Plasma CCN2/connective tissue growth factor is associated with right ventricular dysfunction in patients with neuroendocrine tumors

<p>Abstract</p> <p>Background</p> <p>Carcinoid heart disease, a known complication of neuroendocrine tumors, is characterized by right heart fibrotic lesions. Carcinoid heart disease has traditionally been defined by the degree of valvular involvement. Right ventricular (RV) dysfunction due to mural involvement may also be a manifestation. Connective tissue growth factor (CCN2) is elevated in many fibrotic disorders. Its role in carcinoid heart disease is unknown. We sought to investigate the relationship between plasma CCN2 and valvular and mural involvement in carcinoid heart disease.</p> <p>Methods</p> <p>Echocardiography was performed in 69 patients with neuroendocrine tumors. RV function was assessed using tissue Doppler analysis of myocardial systolic strain. Plasma CCN2 was analyzed using an enzyme-linked immunosorbent assay. Mann-Whitney U, Kruskal-Wallis, Chi-squared and Fisher's exact tests were used to compare groups where appropriate. Linear regression was used to evaluate correlation.</p> <p>Results</p> <p>Mean strain was -21% ± 5. Thirty-three patients had reduced RV function (strain > -20%, mean -16% ± 3). Of these, 8 had no or minimal tricuspid and/or pulmonary regurgitation (TR/PR). Thirty-six patients had normal or mildly reduced RV function (strain ≤ -20%, mean -25% ± 3). There was a significant inverse correlation between RV function and plasma CCN2 levels (r = 0.47, p < 0.001). Patients with reduced RV function had higher plasma CCN2 levels than those with normal or mildly reduced RV function (p < 0.001). Plasma CCN2 ≥ 77 μg/L was an independent predictor of reduced RV function (odds ratio 15.36 [95% CI 4.15;56.86]) and had 88% sensitivity and 69% specificity for its detection (p < 0.001). Plasma CCN2 was elevated in patients with mild or greater TR/PR compared to those with no or minimal TR/PR (p = 0.008), with the highest levels seen in moderate to severe TR/PR (p = 0.03).</p> <p>Conclusions</p> <p>Elevated plasma CCN2 levels are associated with RV dysfunction and valvular regurgitation in NET patients. CCN2 may play a role in neuroendocrine tumor-related cardiac fibrosis and may serve as a marker of its earliest stages.</p

Real-time conformational changes in LacY

Galactoside/H(+) symport across the cytoplasmic membrane of Escherichia coli is catalyzed by lactose permease (LacY), which uses an alternating access mechanism with opening and closing of deep cavities on the periplasmic and cytoplasmic sides. In this study, conformational changes in LacY initiated by galactoside binding were monitored in real time by Trp quenching/unquenching of bimane, a small fluorophore covalently attached to the protein. Rates of change in bimane fluorescence on either side of LacY were measured by stopped flow with LacY in detergent or in proteoliposomes and were compared with rates of galactoside binding. With LacY in proteoliposomes, the periplasmic cavity is tightly sealed and the substrate-binding rate is limited by the rate of opening of this cavity. Rates of opening, measured as unquenching of bimane fluorescence, are 20–30 s(−1), independent of sugar concentration and essentially the same in detergent or in proteoliposomes. On the cytoplasmic side of LacY in proteoliposomes, slow bimane quenching (i.e., closing of the cavity) is observed at a rate that is also independent of sugar concentration and similar to the rate of sugar binding from the periplasmic side. Therefore, opening of the periplasmic cavity not only limits access of sugar to the binding site of LacY but also controls the rate of closing of the cytoplasmic cavity

Probing of the rates of alternating access in LacY with Trp fluorescence

Sugar/H+ symport by lactose permease (LacY) utilizes an alternating access mechanism in which sugar and H+ binding sites in the middle of the molecule are alternatively exposed to either side of the membrane by sequential opening and closing of inward- and outward-facing hydrophilic cavities. Here, we introduce Trp residues on either side of LacY where they are predicted to be in close proximity to side chains of natural Trp quenchers in either the inward- or outward-facing conformers. In the inward-facing conformer, LacY is tightly packed on the periplasmic side, and Trp residues placed at positions 245 (helix VII) or 378 (helix XII) are in close contact with His-35 (helix I) or Lys-42 (helix II), respectively. Sugar binding leads to unquenching of Trp fluorescence in both mutants, a finding clearly consistent with opening of the periplasmic cavity. The pH dependence of Trp-245 unquenching exhibits a pKa of 8, typical for a His side chain interacting with an aromatic group. As estimated from stopped-flow studies, the rate of sugar-induced opening is ≈100 s−1. On the cytoplasmic side, Phe-140 (helix V) and Phe-334 (helix X) are located on opposite sides of a wide-open hydrophilic cavity. In precisely the opposite fashion from the periplasmic side, mutant Phe-140→Trp/Phe-334→His exhibits sugar-induced Trp quenching. Again, quenching is pH dependent (pKa = 8), but remarkably, the rate of sugar-induced quenching is only ≈0.4 s−1. The results provide yet another strong, independent line of evidence for the alternating access mechanism and demonstrate that the methodology described provides a sensitive probe to measure rates of conformational change in membrane transport proteins