Stretch Current‐Induced Abnormal Impulses in CaMKIIδ Knockout Mouse Ventricular Myocytes

CaMKII and Arrhythmias. Background: CaMKII activation is proarrhythmic in heart failure where myocardium is stretched. However, the arrhythmogenic role of CaMKII in stretched ventricle has not been well understood. Objective: We tested abnormal impulse inducibility by stretch current in myocytes isolated from CaMKIIδ knockout (KO) mouse left ventricle (LV) where CaMKII activity is reduced by ≈ 62%. Methods and Results: Action potentials were recorded by whole‐cell patch clamp, and abnormal impulses were induced in LV myocytes by a simulation of stretch‐activated channel (SAC) current. SAC activation failed to induce abnormal impulses in wild type (WT) myocytes but steadily produced early after‐depolarizations and automaticity in KO myocytes in which an increase in L‐type calcium channel (LTCC) current (I_Ca) and a reduction of sarcoplasmic reticulum Ca²⁺ leak and action potential duration (APD) were observed. The abnormal impulses were not suppressed by CaMKII inhibitor AIP whereas a low concentration of nifedipine eliminated abnormal impulses without shortening APD, implicating I_Ca in promoting stretch‐induced abnormal impulses. In addition, APD prolongation by LTCC opener S(‐)Bay K 8644 or isoproterenol facilitated abnormal impulse induction in WT ventricular myocytes even in the presence of CaMKII inhibitor AIP, whereas APD prolongation by K⁺ channel blocker 4‐aminopyridine promoted abnormal impulses in KO myocytes but not in WT myocytes. Conclusion: I_Ca activation plays a central role in stretch‐induced abnormal impulses and APD prolongation is arrhythmogenic only when I_Ca is highly activated. At increased I_Ca activation, CaMKII inhibition cannot suppress abnormal impulse induction. (J Cardiovasc Electrophysiol, Vol. 24, pp. 457‐463, April 2013)     

Rate-dependent prolongation of action potential duration in isolated rat ventricular myocytes

Rate-dependent alterations of action potential duration (APD) in rat ventricular myocytes were investigated. Action potentials of the isolated myocytes were recorded with patch electrodes containing EGTA (11 mM), and showed a marked rate-dependent prolongation in the APD (0.2–5 Hz). This prolongation was significantly inhibited in the presence of 4-aminopyridine (4-AP), a blocker of the transient outward K⁺ current (I_to). Thus, the rate-dependent decrease in I_to may underlie the change in APD. In contrast, the action potentials recorded from rat ventricular papillary muscles with conventional microelectrodes did not show rate-dependent alterations in the APD, i.e., the APD remained practically unaltered at the frequency range of 0.2–5 Hz. These results suggest that the rate-dependent prolongation of APD (due to rate-dependent blockade of I_to) becomes evident when the intracellular Ca²⁺ was chelated by the internal application of EGTA via patch pipette. We speculate that the rate-dependent prolongation of APD (via decreases in I_to) is masked in the ventricular papillary muscles, probably due to rate-dependent decreases in the inward current (e.g., electrogenic Na⁺–Ca²⁺ exchange current) that is regulated by the intracellular calcium.     

Modulation of sarcoplasmic reticulum Ca<Superscript>2+</Superscript> cycling in systolic and diastolic heart failure associated with aging

Hypertension, atherosclerosis, and resultant chronic heart failure (HF) reach epidemic proportions among older persons, and the clinical manifestations and the prognoses of these worsen with increasing age. Thus, age per se is the major risk factor for cardiovascular disease. Changes in cardiac cell phenotype that occur with normal aging, as well as in HF associated with aging, include deficits in ß-adrenergic receptor (ß-AR) signaling, increased generation of reactive oxygen species (ROS), and altered excitation–contraction (EC) coupling that involves prolongation of the action potential (AP), intracellular Ca²⁺ (Ca _i ²⁺ ) transient and contraction, and blunted force- and relaxation-frequency responses. Evidence suggests that altered sarcoplasmic reticulum (SR) Ca²⁺ uptake, storage, and release play central role in these changes, which also involve sarcolemmal L-type Ca²⁺ channel (LCC), Na⁺–Ca²⁺ exchanger (NCX), and K⁺ channels. We review the age-associated changes in the expression and function of Ca²⁺ transporting proteins, and functional consequences of these changes at the cardiac myocyte and organ levels. We also review sexual dimorphism and self-renewal of the heart in the context of cardiac aging and HF.     

