Evaluation of the Cardiotoxicity of Mitragynine and Its Analogues Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Introduction

Mitragynine is a major bioactive compound of Kratom, which is derived from the leave extracts of Mitragyna speciosa Korth or Mitragyna speciosa (M. speciosa), a medicinal plant from South East Asia used legally in many countries as stimulant with opioid-like effects for the treatment of chronic pain and opioid-withdrawal symptoms. Fatal incidents with Mitragynine have been associated with cardiac arrest. In this study, we determined the cardiotoxicity of Mitragynine and other chemical constituents isolated using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).

Methods and Results

The rapid delayed rectifier potassium current (I _Kr), L-type Ca²⁺ current (I _Ca,L) and action potential duration (APD) were measured by whole cell patch-clamp. The expression of KCNH2 and cytotoxicity was determined by real-time PCR and Caspase activity measurements. After significant I _Kr suppression by Mitragynine (10 µM) was confirmed in hERG-HEK cells, we systematically examined the effects of Mitragynine and other chemical constituents in hiPSC-CMs. Mitragynine, Paynantheine, Speciogynine and Speciociliatine, dosage-dependently (0.1∼100 µM) suppressed I _Kr in hiPSC-CMs by 67% ∼84% with IC₅₀ ranged from 0.91 to 2.47 µM. Moreover, Mitragynine (10 µM) significantly prolonged APD at 50 and 90% repolarization (APD50 and APD90) (439.0±11.6 vs. 585.2±45.5 ms and 536.0±22.6 vs. 705.9±46.1 ms, respectively) and induced arrhythmia, without altering the L-type Ca²⁺ current. Neither the expression,and intracellular distribution of KCNH2/Kv11.1, nor the Caspase 3 activity were significantly affected by Mitragynine.

Conclusions

Our study indicates that Mitragynine and its analogues may potentiate Torsade de Pointes through inhibition of I _Kr in human cardiomyocytes.     

Differential Expression of hERG1 Channel Isoforms Reproduces Properties of Native I Kr and Modulates Cardiac Action Potential Characteristics

Background

The repolarizing cardiac rapid delayed rectifier current, I _Kr, is composed of ERG1 channels. It has been suggested that two isoforms of the ERG1 protein, ERG1a and ERG1b, both contribute to I _Kr. Marked heterogeneity in the kinetic properties of native I _Kr has been described. We hypothesized that the heterogeneity of native I _Kr can be reproduced by differential expression of ERG1a and ERG1b isoforms. Furthermore, the functional consequences of differential expression of ERG1 isoforms were explored as a potential mechanism underlying native heterogeneity of action potential duration (APD) and restitution.

Methodology/Principal Findings

The results show that the heterogeneity of native I _Kr can be reproduced in heterologous expression systems by differential expression of ERG1a and ERG1b isoforms. Characterization of the macroscopic kinetics of ERG1 currents demonstrated that these were dependent on the relative abundance of ERG1a and ERG1b. Furthermore, we used a computational model of the ventricular cardiomyocyte to show that both APD and the slope of the restitution curve may be modulated by varying the relative abundance of ERG1a and ERG1b. As the relative abundance of ERG1b was increased, APD was gradually shortened and the slope of the restitution curve was decreased.

Conclusions/Significance

Our results show that differential expression of ERG1 isoforms may explain regional heterogeneity of I _Kr kinetics. The data demonstrate that subunit dependent changes in channel kinetics are important for the functional properties of ERG1 currents and hence I _Kr. Importantly, our results suggest that regional differences in the relative abundance of ERG1 isoforms may represent a potential mechanism underlying the heterogeneity of both APD and APD restitution observed in mammalian hearts.     

Increased Response to β2-Adrenoreceptor Stimulation Augments Inhibition of IKr in Heart Failure Ventricular Myocytes

Background

Increasing evidence indicates that the rapid component of delayed rectifier potassium current (I_Kr) is modulated by α- and β-adrenergic stimulation. However, the role and mechanism regulating I_Kr through β₂-adrenoreceptor (β-AR) stimulation in heart failure (HF) are unclear.

