Human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs) have emerged as an exciting new tool for cardiac research and can serve as a preclinical platform for drug development and disease ...modeling studies. However, these aspirations are limited by current culture methods in which hPSC‐CMs resemble fetal human cardiomyocytes in terms of structure and function. Herein we provide a novel in vitro platform that includes patterned extracellular matrix with physiological substrate stiffness and is amenable to both mechanical and electrical analysis. Micropatterned lanes promote the cellular and myofibril alignment of hPSC‐CMs while the addition of micropatterned bridges enable formation of a functional cardiac syncytium that beats synchronously over a large two‐dimensional area. We investigated the electrophysiological properties of the patterned cardiac constructs and showed they have anisotropic electrical impulse propagation, as occurs in the native myocardium, with speeds 2x faster in the primary direction of the pattern as compared to the transverse direction. Lastly, we interrogated the mechanical function of the pattern constructs and demonstrated the utility of this platform in recording the strength of cardiomyocyte contractions. This biomimetic platform with electrical and mechanical readout capabilities will enable the study of cardiac disease and the influence of pharmaceuticals and toxins on cardiomyocyte function. The platform also holds potential for high throughput evaluation of drug safety and efficacy, thus furthering our understanding of cardiovascular disease and increasing the translational use of hPSC‐CMs.
A biomimetic cell culture platform that combines micropatterning with a physiologically relevant substrate stiffness was used with cardiomyocytes derived from pluripotent stem cells (hPSC‐CMs) to promote elongation and alignment of their internal cytoarchitecture and results in anisotropic conduction. This cell culture platform enables structural, mechanical, and electrical measurements to be made. Such multiparametric readouts are essential in order to increase the therapeutic and clinical relevance of hPSC‐CMs.
Calcium channel antagonists have diverse effects on cardiac electrophysiology. We studied the effects of verapamil, diltiazem, and nifedipine on HERG K+ channels that encode IKr in native heart ...cells. In our experiments, verapamil caused high-affinity block of HERG current (IC50=143.0 nmol/L), a value close to those reported for verapamil block of L-type Ca2+ channels, whereas diltiazem weakly blocked HERG current (IC50=17.3 micromol/L), and nifedipine did not block HERG current. Verapamil block of HERG channels was use and frequency dependent, and verapamil unbound from HERG channels at voltages near the normal cardiac cell resting potential or with drug washout. Block of HERG current by verapamil was reduced by lowering pHO, which decreases the proportion of drug in the membrane-permeable neutral form. N-methyl-verapamil, a membrane-impermeable, permanently charged verapamil analogue, blocked HERG channels only when applied intracellularly. Verapamil antagonized dofetilide block of HERG channels, which suggests that they may share a common binding site. The C-type inactivation-deficient mutations, Ser620Thr and Ser631Ala, reduced verapamil block, which is consistent with a role for C-type inactivation in high-affinity drug block, although the Ser620Thr mutation decreased verapamil block 20-fold more than the Ser631Ala mutation. Our findings suggest that verapamil enters the cell membrane in the neutral form to act at a site within the pore accessible from the intracellular side of the cell membrane, possibly involving the serine at position 620. Thus, verapamil shares high-affinity HERG channel blocking properties with other class III antiarrhythmic drugs, and this may contribute to its antiarrhythmic mechanism.
Background and purpose:
Fluoxetine (Prozac®) is a widely prescribed drug in adults and children, and it has an active metabolite, norfluoxetine, with a prolonged elimination time. Although uncommon, ...Prozac causes QT interval prolongation and arrhythmias; a patient who took an overdose of Prozac exhibited a prolonged QT interval (QTc 625 msec). We looked for possible mechanisms underlying this clinical finding by analysing the effects of fluoxetine and norfluoxetine on ion channels in vitro.
Experimental approach:
We studied the effects of fluoxetine and norfluoxetine on the electrophysiology and cellular trafficking of hERG K+ and SCN5A Na+ channels heterologously expressed in HEK293 cells.
