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Sian E Harding

Publications and source records attributed to Sian E Harding.

At least 19 recordsLinked to original sources
PubMed2006

A novel Z-groove index characterizing myocardial surface structure.

OBJECTIVE: The role of t-tubule structures in excitation-contraction coupling of ventricular myocytes has been investigated by disruption using prolonged culture, or osmotic shock with formamide. We have used a new method, the Scanning Ion Conductance Microscope (SICM), to investigate in more detail the changes in surface structure of live myocytes during these interventions and to relate them to contractile effects. METHODS: Freshly isolated ventricular myocytes from adult rat hearts were either incubated with formamide, then washed to produce osmotic shock, or put into culture for 2, 4 and 7 days. Contractile characteristics of single myocytes were then measured using the IonOptix system, and in parallel imaged using the SICM which produces a 3-dimensional topographical representation of the cell surface. Loss of t-tubules was quantitated with confocal microscopy after staining with the membrane dye di-8-ANNEPS, and sarcomere structure revealed by immunocytochemical detection of alpha-actinin. RESULTS: Detubulation was produced by either method, with formamide equivalent to 4 day culture in quantitative measures of ANNEPS t-tubule/membrane ratio. SICM images confirmed the loss of t-tubule indentations. Disruption of the Z-groove structure and flattening of the surface were also noted with formamide and, to a lesser extent, culture. A novel Z-groove index was introduced to describe this effect more quantitatively. Contraction and relaxation were impaired by the detubulation methods, but formamide had a markedly greater depressant effect on contraction amplitude than equivalent detubulation by culture. CONCLUSION: Changes in contraction amplitude after detubulation with formamide were more closely related to the alteration in Z-groove structure than loss of t-tubules alone. As well as disrupting t-tubule-induced excitation and calcium movements, formamide may alter the transmission of contraction in the myocyte by interference with sarcomere attachment at the Z-line.

Animals↗
PubMed2006

The human embryonic stem cell-derived cardiomyocyte as a pharmacological model.

Embryonic stem (ES) cells are specialised cells derived from the early embryo, which are capable of both sustained propagation in the undifferentiated state as well as subsequent differentiation into the majority of cell lineages. Human ES cells are being developed for clinical tissue repair, but a number of problems must be addressed before this becomes a reality. However, they also have potential for translational benefit through its use as a test system for screening pharmaceutical compounds. In the cardiac field, present model systems are not ideal for either screening or basic pharmacological/physiological studies. Cardiomyocytes produced from human ES differentiation have advantages for these purposes over the primary isolated cells or the small number of cell lines available. This review describes the methodology for obtaining cardiomyocytes from human embryonic stem cell-derived cardiomyocyte (hESCM), for increasing the proportion of cardiomyocytes in the preparation and for isolating single embryonic stem cell-derived cardiomyocyte (ESCM) from clusters. Their morphological, contractile and electrophysiological characteristics are compared to mature and immature primary cardiomyocytes. The advantages and disadvantages of the hESCM preparation for long term culture and genetic manipulation are described. Basic pharmacological studies on adrenoceptors and muscarinic receptors in hESCM have been performed, and have given stable and reproducible responses. Prolongation of repolarisation can be detected using hESCM cultured on multielectrode arrays (MEA). Human ESCM have a clear potential to improve model systems available for both basic scientific studies and pharmaceutical screening of cardiac target compounds.

Adrenergic Agonists↗
PubMed2006

Iron particles for noninvasive monitoring of bone marrow stromal cell engraftment into, and isolation of viable engrafted donor cells from, the heart.