Methodology/Principal Findings

In the present study, we investigated the effects of fenoterol, a highly selective β₂-AR agonist, on I_Kr in left ventricular myocytes obtained from control and guinea pigs with HF induced by descending aortic banding. I_Kr was measured by using whole cell patch clamp technique. In control myocytes, superfusion of fenoterol (10 µM) caused a 17% decrease in I_Kr. In HF myocytes, the same concentration of fenoterol produced a significantly greater decrease (33%) in I_Kr. These effects were not modified by the incubation of myocytes with CGP-20712A, a β₁-AR antagonist, but were abolished by pretreatment of myocytes with ICI-118551, a β₂-AR antagonist. An inhibitory cAMP analog, Rp-cAMPS and PKA inhibitor significantly attenuated fenoterol-induced inhibition of I_Kr in HF myocytes. Moreover, fenoterol markedly prolonged action potential durations at 90% (APD₉₀) repolarization in HF ventricular myocytes.

Conclusions

The results indicate that inhibition of I_Kr induced by β₂-AR stimulation is increased in HF. The inhibitory effect is likely to be mediated through a cAMP/PKA pathway in HF ventricular myocytes.     

Maximum diastolic potential of human induced pluripotent stem cell-derived cardiomyocytes depends critically on I(Kr)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions, the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (n = 103; 37°C) and to examine the response to 5 µM E-4031 (n = 21) or BaCl₂ (n = 22). Patch-clamp techniques were used to record I_Kr and I_K1 from cells enzymatically dissociated from BC (n = 49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (p<0.001) and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC, E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl₂, at concentrations that selectively block I_K1 (50–100 µM), failed to depolarize the majority of clusters (13/22). Patch-clamp experiments revealed very low or negligible I_K1 in 53% (20/38) of the cells studied, but presence of I_Kr in all (11/11). Consistent with the electrophysiological data, RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of I_K1 in the majority of cells, but robust expression of I_Kr. In contrast to recently reported studies, our data point to major deficiencies of hiPSC-CM, with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd). The vast majority have a maximum diastolic potential that depends critically on I_Kr due to the absence of I_K1. Thus, efforts should be directed at producing more specialized and mature hiPSC-CM for future therapeutic applications.     

Long-Term Prognostic Value of Restitution Slope in Patients with Ischemic and Dilated Cardiomyopathies

Background

An action potential duration (APD) restitution curve with a steep slope ≥1 has been associated with increased susceptibility for malignant ventricular arrhythmias. We aimed to evaluate the “restitution hypothesis” and tested ventricular APD restitution slope as well as effective refractory period (ERP)/APD ratio for long-term prognostic value in patients with ischemic (ICM) or dilated cardiomyopathy (DCM).

Methodology/Principal Findings

Monophasic action potentials were recorded in patients with ICM (n = 32) and DCM (n = 42) undergoing routine programmed ventricular stimulation (PVS). Left ventricular ejection fraction was 32±7% and 28±9%, respectively. APD and ERP were measured at baseline stimulation (S₁) and upon introduction of one to three extrastimuli (S₂–S₄). ERP/APD ratios and the APD restitution curve were calculated and the maximum restitution slope was determined. After a mean follow-up of 6.1±3.0 years, the combined end-point of mortality and and/or implantable cardioverter-defibrillator shock was not predicted by restitution slope or ERP/APD ratios. Comparing S₂ vs. S₃ vs. S₄ extrastimuli for restitution slope (1.5±0.6 vs. 1.4±0.4 vs. 1.3±0.5; p = NS), additional extrastimuli did not lead to a steepening restitution slope. ERP/APD ratio decreased with additional extrastimuli (0.98±0.09 [S₁] vs. 0.97±0.10 [S₂] vs. 0.93±0.11 [S₃]; p = 0.03 S₁ vs. S₃). Positive PVS was strongly predictive of outcome (p = 0.006).

