Eva Delpón

Mutations in the cardiac L-type calcium channel associated with inherited J-wave syndromes and sudden cardiac death

BACKGROUND L-type calcium channel (LTCC) mutations have been associated with Brugada syndrome (BrS), short QT (SQT) syndrome, and Timothy syndrome (LQT8). Little is known about the extent to which LTCC mutations contribute to the J-wave syndromes associated with sudden cardiac death. OBJECTIVE The purpose of this study was to identify mutations in the α1, β2, and α2δ subunits of LTCC (Ca(v)1.2) among 205 probands diagnosed with BrS, idiopathic ventricular fibrillation (IVF), and early repolarization syndrome (ERS). CACNA1C, CACNB2b, and CACNA2D1 genes of 162 probands with BrS and BrS+SQT, 19 with IVF, and 24 with ERS were screened by direct sequencing. METHODS/RESULTS Overall, 23 distinct mutations were identified. A total of 12.3%, 5.2%, and 16% of BrS/BrS+SQT, IVF, and ERS probands displayed mutations in α1, β2, and α2δ subunits of LTCC, respectively. When rare polymorphisms were included, the yield increased to 17.9%, 21%, and 29.1% for BrS/BrS+SQT, IVF, and ERS probands, respectively. Functional expression of two CACNA1C mutations associated with BrS and BrS+SQT led to loss of function in calcium channel current. BrS probands displaying a normal QTc had additional variations known to prolong the QT interval. CONCLUSION The study results indicate that mutations in the LTCCs are detected in a high percentage of probands with J-wave syndromes associated with inherited cardiac arrhythmias, suggesting that genetic screening of Ca(v) genes may be a valuable diagnostic tool in identifying individuals at risk. These results are the first to identify CACNA2D1 as a novel BrS susceptibility gene and CACNA1C, CACNB2, and CACNA2D1 as possible novel ERS susceptibility genes.

Functional Effects of KCNE3 Mutation and Its Role in the Development of Brugada Syndrome

Background— The Brugada syndrome, an inherited syndrome associated with a high incidence of sudden cardiac arrest, has been linked to mutations in 4 different genes, leading to a loss of function in sodium and calcium channel activity. Although the transient outward current (Ito) is thought to play a prominent role in the expression of the syndrome, mutations in Ito-related genes have not been identified as yet. Methods and Results— One hundred five probands with the Brugada syndrome were screened for ion channel gene mutations using single-strand conformation polymorphism electrophoresis and direct sequencing. A missense mutation (R99H) in KCNE3 (MiRP2) was detected in 1 proband. The R99H mutation was found 4/4 phenotype-positive and 0/3 phenotype-negative family members. Chinese hamster ovary-K1 cells were cotransfected using wild-type (WT) or mutant KCNE3 and either WT KCND3 or KCNQ1. Whole-cell patch clamp studies were performed after 48 hours. Interactions between Kv4.3 and KCNE3 were analyzed in coimmunoprecipitation experiments in human atrial samples. Cotransfection of R99H-KCNE3 with KCNQ1 produced no alteration in tail current magnitude or kinetics. However, cotransfection of R99H KCNE3 with KCND3 resulted in a significant increase in the Ito intensity compared with WT KCNE3+KCND3. Using tissues isolated from the left atrial appendages of human hearts, we also demonstrate that Kv4.3 and KCNE3 can be coimmunoprecipitated. Conclusions— These results provide definitive evidence for a functional role of KCNE3 in the modulation of Ito in the human heart and suggest that mutations in KCNE3 can underlie the development of the Brugada syndrome.

