Abstract 18007: A Novel Cell Penetrating Peptide, Cardiac Targeting Peptide, Appears to Utilize Cardiac Channels for Transduction
Background: Cell penetrating peptides (CPP) are small peptides able to carry cargo across cellular membranes resulting in internalization of the intact cargo. Our prior work led to identification of a 12-amino acid peptide, APWHLSSQYSRT, a tissue-specific CPP, which we termed Cardiac Targeting Peptide (CTP), because of its ability to efficiently transduce the mouse heart tissue after an intravenous injection. CTP has the potential to be a novel delivery tool for the heart for both diagnostic and therapeutic purposes. We present the result of studies undertaken to determine CTP’s mechanism of transduction.
Methods and Results: Neonatal mouse hearts were harvested and cardiomyocytes (CMCs) isolated. Mouse embryonic fibroblasts (MEFs) were used as negative controls. CTP was synthesized dually labeled with 6-carboxyfluoroscein and Rhodamine, the latter via an ester link, susceptible to cleavage only by intracellular esterases. Hence, internalization and red fluorescence could not result from membrane attachment or fixation artifact alone. Cells were incubated with 25uM of CTP and showed robust transduction of beating CMCs within 10 minutes, with essentially no uptake by MEFs. This transduction was significantly enhanced by increasing extracellular K+ ion concentration to 20mM (p-value=0.03;Figure 1), without any change by calcium channel inhibitor Verapamil (Vpml). Increased CTP uptake secondary to cell death/apoptosis was ruled out by restoring extracellular K+ concentration to normal levels and resumption of CMC contractile function. Rhodamine with cleaved ester link alone was not internalized by CMCs.
Conclusions: CTP rapidly enters CMCs in a cell specific manner. This transduction is enhanced by increasing the Na-K ATPase pump activity by increasing extracellular K+ concentration, implying that CTP is using this ion channel for cell entry. Further studies utilizing Na-K channel inhibitors and agonists are needed to confirm our findings.
Author Disclosures: T. Feinstein: None. C.W. Lo: None. M. Zahid: Ownership Interest; Significant; Holds patent on Cardiac Targeting Peptide.
- © 2016 by American Heart Association, Inc.