Conclusions/Significance

Neither ventricular APD restitution slope nor ERP/APD ratios predict outcome in patients with ICM or DCM.     

Amiodarone Inhibits Apamin-Sensitive Potassium Currents

Background

Apamin sensitive potassium current (I _KAS), carried by the type 2 small conductance Ca²⁺-activated potassium (SK2) channels, plays an important role in post-shock action potential duration (APD) shortening and recurrent spontaneous ventricular fibrillation (VF) in failing ventricles.

Objective

To test the hypothesis that amiodarone inhibits I _KAS in human embryonic kidney 293 (HEK-293) cells.

Methods

We used the patch-clamp technique to study I _KAS in HEK-293 cells transiently expressing human SK2 before and after amiodarone administration.

Results

Amiodarone inhibited I_KAS in a dose-dependent manner (IC₅₀, 2.67±0.25 µM with 1 µM intrapipette Ca²⁺). Maximal inhibition was observed with 50 µM amiodarone which inhibited 85.6±3.1% of I_KAS induced with 1 µM intrapipette Ca²⁺ (n = 3). I_KAS inhibition by amiodarone was not voltage-dependent, but was Ca²⁺-dependent: 30 µM amiodarone inhibited 81.5±1.9% of I _KAS induced with 1 µM Ca²⁺ (n = 4), and 16.4±4.9% with 250 nM Ca²⁺ (n = 5). Desethylamiodarone, a major metabolite of amiodarone, also exerts voltage-independent but Ca²⁺ dependent inhibition of I _KAS.

Conclusion

Both amiodarone and desethylamiodarone inhibit I _KAS at therapeutic concentrations. The inhibition is independent of time and voltage, but is dependent on the intracellular Ca²⁺ concentration. SK2 current inhibition may in part underlie amiodarone''s effects in preventing electrical storm in failing ventricles.     

Role of Sarcoplasmic Reticulum Calcium in Development of Secondary Calcium Rise and Early Afterdepolarizations in Long QT Syndrome Rabbit Model

Background

L-type calcium current reactivation plays an important role in development of early afterdepolarizations (EADs) and torsades de pointes (TdP). Secondary intracellular calcium (Ca_i) rise is associated with initiation of EADs.

Objective

To test whether inhibition of sarcoplasmic reticulum (SR) Ca²⁺ cycling suppresses secondary Ca_i rise and genesis of EADs.

Methods

Langendorff perfusion and dual voltage and Ca_i optical mapping were conducted in 10 rabbit hearts. Atrioventricular block (AVB) was created by radiofrequency ablation. After baseline studies, E4031, SR Ca²⁺ cycling inhibitors (ryanodine plus thapsigargin) and nifedipine were then administrated subsequently, and the protocols were repeated.

Results

At baseline, there was no spontaneous or pacing-induced TdP. After E4031 administration, action potential duration (APD) was significantly prolonged and the amplitude of secondary Ca_i rise was enhanced, and 7 (70%) rabbits developed spontaneous or pacing-induced TdP. In the presence of ryanodine plus thapsigargin, TdP inducibility was significantly reduced (2 hearts, 20%, p = 0.03). Although APD was significantly prolonged (from 298 ± 30 ms to 457 ± 75 ms at pacing cycle length of 1000 m, p = 0.007) by ryanodine plus thapsigargin, the secondary Ca_i rise was suppressed (from 8.8 ± 2.6% to 1.2 ± 0.9%, p = 0.02). Nifedipine inhibited TdP inducibility in all rabbit hearts.

Conclusion

In this AVB and long QT rabbit model, inhibition of SR Ca²⁺ cycyling reduces the inducibility of TdP. The mechanism might be suppression of secondary Ca_i rise and genesis of EADs.     

