Normal Ventricular Myocytes Research Articles

A Compartmentalized Mathematical Model of the β1- and β2-Adrenergic Signaling Systems in Ventricular Myocytes from Mouse in Heart Failure.

Mouse models of heart failure are extensively used to research human cardiovascular diseases. In particular, one of the most common is the mouse model of heart failure resulting from transverse aortic constriction (TAC). Despite this, there are no comprehensive compartmentalized mathematical models that describe the complex behavior of the action potential, [Ca2+]i transients, and their regulation by β1- and β2-adrenergic signaling systems in failing mouse myocytes. In this paper, we develop a novel compartmentalized mathematical model of failing mouse ventricular myocytes after TAC procedure. The model describes well the cell geometry, action potentials, [Ca2+]i transients, and β1- and β2-adrenergic signaling in the failing cells. Simulation results obtained with the failing cell model are compared to those from the normal ventricular myocytes. Exploration of the model reveals the sarcoplasmic reticulum Ca2+ load mechanisms in failing ventricular myocytes. We also show a larger susceptibility of the failing myocytes to early and delayed afterdepolarizations and to a proarrhythmic behavior of Ca2+ dynamics upon stimulation with isoproterenol. The mechanisms of the proarrhythmic behavior suppression is investigated and sensitivity analysis is performed. The developed model can explain the existing experimental data on failing mouse ventricular myocytes and make experimentally testable predictions of a failing myocyte's behavior.

Mechanisms of phase-3 early afterdepolarizations and triggered activities in ventricular myocyte models.

Early afterdepolarizations (EADs) are abnormal depolarizations during the repolarizing phase of the action potential, which are associated with cardiac arrhythmogenesis. EADs are classified into phase‐2 and phase‐3 EADs. Phase‐2 EADs occur during phase 2 of the action potential, with takeoff potentials typically above −40 mV. Phase‐3 EADs occur during phase 3 of the action potential, with takeoff potential between −70 and −50 mV. Since the amplitude of phase‐3 EADs can be as large as that of a regular action potential, they are also called triggered activities (TAs). This also makes phase‐3 EADs and TAs much more arrhythmogenic than phase‐2 EADs since they can propagate easily in tissue. Although phase‐2 EADs have been widely observed, phase‐3 EADs and TAs have been rarely demonstrated in isolated ventricular myocytes. Here we carry out computer simulations of three widely used ventricular action potential models to investigate the mechanisms of phase‐3 EADs and TAs. We show that when the T‐type Ca2+ current (ICa,T) is absent (e.g., in normal ventricular myocytes), besides the requirement of increasing inward currents and reducing outward currents as for phase‐2 EADs, the occurrence of phase‐3 EADs and TAs requires a substantially large increase of the L‐type Ca2+ current and the slow component of the delayed rectifier K+ current. The presence of ICa,T (e.g., in neonatal and failing ventricular myocytes) can greatly reduce the thresholds of these two currents for phase‐3 EADs and TAs. This implies that ICa,T may play an important role in arrhythmogenesis in cardiac diseases.

Cardiac-specific overexpression of caveolin-3 preserves t-tubular ICa during heart failure in mice.

