Adult Guinea Pig Ventricular Myocytes Research Articles

Macrophage-Dependent Interleukin-6-Production and Inhibition of IK Contributes to Acquired QT Prolongation in Lipotoxic Guinea Pig Heart.

In the heart, the delayed rectifier K current, IK, composed of the rapid (IKr) and slow (IKs) components contributes prominently to normal cardiac repolarization. In lipotoxicity, chronic elevation of pro-inflammatory cytokines may remodel IK, elevating the risk for ventricular arrythmias and sudden cardiac death. We investigated whether and how the pro-inflammatory interleukin-6 altered IK in the heart, using electrophysiology to evaluate changes in IK in adult guinea pig ventricular myocytes. We found that palmitic acid (a potent inducer of lipotoxicity), induced a rapid (~24 h) and significant increase in IL-6 in RAW264.7 cells. PA-diet fed guinea pigs displayed a severely prolonged QT interval when compared to low-fat diet fed controls. Exposure to isoproterenol induced torsade de pointes, and ventricular fibrillation in lipotoxic guinea pigs. Pre-exposure to IL-6 with the soluble IL-6 receptor produced a profound depression of IKr and IKs densities, prolonged action potential duration, and impaired mitochondrial ATP production. Only with the inhibition of IKr did a proarrhythmic phenotype of IKs depression emerge, manifested as a further prolongation of action potential duration and QT interval. Our data offer unique mechanistic insights with implications for pathological QT interval in patients and vulnerability to fatal arrhythmias.

Interleukin-6 inhibition of hERG underlies risk for acquired long QT in cardiac and systemic inflammation.

Increased proinflammatory interleukin-6 (IL-6) levels are associated with acquired long QT-syndrome (LQTS) in patients with systemic inflammation, leading to higher risks for life-threatening polymorphic ventricular tachycardia such as Torsades de Pointes. However, the functional and molecular mechanisms of this association are not known. In most cases of acquired LQTS, the target ion channel is the human ether-á-go-go-related gene (hERG) encoding the rapid component of the delayed rectifier K current, IKr, which plays a critical role in cardiac repolarization. Here, we tested the hypothesis that IL-6 may cause QT prolongation by suppressing IKr. Electrophysiological and biochemical assays were used to assess the impact of IL-6 on the functional expression of IKr in HEK293 cells and adult guinea-pig ventricular myocytes (AGPVM). In HEK293 cells, IL-6 alone or in combination with the soluble IL-6 receptor (IL-6R), produced a significant depression of IKr peak and tail current densities. Block of IL-6R or Janus kinase (JAK) reversed the inhibitory effects of IL-6 on IKr. In AGPVM, IL-6 prolonged action potential duration (APD) which was further prolonged in the presence of IL-6R. Similar to heterologous cells, IL-6 reduced endogenous guinea pig ERG channel mRNA and protein expression. The data are first to demonstrate that IL-6 inhibition of IKr and the resulting prolongation of APD is mediated via IL-6R and JAK pathway activation and forms the basis for the observed clinical QT interval prolongation. These novel findings may guide the development of targeted anti-arrhythmic therapeutic interventions in patients with LQTS and inflammatory disorders.

Toward a new Therapeutic Strategy in the Treatment of Timothy Syndrome

Timothy Syndrome (TS) is a multisystem disorder, featuring neurological impairment and life-threatening arrhythmias which are often fatal in early childhood. The basis of TS is a single point mutation (either G406R or G402S) within the CaV1.2 L-type Ca2+ channel. These mutations can occur in either of the mutually exclusive exons 8 or 8a, such that the expression level of each exon contributes significantly to the overall severity of the disease. We have modeled this dependence on gene dosage in an established adult ventricular myocyte model, and found that there is a non-linear dependence of the action potential duration on the fraction of TS channels, resulting in a threshold for arrhythmogenesis. To explore this experimentally at the cell-network level, we variably expressed TS channels in cultured adult guinea-pig ventricular myocytes. Indeed, conservative expression of TS channels yielded graded action-potential prolongation, but a small further increase led to significant arrhythmia. Such non-linear dependence on channel expression imparts an important principle for therapeutics: a small shift in the complement of mutant versus wild type channels may impart a significant clinical improvement. A possible therapeutic strategy would therefore employ methods to alter the splice expression pattern of the mutually exclusive exons 8 and 8a. Here, we demonstrate just such a shift in the expression pattern of exons 8 versus 8a in cultured myocytes. This represents an exciting new treatment strategy, where suppression of the TS containing exon is accompanied by a corresponding increase in non-TS channels. The potential benefits for TS patients are significant and may serve as a model system for developing a new therapeutic strategy for any channelopathy in which the mutation occurs within a mutually exclusive exon.