Cardiac resynchronization therapy to prevent life-threatening arrhythmias in patients with congestive heart failure

Various clinical data demonstrate that cardiac resynchronization therapy (CRT) provides a favorable structural as well as electrical remodeling. The CArdiac Resynchronization–Heart Failure study, which tested the pure effect of CRT (using CRT devices without the capability of defibrillation) clearly showed a significant reduction in the total mortality by partly preventing sudden cardiac death. The antiarrhythmic effects of CRT are explained, at least in part, by ionic and genetic modulation of ventricular myocytes. It has been revealed in animal experiments to mimic disorganized ventricular contraction that CRT reverses down-regulation of certain K⁺ channels and abnormal Ca²⁺ homeostasis in the failing heart. However, CRT can be proarrhythmic in some particular cases especially in the early phase of this therapy. According to our study, proarrhythmic effects after CRT can be observed in approximately 10% of patients. The relatively high incidence of the proarrhythmic effects of CRT may promote a trend toward selecting CRT-D rather than CRT-P.     

Calcium release near l-type calcium channels promotes beat-to-beat variability in ventricular myocytes from the chronic AV block dog

Beat-to-beat variability of ventricular repolarization (BVR) has been proposed as a strong predictor of Torsades de Pointes (TdP). BVR is also observed at the myocyte level, and a number of studies have shown the importance of calcium handling in influencing this parameter. The chronic AV block (CAVB) dog is a model of TdP arrhythmia in cardiac hypertrophy, and myocytes from these animals show extensive remodeling, including of Ca^2 + handling. This remodeling process also leads to increased BVR. We aimed to determine the role that (local) Ca^2 + handling plays in BVR.In isolated LV myocytes an exponential relationship was observed between BVR magnitude and action potential duration (APD) at baseline. Inhibition of Ca^2 + release from sarcoplasmic reticulum (SR) with thapsigargin resulted in a reduction of [Ca^2 +]_i, and of both BVR and APD. Increasing I_CaL in the presence of thapsigargin restored APD but BVR remained low. In contrast, increasing I_CaL with preserved Ca^2 + release increased both APD and BVR. Inhibition of Ca^2 + release with caffeine, as with thapsigargin, reduced BVR despite maintained APD. Simultaneous inhibition of Na⁺/Ca^2 + exchange and I_CaL decreased APD and BVR to similar degrees, whilst increasing diastolic Ca^2 +. Buffering of Ca^2 + transients with BAPTA reduced BVR for a given APD to a greater extent than buffering with EGTA, suggesting subsarcolemmal Ca^2 + transients modulated BVR to a larger extent than the cytosolic Ca^2 + transient.In conclusion, BVR in hypertrophied dog myocytes, at any APD, is strongly dependent on SR Ca^2 + release, which may act through modulation of the l-type Ca^2 + current in a subsarcolemmal microdomain.     

Remodeling of outward K+ currents in pressure-overload heart failure

Objectives: Outward K⁺ currents are critical determinants of action potential repolarization and the site of action of a number of electrophysiologically active drugs. Further, expression and processing of the channels underlying these currents is altered in heart disease. Here, we investigated the native transmural gradient of outward K⁺ currents in murine left ventricle (LV) and delineated disease‐related remodeling of these currents in heart failure (HF). Methods: Pressure‐overload heart failure was induced in mice by thoracic aortic constriction. Outward K⁺ currents were recorded using the whole‐cell patch clamp technique in acutely dissociated ventricular myocytes. Results: Unambiguous gradients of outward K⁺ current density and Kv4.2 protein abundance were observed across the wall of the LV, with significantly larger current density and protein levels in subepicardial (SEP) myocytes, compared with subendocardial (SEN) myocytes. Voltage dependences of current activation and inactivation were similar in SEP and SEN myocytes. In failing LV, however, outward K⁺ current density was significantly decreased in SEP but not in SEN cells leading to elimination of the native transmural gradient. In failing LV, the voltage dependences of K⁺ current activation and inactivation were not altered. However, current inactivation (decay) was significantly accelerated and recovery from inactivation was significantly slowed. Consistent with this, Western blot analysis revealed a decrease in KChIP2 protein abundance in failing LV. Conclusions: This is the first report of HF‐related remodeling of outward K⁺ currents in murine LV. Similar to humans, disease‐related remodeling occurs differentially across the murine ventricular wall, leading to loss of the native gradient of repolarization. Together with slowed recovery from inactivation, these alterations likely promote abnormal impulse conduction, a major proarrhythmic mechanism.     

