Phosphonates are rare and unusually bioactive natural products. However, most bacterial phosphonate biosynthetic capacity is dedicated to tailoring cell surfaces with molecules like 2-aminoethylphosphonate (AEP). Although phosphoenolpyruvate mutase (Ppm)-catalyzed installation of C-P bonds is known, subsequent phosphonyl tailoring (Pnt) pathway steps remain enigmatic. Here we identify nucleotidyltransferases in over two-thirds of phosphonate biosynthetic gene clusters, including direct fusions to ~60% of Ppm enzymes. We characterize two putative phosphonyl tailoring cytidylyltransferases (PntCs) that prefer AEP over phosphocholine (P-Cho) – a similar substrate used by the related enzyme LicC, which is a virulence factor in Streptococcus pneumoniae. PntC structural analyses reveal steric discrimination against phosphocholine. These findings highlight nucleotidyl activation as a predominant chemical logic in phosphonate biosynthesis and set the stage for probing diverse phosphonyl tailoring pathways.

Currently, the early pre-clinical detection of left ventricular (LV) dysfunction is difficult as biomarkers are not specific for the cardiomyopathic process. The underlying molecular mechanisms leading to heart failure remain elusive, highlighting the need for identification of cardiac-specific markers. The growth hormone secretagogue receptor (GHSR) and its ligand ghrelin are present in cardiac tissue and are known to contribute to myocardial energetics. Here, we examined tissue ghrelin-GHSR levels as specific markers of cardiac dysfunction in patients who underwent cardiac transplantation. Samples of cardiac tissue were obtained from 10 cardiac transplant patients at the time of organ harvesting, and during serial post-transplant biopsies. Quantitative fluorescence microscopy using a novel fluorescent ghrelin analog was used to measure levels of GHSR, and immunofluorescence was used to measure levels of ghrelin, B-type natriuretic peptide (BNP) and tissue markers of cardiomyocyte contractility and growth. GHSR and ghrelin expression levels were highly variable in the explanted heart, less in the grafted heart biopsies. GHSR and ghrelin were strongly positively correlated, and both markers were negatively correlated with LV ejection fraction. Ghrelin had stronger positive correlations than BNP with the signaling markers for contractility and growth. These data suggest that GHSR-ghrelin have potential use as an integrated marker of cardiac dysfunction. Interestingly, tissue ghrelin appeared to be a more sensitive indicator than BNP to the biochemical processes that are characteristic of heart failure. This work allows for further use of ghrelin-GHSR to interrogate cardiac-specific biochemical mechanisms in pre-clinical stages of HF.

The hormone ghrelin and its receptor, the growth hormone secretagogue receptor (GHSR) are expressed in myocardium. GHSR binding activates signalling pathways coupled to cardiomyocyte survival and contractility. These properties have made the ghrelin-GHSR axis a candidate for a biomarker of cardiac function. The dynamics of ghrelin-GHSR are altered significantly in late stages of heart failure (HF) and cardiomyopathy, when left ventricular (LV) function is failing. We examined the relationship of GHSR with ghrelin in cardiac tissue from patients with valvular disease with no detectable changes in LV function. Biopsy samples from the left ventricle and left atrium were obtained from 25 patients with valvular disease (of whom 13 also had coronary artery disease) and preserved LV ejection fraction, and compared to control samples obtained via autopsy. Using quantitative confocal fluorescence microscopy, levels of GHSR were determined using [Dpr3(n-octanoyl),Lys19(sulfo-Cy5)]ghrelin(1-19), and immunofluorescence determined ghrelin, the heart failure marker natriuretic peptide type-B (BNP), and contractility marker sarcoplasmic reticulum ATPase pump (SERCA2a). A positive correlation between GHSR and ghrelin was apparent in only diseased tissue. Ghrelin and BNP significantly correlated in the left ventricle and strongly colocalized to the same intracellular compartment in diseased and control tissue. GHSR, ghrelin, and BNP all strongly and significantly correlated with SERCA2a in the left ventricle of diseased tissue only. Our results suggest that the dynamics of the myocardial ghrelin-GHSR axis is altered in cardiovascular disease in the absence of measurable changes in heart function, and might accompany a regional shift in endocrine programming.

The hormone ghrelin and its receptor, the growth hormone secretagogue receptor (GHSR) are expressed in myocardium. GHSR binding activates signalling pathways coupled to cardiomyocyte survival and contractility. These properties have made the ghrelin-GHSR axis a candidate for a biomarker of cardiac function. The dynamics of ghrelin-GHSR are altered significantly in late stages of heart failure and cardiomyopathy, when left ventricular (LV) function is failing. We examined the relationship of GHSR with ghrelin in cardiac tissue from patients with valvular disease with no detectable changes in LV function. Biopsy samples from the LV and left atrium (LA) were obtained from 25 patients with valvular disease (of whom 13 also had coronary artery disease) and preserved LV ejection fraction, and compared control samples obtained via autopsy. Using quantitative confocal fluorescence microscopy, levels of GHSR were determined using a fluorescent peptide analog of ghrelin, Cy5-ghrelin(1-19); ghrelin, the heart failure marker natriuretic peptide type-b (BNP), and contractility marker sarcoplasmic reticulum ATPase pump (SERCA2a) were measured by immunofluorescence. A positive correlation between GHSR and ghrelin was apparent in only diseased tissue. Ghrelin and BNP significantly correlated in the LV and strongly co-localized to the same intracellular compartment in both diseased and control tissue. GHSR, ghrelin and BNP all strongly and significantly correlated with SERCA2a in the LV of diseased tissue only. Our results suggest that the dynamics of the myocardial ghrelin/GHSR axis is altered in cardiovascular disease in the absence of measurable changes in heart function, and may accompany a regional shift in endocrine programming.

Background: Currently, the early pre-clinical detection of left ventricular (LV) dysfunction is difficult as biomarkers are not specific for the cardiomyopathic process. The underlying molecular mechanisms leading to heart failure remain elusive, highlighting the need for identification of cardiac-specific markers. The growth hormone secretagogue receptor (GHSR) and its ligand ghrelin are present in cardiac tissue and are known to contribute to myocardial energetics. Here, we examined tissue ghrelin-GHSR levels as specific markers of cardiac dysfunction in patients who underwent cardiac transplantation. Methods and Results: Samples of cardiac tissue were obtained from 10 cardiac transplant patients at the time of organ harvesting, and during serial post-transplant biopsies. Quantitative fluorescence microscopy using a novel fluorescent ghrelin analog was used to measure levels of GHSR, and immunofluorescence was used to measure levels of ghrelin, b-type natriuretic peptide (BNP) and tissue markers of cardiomyocyte contractility and growth. GHSR and ghrelin expression levels were highly variable in the explanted heart, less in the grafted heart biopsies. GHSR and ghrelin were strongly positively correlated, and both markers were negatively correlated with LV ejection fraction. Ghrelin had stronger positive correlations than BNP with the signaling markers for contractility and growth. Conclusions: These data suggest that GHSR-ghrelin have potential use as an integrated marker of cardiac dysfunction. Interestingly, tissue ghrelin appeared to be a more sensitive indicator than BNP to the biochemical processes that are characteristic of heart failure. This work allows for further use of ghrelin-GHSR to interrogate cardiac-specific biochemical mechanisms in pre-clinical stages of HF. Precis: This study shows the relationships between GHSR, ghrelin, and signaling molecules with relation to heart function in human heart failure with tissue from diseased heart and healthy heart biopsies.