Stem cells offer a promising approach to the treatment of myocardial infarction and prevention of heart failure. We have used iron labeling of bone marrow stromal cells (BMSCs) to noninvasively track cell location in the infarcted rat heart over 16 weeks using cine-magnetic resonance imaging (cine-MRI) and to isolate the BMSCs from the grafted hearts using the magnetic properties of the donor cells. BMSCs were isolated from rat bone marrow, characterized by flow cytometry, transduced with lentiviral vectors expressing green fluorescent protein (GFP), and labeled with iron particles. BMSCs were injected into the infarct periphery immediately following coronary artery ligation, and rat hearts were imaged at 1, 4, 10, and 16 weeks postinfarction. Signal voids caused by the iron particles in the BMSCs were detected in all rats at all time points. In mildly infarcted hearts, the volume of the signal void decreased over the 16 weeks, whereas the signal void volume did not decrease significantly in severely infarcted hearts. High-resolution three-dimensional magnetic resonance (MR) microscopy identified hypointense regions at the same position as in vivo. Donor cells containing iron particles and expressing GFP were identified in MR-targeted heart sections after magnetic cell separation from digested hearts. In conclusion, MRI can be used to track cells labeled with iron particles in damaged tissue for at least 16 weeks after injection and to guide tissue sectioning by accurately identifying regions of cell engraftment. The magnetic properties of the iron-labeled donor cells can be used for their isolation from host tissue to enable further characterization.

Animals↗
PubMed2006

Functional characterization of embryonic stem cell-derived cardiomyocytes using scanning ion conductance microscopy.

We report here the novel use of scanning ion conductance microscopy (SICM) to produce surface images of embryonic stem cell-derived cardiomyocytes (ESCM) to identify individual contracting cardiomyocytes among different cell types. By measuring amplitude and rhythm we can quantitate contraction of ESCM. This method gives, within the same experiment, an assessment of the number and position of ESCM within the layer of mixed cell types, as well as an accurate measure of the response of individual ESCM. Using different modulators of contraction as examples we showed how SICM could be used for recording their responses. We subsequently demonstrated that this model can be used to investigate the protective effect of antiarrhythmogenic drugs.

Animals↗
PubMed2004

Effect of overexpressed adenylyl cyclase VI on beta 1- and beta 2-adrenoceptor responses in adult rat ventricular myocytes.

1. Adenylyl cyclase VI (ACVI) is one of the most abundantly expressed beta adrenergic receptor (betaAR)-coupled cyclases responsible for cyclic AMP (cAMP) production within the mammalian myocardium. We investigated the role of ACVI in the regulation of cardiomyocyte contractility and whether it is functionally coupled with beta(1) adrenergic receptor (beta(1)AR). 2. Recombinant adenoviruses were generated for ACVI and for antisense to ACVI (AS). Adult rat ventricular myocytes were transfected with ACVI virus, AS or both (SAS). Adenovirus for green fluorescent protein (GFP) served as control. Myocyte contraction amplitudes (% shortening) and relaxation times (R50) were analysed. ACVI function was determined using cAMP assays. 3. ACVI-transfected cells demonstrated a strong 139 kDa ACVI protein band compared to controls. ACVI myocytes had higher steady-state intracellular cAMP levels than GFP myocytes when unstimulated (GFP vs ACVI=6.60+/-0.98 vs 14.2+/-2.1 fmol cAMP/viable cell, n=4, P<0.05) and in the presence of 1 microm isoprenaline or 10 microm forskolin. 4. ACVI myocytes had increased basal contraction (% shortening: GFP vs ACVI: 1.90+/-1.36 vs 3.91+/-2.29, P<0.0001) and decreased basal R50 (GFP vs ACVI: 62.6+/-24.2 ms (n=50) vs 45.0+/-17.2 ms (n=248), P<0.0001). ACVI myocyte responses were increased for forskolin (E(max): GFP=6.70+/-1.59 (n=6); ACVI=9.06+/-0.69 (n=14), P<0.01) but not isoprenaline. 5. ACVI myocyte responses were increased (E(max): GFP vs ACVI=3.16+/-0.77 vs 5.10+/-0.60, P<0.0001) to xamoterol (a partial beta(1)AR-selective agonist) under beta(2)AR blockade (+50 nm ICI 118, 551). AS decreased both control and ACVI-stimulated xamoterol responses (E(max): AS=2.59+/-1.42, SAS=1.38+/-0.5). ACVI response was not mimicked by IBMX. Conversely, response through beta(2) adrenergic receptor (beta(2)AR) was decreased in ACVI myocytes. 6. In conclusion, ACVI overexpression constitutively increases myocyte contraction amplitudes by raising cAMP levels. Native ACVI did not contribute to basal cAMP production or contraction amplitude and only to a minor extent to the forskolin response. beta(1)AR but not beta(2)AR coupling was dependent on ACVI.