Novel fluorescence resonance energy transfer-based reporter reveals differential calcineurin activation in neonatal and adult cardiomyocytes.

Novel fluorescence resonance energy transfer-based genetically encoded reporters of calcineurin are constructed by fusing the two subunits of calcineurin with P2A-based linkers retaining the expected native conformation of calcineurin. Calcineurin reporters display robust responses to calcium transients in HEK293 cells. The sensor responses are correlated with NFATc1 translocation dynamics in HEK293 cells. The sensors are uniformly distributed in neonatal myocytes and respond efficiently to single electrically evoked calcium transients and show cumulative activation at frequencies of 0.5 and 1 Hz. In adult myocytes, the calcineurin sensors appear to be localized to the cardiac z-lines, and respond to cumulative calcium transients at frequencies of 0.5 and 1 Hz. The phosphatase calcineurin is a central component of many calcium signalling pathways, relaying calcium signals from the plasma membrane to the nucleus. It has critical functions in a multitude of systems, including immune, cardiac and neuronal. Given the widespread importance of calcineurin in both normal and pathological conditions, new tools that elucidate the spatiotemporal dynamics of calcineurin activity would be invaluable. Here we develop two separate genetically encoded fluorescence resonance energy transfer (FRET)-based sensors of calcineurin activation, DuoCaN and UniCaN. Both sensors showcase a large dynamic range and rapid response kinetics, differing primarily in the linker structure between the FRET pairs. Both sensors were calibrated in HEK293 cells and their responses correlated well with NFAT translocation to the nucleus, validating the biological relevance of the sensor readout. The sensors were subsequently expressed in neonatal rat ventricular myocytes and acutely isolated adult guinea pig ventricular myocytes. Both sensors demonstrated robust responses in myocytes and revealed kinetic differences in calcineurin activation during changes in pacing rate for neonatal versus adult myocytes. Finally, mathematical modelling combined with quantitative FRET measurements provided novel insights into the kinetics and integration of calcineurin activation in response to myocyte Ca transients. In all, DuoCaN and UniCaN stand as valuable new tools for understanding the role of calcineurin in normal and pathological signalling.

Endostatin inhibits T-type Ca2+ channel current in guinea pig ventricular myocyte.

Endostatin, a fragment of collagen XVIII, is known as an endogenous angiogenesis inhibitor, and its serum concentration increases in various cardiovascular diseases. T-type Ca2+ channel, low voltage-activated Ca2+ channel, is not expressed in adult ventricular myocytes. Re-expression of T-type Ca2+ channels in cardiac myocytes is thought to be involved in the development of cardiac hypertrophy. We examined the effects of endostatin on T-type Ca2+ channel current by whole-cell patch clamp technique in freshly isolated adult guinea pig ventricular myocytes, which exceptionally express T-type Ca2+ channels. Although endostatin 300 ng/ml had no effect on L-type Ca2+ current, it significantly inhibited T-type Ca2+ current. These data indicate that endostatin can be an endogenous inhibitor of T-type Ca2+ channels in the cardiac myocytes.

Live Cell Biochemistry Implicates Protein Kinase a Modulation of L-Type CaV1.4 Channels

The regulation of L-type Ca2+ channels by protein kinase A (PKA), though biologically crucial, has long remained mechanistically storied and complex, as studied in native cells at one extreme, and through in vitro biochemistry at the other. Here, we adopt a different tactic and focus initially on an intermediate context, using ideas drawn from synthetic biology and live-cell biochemistry. We set out to create a form of PKA modulation in L-type channels, based on our recent findings that: (a) calmodulin (CaM) competes for binding at a channel C-terminal ‘IQ’ domain with an ‘ICDI’ module in the C-terminal extremity of L-type CaV1.3/1.4 channels, and (b) dislodging CaM profoundly suppresses peak channel opening by severalfold and eliminates their Ca2+-dependent inactivation (CDI) (Adams et al (2014), Cell in press). We reasoned that implanting a synthetic phosphorylation site in ICDI might weaken IQ interaction in a PKA-sensitive manner, allowing channels to rebind CaM and undergo CDI. Cognizant that full-bore PKA signaling is best conserved within certain native rather than model cells, we performed live-cell FRET interaction assays (IQ versus ICDI) in adult guinea-pig ventricular myocytes renown for strong PKA signaling. To our surprise in control experiments, we discovered that IQ interaction with the wild-type ICDI of L-type CaV1.4 channels is already sharply attenuated by PKA activation, whereas ICDI modules from other L-type isoforms showed no such modulability. Accordingly, we synthesized chimeric L-type CaV1.3 channels fused to the CaV1.4 ICDI module, and endowed such channels with robust forskolin-dependent enhancement of CDI, as observed in HEK293 cells. For wild-type CaV1.4 channels, we now also resolved analogous forskolin activatable CDI. This effect, discovered through a synthetic live-cell biochemical approach, might underlie the dopaminergic regulation of CaV1.4 implicated in circadian control within the retina.

