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Cardiac-specific overexpression of wild-type [[alpha].sub.1B]-adrenergic receptors ([[alpha].sub.1B]-AR) in mice predisposes to dilated cardiomyopathy and sudden death. Although [alpha]-adrenergic stimulation is thought to contribute to induction of arrhythmias in heart failure, the electrophysiological consequences of chronic [[alpha].sub.1]-adrenergic activation have not been clearly defined. Thus we characterized ventricular repolarization and monitored incidence of spontaneous arrhythmias in end-stage heart failure [[alpha].sub.1B]-AR mice (9-12 mo) and younger [[alpha].sub.1B]-AR mice (2-3 too) that do not present signs of heart failure. Compared with aged-matched controls, the corrected QT interval was 34% longer in the 9-to 12-mo [[alpha].sub.1B]-AR mice, and the action potential durations were also significantly prolonged in these mice. These changes were associated with a decrease in the density of the outward [K.sup.+] currents, [Ca.sup.2+]-independent transient, ultrarapid delayed rectifier, and steady state (at +30 mV, reduction of 68, 64, and 41%, respectively), and underlying [K.sup.+] channel expression. Electrocardiogram (ECG) recordings revealed that older [[alpha].sub.1B]-AR mice exhibited spontaneous ventricular arrhythmias. The alterations in repolarization can contribute to these rhythm abnormalities and are likely caused by chronic [[alpha].sub.1B]-AR activity. Additional data obtained in 2- to 3-mo [[alpha].sub.1B]-AR mice clearly showed that electrical remodeling was already observed in younger transgenic animals. However, it appeared to be slightly less pronounced than in older mice. These results suggest that there are two waves of remodeling: one due to chronic [[alpha].sub.1B]-AR activity, and a second due to heart failure. Taken together, these data provide strong evidence for a pathological role of chronic [[alpha].sub.1B]-AR activity in the development of repolarization defects and ventricular arrhythmias. mouse ventricle; arrhythmias; [K.sup.+] currents

We have previously demonstrated that in a rat model of trauma-hemorrhage (T-H), glucosamine administration during resuscitation improved cardiac function, reduced circulating levels of inflammatory cytokines, and increased tissue levels of O-linked N-acetylglucosamine (O-GlcNAc) on proteins. The mechanism(s) by which glucosamine mediated its protective effect were not determined; therefore, the goal of this study was to test the hypothesis that glucosamine treatment attenuated the activation of the nuclear factor-[kappa]B (NF-[kappa]B) signaling pathway in the heart via an increase in protein O-GlcNAc levels. Fasted male rats were subjected to T-H by bleeding to a mean arterial blood pressure of 40 mmHg for 90 min followed by resuscitation. Glucosamine treatment during resuscitation significantly attenuated the T-H-induced increase in cardiac levels of TNF-[alpha] and IL-6 mRNA, I[kappa]B-[alpha] phosphorylation, NF-[kappa]B, NF-[kappa]B DNA binding activity, ICAM-1, and MPO activity. LPS (2 [micro]g/ml) increased the levels of I[kappa]B-[alpha] phosphorylation, TNF-[alpha], ICAM-1, and NF-[kappa]B in primary cultured cardiomyocytes, which was significantly attenuated by glucosamine treatment and overexpression of O-GlcNAc transferase; both interventions also significantly increased O-GlcNAc levels. In contrast, the transfection of neonatal rat ventricular myocytes with OGT small-interfering RNA decreased O-GlcNAc transferase and O-GlcNAc levels and enhanced the LPS-induced increase in I[kappa]B-[alpha] phosphorylation. Glucosamine treatment of macrophage cell line RAW 264.7 also increased O-GIcNAc levels and attenuated the LPS-induced activation of NF-[kappa]B. These results demonstrate that the modulation of O-GlcNAc levels alters the response of cardiomyocytes to the activation of the NF-[kappa]B pathway, which may contribute to the glucosamine-mediated improvement in cardiac function following hemorrhagic shock. nuclear factor-[kappa]B; cytokines; neonatal rat ventricular myocytes; O-linked N-acetylglucosamine