Hydrogen Sulfide Suppresses Outward Rectifier Potassium Currents in Human Pluripotent Stem Cell-Derived Cardiomyocytes

Aim

Hydrogen sulfide (H₂S) is a promising cardioprotective agent and a potential modulator of cardiac ion currents. Yet its cardiac effects on humans are poorly understood due to lack of functional cardiomyocytes. This study investigates electrophysiological responses of human pluripotent stem cells (hPSCs) derived cardiomyocytes towards H₂S.

Methods and Results

Cardiomyocytes of ventricular, atrial and nodal subtypes differentiated from H9 embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) were electrophysiologically characterized. The effect of NaHS, a donor of H₂S, on action potential (AP), outward rectifier potassium currents (I _Ks and I _Kr), L-type Ca²⁺ currents (I _CaL) and hyperpolarization-activated inward current (I _f) were determined by patch-clamp electrophysiology and confocal calcium imaging. In a concentration-dependent manner, NaHS (100 to 300 µM) consistently altered the action potential properties including prolonging action potential duration (APD) and slowing down contracting rates of ventricular-and atrial-like cardiomyocytes derived from both hESCs and hiPSCs. Moreover, inhibitions of slow and rapid I _K (I _Ks and I _Kr), I _CaL and I _f were found in NaHS treated cardiomyocytes and it could collectively contribute to the remodeling of AP properties.

Conclusions

This is the first demonstration of effects of H₂S on cardiac electrophysiology of human ventricular-like, atrial-like and nodal-like cardiomyocytes. It reaffirmed the inhibitory effect of H₂S on I _CaL and revealed additional novel inhibitory effects on I _f, I _Ks and I _Kr currents in human cardiomyocytes.     

Diclofenac Prolongs Repolarization in Ventricular Muscle with Impaired Repolarization Reserve

Background

The aim of the present work was to characterize the electrophysiological effects of the non-steroidal anti-inflammatory drug diclofenac and to study the possible proarrhythmic potency of the drug in ventricular muscle.

Methods

Ion currents were recorded using voltage clamp technique in canine single ventricular cells and action potentials were obtained from canine ventricular preparations using microelectrodes. The proarrhythmic potency of the drug was investigated in an anaesthetized rabbit proarrhythmia model.

Results

Action potentials were slightly lengthened in ventricular muscle but were shortened in Purkinje fibers by diclofenac (20 µM). The maximum upstroke velocity was decreased in both preparations. Larger repolarization prolongation was observed when repolarization reserve was impaired by previous BaCl₂ application. Diclofenac (3 mg/kg) did not prolong while dofetilide (25 µg/kg) significantly lengthened the QT_c interval in anaesthetized rabbits. The addition of diclofenac following reduction of repolarization reserve by dofetilide further prolonged QT_c. Diclofenac alone did not induce Torsades de Pointes ventricular tachycardia (TdP) while TdP incidence following dofetilide was 20%. However, the combination of diclofenac and dofetilide significantly increased TdP incidence (62%). In single ventricular cells diclofenac (30 µM) decreased the amplitude of rapid (I_Kr) and slow (I_Ks) delayed rectifier currents thereby attenuating repolarization reserve. L-type calcium current (I_Ca) was slightly diminished, but the transient outward (I_to) and inward rectifier (I_K1) potassium currents were not influenced.

Conclusions

Diclofenac at therapeutic concentrations and even at high dose does not prolong repolarization markedly and does not increase the risk of arrhythmia in normal heart. However, high dose diclofenac treatment may lengthen repolarization and enhance proarrhythmic risk in hearts with reduced repolarization reserve.     