Calmodulin mutations associated with long QT syndrome prevent inactivation of cardiac L-type Ca2 + currents and promote proarrhythmic behavior in ventricular myocytes

Recent work has identified missense mutations in calmodulin (CaM) that are associated with severe early-onset long-QT syndrome (LQTS), leading to the proposition that altered CaM function may contribute to the molecular etiology of this subset of LQTS. To date, however, no experimental evidence has established these mutations as directly causative of LQTS substrates, nor have the molecular targets of CaM mutants been identified. Here, therefore, we test whether expression of CaM mutants in adult guinea-pig ventricular myocytes (aGPVM) induces action-potential prolongation, and whether affiliated alterations in the Ca2+ regulation of L-type Ca2+ channels (LTCC) might contribute to such prolongation. In particular, we first overexpressed CaM mutants in aGPVMs, and observed both increased action potential duration (APD) and heightened Ca2+ transients. Next, we demonstrated that all LQTS CaM mutants have the potential to strongly suppress Ca2+/CaM-dependent inactivation (CDI) of LTCCs, whether channels were heterologously expressed in HEK293 cells, or present in native form within myocytes. This attenuation of CDI is predicted to promote action-potential prolongation and boost Ca2+ influx. Finally, we demonstrated how a small fraction of LQTS CaM mutants (as in heterozygous patients) would nonetheless suffice to substantially diminish CDI, and derange electrical and Ca2+ profiles. In all, these results highlight LTCCs as a molecular locus for understanding and treating CaM-related LQTS in this group of patients.

Differential Regulation of Slow and Rapid Delayed Rectifier Potassium Currents by cGMP Dependent Nitric Oxide Signalling Pathways in Isolated Adult Guinea Pig Ventricular Myocytes

Nitric oxide (NO) signalling pathways are reported to regulate cardiac repolarisation. This was investigated using BAY 60-2770, which activates soluble guanylyl cyclase (sGC) in a NO and haem independent manner to generate cGMP. Action potentials (APs) and currents were recorded from isolated guinea pig ventricular myocytes at 37oC using the perforated patch clamp technique. APs were stimulated at 2 Hz. The slow and rapid delayed rectifier potassium currents, IKs and IKr were measured as tail currents elicited by a voltage step from +40 mV to −50 mV. The following experiments were carried out in the presence of 100 µM IBMX (3-Isobutyl-1-methylxanthine) a non-selective phosphodiesterase (PDE) inhibitor. 1 µM BAY 60-2770 lengthened APD90 by 15.7 ms (p<0.05, n=7), suggesting that sGC activation and an increase in cGMP inhibits repolarising currents. Further experiments showed that IKs was inhibited by BAY 60-2770. IBMX alone enhanced IKs by 77 ± 9 % (p<0.001, n=8) and subsequent addition of BAY 60-2770 reduced the IBMX dependent enhancement of IKs by 68 ± 2 % (p<0.001, n=8). This suggests that PDE inhibition permits IKs to be inhibited by cGMP dependent NO signalling pathways. The sGC mediated inhibition of IKs by BAY 60-2770 is unlikely to be due to phosphorylation by protein kinase G (PKG) because the inhibition remained in the presence of 100 nM KT-5823, a PKG inhibitor and because PKG activation with 100 µM 8-bromo-cGMP had no effect on IKs. IKr was not regulated by PDE inhibition, sGC activation or PKG activation. Overall, these findings suggest that cGMP dependent NO signalling pathways regulate IKs, but not IKr, via a PKG independent mechanism.