Methylene Blue Counteracts H<Subscript>2</Subscript>S-Induced Cardiac Ion Channel Dysfunction and ATP Reduction

We have previously demonstrated that methylene blue (MB) counteracts the effects of hydrogen sulfide (H₂S) cardiotoxicity by improving cardiomyocyte contractility and intracellular Ca²⁺ homeostasis disrupted by H₂S poisoning. In vivo, MB restores cardiac contractility severely depressed by sulfide and protects against arrhythmias, ranging from bundle branch block to ventricular tachycardia or fibrillation. To dissect the cellular mechanisms by which MB reduces arrhythmogenesis and improves bioenergetics in myocytes intoxicated with H₂S, we evaluated the effects of H₂S on resting membrane potential (E_m), action potential (AP), Na⁺/Ca²⁺ exchange current (I_NaCa), depolarization-activated K⁺ currents and ATP levels in adult mouse cardiac myocytes and determined whether MB could counteract the toxic effects of H₂S on myocyte electrophysiology and ATP. Exposure to toxic concentrations of H₂S (100 µM) significantly depolarized E_m, reduced AP amplitude, prolonged AP duration at 90% repolarization (APD₉₀), suppressed I_NaCa and depolarization-activated K⁺ currents, and reduced ATP levels in adult mouse cardiac myocytes. Treating cardiomyocytes with MB (20 µg/ml) 3 min after H₂S exposure restored E_m, APD₉₀, I_NaCa, depolarization-activated K⁺ currents, and ATP levels toward normal. MB improved mitochondrial membrane potential (?ψ_m) and oxygen consumption rate in myocytes in which Complex I was blocked by rotenone. We conclude that MB ameliorated H₂S-induced cardiomyocyte toxicity at multiple levels: (1) reversing excitation–contraction coupling defects (Ca²⁺ homeostasis and L-type Ca²⁺ channels); (2) reducing risks of arrhythmias (E_m, APD, I_NaCa and depolarization-activated K⁺ currents); and (3) improving cellular bioenergetics (ATP, ?ψ_m).     

IKs restricts excessive beat-to-beat variability of repolarization during beta-adrenergic receptor stimulation

In vivo studies have suggested that increased beat-to-beat variability of ventricular repolarization duration (BVR) is a better predictor of drug-induced torsades de pointes than repolarization prolongation alone. Cellular BVR and its dynamics before proarrhythmic events are poorly understood. We investigated differential responses of BVR in single myocytes during I_Ks blockade versus I_Kr blockade and late-I_Na augmentation, under the influence of β-adrenergic receptor stimulation. Transmembrane action potentials were recorded from isolated canine left-ventricular midmyocytes at various pacing rates. I_Ks was blocked by HMR1556, I_Kr by dofetilide. Late I_Na was augmented by sea anemone toxin-II. Isoproterenol was added for β-adrenergic receptor stimulation. BAPTA-AM buffered intracellular Ca²⁺. SEA0400 partially inhibited the Na⁺-Ca²⁺ exchanger. BVR was quantified as variability of action-potential duration at 90% repolarization: Σ(|APD90; i + 1 minus APD90; i|) / [nbeats × √ 2] for 30 consecutive action potentials. Baseline BVR was significantly increased by I_Kr blockade and late-I_Na augmentation, especially at slow pacing rates. β-adrenergic stimulation restabilized these BVR changes. In contrast, I_Ks blockade caused very little change in repolarization when compared to baseline conditions, but predisposed the myocyte to increased BVR during β-adrenergic stimulation, especially at fast rates. BAPTA-AM and SEA0400 reduced this excessive BVR and eliminated early afterdepolarizations. In conclusion, β-adrenergic receptor stimulation exaggerates BVR during I_Ks blockade, indicating a BVR-stabilizing role of β-adrenergic-sensitive I_Ks. Loss of I_Ks plus overriding of Ca²⁺-dependent membrane currents, including inward Na⁺-Ca²⁺ exchange current, conspire to proarrhythmic BVR under these conditions.     

