Hypoxia induces dilated cardiomyopathy in the chick embryo:mechanism, intervention, and long-term consequences

Background - Intrauterine growth restriction is associated with an increased future risk for developing cardiovascular diseases. Hypoxia in utero is a common clinical cause of fetal growth restriction. We have previously shown that chronic hypoxia alters cardiovascular development in chick embryos. The aim of this study was to further characterize cardiac disease in hypoxic chick embryos. Methods - Chick embryos were exposed to hypoxia and cardiac structure was examined by histological methods one day prior to hatching (E20) and at adulthood. Cardiac function was assessed in vivo by echocardiography and ex vivo by contractility measurements in isolated heart muscle bundles and isolated cardiomyocytes. Chick embryos were exposed to vascular endothelial growth factor (VEGF) and its scavenger soluble VEGF receptor-1 (sFlt-1) to investigate the potential role of this hypoxia-regulated cytokine. Principal Findings - Growth restricted hypoxic chick embryos showed cardiomyopathy as evidenced by left ventricular (LV) dilatation, reduced ventricular wall mass and increased apoptosis. Hypoxic hearts displayed pump dysfunction with decreased LV ejection fractions, accompanied by signs of diastolic dysfunction. Cardiomyopathy caused by hypoxia persisted into adulthood. Hypoxic embryonic hearts showed increases in VEGF expression. Systemic administration of rhVEGF165 to normoxic chick embryos resulted in LV dilatation and a dose-dependent loss of LV wall mass. Lowering VEGF levels in hypoxic embryonic chick hearts by systemic administration of sFlt-1 yielded an almost complete normalization of the phenotype. Conclusions/Significance - Our data show that hypoxia causes a decreased cardiac performance and cardiomyopathy in chick embryos, involving a significant VEGF-mediated component. This cardiomyopathy persists into adulthood.

Changes in nucleic acid and protein levels in atrophying skeletal muscle in cancer cachexia

Background: To investigate factors responsible for muscle loss in cachexia changes in nucleic acid and protein levels have been determined and compared with those induced by a tumour-produced cachectic factor, proteolysis-inducing factor (PIF). Materials and Methods: Mice were transplanted with the MAC16 tumour, while non-tumour bearing mice received PIF (1.5 mg/kg; i.v.) over a 24 h period. Results: There was an exponential decrease in RNA and protein in gastrocnemius muscle with weight loss without an effect on the DNA content. Levels of myosin followed the decrease in total protein, while actin levels remained constant. There was also a significant loss of protein from soleus muscle and spleen, but not from heart, liver and kidney. PIF also produced a significant loss of RNA and protein in spleen and reduced the protein content of soleus muscle. Conclusion: This suggests that PIF may be responsible for changes in protein and RNA content of tissues with the development of cachexia.

Local delivery of the KCa3.1 blocker, TRAM-34, prevents acute angioplasty-induced coronary smooth muscle phenotypic modulation and limits stenosis

Objective-We previously demonstrated that upregulation of intermediate-conductance Ca2+ -activated K+ channels (KCa 3.1) is necessary for mitogen-induced phenotypic modulation in isolated porcine coronary smooth muscle cells (SMCs). The objective of the present study was to determine the role of KCa3.1 in the regulation of coronary SMC phenotypic modulation in vivo using a swine model of postangioplasty restenosis. Methods and Results-Balloon angioplasty was performed on coronary arteries of swine using either noncoated or balloons coated with the specific KCa3.1 blocker TRAM-34. Expression of KCa3.1, c-jun, c-fos, repressor element-1 silencing transcription factor (REST), smooth muscle myosin heavy chain (SMMHC), and myocardin was measured using qRT-PCR in isolated medial cells 2 hours and 2 days postangioplasty. KCa3.1, c-jun, and c-fos mRNA levels were increased 2 hours postangioplasty, whereas REST expression decreased. SMMHC expression was unchanged at 2 hours, but decreased 2 days postangioplasty. Use of TRAM-34 coated balloons prevented KCa3.1 upregulation and REST downregulation at 2 hours, SMMHC and myocardin downregulation at 2 days, and attenuated subsequent restenosis 14 and 28 days postangioplasty. Immunohistochemical analysis demonstrated corresponding changes at the protein level. Conclusion-Blockade of KCa3.1 by delivery of TRAM-34 via balloon catheter prevented smooth muscle phenotypic modulation and limited subsequent restenosis. © 2008 American Heart Association, Inc.