New Findings What is the central question of this study? What is the cellular basis of the protection conferred on the heart by overexpression of caveolin‐3 (Cav‐3 OE) against many of the features of heart failure normally observed in vivo? What is the main finding and its importance? Cav‐3 overexpression has little effect in normal ventricular myocytes but reduces cellular hypertrophy and preserves t‐tubular I Ca, but not local t‐tubular Ca2+ release, in heart failure induced by pressure overload in mice. Thus Cav‐3 overexpression provides specific but limited protection following induction of heart failure, although other factors disrupt Ca2+ release. Caveolin‐3 (Cav‐3) is an 18 kDa protein that has been implicated in t‐tubule formation and function in cardiac ventricular myocytes. During cardiac hypertrophy and failure, Cav‐3 expression decreases, t‐tubule structure is disrupted and excitation–contraction coupling (ECC) is impaired. Previous work has suggested that Cav‐3 overexpression (OE) is cardio‐protective, but the effect of Cav‐3 OE on these cellular changes is unknown. We therefore investigated whether Cav‐3 OE in mice is protective against the cellular effects of pressure overload induced by 8 weeks’ transverse aortic constriction (TAC). Cav‐3 OE mice developed cardiac dilatation, decreased stroke volume and ejection fraction, and hypertrophy and pulmonary congestion in response to TAC. These changes were accompanied by cellular hypertrophy, a decrease in t‐tubule regularity and density, and impaired local Ca2+ release at the t‐tubules. However, the extent of cardiac and cellular hypertrophy was reduced in Cav‐3 OE compared to WT mice, and t‐tubular Ca2+ current (I Ca) density was maintained. These data suggest that Cav‐3 OE helps prevent hypertrophy and loss of t‐tubular I Ca following TAC, but that other factors disrupt local Ca2+ release.

The Function of Stromal Interaction Molecule 1 (STIM1) in Heart

Stromal interaction molecule 1 (STIM1), an ER/SR Ca2+ sensor, has been well described as an activator of store-operated calcium channel (SOCC) Orai1 or the equivalents, upon SR/ER (Sarco/endoplasmic reticulum) Ca2+ depletion in many cell types. In heart, however, we found that the canonical actions of STIM1 do not apply. For example, STIM1 appears to be organized in puncta and linear segments apparently at the junctional SR (jSR) whether or not the SR is loaded with Ca2+. In normal ventricular myocytes the mobility of STIM1 was not changed by SR Ca2+ load when evaluated by fluorescence recovery after photobleaching (FRAP). Furthermore, in ventricular myocytes, STIM1 function does not depend on Orai1 while in sinoatrial node (SAN) cells, it does. In the ventricular myocytes, we found that STIM1 binds to non-phosphorylated phospholamban, an endogenous inhibitor of SERCA2a (Sarco/endoplasmic reticulum Ca2+ ATPase). By this means increased STIM1 levels activate (or “dis-inhibit”) SERCA2a in ventricular myocytes. In the SAN cells, however, STIM1 is involved in SAN automaticity regulation through Orai1 (and possibly by other means). Consistent with these findings, STIM1 null cells show decreased SR Ca2+ content in SAN and STIM1 overexpressing cells show increased SR Ca2+ content in ventricular myocytes. We therefore conclude that in heart, STIM1 contributes to the regulation of SR Ca2+ content through binding to different partners (including Orai1 and phospholamban) in ventricular myocytes and SAN. The kinetics and more complete physiological and pathophysiological influences of STIM1 on SR Ca2+ content in heart are under active investigation.

Different effects of lysophosphatidic acid on L-type calcium current in neonatal rat ventricular myocytes with and without H2O2 treatment

L-type calcium current (ICa-L) alterations are implicated in various cardiac diseases, and the lysophosphatidic acid (LPA) level increases in several ischemic heart diseases. We investigated the effects of LPA on ICa-L in normal and H2O2-treated neonatal rat ventricular myocytes. LPA treatment (24h) increased the action potential duration (APD) and ICa-L in normal ventricular myocytes, but it decreased these parameters in H2O2-treated myocytes. LPA increased the single-channel open probability of L-type calcium channels in both normal and H2O2-treated myocytes. LPA activated calcineurin (CaN) and induced the cytoplasm-to-nucleus translocation of nuclear factor of activated T-cells (NFAT) in H2O2-treated cardiomyocytes. In H2O2-treated cardiomyocytes, LPA decreased Cav1.2 mRNA and protein expression levels at 4 and 8h, respectively. A CaN inhibitor (FK-506) prevented LPA-induced APD, ICa-L, and Cav1.2 mRNA and protein down-regulation. The LPA-induced ICa-L increase in normal cardiomyocytes was CaN–NFAT signaling-independent, and LPA did not affect Cav1.2 mRNA or protein expression. In conclusion, LPA increases the ICa-L in normal ventricular myocytes by increasing the single-channel open probability of L-type calcium channels, and LPA decreases ICa-L in H2O2-treated cardiomyocytes via the CaN–NFAT pathway.

