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Biomedical subjects

David G Allen

Publications and source records attributed to David G Allen.

At least 19 recordsLinked to original sources
PubMed2006

Intracellular calcium handling in ventricular myocytes from mdx mice.

Duchenne muscular dystrophy (DMD) is a lethal degenerative disease of skeletal muscle, characterized by the absence of the cytoskeletal protein dystrophin. Some DMD patients show a dilated cardiomyopathy leading to heart failure. This study explores the possibility that dystrophin is involved in the regulation of a stretch-activated channel (SAC), which in the absence of dystrophin has increased activity and allows greater Ca(2+) into cardiomyocytes. Because cardiac failure only appears late in the progression of DMD, we examined age-related effects in the mdx mouse, an animal model of DMD. Ca(2+) measurements using a fluorescent Ca(2+)-sensitive dye fluo-4 were performed on single ventricular myocytes from mdx and wild-type mice. Immunoblotting and immunohistochemistry were performed on whole hearts to determine expression levels of key proteins involved in excitation-contraction coupling. Old mdx mice had raised resting intracellular Ca(2+) concentration ([Ca(2+)](i)). Isolated ventricular myocytes from young and old mdx mice displayed abnormal Ca(2+) transients, increased protein expression of the ryanodine receptor, and decreased protein expression of serine-16-phosphorylated phospholamban. Caffeine-induced Ca(2+) transients showed that the Na(+)/Ca(2+) exchanger function was increased in old mdx mice. Two SAC inhibitors streptomycin and GsMTx-4 both reduced resting [Ca(2+)](i) in old mdx mice, suggesting that SACs may be involved in the Ca(2+)-handling abnormalities in these animals. This finding was supported by immunoblotting data, which demonstrated that old mdx mice had increased protein expression of canonical transient receptor potential channel 1, a likely candidate protein for SACs. SACs may play a role in the pathogenesis of the heart failure associated with DMD. Early in the disease process and before the onset of clinical symptoms increased, SAC activity may underlie the abnormal Ca(2+) handling in young mdx mice.

Age Factors↗
PubMed2006

Streptomycin reduces stretch-induced membrane permeability in muscles from mdx mice.

It is well-known that muscles from mdx mice are more susceptible to membrane damage from eccentric contractions than wild-type muscles. The present study tested the hypothesis that the stretch-induced membrane permeability in dystrophic muscle is due to Ca(2+) entry through stretch-activated channels (SACs) and the subsequent activation of Ca(2+) -dependent degradative pathways. Eccentric contractions were carried out on muscles from mdx and wild-type mice, both on isolated muscles and on intact mice subjected to downhill running on a treadmill. In isolated muscles the SAC blockers, streptomycin and GsMTx4, improved force and significantly reduced the uptake of procion orange dye into fibres from mdx muscles, which increased progressively over 60 min after the eccentric contractions. In experiments on intact mdx mice, streptomycin also partially prevented the reduced force and the increased membrane permeability (Evans Blue Dye uptake). The results suggest that Ca(2+) entry through SACs activates Ca(2+) -dependent pathways, which are the main cause of the increased membrane permeability in mdx muscle.

Animals↗
PubMed2006

AICAR inhibits the Na+/H+ exchanger in rat hearts--possible contribution to cardioprotection.

