Altered myocardial expression of ghrelin and its receptor (GHSR-1a) in patients with severe heart failure

Characterization of a far-red analog of ghrelin for imaging GHS-R in P19-derived cardiomyocytes

“…GHS-R expression is up-regulated in left ventricular tissue from patients with congestive heart failure [3] and atherosclerosis [8], as well as in the hearts of rats during the hyperdynamic phase of sepsis [28]. These results indicate that GHS-R signaling is altered in cardiovascular disease, and point toward the use of ghrelin in pharmacologic therapy of heart disease.…”

“…Lys 19 (mtt) was selectively deprotected using 2% trifluoroacetic acid, 5% triisopropylsilane in dichloromethane, followed by coupling to Cy5 succinimidyl ester (0.025 mmol, 19 mg) in the presence of N,N-diisopropylethylamine (0.6 mmol, 100 L). Once fully functionalized, Dpr 3 (octanoyl), Lys 19 (Cy5) ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) was cleaved from the resin using 95% trifluoroacetic acid, 2.5% triisopropylsilane and 2.5% water (2 mL). The crude peptide was precipitated using ice cold tert-butylmethylether (20 mL) and the dark blue crude solid was then isolated by centrifugation.…”

“…The following day, 10 M Cy5-ghrelin(1-19) or 10 M Cy5-des-acyl ghrelin(1-19) was added to the dishes (500 L/well), and cells were incubated at 37 • C for 30 min, and subsequently rinsed in serum-free media. For blocking studies, HEK/GHS-R1a cells were incubated with 10 M Cy5-ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) in the presence of 40 M unlabeled ghrelin . Live cell images were captured using an inverted fluorescent microscope and final images were prepared using ImageJ.…”

“…Adult male or female C57BL/6 mice were sacrificed using CO 2 and the hearts were immediately removed and fixed in 4% paraformaldehyde (in PBS) overnight. Whole hearts were then equilibrated in 30% sucrose in PBS, snap-frozen in Tissue-Tek OCT using dry-ice cooled ethanol, and subsequently cryosectioned at 8 m. To assess binding of both acylated and non-acylated ghrelin, sections were incubated for 30 min at room temperature with various concentrations (0-20 M) of Cy5-ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) or Cy5-des-acyl ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) alone. To determine the specificity of binding, sections were incubated with 10 M Cy5-ghrelin(1-19) and either 100 M ghrelin (1-28) or 100 M hexarelin.…”

“…These results indicate that GHS-R signaling is altered in cardiovascular disease, and point toward the use of ghrelin in pharmacologic therapy of heart disease. Therefore, given the importance of GHS-R signaling and expression in the heart, we are developing ghrelin analogs for the purpose of generating agents for in situ and in vivo imaging of cardiac GHS-R. We have previously designed a fluorescent ghrelin analog consisting of a fluorescein moiety attached through a lysine to the N-terminal 18 amino acids of ghrelin (fluorescein-ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)) [20] and have shown that it specifically binds to GHS-R1a in mouse heart tissue.…”