Adenylyl Cyclases↗
PubMed2004

Overexpression of beta 1-adrenoceptors in adult rat ventricular myocytes enhances CGP 12177A cardiostimulation: implications for 'putative' beta 4-adrenoceptor pharmacology.

1. CGP 12177A mediates cardiostimulation by activation of the 'putative' beta(4)-adrenoceptor; however, it has recently been reported that disruption of the beta(1)-adrenoceptor gene abolishes this effect. We have adenovirally overexpressed beta(1)-adrenoceptors in isolated, cultured adult rat ventricular cardiomyocytes and observed the inotropic potency of isoprenaline and CGP 12177A (in the presence of 1 microm propranolol). 2. Isoprenaline was a full inotropic agonist at rat ventricular myocytes (pD(2) 7.69+/-0.12). CGP 12177A was a nonconventional partial agonist (pD(2) 6.34+/-0.09), increasing inotropy and lusitropy, with an intrinsic activity of 0.34 and antagonised by bupranolol. 3. beta(1)-adrenoceptor overexpression enhanced the inotropic potency of isoprenaline by 11.7-fold (pD(2) 8.76+/-0.14) and CGP 12177A by 5.9-fold (7.11+/-0.10), respectively. Green fluorescent protein (GFP) overexpression did not alter the potency of isoprenaline or CGP 12177A (pD(2) 7.41+/-0.24 and pD(2) 6.60+/-0.50, respectively). 4. The cardiostimulant effects of CGP 12177A were enhanced by IBMX (phosphodiesterase inhibitor) and decreased by Rp-cAMPS (cAMP antagonist). CGP 12177A also increased cAMP levels. CGP 12177A but not isoprenaline initiated arrhythmias at lower concentrations following beta(1)-adrenoceptor overexpression. 5. (125)I-Cyanopindolol saturation binding in Adv.beta(1) myocytes demonstrated approximately 18-fold increase in beta(1)-adrenoceptors. (3)H-CGP 12177A saturation binding, in the presence of propranolol, increased approximately 5-fold following overexpression of beta(1)-adrenoceptors. 6. This study demonstrates enhanced cardiostimulation by CGP 12177A (in the presence of propranolol) in rat ventricular myocytes overexpressing beta(1)-adrenoceptors, mediated by a Gs/cAMP signalling pathway. 'Putative' beta(4)-adrenoceptor pharmacology appears to be mediated by activation of a novel affinity state of the beta(1)-adrenoceptor.

Adrenergic beta-1 Receptor Agonists↗
PubMed2004

Role of interleukin 6 in myocardial dysfunction of meningococcal septic shock.