AICAR (5-amino-1-beta-D: -ribofuranosyl-imidazole-4-carboxamide) is an adenosine analog which improves the recovery of the heart after ischemia. In some tissues AICAR enters cells and stimulates AMP-activated protein kinase (AMPK). We explored the mechanism of cardioprotection in isolated rat hearts. We confirmed that AICAR (0.5 mM) applied 10 min prior to a 30-min period of ischemia and present throughout ischemia and reperfusion caused a substantial improvement in the recovery of developed pressure on reperfusion. However, adenosine (100 microM) produced no improvement, suggesting that the mechanism of action of AICAR was not increased endogenous adenosine production. Measurements of intracellular sodium concentration ([Na(+)](i)) showed that AICAR prevented the rapid rise of [Na(+)](i), which normally occurs on reperfusion. Inhibitors of the cardiac sodium-hydrogen exchanger (NHE1) also protect the heart from ischemic damage and also prevent the rapid rise of [Na(+)](i) on reperfusion, suggesting that AICAR might cause the inhibition of NHE1. We tested this possibility on isolated rat ventricular myocytes in which the recovery of pH(i) after NH(4)Cl exposure provides a measure of NHE1 activity. AICAR (0.5 micromM) inhibited NHE1 activity in response to an acid load by about 80%. To test whether the AICAR-induced inhibition of NHE1 arose through adenosine, we used the adenosine receptor blocker 8-sulfophenyltheophylline (8-SPT) and found that it had no measureable effect. To test whether the AICAR-induced inhibition of NHE1 might occur through the activation of AMPK, we measured the activity of two isoforms of AMPK. Surprisingly, activity was reduced, whereas in many other tissues AICAR increases AMPK activity. Furthermore, this effect of AMPK was blocked by 8-SPT, suggesting that the inhibition of AMPK arose through an adenosine-receptor-related pathway. We conclude that AICAR inhibits NHE1 through an unidentified pathway. This inhibition may make a contribution to the cardioprotective effects of AICAR.

AMP-Activated Protein Kinases↗
PubMed2006

Molecular insights from a novel cardiac troponin I mouse model of familial hypertrophic cardiomyopathy.

Gene mutations in cardiac troponin I (cTnI) account for up to 5% of genotyped families with familial hypertrophic cardiomyopathy (FHC). Little is known about how cTnI mutations cause disease. Five lines of transgenic mice were generated which overexpress the human disease-causing cTnI gene mutation, Gly203Ser (designated cTnI-G203S), in a cardiac-specific manner. Mice were compared to transgenic mice that overexpress normal cTnI (cTnI-wt) and non-transgenic littermates (NTG). cTnI-G203S mice developed all the characteristic features of FHC by age 21 weeks. Left ventricular hypertrophy was observed on echocardiography (1.25+/-0.05 mm vs. 0.86+/-0.02 mm in cTnI-wt, P<0.01), associated with a significant 4-fold increase in RNA markers of hypertrophy, ANF and BNP. Myocyte hypertrophy, myofiber disarray and interstitial fibrosis were observed in cTnI-G203S mice. Expression of the cTnI-G203S mutation in neonatal cardiomyocytes resulted in a significant increase in myocyte volume, and reduced interactions with both troponins T and C. Ca2+ cycling was abnormal in adult cardiomyocytes extracted from cTnI-G203S mice, with a prolonged decay constant in Ca2+ transients and a reduced decay constant in response to caffeine treatment. Mice with the cTnI-G203S gene mutation develop all the phenotypic features of human FHC. The cTnI-G203S mutation disrupts interactions with partner proteins, and results in intracellular Ca2+ dysregulation early in life, suggesting a pathogenic role in development of FHC.

Animals↗
PubMed2006

Muscle damage in mdx (dystrophic) mice: role of calcium and reactive oxygen species.

1. Duchenne muscular dystrophy (DMD) is a lethal, degenerative muscle disease caused by a genetic mutation that leads to the complete absence of the cytoskeletal protein dystrophin in muscle fibres. 2. The present review provides an overview of some of the physiological pathways that may contribute to muscle damage and degeneration in DMD, based primarily on experimental findings in the mdx mouse, an animal model of this disease. 3. A rise in intracellular calcium is widely thought to be an important initiating event in the dystrophic pathogenesis. The pathway(s) leading to increased intracellular calcium in dystrophin deficient muscle is uncertain, but recent work from our laboratory provides evidence that stretch-activated channels are an important source of the calcium influx. Other possible routes of calcium entry are also discussed. 4. The consequences of elevated cytosolic calcium may include activation of proteases, such as calpain, and increased production of reactive oxygen species (ROS), which can cause protein and membrane damage. 5. Another possible cause of damage in dystrophic muscle involves inflammatory pathways, such as those mediated by neutrophils, macrophages and associated cytokines. There is recent evidence that increased ROS may be important in both the activation of and the damage caused by this inflammatory pathway in mdx muscle.

