Abstract
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-N-Hydroxysuccinimide (Cy5.5) labeled CTP were undertaken in wild-type mice. Cardiac targeting peptide was labeled with Technetium 99m (99mTc) 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. 99mTc-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. 99mTc-HYNIC-CTP targets the normal murine heart with substantially improved targeting compared with 99mTc 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.
Highlights
The Achilles heel of cardiac therapeutics, as well as diagnostics, has been a lack of cardiac-specific vectors
We show an application of cardiac targeting peptide (CTP) to target Technetium 99m (99m Tc) to the heart for imaging purposes
We show that fluorescently labeled CTP is rapidly and successfully internalized by human-induced pluripotent stem cell-derived beating CMCs, indicating that our findings are not species-limited to the mouse heart, but they have potentials for human application
Summary
The Achilles heel of cardiac therapeutics, as well as diagnostics, has been a lack of cardiac-specific vectors. Biomolecules 2018, 8, 147 and using cardiac-specific promoters, but take from days to a week for expression, have either pre-existing immunity or evoke a rapid immune response on first exposure, generating neutralizing antibodies that preclude repeat therapies [2]. These approaches are limited to the delivery of nucleic acids only. In 1988, two separate groups demonstrated the ability of the trans-activator of transcription (Tat) protein of the human immunodeficiency virus (HIV) to enter cultured cells and to promote viral gene expression [3,4]
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