Biomedical subjects

Sian E Harding

Publications and source records attributed to Sian E Harding.

26 records · Page 2Linked to original sources

PubMed2002

Targeting phospholamban by gene transfer in human heart failure.

BACKGROUND: Myocardial cells from failing human hearts are characterized by abnormal calcium handling, a negative force-frequency relationship, and decreased sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) activity. In this study, we tested whether contractile function can be improved by decreasing the inhibitory effects of phospholamban on SERCA2a with adenoviral gene transfer of antisense phospholamban (asPL). METHODS AND RESULTS: Myocardial cells isolated from 9 patients with end-stage heart failure and 18 donor nonfailing hearts were infected with adenoviruses encoding for either the antisense of phospholamban (Ad.asPL), the SERCA2a gene (Ad.SERCA2a), or the reporter genes beta-galactosidase and green fluorescent protein (Ad.betagal-GFP). Adenoviral gene transfer with Ad.asPL decreased phospholamban expression over 48 hours, increasing the velocity of both contraction and relaxation. Compared with cardiomyocytes infected with Ad.asPL (n=13), human myocytes infected with Ad.betagal-GFP (n=8) had enhanced contraction velocity (20.3 +/- 3.9% versus 8.7 +/- 2.6% shortening/second; P<0.01) and relaxation velocity (26.0 +/- 6.2% versus 8.6 +/- 4.3% shortening/second; P<0.01). The improvement in contraction and relaxation velocities was comparable to cardiomyocytes infected with Ad.SERCA2a. Failing human cardiomyocytes had decreased contraction and Ca2+ release with increasing frequency (0.1 to 2 Hz). Phospholamban ablation restored the frequency response in the failing cardiomyocytes to normal; increasing frequency resulted in enhanced sarcoplasmic reticulum Ca2+ release and contraction. CONCLUSION: These results show that gene transfer of asPL can improve the contractile function in failing human myocardium. Targeting phospholamban may provide therapeutic benefits in human heart failure.

Adenoviridae↗

PubMed2002

Effects of Na(+)/Ca(2+)-exchanger overexpression on excitation-contraction coupling in adult rabbit ventricular myocytes.

The Na(+)/Ca(2+)-exchanger (NCX) is the main mechanism by which Ca(2+) is transported out of the ventricular myocyte. NCX levels are raised in failing human heart, and the consequences of this for excitation-contraction coupling are still debated. We have increased NCX levels in adult rabbit myocytes by adenovirally-mediated gene transfer and examined the effects on excitation-contraction coupling after 24 and 48 h. Infected myocytes were identified through expression of green fluorescent protein (GFP), transfected under a separate promoter on the same viral construct. Control experiments were done with both non-infected myocytes and those infected with adenovirus expressing GFP only. Contraction amplitude was markedly reduced in NCX-overexpressing myocytes at either time point, and neither increasing frequency nor raising extracellular Ca(2+) could reverse this depression. Resting membrane potential and action potential duration were largely unaffected by NCX overexpression, as was peak Ca(2+) entry via the L-type Ca(2+) channel. Systolic and diastolic Ca(2+) levels were significantly reduced, with peak systolic Ca(2+) in NCX-overexpressing myocytes lower than diastolic levels in control cells at 2 m m extracellular Ca(2+). Both cell relengthening and the decay of the Ca(2+) transient were significantly slowed. Sarcoplasmic reticulum (SR) Ca(2+) stores were completely depleted in a majority of myocytes, and remained so despite increasingly vigorous loading protocols. Depressed contractility following NCX overexpression is therefore related to decreased SR Ca(2+) stores and low diastolic Ca(2+) levels rather than reduced Ca(2+) entry.

Adenoviridae↗

PubMed2002

Ion channels in small cells and subcellular structures can be studied with a smart patch-clamp system.

We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipette is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.

Animals↗

PubMed2002

Taurocholate induces changes in rat cardiomyocyte contraction and calcium dynamics.

Obstetric cholestasis is characterized by raised bile acids, and can be complicated by intrauterine death. We have shown that the bile acid taurocholate causes loss of synchronous beating, bradycardia and cessation of contraction in cultured rat cardiomyocytes [Williamson, Gorelik, Eaton, Lab, de Swiet and Korchev (2001) Clin. Sci. 100, 363-369]. The aim of the present study was to investigate the effect of taurocholate on cardiomyocytes further. We demonstrated a reduced rate of contraction and proportion of beating cells when rat cardiomyocytes were exposed to increasing concentrations of taurocholate (0.1-3.0 mM); more marked at higher concentrations (P<0.001). Using scanning ion-conductance microscopy, we also demonstrated reduced amplitude of contraction and calcium transients with taurocholate. Our observations indicate that taurocholate affects calcium release from the sarcoplasmic reticulum and this parallels changes in contractile function. The relationship between the contraction amplitude and calcium transient is not linear, particularly at higher concentrations of taurocholate. We observed different effects in individual cultured neonatal cells; a reversible reduction in rate and amplitude of contraction in some, and irreversible oscillatory (fibrillatory) cessation of beating in others. The effects were more marked with higher concentrations. The contraction amplitude was also reduced in adult cardiomyocytes. The changes were reversible following removal of taurocholate in adult, but not in neonatal, cardiomyocytes exposed to higher concentrations (>0.3 mM) (P<0.001). In conclusion we have demonstrated that the bile acid taurocholate can cause different types of dysrhythmia in individual cardiomyocytes. These results provide further support for the hypothesis that obstetric cholestasis may produce cardiac-related sudden intrauterine death.