Key results:
Voltage clamp analyses employing square pulse or ventricular action potential waveform protocols showed that fluoxetine and norfluoxetine caused direct, concentration‐dependent, block of hERG current (IhERG). Biochemical studies showed that both compounds also caused concentration‐dependent reductions in the trafficking of hERG channel protein into the cell surface membrane. Fluoxetine had no effect on SCN5A channel or HEK293 cell endogenous current. Mutations in the hERG channel drug binding domain reduced fluoxetine block of IhERG but did not alter fluoxetine's effect on hERG channel protein trafficking.
Conclusions and implications:
Our findings show that both fluoxetine and norfluoxetine at similar concentrations selectively reduce IhERG by two mechanisms, (1) direct channel block, and (2) indirectly by disrupting channel protein trafficking. These two effects are not mediated by a single drug binding site. Our findings add complexity to understanding the mechanisms that cause drug‐induced long QT syndrome.
British Journal of Pharmacology (2006) 149, 481–489. doi:10.1038/sj.bjp.0706892
Amino acid sequence variations in SCN5A are known to affect function of wild-type channels and also those with coexisting mutations; therefore, it is important to know the exact sequence and function ...of channels most commonly present in human myocardium. SCN5A was analyzed in control panels of human alleles, demonstrating that the existing clones (hH1, hH1a, hH1b) each contained a rare variant and thus none represented the common sequence. Confirming prior work, the H558R polymorphism was present in approximately 30% of subjects. Quantitative mRNA analysis from human hearts showed that a shorter 2015 amino acid splice variant lacking glutamine at position 1077 (Q1077del) made up 65% of the transcript in every heart examined. Age, sex, race, or structural heart disease did not affect this proportion of Q1077del. Estimated population frequencies for the four common variants were 25% SCN5A, 10% H558R, 45% Q1077del, and 20% H558R;Q1077del, where the reference sequence SCN5A is GenBank AC137587. When expressed in HEK-293 cells, these common variants had a more positive mid-point of the voltage dependence of inactivation than the standard clone hH1. Also, channels containing Q1077 expressed smaller currents. When H558R was present with Q1077 (H558R), current expression was profoundly reduced despite normal trafficking to the cell surface. Thus, four variant sequences for SCN5A are commonly present in human myocardium and they exhibit functional differences among themselves and with the previous standard clone. These results have implications for the choice of background sequence for experiments with heterologous expression systems, and possibly implications for electrophysiological function in vivo.
Fatal arrhythmias from occult long QT syndrome may be responsible for some cases of sudden infant death syndrome (SIDS). Because patients who have long QT syndrome with sodium channel gene (SCN5A) ...defects have an increased frequency of cardiac events during sleep, and a recent case is reported of a sporadic SCN5A mutation in an infant with near SIDS, SCN5A has emerged as the leading candidate ion channel gene for SIDS.
To determine the prevalence and functional properties of SCN5A mutations in SIDS.
Postmortem molecular analysis of 93 cases of SIDS or undetermined infant death identified by the Medical Examiner's Office of the Arkansas State Crime Laboratory between September 1997 and August 1999. Genomic DNA was extracted from frozen myocardium and subjected to SCN5A mutational analyses. Missense mutations were incorporated into the human heart sodium channel alpha subunit by mutagenesis, transiently transfected into human embryonic kidney cells, and characterized electrophysiologically.
Molecular and functional characterization of SCN5A defects.
Two of the 93 cases of SIDS possessed SCN5A mutations: a 6-week-old white male with an A997S missense mutation in exon 17 and a 1-month old white male with an R1826H mutation in exon 28. These 2 distinct mutations occurred in highly conserved regions of the sodium channel and were absent in 400 control patients (800 alleles). Functionally, the A997S and R1826H mutant channels expressed a sodium current characterized by slower decay and a 2- to 3-fold increase in late sodium current.