In Vivo Human Left-to-Right Ventricular Differences in Rate Adaptation Transiently Increase Pro-Arrhythmic Risk following Rate Acceleration

Left-to-right ventricular (LV/RV) differences in repolarization have been implicated in lethal arrhythmias in animal models. Our goal is to quantify LV/RV differences in action potential duration (APD) and APD rate adaptation and their contribution to arrhythmogenic substrates in the in vivo human heart using combined in vivo and in silico studies. Electrograms were acquired from 10 LV and 10 RV endocardial sites in 15 patients with normal ventricles. APD and APD adaptation were measured during an increase in heart rate. Analysis of in vivo electrograms revealed longer APD in LV than RV (207.8±21.5 vs 196.7±20.1 ms; P<0.05), and slower APD adaptation in LV than RV (time constant τ^s = 47.0±14.3 vs 35.6±6.5 s; P<0.05). Following rate acceleration, LV/RV APD dispersion experienced an increase of up to 91% in 12 patients, showing a strong correlation (r² = 0.90) with both initial dispersion and LV/RV difference in slow adaptation. Pro-arrhythmic implications of measured LV/RV functional differences were studied using in silico simulations. Results show that LV/RV APD and APD adaptation heterogeneities promote unidirectional block following rate acceleration, albeit being insufficient for establishment of reentry in normal hearts. However, in the presence of an ischemic region at the LV/RV junction, LV/RV heterogeneity in APD and APD rate adaptation promotes reentrant activity and its degeneration into fibrillatory activity. Our results suggest that LV/RV heterogeneities in APD adaptation cause a transient increase in APD dispersion in the human ventricles following rate acceleration, which promotes unidirectional block and wave-break at the LV/RV junction, and may potentiate the arrhythmogenic substrate, particularly in patients with ischemic heart disease.     

Impaired Inactivation of L-Type Ca2+ Current as a Potential Mechanism for Variable Arrhythmogenic Liability of HERG K+ Channel Blocking Drugs

The proarrhythmic effects of new drugs have been assessed by measuring rapidly activating delayed-rectifier K⁺ current (I_Kr) antagonist potency. However, recent data suggest that even drugs thought to be highly specific I_Kr blockers can be arrhythmogenic via a separate, time-dependent pathway such as late Na⁺ current augmentation. Here, we report a mechanism for a quinolone antibiotic, sparfloxacin-induced action potential duration (APD) prolongation that involves increase in late L-type Ca²⁺ current (I_CaL) caused by a decrease in Ca²⁺-dependent inactivation (CDI). Acute exposure to sparfloxacin, an I_Kr blocker with prolongation of QT interval and torsades de pointes (TdP) produced a significant APD prolongation in rat ventricular myocytes, which lack I_Kr due to E4031 pretreatment. Sparfloxacin reduced peak I_CaL but increased late I_CaL by slowing its inactivation. In contrast, ketoconazole, an I_Kr blocker without prolongation of QT interval and TdP produced reduction of both peak and late I_CaL, suggesting the role of increased late I_CaL in arrhythmogenic effect. Further analysis showed that sparfloxacin reduced CDI. Consistently, replacement of extracellular Ca²⁺ with Ba²⁺ abolished the sparfloxacin effects on I_CaL. In addition, sparfloxacin modulated I_CaL in a use-dependent manner. Cardiomyocytes from adult mouse, which is lack of native I_Kr, demonstrated similar increase in late I_CaL and afterdepolarizations. The present findings show that sparfloxacin can prolong APD by augmenting late I_CaL. Thus, drugs that cause delayed I_CaL inactivation and I_Kr blockage may have more adverse effects than those that selectively block I_Kr. This mechanism may explain the reason for discrepancies between clinically reported proarrhythmic effects and I_Kr antagonist potencies.     