Ranolazine improves diastolic dysfunction in isolated myocardium from failing human hearts--role of late sodium current and intracellular ion accumulation

The goal of this study was to test the hypothesis that the novel anti-ischemic drug ranolazine, which is known to inhibit late I_Na, could reduce intracellular [Na⁺]_i and diastolic [Ca²⁺]_i overload and improve diastolic function. Contractile dysfunction in human heart failure (HF) is associated with increased [Na⁺]_i and elevated diastolic [Ca²⁺]_i. Increased Na⁺ influx through voltage-gated Na⁺ channels (late I_Na) has been suggested to contribute to elevated [Na⁺]_i in HF. In isometrically contracting ventricular muscle strips from end-stage failing human hearts, ranolazine (10 µmol/L) did not exert negative inotropic effects on twitch force amplitude. However, ranolazine significantly reduced frequency-dependent increase in diastolic tension (i.e., diastolic dysfunction) by ~ 30% without significantly affecting sarcoplasmic reticulum (SR) Ca²⁺ loading. To investigate the mechanism of action of this beneficial effect of ranolazine on diastolic tension, Anemonia sulcata toxin II (ATX-II, 40 nmol/L) was used to increase intracellular Na⁺ loading in ventricular rabbit myocytes. ATX-II caused a significant rise in [Na⁺]_i typically seen in heart failure via increased late I_Na. In parallel, ATX-II significantly increased diastolic [Ca²⁺]_i. In the presence of ranolazine the increases in late I_Na, as well as [Na⁺]_i and diastolic [Ca²⁺]_i were significantly blunted at all stimulation rates without significantly decreasing Ca²⁺ transient amplitudes or SR Ca²⁺ content. In summary, ranolazine reduced the frequency-dependent increase in diastolic tension without having negative inotropic effects on contractility of muscles from end-stage failing human hearts. Moreover, in rabbit myocytes the increases in late I_Na, [Na⁺]_i and [Ca²⁺]_i caused by ATX-II, were significantly blunted by ranolazine. These results suggest that ranolazine may be of therapeutic benefit in conditions of diastolic dysfunction due to elevated [Na⁺]_i and diastolic [Ca²⁺]_i.     

Differential alteration of cardiotonic effects of EMD 57033 and β-adrenoceptor agonists in volume-overload rabbit ventricular myocytes

Background: We investigated the effects of EMD 57033, a prototype Ca²⁺ sensitizer, and β-adrenoceptor agonists in ventricular myocytes isolated from the volume-overload (V-O) heart failure model of the rabbit. Methods and Results: V-O cardiac hypertrophy was induced in rabbits by the formation of an arterio-venous shunt between the carotid artery and jugular vein 12 to 15 weeks after the operation. Ventricular myocytes were enzymically isolated from normal and V-O rabbit hearts. The myocyte was loaded with a fluorescence Ca²⁺ dye, indo-1, and Ca²⁺ transients, and cell lengths were measured simultaneously. V-O myocytes were significantly larger than control myocytes. Duration of Ca²⁺ transients and cell shortening was significantly longer in the V-O myocytes than in control myocytes. Effects of cardiotonic interventions, including EMD 57033, isoproterenol, and dobutamine, on Ca²⁺ transients and cell shortening in V-O myocytes were compared with those in control rabbit myocytes. Isoproterenol and dobutamine increased the systolic cell shortening and peak Ca²⁺ transients and abbreviated the duration of cell shortening and Ca²⁺ transients. These responses were markedly attenuated in V-O myocytes. By contrast, the response of cell shortening to EMD 57033 was unaltered, and the Ca²⁺ sensitizing effect of EMD 57033 was rather enhanced in V-O myocytes. Conclusion: Our results indicate that the effectiveness of Ca²⁺ sensitizers is maintained in the V-O rabbit hypertrophy and heart failure model in contrast to the blunted response to β-adrenoceptor agonists, which provides an insight on therapeutic strategy with Ca²⁺ sensitizers for the treatment of contractile dysfunction in congestive heart failure.     