BACKGROUND: Myocardial failure has a central role in the complex pathophysiology of septic shock and contributes to organ failure and death. During the sepsis-induced inflammatory process, specific factors are released that depress myocardial contractile function. We aimed to identify these mediators of myocardial depression in meningococcal septic shock. METHODS: We combined gene-expression profiling with protein and cellular methods to identify a serum factor causing cardiac dysfunction in meningococcal septic shock. We identified genes that were significantly upregulated in blood after exposure to meningococci. We then selected for further analysis those genes whose protein products had properties of a myocardial depressant factor--specifically a 12-25 kDa heat-stable protein that is released into serum shortly after onset of meningococcal infection. FINDINGS: We identified 174 significantly upregulated genes in meningococcus-infected blood: six encoded proteins that were of the predicted size and had characteristics of a myocardial depressant factor. Of these, interleukin 6 caused significant myocardial depression in vitro. Removal of interleukin 6 from serum samples of patients with meningococcaemia and from supernatants of inflammatory cells stimulated by meningococci in vitro abolished the negative inotropic activity. Furthermore, concentrations in serum of interleukin 6 strongly predicted degree of myocardial dysfunction and severity of disease in children with meningococcal septic shock. INTERPRETATION: Interleukin 6 is a mediator of myocardial depression in meningococcal disease. This cytokine and its downstream mediators could be a target for future treatment strategies.

Adult↗
PubMed2004

Loss of beta-adrenoceptor response in myocytes overexpressing the Na+/Ca(2+)-exchanger.

Increased Na+/Ca(2+)-exchanger (NCX) and altered beta-adrenoceptor (betaAR) responses are observed in failing human heart. To determine the possible interaction between these changes, we investigated the effect of NCX overexpression on responses to isoproterenol in adult rat ventricular myocytes. Responses to isoproterenol were largely mediated through the beta1AR in control myocytes. Adenovirally-mediated overexpression of NCX, at levels, which did not alter basal contraction of myocytes, markedly depressed the isoproterenol concentration-response curve. Responses to isoproterenol could be restored to normal by beta2AR blockade, suggesting a beta2AR-mediated inhibition of beta1AR signalling. Pertussis toxin normalised isoproterenol responses in NCX cells, indicating that beta2AR effects were mediated by Gi. Negative-inotropic effects of high concentrations of ICI 118,551, previously shown to be due to beta2AR-Gi coupling, were increased in NCX cells. We conclude that NCX upregulation can markedly alter the consequences of betaAR stimulation and that this may contribute to the alterations in betaAR response seen in failing human heart.

Adrenergic beta-Antagonists↗
PubMed2004

Gene transfer in cardiac myocytes.

Congestive heart failure (CHF) represents an enormous clinical problem and remains a leading cause of death despite advances in treatment. New treatments significantly impact mortality and disease course; they do not cure the underlying pathology. Gene transfer, the ability to genetically reprogram the heart in relevant cardiovascular disease models, allows testing the role of specific molecular pathways in disease pathogenesis. Potential therapeutic intervention targets can be then identified and approached with the full spectrum of therapeutic options, including traditional pharmacology, targeted synthesis of small molecule agonists or antagonists, biological agents (cells, antibodies, genetic material), or gene-based therapy. Lessons gleaned from gene transfer experiments on local modulation of cardiac genetic programs will guide attempts to transform early investigations into established therapy.

Female↗
PubMed2003

Cardiostimulant and cardiodepressant effects through overexpressed human beta2-adrenoceptors in murine heart: regional differences and functional role of beta1-adrenoceptors.

