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.

NF-κB mediates proteolysis-inducing factor induced protein degradation and expression of the ubiquitin-proteasome system in skeletal muscle

Loss of skeletal muscle in cancer cachexia has a negative effect on both morbidity and mortality. The role of nuclear factor-κB (NF-κB) in regulating muscle protein degradation and expression of the ubiquitin-proteasome proteolytic pathway in response to a tumour cachectic factor, proteolysis-inducing factor (PIF), has been studied by creating stable, transdominant-negative, muscle cell lines. Murine C2C12 myoblasts were transfected with plasmids with a CMV promoter that had mutations at the serine phosphorylation sites required for degradation of I-κBα, an NF-κB inhibitory protein, and allowed to differentiate into myotubes. Proteolysis-inducing factor induced degradation of I-κBα, nuclear accumulation of NF-κB and an increase in luciferase reporter gene activity in myotubes containing wild-type, but not mutant, I-κBα, proteins. Proteolysis-inducing factor also induced total protein degradation and loss of the myofibrillar protein myosin in myotubes containing wild-type, but not mutant, plasmids at the same concentrations as those causing activation of NF-κB. Proteolysis-inducing factor also induced increased expression of the ubiquitin-proteasome pathway, as determined by 'chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the β-subunits of the proteasome, protein expression of 20S α-subunits and the 19S subunits MSSI and p42, as well as the ubiquitin conjugating enzyme, E214k, in cells containing wild-type, but not mutant, I-κBα. The ability of mutant I-κBα to inhibit PIF-induced protein degradation, as well as expression of the ubiquitin-proteasome pathway, confirms that both of these responses depend on initiation of transcription by NF-κB. © 2005 Cancer Research UK.

Development of an in-vitro model system to investigate the mechanism of muscle protein catabolism induced by proteolysis-inducing factor

The mechanism of muscle protein catabolism induced by proteolysis-inducing factor, produced by cachexia-inducing murine and human tumours has been studied in vitro using C2C12 myoblasts and myotubes. In both myoblasts and myotubes protein degradation was enhanced by proteolysis-inducing factor after 24 h incubation. In myoblasts this followed a bell-shaped dose-response curve with maximal effects at a proteolysis-inducing factor concentration between 2 and 4 nM, while in myotubes increased protein degradation was seen at all concentrations of proteolysis-inducing factor up to 10 nM, again with a maximum of 4 nM proteolysis-inducing factor. Protein degradation induced by proteolysis-inducing factor was completely attenuated in the presence of cycloheximide (1 μM), suggesting a requirement for new protein synthesis. In both myoblasts and myotubes protein degradation was accompanied by an increased expression of the α-type subunits of the 20S proteasome as well as functional activity of the proteasome, as determined by the 'chymotrypsin-like' enzyme activity. There was also an increased expression of the 19S regulatory complex as well as the ubiquitin-conjugating enzyme (E214k), and in myotubes a decrease in myosin expression was seen with increasing concentrations of proteolysis-inducing factor. These results show that proteolysis-inducing factor co-ordinately upregulates both ubiquitin conjugation and proteasome activity in both myoblasts and myotubes and may play an important role in the muscle wasting seen in cancer cachexia. © 2002 Cancer Research UK.

General features of Multiple System Atrophy

Multiple system atrophy (MSA) is a rare movement disorder and a member of a group of neurodegenerative diseases referred to collectively as the ‘parkinsonian syndromes’. Characteristic of these syndromes is that the patient exhibits symptoms of ‘parkinsonism’, viz., a range of problems involving movement, most typically manifest in Parkinson’s disease (PD) itself1, but also seen in progressive supranuclear palsy (PSP), and to some extent in dementia with Lewy bodies (DLB). MSA is a relatively ‘new’ descriptive term and is derived from three previously described diseases, viz., olivopontocerebellar atrophy, striato-nigral degeneration, and Shy-Drager syndrome. The classical symptoms of MSA include parkinsonism, ataxia, and autonomic dysfunction.6 Ataxia describes a gross lack of coordination of muscle movements while autonomic dysfunction involves a variety of systems that regulate unconscious bodily functions such as heart rate, blood pressure, bladder function, and digestion. Although primarily a neurological disorder, patients with MSA may also develop visual signs and symptoms that could be useful in differential diagnosis. The most important visual signs may include oculomotor dysfunction and problems in pupil reactivity but are less likely to involve aspects of primary vision such as visual acuity, colour vision, and visual fields. In addition, the eye-care practitioner can contribute to the management of the visual problems of MSA and therefore, help to improve quality of life of the patient. Hence, this first article in a two-part series describes the general features of MSA including its prevalence, signs and symptoms, diagnosis, pathology, and possible causes.