Induced Overexpression of Na+/Ca2+ Exchanger Does Not Aggravate Myocardial Dysfunction Induced by Transverse Aortic Constriction

BackgroundAlterations in expression and activity of cardiac Na⁺/Ca²⁺ exchanger (NCX1) have been implicated in the pathogenesis of heart failure.Methods and ResultsUsing transgenic mice in which expression of rat NCX1 was induced at 5 weeks of age, we performed transverse aortic constriction (TAC) at 8 weeks and examined cardiac and myocyte function at 15–18 weeks after TAC (age 23–26 weeks). TAC induced left ventricular (LV) and myocyte hypertrophy and increased myocardial fibrosis in both wild-type (WT) and NCX1-overexpressed mice. NCX1 and phosphorylated ryanodine receptor expression was increased by TAC, whereas sarco(endo)plasmic reticulum Ca²⁺-ATPase levels were decreased by TAC. Action potential duration was prolonged by TAC, but to a greater extent in NCX1 myocytes. Na⁺/Ca²⁺ exchange current was similar between WT-TAC and WT-sham myocytes, but was higher in NCX1-TAC myocytes. Both myocyte contraction and [Ca²⁺]_i transient amplitudes were reduced in WT-TAC myocytes, but restored to WT-sham levels in NCX1-TAC myocytes. Despite improvement in single myocyte contractility and Ca²⁺ dynamics, induced NCX1 overexpression in TAC animals did not ameliorate LV hypertrophy, increase ejection fraction, or enhance inotropic (maximal first derivative of LV pressure rise, +dP/dt) responses to isoproterenol.ConclusionsIn pressure-overload hypertrophy, induced overexpression of NCX1 corrected myocyte contractile and [Ca²⁺]_i transient abnormalities but did not aggravate or improve myocardial dysfunction.     

Activated human platelet products induce proarrhythmic effects in ventricular myocytes

Sudden cardiac death remains one of the most prevalent modes of death and is mainly caused by ventricular fibrillation (VF) in the setting of acute ischemia resulting from coronary thrombi. Animal experiments have shown that platelet activation may increase susceptibility of ischemic myocardium to VF, but the mechanism is unknown. In the present study, we evaluated the effects of activated blood platelet products (ABPPs) on electrophysiological properties and intracellular Ca²⁺ (Ca²⁺_i) homeostasis. Platelets were collected from healthy volunteers. After activation, their secreted ABPPs were added to superfusion solutions. Rabbit ventricular myocytes were freshly isolated, and membrane potentials and Ca²⁺_i were recorded using patch-clamp methodology and indo-1 fluorescence measurements, respectively. ABPPs prolonged action potential duration and induced early and delayed afterdepolarizations. ABPPs increased L-type Ca²⁺ current (I_Ca,L) density, but left densities of sodium current, inward rectifier K⁺ current, transient outward K⁺ current, and rapid component of the delayed rectifier K⁺ current unchanged. ABPPs did not affect kinetics or (in)activation properties of membrane currents. ABPPs increased systolic Ca²⁺_i, Ca²⁺_i transient amplitude, and sarcoplasmic reticulum Ca²⁺ content. ABPPs did not affect the Na⁺− Ca²⁺ exchange current (I_NCX) in Ca²⁺-buffered conditions. Products secreted from activated human platelets induce changes in I_Ca,L and Ca²⁺_i, which result in action potential prolongation and the occurrence of early and delayed afterdepolarizations in rabbit myocytes. These changes may trigger and support reentrant arrhythmias in ischemia models of coronary thrombosis.     

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 Ca^2 + regulation of L-type Ca^2 + 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 Ca^2 + transients. Next, we demonstrated that all LQTS CaM mutants have the potential to strongly suppress Ca^2 +/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 Ca^2 + 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 Ca^2 + profiles. In all, these results highlight LTCCs as a molecular locus for understanding and treating CaM-related LQTS in this group of patients.     