(-)-Isoprenaline enhances cardiac contractility through beta-adrenoceptors. However, in cardiac tissue from transgenic mice with a 200-400-fold cardiac overexpression of the human beta(2)-adrenoceptor (TG4) we observed a pronounced cardiodepression at high (-)-isoprenaline concentrations. Here, we investigated the functional role of the coexisting beta(1)-, beta(2)-, and beta(3)-adrenoceptor subtypes in several regions of the TG4 heart, and in particular their contribution to the negative inotropic effect. In paced TG4 left atria, (-)-isoprenaline produced bell-shaped concentration-effect curves increasing (-logEC(50)M=9.0) and decreasing (-logIC(50)M=6.4) contractile force. These effects were unaffected by the beta(1)-selective CGP 20712A (300 nM). The beta(2)-selective inverse agonist ICI 118,551 (30-1,000 nM) antagonised in surmountable manner both the positive and negative inotropic effects of (-)-isoprenaline with similar concentration-dependence, consistent with an exclusive mediation through beta(2)-adrenoceptors. The beta(3)-adrenoceptor-selective agonist BRL37344 (1 nM-10 microM) failed to produce significant inotropic effects in TG4 left atria. Subsequently, we measured left atrial action potentials accompanying the inotropic changes induced by (-)-isoprenaline. Action potentials tended to have shorter duration in left atria from TG4 mice than from non-transgenic littermate mice. However, (-)-isoprenaline prolonged the duration of 30% repolarisation in atria from non-transgenic littermate but not from TG4 mice, while 90% repolarisation was abbreviated in both groups of atria. Negative inotropic effects of (-)-isoprenaline were also observed in right ventricular preparations. Pertussis toxin-treatment of the mice abolished the negative inotropic effects in left atria and reduced cardiodepression in right ventricle, indicating an involvement of beta(2)-adrenoceptor coupling to PTX-sensitive G-proteins. In additional experiments, designed to study the native murine beta(1)-adrenoceptor function, we used the physiological beta(1)-adrenoceptor agonist (-)-noradrenaline. In the presence of 600 nM ICI 118,551 we failed to find a functional role of the beta(1)-adrenoceptors in left atria, and detected only a marginal contribution to the positive chronotropic effect in right atria. We also investigated the effects of the non-conventional partial agonist (-)-CGP 12177 (0.2 nM-6 microM), which in wild-type mice causes tachycardia through beta(1)-adrenoceptors. In TG4 right atria, however, (-)-CGP 12177-evoked tachycardia was resistant to blockade by CGP 20712A but antagonised by ICI 118,551, consistent with mediation through human beta(2)-adrenoceptors. The results from TG4 mice suggest that the positive and negative inotropic effects of (-)-isoprenaline are mediated through human overexpressed beta(2)-adrenoceptors coupled to G(s) protein and G(i) protein, respectively. The (-)-isoprenaline-evoked shortening of the atrial action potential combined with reduced responses of L-type Ca(2+) current may contribute to the negative inotropic effects. The function of murine cardiac beta(1)-adrenoceptors is suppressed by overexpressed human beta(2)-adrenoceptors.

Action Potentials↗
PubMed2003

Evidence for protein phosphatase inhibitor-1 playing an amplifier role in beta-adrenergic signaling in cardiac myocytes.

The protein phosphatase inhibitor-1 (PPI-1) inhibits phosphatase type-1 (PP1) only when phosphorylated by protein kinase A and could play a pivotal role in the phosphorylation/dephosphorylation balance. Rat cardiac PPI-1 was cloned by reverse transcriptase-polymerase chain reaction, expressed in Eschericia coli, evaluated in phosphatase assays, and used to generate an antiserum. An adenovirus was constructed encoding PPI-1 and green fluorescent protein (GFP) under separate cytomegalovirus promotors (AdPPI-1/GFP). A GFP-only virus (AdGFP) served as control. Engineered heart tissue (EHT) from neonatal rat cardiomyocytes and adult rat cardiac myocytes (ARCMs) were used as model systems. PPI-1 expression was determined in human ventricular samples by Northern blots. Compared with AdGFP, AdPPI-1/GFP-infected neonatal rat cardiomyocytes displayed a 73% reduction in PP1 activity. EHTs infected with AdPPI-1/GFP exhibited a fivefold increase in isoprenaline sensitivity. AdPPI-1/GFP-infected ARCMs displayed enhanced cell shortening as well as enhanced phospholamban phosphorylation when stimulated with 1 nM isoprenaline. PPI-1 mRNA levels were reduced by 57+/-12% in failing hearts with dilated and ischemic cardiomyopathy (n=8 each) compared with nonfailing hearts (n=8). In summary, increased PPI-1 expression enhances myocyte sensitivity to isoprenaline, indicating that PPI-1 acts as an amplifier in beta-adrenergic signaling. Decreased PPI-1 in failing human hearts could participate in desensitization of the cAMP pathway.