Acute reversal of phospholamban inhibition facilitates the rhythmic whole-cell propagating calcium waves in isolated ventricular myocytes

Phospholamban (PLB) inhibits the activity of cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a). Phosphorylation of PLB during sympathetic activation reverses SERCA2a inhibition, increasing SR Ca2+ uptake. However, sympathetic activation also modulates multiple other intracellular targets in ventricular myocytes (VMs), making it impossible to determine the specific effects of the reversal of PLB inhibition on the spontaneous SR Ca2+ release. Therefore, it remains unclear how PLB regulates rhythmic activity in VMs.Here, we used the Fab fragment of 2D12, a monoclonal anti-PLB antibody, to test how acute reversal of PLB inhibition affects the spontaneous SR Ca2+ release in normal VMs. Ca2+ sparks and spontaneous Ca2+ waves (SCWs) were recorded in the line-scan mode of confocal microscopy using the Ca2+ fluorescent dye Fluo-4 in isolated permeabilized mouse VMs. Fab, which reverses PLB inhibition, significantly increased the frequency, amplitude, and spatial/temporal spread of Ca2+ sparks in VMs exposed to 50nM free [Ca2+]. At physiological diastolic free [Ca2+] (100–200nM), Fab facilitated the formation of whole-cell propagating SCWs. At higher free [Ca2+], Fab increased the frequency and velocity, but decreased the decay time of the SCWs. cAMP had little additional effect on the frequency or morphology of Ca2+ sparks or SCWs after Fab addition. These findings were complemented by computer simulations. In conclusion, acute reversal of PLB inhibition alone significantly increased the spontaneous SR Ca2+ release, leading to the facilitation and organization of whole-cell propagating SCWs in normal VMs. PLB thus plays a key role in subcellular Ca2+ dynamics and rhythmic activity of VMs.

Stem Cell–Derived Cardiomyocytes as a Tool for Studying Proarrhythmia

Proarrhythmia is a leading cause of hospitalization for adverse drug events, and screening for proarrhythmic potential resulting from QT interval prolongation has become a major component of the development process for any new drug candidate. Conventional wisdom, now embedded in regulatory guidelines from the Food and Drug Administration and other regulatory agencies, holds that drug block of the rapid component of the cardiac delayed-rectifier potassium current, I Kr, is the major proximate mechanism underlying prolongation of cardiac action potentials, an effect that leads to early afterdepolarizations in vitro and is manifest on the surface ECG as QT prolongation and occasionally torsades de pointes.1,2 As a result, regulatory guidance suggests an early look at I Kr blocking potency of new drug entities, even recognizing that other mechanisms, some newly described,3,4 may contribute. Article see p 1677 However, the correlation between I Kr block and QT interval prolongation is imperfect. Multiple mechanisms may underlie this “disconnect,” and a reasonable generalization is that I Kr is but one (albeit important) component of normal cardiac repolarization, and abnormalities in expression or function of other components of repolarization may, in turn, modulate the net effect of I Kr block on action potentials in cardiac myocytes and on QT interval and arrhythmia susceptibility in the whole heart. The logical extension of this line of reasoning is that screening for proarrhythmic potential should include the evaluation of not only drug effects on individual ionic currents such as I Kr contributing to repolarization but also the integrated effect of drugs on action potentials in vitro and on the QT interval in animal models and in humans. In conventional drug development programs, this is commonly accomplished by comparing the effects of a candidate drug molecule on QT intervals in human subjects with …

Regulatory Effect of Connexin 43 on Basal Ca2+ Signaling in Rat Ventricular Myocytes

