Calcitriol Suppressed Isoproterenol-induced Proliferation of Cardiac Fibroblasts via Integrin ß3/FAK/Akt Pathway.

OBJECTIVE: Cardiac fibroblasts (CFs) proliferation and extracellular matrix deposition are important features of cardiac fibrosis. Various studies have indicated that vitamin D displays an anti-fibrotic property in chronic heart diseases. This study explored the role of vitamin D in the growth of CFs via an integrin signaling pathway. METHODS: MTT and 5-ethynyl-2'-deoxyuridine assays were performed to determine cell viability. Western blotting was performed to detect the expression of proliferating cell nuclear antigen (PCNA) and integrin signaling pathway. The fibronectin was observed by ELISA. Immunohistochemical staining was employed to evaluate the expression of integrin ß3. RESULTS: The PCNA expression in the CFs was enhanced after isoproterenol (ISO) stimulation accompanied by an elevated expression of integrin beta-3 (ß3). The blockade of the integrin ß3 with a specific integrin ß3 antibody reduced the PCNA expression induced by the ISO. Decreasing the integrin ß3 by siRNA reduced the ISO-triggered phosphorylation of FAK and Akt. Both the FAK inhibitor and Akt inhibitor suppressed the PCNA expression induced by the ISO in the CFs. Calcitriol (CAL), an active form of vitamin D, attenuated the ISO-induced CFs proliferation by downregulating the integrin ß3 expression, and phosphorylation of FAK and Akt. Moreover, CAL reduced the increased levels of fibronectin and hydroxyproline in the CFs culture medium triggered by the ISO. The administration of calcitriol decreased the integrin ß3 expression in the ISO-induced myocardial injury model. CONCLUSION: These findings revealed a novel role for CAL in suppressing the CFs growth by the downregulation of the integrin ß3/FAK/Akt pathway.

Efficacy of Long-term Selenium Supplementation in the Treatment of Chronic Keshan Disease with Congestive Heart Failure.

Few effective treatments for chronic Keshan disease have been available till now. The efficacy of long-term selenium supplementation in the treatment of chronic Keshan disease with congestive heart failure is inconclusive. This study aimed to determine whether selenium supplementation is associated with a decreased risk of cardiac death in chronic Keshan disease with congestive heart failure by ten years of follow-up. A retrospective long-term follow-up analysis was performed on a monitored cohort consisting of 302 chronic Keshan disease patients with a mean age of 40.8±11.4 years. Of the 302 chronic Keshan disease patients, 170 (56.3%) were given selenium supplementation until the end point of follow-up. Cox proportional hazards regression models were used to identify the independent predictors of cardiac events. Our results showed that during the follow-up, there were 101 deaths of patients with chronic Keshan disease in the selenium supplementation group (101/170, 59.4%) and 98 in non-selenium supplementation group (98/132, 74.2%). Multivariate analyses suggested that selenium supplementation was associated with a decreased risk of cardiac death (HR 0.39, 95% CI 0.28-0.53) after adjustment for baseline age, sex, cigarette smoking, family history of Keshan disease, body mass index (BMI), heart rate, electrocardiogram (ECG) abnormalities, blood pressure, initial cardiothoracic ratio, left ventricular ejection fractions (LVEF) and whole-blood selenium concentration. Our ten-year follow-up analysis indicated that selenium supplementation, specifically combined with the use of angiotensin-converting enzyme inhibitor and beta blocker therapy, improved the survival of patients with chronic Keshan disease with congestive heart failure. BMI, selenium deficiency, male, combined ECG abnormalities, LVEF, and fast heart rate increased the risk of cardiac events.

[Progressive Risks of Latent Keshan Disease: a Long Term Follow-up Study].

OBJECTIVE: To observe ten-year prognosis of patients with latent Keshan disease (KD) and to determine its associated risk factors. METHODS: A total of 448 patients with newly diagnosed latent KD were monitored and followed up for 10 years. Their ECG abnormalities were classified as major or minor using the Minnesota Code. COX proportional hazards regression models were established to identify risk factors associated with the development of chronic KD. RESULTS: A final sample of 414 cases was included in analyses, with an average of (112.9 ± 17.5) months of follow-up. At the end of follow-up, 92 (22. 2%) patients developed chronic KD. Older age (> 15 years), male, family history of KD, smoking, lower level of blood selenium (< 60 µg/L), major ECG abnormalities, and 18.5 kg/m² ≤ body mass index (BMI) 23.9 kg/m² were associated with higher cumulative incidence of chronic KD. The COX regression models showed that major ECG abnormalities, BMI, selenium deficiency, hypertension, and ventricular premature complex (VPC) abnormalities contributed to increased risk of chronic KD. A positive linear correlation (r = 0.719, P < 0.01) between GPx activity and blood selenium concentration was found. CONCLUSION: Major ECG abnormalities, BMI, selenium deficiency, hypertension and VPC abnormalities are associated with the development of chronic KD.

