The various cardiac regions have specific action potential properties appropriate to their electrical specialization, resulting
from a specific pattern of ion-channel functional expression. The ...present study addressed regionally defined differential
ion-channel expression in the non-diseased human heart with a genomic approach. High-throughput real-time RT-PCR was used
to quantify the expression patterns of 79 ion-channel subunit transcripts and related genes in atria, ventricular epicardium
and endocardium, and Purkinje fibres isolated from 15 non-diseased human donor hearts. Two-way non-directed hierarchical clustering
separated atria, Purkinje fibre and ventricular compartments, but did not show specific patterns for epicardium versus endocardium, nor left- versus right-sided chambers. Genes that characterized the atria ( versus ventricles) included Cx40, Kv1.5 and Kir3.1 as expected, but also Cav1.3, Cav3.1, Cavα2δ2, Navβ1, TWIK1, TASK1 and HCN4.
Only Kir2.1, RyR2, phospholamban and Kv1.4 showed higher expression in the ventricles. The Purkinje fibre expression-portrait
( versus ventricle) included stronger expression of Cx40, Kv4.3, Kir3.1, TWIK1, HCN4, ClC6 and CALM1, along with weaker expression
of mRNA encoding Cx43, Kir2.1, KChIP2, the pumps/exchangers Na + ,K + -ATPase, NCX1, SERCA2, and the Ca 2 + -handling proteins RYR2 and CASQ2. Transcripts that were more strongly expressed in epicardium ( versus endocardium) included Cav1.2, KChIP2, SERCA2, CALM3 and calcineurin-α. Nav1.5 and Navβ1 were more strongly expressed in the
endocardium. For selected genes, RT-PCR data were confirmed at the protein level. This is the first report of the global portrait
of regional ion-channel subunit-gene expression in the non-diseased human heart. Our data point to significant regionally
determined ion-channel expression differences, with potentially important implications for understanding regional electrophysiology,
arrhythmia mechanisms, and responses to ion-channel blocking drugs. Concordance with previous functional studies suggests
that regional regulation of cardiac ion-current expression may be primarily transcriptional.
The Iroquois homeobox (Irx) homeodomain transcription factors are important for several aspects of embryonic development. In the developing heart, individual Irx genes are important for certain ...postnatal cardiac functions, including cardiac repolarization (Irx5) and rapid ventricular conduction (Irx3). Irx genes are expressed in dynamic and partially overlapping patterns in the developing heart. Here we show in mice that Irx3 and Irx5 have redundant function in the endocardium to regulate atrioventricular canal morphogenesis and outflow tract formation. Our data suggest that direct transcriptional repression of Bmp10 by Irx3 and Irx5 in the endocardium is required for ventricular septation. A postnatal deletion of Irx3 and Irx5 in the myocardium leads to prolongation of atrioventricular conduction, due in part to activation of expression of the Na(+) channel protein Nav1.5. Surprisingly, combined postnatal loss of Irx3 and Irx5 results in a restoration of the repolarization gradient that is altered in Irx5 mutant hearts, suggesting that postnatal Irx3 activity can be repressed by Irx5. Our results have uncovered complex genetic interactions between Irx3 and Irx5 in embryonic cardiac development and postnatal physiology.
I
current, a major component of cardiac repolarization, is mediated by human
-Related Gene (hERG, K
11.1) potassium channels. The blockage of these channels by pharmacological compounds is associated ...to drug-induced long QT syndrome (LQTS), which is a life-threatening disorder characterized by ventricular arrhythmias and defects in cardiac repolarization that can be illustrated using cardiomyocytes derived from human-induced pluripotent stem cells (hiPS-CMs). This study was meant to assess the modification in hiPS-CMs excitability and contractile properties by BeKm-1, a natural scorpion venom peptide that selectively interacts with the extracellular face of hERG, by opposition to reference compounds that act onto the intracellular face. Using an automated patch-clamp system, we compared the affinity of BeKm-1 for hERG channels with some reference compounds. We fully assessed its effects on the electrophysiological, calcium handling, and beating properties of hiPS-CMs. By delaying cardiomyocyte repolarization, the peptide induces early afterdepolarizations and reduces spontaneous action potentials, calcium transients, and contraction frequencies, therefore recapitulating several of the critical phenotype features associated with arrhythmic risk in drug-induced LQTS. BeKm-1 exemplifies an interesting reference compound in the integrated hiPS-CMs cell model for all drugs that may block the hERG channel from the outer face. Being a peptide that is easily modifiable, it will serve as an ideal molecular platform for the design of new hERG modulators displaying additional functionalities.
Human genetically inherited cardiac diseases have been studied mainly in heterologous systems or animal models, independent of patients' genetic backgrounds. Because sources of human cardiomyocytes ...(CMs) are extremely limited, the use of urine samples to generate induced pluripotent stem cell-derived CMs would be a noninvasive method to identify cardiac dysfunctions that lead to pathologies within patients' specific genetic backgrounds. The objective was to validate the use of CMs differentiated from urine-derived human induced pluripotent stem (UhiPS) cells as a new cellular model for studying patients' specific arrhythmia mechanisms.