PITX2 Insufficiency Leads to Atrial Electrical and Structural Remodeling Linked to Arrhythmogenesis

Background— Pitx2 is a homeobox transcription factor that plays a pivotal role in early left/right determination during embryonic development. Pitx2 loss-of-function mouse mutants display early embryonic lethality with severe cardiac malformations, demonstrating the importance of Pitx2 during cardiogenesis. Recently, independent genome-wide association studies have provided new evidence for a putative role of PITX2 in the adult heart. These studies have independently reported several risk variants close to the PITX2 locus on chromosome 4q25 that are strongly associated with atrial fibrillation in humans. Methods and Results— We show for the first time that PITX2C expression is significantly decreased in human patients with sustained atrial fibrillation, thus providing a molecular link between PITX2 loss of function and atrial fibrillation. In addition, morphological, molecular, and electrophysiological characterization of chamber-specific Pitx2 conditional mouse mutants reveals that atrial but not ventricular chamber-specific deletion of Pitx2 results in differences in the action potential amplitude and resting membrane potential in the adult heart as well as ECG characteristics of atrioventricular block. Lack of Pitx2 in atrial myocardium impairs sodium channel and potassium channel expression, mediated in part by miRNA misexpression. Conclusions— This study thus identifies Pitx2 as an upstream transcriptional regulator of atrial electric function, the insufficiency of which results in cellular and molecular changes leading to atrial electric and structural remodeling linked to arrhythmogenesis.

Molecular Determinants of Stereoselective Bupivacaine Block of hKv1.5 Channels

Enantiomers of local anesthetics are useful probes of ion channel structure that can reveal three-dimensional relations for drug binding in the channel pore and may have important clinical consequences. Bupivacaine block of open hKv1.5 channels is stereoselective, with the R(+)-enantiomer being 7-fold more potent than the S(-)-enantiomer (Kd = 4.1 mumol/L versus 27.3 mumol/L). Using whole-cell voltage clamp of hKv1.5 channels and site-directed mutants stably expressed in Ltk- cells, we have identified a set of amino acids that determine the stereoselectivity of bupivacaine block. Replacement of threonine 505 by hydrophobic amino acids (isoleucine, valine, or alanine) abolished stereoselective block, whereas a serine substitution preserved it [Kd = 60 mumol/L and 7.4 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. A similar substitution at the internal tetraethylammonium binding site (T477S) reduced the affinity for both enantiomers similarly, thus preserving the stereoselectivity [Kd = 45.5 mumol/L and 7.8 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. Replacement of L508 or V512 by a methionine (L508M and V512M) abolished stereoselective block, whereas substitution of V512 by an alanine (V512A) preserved it. Block of Kv2.1 channels, which carry valine, leucine, and isoleucine residues at T505, L508, and V512 equivalent sites, respectively, was not stereoselective [Kd = 8.3 mumol/L and 13 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. These results suggest that (1) the bupivacaine binding site is located in the inner mouth of the pore, (2) stereoselective block displays subfamily selectivity, and (3) a polar interaction with T505 combined with hydrophobic interactions with L508 and V512 are required for stereoselective block.

Losartan and Its Metabolite E3174 Modify Cardiac Delayed Rectifier K+ Currents

BACKGROUND The effects of type 1 angiotensin II receptor antagonist losartan and its metabolite E3174 on transmembrane action potentials, hKv1.5, HERG, and I(Ks) currents were analyzed. METHODS AND RESULTS Guinea pig ventricular action potentials were recorded with microelectrode techniques and hKv1.5 and HERG currents with the whole-cell patch-clamp technique. I(Ks) was recorded in guinea pig ventricular myocytes with the perforated-nystatin-patch configuration. Losartan and E3174 transiently increased the hKv1.5 current by 8.0+/-1.4% and 7.4+/-1.6%, respectively. Thereafter, they produced a voltage-dependent block, E3174 being more potent than losartan (P<0.05) for this effect. Losartan decreased HERG currents elicited at 0 mV (23.3+/-4.8%), whereas E3174 increased the current (30.5+/-6.2%). Both drugs shifted the midpoint of the activation curve of HERG channels to more negative potentials. In ventricular myocytes, losartan and E3174 inhibited the I(Ks) (18.4+/-3.2% and 6. 5+/-0.7%, respectively). Losartan-induced block was voltage-independent, whereas E3174 shifted the midpoint of the activation curve to more negative potentials. Losartan lengthened the duration of the action potentials at both 50% and 90% of repolarization, whereas E3174 slowed only the final phase of the repolarization process. CONCLUSIONS These results demonstrated that at therapeutic concentrations, both losartan and E3174 modified the cardiac delayed rectifier hKv1.5, HERG, and Ks currents.