Regional and frequency-dependent changes in action potentials and transient outward K<Superscript>+</Superscript> currents in ventricular myocytes from J-2-K cardiomyopathic hamsters

Abstract. Objectives Although lengthening of action potential duration (APD) and decreased transient outward K⁺ currents (I_to) have been observed in ventricular myocytes from cardiomyopathic hamsters, epi- and endo-cardial differences in I_to and their roles in frequency-dependent changes in APD have not been claried. Methods The patch-clamp technique of whole-cell conguration was used to record membrane potentials and currents in epicardial and endocardial myocytes of the J-2 hamster germline without (control) and with cardiomyopathy (CM). Results In control, APD in endocardial myocytes was longer than that in epicardial myocytes at 0.1 Hz. APD significantly lengthened with increased frequencies of stimulation from 0.1 to 6.0 Hz in both groups with the longer APD in endocardial myocytes. In CM, APD lengthened in epicardial myocytes exceeding the endocardial APD without a frequency-dependent prolongation. Pretreatment with 4 mM 4-aminopyridine completely abolished the frequency-dependent changes and abolished APD differences between epicardial and endocardial myocytes, and between control and CM hamsters. The transient outward K⁺ current (I_to) significantly decreased in epicardial myocytes from CM hamsters compared with that of control (17.5 ± 1.5 pA/pF in control vs. 9.5 ± 2.5 pA/pF in CM at +60 mV) with altered recovery from inactivation, without changes in the endocardial I_to. Moreover, the inward rectifier K⁺ current decreased in epicardial myocytes from CM hamsters and the L-type Ca2⁺ current reduced in both regions from CM compared to control. Conclusion Results indicate that differences in APD between epi- and endocardial myocytes in CM hamsters are mainly caused by a decreased current density and altered recovery from inactivation of I_to in epicardial myocytes.     

Diastolic and systolic heart failure: Different stages or distinct phenotypes of the heart failure syndrome?

It remains uncertain if diastolic heart failure (DHF) is a distinct HF phenotype or a precursor stage of systolic HF (SHF). The unimodal distribution of left ventricular ejection fraction (LVEF) in HF, depressed LV long-axis shortening in DHF, and progression to eccentric LV remodeling in hypertension favor DHF and SHF as successive stages. These arguments are countered by the bimodal distribution of LVEF after correction for gender, by the preserved LV twist in DHF and by the low incidence of eccentric LV remodeling in hypertension. Clinical features, LV anatomy and histology, cardiomyocyte stiffness, myocardial effects of diabetes, and the response to HF therapy support DHF and SHF as distinct phenotypes. Comparison of the myocardial signal transduction cascades that drive LV remodeling in DHF and SHF may solve the controversy. This review analyzes arguments supporting DHF and SHF as successive stages or distinct phenotypes of the HF syndrome.     

ATX-II effects on the apparent location of M cells in a computational model of a human left ventricular wedge

Introduction: The apparent location of the myocytes (M cells) with the longest action potential duration (APD) in a canine left ventricular (LV) wedge have been reported to shift after application of a sea anemone toxin, ATX‐II. This toxin slows inactivation of I_Na and thus prolongs APD. Thus, M cells may exhibit dynamic functional states, rather than being a static, anatomically discrete, myocyte population. In this study, we attempted to further define and understand this phenomenon using a mathematical model of the human ventricular myocyte action potential incorporated into an in silico “wedge” preparation. Our simulations demonstrate that even under conditions of a fixed population and ratio of epicardial, M, and endocardial myocytes, the apparent anatomical position (transmural location) of the myocytes with the longest APD can shift following ATX‐II treatment. This arises because the ATX‐II effect, modeled as a small increase in the late or persistent Na⁺ current, and consequent prolongation of APD significantly changes the electrotonic interactions between ventricular myocytes in this LV wedge preparation. Methods and Results: This LV wedge model is based on bidomain equations. It corresponds to a rectangular tissue immersed in a passive and isotropic medium that represents the superfusion bath. In this theoretical work, the three known different and discrete populations of myocytes in the human left ventricle have been included: the epicardial, M, and endocardial cells. The effects of ATX‐II on I_Na were simulated by altering the voltage‐dependent steady‐state inactivation of the parameters h (fast gate) and j (slow gate). As a result, in these ATX‐II simulations a persistent late Na⁺ current was generated in all three types of ventricular myocytes. However, the APDs were prolonged in a heterogeneous pattern. Our simulations demonstrate that after the ATX‐II effects develop, alterations in transmural electrotonic interactions can produce changes in the transmural location of myocytes with the longest APD. Conclusions: The combination of intercellular electrotonic interactions, which tend to reduce and smooth out the discrete transmural APD variations, and the heterogeneous effects of ATX‐II, which preferentially prolong the APD of M cells, can shift the location of the ventricular myocytes. This shift results in significantly altered transmural patterns of action potential durations, which would be expected to change localized refractory period and excitability. These cellular changes give rise to alterations in the corresponding surface electrograms and may change the overall substrates for conduction and rhythm disturbances.     