Structural and functional plasticity in long-term cultures of adult ventricular myocytes

Cultured heart cells have long been valuable for characterizing biological mechanism and disease pathogenesis. However, these preparations have limitations, relating to immaturity in key properties like excitation–contraction coupling and β-adrenergic stimulation. Progressive attenuation of the latter is intimately related to pathogenesis and therapy in heart failure. Highly valuable would be a long-term culture system that emulates the structural and functional changes that accompany disease and development, while concurrently permitting ready access to underlying molecular events. Accordingly, we here produce functional monolayers of adult guinea-pig ventricular myocytes (aGPVMs) that can be maintained in long-term culture for several weeks. At baseline, these monolayers exhibit considerable myofibrillar organization and a significant contribution of sarcoplasmic reticular (SR) Ca2+ release to global Ca2+ transients. In terms of electrical signaling, these monolayers support propagated electrical activity and manifest monophasic restitution of action-potential duration and conduction velocity. Intriguingly, β-adrenergic stimulation increases chronotropy but not inotropy, indicating selective maintenance of β-adrenergic signaling. It is interesting that this overall phenotypic profile is not fixed, but can be readily enhanced by chronic electrical stimulation of cultures. This simple environmental cue significantly enhances myofibrillar organization as well as β-adrenergic sensitivity. In particular, the chronotropic response increases, and an inotropic effect now emerges, mimicking a reversal of the progression seen in heart failure. Thus, these aGPVM monolayer cultures offer a valuable platform for clarifying long elusive features of β-adrenergic signaling and its plasticity.

The Role of CGMP Dependent Nitric Oxide Signalling on Cardiac Repolarisation in Adult Guinea Pig Ventricular Myocytes

Recently, roles for nitric oxide (NO) signalling pathways have been linked with abnormalities in cardiac repolarisation and cardiac arrhythmias. Studies from isolated heart preparations have demonstrated an antifibrillatory effect following vagus nerve stimulation, which is NO dependent. The cellular and molecular basis for this protective effect is unknown. In this study, cGMP dependent regulation of repolarisation was investigated using BAY 60-2770, a novel NO and haem independent soluble guanylyl cyclase (sGC) activator. Action potentials and currents were recorded from isolated guinea pig ventricular myocytes using the perforated patch clamp technique. Cellular cGMP was quantified by radioimmunoassay and expressed as a fold change relative to basal levels. 1 μM BAY 60-2770 increased cellular cGMP by 2.2 fold and by 7.1 fold in the presence of 100 μM IBMX (3-Isobutyl-1-methylxanthine), a non-selective phosphodiesterase (PDE) inhibitor. In vitro assays on purified sGC have shown that ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one) potentiates the action of BAY 60-2770. This was corroborated in our experiments, BAY 60-2770 + 10 μM ODQ increased cGMP by 27.9 fold. Despite substantial increases in cellular cGMP, changes in times to 90% repolarisation (APD90) were modest. BAY 60-2770 shortened APD90 by 11.3 ms and adding ODQ to BAY 60-2770 caused no further APD90 shortening. In contrast, when cGMP hydrolysis was blocked by IBMX, BAY 60-2770 lengthened APD90 by 15.7 ms. Preliminary findings indicate that delayed rectifier potassium currents are modulated by these pathways. Overall, these findings demonstrate the complex interplay between cGMP and cAMP mediated effects on the ion channels regulating cardiac repolarisation. The modest effect of BAY 60-2770 + ODQ on APD90, despite a substantial increase in cellular cGMP, highlights that PDEs limit cGMP accumulation close to ion channels in the sarcolemma, thus compartmentalising cGMP signalling.

Manipulability of β-Adrenergic Responsiveness in Adult Guniea-Pig Cardiomyocyte Cultures

β-adrenergic stimulation of cardiomyocytes induces vital upregulation of both chronotropy (heart rate) and ionotropy (contractility). Progressive attenuation of this regulation constitutes critical pathogenic and therapeutic dimensions of heart failure (Gene Ther19:686). Yet, key features of this pathway and its dysfunction in disease remain unclear. Highly valuable would be a long-term cultured myocyte system that emulates the strong-to-weak progression of β-adrenergic responsiveness between normal and diseased states, while permitting ready manipulation and measurement of underlying molecular events. Here, we used improved culture protocols to produce functional monolayers of adult guinea-pig ventricular myocytes (aGPVMs) that can be maintained for several weeks. At baseline, these monolayers support propagated electrical activity and exhibit monophasic restitution of action-potential duration and conduction velocity. Intriguingly, β-adrenergic stimulation (0.5 μM isoproterenol, iso) upregulates chronotropy without change in inotropy (a, Ca2+ transients), indicating selective regression of adrenergic signaling. By contrast, after several-day electrical stimulation of cultures, β-adrenergic stimulation not only augments chronotropy, but robustly enhances inotropy (b). Thus we have generated a cultured myocyte system whose β-adrenergic responsiveness can be easily manipulated, offering a valuable platform for clarifying long elusive features of adrenergic signaling and its plasticity in disease.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Normalizing Action-Potential Morphology in Long-Term Cultures of Adult Guinea-Pig Ventricular Cardiomyocytes by Ca VBETA Expression

