microscopy. TriCEPs technology was utilized to study the mechanism of transduction. Bio-distribution studies showed peak uptake of CTP at 15 min. Tc-HYNIC-CTP showed heart-specific uptake. Robust transduction of beating human iPSC-derived CMCs was seen. TriCEPs experiments revealed five candidate binding partners for CTP, with Kcnh5 being felt to be the most likely candidate as it showed a trend towards being competed out by siRNA knockdown. Transduction efficiency was enhanced by increasing extracellular potassium concentration, and with Quinidine, a Kcnh5 inhibitor, that blocks the channel in an open position. We demonstrate that CTP transduces the normal heart as early as 15 min. Tc-HYNIC-CTP targets the normal murine heart with substantially improved targeting compared with Tc Sestamibi. Cardiac targeting peptide's transduction ability is not species limited and has human applicability. Cardiac targeting peptide appears to utilize Kcnh5 to gain cell entry, a phenomenon that is affected by pre-treatment with Quinidine and changes in potassium levels.
我们之前的工作确定了一种针对心脏的 12 个氨基酸肽，称为心脏靶向肽（CTP）。现在，我们定量评估了 CTP 的生物分布，展示了一种用于鼠心脏成像的临床应用，并研究了其转导机制。在野生型小鼠中进行了 Cy5.5-羟基琥珀酰亚胺（Cy5.5）标记的 CTP 的生物分布研究。使用螯合剂肼基烟酰胺（HYNIC）对心脏靶向肽进行了锝 99m（Tc）标记，并使用微单光子发射计算机断层扫描/计算机断层扫描（SPECT/CT）进行成像。将双标记的 CTP 与人类诱导多能干细胞（iPSC）衍生的心肌细胞（CMCs）孵育，并使用共聚焦显微镜进行成像。利用 TriCEPs 技术研究转导机制。生物分布研究显示，CTP 的摄取峰值出现在 15 分钟。Tc-HYNIC-CTP 显示出心脏特异性摄取。可以看到跳动的人类 iPSC 衍生的 CMC 有很强的转导。TriCEPs 实验揭示了 CTP 的五个候选结合伴侣，其中 Kcnh5 被认为是最有可能的候选者，因为它显示出通过 siRNA 敲低被竞争出的趋势。通过增加细胞外钾浓度和使用奎尼丁（一种抑制开放状态下通道的 Kcnh5 抑制剂）可以增强转导效率。我们证明 CTP 在 15 分钟内即可转导正常心脏。Tc-HYNIC-CTP 与 Tc Sestamibi 相比，靶向正常鼠心脏的效果有了显著提高。心脏靶向肽的转导能力不受物种限制，具有人类适用性。心脏靶向肽似乎利用 Kcnh5 进入细胞，这种现象受奎尼丁预处理和钾水平变化的影响。