Toll-like receptor 4 activation promotes cardiac arrhythmias by decreasing the transient outward potassium current (Ito) through an IRF3-dependent and MyD88-independent pathway

Cardiac arrhythmias are one of the main causes of death worldwide. Several studies have shown that inflammation plays a key role in different cardiac diseases and Toll-like receptors (TLRs) seem to be involved in cardiac complications. In the present study, we investigated whether the activation of TLR4 induces cardiac electrical remodeling and arrhythmias, and the signaling pathway involved in these effects. Membrane potential was recorded in Wistar rat ventricle. Ca^2 + transients, as well as the L-type Ca^2 + current (I_CaL) and the transient outward K⁺ current (I_to), were recorded in isolated myocytes after 24 h exposure to the TLR4 agonist, lipopolysaccharide (LPS, 1 μg/ml). TLR4 stimulation in vitro promoted a cardiac electrical remodeling that leads to action potential prolongation associated with arrhythmic events, such as delayed afterdepolarization and triggered activity. After 24 h LPS incubation, I_to amplitude, as well as Kv4.3 and KChIP2 mRNA levels were reduced. The I_to decrease by LPS was prevented by inhibition of interferon regulatory factor 3 (IRF3), but not by inhibition of interleukin-1 receptor-associated kinase 4 (IRAK4) or nuclear factor kappa B (NF-κB). Extrasystolic activity was present in 25% of the cells, but apart from that, Ca^2 + transients and I_CaL were not affected by LPS; however, Na⁺/Ca^2 + exchanger (NCX) activity was apparently increased. We conclude that TLR4 activation decreased I_to, which increased AP duration via a MyD88-independent, IRF3-dependent pathway. The longer action potential, associated with enhanced Ca^2 + efflux via NCX, could explain the presence of arrhythmias in the LPS group.     

Role of mitochondrial dysfunction in cardiac glycoside toxicity

Cardiac glycosides, which inhibit the plasma membrane Na⁺ pump, are one of the four categories of drug recommended for routine use to treat heart failure, yet their therapeutic window is limited by toxic effects. Elevated cytoplasmic Na⁺ ([Na⁺]_i) compromises mitochondrial energetics and redox balance by blunting mitochondrial Ca²⁺ ([Ca²⁺]_m) accumulation, and this impairment can be prevented by enhancing [Ca²⁺]_m. Here, we investigate whether this effect underlies the toxicity and arrhythmogenic effects of cardiac glycosides and if these effects can be prevented by suppressing mitochondrial Ca²⁺ efflux, via inhibition of the mitochondrial Na⁺/Ca²⁺ exchanger (mNCE). In isolated cardiomyocytes, ouabain elevated [Na⁺]_i in a dose-dependent way, blunted [Ca²⁺]_m accumulation, decreased the NADH/NAD + redox potential, and increased reactive oxygen species (ROS). Concomitant treatment with the mNCE inhibitor CGP-37157 ameliorated these effects. CGP-37157 also attenuated ouabain-induced cellular Ca²⁺ overload and prevented delayed afterdepolarizations (DADs). In isolated perfused hearts, ouabain's positive effects on contractility and respiration were markedly potentiated by CGP-37157, as were those mediated by β-adrenergic stimulation. Furthermore, CGP-37157 inhibited the arrhythmogenic effects of ouabain in both isolated perfused hearts and in vivo. The findings reveal the mechanism behind cardiac glycoside toxicity and show that improving mitochondrial Ca²⁺ retention by mNCE inhibition can mitigate these effects, particularly with respect to the suppression of Ca²⁺-triggered arrhythmias, while enhancing positive inotropic actions. These results suggest a novel strategy for the treatment of heart failure.     

