THE GROWTH HORMONE SECRETAGOGUE RECEPTOR, GHRELIN, AND BNP IN HUMAN HEART DISEASE WITH PRESERVED EJECTION FRACTION

Heart disease (HD) diagnosis is based on clinical features, imaging, and circulating biomarkers, notably B-type natriuretic peptide (BNP), the gold standard marker of heart failure. However, a cardiac-specific biomarker detecting contractile dysfunction at early stages is lacking. The growth hormone secretagogue receptor (GHSR) and its ligand ghrelin may be cardiac-specific biomarkers. We have characterized a fluorescent analog of ghrelin, Cy5-ghrelin (1-19) that detects changes in cardiac GHSR in situ in diabetic cardiomyopathy and heart failure. We are now using this tool to map changes in regional and cellular distribution of GHSR in HD. We obtained tissue from the left ventricle (LV) and right atrium (RA) from 10 cardiac surgery patients with preserved ejection fraction (pEF) and from 10 deceased donors with no history of HD. GHSR levels were measured by quantitative fluorescence microscopy using Cy5-ghrelin(1-19). Ghrelin and BNP were measured by immunofluorescence. High-resolution images were acquired using a Nikon A1R confocal microscope and fluorescence intensities were quantified using ImageJ FIJI's built-in algorithms (Shanbhag for GHSR or RenyiEntropy for ghrelin and BNP). Values of fluorescence intensities were compared based on individual algorithms and colocalization analysis was conducted with the NIS-Elements software. Data were analysed by t-test, one-way ANOVA, and Pearson correlation coefficient (PCC) with significance set at p < 0.05. There were significantly lower levels of GHSR in RA tissue samples from cardiac surgery patients (p=0.041) and no significant differences in ghrelin or BNP expression (Figure 1). There was strong colocalization of ghrelin and BNP in both healthy (PCC=0.935) and diseased (PCC=0.856) tissue samples. In healthy tissue, colocalization appeared as large, discretely defined punctate areas near the nucleus, whereas in the tissue from cardiac surgery patients, ghrelin and BNP signals were more diffuse and contained in smaller areas surrounding the nucleus (Figure 2, arrows). We have shown expression of GHSR, ghrelin and BNP in both healthy and diseased cardiac tissue with only GHSR significantly changing in HD. This may indicate GHSR expression is decreased in HD with pEF whereas BNP (elevated in end stage HF) and ghrelin are not changed. The strong correlation between ghrelin and BNP indicates that these markers may be stored in the same intracellular compartment near the nucleus where they are released upon cellular stress induced by HD. This is the first study showing colocalization between ghrelin and BNP in human cardiac tissue. We are currently stratifying these results based on sex, type of HD, and region of the heart.View Large Image Figure ViewerDownload Hi-res image Download (PPT)