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.

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.

Evaluation of the Heart of Birmingham teaching Primary Care Trust (HoBtPCT): My Choice Weight Management Programme:report commissioned by Heart of Birmingham teaching Primary Care Trust

This report details an evaluation of the My Choice Weight Management Programme undertaken by a research team from the School of Pharmacy at Aston University. The My Choice Weight Management Programme is delivered through community pharmacies and general practitioners (GPs) contracted to provide services by the Heart of Birmingham teaching Primary Care Trust. It is designed to support individuals who are ‘ready to change’ by enabling the individual to work with a trained healthcare worker (for example, a healthcare assistant, practice nurse or pharmacy assistant) to develop a care plan designed to enable the individual to lose 5-10% of their current weight. The Programme aims to reduce adult obesity levels; improve access to overweight and obesity management services in primary care; improve diet and nutrition; promote healthy weight and increased levels of physical activity in overweight or obese patients; and support patients to make lifestyle changes to enable them to lose weight. The Programme is available for obese patients over 18 years old who have a Body Mass Index (BMI) greater than 30 kg/m2 (greater than 25 kg/m2 in Asian patients) or greater than 28 kg/m2 (greater than 23.5 kg/m2 in Asian patients) in patients with co-morbidities (diabetes, high blood pressure, cardiovascular disease). Each participant attends weekly consultations over a twelve session period (the final iteration of these weekly sessions is referred to as ‘session twelve’ in this report). They are then offered up to three follow up appointments for up to six months at two monthly intervals (the final of these follow ups, taking place at approximately nine months post recruitment, is referred to as ‘session fifteen’ in this report). A review of the literature highlights the dearth of published research on the effectiveness of primary care- or community-based weight management interventions. This report may help to address this knowledge deficit. A total of 451 individuals were recruited on to the My Choice Weight Management Programme. More participants were recruited at GP surgeries (n=268) than at community pharmacies (n=183). In total, 204 participants (GP n=102; pharmacy n=102) attended session twelve and 82 participants (GP n=22; pharmacy 60) attended session fifteen. The unique demographic characteristics of My Choice Weight Management Programme participants – participants were recruited from areas with high levels of socioeconomic deprivation and over four-fifths of participants were from Black and Minority Ethnic groups; populations which are traditionally underserved by healthcare interventions – make the achievements of the Programme particularly notable. The mean weight loss at session 12 was 3.8 kg (equivalent to a reduction of 4.0% of initial weight) among GP surgery participants and 2.4 kg (2.8%) among pharmacy participants. At session 15 mean weight loss was 2.3 kg (2.2%) among GP surgery participants and 3.4 kg (4.0%) among pharmacy participants. The My Choice Weight Management Programme improved the general health status of participants between recruitment and session twelve as measured by the validated SF-12 questionnaire. While cost data is presented in this report, it is unclear which provider type delivered the Programme more cost-effectively. Attendance rates on the Programme were consistently better among pharmacy participants than among GP participants. The opinions of programme participants (both those who attended regularly and those who failed to attend as expected) and programme providers were explored via semi-structured interviews and, in the case of the participants, a selfcompletion postal questionnaire. These data suggest that the Programme was almost uniformly popular with both the deliverers of the Programme and participants on the Programme with 83% of questionnaire respondents indicating that they would be happy to recommend the Programme to other people looking to lose weight. Our recommendations, based on the evidence provided in this report, include: a. Any consideration of an extension to the study also giving comparable consideration to an extension of the Programme evaluation. The feasibility of assigning participants to a pharmacy provider or a GP provider via a central allocation system should also be examined. This would address imbalances in participant recruitment levels between provider type and allow for more accurate comparison of the effectiveness in the delivery of the Programme between GP surgeries and community pharmacies by increasing the homogeneity of participants at each type of site and increasing the number of Programme participants overall. b. Widespread dissemination of the findings from this review of the My Choice Weight Management Project should be undertaken through a variety of channels. c. Consideration of the inclusion of the following key aspects of the My Choice Weight Management Project in any extension to the Programme: i. The provision of training to staff in GP surgeries and community pharmacies responsible for delivery of the Programme prior to patient recruitment. ii. Maintaining the level of healthcare staff input to the Programme. iii. The regular schedule of appointments with Programme participants. iv. The provision of an increased variety of printed material. d. A simplification of the data collection method used by the Programme commissioners at the individual Programme delivery sites.