Cardiac remodeling is thought to be the major cause of chronic heart dysfunction after myocardial infarction (MI). However, molecules involved in this process have not been thoroughly elucidated. In this study we investigated the long-term effects of the growth factor midkine (MK) in cardiac remodeling after MI. MI was produced by ligation of the left coronary artery. MK expression was progressively increased after MI in wild-type mice, and MK-deficient mice showed a higher mortality. Exogenous MK improved survival and ameliorated left ventricular dysfunction and fibrosis not only of MK-deficient mice but also of wild-type mice. Angiogenesis in the peri-infarct zone was also enhanced. These in vivo changes induced by exogenous MK were associated with the activation of phosphatidylinositol 3-kinase (PI3K)/Akt and MAPKs (ERK, p38) and the expression of syndecans in the left ventricular tissue. In vitro experiments using human umbilical vein endothelial cells confirmed the potent angiogenic action of MK via the PI3K/Akt pathway. These results suggest that MK prevents the cardiac remodeling after MI and improves the survival most likely through an enhancement of angiogenesis. MK application could be a new therapeutic strategy for the treatment of ischemic heart failure. growth factor

The time-varying elastance concept provides a comprehensive description of the intrinsic mechanical properties of the left ventricle that are assumed to be load independent. Based on pressure-volume measurements obtained with combined pressure conductance catheterization in six open-chest anesthetized sheep, we show that the time to reach end systole (defined as maximal elastance) is progressively prolonged for increasing ventricle pressures, which challenges the original (load-independent) time-varying elastance concept. Therefore, we developed a method that takes into account load dependency by normalization of time course of the four cardiac phases (isovolumic contraction, ejection, isovolumic relaxation, filling) individually. With this normalization, isophase lines are obtained that connect points in pressurevolume loops of different beats at the same relative time in each of the four cardiac phases, instead of isochrones that share points at the same time in a cardiac cycle. The results demonstrate that pressure curves can be predicted with higher accuracy, if elastance curves are estimated using isophase lines instead of using isochrones [root-meansquare error (RMSE): 3.8 [+ or -] 1.0 vs. 14.0 [+ or -] 7.4 mmHg (P < 0.001), and variance accounted for (VAF): 94.8 + 1.3 vs. 78.6 [+ or -] 14.8% (P < 0.001)]. Similar results were found when the intercept volume was assumed to be time varying [RMSE: 1.7 [+ or -] 0.3 vs. 13.4 [+ or -] 7.4 mmHg (P < 0.001), and VAF: 97.4 + 0.5 vs. 81.8 [+ or -] 15.5% (P < 0.001)]. In conclusion, phase-dependent time normalization reduces cardiac load dependency of timing and increases accuracy in estimating time-varying elastance. ventricular loading; cardiac time course; isochrones; isophase lines

Tricyclic antidepressants such as amitriptyline (AMT) have been reported to have adverse side effects on cardiac performance. AMT effects on Ca handling in ventricular myocytes, however, are not well understood. Therefore, we investigated AMT action on sarcoplasmic reticulum (SR) Ca release in ventricular myocytes, ryanodine receptor (RyR) activity, and Ca uptake by SR microsomes. In permeabilized myocytes, AMT transiently increased free luminal Ca concentration ([Ca]) followed by marked depletion. AMT (10 [micro]M) caused a rapid and a transient increase of Ca spark frequency, followed by a significant suppression of spark activity. The latter was associated with a decrease of Ca spark amplitude and SR Ca load to 87 and 60%, respectively. AMT (10 [micro]M) completely abolished propagation of spontaneous Ca waves. Higher concentrations of AMT (0.1-1 mM) evoked SR Ca release reminiscent of the effect of caffeine (20 mM) and caused almost complete depletion of SR Ca content. Studies on single calsequestrin-free RyR channels revealed that AMT increased the mean open time and open probability ([P.sub.o]) in a dose-dependent fashion (dissociation constant = 4.2 [micro]M). High concentrations of AMT (>25 [micro]M) evoked frequent long openings with [P.sub.o] reaching very high levels (>0.70). In studies with cardiac SR microsomes, AMT slowed the rate of ATP-dependent Ca uptake. We conclude that AMT affects SR Ca handling in ventricular myocytes by multiple mechanisms, including direct stimulation of RyRs and inhibition of SR Ca uptake. These effects could contribute to AMT cardiotoxicity. heart; excitation-contraction coupling; ryanodine receptor; calcium sparks; calsequestrin; antidepressants