BackgroundIt has been found that gap junction-associated intracellular Ca2+ [Ca2+]i disturbance contributes to the arrhythmogenesis and hyperconstriction in diseased heart. However, whether functional gaps are also involved in the regulation of normal Ca2+ signaling, in particular the basal [Ca2+]i activities, is unclear.Methods and ResultsGlobal and local Ca2+ signaling and gap permeability were monitored in cultured neonatal rat ventricular myocytes (NRVMs) and freshly isolated mouse ventricular myocytes by Fluo4/AM and Lucifer yellow (LY), respectively. The results showed that inhibition of gap communication by heptanol, Gap 27 and flufenamic acid or interference of connexin 43 (Cx43) with siRNA led to a significant suppression of LY uptake and, importantly, attenuations of global Ca2+ transients and local Ca2+ sparks in monolayer NRVMs and Ca2+ sparks in adult ventricular myocytes. In contrast, overexpression of rat-Cx43 in NRVMs induced enhancements in the above measurements, and so did in HEK293 cells expressing rat Cx43. Additionally, membrane-permeable inositol 1,4,5-trisphosphate (IP3 butyryloxymethyl ester) and phenylephrine, an agonist of adrenergic receptor, could relieve the inhibited Ca2+ signal and LY uptake by gap uncouplers, whereas blockade of IP3 receptor with xestospongin C or 2-aminoethoxydiphenylborate mimicked the effects of gap inhibitors. More importantly, all these gap-associated effects on Ca2+ signaling were also found in single NRVMs that only have hemichannels instead of gap junctions. Further immunostaining/immunoblotting single myocytes with antibody against Cx43 demonstrated apparent increases in membrane labeling of Cx43 and non-junctional Cx43 in overexpressed cells, suggesting functional hemichannels exist and also contribute to the Ca2+ signaling regulation in cardiomyocytes.ConclusionsThese data demonstrate that Cx43-associated gap coupling plays a role in the regulation of resting Ca2+ signaling in normal ventricular myocytes, in which IP3/IP3 receptor coupling is involved. This finding may provide a novel regulatory pathway for mediation of spontaneous global and local Ca2+ activities in cardiomyocytes.

The new HNO donor, 1-nitrosocyclohexyl acetate, increases contractile force in normal and β-adrenergically desensitized ventricular myocytes

Contractile dysfunction and diminished response to β-adrenergic agonists are characteristics for failing hearts. Chemically donated nitroxyl (HNO) improves contractility in failing hearts and thus may have therapeutic potential. Yet, there is a need for pharmacologically suitable donors. In this study we tested whether the pure and long acting HNO donor, 1-nitrosocyclohexyl acetate (NCA), affects contractile force in normal and pathological ventricular myocytes (VMs) as well as in isolated hearts. VMs were isolated from mice either subjected to isoprenaline-infusion (ISO; 30 μg/g per day) or to vehicle (0.9% NaCl) for 5 days. Sarcomere shortening and Ca 2+ transients were simultaneously measured using the IonOptix system. Force of contraction of isolated hearts was measured by a Langendorff-perfusion system. NCA increased peak sarcomere shortening by + 40–200% in a concentration-dependent manner (EC 50 ∼55 μM). Efficacy and potency did not differ between normal and chronic ISO VMs, despite the fact that the latter displayed a markedly diminished inotropic response to acute β-adrenergic stimulation with ISO (1 μM). NCA (60 μM) increased peak sarcomere shortening and Ca 2+ transient amplitude by ∼200% and ∼120%, respectively, suggesting effects on both myofilament Ca 2+ sensitivity and sarcoplasmic reticulum (SR) Ca 2+ cycling. Importantly, NCA did not affect diastolic Ca 2+ or SR Ca 2+ content, as assessed by rapid caffeine application. NCA (45 μM) increased force of contraction by 30% in isolated hearts. In conclusion, NCA increased contractile force in normal and β-adrenergically desensitized VMs as well as in isolated mouse hearts. This profile warrants further investigations of this HNO donor in the context of heart failure.