The effects of metformin on the vascular system

Macrovascular contraction and relaxation effects of metformin were measured using a Mulvany Halpern myograph. Mouse aortic ring sections were treated for 1 and 4 hours in vitro with metformin at 10-5M, and for 2, 4 and 8 weeks in vivo with metformin at 250mg/kg/day. The rings were contacted with increasing concentrations of noradrenaline (10-9M, 10-8M, 10-7M, 10-6M) in the absence and presence of metformin. Maximally contracted tissue was then relaxed using increasing acetylcholine concentrations (10-9M, 10-8M, 10-7M, 10-6M). Meformin increased the sensitivity of the aorta to noradrenaline-induced contraction. The maximal effect in vitro was seen after 4 hours giving a 221% increase in contraction after 4 hours at noradrenaline 10-6M. Acetylcholine-stimulated relaxation via endothelium also increased with metformin after 4 hours by 36.85%. The maximal effect of metformin treatment in vivo was seen on aortic contraction after 8 weeks: the effect of melformin treatment on relaxation was less marked at this time. Metformin also increased passive tension generated by the aortic vessel wall after 4 hours, which was reversed by administration of papaverine, which acts directly on vascular smooth muscle. Metformin was shown not to alter nitric oxide production by the mouse aortic wall after 1 and 4 hours in vitro. Metformin lowered basal calcium concentrations, as measured by FURA/2AM, generating a slow sustained increase in calcium release induced by noradrenaline during contraction. This research programme has shown that metformin can increase both the contraction and relaxation capabilities of aortic sections treated both in vitro and in vivo with therapeutic concentrations of metformin at 10-5M. Metformin has been shown to act directly in the vascular wall to alter vascular contractility via effects on both vascular smooth muscle and endothelium, and to influence calcium movements independently of nitric oxide.

The effect of cis-5,8,11,14,17-eicosapentaenoic acid upon the contractile mechanisms linked to calcium influx and the mobilisation of intracellular calcium in aortic smooth muscle

Epidemiological studies previously identified cis-5,8,11,14,17-eicosapentaenoic acid (EPA) as the biologically active component of fish oil of benefit to the cardiovascular system. Although clinical investigations demonstrated its usefulness in surgical procedures, its mechanism of action still remained unclear. It was shown in this thesis, that EPA partially blocked the contraction of aortic smooth muscle cells to the vasoactive agents KCl and noradrenaline. The latter effect was likely caused by reducing calcium influx through receptor-operated channels, supporting a recent suggestion by Asano et al (1997). Consistently, EPA decreased noradrenaline-induced contractures in aortic tissue, in support of previous reports (Engler, 1992b). The observed effect of EPA on cell contractions to KCl was not simple due to blocking calcium influx through L-type channels, consistent with a previous suggestion by Hallaq et al (1992). Moreover, EPA caused a transient increase in [Ca2+]i in the absence of extracellular calcium. To resolve this it was shown that EPA increased inositol phosphate formation which, it is suggested, caused the release of calcium from an inositol phosphate-dependent internal binding site, possibly that of an intracellular membrane or superficial sarcoplasmic reticulum, producing the transient increase in [Ca2+]i. As it was shown that the cellular contractile filaments were not desensitised to calcium by EPA, it is suggested that the transient increase in [Ca2+]i subsequently blocks further cell contraction to KCl by activating membrane-associated potassium channels. Activation of potassium channels induces the cellular efflux of potassium ions, thereby hyperpolarising the plasma membrane and moving the membrane potential farther from the activation range for calcium channels. This would prevent calcium influx in the longer term and could explain the initial observed effect of EPA to block cell contraction to KCl.