Cells obtained from urine samples of a patient with long QT syndrome who harbored the HERG A561P gene mutation and his asymptomatic noncarrier mother were reprogrammed using the episomal-based method. UhiPS cells were then differentiated into CMs using the matrix sandwich method.UhiPS-CMs showed proper expression of atrial and ventricular myofilament proteins and ion channels. They were electrically functional, with nodal-, atrial- and ventricular-like action potentials recorded using high-throughput optical and patch-clamp techniques. Comparison of HERG expression from the patient's UhiPS-CMs to the mother's UhiPS-CMs showed that the mutation led to a trafficking defect that resulted in reduced delayed rectifier K(+) current (IKr). This phenotype gave rise to action potential prolongation and arrhythmias.
UhiPS cells from patients carrying ion channel mutations can be used as novel tools to differentiate functional CMs that recapitulate cardiac arrhythmia phenotypes.
Studies on animal models have shown that Irx5 is an important regulator of cardiac development and that it regulates ventricular electrical repolarization gradient in the adult heart. Mutations in ...IRX5 have also been linked in humans to cardiac conduction defects. In order to fully characterize the role of IRX5 during cardiac development and in cardiomyocyte function, we generated three genetically-modified human induced pluripotent stem cell lines: two knockout lines (heterozygous and homozygous) and a knockin HA-tagged line (homozygous).
Patients with HIV present with a higher prevalence of QT prolongation, of which molecular bases are still not clear. Among HIV proteins, Tat serves as a transactivator that stimulates viral genes ...expression and is required for efficient HIV replication. Tat is actively secreted into the blood by infected T-cells and affects organs such as the heart. Tat has been shown to alter cardiac repolarization in animal models but how this is mediated and whether this is also the case in human cells is unknown. In the present study, we show that Tat transfection in heterologous expression systems led to a decrease in hERG (underlying cardiac I
) and human KCNE1-KCNQ1 (underlying cardiac I
) currents and to an acceleration of their deactivation. This is consistent with a decrease in available phosphatidylinositol-(4,5)-bisphosphate (PIP
). A mutant Tat, unable to bind PIP
, did not reproduce the observed effects. In addition, WT-Tat had no effect on a mutant KCNQ1 which is PIP
-insensitive, further confirming the hypothesis. Twenty-four-hour incubation of human induced pluripotent stem cells-derived cardiomyocytes with Wild-type Tat reduced I
and accelerated its deactivation. Concordantly, this Tat incubation led to a prolongation of the action potential (AP) duration. Events of AP alternans were also recorded in the presence of Tat, and were exacerbated at a low pacing cycle length. Altogether, these data obtained on human K
channels both in heterologous expression systems and in human cardiomyocytes suggest that Tat sequesters PIP
, leading to a reduction of I
and I
, and provide a molecular mechanism for QT prolongation in HIV-infected patients.
Rapid electrical conduction in the His–Purkinje system tightly controls spatiotemporal activation of the ventricles. Although recent work has shed much light on the regulation of early specification ...and morphogenesis of the His–Purkinje system, less is known about how transcriptional regulation establishes impulse conduction properties of the constituent cells. Here we show that Iroquois homeobox gene 3 (Irx3) is critical for efficient conduction in this specialized tissue by antithetically regulating two gap junction–forming connexins (Cxs). Loss of Irx3 resulted in disruption of the rapid coordinated spread of ventricular excitation, reduced levels of Cx40, and ectopic Cx43 expression in the proximal bundle branches. Irx3 directly represses Cx43 transcription and indirectly activates Cx40 transcription. Our results reveal a critical role for Irx3 in the precise regulation of intercellular gap junction coupling and impulse propagation in the heart.
Catecholamine-induced QT prolongation (CIQTP) is an inherited cardiac disease characterized by a normal baseline ECG and a risk of sudden cardiac death by ventricular arrhythmia due to a QT ...prolongation that only appears during catecholergic stimulation, especially mental stress. Induced pluripotent stem cells (hiPSCs) were generated from peripheral blood mononuclear cells collected from two CIQTP-affected patients from two different families. These two hiPSC lines are a valuable model to study biological alterations due to CIQTP.
Four human induced pluripotent stem cell (hiPSC) lines have been generated from healthy control European donors, and validated. This resource represents a useful tool for stem cell-based research, as ...references for developmental studies and disease modeling linked to any type of human tissue and organ, in an ethnical-, sex- and age-matched context. They providea reliable in-vitro model for single cell- and tissue-based investigations, and are also a valuable tool for genome editing-based studies.
Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an exercise and emotional stress-induced life-threatening inherited heart rhythm disorder, characterized by an abnormal cellular ...calcium homeostasis. Most reported cases have been linked to mutations in the gene encoding the type 2 ryanodine receptor gene, RYR2. We generated induced pluripotent stem cells (hiPSCs) from peripheral blood mononuclear cells (PBMC) from three CPVT-affected patients, two of them carrying p.R4959Q mutation and one carrying p.Y2476D mutation. These generated hiPSC lines are a useful model to study pathophysiological consequences of RYR2 dysfunction in humans and the molecular basis of CPVT.
PDF