Late sodium current contributes to diastolic cell Ca2+ accumulation in chronic heart failure

We elucidate the role of late Na+ current (INaL) for diastolic intracellular Ca2+ (DCa) accumulation in chronic heart failure (HF). HF was induced in 19 dogs by multiple coronary artery microembolizations; 6 normal dogs served as control. Ca2+ transients were recorded in field-paced (0.25 or 1.5 Hz) fluo-4-loaded ventricular myocytes (VM). INaL and action potentials were recorded by patch-clamp. Failing VM, but not normal VM, exhibited (1) prolonged action potentials and Ca2+ transients at 0.25 Hz, (2) substantial DCa accumulation at 1.5 Hz, and (3) spontaneous Ca2+ releases, which occurred after 1.5 Hz stimulation trains in ~31% cases. Selective INaL blocker ranolazine (10 microM) or the prototypical Na+ channel blocker tetrodotoxin (2 microM) reversibly improved function of failing VM. The DCa accumulation and the beneficial effect of INaL blockade were reproduced in silico using an excitation-contraction coupling model. We conclude that INaL contributes to diastolic Ca2+ accumulation and spontaneous Ca2+ release in HF.

Different Na+K+-ATPase signal pathways was involved in the increase of [Ca2+]i induced by strophanthidin in normal and failing isolated guinea pig ventricular myocytes

To determine whether different Na+/K+-ATPase signal transduction pathways have positive inotropic effects on normal ventricular myocytes (NC) and failing ventricular myocytes (FC), and are involved in an increase of [Ca2+]i induced by strophanthidin (Str). A guinea pig model of congestive heart failure was made by constricting descending aorta. The left ventricular myocytes were enzymatically isolated. The effects of 25 micromol/L Str with different signal-transducing inhibitors on contractility and the calcium transient of NC or FC from guinea pigs were simultaneously assessed and compared with those in the 25 micromol/L Str-only group by a video-based, motion-edge detection system. Str at 1, 10, and 25 micromol/L in NC and Str at 0.1, 1, 10, and 25 micromol/L) in FC elevated the calcium transient amplitude and increased the positive inotropic effects in a concentration-dependent manner, respectively. At the same concentration, the effects of Str were more potent in FC than in NC. In FC, both the mitogen-activated protein kinase (MAPK) and reactive oxygen species (ROS) signal transduction pathway of Na+/K+-ATPase were involved in the increase of the calcium transient induced by Str, but only activation of the MAPK pathway increased the calcium transient in NC. However, only the ROS pathway was involved in positive inotropic effects both in NC and FC. The present study suggests that Na+/K+-ATPase signaling pathways involved in the inotropic effects of Str in NC and FC are consistent, and Na+/K+-ATPase signaling pathways involved in the increase of [Ca2+]i by Str in NC and FC are different.

The Effect of Amiodarone on K<sup>+</sup> Channel Current in the Normal and Hypertrophied Rat Heart

The basis for the unique effectiveness of amiodarone treatment on cardiac arrhythmias is incompletely understood. We investigated and compared the effects of amiodarone on K + channels of hypertrophied ventricular myocytes with normal ventricular myocytes in rats. The pressure overload hypertrophy models of rats were established by partial ligation of ascending aorta for 4 weeks. Ventricular myocytes were exposed to 1, 10 and 50µmol/l amiodarone, and whole cell patch-clamp technique was used to study the effects of amiodarone on outward currents, such as delayed rectifier outward K + current (IK), slowly activating delayed rectifier outward K + current (IKs), transient outward K + current (Ito )a nd rectifier K + current (IK1). Compared with the control group, the current density of IK ,I K1 and Ito were all decreased in hypertrophied myocytes group. Hyper-concentration of amiodarone (10 and 50µmol/l) inhibited Ito of the control group (37.5% ± 5.8% and 54.3% ± 5.7%) and the hypertrophied myocytes group (9.3% ± 2.3% and 22.8% ± 3.0%), but had effect on IK1 neither the control nor hypertrophied myocytes group. 10µmol/l amiodarone inhibited IKs 23.3% ± 6.2% in the control group and 50.1% ± 9.8% in the hypertrophied myocytes group. Ito and IKs appeared difference affection of amiodarone’s action on both groups. IKs in hypertrophy group was higher than that in control, whereas Ito was lower in hypertrophy group. We concluded amiodarone do inhibit Ito and IK in rat normal and hypertrophied cardiomyocytes. Amiodarone application should be treated difference between hypertrophied heart and normal heart.

