Fig. 2.

Identification of Nucleolin as a candidate regulator of cardiac growth. A: abundance of chromatin-bound Nucleolin was quantified by calculating the area under the curve (from 18 replicates: 6 replicates in each of 3 conditions) for each of two Nucleolin peptides used for identification and quantitation. Shown are the extracted ion chromatograms of the Nucleolin peptide VEGSEPTTPFNLFIGNLNPNK; the x-axis indicates chromatographic time. B: replicates from the three biological conditions (basal, hypertrophy, and failure) were averaged to give average abundance values for each condition which, as shown in the trend plot (panel C; this plot includes mean value of two peptides used for detection/quantitation), demonstrate an increase in Nucleolin during hypertrophy that persists in the failing heart. D: increased levels of chromatin-bound Nucleolin (Ncl) from mice in cardiac hypertrophy and failure were confirmed via Western blotting, supporting mass spectrometry results. IB, immunoblot. Total cellular (nuclear and cytoplasmic) levels of Nucleolin was also examined in mice euthanized in stages of hypertrophy or failure (E; *P < 0.01.), as determined by heart weight (HW) to body weight (BW) ratio and ejection fraction (ECHO), by qPCR analysis showing a decrease in the hypertrophic myocardium, accompanied by changes in pre-rRNA (ITS) and mature (18S) rRNA using primers that uniquely detect these species (F and G). These results suggest that while chromatin bound Nucleolin increases during cardiac hypertrophy, total Nucleolin mRNA levels in the cell decrease. To examine the role of Nucleolin in the cardiomyocyte, isolated neonatal rat ventricular myocytes (NRVMs) were treated with Nucleolin siRNA or lipofectamine control. Western blot analysis confirmed that loss of Nucleolin results in an increase in the heterochromatin mark H3K9Me3 (blots are indicative of 3 independent experiments, n = 3 in each; H) but had no effect on cell size under basal conditions when quantified via phalloidin staining (I). Number of cells measured is indicated in each bar. F: rRNA transcription and maturation was examined using primers that detect pre-rRNA (ITS) and mature rRNA (18S) by qRT-PCR. J: loss of Nucleolin in NRVMs also led to a decrease in both rRNA transcription and maturation. n = 3/group. Asterisks indicate a P value < 0.05. K: strikingly, loss of Nucleolin in rat ventricular myocytes also led to an increase in the expression of β-MHC, ANF and Serca2a, suggesting that Nucleolin regulates some of the mRNA-encoding genes known to control pathological cardiac growth. L: additionally, while the fraction of Nucleolin bound to chromatin increases in a mouse model of pressure-overload hypertrophy, total cellular levels of Nucleolin were altered in an agonist-dependent manner in the setting of hypertrophy in NRVMs (n = 3/group). Error bars indicate SE. ISO, isoproterenol; ET-1, endothelin-1; PE, phenylephrine.