Cardiac electrophysiological effects of nitric oxide

Nitric oxide (NO) synthetized by essentially all cardiac cell types plays a key role in the regulation of cardiac function. Recent evidence shows that NO modulates the activity of cardiac ion channels implicated in the genesis of the cardiac action potential and exerts anti-arrhythmic properties under some circumstances. We review the effects of NO on cardiac ion channels and the signalling pathways, including cGMP-dependent (protein kinase G and cGMP-regulated phosphodiesterases) and cGMP-independent mechanisms (S-nitrosylation and direct effects on G proteins) and finally the role of NO in the genesis of cardiac arrhythmias during ischemia-reperfusion, heart failure, long QT syndrome, atrial fibrillation, and sudden cardiac death.

Spironolactone and Its Main Metabolite, Canrenoic Acid, Block Human Ether-a-Go-Go-Related Gene Channels

Background—It has been demonstrated that spironolactone (SP) decreases the QT dispersion in chronic heart failure. In this study, the effects of SP and its metabolite, canrenoic acid (CA), on human ether-a-go-go–related gene (HERG) currents were analyzed. Methods and Results—HERG currents elicited in stably transfected Chinese hamster ovary cells were measured with the whole-cell patch-clamp technique. SP decreased HERG currents in a concentration-dependent manner (IC50=23.0±1.5 &mgr;mol/L) and shifted the midpoint of the activation curve to more negative potentials (Vh=−13.1±3.4 versus −18.9±3.6 mV, P <0.05) without modifying the activation and deactivation kinetics. SP-induced block (1 &mgr;mol/L) appeared at the range of membrane potentials coinciding with that of channel activation, and thereafter, it remained constant, reaching 24.7±3.8% at +60 mV (n=6, P <0.05). CA (0.01 nmol/L to 500 &mgr;mol/L) blocked HERG channels in a voltage- and frequency-independent manner. CA at 1 nmol/L shifted the midpoint of the activation curve to −19.9±1.8 mV and accelerated the time course of channel activation (&tgr;=1064±125 versus 820±93 ms, n=11, P <0.01). The envelope of the tail test demonstrated that at the very beginning of the pulses to +40 mV (25 ms), a certain amount of block was apparent (31.3±9.9%). CA did not modify the voltage-dependence of HERG channel inactivation (Vh=−60.8±5.6 versus −62.9±3.1 mV, n=6, P >0.05) or the kinetics of the reactivation process at any potential tested. CA and aldosterone also blocked the native IKr in guinea-pig ventricular myocytes. Conclusions—At concentrations reached after administration of therapeutic doses of SP, CA blocked the HERG channels by binding to both the closed and open states of the channel.

IKur/Kv1.5 channel blockers for the treatment of atrial fibrillation

Atrial fibrillation (AF) is the most common sustained arrhythmia. Anti-arrhythmic drugs remain the mainstay of therapy, but the available class I and III anti-arrhythmic drugs are only moderately effective in long-term restoring/maintaining sinus rhythm (SR) and can produce potentially fatal ventricular pro-arrhythmia. In an attempt to identify safer and more effective anti-arrhythmic drugs, drug discovery efforts have focused on ‘atrial selective drugs’ that target cardiac ion channel(s) that are exclusively or predominantly expressed in the atria. The ultra-rapid activating delayed rectifier K+ current (IKur), carried by Kv1.5 channels, is a major repolarizing current in human atria, but seems to play no role in the ventricle. This finding offers the possibility of developing selective IKur blockers to restore and maintain SR without a risk of ventricular pro-arrhythmia. Several IKur blockers are now being developed but clinical data are still limited, so the precise role of these agents in the treatment of AF remains to be defined. In this review we analyze the possible advantages and disadvantages of the developmental IKur blockers as they represent the first step for the development of potential atrial selective drugs for a more effective and safer treatment and prevention of AF.