Remodeling of Gap Junctions in Ischemic and Nonischemic Forms of Heart Disease

Electrical activation of the myocardium to produce effective pumping of blood depends on the orderly coordinated spatial and temporal transfer of current from one cell to another via gap junctions. Normal ventricular myocytes are extensively coupled by gap junctions and have the capacity to rapidly increase the amount of connexin within gap junction plaques to meet physiological demands for enhanced cell-cell communication. However, myocytes can also rapidly uncouple in response to injury or disease. In general, both acute and chronic forms of heart disease caused by diverse etiologies are associated with changes in the expression of connexins and remodeling of gap junctions. Such remodeling may have both adaptive and maladaptive consequences and contribute to major clinical processes such as heart failure and sudden cardiac death. Our laboratory has investigated mechanisms regulating cell-cell electrical coupling in the heart under physiological and pathophysiological conditions. This review is focused on selected aspects of this work pertaining to changes in coupling in response to acute and chronic ischemic heart disease and in familial cardiomyopathies caused by mutations in genes encoding desmosomal proteins.

Internalization and Dephosphorylation of Connexin43 in Hypertrophied Right Ventricles of Rats With Pulmonary Hypertension

Altered expression and distribution of gap junctions might provide substrates for abnormal conduction and arrhythmogenesis in the heart, but little is known about the regulation of gap junctions under pathological conditions. The organization and phosphorylation state of connexin43 (Cx43) in ventricular hypertrophy will be investigated. Right ventricular (RV) hypertrophy was induced in rats by treatment with monocrotaline. Subcellular Cx43 distribution was assessed by immunoconfocal and electron microscopy. Immunolabeling of Cx43 was confined to the intercalated disks in the normal ventricular myocytes of control rats, but hypertrophied RV cells from monocrotaline-treated rats showed dispersion of Cx43 immunolabeling over the cell surface and in the cytoplasm; cytoplasmic Cx43 was increased by approximately 7-fold (n=15). The Cx43 internalization was confirmed by the double staining of monocrotaline-treated RV tissues for Cx43/wheat germ agglutinin (WGA) and Cx43/zonula occludens protein-1 (ZO-1). Electron microscopy of hypertrophied RVs showed an increase in annular gap junctions immunolabeled with Cx43. Immunoblotting revealed a significant increase in non-phosphorylated Cx43 in hypertrophied RVs (by approximately 5-fold, n=8) without changes in the total amount of Cx43. The accumulation of non-phosphorylated Cx43 in hypertrophied RVs was also recognized by immunoconfocal-microscopy with an isoform-specific antibody. Ventricular hypertrophy is associated with the dephosphorylation of Cx43 and its translocation from the intercalated disks to intracellular pools, suggesting accelerated gap junction degradation.

An Intimate Relationship

We last offered our ideas on the role of reverse EC coupling in the initiation of arrhythmias in 2001.1 At that time we emphasized that spurious Ca2+ release or oscillations in Ca2+ levels could serve as possible triggers for arrhythmias. Since then much has been done to confirm this role for intracellular Ca2+. At that time we did not discuss the role of Ca2+ cycling in the maintenance or conversion of stable tachycardias to VF. However, in consideration of the new data on the role of APD restitution in the initiation of VF (increase in wavebreaks; eg, see Garfinkel et al2), we turn our attention now to the role Ca2+ cycling in the myocyte and its impact on APD restitution relations in the isolated rabbit ventricular cell.3 Goldhaber et al consider the role of Ca2+ using different types of APD restitution protocols to emphasize the dynamic nature of intracellular Ca2+ changes and its subsequent impact on the myocyte APD. In their study APD alternans is coupled to Ca2+ cycling, which in itself is not new since others have observed that APD alternans demonstrates a hystersis and is inhibited with BAPTA-AM buffering.4 Some have suggested that repolarization alternans is more closely associated …