Adenoviridae↗
PubMed2003

Changes in sarcolemmal Ca entry and sarcoplasmic reticulum Ca content in ventricular myocytes from patients with end-stage heart failure following myocardial recovery after combined pharmacological and ventricular assist device therapy.

AIMS: Support with left ventricular assist devices (LVAD) improves cardiac performance in patients with end-stage heart failure. In some cases this strategy, combined with pharmacological treatment, has led to a clinical improvement which remained after LVAD explant. This study defines changes in Ca handling at the cellular level in failing left ventricular tissue taken at LVAD implant (LVAD core) and LVAD removal (post-LVAD). METHODS AND RESULTS: We studied cell size and Ca regulation in enzymatically dissociated cardiac myocytes. We used confocal microscopy and electrophysiological techniques to investigate the SR Ca content and major Ca movements across the sarcolemma during the action potential. We firstly recorded a significant reduction in cell capacitance and cell volume consistent with regression of cellular hypertrophy in post-LVAD myocytes compared with LVAD core myocytes. Ca entry via sarcolemmal Ca channels during the action potential using action potential voltage-clamping was significantly increased in post-LVAD myocytes compared with LVAD cores myocytes. Finally, SR Ca content (assessed by integrating the caffeine-induced Na/Ca exchanger transient inward current) in post-LVAD myocytes was also significantly increased compared with LVAD cores myocytes. CONCLUSIONS: These results show that in myocytes from patients after LVAD support there is more Ca entry to trigger Ca release and more SR Ca content, leading to improved contractile function.

Calcium↗
PubMed2003

Contractile effects of adenovirally-mediated increases in SERCA2a activity: a comparison between adult rat and rabbit ventricular myocytes.

Adenoviral vectors have been successfully used to increase the activity of the sarcoplasmic reticulum Ca(2+)-ATPase in adult ventricular myocytes and to produce functional improvements in contractility in vivo and in vitro. While in vivo experiments are often performed in rat, in vitro manipulation of myocytes has been confined to rabbit and human cells. In the present study we make quantitative comparisons between cultured adult rat and rabbit myocytes in their responses to SERCA2a overexpression using adenoviral vectors. We also compare the strategy of SERCA2a overexpression with that of phospholamban down-regulation, using adenovirus carrying antisense message, as a means to increase SERCA2a activity and enhance contraction and relaxation. Adult myocytes were cultured for 48 h with either vector, and contraction assessed in 2 mM Ca2+, 37 degrees C, at a range of stimulation frequencies. Contraction amplitude was enhanced to a similar degree in either rat or rabbit myocytes at most stimulation frequencies, with SERCA2a overexpression and phospholamban down-regulation approximately equally effective. The maximum effect of either vector was less than that of beta-adrenoceptor agonists. Relaxation was accelerated in rabbit myocytes more strongly than in rat. Phospholamban antisense was slightly less effective than SERCA2a overexpression on relaxation times in rabbit. Increasing stimulation frequency also accelerated relaxation in rat myocytes: this effect was greater than, and additive with, that of SERCA2a overexpression. We conclude that, despite some species-dependent modification, the effects of increased SERCA2a activity are broadly similar in rat and rabbit. Both SERCA2a overexpression and phospholamban down-regulation are effective strategies, and neither appears to produce supraphysiological stimulatory effects on contraction or relaxation.

Adenoviridae↗
PubMed2002

Interaction between increased SERCA2a activity and beta -adrenoceptor stimulation in adult rabbit myocytes.

Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a overexpression and phospholamban depletion have been shown to have beneficial effects on contractility in heart failure. However, the high sympathetic tone during development of failure may interact with increases in SERCA2a activity in potentially deleterious ways. We used adenoviral vectors to overexpress SERCA2a or partially downregulate phospholamban in adult rabbit ventricular myocytes in culture and studied the responses of these cells to beta-adrenoceptor stimulation. SERCA2a overexpression and phospholamban depletion had quantitatively similar effects on basal contraction amplitude and in accelerating relaxation. Increasing SERCA2a activity by either strategy had little effect on the increase in contraction amplitude or incidence of arrhythmias with increasing isoproterenol. Maximum acceleration of relaxation by beta-adrenoceptor stimulation was similar to that produced by SERCA2a overexpression. Isoproterenol treatment of SERCA2a-overexpressing or phospholamban-deficient myocytes produced a further modest decrease in relaxation time, with similar final values in both groups. We find no evidence for Ca(2+) overload induced by SERCA2a overexpression alone or in combination with catecholamines.

Adrenergic beta-Agonists↗
PubMed2002

Specific beta(2)AR blocker ICI 118,551 actively decreases contraction through a G(i)-coupled form of the beta(2)AR in myocytes from failing human heart.

BACKGROUND: We have observed direct (noncatecholamine-blocking) negative inotropic effects of the selective beta(2)-adrenoceptor (AR) antagonist ICI 118,551 in myocytes from failing human ventricle. In this study we characterize the effect in parallel in human myocytes and in myocytes from animal models where beta(2)ARs or G(i) proteins are overexpressed. METHODS AND RESULTS: Enzymatically isolated, superfused ventricular myocytes were exposed to betaAR agonists and antagonists/inverse agonists, and contraction amplitude was measured. ICI 118,551 decreased contraction in ventricular myocytes from failing human hearts by 45.3+/-4.1% (n=20 hearts/31 myocytes, P<0.001) but had little effect in nonfailing hearts (4.9+/-4%, n=5 myocytes/3 hearts). Effects were significantly larger in patients classified as end-stage. Transgenic mice with high beta(2)AR number and increased G(i) levels had normal basal contractility but showed a similar negative inotropic response to ICI 118,551. Overexpression of human beta(2)AR in rabbit myocytes using adenovirus potentiated the negative inotropic effect of ICI 118,551. In human, rabbit, and mouse myocytes, the negative inotropic effects were blocked after treatment of cells with pertussis toxin to inactivate G(i), and overexpression of G(i)alpha(2) induced the effect de novo in normal rat myocytes. CONCLUSIONS: We hypothesize that ICI 118,551 binding directs the beta(2)AR to a G(i)-coupled form and away from the G(s)-coupled form (ligand-directed trafficking). ICI 118,551 effectively acts as an agonist at the G(i)-coupled beta(2)AR, producing a direct negative inotropic effect. Conditions where beta(2)ARs are present and G(i) is raised (failing human heart, TGbeta(2) mouse heart) predispose to the appearance of the negative inotropic effect.

Adrenergic beta-2 Receptor Antagonists↗
PubMed2002

High-resolution scanning patch-clamp: new insights into cell function.

Cell specialization is often governed by the spatial distribution of ion channels and receptors on the cell surface. So far, little is known about functional ion channel localization. This is due to a lack of satisfactory methods for investigating ion channels in an intact cell and simultaneously determining the channels' positions accurately. We have developed a novel high-resolution scanning patch-clamp technique that enables the study of ion channels, not only in small cells, such as sperm, but in submicrometer cellular structures, such as epithelial microvilli, fine neuronal dendrites, and, particularly, T-tubule openings of cardiac myocytes. In cardiac myocytes, as in most excitable cells, action potential propagation depends essentially on the properties of ion channels that are functionally and spatially coupled. We found that the L-type calcium and chloride channels are distributed and colocalized in the region of T-tubule openings, but not in other regions of the myocyte. In addition, chloride channels were found in narrowly defined regions of Z-grooves. This finding suggests a new synergism between these types of channels that may be relevant for action potential propagation along the T-tubule system and excitation-contraction coupling.

Animals↗