Rationale Hyperamylinemia is common in patients with obesity and insulin resistance, coincides with hyperinsulinemia, and results in amyloid deposition. Amylin amyloids are generally considered a pancreatic disorder in type-2 diabetes. However, elevated circulating levels of amylin may also lead to amylin accumulation and proteotoxicity in peripheral organs, including the heart. Objective To test whether amylin accumulates in the heart of obese and type-2 diabetic patients and to uncover the effects of amylin accumulation on cardiac morphology and function. Methods and Results We compared amylin deposition in failing and non-failing hearts from lean, obese, and type-2 diabetic humans using immunohistochemistry and western blots. We found significant accumulation of large amylin oligomers, fibrils and plaques in failing hearts from obese and diabetic patients, but not in normal hearts and failing hearts from lean, non-diabetic humans. Small amylin oligomers were even elevated in non-failing hearts from overweight/obese patients suggesting an early state of accumulation. Using a rat model of hyperamylinemia transgenic for human amylin, we observed that amylin oligomers attach to the sarcolemma, leading to myocyte Ca2+ dysregulation, pathological myocyte remodeling, and diastolic dysfunction, starting from pre-diabetes. In contrast, pre-diabetic rats expressing the same level of wild-type rat amylin, a non-amyloidogenic isoform, exhibited normal heart structure and function. Conclusions Hyperamylinemia promotes amylin deposition in the heart causing alterations of cardiac myocyte structure and function. We propose that detection and disruption of cardiac amylin buildup may be both a predictor of heart dysfunction and a novel therapeutic strategy in diabetic cardiomyopathy.

Rationale Calmodulin (CaM) associates with cardiac ryanodine receptors (RyR2) as an important regulator. Defective CaM-RyR2 interaction may occur in heart failure (HF), cardiac hypertrophy, and catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the in situ binding properties for CaM-RyR2 are unknown. Objective We sought to measure the in situ binding affinity and kinetics for CaM-RyR2 in normal and HF ventricular myocytes, estimate the percentage of Z-line localized CaM that is RyR2-bound and test cellular function of defective CaM-RyR2 interaction. Methods & Results Using FRET (fluorescence resonance energy transfer) in permeabilized myocytes, we specifically resolved RyR2-bound CaM from other potential binding targets, and measured CaMRyR2 binding affinity in situ (Kd =10-20 nM). Using RyR2ADA/+ knock-in (KI) mice, in which half of the CaM-RyR2 binding is suppressed, we estimated that >90% of Z-line CaM is RyR2-bound. Functional tests indicated a higher propensity for Ca2+ waves production and stress induced ventricular arrhythmia in RyR2ADA/+ mice. In a post myocardial infarction (MI) rat HF model, we detected a decrease in the CaMRyR2 binding affinity (Kd ≈ 51nM, ~3 fold increase) and unaltered FKBP12.6-RyR2 binding affinity (Kd ≈ 0.8nM). Conclusions CaM binds to RyR2 with high affinity in cardiac myocytes. Physiologically, CaM is bound to >70% of RyR2 monomers and inhibits SR Ca2+ release. RyR2 is the major binding site for CaM along the Z-line in cardiomyocytes and dissociating CaM from RyR2 can cause severe ventricular arrhythmia. In HF, RyR2 shows decreased CaM affinity, but unaltered FKBP12.6 affinity.

We have previously demonstrated that intermittent high-altitude (IHA) hypoxia significantly attenuates ischemia-reperfusion (I/R) injury-induced excessive increase in resting intracellular Ca(2+) concentrations ([Ca(2+)](i)). Because the sarcoplasmic reticulum (SR) and Na(+)/Ca(2+) exchanger (NCX) play crucial roles in regulating [Ca(2+)](i) and both are dysfunctional during I/R, we tested the hypothesis that IHA hypoxia may prevent I/R-induced Ca(2+) overload by maintaining Ca(2+) homeostasis via SR and NCX mechanisms. We thus determined the dynamics of Ca(2+) transients and cell shortening during preischemia and I/R injury in ventricular cardiomyocytes from normoxic and IHA hypoxic rats. IHA hypoxia did not affect the preischemic dynamics of Ca(2+) transients and cell shortening, but it significantly suppressed the I/R-induced increase in resting [Ca(2+)](i) levels and attenuated the depression of the Ca(2+) transients and cell shortening during reperfusion. Moreover, IHA hypoxia significantly attenuated I/R-induced depression of the protein contents of SR Ca(2+) release channels and/or ryanodine receptors (RyRs) and SR Ca(2+) pump ATPase (SERCA2) and SR Ca(2+) release and uptake. In addition, a delayed decay rate time constant of Ca(2+) transients and cell shortening of Ca(2+) transients observed during ischemia was accompanied by markedly inhibited NCX currents, which were prevented by IHA hypoxia. These findings indicate that IHA hypoxia may preserve Ca(2+) homeostasis and contraction by preserving RyRs and SERCA2 proteins as well as NCX activity during I/R.