Glutaredoxin-1 up-regulation induces soluble vascular endothelial growth factor receptor 1, attenuating post-ischemia limb revascularization

Glutaredoxin-1 (Glrx) is a cytosolic enzyme that regulates diverse cellular function by removal of GSH adducts from S-glutathionylated proteins including signaling molecules and transcription factors. Glrx is up-regulated during inflammation and diabetes. Glrx overexpression inhibits VEGF-induced endothelial cell (EC) migration. The aim was to investigate the role of up-regulated Glrx in EC angiogenic capacities and in vivo revascularization in the setting of hind limb ischemia. Glrx overexpressing EC from Glrx transgenic mice (TG) showed impaired migration and network formation and secreted higher level of soluble VEGF receptor 1 (sFlt), an antagonizing factor to VEGF. After hind limb ischemia surgery Glrx TG mice demonstrated impaired blood flow recovery, associated with lower capillary density and poorer limb motor function compared to wild type littermates. There were also higher levels of anti-angiogenic sFlt expression in the muscle and plasma of Glrx TG mice after surgery. Non-canonical Wnt5a is known to induce sFlt. Wnt5a was highly expressed in ischemic muscles and EC from Glrx TG mice, and exogenous Wnt5a induced sFlt expression and inhibited network formation in human microvascular EC. Adenoviral Glrx-induced sFlt in EC was inhibited by a competitive Wnt5a inhibitor. Furthermore, Glrx overexpression removed GSH adducts on p65 in ischemic muscle and EC, and enhanced nuclear factor kappa B (NF-kB) activity which was responsible for Wnt5a-sFlt induction. Taken together, up-regulated Glrx induces sFlt in EC via NF-kB -dependent Wnt5a, resulting in attenuated revascularization in hind limb ischemia. The Glrx-induced sFlt may be a part of mechanism of redox regulated VEGF signaling.

The effect of whole body vibration on oxygen uptake and electromyographic signal of the rectus femoris muscle during static and dynamic squat

Whole Body Vibrations consist of a vibration stimulus mechanically transferred to the body. The impact of vibration treatment on specific muscular activity, neuromuscular, and postural control has been widely studied. We investigated whole body vibration (WBV) effect on oxygen uptake and electromyographic signal of the rectus femoris muscle during static and dynamic squat. Fourteen healthy subjects performed a static and dynamic squat with and without vibration. During the vibration exercises, a significant increase was found in oxygen uptake (P=0.05), which increased by 44% during the static squat and 29.4% during the dynamic squat. Vibration increased heart rate by 11.1 ± 9.1 beats.min-1 during the static squat and 7.9 ± 8.3 beats.min-1 during the dynamic squat. No significant changes were observed in rate of perceived exertion between the exercises with and without vibration. The results indicate that the static squat with WBV produced higher neuromuscular and cardiorespiratory system activation for exercise duration ?60 sec. Otherwise, if the single bout duration was higher than 60 sec, the greater cardiorespiratory system activation was achieved during the dynamic squat with WBV while higher neuromuscular activation was still obtained with the static exercise.

Glutathione adducts induced by ischemia and deletion of glutaredoxin-1 stabilize HIF-1α and improve limb revascularization

Reactive oxygen species (ROS) are increased in ischemic tissues and necessary for revascularization; however, the mechanism remains unclear. Exposure of cysteine residues to ROS in the presence of glutathione (GSH) generates GSH-protein adducts that are specifically reversed by the cytosolic thioltransferase, glutaredoxin-1 (Glrx). Here, we show that a key angiogenic transcriptional factor hypoxia-inducible factor (HIF)-1α is stabilized by GSH adducts, and the genetic deletion of Glrx improves ischemic revascularization. In mouse muscle C2C12 cells, HIF-1α protein levels are increased by increasing GSH adducts with cell-permeable oxidized GSH (GSSG-ethyl ester) or 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanyl thiocarbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), an inhibitor of glutathione reductase. A biotin switch assay shows that GSSG-ester-induced HIF-1α contains reversibly modified thiols, and MS confirms GSH adducts on Cys520 (mouse Cys533). In addition, an HIF-1α Cys520 serine mutant is resistant to 2-AAPA–induced HIF-1α stabilization. Furthermore, Glrx overexpression prevents HIF-1α stabilization, whereas Glrx ablation by siRNA increases HIF-1α protein and expression of downstream angiogenic genes. Blood flow recovery after femoral artery ligation is significantly improved in Glrx KO mice, associated with increased levels of GSH-protein adducts, capillary density, vascular endothelial growth factor (VEGF)-A, and HIF-1α in the ischemic muscles. Therefore, Glrx ablation stabilizes HIF-1α by increasing GSH adducts on Cys520 promoting in vivo HIF-1α stabilization, VEGF-A production, and revascularization in the ischemic muscles