Animals↗
PubMed2006

Whiteness and difference in nursing.

This paper uses a semiotic, performative theory of language and post-colonial theory to argue that nursing's representations of 'multiculturalism' need to be grounded in a theory of whiteness, an historicized understanding of how ethnic/cultural differences come to be represented in the ways they are and informed by Foucault's notions of power/knowledge. Using nursing education and 'cultural compentency' as examples, the paper draws on a range of literatures to suggest more critical and politically productive ways of approaching difference from within nursing's largely white interpretive framework.

Authoritarianism↗
PubMed2005

The activity-induced reduction of myofibrillar Ca2+ sensitivity in mouse skeletal muscle is reversed by dithiothreitol.

The aim of this study was to further characterize the reduction of myofibrillar Ca2+ sensitivity in mouse muscle which has been observed after fatigue at 37 degrees C. Muscle bundles and single fibres were isolated from mouse flexor digitorum brevis muscle and studied at 37 degrees C. The single fibres were injected with the Ca2+ indicator indo-1. Muscle fatigue was produced by 0.4 s tetani repeated at 4 s intervals until force had fallen to less than 50% of initial. Excitation-contraction coupling was assessed by measuring the cytosolic calcium concentration ([Ca2+]i) during tetani, and the maximum Ca2+-activated force and the myofibrillar Ca2+ sensitivity were estimated from a series of tetani at different stimulation frequencies. Two main results were found. (i) The reduction of Ca2+ sensitivity only occurred when the muscle was intensely stimulated leading to fatigue. When the muscle was rested for 10 min at 37 degrees C there was no significant change in Ca2+ sensitivity. (ii) If the membrane-permeant thiol-specific reducing agent dithiothreitol (0.5 mm) was applied to the muscle for 2 min following the fatigue protocol, the reduction in Ca2+ sensitivity was reversed. Dithiothreitol had no effect on Ca2+ sensitivity in unfatigued preparations. There was no effect of fatigue or dithiothreitol on tetanic [Ca2+]i or on the maximum Ca2+-activated force. These results suggest that intense activity of skeletal muscle at 37 degrees C causes the production of reactive oxygen species which oxidize a target protein. We propose that critical sulphydryl groups on the target protein(s) are converted to disulphide bonds and this reaction reduces Ca2+ sensitivity.

Animals↗
PubMed2005

Conversational silence, coercion, equality: the role of language in influencing who gets identified as abused.

Various methods of ascertaining self-reported exposure to intimate partner violence (IPV) in health care and research settings have been evaluated to identify women who interpret themselves as abused for clinical and research intervention. However, few interpretive frameworks have been proposed to explain factors that may influence the success of this ascertainment process, including the contribution of language in facilitating women's interpretations of situations as abusive across social, cultural and historical contexts. This omission is substantial, given that IPV is context-specific, involving interaction between individuals of diverse linguistic and cultural backgrounds and their sociocultural environment. In the first part of this paper, we outline hermeneutics, one interpretive theoretical tradition to describe approaches to interpreting IPV. Hermeneutics is a linguistic philosophy that focuses on questions of how people understand spoken language, written text, and themselves through language across sociocultural environments. Hermeneutics acknowledges conditions and situations that facilitate opportunities for broad shared understanding and vocabularies about violence between communities, professionals and abused women, which in turn may reduce harm to women and negotiate action women may want to take in response to situations they interpret as abusive. In the second and third parts of the paper, we compare and contrast the strengths and limitations of three common approaches for asking women about IPV in health care and research settings, and outline a multi-dimensional IPV ascertainment tool that incorporates the three asking approaches to facilitate professionals bringing broad definitions of and vocabularies about abuse to encounters with women. This paper provides health care researchers, clinicians and policy makers with a framework for understanding the potential influence of language on women's interpretations of IPV, including the role of community and professional conversational silence, coercion and equality in influencing women's interpretations. We look at the influence of language about intimate partner violence in the United States on women's interpretations of abuse, although the basic constructs presented here could be applied in other countries and settings.