Mechanism of protein catobolism in skeletal muscle during cancer cachexia

Cancer cachexia is characterised by selective depletion of skeletal muscle protein reserves. The ubiquitin-proteasome proteolytic pathway has been shown to be responsible for muscle wasting in a range of cachectic conditions including cancer cachexia. To establish the importance of this pathway in muscle wasting during cancer (and sepsis), a quantitative competitive RT-PCR (QcRT-PCR) method was developed to measure the mRNA levels of the proteasome sub units C2a and C5ß and the ubiquitin-conjugating enzyme E214k. Western blotting was also used to measure the 20S proteasome and E214k protein expression. In vivo studies in mice bearing a cachexia inducing murine colon adenocarcinoma (MAC16) demonstrated the effect of progressive weight loss on the mRNA and protein expression for 20S proteasome subunits, as well as the ubiquitin-conjugating enzyme, E214k, in gastrocnemius and pectoral muscles. QcRT-PCR measurements showed a good correlation between expression of the proteasome subunits (C2 and CS) and the E214k enzyme mRNA and weight loss in gastrocnemius muscle, where expression increased with increasing weight loss followed by a decrease in expression at higher weight losses (25-27%). Similar results were obtained in pectoral muscles, but with the expression being several fold lower in comparison to that in gastrocnemius muscle, reflecting the different degrees of protein degradation in the two muscles during the process of cancer cachexia. Western blot analysis of 20S and E214k protein expression followed a similar pattern with respect to weight loss as that found with mRNA. In addition, mRNA and protein expression of the 20S proteasome subunits and E214k enzyme was measured in biopsies from cachectic cancer patients, which also showed a good correlation between weight loss and proteasome expression, demonstrating a progressive increase in expression of the proteasome subunits and E214k mRNA and protein in cachectic patients with progressively increasing weight loss.The effect of the cachexia-inducing tumour product PIF (proteolysis inducing factor) and 15-hydroxyeicosatetraenoic acid (15-HETE), the arachidoinic acid metabolite (thought to be the intracellular transducer of PIF action) has also been determined. Using a surrogate model system for skeletal muscle, C2C12 myotubes in vitro, it was shown that both PIF and 15-HETE increased proteasome subunit expression (C2a and C5ß) as well as the E214k enzyme. This increase gene expression was attenuated by preincubation with EPA or the 15-lipoxygenase inhibitor CV-6504; immunoblotting also confirmed these findings. Similarly, in sepsis-induced cachexia in NMRI mice there was increased mRNA and protein expression of the 20S proteasome subunits and the E214k enzyme, which was inhibited by EPA treatment. These results suggest that 15-HETE is the intracellular mediator for PIF induced protein degradation in skeletal muscle, and that elevated muscle catabolism is accomplished through upregulation of the ubiquitin-proteasome-proteolytic pathway. Furthermore, both EPA and CV -6504 have shown anti-cachectic properties, which could be used in the future for the treatment of cancer cachexia and other similar catabolic conditions.

Behavioural correlates of ocular accomodation and the autonomic nervous system

The binding issue of th is thesis was the examination of workload, induced by relinotopic and spatiotopic stimuli, on both the ocu lomotor and cardiovascular systems together with investigating the covariation between the two systems - the 'eye-heart' link. Further, the influence of refractive error on ocular accommodation and cardiovascular function was assessed. A clinical evaluation was undertaken to assess the newly available open-view infrared Shin-Nippon NVision-K 5001 optometer, its benefit being the capability to measure through pupils = 2.3 mm. Measurements of refractive error taken with the NVision-K were found to be both accurate (Difference in Mean Spherical Equivalent: 0.14 ± 0.35 D; p = 0.67) and repeatable when compared to non-cycloplegic subjective refraction. Due to technical difficulties, however, the NVision-K could not be used for the purpose of the thesis, as such, measures of accommodation were taken using the continuously recording Shin-Nippon SRW-5000 openview infrared optometer, coupled with a piezo-electric finger pulse transducer to measure pulse. Heart rate variability (HRV) was spectrally analysed to determine the systemic sympathetic and parasympathetic components of the autonomic nervous system (ANS). A large sample (n = 60), cross-sectional study showed late-onset myopes (LOMs) display less accurate responses when compared to other refractive groups at high accommodative demand levels (3 .0 0 and 4.0D). Tonic accommodation (TA) was highest in the hypermetropes, fo llowed by emmetropes and early-onset myopes while the LOM subjects demonstrated statistically significant lower levels of TA. The root-meansquare (RMS) value of the accommodative response was shown to amplify with increased levels of accommodative demand. Changes in refractive error only became significant between groups at higher demand levels (3.0 D and 4.0 D) with the LOMs showing the largest magnification in oscilIations. Examination of the stimulus-response cross-over point with the unit ratio line and TA showed a correlation between the two (r = 0.45, p = 0.001), where TA is approximately twice the dioptric value of the stimulus-response cross-over point. Investigation of the relationship between ocular accommodation and systemic ANS function demonstrated covariation between the systems. Subjects with a faster heart rate (lower heart period) tended to have a higher TA value (r = -0.27, p < 0.05). Further, an increase in accommodative demand accompanies a faster heart rate. The influence of refractive error on the cardiovascular response to changes in accommodative demand, however, was equivocal. Examination of the microfluctuations ofacconunodation demonstrated a correlation between the temporal frequency location of the accommodative high Frequency component (HFC) and the arterial pulse frequency. The correlation was present at a range of accommodative demands from 0.0 D to 4.0 D and in all four refractive groups, suggesting that the HFC was augmented by physiological factors. Examination of the effect of visual cognition on ocular accommodation and the ANS confirmed that increasing levels of cognition affect the accommodative mechanism. The accommodative response shifted away from the subject at both near and far. This shift in accommodative response accompanied a decay in the systemic parasympathetic innervation to the heart. Differences between refractive groups also existed with LOMs showing less accurate responses compared to emmetropes. This disparity, however, appeared to be augmented by the systemic sympathetic nervous system. The investigations discussed explored Ihe role of oculomotor and cardiovascular fu nction in workload enviromnents, providing evidence for a behavioural link between the cardiovascular and oculomotor systems.