Animals↗

PubMed2002

Dissociation of hypertrophic growth from changes in myocyte contractile function.

Increases in myocyte size and impairment of contractile function are both features of the pathophysiologic hypertrophic process. In this short review, we re-present evidence, both in human disease and in animal models, that the 2 phenomena can be dissociated in both time and relative degree. Additionally, in an animal model, physiologic growth concurrent with pathophysiologic hypertrophy induces similar changes in myocyte macroarchitecture but without the deleterious consequences for contractile function.

Animals↗

PubMed2002

Altered mechanical properties and intracellular calcium signaling in cardiomyocytes from annexin 6 null-mutant mice.

Annexin 6 is one of a widely expressed family of calcium-binding proteins found in most mammalian tissues, including the heart. Several studies have implicated annexin 6 in the regulation of intracellular Ca2+ signaling, and it has been shown in vitro to act as a modulator of the sarcoplasmic reticulum Ca2+-release channel, cardiac L-type calcium channel, and Na+/Ca2+ exchanger. To investigate the role of annexin 6 in intact cardiomyocytes, we used mice containing a targeted disruption of the annexin 6 gene. Compared with controls, the myocytes of annexin 6 null-mutant mice demonstrated a significant increase in the rates of shortening and relengthening. Intracellular Ca2+ transients in fura-2-loaded cardiomyocytes induced by caffeine showed a normal baseline and amplitude, whereas the rate of decay was doubled in annexin 6-/- myocytes compared with control mice. These results show that annexin 6 knockout in the mouse leads to an increase in myocyte contractility and faster diastolic Ca2+ removal from the cytoplasm. In light of published findings showing annexin 6 to be down-regulated in end-stage heart failure, these results are consistent with a role for annexin 6 as a negative inotropic factor in the regulation of cardiomyocyte mechanics.

Animals↗

PubMed2002

Characterization of a myocardial depressant factor in meningococcal septicemia.

OBJECTIVE: Identification and characterization of myocardial depressant factors present in meningococcal septicemia. DESIGN: Laboratory investigation of myocardial depression that used isolated cardiac myocytes as an model of cardiac contractile function. SETTING: University hospital and laboratories. PATIENTS: Children with severe meningococcal septic shock requiring intensive care. ANIMALS: Myocytes obtained from adult male Sprague-Dawley rats. INTERVENTIONS: Serum samples obtained from the acute phase of sepsis were evaluated for the presence of myocardial depressant activity. Further characterization of the myocardial depressant factor was undertaken by using cell culture supernatants from whole blood and peripheral blood mononuclear cells that had been exposed to heat-killed meningococci. MEASUREMENTS AND MAIN RESULTS: Myocardial depressant activity was measured by using isolated rat left-ventricular myocytes. Changes in amplitude of contraction and in the speed of contraction and relaxation were determined after cells were exposed to various stimuli. Serum from patients with meningococcal disease had myocardial depressant activity. This activity was also present in whole blood and peripheral blood mononuclear cells exposed to meningococci. Myocardial depressant activity was found to be heat stable, proteinaceous, and of a molecular weight range of 10-25 kDa. The activity did not elevate concentrations of cyclic guanylic acid. Lipopolysaccharide-binding protein augmented the release of myocardial depressant factor by peripheral blood mononuclear cells exposed to meningococci. CONCLUSIONS: Myocardial depression in meningococcal sepsis is mediated in part by circulating myocardial depressant factors. Myocardial depressant factors are also released when whole blood or peripheral blood mononuclear cells of healthy donors are exposed to heat-killed meningococci. Release of the factors appears to be mediated through endotoxin-induced activation of peripheral blood mononuclear cells, since lipopolysaccharide-binding protein augments release in a dose-responsive manner. Partial physicochemical characterization of the factors has been achieved.

Adolescent↗

PubMedDate not supplied

beta-adrenoceptor blockers as agonists: coupling of beta2-adrenoceptors to multiple G-proteins in the failing human heart.

Beta blockers have been shown in clinical trials to improve cardiac function and reduce mortality of heart failure patients. However, these agents require careful titration since they can produce an initial decrease in cardiac output. The authors have recently shown that beta blockers, including some used clinically, can directly depress contraction of myocardium from the failing (but not nonfailing) human heart. This occurs on single ventricular myocytes and is therefore completely independent of any inhibition of endogenous catecholamines. The effect appears to be mediated primarily by the beta2-adrenoceptor (AR) and is dependent on the inhibitory guanine nucleotide binding protein, Gi. Using a transgenic mouse model, as well as adenoviral vectors to overexpress Gi or the human beta2AR in adult myocytes of various species, the authors demonstrate that agents that are blockers for bAR/Gs coupling can be agonists at a beta2AR/Gi-coupled form of the receptor. The negative effect of beta blockers could contribute to the initial cardiodepression that is observed when introducing these agents in heart failure patients. However, in the long term, beta2AR/Gi coupling may enhance the ability of beta blockers to protect and improve the function of the failing heart.

Adrenergic beta-2 Receptor Antagonists↗