in-vivo-evaluation-of-mucociliary-clearance-in-mice

In vivo Evaluation of Mucociliary Clearance in Mice

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a ...

in-vivo-imaging-transduction-efficiencies-cardiac-targeting

In Vivo Imaging of Transduction Efficiencies of Cardiac Targeting Peptide

Since the initial description of protein transduction domains, also known as cell penetrating peptides, over 25 years ago, there has been intense interest ...

University of Pittsburgh School of Medicine

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.

Kyle S. Feldman

Department of Developmental Biology

University of Pittsburgh School of Medicine

In vivo Evaluation of Mucociliary Clearance in Mice

In Vivo Imaging of Transduction Efficiencies of Cardiac Targeting Peptide

Kyle S. Feldman

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Developmental Biology

University of Pittsburgh School of Medicine

In vivo Evaluation of Mucociliary Clearance in Mice

In Vivo Imaging of Transduction Efficiencies of Cardiac Targeting Peptide

Department of Developmental Biology