Mechanism of muscle protein degradation in Cancer Cachexia

A protein-mobilising factor of estimated molecular weight 24 KDa (p24) was purified both from the cachexia-inducing MAC 16 tumour and the urine of cachectic cancer patients by a combination of ammonium sulphate precipitation and affinity chromatography using a monoclonal antibody developed against the murine material. Administration of p24 to non tumour-bearing mice caused a decrease in body weight 24 h after the first injection, which was attenuated by prior treatment with the monoclonal antibody. Loss of body weight was accompanied by an accelerated loss of skeletal muscle protein, as determined by the release of tyrosine from this tissue. This was associated with an increased release of PGE2 and both protein degradation and PGE2 release were attenuated by the monoclonal antibody. Loss of protein mass arose from both a decrease in the rate of protein synthesis and an elevation of protein breakdown; the latter due to an activation of the ubiquitin-proteasome proteolytic system. In isolated muscle, p24 was capable of promoting protein breakdown and this was also associated with increased PGE2 levels. Both tyrosine and PGE2 release, were inhibited by PGE2 inhibitors and a specific inhibitor of cPLA2. When added to muscle cells in culture, p24 caused an elevation in the rates of total and myofibrillar protein breakdown and a depression in the rate of protein synthesis which was inhabitable by short-term incubation in insulin, suggesting that p24 may inhibit protein synthesis by causing an arrest in the translational process.

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.

Mechanism of induction of muscle protein degradation by angiotensin II

Angiotensin I and II have been shown to directly induce protein degradation in skeletal muscle through an increased activity and expression of the ubiquitin-proteasome proteolytic pathway. This investigation determines the role of the nuclear transcription factor nuclear factor-κB (NF-κB) in this process. Using murine myotubes as a surrogate model system both angiotensin I and II were found to induce activation of protein kinase C (PKC), with a parabolic dose-response curve similar to the induction of total protein degradation. Activation of PKC was required for the induction of proteasome expression, since calphostin C, a highly specific inhibitor of PKC, attenuated both the increase in total protein degradation and in proteasome expression and functional activity increased by angiotensin II. PKC is known to activate I-κB kinase (IKK), which is responsible for the phosphorylation and subsequent degradation of I-κB. Both angiotensin I and II induced an early decrease in cytoplasmic I-κB levels followed by nuclear accumulation of NF-κB. Using an NF-κB luciferase construct this was shown to increase transcriptional activation of NF-κB regulated genes. Maximal luciferase expression was seen at the same concentrations of angiotensin I/II as those inducing protein degradation. Total protein degradation induced by both angiotensin I and II was attenuated by resveratrol, which prevented nuclear accumulation of NF-κB, confirming that activation of NF-κB was responsible for the increased protein degradation. These results suggest that induction of proteasome expression by angiotensin I/II involves a signalling pathway involving PKC and NF-κB. © 2005 Elsevier Inc. All rights reserved.