Approximately 2% of this prospective, population-based cohort of SIDS cases had an identifiable SCN5A channel defect, suggesting that mutations in cardiac ion channels may provide a lethal arrhythmogenic substrate in some infants at risk for SIDS.
The purpose of this study was to determine the prevalence and spectrum of nonsynonymous polymorphisms (amino acid variants) in the cardiac sodium channel among healthy subjects.
Pathogenic mutations ...in the cardiac sodium channel gene, SCN5A, cause approximately 15 to 20% of Brugada syndrome (BrS1), 5 to 10% of long QT syndrome (LQT3), and 2 to 5% of sudden infant death syndrome.
Using single-stranded conformation polymorphism, denaturing high-performance liquid chromatography, and/or direct DNA sequencing, mutational analysis of the protein-encoding exons of SCN5A was performed on 829 unrelated, anonymous healthy subjects: 319 black, 295 white, 112 Asian, and 103 Hispanic.
In addition to the four known common polymorphisms (R34C, H558R, S1103Y, and R1193Q), four relatively ethnic-specific polymorphisms were identified: R481W, S524Y, P1090L, and V1951L. Overall, 39 distinct missense variants (28 novel) were elucidated. Nineteen variants (49%) were found only in the black cohort. Only seven variants (18%) localized to transmembrane-spanning domains. Four variants (F1293S, R1512W, and V1951L cited previously as BrS1-causing mutations and S1787N previously published as a possible LQT3-causing mutation) were identified in this healthy cohort.
This study provides the first comprehensive determination of the prevalence and spectrum of cardiac sodium channel variants in healthy subjects from four distinct ethnic groups. This compendium of SCN5A variants is critical for proper interpretation of SCN5A genetic testing and provides an essential hit list of targets for future functional studies to determine whether or not any of these variants mediate genetic susceptibility for arrhythmias in the setting of either drugs or disease.
We have established stably transfected HEK 293 cell lines expressing high levels of functional human ether-a go-go-related gene (HERG) channels. We used these cells to study biochemical ...characteristics of HERG protein, and to study electrophysiological and pharmacological properties of HERG channel current at 35°C. HERG-transfected cells expressed an mRNA band at 4.0
kb. Western blot analysis showed two protein bands (155 and 135 kDa) slightly larger than the predicted molecular mass (127 kDa). Treatment with
N-glycosidase F converted both bands to smaller molecular mass, suggesting that both are glycosylated, but at different levels. HERG current activated at voltages positive to −50
mV, maximum current was reached with depolarizing steps to −10
mV, and the current amplitude declined at more positive voltages, similar to HERG channel current expressed in other heterologous systems. Current density at 35°C, compared with 23°C, was increased by more than twofold to a maximum of 53.4
±
6.5 pA/pF. Activation, inactivation, recovery from inactivation, and deactivation kinetics were rapid at 35°C, and more closely resemble values reported for the rapidly activating delayed rectifier K
+ current (
I
Kr) at physiological temperatures. HERG channels were highly selective for K
+. When we used an action potential clamp technique, HERG current activation began shortly after the upstroke of the action potential waveform. HERG current increased during repolarization to reach a maximum amplitude during phases 2 and 3 of the cardiac action potential. HERG contributed current throughout the return of the membrane to the resting potential, and deactivation of HERG current could participate in phase 4 depolarization. HERG current was blocked by low concentrations of E-4031 (IC
50 7.7 nM), a value close to that reported for
I
Kr in native cardiac myocytes. Our data support the postulate that HERG encodes a major constituent of
I
Kr and suggest that at physiological temperatures HERG contributes current throughout most of the action potential and into the postrepolarization period.