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Backgroundgalectin-1 has been implicated in tumor invasion and metastasis and is frequently over-expressed in epithelial ovarian cancer (EOC), but its potential as a biomarker remains unclear. In this novel study, we have explored the possible use of galectin-1 as a biomarker for EOC.Methodsgalectin-1 in sera was evaluated by ELISA in a pilot panel of EOC patients, healthy volunteers, patients with benign gynecologic tumors or other gynecologic malignancies. We examined galectin-1 expression in EOC tumor samples by Western Blot, qRT-PCR and immunohistochemistry. In vitro experiments were conducted to elucidate the biologic role of galectin-1 in EOC progression using over-expression of galectin-1 in OVCAR-3 cells. We also looked for the association of galectin-1 expression with clinic pathological variables and survival outcomes in EOC.ResultsA significant difference was detected in serum galectin-1 between EOC patients with non-metastatic and those with metastatic disease, but not between EOC patients and healthy volunteers. It increased in recurrent cases and decreased after debulking surgery. Both of galectin-1 mRNA and protein levels were increased in 90 % of the examined EOC tissue samples, compared with a wedge resection of a normal ovary. High galectin-1 in peritumor stroma was primarily detected in advanced stages of EOC. Over expression of galectin-1 significantly increased the ability of OVCAR-3 cells’ migration and invasion.ConclusionsOur results suggest that galectin-1 might play a role in tumor progression and be associated with poor outcome in EOC. It could be a novel prognostic and progression biomarker in EOC patients.

Background/Aims: Our study aims to investigate the role, effect and mechanisms of ESRP1 (epithelial splicing regulatory protein 1) in epithelial-mesenchymal transition (EMT) in epithelial ovarian cancer (EOC). Methods: Microarray and immunohistochemical analysis of ESRP1 expression were performed in EOC cases. The correlations between ESRP1 expression and clinical factors on EOC were assessed. Lentivirus-mediated RNA interference and EGFP vector which contains ESRP1 gene were used to down-regulate and up-regulate ESRP1 expression in human EOC cell lines. Roles of ESRP1 in cell growth, migration and invasion of EOC cells were also measured by Cell Counting Kit-8 and Transwell systems in vitro and by a nude mice intraperitoneal transplantation model in vivo. Results: By the analysis of Gene Expression Omnibus (GEO) (p<0.05) and our own microarray data (p<0.001), ESRP1 expression in EOC was significantly different from normal ovarian tissue. It was abundant in the nuclei of cancer cells and in malignant lesions. However, it was weakly expressed or negative in both normal and benign lesions. High ESRP1 expression in EOC was associated with poor clinical outcomes. Decreased ESRP1 expression significantly increased cell migration and invasion both in vivo and in vitro. Snail strongly repressed ESRP1 transcription through binding to the ESRP1 promoter in EOC cells. Furthermore, ESRP1 regulated the expression of CD44s. Down-regulated ESRP1 resulted in an isoform switching from CD44v to CD44s, which modulated epithelial-mesenchymal transition (EMT) program in EOC. Up-regulatin of ESRP1 was detected in mesenchymal to epithelial transition (MET) in vivo. Conclusions: ESRP1 regulates CD44 alternative splicing during the EMT process which plays an important role in EOC carcinogenesis. In addition, ESRP1 is associated with disease prognosis in EOC.Jie Tang, MD, Ph.D, Professor 283 Tongzipo Road, Yuelu District,

The detection of all glands during total parathyroidectomy (TPTX) in secondary hyperparathyroidism (SHPT) patients is often difficult due to their variability in number and location. The objective of this study was to evaluate the feasibility of near-infrared fluorescence (NIRF) imaging using indocyanine green (ICG) for intraoperative parathyroid gland (PTG) localization in SHPT patients. Twenty-nine patients with SHPT were divided into two groups with or without intraoperative NIRF imaging. ICG was administered in patients undergoing intraoperative imaging, and the fluorescence of PTGs was assessed. Clinical and histopathologic variables were analyzed to determine factors associated with ICG uptake. Comparisons between NIRF and preoperative imaging, as well as differences between groups with or without NIRF imaging, were carried out to evaluate the efficacy of this technique. Most PTGs could be clearly identified, including one ectopic gland. The sensitivity of NIRF imaging is 91.1% in contrast to 81.82% for ultrasonography (US), 62.34% for 99mTc-MIBI and 85.71% for computed tomography (CT). In addition, intraoperative NIRF imaging can reduce the operation time and improve the complete resection rate compared with the group not using it. Intraoperative NIRF imaging using ICG during TPTX is technically feasible and reliable for assisting surgeons in detecting and confirming PTGs.

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