β- and α-adrenergic cross-signaling for L-type Ca current is impaired in transgenic mice with constitutive activation of εPKC

It is well established that β-adrenoceptor stimulation activates PKA and α 1-adrenoceptor stimulation activates PKC. In normal ventricular myocytes, acute activation of α 1-adrenoceptors inhibits β-adrenoceptor stimulated L-type Ca current ( I Ca-L) and direct activation of εPKC leads to I Ca-L inhibition. Because increased PKC activity has been observed chronically in in vivo setting such as failing human heart, we hypothesized that chronic in vivo activation of εPKC alters I Ca-L and its response to adrenergic stimulation. Therefore, we investigated the interaction between β- and α 1-adrenoceptors vis-à-vis I Ca-L in myocytes from transgenic mice (TG) with cardiac specific constitutive activation of εPKC (εPKC agonist). Whole-cell I Ca-L was recorded from εPKC agonist TG mice and age-matched non-TG (NTG) littermates under: (1) basal condition, (2) β-adrenoceptor agonist, isoproterenol (ISO), and (3) ISO plus α 1-adrenoceptor agonist, methoxamine. The present results are the first to demonstrate that chronic in vivo activation of εPKC leads to reduced basal I Ca-L density. β-adrenoceptor activation of I Ca-L is blunted in εPKC agonist TG mice. α-adrenoceptor cross-talk with β-adrenoceptor signaling pathways vis-à-vis L-type Ca channels is impaired in εPKC agonist TG mice. The diminished response to ISO and methoxamine suggests a protective feedback regulatory mechanism in εPKC agonist TG mice and could be vital in the settings of excessive release of catecholamines during heart failure.

AT 2 , Judgment Day

As reviewed recently in these pages,1 the vasoconstrictor angiotensin II (Ang II) is one of the circulating peptide growth factors, and clinically the most relevant, that fulfill many of Koch’s postulates for having additional effects locally in the myocardium. These criteria include local production of Ang II within the heart, its upregulation there by hypertrophic signals, its release from cardiac cells after mechanical load, the presence of its receptors on the surface of cardiac cells, and the responsiveness of both cardiac myocytes and cardiac fibroblasts to provocation by this peptide.1,2 This local circuit of Ang II production, release, binding, and transduction within the myocardium itself is the most cogent explanation for the effectiveness of ACE inhibitors in heart failure, even at doses that do not alter loading conditions.1,2 See p 346 Three related receptors exist for Ang II: AT1a, AT1b, and AT2, which communicate with a complex cell-wide web of signaling cascades through GTP-binding (G) proteins as the proximal transducer. The operation of a local circuit for Ang II within the myocardium poses two questions, which have attracted much recent experimental effort, and an even larger share of controversy. First, given that multiple receptors exist for Ang II, which type or types are responsible for pathobiology provoked by the local circuit? Second, given that cardiac muscle and nonmuscle cells are both potential targets for Ang II, what is the respective role of each? Genetic models that altogether lack the receptors individually provide an invaluable experimental tool beyond what is achievable by use of pharmacological inhibitors alone. Surprisingly, the Ang II receptor …

Sarcoplasmic reticulum Ca(2+) release causes myocyte depolarization. Underlying mechanism and threshold for triggered action potentials.