Clenbuterol, a compound classified as a [[beta].sub.2]-adrenoceptor (AR) agonist, has been employed in combination with left ventricular assist devices (LVADs) to treat patients with severe heart failure. Previous studies have shown that chronic administration of clenbuterol affects cardiac excitation-contraction coupling. However, the acute effects of clenbuterol and the signaling pathway involved remain undefined. We investigated the acute effects of clenbuterol on isolated ventricular myocyte sarcomere shortening, [Ca.sup.2+] transients, and L-type [Ca.sup.2+] current and compared these effects to two other clinically used [[beta].sub.2]-AR agonists: fenoterol and salbutamol. Clenbuterol (30 [micro]M) produced a negative inotropic response, whereas fenoterol showed a positive inotropic response. Salbutamol had no significant effects. Clenbuterol reduced [Ca.sup.2+] transient amplitude and L-type [Ca.sup.2+] current. Selective [[beta].sub.1]-AR blockade did not affect the action of clenbuterol on sarcomere shortening but significantly reduced contractility in the presence of fenoterol and salbutamol (P < 0.05). Incubation with 2 [micro]g/ml pertussis toxin significantly reduced the negative inotropic effects of 30 [micro]M clenbuterol. In addition, overexpression of inhibitory G protein ([G.sub.i]) by adenoviral transfection induced a stronger clenbuterol-mediated negative inotropic effect, suggesting the involvement of the [G.sub.i] protein. We conclude that clenbuterol does not increase and, at high concentrations, significantly depresses contractility of isolated ventricular myocytes, an effect not seen with fenoterol or salbutamol. In its negative inotropism, clenbuterol predominantly acts through [G.sub.i], and the consequent downstream signaling pathways activation may explain the beneficial effects observed during chronic administration of clenbuterol in patients treated with LVADs. [beta]-adrenoceptor; G proteins; heart failure; cardiac myocytes

To fully characterize the mechanisms of defibrillation, it is necessary to understand the response, within the three-dimensional (3D) volume of the ventricles, to shocks given in diastole. Studies that have examined diastolic responses conducted measurements on the epicardium or on a transmural surface of the left ventricular (LV) wall only. The goal of this study was to use optical imaging experiments and 3D bidomain simulations, including a model of optical mapping, to ascertain the shock-induced virtual electrode and activation patterns throughout the rabbit ventricles following diastolic shocks. We tested the hypothesis that the locations of shock-induced regions of hyperpolarization govern the different diastolic activation patterns for shocks of reversed polarity. In model and experiment, uniform-field monophasic shocks of reversed polarities (cathode over the right ventricle is RV-, reverse polarity is LV-) were applied to the ventricles in diastole. Experiments and simulations revealed that RV- shocks resulted in longer activation times compared with LV- shocks of the same strength. 3D simulations demonstrated that RV- shocks induced a greater volume of hyperpolarization at shock end compared with LV- shocks; most of these hyperpolarized regions were located in the LV. The results of this study indicate that ventricular geometry plays an important role in both the location and size of the shock-induced virtual anodes that determine activation delay during the shock and subsequently affect shock-induced propagation. If regions of hyperpolarization that develop during the shock are sufficiently large, activation delay may persist until shock end. virtual electrodes; optical mapping; electric shocks; defibrillation