SCN5A encodes the alpha-subunit of the ion channel that carries Na current in human heart. From a human cardiac cDNA library we recloned SCN5A. The new clone hH1b differed from existing clones hH1 in ...four and from hH1a in three positions. The common polymorphism H558R was uniquely present in hH1b. Voltage clamp study showed minor but potentially important kinetic differences between hH1b and the other clones. More dramatically, when the LQT3 mutation M1766L was introduced into the different clones, Na current was markedly reduced in the hH1 and hH1a backgrounds, whereas in hH1b the Na current was not reduced. Immunocytochemistry experiments showed a trafficking defect for M1766L Na channels in hH1 and hH1a but not in hH1b. The double-mutation M1766L/H558R in the hH1a background restored normal trafficking and current including persistent late current, suggesting the disease phenotype was the result of a "double hit" that included the common polymorphism, H558R. These results show that the choice of background clone must be carefully considered in mutagenesis studies. This also represents an example of intragenic complementation, the first for such a large protein.
Electrophysiological remodeling of ion channels in heart failure causes action potential prolongation and plays a role in arrhythmia mechanism. The importance of down-regulation of potassium currents ...is well-known, but a role for Na current (
I
Na) in heart failure is less well established. We studied
I
Na in heart failure ventricular cells from a canine pacing model of heart failure and also from explanted failing human hearts. Peak
I
Na density was significantly decreased by 39% and 57% in the dog model and in human heart failure, respectively. The kinetics of peak
I
Na were not different in heart failure. Late
I
Na was measured 750 ms after the initial depolarization as the saxitoxin (STX)-sensitive current and also as the current remaining after contaminating currents were blocked. Late
I
Na as a percentage of the peak
I
Na was significantly increased in both conditions. In dogs, STX sensitive late
I
Na was 0.5 ± 0.1%
n = 16 cells from eight normal hearts and 3.4 ± 1.4%
n = 12 cells from seven failing hearts; in humans, it was 0.2 ± 0.1%
n = 4 cells from two normal hearts and 2.4 ± 0.5%
n = 10 cells from three human failing hearts (–40 mV). Quantitative measures of mRNA including RNase protection assays and real time quantitative PCR in the dog model showed no differences for different α subunit isoforms (NaV1.1, 1.3, 1.5) and for the β1 and β2 subunits. This suggests neither α subunit isoform switching nor altered β subunit expression is a mechanism for increased late
I
Na. We conclude that a peak
I
Na is decreased, and non-inactivating late
I
Na is increased in heart failure and this may contribute to action potential prolongation and the generation of arrhythmia.
To perform a molecular autopsy of the
RyR2-encoded cardiac ryanodine receptor/calcium release channel in medical examiner/coroner's cases of sudden unexplained death (SUD).
From September 1998 to ...March 2004, 49 cases of SUD were referred by medical examiners/coroners to the Sudden Death Genomics Laboratory at the Mayo Clinic in Rochester, Minn, for a cardiac channel molecular autopsy. Mutational analysis of 18 exons of
RyR2 implicated previously in the pathogenesis of catecholaminergic polymorphic ventricular tachycardia (CPVT) was performed on genomic DNA using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing.
This cohort of 49 cases of SUD included 30 males, 13 with a family history of syncope, cardiac arrest, or sudden cardiac death (mean ± SD age at death, 14.2±10.9 years). Six distinct
RyR2 missense mutations (3 novel) were discovered in 7 cases (14%, 6 males, mean ± SD age at death, 13.6±11.2 years) of SUD. The activities at the time of SUD were exertion (3), emotion (1), and unknown (3). The mutations, R420W, S2246L, N4097S, E4146K, T4158P, and R4497C, involved nonconservative amino acid substitutions in highly conserved residues across species and were not seen in 400 reference alleles.
This study represents the first molecular autopsy of
RyR2 in medical examiner-referred cases of SUD. A targeted analysis of only 18 of the 105 protein-encoding exons of the cardiac ryanodine receptor/calcium release channel revealed potential CPVT1-causing
RyR2 mutations in 1 of every 7 cases of SUD. These findings suggest that postmortem genetic testing of
RyR2 should be considered as a part of the comprehensive med-icolegal autopsy investigation of a SUD case and that this potentially heritable and often elusive arrhythmia syndrome be scrutinized carefully in family members of those who experience SUD.
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