Characterization of a far-red analog of ghrelin for imaging GHS-R in P19-derived cardiomyocytes.
Ghrelin and its receptor, the growth hormone secretagogue receptor (GHS-R), are expressed in the heart, and may function to promote cardiomyocyte survival, differentiation and contractility. Previously, we had generated a truncated analog of ghrelin conjugated to fluorescein isothiocyanate for the purposes of determining GHS-R expression in situ. We now report the generation and characterization of a far-red ghrelin analog, [Dpr(3)(octanoyl), Lys(19)(Cy5)]ghrelin (1-19), and show that it can be used to image changes in GHS-R in developing cardiomyocytes. We also generated the des-acyl analog, des-acyl [Lys(19)(Cy5)]ghrelin (1-19) and characterized its binding to mouse heart sections. Receptor binding affinity of Cy5-ghrelin as measured in HEK293 cells overexpressing GHS-R1a was within an order of magnitude of that of fluorescein-ghrelin and native human ghrelin, while the des-acyl Cy5-ghrelin did not bind GHS-R1a. Live cell imaging in HEK293/GHS-R1a cells showed cell surface labeling that was displaced by excess ghrelin. Interestingly, Cy5-ghrelin, but not the des-acyl analog, showed concentration-dependent binding in mouse heart tissue sections. We then used Cy5-ghrelin to track GHS-R expression in P19-derived cardiomyocytes. Live cell imaging at different time points after DMSO-induced differentiation showed that GHS-R expression preceded that of the differentiation marker aMHC and tracked with the contractility marker SERCA 2a. Our far-red analog of ghrelin adds to the tools we are developing to map GHS-R in developing and diseased cardiac tissues.
Departments of Pathology, Chemistry, Medical Imaging, Medical Biophysics, and Oncology, Western University, Richmond Street, London, Ontario, Canada; Imaging Program, Lawson Health Research Institute, 268 Grosvenor Street, London, Ontario N6A 4V2, Canada.
Gregory A F
GA
Douglas
Departments of Pathology, Chemistry, Medical Imaging, Medical Biophysics, and Oncology, Western University, Richmond Street, London, Ontario, Canada; Imaging Program, Lawson Health Research Institute, 268 Grosvenor Street, London, Ontario N6A 4V2, Canada.
Rebecca
R
McGirr
Departments of Pathology, Chemistry, Medical Imaging, Medical Biophysics, and Oncology, Western University, Richmond Street, London, Ontario, Canada; Imaging Program, Lawson Health Research Institute, 268 Grosvenor Street, London, Ontario N6A 4V2, Canada.
Carlie L
CL
Charlton
Departments of Pathology, Chemistry, Medical Imaging, Medical Biophysics, and Oncology, Western University, Richmond Street, London, Ontario, Canada; Imaging Program, Lawson Health Research Institute, 268 Grosvenor Street, London, Ontario N6A 4V2, Canada.
Dov B
DB
Kagan
Departments of Pathology, Chemistry, Medical Imaging, Medical Biophysics, and Oncology, Western University, Richmond Street, London, Ontario, Canada; Imaging Program, Lawson Health Research Institute, 268 Grosvenor Street, London, Ontario N6A 4V2, Canada.
Lisa M
LM
Hoffman
Departments of Pathology, Chemistry, Medical Imaging, Medical Biophysics, and Oncology, Western University, Richmond Street, London, Ontario, Canada; Imaging Program, Lawson Health Research Institute, 268 Grosvenor Street, London, Ontario N6A 4V2, Canada.
Leonard G
LG
Luyt
Departments of Pathology, Chemistry, Medical Imaging, Medical Biophysics, and Oncology, Western University, Richmond Street, London, Ontario, Canada; Imaging Program, Lawson Health Research Institute, 268 Grosvenor Street, London, Ontario N6A 4V2, Canada. Electronic address: sdhanvan@lawsonimaging.ca.
Savita
S
Dhanvantari
D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation; D005260:Female; D054439:Ghrelin / Q000031:analogs & derivatives* / Q000378:metabolism; D057809:HEK293 Cells / Q000378:metabolism; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D057054:Molecular Imaging / Q000379:methods*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D010446:Peptide Fragments; D054440:Receptors, Ghrelin / Q000032:analysis* / Q000378:metabolism
C000597542:(Dpr3(octanoyl)Lys19(Cy5))ghrelin(1-19); D054439:Ghrelin; D010446:Peptide Fragments; D054440:Receptors, Ghrelin; C422650:ghrelin, des-n-octanoyl
Cardiomyocytes; Differentiation; Fluorescence; GHS-R; Ghrelin; P19 cells