α1-Syntrophin Variant Identified in Drug-Induced Long QT Syndrome Increases Late Sodium Current

Drug-induced long-QT syndrome (diLQTS) is often due to drug block of I_Kr, especially in genetically susceptible patients with subclinical mutations in the I_Kr-encoding KCHN2. Few variants in the cardiac Na_V1.5 Na⁺ channel complex have been associated with diLQTS. We tested whether a novel SNTA1 (α1-syntrophin) variant (p.E409Q) found in a patient with diLQTS increases late sodium current (I_Na-L), thereby providing a disease mechanism. Electrophysiological studies were performed in HEK293T cells co-expressing human Na_V1.5/nNOS/PMCA4b with either wild type (WT) or SNTA1 variants (A390V-previously reported in congenital LQTS; and E409Q); and in adult rat ventricular cardiomyocytes infected with SNTA1 expressing adenoviruses (WT or one of the two SNTA1 variants). In HEK293T cells and in cardiomyocytes, there was no significant difference in the peak I_Na densities among the SNTA1 WT and variants. However, both variants increased I_Na-L (% of peak current) in HEK293T cells (0.58±0.10 in WT vs. 0.90±0.11 in A390V, p = 0.048; vs. 0.88±0.07 in E409Q, p = 0.023). In cardiomyocytes, I_Na-L was significantly increased by E409Q, but not by A390V compared to WT (0.49±0.14 in WT vs.0.94±0.23 in A390V, p = 0.099; vs. 1.12±0.24 in E409Q, p = 0.019). We demonstrated that a novel SNTA1 variant is likely causative for diLQTS by augmenting I_Na-L. These data suggest that variants within the Na_V1.5-interacting α1-syntrophin are a potential mechanism for diLQTS, thereby expanding the concept that variants within congenital LQTS loci can cause diLQTS.     

Characterization of the inhibitory effects of erythromycin and clarithromycin on the HERG potassium channel

Both erythromycin and clarithromycin have been reported to cause QT prolongation and the cardiac arrhythmia torsade de pointes in humans, however direct evidence documenting that these drugs produce this effect by blocking human cardiac ion channels is lacking. The goal of this study was to test the hypothesis that these macrolide antibiotics significantly block the delayed rectifier current (I_Kr) encoded by HERG (the human ether-a-go-go-related gene) at drug concentrations, temperature and ionic conditions mimicking those occurring in human subjects. Potassium currents in HEK 293 cells stably transfected with HERG were recorded using a whole cell voltage clamp method. Exposure of cells to erythromycin reduced the HERG encoded potassium current in a concentration dependent manner with an IC₅₀ of 38.9 ± 1.2 M and Hill Slope factor of 0.4 ± 0.1. Clarithromycin produced a similar concentration-dependent block with an IC₅₀ of 45.7 ± 1.1 M and Hill Slope factor of 1.0 ± 0.1. Erythromycin (25–250 M) and clarithromycin (5 or 25 M) also produced a significant decrease in the integral of the current evoked by an action potential shaped voltage clamp protocol. The results of this study document that both erythromycin and clarithromycin significantly inhibit the HERG potassium current at clinically relevant concentrations.     

Resveratrol Attenuates the Na+-Dependent Intracellular Ca2+ Overload by Inhibiting H2O2-Induced Increase in Late Sodium Current in Ventricular Myocytes

Background/Aims

Resveratrol has been demonstrated to be protective in the cardiovascular system. The aim of this study was to assess the effects of resveratrol on hydrogen peroxide (H₂O₂)-induced increase in late sodium current (I _Na.L) which augmented the reverse Na⁺-Ca²⁺ exchanger current (I _NCX), and the diastolic intracellular Ca²⁺ concentration in ventricular myocytes.

Methods

I _Na.L, I _NCX, L-type Ca²⁺ current (I _Ca.L) and intracellular Ca²⁺ properties were determined using whole-cell patch-clamp techniques and dual-excitation fluorescence photomultiplier system (IonOptix), respectively, in rabbit ventricular myocytes.