Coercion↗
PubMed2005

Reactive oxygen species reduce myofibrillar Ca2+ sensitivity in fatiguing mouse skeletal muscle at 37 degrees C.

The mechanisms of muscle fatigue were studied in small muscle bundles and single fibres isolated from the flexor digitorum brevis of the mouse. Fatigue caused by repeated isometric tetani was accelerated at body temperature (37 degrees C) when compared to room temperature (22 degrees C). The membrane-permeant reactive oxygen species (ROS) scavenger, Tiron (5 mM), had no effect on the rate of fatigue at 22 degrees C but slowed the rate of fatigue at 37 degrees C to that observed at 22 degrees C. Single fibres were microinjected with indo-1 to measure intracellular calcium. In the accelerated fatigue at 37 degrees C the tetanic [Ca2+](i) did not change significantly and the decline of maximum Ca2+-activated force was similar to that observed at 22 degrees C. The cause of the greater rate of fatigue at 37 degrees C was a large fall in myofibrillar Ca2+ sensitivity. In the presence of Tiron, the large fall in Ca2+ sensitivity was abolished and the usual decline in tetanic [Ca2+](i) was observed. This study confirms the importance of ROS in fatigue at 37 degrees C and shows that the mechanism of action of ROS is a decline in myofibrillar Ca2+ sensitivity.

Animals↗
PubMed2005

Fibroblasts can be genetically modified to produce excitable cells capable of electrical coupling.

BACKGROUND: Cardiac conduction occurs in an electrical syncytium of excitable cells connected by gap junctions. Disruption of these electrophysiological properties causes conduction slowing or block. Depending on the location of affected cells within the heart, this has the potential to result in clinical syndromes such as atrioventricular block. With a view to developing gene therapy strategies for repairing cardiac conduction defects, we sought to establish whether the phenotype of fibroblasts can be modified by gene transfer to produce cells capable of electrical excitation and coupling. METHODS AND RESULTS: High-titer lentiviral vectors encoding MyoD, a myogenic transcription factor, and connexin43, a gap junction protein, were produced by established methods. Human dermal fibroblasts (HDFs) were efficiently (>80%) transduced at a multiplicity of infection of 50. HDFs transduced with the MyoD-encoding vector underwent myogenic conversion, as evidenced by myotube formation and detection of muscle-specific proteins. Importantly, calcium transients indicative of membrane excitability were observed in MyoD-induced myotubes after loading with a calcium-sensitive dye and electrical stimulation. Transients from adjacent myotubes displayed different excitation thresholds, indicating an absence of coupling between cells, consistent with skeletal muscle biology. In contrast, simultaneous transduction of HDFs with MyoD and connexin43-encoding vectors resulted in the appearance of transients in adjacent myotubes with identical thresholds, indicative of electrical coupling. Notably, dye transfer studies confirmed gap junctional intercellular communication. CONCLUSIONS: Fibroblasts can be genetically modified to produce excitable cells capable of electrical coupling. These observations strengthen the prospect of developing gene-based strategies for repairing cardiac conduction defects.

Animals↗
PubMed2005

The development of a multidimensional measure of job market cognitions: the Employment Opportunity Index (EOI).

The purpose of this research was to develop a multidimensional measure of job market perceptions based on a meta-analysis. Item sets were developed to operationally define the dimensions and were tested among 3 samples. Results of a series of exploratory and confirmatory factor analyses in 3 samples indicated that the 5 scales have satisfactory psychometric properties, construct, and criterion-related validity. The 5 dimensions accounted for significant and relatively large amounts of turnover variance, even after a number of standard turnover predictors had been taken into account. The results suggest the presence of job search microprocesses in the employee turnover process. These microprocesses are described and integrated into current thinking about the turnover process.

Adult↗
PubMed2005

Turnover intentions and voluntary turnover: the moderating roles of self-monitoring, locus of control, proactive personality, and risk aversion.