Theoretical investigation of action potential duration dependence on extracellular Ca in human cardiomyocytes

Reduction in [Ca²⁺]_o prolongs the AP in ventricular cardiomyocytes and the QT_c interval in patients. Although this phenomenon is relevant to arrhythmogenesis in the clinical setting, its mechanisms are counterintuitive and incompletely understood. To evaluate in silico the mechanisms of APD modulation by [Ca²⁺]_o in human cardiomyocytes. We implemented the Ten Tusscher-Noble-Noble-Panfilov model of the human ventricular myocyte and modified the formulations of the rapidly and slowly activating delayed rectifier K⁺ currents (I_Kr and I_Ks) and L-type Ca²⁺ current (I_CaL) to incorporate their known sensitivity to intra- or extracellular Ca²⁺. Simulations were run with the original and modified models at variable [Ca²⁺]_o in the clinically relevant 1 to 3 mM range. The original model responds with APD shortening to decrease in [Ca²⁺]_o, i.e. opposite to the experimental observations. Incorporation of Ca²⁺ dependency of K⁺ currents cannot reproduce the inverse relation between APD and [Ca²⁺]_o. Only when I_CaL inactivation process was modified, by enhancing its dependency on Ca²⁺, simulations predict APD prolongation at lower [Ca²⁺]_o. Although Ca²⁺-dependent I_CaL inactivation is the primary mechanism, secondary changes in electrogenic Ca²⁺ transport (by Na⁺/Ca²⁺ exchanger and plasmalemmal Ca²⁺-ATPase) contribute to the reversal of APD dependency on [Ca²⁺]_o. This theoretical investigation points to Ca²⁺-dependent inactivation of I_CaL as a mechanism primarily responsible for the dependency of APD on [Ca²⁺]_o. The modifications implemented here make the model more suitable to analyze repolarization mechanisms when Ca²⁺ levels are altered.     

SR-targeted CaMKII inhibition improves SR Ca²+ handling, but accelerates cardiac remodeling in mice overexpressing CaMKIIδC

Cardiac myocyte overexpression of CaMKIIδ_C leads to cardiac hypertrophy and heart failure (HF) possibly caused by altered myocyte Ca²⁺ handling. A central defect might be the marked CaMKII-induced increase in diastolic sarcoplasmic reticulum (SR) Ca²⁺ leak which decreases SR Ca²⁺ load and Ca²⁺ transient amplitude. We hypothesized that inhibition of CaMKII near the SR membrane would decrease the leak, improve Ca²⁺ handling and prevent the development of contractile dysfunction and HF. To test this hypothesis we crossbred CaMKIIδ_C overexpressing mice (CaMK) with mice expressing the CaMKII-inhibitor AIP targeted to the SR via a modified phospholamban (PLB)-transmembrane-domain (SR-AIP). There was a selective decrease in the amount of activated CaMKII in the microsomal (SR/membrane) fraction prepared from these double-transgenic mice (CaMK/SR-AIP) mice. In ventricular cardiomyocytes from CaMK/SR-AIP mice, SR Ca²⁺ leak, assessed both as diastolic Ca²⁺ shift into SR upon tetracaine in intact myocytes or integrated Ca²⁺ spark release in permeabilized myocytes, was significantly reduced. The reduced leak was accompanied by enhanced SR Ca²⁺ load and twitch amplitude in double-transgenic mice (vs. CaMK), without changes in SERCA expression or NCX function. However, despite the improved myocyte Ca²⁺ handling, cardiac hypertrophy and remodeling was accelerated in CaMK/SR-AIP and cardiac function worsened. We conclude that while inhibition of SR localized CaMKII in CaMK mice improves Ca²⁺ handling, it does not necessarily rescue the HF phenotype. This implies that a non-SR CaMKIIδ_C exerts SR-independent effects that contribute to hypertrophy and HF, and this CaMKII pathway may be exacerbated by the global enhancement of Ca transients.