Spontaneous sarcoplasmic reticulum (SR) Ca(2+) release causes delayed afterdepolarizations (DADs) via Ca(2+)-induced transient inward currents (I:(ti)). However, no quantitative data exists regarding (1) Ca(2+) dependence of DADs, (2) Ca(2+) required to depolarize the cell to threshold and trigger an action potential (AP), or (3) relative contributions of Ca(2+)-activated currents to DADs. To address these points, we evoked SR Ca(2+) release by rapid application of caffeine in indo 1-AM-loaded rabbit ventricular myocytes and measured caffeine-induced DADs (cDADs) with whole-cell current clamp. The SR Ca(2+) load of the myocyte was varied by different AP frequencies. The cDAD amplitude doubled for every 88+/-8 nmol/L of Delta[Ca(2+)](i) (simple exponential), and the Delta[Ca(2+)](i) threshold of 424+/-58 nmol/L was sufficient to trigger an AP. Blocking Na(+)-Ca(2+) exchange current (I(Na/Ca)) by removal of [Na](o) and [Ca(2+)](o) (or with 5 mmol/L Ni(2+)) reduced cDADs by >90%, for the same Delta[Ca(2+)](i). In contrast, blockade of Ca(2+)-activated Cl(-) current (I(Cl(Ca))) with 50 micromol/L niflumate did not significantly alter cDADs. We conclude that DADs are almost entirely due to I(Na/Ca), not I(Cl(Ca)) or Ca(2+)-activated nonselective cation current. To trigger an AP requires 30 to 40 micromol/L cytosolic Ca(2+) or a [Ca(2+)](i) transient of 424 nmol/L. Current injection, simulating I(ti)s with different time courses, revealed that faster I:(ti)s require less charge for AP triggering. Given that spontaneous SR Ca(2+) release occurs in waves, which are slower than cDADs or fast I(ti)s, the true Delta[Ca(2+)](i) threshold for AP activation may be approximately 3-fold higher in normal myocytes. This provides a safety margin against arrhythmia in normal ventricular myocytes.

Low insulin and high glucose induce abnormal relaxation in cultured adult rat ventricular myocytes.

One of the most prominent myocardial defects associated with diabetes is abnormal diastole. We have recently reported that this dysfunction involves prolonged relaxation (relengthening) in isolated ventricular myocytes that occurs within days after the induction of diabetes. The present study was designed to evaluate the role of insulin and glucose int he etiology of this dysfunction with a serum-free myocyte culture system. Adult rat ventricular myocytes were cultured for 1-4 days in a "diabetic-like" medium containing five times less insulin and approximately five times more glucose than in our normal medium. Mechanical properties and Ca2+ transients (fura 2) were evaluated with a high-resolution (120-Hz) video-based edge-detection/spectro-fluormetric system. The cells were field stimulated to contract at slow and physiologically relevant rates, and indexes of contraction and relaxation were evaluated. Relengthening was markedly longer in myocytes cultured in low-insulin-high-glucose (LIHG) medium compared with those in normal medium, whereas contraction was unaffected. Intracellular Ca2+ transients showed slower rates of decay in myocytes cultured in LIHG medium. These data demonstrate that maintaining normal ventricular myocytes in an LIHG environment prolongs relaxation in a manner similar to the effects of in vivo diabetes. Furthermore, the abnormal relaxation is inducible in 1 day, suggesting rapid alterations in processes regulating relaxation, which likely include impaired Ca2+ sequestration and/or extrusion.

Effects of halothane and ketamine on activation and inactivation of myocardial calcium current.

We evaluated the effects of clinically relevant concentrations of halothane (1%) and ketamine (10(-4) M) on activation, inactivation, and recovery from inactivation of voltage-gated sarcolemmal calcium current (ICa) in single guinea pig ventricular myocytes, using the whole cell voltage clamp. Both anesthetics had qualitatively similar effects. The potential at half-activation was shifted from -18 to -23 mV for halothane (p < 0.03) and from -17 to -21 mV for ketamine (p = 0.005). There was no change in the slope of the activation curve for either anesthetic. The potential at half-inactivation was shifted from -29 to -40 mV with exposure to halothane (p < 0.001) and from -27 to -33 mV (p < 0.001) with exposure to ketamine. There was no change in the slope of the inactivation curve with either agent. The changes in time constant of recovery from steady-state inactivation with halothane did not reach statistical significance (178 vs. 207 ms, p = 0.20) and was significantly prolonged with exposure to ketamine (106 vs. 157 ms, p = 0.005). The two anesthetics show parallel shifts in activation, inactivation, and recovery from inactivation of ICa in ventricular myocardial cells. These findings in normal ventricular myocytes may help interpret the interactions of these anesthetics with other types of heart muscle, such as ischemic and immature myocardium.