Repeated restraint stress (RRS; 3 h of restraint on 3 consecutive days) in rodents produces temporary hypophagia, but a long-term downregulation of body weight. The mild stress (MS) of an intraperitoneal injection of saline and housing in a novel room for 2 h also inhibits food intake and weight gain, but the effects are smaller than for RRS. Previous exposure to RRS exaggerates hypophagia, glucocorticoid release, and anxiety-type behavior caused by MS. Here we tested the involvement of brain stem corticotrophin-releasing factor receptors (CRFR) in mediating energetic and glucocorticoid responses to RRS or MS and in promoting stress hyperresponsiveness in RRS rats. Administration of 1.3 nmol [alpha]hCRF(9-41), a nonspecific CRFR antagonist, exaggerated hypophagia and weight loss in both RRS and MS rats, whereas 0.26 nmol had no effect in RRS or MS rats. In contrast, 2 nmol of the nonspecific antagonist astressin had no effect on weight loss or hypersensitivity to subsequent MS in RRS rats, but blocked weight loss and inhibition of food intake caused by MS alone. MS rats infused with 3 nmol antisauvagine-30, a CRFR2 antagonist, did not lose weight in the 48 h after MS, but 0.3 nmol did not prevent weight loss in MS rats. These data suggest that inhibition of food intake and weight loss induced by RRS or by MS involve different pathways, with hindbrain CRFR mediating the effect of MS on body weight and food intake. Hindbrain CRFR do not appear to influence stressinduced corticosterone release in RRS rats. brain stem; [alpha]hCRF(9-41); astressin; antisauvagine-30; rats

The objective of this study was to test the hypothesis that the mechanism mediating left ventricular (LV) dysfunction in the aging rat heart involves, in part, changes in cardiac cytoskeletal components. Our results show that there were no significant differences in heart rate, LV pressure, or LV diameter between conscious, instrumented young [5.9 [+ or -] 0.3 mo (n = 9)] and old rats [30.6 [+ or -] 0.1 mo (n = 10)]. However, the first derivative of LV pressure (LV dP/dt) was reduced (8,309 [+ or -] 790 vs. 11,106 [+ or -] 555 mmHg/s, P < 0.05) and isovolumic relaxation time ([tau]) was increased (8.7 [+ or -] 0.7 vs. 6.3 [+ or -] 0.6 ms, P < 0.05) in old vs. young rats, respectively. The differences in baseline LV function in young and old rats, which were modest, were accentuated after [beta]-adrenergic receptor stimulation with dobutamine (20 p,g/kg), which increased LV dP/dt by 170 [+ or -] 9% in young rats, significantly more (P < 0.05) than observed in old rats (115 [+ or -] 5%). Volume loading in anesthetized rats demonstrated significantly impaired LV compliance in old rats, as measured by the LV end-diastolic pressure and dimension relationship. In old rat hearts, there was a significant (P < 0.05) increase in the percentage of LV collagen (2.4 [+ or -] 0.2 vs. 1.3 [+ or -] 0.2%), [alpha]-tubulin (92%), and [beta]-tubulin (2.3-fold), whereas intact desmin decreased by 51%. Thus the cardiomyopathy of aging in old, conscious rats may be due not only to increases in collagen but also to alterations in cytoskeletal proteins. aging cardiomyopathy; left ventricular systolic function; left ventricular diastolic function; tubulin