Effects of levobupivacaine, ropivacaine and bupivacaine on HERG channels: stereoselective bupivacaine block

Levobupivacaine and ropivacaine are the pure S(−) enantiomers of N‐butyl‐ and N‐propyl‐2′,6′‐pipecoloxylidide, developed as less cardiotoxic alternatives to bupivacaine. In the present study, we have analysed the effects of levobupivacaine, ropivacaine and bupivacaine on HERG channels stably expressed in CHO cells. The three drugs blocked HERG channels in a concentration‐, time‐ and state‐dependent manner. Block measured at the end of 5 s pulses to −10 mV induced by 20 μM bupivacaine (52.7±2.0%, n=15) and ropivacaine (55.5±2.7%, n=13) was similar (P>0.05) and both lower than that induced by levobupivacaine (67.5±4.2%, n=11) (P<0.05). Dextrobupivacaine (20 μM) was less potent (47.2±5.2%, n=10) than levobupivacaine (P<0.05), indicating stereoselective HERG channel block. Block induced by the three local anaesthetics exhibited a steep voltage dependence in the range of channel activation. In all cases, block measured at the maximum peak current at a test potential of 0 mV after promoting recovery from inactivation (I→O) was lower than that observed at the end of 5‐s pulses (I+O). Levobupivacaine, ropivacaine and bupivacaine accelerated HERG inactivation kinetics, slowed the recovery from inactivation and shifted the inactivation curve towards more negative membrane potentials. The three local anaesthetics induced a rapid time‐dependent decline after using a protocol that quickly activates HERG channels. All these results suggest that: (1) these drugs bind to the open and the inactivated states of HERG channels, (2) they stabilize HERG channels in the inactivated state, and (3) block induced by bupivacaine enantiomers is stereoselective.

Propafenone Preferentially Blocks the Rapidly Activating Component of Delayed Rectifier K+ Current in Guinea Pig Ventricular Myocytes : Voltage-Independent and Time-Dependent Block of the Slowly Activating Component

The effects of propafenone on the delayed rectifier K+ current were studied in guinea pig ventricular myocytes by using the patch-clamp technique. In these myocytes, this current consists of at least two components: a La(3+)-sensitive component activating rapidly with moderate depolarizations and a La(3+)-resistant current slowly activating at more positive potentials. In the absence of La3+ (when both components are present), propafenone inhibited the delayed outward current, its effects being more marked after weak than after strong depolarizations. Propafenone-induced block of the tail currents elicited on return to -30 mV was more marked after short than after long depolarizing pulses. In the presence of 1 mumol/L propafenone, the envelope-of-tails test was satisfied, thus indicating that at this concentration propafenone completely blocks the rapidly activating component. In the presence of La3+ (when only the slow component is present), the steady state inhibition induced by 5 mumol/L propafenone on both the maximum activated and the tail currents was independent of the test pulse voltage. Development of propafenone-induced block on the slowly activating component was very fast and linked to channel opening. In addition, the blockade appeared to be use dependent, with the rate constant of the onset kinetics at 2 Hz being 0.44 +/- 0.1 pulse-1. The recovery process from propafenone-induced block exhibited a time constant of 2.5 +/- 0.4 s. These results indicated that propafenone preferentially inhibits the rapidly activating component of the delayed rectifier and that it blocks in a voltage-independent and time-dependent manner the slow component of this current.