Adult guinea-pig ventricular myocytes (aGPVMs) exhibit enduring action-potential plateaus similar to those in humans, making this system advantageous for investigating human phase 2 phenomena. Long-term cultures of aGPVMs (Canadian J Physiol Pharmacol7:740) further offer the promise of genetic manipulation by somatic gene transfer, but have been infrequently exploited. Here, we confirm that aGPVM cultures form monolayers featuring organized myofibrils and robust contraction. Beyond this, we now demonstrate that point pacing of di-4-ANEPPS stained monolayers enable optical measurements of action potentials. At 2-Hz pacing, action potentials indeed lasted ∼250 ms, but displayed attenuated plateau phases (a, gray trace). To normalize phase 2, and to demonstrate genetic manipulability, we virally expressed Ca2+ channel CaVβ subunits that enhance L-type channels in heterologous systems. CaVβ expression in fact restored a full-bore plateau phase, as shown by the black trace in a. To confirm a role of Ca2+ current, we obtained single-channel recordings of native L-type channels. CaVβ subunits strikingly increased open probability, while decreasing inactivation (b, control; c, CaVβ expression), thus explaining the normalization of action-potential morphology. Overall, long-term cultured aGPVMs offer a powerful model system for electrophysiological exploration.

Dynamic Partnership between KCNQ1 and KCNE1 and Influence on Cardiac IKs Current Amplitude by KCNE2

Cardiac slow delayed rectifier (IKs) channel is composed of KCNQ1 (pore-forming) and KCNE1 (auxiliary) subunits. Although KCNE1 is an obligate IKs component that confers the uniquely slow gating kinetics, KCNE2 is also expressed in human heart. In vitro experiments suggest that KCNE2 can associate with the KCNQ1-KCNE1 complex to suppress the current amplitude without altering the slow gating kinetics. Our goal here is to test the role of KCNE2 in cardiac IKs channel function. Pulse-chase experiments in COS-7 cells show that there is a KCNE1 turnover in the KCNQ1-KCNE1 complex, supporting the possibility that KCNE1 in the IKs channel complex can be substituted by KCNE2 when the latter is available. Biotinylation experiments in COS-7 cells show that although KCNE1 relies on KCNQ1 coassembly for more efficient cell surface expression, KCNE2 can independently traffic to the cell surface, thus becoming available for substituting KCNE1 in the IKs channel complex. Injecting vesicles carrying KCNE1 or KCNE2 into KCNQ1-expressing oocytes leads to KCNQ1 modulation in the same manner as KCNQ1+KCNEx (where x=1 or 2) cRNA coinjection. Thus, free KCNEx peptides delivered to the cell membrane can associate with existing KCNQ1 channels to modulate their function. Finally, adenovirus-mediated KCNE2 expression in adult guinea pig ventricular myocytes exhibited colocalization with native KCNQ1 protein and reduces the native IKs current density. We propose that in cardiac myocytes the IKs current amplitude is under dynamic control by the availability of KCNE2 subunits in the cell membrane.