The impact of ischemic preconditioning (IPC) on contraction, [Ca.sup.2+] homeostasis, and cell survival was compared in isolated ventricular myocytes from young adult (~3 mo) and aged (~24 mo) male Fischer-344 rats. Myocytes were field stimulated at 4 Hz (37[degrees]C). Contraction (edge detector) and intracellular [Ca.sup.2+] (fura-2) were measured simultaneously. Viability was assessed with trypan blue. All cells were exposed to 30 min of simulated ischemia followed by reperfusion. Some cells were preconditioned by exposure to 5 min of simulated ischemia before prolonged ischemia. Pretreatment with IPC abolished postischemic contractile depression, inhibited diastolic contracture, and increased [Ca.sup.2+] transient amplitudes in reperfusion in young adult and aged cells. IPC did not affect the modest rise in diastolic [Ca.sup.2+] in ischemia in young adult myocytes. However, IPC abolished the marked rise in diastolic [Ca.sup.2+] observed in ischemia and early reperfusion in aged myocytes. IPC also suppressed mechanical alternans in ischemia in aged cells, but younger myocytes showed little evidence of mechanical alternans whether or not cells were preconditioned. IPC markedly improved cell viability in reperfusion in young adult but not aged cells. These results suggest that IPC augments the recovery of contractile function in reperfusion by increasing [Ca.sup.2+] transient amplitudes in ventricular myocytes from young adult and aged rats. IPC reduced diastolic [Ca.sup.2+] accumulation in ischemia in aged myocytes, which may diminish the severity of mechanical alternans in aged cells. Nonetheless, the efficacy of IPC is compromised in aging, as IPC did not improve survival of aged myocytes exposed to ischemia and reperfusion. cardioprotection; reperfusion; mechanical alternans; senescence

Therapies for heart disease are based largely on our understanding of the adult myocardium. The dramatic differences in action potential (AP) shape between neonatal and adult cardiac myocytes, however, indicate that a different set of molecular interactions in neonatal myocytes necessitates different treatment for newborns. Computational modeling is useful for synthesizing data to determine how interactions between components lead to systems-level behavior, but this technique has not been used extensively to study neonatal heart cell function. We created a mathematical model of the neonatal (day 1) mouse myocyte by modifying, on the basis of experimental data, the densities and/or formulations of ion transport mechanisms in an adult cell model. The new model reproduces the characteristic AP shape of neonatal cells, with a brief plateau phase and longer duration than the adult (action potential duration at 80% repolarization = 60.1 vs. 12.6 ms). The simulation results are consistent with experimental data, including 1) decreased density and altered inactivation of transient outward [K.sup.+] currents, 2) increased delayed rectifier [K.sup.+] currents, 3) [Ca.sup.2+] entry through T-type as well as L-type [Ca.sup.2+] channels, 4) increased [Ca.sup.2+] influx through [Na.sup.+]/[Ca.sup.2+] exchange, and 5) [Ca.sup.2+] transients resulting from transmembrane [Ca.sup.2+] entry rather than release from the sarcoplasmic reticulum (SR). Simulations performed with the model generated novel predictions, including increased SR [Ca.sup.2+] leak and elevated intracellular [Na.sup.+] concentration in neonatal compared with adult myocytes. This new model can therefore be used for testing hypotheses and obtaining a better quantitative understanding of differences between neonatal and adult physiology. ionic currents; excitation-contraction coupling; cardiac development; cardiac myocyte