[The establishment and identification of GPx-1(P198L) gene systemic expression transgenic mice].

OBJECTIVE: To generate systemic expression human cellular glutathione peroxidase-1 (GPx-1) (198Leu) transgenic mice model in order to investigate the functional variants in GPx-1 gene in oxidative stress-related diseases. METHODS: After linearization with BamnH I and Acc I, the transgenic construct GPx-1 (198Leu) was microinjected into the zygotes of C57BL/6J mice to generate transgenic mice, whose genotype was detected by PCR with specific primers. The GPx-1 gene expression profile was determined by Western blotting. RESULTS: 13 transgenic founder mice were successfully generated. Western blotting result showed that the protein expression level of 4 transgenic mice in hearts were higher than that of wild type mice. CONCLUSION: Human GPx-1PSL transgenic mice was successfully established. This kind of animal model is of significance for making further researches on oxidative stress-related diseases.

Suppression of PKCε-mediated mitochondrial connexin 43 phosphorylation at serine 368 is involved in myocardial mitochondrial dysfunction in a rat model of dilated cardiomyopathy.

Mitochondrial connexin 43 (Cx43) is important in cardioprotection by ischemic preconditioning; however, whether mitochondrial Cx43 is involved in mitochondrial dysfunction in the pathogenesis of dilated cardiomyopathy (DCM) remains to be elucidated. The present study was performed to investigate the changes in expression and the phosphorylation state of mitochondrial Cx43 in a rat model of DCM, and to determine whether the altered phosphorylation state of mitochondrial Cx43 was involved in mitochondrial dysfunction. A rat model of DCM was generated by daily oral administration of furazolidone (FZD) for 30 weeks. Reverse transcription polymerase chain reaction and western blot analysis revealed a decrease in the overall expression of Cx43, accompanied by reduced levels of serine 368­phosphorylated­Cx43 immunoreactivity in the myocardium and myocardial mitochondria. In addition, the mitochondrial membrane potential and the activities of cytochrome c oxidase, succinate dehydrogenase and protein kinase C (PKC) Îµ were all significantly reduced compared with those of the control group. Phorbol­12­myristate­13­acetate (PMA), a specific PKC activator, partially reversed the FZD­induced mitochondrial Cx43 dephosphorylation at serine 368 and mitochondrial dysfunction in the cardiomyocytes. However, pretreatment with 18ß­glycerrhetinic acid, a connexin channel inhibitor, eliminated the mitochondrial protective effect of PMA in the cardiomyocytes sparsely plated without cell to cell contact. These results suggested that dephosphorylation of mitochondrial Cx43 at serine 368, due to the suppression of PKCε activity, may be a novel mechanism for mitochondrial dysfunction in the pathogenesis of DCM.

[Effects of p66shc adapter protein and estrogen on cardiomyocyte apoptosis induced by angiotensin II].

OBJECTIVE: To explore the role of p66shc in cardiomyocyte apoptosis induced by angiotensin (Ang) II and the effect of estrogen pretreatment. METHODS: Neonatal rat cardiomyocytes were randomly divided into five groups: normal control, 10(-11) mol/L Ang II, 10(-9) mol/L Ang II, 10(-7) mol/L Ang II, and 10(-7) mol/L Ang II + estrogen treated groups. The cell viability was measured by MTT. The level of reactive oxygen species (ROS) and cell apoptosis rate were measured by flow cytometry. Mitochondrial membrane potential (MMP) was detected using a fluorescence microplate reader, and the protein expression of phosphorylated and total p66shc were detected using Western blot. RESULTS: With the increase of Ang II concentrations, cell viabilities and MMP levels decreased, whereas, the levels of ROS and cell apoptosis rates increased (P < 0.05). Pretreatment with estrogen significantly attenuated the cardiomyocyte injury induced by Ang II (P < 0.05). The protein expression of phosphorylated p66shc in the whole cell lysates and total p66shc in the mitochondria increased in a dose-dependent manner when cardiomyocytes were exposed to Ang II (P < 0.05). Pretreatment with estrogen significantly down-regulated the protein expression of phosphorylated p66shc in the whole cell lysates and total p66shc in the mitochondria (P < 0.05). CONCLUSION: p66shc is involved in cardiomyocyte apoptosis induced by Ang II, and estrogen could attenuate Ang II induced cardiomyocyte injury through down-regulating the protein expression of p66shc.