Rem Selectively Abolishes β1-adrenergic Regulation Of CaV1.2 Channels In Heart

β-adrenergic modulation of cardiac CaV1.2 channels is critical for sympathetic regulation of the heartbeat, and its disruption is a harmful hallmark of heart failure. RGK (Rem, Rem2, Rad, Gem/Kir) GTPases potently inhibit CaV channels by interacting with their auxiliary β subunits. Intriguingly, RGK proteins are present in heart, and their levels are elevated in heart failure. We examined the impact of the RGK GTPase, Rem, on CaV1.2 channels in heart cells and assessed whether there was crosstalk with the β-adrenergic modulation of the channel. Cultured adult guinea pig ventricular myocytes expressed robust CaV1.2 currents (ICa,L) (15.08 pA/pF) and responded to β1-adrenergic activation (1 μM isoproterenol + 1 μM ICI118,551) with a sharp, three-fold increase in current density. Isochronal cardiac cells expressing YFP-Rem, achieved through adenovirus infection, displayed a markedly lower basal current density (5.85 pA/pF). Nevertheless, the effect of Rem in heart is quantitatively smaller than seen in recombinant channels expressed in HEK 293 cells, which feature a virtual ablation of ICa,L. Surprisingly, the remaining Rem-insensitive ICa,L in guinea pig heart cells was essentially unresponsive to β1-adrenergic stimulation. This was not due to disruption of the signaling pathway because isoproterenol-mediated increase in cardiac IKs remained unchanged. Intriguingly, the Rem insensitive ICa,L remained responsive to forskolin. These results reveal an unexpected crosstalk between RGK GTPases and β-adrenergic signaling pathway at the level of cardiac ICa,L, and suggests that Rem selectively inhibits spatially distinct CaV1.2 channels in single heart cells.

Identifying The Site Of The Source Of Reactive Oxygen Species Within The Mitochondria After Transient Exposure Of Cardiac Myocytes To Hydrogen Peroxide

Oxidative stress is a feature of cardiovascular disease. Hydrogen peroxide (H2O2) can act as a signaling molecule to mediate cardiovascular pathology. We have previously shown that transient exposure of adult guinea pig ventricular myocytes to H2O2 leads to further production of reactive oxygen species (ROS) from the mitochondria. We have demonstrated that exposure of myocytes to 30μM H2O2 for 5 min then 10U/ml catalase for 5 min to degrade the H2O2 caused a 65.4±8.4% further increase in superoxide by the mitochondria (n=47). We tested whether transient exposure to H2O2 altered protein synthesis in the myocytes. Exposure of myocytes to 30μM H2O2 for 5 min followed by 10U/ml catalase for 5 min caused a 2-fold increase in protein synthesis measured as 3H-Leucine incorporation (n=10). This suggests that a transient exposure to H2O2 may be sufficient to induce cardiac hypertrophy. We now wish to identify the site of ROS production in the mitochondria. Superoxide was assessed with the fluorescent indicator dihydroethidium (DHE). Exposing myocytes to 1μM DPI, which binds prior to the ROS generation site of complex I, followed by transient exposure to H2O2 resulted in complete attenuation of the increase in DHE signal after exposure to H2O2. Exposing myocytes to 1μM rotenone, which binds after the ROS generation site of complex I, followed by transient exposure to H2O2 resulted in a 45% reduction in the increase in DHE signal after exposure to H2O2. These data suggest the source of ROS production is distal to complex I. Identifying the site of production of ROS may represent a possible therapeutic target to prevent the development of cardiac hypertrophy associated with a transient exposure to H2O2.

Can optical recordings of membrane potential be used to screen for drug-induced action potential prolongation in single cardiac myocytes?

Introduction Potential-sensitive dyes have primarily been used to optically record action potentials (APs) in whole heart tissue. Using these dyes to record drug-induced changes in AP morphology of isolated cardiac myocytes could provide an opportunity to develop medium throughout assays for the pharmaceutical industry. Ideally, this requires that the dye has a consistent and rapid response to membrane potential, is insensitive to movement, and does not itself affect AP morphology. Materials and methods We recorded the AP from isolated adult guinea-pig ventricular myocytes optically using di-8-ANEPPS in a single-excitation dual-emission ratiometric system, either separately in electrically field stimulated myocytes, or simultaneously with an electrical AP recorded with a patch electrode in the whole-cell bridge mode. The ratio of di-8-ANEPPS fluorescence signal was calibrated against membrane potential using a switch-clamp to voltage clamp the myocyte. Results Our data show that the ratio of the optical signals emitted at 560/620 nm is linearly related to voltage over the voltage range of an AP, producing a change in ratio of 7.5% per 100 mV, is unaffected by cell movement and is identical to the AP recorded simultaneously with a patch electrode. However, the APD 90 recorded optically in myocytes loaded with di-8-ANEPPS was significantly longer than in unloaded myocytes recorded with a patch electrode (355.6 ± 13.5 vs. 296.2 ± 16.2 ms; p < 0.01). Despite this effect, the apparent IC 50 for cisapride, which prolongs the AP by blocking I Kr, was not significantly different whether determined optically or with a patch electrode (91 ± 46 vs. 81 ± 20 nM). Discussion These data show that the optical AP recorded ratiometrically using di-8-ANEPPS from a single ventricular myocyte accurately follows the action potential morphology. This technique can be used to estimate the AP prolonging effects of a compound, although di-8-ANEPPS itself prolongs APD 90. Optical dyes require less technical skills and are less invasive than conventional electrophysiological techniques and, when coupled to ventricular myocytes, decreases animal usage and facilitates higher throughput assays.