To investigate how intercellular coupling can be changed during [Ca.sup.2+] overloading of ventricular muscle, we studied [Ca.sup.2+] signals in individual cells and the histochemistry of the major gap junction channel, connexin43 (Cx43), using multicellular preparations. Papillary muscles were obtained from guinea pig ventricles and loaded with rhod-2. Sequential [Ca.sup.2+] images of surface cells were obtained with a confocal microscope. In intact muscles, all cells showed simultaneous [Ca.sup.2+] transients in response to field stimulation over a field of view of 0.3 x 0.3 [mm.sup.2]. In severely [Ca.sup.2+]-overloaded muscles, obtained by high-frequency stimulation in nonflowing Krebs solution, cells became less responsive to stimulation. Furthermore, nonsimultaneous but serial onsets of [Ca.sup.2+] transients were often detected, suggesting a propagation delay of action potentials. The time lag of the onset between two aligned cells was sometimes as long as 100 ms. Similar lags were also observed in muscles with gap junction channels inhibited by heptanol. To investigate whether the phosphorylation state of Cx43 is affected in [Ca.sup.2+]-overloaded muscles, the distributions of phosphorylated and nonphosphorylated Cx43 were determined using specific antibodies. Most of the Cx43 was phosphorylated in the nonoverloaded muscles, whereas nonphosphorylated Cx43 was significantly elevated in severely [Ca.sup.2+]-overloaded muscles. Our results suggest that the propagation delay of action potential within a small area, a few square millimeters, can be a cause of abnormal conduction and a microreentry in [Ca.sup.2+]-overloaded heart. Inactivation of [Na.sup.+] channels and inhibition of gap junctional communication may underlie the cell-to-cell propagation delay. [Ca.sup.2+] transient; connexin43; propagation delay; gap junction; arrhythmia

This study examined the impact of age on contractile function, [Ca.sup.2+] homeostasis, and cell viability in isolated myocytes exposed to simulated ischemia and reperfusion. Ventricular myocytes were isolated from anesthetized young adult (3 too) and aged (24 too) male Fischer 344 rats. Cells were fieldstimulated at 4 Hz (37[degrees]C), exposed to simulated ischemia, and reperfused with Tyrode solution. Cell shortening and intracellular [Ca.sup.2+] were measured simultaneously with an edge detector and fura-2. Cell viability was assessed by Trypan blue exclusion. Ischemia (20-45 min) depressed amplitudes of contraction equally in isolated myocytes from young adult and aged animals. The degree of postischemic contractile depression (stunning) was comparable in both groups. [Ca.sup.2+] transient amplitudes were depressed in early reperfusion in young adult and aged cells and then recovered to preischemic levels in both groups. Cell viability also declined equally in reperfusion in both groups. In short, some cellular responses to simulated ischemia and reperfusion were similar in both groups. Even so, aged myocytes exhibited a much greater and more prolonged accumulation of diastolic [Ca.sup.2+] in ischemia and in early reperfusion compared with myocytes from younger animals. In addition, the degree of mechanical alternans in ischemia increased significantly with age. The observation that there is an age-related increase in accumulation of diastolic [Ca.sup.2+] in ischemia and early reperfusion may account for the increased sensitivity to ischemia and reperfusion injury in the aging heart. The occurrence of mechanical alternans in ischemia may contribute to contractile dysfunction in ischemia in the aging heart. calcium transients; cell shortening; senescence

Endothelin (ET)-I regulates the contractility and growth of the heart by binding G protein-coupled receptors of the ET type A receptor (E[T.sub.A])/ET type B (E[T.sub.B]) receptor family. E[T.sub.A], the predominant ET-1 receptor subtype in myocardium, is thought to localize preferentially within cardiac T tubules, but the consequences of mislocalization are not fully understood. Here we examined the effects of the overexpression of ETA in conjunction with T-tubule loss in cultured adult rat ventricular myocytes. In adult myocytes cultured for 3 to 4 days, the normally robust positive inotropic effect (PIE) of ET-1 was lost in parallel with T-tubule degeneration and a decline in ETA protein levels. In these T tubule-compromised myocytes, an overexpression of ETA using an adenoviral vector did not rescue the responsiveness q to ET-1, despite the robust expression in the surface sarcolemma. The 1 inclusion of the actin polymerization inhibitor cytochalasin D (CD) j during culture prevented gross morphological changes including a 1 loss of T tubules and a rounding of intercalated discs, but CD alone did not rescue the responsiveness to ET-1 or prevent ETA down regulation. The rescue of a normal PIE in 3- to 4-day cultured myocytes T required both an increased expression of ETA and intact T tubules ' (preserved with CD). Therefore, the activation of ETA localized in T tubules was associated with a strong PIE, whereas the activation of q ETA in surface sarcolemma was not. The results provide insight into i the pathological cardiac conditions in which ETA is upregulated and 2 T-tubule morphology is altered. cytochalasin D; inotropism; endothelin type A receptor; heart failure 1