Selenium deficiency is associated with endoplasmic reticulum stress in a rat model of cardiac malfunction.

The relationship between selenium (Se) deficiency-induced cardiac malfunction and endoplasmic reticulum (ER) stress is poorly understood. In the present study, 18 weaning Sprague Dawley rats were randomly fed with three different Se diets, and myocardial glutathione peroxidase (GPx) activity was measured by an enzyme activity assay. Cardiac function was evaluated by hemodynamic parameters. ER stress markers immunoglobulin-binding protein (BiP)/glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) were detected by western blotting. Our data showed that myocardial GPx activity and cardiac function were conspicuously impaired in Se-deficient rats. Expression of GRP78 and CHOP was significantly upregulated by treatment of Se deficiency. Improvements in myocardial GPx activity and cardiac function, as well as decreases in expression of GRP78 and CHOP, were observed after Se supplementation. Consequently, our data show that ER stress was involved in Se deficiency-induced cardiac dysfunction.

[Two-dimensional gel electrophoresis map of serum proteins in patients with chronic Keshan disease].

OBJECTIVE: To identify the different serum proteins expressed in patients with Keshan disease (KD). METHODS: Two-dimensional gel electrophoresis (2-DE) was performed with serum samples from the patients with chronic KD and healthy controls to separate serum proteins. The gels were stained by sliver and scanned by Umax scanner. The data were analyzed by ImageMaster 2D software. KD related proteins were identified through searching the ExPASy SWISS-2DPAGE database. RESULTS: Stable two-dimensional gel electrophoresis maps were established for serum samples of KD patients and healthy controls. A total of 808 and 814 protein spots were observed in KD patients and healthy controls, respectively. The two maps had 96.5475% identical protein spots and 44 differentially expressed protein spots. Eleven protein spots were expressed exclusively in KD patients and 12 protein spots only appeared in healthy controls. About 21 proteins were expressed in both groups but varied in quantities (14 proteins were over-expressed by more than 3 times and 7 proteins were under-expressed by more than 3 times in KD patients, P < 0.01). Among the 353 protein spots matched with the ExPASy-SWISS-2DPAGE databank, No. 1177 protein appeared in the KD patient was found to have the closest match with P02774 2-D0004T6 known as vitamin D binding protein (VDBP). CONCLUSION: There is a significant difference in serum protein expression between KD patients and normal people. VDBP might play a role in cardiac muscle damage via inflammatory immune reactions.

Mitochondrial-related gene expression profiles suggest an important role of PGC-1alpha in the compensatory mechanism of endemic dilated cardiomyopathy.

Keshan disease (KD) is an endemic dilated cardiomyopathy with unclear etiology. In this study, we compared mitochondrial-related gene expression profiles of peripheral blood mononuclear cells (PBMCs) derived from 16 KD patients and 16 normal controls in KD areas. Total RNA was isolated, amplified, labeled and hybridized to Agilent human 4 × 44k whole genome microarrays. Mitochondrial-related genes were screened out by the Third-Generation Human Mitochondria-Focused cDNA Microarray (hMitChip3). Quantitative real-time PCR, immunohistochemical and biochemical parameters related mitochondrial metabolism were conducted to validate our microarray results. In KD samples, 34 up-regulated genes (ratios ≥ 2.0) were detected by significance analysis of microarrays and ingenuity systems pathway analysis (IPA). The highest ranked molecular and cellular functions of the differentially regulated genes were closely related to amino acid metabolism, free radical scavenging, carbohydrate metabolism, and energy production. Using IPA, 40 significant pathways and four significant networks, involved mainly in apoptosis, mitochondrion dysfunction, and nuclear receptor signaling were identified. Based on our results, we suggest that PGC-1alpha regulated energy metabolism and anti-apoptosis might play an important role in the compensatory mechanism of KD. Our results may lead to the identification of potential diagnostic biomarkers for KD in PBMCs, and may help to understand the pathogenesis of KD.