Results

Resveratrol (10, 20, 40 and 80 µM) decreased I _Na.L in myocytes both in the absence and presence of H₂O₂ (300 µM) in a concentration dependent manner. Ranolazine (3–9 µM) and tetrodotoxin (TTX, 4 µM), I _Na.L inhibitors, decreased I _Na.L in cardiomyocytes in the presence of 300 µM H₂O₂. H₂O₂ (300 µM) increased the reverse I _NCX and this increase was significantly attenuated by either 20 µM resveratrol or 4 µM ranolazine or 4 µM TTX. In addition, 10 µM resveratrol and 2 µM TTX significantly depressed the increase by 150 µM H₂O₂ of the diastolic intracellular Ca²⁺ fura-2 fluorescence intensity (FFI), fura-fluorescence intensity change (△FFI), maximal velocity of intracellular Ca²⁺ transient rise and decay. As expected, 2 µM TTX had no effect on I _Ca.L.

Conclusion

Resveratrol protects the cardiomyocytes by inhibiting the H₂O₂-induced augmentation of I _Na.L.and may contribute to the reduction of ischemia-induced lethal arrhythmias.     

Spatially Discordant Alternans and Arrhythmias in Tachypacing-Induced Cardiac Myopathy in Transgenic LQT1 Rabbits: The Importance of IKs and Ca2+ Cycling

BackgroundRemodeling of cardiac repolarizing currents, such as the downregulation of slowly activating K⁺ channels (I_Ks), could underlie ventricular fibrillation (VF) in heart failure (HF). We evaluated the role of I_ks remodeling in VF susceptibility using a tachypacing HF model of transgenic rabbits with Long QT Type 1 (LQT1) syndrome.ConclusionsCompared with LMC-TICM, LQT1-TICM rabbits exhibit steepened APD restitution and complex DA modulated by Ca²⁺. Our results strongly support the contention that the downregulation of I_Ks in HF increases Ca²⁺ dependent alternans and thereby the risk of VF.     

Modeling of Longitudinal Changes in Left Ventricular Dimensions among Female Adolescent Runners

Purpose

Left ventricular (LV) enlargement has been linked to sudden cardiac death among young athletes. This study aimed to model the effect of long-term incessant endurance training on LV dimensions in female adolescent runners.

Methods

Japanese female adolescent competitive distance runners (n = 36, age: 15 years, height: 158.1 ± 4.6 cm, weight: 44.7 ± 6.1 kg, percent body fat: 17.0 ± 5.2%) underwent echocardiography and underwater weighing every 6 months for 3 years. Since the measurement occasions varied across subjects, multilevel analysis was used for curvilinear modeling of changes in running performance (velocities in 1500 m and 3000 m track race), maximal oxygen uptake (VO₂max), body composition, and LV dimensions.

Results

Initially, LV end-diastolic dimension (LVEDd) and LV mass were 47.0 ± 3.0 mm and 122.6 ± 15.7 g, respectively. Running performance and VO₂max improved along with the training duration. The trends of changes in fat-free mass (FFM) and LVEDd were similarly best described by quadratic polynomials. LVEDd did not change over time in the model including FFM as a covariate. Increases in LV wall thicknesses were minimal and independent of FFM. LV mass increased according to a quadratic polynomial trend even after adjusting for FFM.

Conclusions

FFM was an important factor determining changes in LVEDd and LV mass. Although running performance and VO₂max were improved by continued endurance training, further LV cavity enlargement hardly occurred beyond FFM gain in these adolescent female runners, who already demonstrated a large LVEDd.     

Impaired cardiac and peripheral hemodynamic responses to inhaled β2-agonist in cystic fibrosis

Background

Pulmonary system dysfunction is a hallmark of cystic fibrosis (CF) disease. In addition to impaired cystic fibrosis transmembrane conductance regulator protein, dysfunctional β₂-adrenergic receptors (β₂AR) contribute to low airway function in CF. Recent observations suggest CF may also be associated with impaired cardiac function that is demonstrated by attenuated cardiac output (Q), stroke volume (SV), and cardiac power (CP) at both rest and during exercise. However, β₂AR regulation of cardiac and peripheral vascular tissue, in-vivo, is unknown in CF. We have previously demonstrated that the administration of an inhaled β-agonist increases SV and Q while also decreasing SVR in healthy individuals. Therefore, we aimed to assess cardiac and peripheral hemodynamic responses to the selective β₂AR agonist albuterol in individuals with CF.