Dopamine is used to treat heart failure, particularly after cardiac surgery in infants, but the mechanisms of action are unclear. We investigated differences in the effect of dopamine on L-type calcium current ([I.sub.ca]) between newborn (NB, 1-4 days) and adult (AD, 3-4 mo) rabbit ventricular myocytes. Myocytes were enzymatically dissociated from NB and AD rabbit hearts.[I.sub.ca] was recorded by using the whole cell patch-clamp technique, mRNA levels of cardiac dopamine receptor type 1 (D1), type 2 (D2), and [beta]-adrenergic receptors ([beta]-ARs) were measured by real-time RT-PCR. Dopamine (100 [micro]M) increased [I.sub.ca] more in NB ([E.sub.max 87 [+ or -] 10%) than in AD ventricular cells ([E.max. 21 [+ or -] 3%). Further investigation of this difference showed that mRNA levels of the D1 receptor were significantly higher in NB, and, with [beta]-AR blockade, dopamine increased [I.sub.Ca] more in NB than AD cells. Additionally, SKF-38393 (selective D1 receptor agonist) significantly increased[I.sub.Ca] by 55 [+ or -] 4% in NB (P < 0.05, n = 4) and by 11 [+ or -] 1% in AD (P < 0.05, n = 6). Dopamine in the presence of SCH-23390 (D1 receptor antagonist) increased [I.sub.Ca] in NB cells by 67 [+ or -] 5% and by 22 [+ or -] 2% in AD cells, suggesting a role for [beta]-AR stimulation. Selective blockade of [[beta].sub.1]- or [[beta].sub.2]-receptors (with block of D 1 receptors) showed that the [beta]-AR action of dopamine in the NB was largely mediated via [[beta].sub.2]-AR activation. Dopamine produces a larger increase in [I.sub.Ca] in NB cardiomyocytes compared with ADs. The mechanism of action is not only through [[beta].sub.2]-ARs but also due to higher expression of cardiac D1 receptor in NB. development; [beta]-adrenergic receptors; ventricle

The onset of ventricular fibrillation (VF) has been associated with steep action potential duration restitution in both clinical and computational studies. Recently, detailed clinical restitution properties in cardiac patients were reported showing a substantial degree of heterogeneity in restitution slopes at the epicardium of the ventricles. The aim of the present study was to investigate the effect of heterogeneous restitution properties in a three-dimensional model of the ventricles using these clinically measured restitution data. We used a realistic model of the human ventricles, including detailed descriptions of cell electrophysiology, ventricular anatomy, and fiber direction anisotropy. We extended this model by mapping the clinically observed epicardial restitution data to our anatomic representation using a diffusion-based algorithm. Restitution properties were then fitted by regionally varying parameters of the electrophysiological model. We studied the effects of restitution heterogeneity on the organization of VF by analyzing filaments and the distributions of excitation periods. We found that the number of filaments and the excitation periods were both dependent on the extent of heterogeneity. An increased level of heterogeneity leads to a greater number of filaments and a broader distribution of excitation periods, thereby increasing the complexity and dynamics of VF. Restitution heterogeneity may play an important role in providing a substrate for cardiac arrhythmias. action potential duration-restitution-heterogeneity; reentrant arrhythmias; ventricular fibrillation; human ventricular myocytes; computer simulation