Comparative Pharmacology of Guinea Pig Cardiac Myocyte and Cloned hERG (IKr) Channel

This study used whole-cell, patch clamp techniques on isolated guinea pig ventricular myocytes and HEK293 cells expressing cloned human ether-a-go-go-related gene (hERG) to examine the action of drugs causing QT interval prolongation and torsades de pointes (TdP) in man. Similarities and important differences in drug actions on cardiac myocytes and cloned hERG I(Kr) channels were established. Qualitative actions of the drugs on cardiac myocytes corresponded with results obtained from Purkinje fibers and measurement of QT interval prolongation in animal and human telemetry studies. Adult guinea pig ventricular myocytes were isolated by enzymatic digestion. Cells were continuously perfused with Tyrode's solution at 33-35 degrees C. Recordings were made using the whole-cell, patch clamp technique. Action potentials (APs) were elicited under current clamp. Voltage clamp was used to study the effect of drugs on I(Kr) (rapidly activating delayed rectifier potassium current), I(Na) (sodium current), and I(Ca) (L-type calcium current). Dofetilide increased the myocyte action potential duration (APD) in a concentration-dependent manner, with a pIC50 of 7.3. Dofetilide 1 microM elicited early afterdepolarizations (EADs) but had little affect on I(Ca) or I(Na). E-4031 increased APD in a concentration-dependent manner, with a pIC50 of 7.2. In contrast, 10 microM loratadine, desloratadine, and cetirizine had little effect on APD or I(Kr). Interestingly, cisapride displayed a biphasic effect on myocyte APD and inhibited I(Ca) at 1 microM. Even at this high concentration, cisapride did not elicit EADs. A number of AstraZeneca compounds were tested on cardiac myocytes, revealing a mixture of drug actions that were not observed in hERG currents in HEK293 cells. One compound, particularly AR-C0X, was a potent blocker of myocyte AP (pIC50 of 8.4). AR-C0X also elicited EADs in cardiac myocytes. The potencies of the same set of drugs on the cloned hERG channel also were assessed. The pIC50 values for dofetilide, E-4031, terfenadine, loratadine, desloratadine, and cetirizine were 6.8, 7.1, 7.3, 5.1, 5.2, and <4, respectively. Elevation of temperature from 22 to 35 degrees C significantly enhanced the current kinetics and amplitudes of hERG currents and resulted in approximately fivefold increase in E-4031 potency. Our study demonstrates the advantages of cardiac myocytes over heterologously expressed hERG channels in predicting QT interval prolongation and TdP in man. The potencies of some drugs in cardiac myocytes were similar to hERG, but only myocytes were able to detect important changes in APD characteristics and display EADs predictive of arrhythmia development. We observed similar qualitative drug profiles in cardiac myocytes, dog Purkinje fibers, and animal and human telemetry studies. Therefore, isolated native cardiac myocytes are a better predictor of drug-induced QT prolongation and TdP than heterologously expressed hERG channels. Isolated cardiac myocytes, when used with high-throughput patch clamp instruments, may have an important role in screening potential cardiotoxic compounds in the early phase of drug discovery. This would significantly reduce the attrition rate of drugs entering preclinical and/or clinical development. The current kinetics and amplitudes of the cloned hERG channel were profoundly affected by temperature, significantly altering the potency of one drug (E-4031). This finding cautions against routine drug testing at room temperature compared to physiologic temperature when using the cloned hERG channel.

Na+/Ca2+ exchanger plays a key role in inducing apoptosis after hypoxia in cultured guinea pig ventricular myocytes.