This article explores moderators of the relationship between turnover intentions and turnover behavior to better explain why some employees translate intentions into behavior and other employees do not. Individual differences in self-monitoring, locus of control, proactive personality, and risk aversion were examined. Results indicate that self-monitoring and risk aversion moderate the intentions-turnover link. Specifically, the relationship between turnover intentions and turnover is stronger for low self-monitors and those lower in risk aversion. Locus of control moderated the relationship in 1 of 2 samples such that the relationship was stronger for those with an internal locus of control. Proactive personality, however, did not directly moderate the relationship between intentions and turnover behaviors.

Adaptation, Psychological↗
PubMed2004

Effects of stretch-activated channel blockers on [Ca2+]i and muscle damage in the mdx mouse.

The mdx mouse lacks dystrophin and is a model of human Duchenne muscular dystrophy. Single mdx muscle fibres were isolated and subjected to a series of stretched (eccentric) contractions while measuring intracellular calcium concentration ([Ca(2+)](i)) with fluo-3 and confocal microscopy. Following the stretched contractions there was a slow rise in resting [Ca(2+)](i) and after 30 min both the [Ca(2+)](i) during a tetanus (tetanic [Ca(2+)](i)) and the tetanic force were reduced. Two blockers of stretch-activated channels, streptomycin and the spider venom toxin GsMTx4, prevented the rise of resting [Ca(2+)](i) and partially prevented the decline of tetanic [Ca(2+)](i) and force. Reducing extracellular calcium to zero also prevented the rise in resting [Ca(2+)](i) and prevented some of the decline in tetanic [Ca(2+)](i) and force. Patch-clamping experiments identified a stretch-activated channel in both wild-type and mdx myotubes which was blocked by GsMTx4. These data suggest that blockers of stretch-activated channels can ameliorate the force reduction following stretched contractions by reducing the influx of Ca(2+) into the muscle. We therefore tested whether in intact mdx mice streptomycin, added to the drinking water, was capable of reducing muscle damage. mdx mice show a period of muscle damage from 20 to 40 days of life and fibres which regenerate from this damage display central nuclei. We measured the frequency of central nuclei in control mdx mice compared to streptomycin-treated mdx mice and showed that the incidence of central nuclei was significantly reduced by streptomycin treatment. This result suggests that blockers of stretch-activated channels may protect against muscle damage in the intact mdx mouse.

Aniline Compounds↗
PubMed2004

Cyanide inhibits the Na+/Ca2+ exchanger in isolated cardiac pacemaker cells of the cane toad.

The effects of the metabolic inhibition on the activity of the Na+/Ca2+ exchanger (NCX) were studied in single isolated pacemaker cells from the cane toad. Ca2+ influx on NCX (reverse mode) was estimated by measuring the increase in intracellular calcium concentration ([Ca2+]i) in response to extracellular Na+-free solution. After application of 2 mM sodium cyanide for 3-5 min, the peak [Ca2+]i in Na+-free solution was significantly decreased from 377+/-42 nM to 260+/-46 nM, suggesting inhibition of NCX. To study Ca2+ efflux on NCX (forward mode), we recorded the tail currents on repolarization which were abolished by Ni2+ and by Na+-free solution. Cyanide decreased the amplitude of tail currents by 36+/-3%. To investigate the intrinsic properties of NCX during the metabolic inhibition, we used rapid application of caffeine to trigger sarcoplasmic reticulum Ca2+ release, which then stimulates NCX current (I(NCX) ). Both the caffeine-induced peak [Ca2+]i and the peak I(NCX) were reduced by cyanide exposure. When I(NCX) was plotted against [Ca2+], the slope of the decay phase was decreased in the presence of CN- to 44+/-8% of control, indicating that for a given [Ca2+]i there was less I(NCX) produced. These results show that cyanide (CN-) inhibits NCX activity at least partly through changes in the intrinsic properties of NCX. The inhibition of NCX probably contributes to the slower firing rate of pacemaker cells in CN-.