Selective stimulation of [[beta].sub.2]-adrenergic receptors (ARs) in newborn rabbit ventricular myocardium invokes a positive inotropic effect that is lost during postnatal maturation. The underlying mechanisms for this age-related stimulatory response remain unresolved. We examined the effects of [[beta].sub.2]-AR stimulation on L-type [Ca.sup.2+] current ([I.sub.Ca,L]) during postnatal development. [I.sub.Ca,L] was measured (37[degrees]C; either [Ca.sup.2+] or [Ba.sup.2+] as the charge carrier) using the whole-cell patch-clamp technique in newborn (1 to 5 days old) and adult rabbit ventricular myocytes. [Ca.sup.2+] transients were measured concomitantly by dialyzing the cell with indo-1. Activation of [[beta].sub.2]-ARs (with either 100 nM zinterol or 1 [micro]M isoproterenol in the presence of the [[beta].sub.1]-AR antagonist, CGP20712A) stimulated [I.sub.Ca,L] twofold in newborns but not in adults. The [[beta].sub.2]-AR-mediated increase in [Ca.sup.2+] transient amplitude in newborns was due exclusively to the augmentation of [I.sub.Ca, L]. Zinterol increased the rate of inactivation of [I.sub.Ca.L] and increased the [Ca.sup.2+] flux integral. The [[beta].sub.2]-AR inverse agonist, ICI- 118551 (500 nM), but not the [[beta].sub.1]-AR antagonist, CGP20712A (500 nM), blocked the response to zinterol. Unexpectedly, the PKA blockers, H-89 (10 [micro]M), PKI 6-22 amide (10 [micro]M), and Rp-cAMP (100 [micro]M), all failed to prevent the response to zinterol but completely blocked responses to selective [[beta].sub.1]-AR stimulation of [I.sub.Ca,L] in newborns. Our results demonstrate that in addition to the conventional [[beta].sub.1]-AR/cAMP/PKA pathway, newborn rabbit myocardium exhibits a novel [[beta].sub.2]-AR-mediated, PKA-insensitive pathway that stimulates [I.sub.Ca,L]. This striking developmental difference plays a major role in the age-related differences in inotropic responses to [[beta].sub.2]-AR agonists. development; protein kinase A; adenosine 3',5'-cyclic monophosphate

Recombinant lentiviral vectors (LVs) are capable of transducing neonatal rat ventricular myocytes (NRVMs) and providing stable, long-term transgene expression. The goal of the present study was to comprehensively test whether transduction of NRVMs by LVs results in cytotoxicity and to examine the electrophysiological consequences of gene modification of NRVM monolayers by two vectors: one encoding a putatively inert enhanced green fluorescent protein (eGFP) and the other a major ion channel protein, inward rectifier [K.sup.+] channel (Kir) 2.1. Freshly isolated NRVMs were transduced and cultured in monolayers. Immunohistochemistry, Trypan blue exclusion, annexin V binding followed by flow cytometry (FCM), and terminal transferase dUTP nick-end labeling assays were performed to assess for cytotoxicity. Optical mapping studies of action potential propagation in NRVM monolayers were performed to characterize the electrophysiological alterations following transduction. The cytotoxicity assays revealed that transduction had no adverse effects on NRVM cultures. However, eGFP-transduced monolayers exhibited a decrease in conduction velocity (CV) and action potential duration (APD) compared with monolayers transduced with LVs encoding LacZ or devoid of a transgene. In addition, small interfering RNA-mediated knockdown of eGFP expression corrected this phenotype. In contrast, Kir2.1 gene-modified monolayers showed an increase in CV and a predictable decrease in APD. This study demonstrates that LVs transduce NRVMs without cytotoxic effects. However, eGFP has a significant effect on APD and CV in this experimental system and calls into question the widely held belief that GFP is physiologically inert. In addition, LV-mediated overexpression of Kir2.1 opens up the prospect of studying the functional role of inward rectifier [K.sup.+] current in cardiac arrhythmias. lentiviral vectors; enhanced green fluorescent protein; inward rectifier [K.sup.+] channel 2.1