Altered Na(+)/Ca(2+) exchanger (NCX) protein expression or activity is thought to contribute to various aspects of cardiac pathology. In guinea pig ventricular myocytes, NCX-mediated Ca(2+) entry is almost entirely responsible for Ca(2+) overload during hypoxia-reoxygenation. Because Ca(2+) overload is a common initiator of apoptosis, the purpose of this study was to test the hypotheses that NCX activity is critically involved in initiating apoptosis after hypoxia-reoxygenation and that hypoxia-reoxygenation-induced apoptosis can be modulated by changes in NCX protein expression or activity. An NCX antisense oligonucleotide was used to reduce NCX protein expression in cultured adult guinea pig ventricular myocytes. Caspase-3 activation and cytochrome c release were used as markers of apoptosis. Hypoxia-reoxygenation-induced apoptosis was significantly decreased in antisense-treated myocytes compared with untreated control or nonsense-treated myocytes. Pretreatment of cultured myocytes for 24 h with either endothelin-1 or phenylephrine was found to increase both NCX protein expression and evoked NCX activity as well as enhance hypoxia-reoxygenation-induced apoptosis. Control experiments demonstrated that endothelin-1 and phenylephrine did not induce apoptosis on their own nor did they enhance the apoptotic response in a model of Ca(2+)-dependent, NCX-independent apoptosis. Additional control experiments demonstrated that the NCX antisense oligonucleotide did not alter the apoptotic response of myocytes to either H(2)O(2) or isoproterenol. Taken together, these data suggest that the NCX has a critical and specific role in the initiation of apoptosis after hypoxia-reoxygenation in guinea pig myocytes and that hypoxia-reoxygenation-induced apoptosis is quite sensitive to changes in NCX activity.

Microtubules mobility affects the modulation of L-type ICa by muscarinic and β-adrenergic agonists in guinea-pig cardiac myocytes

To investigate the interaction of cytoskeleton with the receptor modulation of ionic currents, we studied the effect of muscarinic and β-adrenergic stimulation in adult guinea-pig ventricular cardiac myocytes treated with paclitaxel and colchicine, two drugs that respectively stabilize or destabilize microtubules. We observed that the stabilization of microtubules with paclitaxel (1 μM for 1–4 h) did not markedly affect either the kinetics of I Ca, or the stimulatory effect of isoproterenol (Iso, 1 μM); however paclitaxel significantly blunted the response to carbachol (CCh, 1 μM). In agreement with the electrophysiological measurements, Iso induced a similar enhancement of intracellular cAMP levels in both control and paclitaxel-treated cells, while the response to CCh 1 μM was significantly reduced in paclitaxel-treated cells. The reduction of muscarinic response induced by paclitaxel was also evident in atrial cells, in which the stimulation of I KACh by CCh 1 μM was reduced to about 10%. Compared to the muscarinic response, paclitaxel did not have significant effect on the purinergic (adenosine 1–10 μM) modulation of I Ca. In contrast to paclitaxel, in colchicine-treated cells, I Ca was not enhanced by β-adrenergic stimulation, but instead reduced by CCh, even in the absence of previous stimulation. In conclusion, our data suggest that microtubule stabilization significantly affects the muscarinic modulation of I Ca, by interacting with the receptor or the G-protein rather than on the intracellular signaling cascade.

G protein modulates thyroid hormone-induced Na(+) channel activation in ventricular myocytes.

To evaluate the effects of liothyronine (3,5,3'-triiodo-L-thyronine, T(3)) on Na(+) channel current (I(Na)) properties, I(Na) was recorded in adult guinea pig ventricular myocytes. T(3) (1 nM) acutely increased whole cell I(Na) and shifted the steady-state I(Na) inactivation curve dose dependently. When the pipette solution contained 100 microM GTP or GTPgammaS, the effect of T(3) on the whole cell I(Na) was increased two- to threefold. This effect was almost completely abolished by pertussis toxin preincubation. In the cell-attached patch, T(3) increased the open probability of single I(Na) by reducing the null probability. In the inside-out patch, T(3) effect was 10 times faster than that in whole cell and cell-attached patches while GTPgammaS was present and could be completely washed out. T(3) alone slightly increased the channel open probability by increasing the closed state to open state rate constant (k(CO)) and reducing the null probability. GTPgammaS exposure only increased the number of functional channels. T(3) and GTPgammaS synergistically enhanced the channel open probability 5.8 +/- 0.5-fold by increasing k(CO), decreasing the open state to absorbing inactivated state rate constant, and greatly reducing the null probability. These results demonstrate that T(3) acts on the cytosolic side of the membrane and acutely activates I(Na). Pertussis toxin-sensitive G protein modulation greatly magnifies the T(3) effects on the channel kinetics and null probability, thereby increasing the channel open probability.