Action Potentials↗
PubMed2004

Stretch-activated channels in stretch-induced muscle damage: role in muscular dystrophy.

1. Stretch-induced muscle injury results in the damage that causes reduced force and increased membrane permeability. This muscle damage is caused, in part, by ionic entry through stretch-activated channels and blocking these channels with Gd3+ or streptomycin reduces the force deficit associated with damage. 2. Dystrophin-deficient muscles are more susceptible to stretch-induced muscle injury and the recovery from injury can be incomplete. We have found that Na+ entry associated with stretch-induced injury is enhanced in dystrophin-deficient muscles and that blockers of stretch-activated channels are capable of preventing ionic entry and reducing muscle damage. 3. A model is presented that proposes links between stretch-induced injury, opening of stretch-activated channels, increased levels of intracellular ions and various forms of muscle damage. Although changes in Na+ accompany stretch-induced muscle injury, we believe that changes in Ca2+ probably have a more central role in the damage process.

Animals↗
PubMed2003

Gadolinium reduces short-term stretch-induced muscle damage in isolated mdx mouse muscle fibres.

Duchenne muscular dystrophy is a lethal muscle disease caused by absence of the protein dystrophin which is part of a glycoprotein complex located on the intracellular surface of the surface membrane. The precise function of dystrophin and the reason why its absence causes severe muscle damage are unclear. Stretch-induced muscle damage is well recognised in normal muscle and is more severe in muscles from animals lacking dystrophin (mdx mice). It has been proposed that stretch-induced damage underlies the progression of damage in muscular dystrophy. In the present study we confirm that single fibres from mdx muscle are more susceptible to stretch-induced damage and show that there is an associated rise in intracellular sodium concentration ([Na+]i) which is greater than in wild-type mice. We show that this rise in [Na+]i can be prevented by Gd3+, which is an established blocker of stretch-activated channels. mdx fibres have a higher than normal resting [Na+]i and this is also reduced by Gd3+. If Gd3+ is applied over the period in which [Na+]i rises following stretched contraction, it prevents one component of the reduced force. The other component of reduced force is caused by inhomogeneity of sarcomeres and can be minimised by stretching the muscle to its new optimum length. These experiments show that part of the short-term damage caused by stretch in mdx fibres can be prevented by blocking stretch-activated channels.

Animals↗
PubMed2003

The cardioprotective effects of Na+/H+ exchange inhibition and mitochondrial KATP channel activation are additive in the isolated rat heart.

The mechanisms of recovery of the isolated rat heart were studied after 30 min of global ischemia. Functional recovery was assessed by the percentage recovery of developed pressure after 30 min reperfusion and by the magnitude of the contracture on reperfusion. After a control ischemia, developed pressure recovered to only 12+/-2% of pre-ischemic control and the reperfusion contracture was very large (81+/-6 mmHg). Activation of the mitochondrial KATP channel with 100 microM diazoxide present throughout ischemia and reperfusion improved recovery of developed pressure to 36+/-3% and reduced the reperfusion contracture (53+/-4 mmHg). Inhibition of the sodium/hydrogen exchanger with 10 microM cariporide caused a larger recovery of developed pressure to 72+/-4% and further reduced the reperfusion contracture (11+/-3 mmHg). The combination of both drugs increased recovery of developed pressure to 96+/-4% and the reperfusion contracture remained small (11+/-5 mmHg). The effectiveness of the timing of exposure to these drugs was explored. When both diazoxide and cariporide were applied 2 min before the end of ischaemia and remained present during reperfusion the recovery of developed pressure was 81+/-4% and the reperfusion contracture was small (12+/-3 mmHg); neither was significantly different to the recovery when both drugs were present throughout ischemia and reperfusion. We conclude that mitochondrial damage, blocked by diazoxide, and the coupled exchanger pathway, blocked by cariporide, are two of the principal damage pathways and functional recovery appears to be complete when both are blocked. The combination of these drugs is also highly effective when given 2 min before the end of ischemia.

Animals↗