Methods

18 CF and 30 control (CTL) subjects participated (ages 22 ± 2 versus 27 ± 2 and BSA = 1.7 ± 0.1 versus 1.8 ± 0.0 m², both p < 0.05). We assessed the following at baseline and at 30- and 60-minutes following nebulized albuterol (2.5mg diluted in 3.0mL of normal saline) inhalation: 12-lead ECG for HR, manual sphygmomanometry for systolic and diastolic blood pressure (SBP and DBP, respectively), acetylene rebreathe for Q and SV. We calculated MAP = DBP + 1/3(SBP–DBP); systemic vascular resistance (SVR) = (MAP/Q)•80; CP = Q•MAP; stroke work (SW) = SV•MAP; reserve (%change baseline to 30- or 60-minutes). Hemodynamics were indexed to BSA (Q_I, SV_I, SW_I, CP_I, SVR_I).

Results

At baseline, CF demonstrated lower SV, SV_I, SW, and SW_I but higher HR than CTL (p < 0.05); other measures did not differ. At 30-minutes, CF demonstrated higher HR and SVR_I, but lower Q, SV, SV_I, CP, CP_I, SW, and SW_I versus CTL (p < 0.05). At 60-minutes, CF demonstrated higher HR, SVR, and SVR_I, whereas all cardiac hemodynamics were lower than CTL (p < 0.05). Reserves of CP, SW, and SVR were lower in CF versus CTL at both 30 and 60-minutes (p < 0.05).

Conclusions

Cardiac and peripheral hemodynamic responsiveness to acute β₂AR stimulation via albuterol is attenuated in individuals with CF, suggesting β₂AR located in cardiac and peripheral vascular tissue may be dysfunctional in this population.     

The Interaction of Caveolin 3 Protein with the Potassium Inward Rectifier Channel Kir2.1: PHYSIOLOGY AND PATHOLOGY RELATED TO LONG QT SYNDROME 9 (LQT9)*

Mutations in CAV3 cause LQT syndrome 9 (LQT9). A previously reported LQT9 patient had prominent U waves on ECG, a feature that has been correlated with Kir2.1 loss of function. Our objective was to determine whether caveolin 3 (Cav3) associates with Kir2.1 and whether LQT9-associated CAV3 mutations affect the biophysical properties of Kir2.1. Kir2.1 current (I_K1) density was measured using the whole-cell voltage clamp technique. WT-Cav3 did not affect I_K1. However, F97C-Cav3 and T78M-Cav3 decreased I_K1 density significantly by ∼60%, and P104L-Cav3 decreased I_K1 density significantly by ∼30% at −60 mV. Immunostained rat heart cryosections and HEK293 cells cotransfected with Kir2.1 and WT-Cav3 both demonstrated colocalization of Kir2.1 and WT-Cav3 by confocal imaging. Cav3 coimmunoprecipitated with Kir2.1 in human ventricular myocytes and in heterologous expression systems. Additionally, FRET efficiency was highly specific, with a molecular distance of 5.6 ± 0.4 nm, indicating close protein location. Colocalization experiments found that Cav3 and Kir2.1 accumulated in the Golgi compartment. On-cell Western blot analysis showed decreased Kir2.1 cell surface expression by 60% when expressed with F97C-Cav3 and by 20% when expressed with P104L-Cav3 compared with WT-Cav3. This is the first report of an association between Cav3 and Kir2.1. The Cav3 mutations F97C-Cav3, P104L-Cav3, and T78M-Cav3 decreased I_K1 density significantly. This effect was related to a reduced cell surface expression of Kir2.1. Kir2.1 loss of function is additive to the increase described previously in late I_Na, prolonging repolarization and leading to arrhythmia generation in Cav3-mediated LQT9.