Experimental Models of Brugada syndrome Sendfeld, Franziska; Selga, Elisabet; Scornik, Fabiana S ...
International journal of molecular sciences,
04/2019, Letnik:
20, Številka:
9
Journal Article
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Brugada syndrome is an inherited, rare cardiac arrhythmogenic disease, associated with sudden cardiac death. It accounts for up to 20% of sudden deaths in patients without structural cardiac ...abnormalities. The majority of mutations involve the cardiac sodium channel gene
and give rise to classical abnormal electrocardiogram with ST segment elevation in the right precordial leads V1 to V3 and a predisposition to ventricular fibrillation. The pathophysiological mechanisms of Brugada syndrome have been investigated using model systems including transgenic mice, canine heart preparations, and expression systems to study different
mutations. These models have a number of limitations. The recent development of pluripotent stem cell technology creates an opportunity to study cardiomyocytes derived from patients and healthy individuals. To date, only a few studies have been done using Brugada syndrome patient-specific iPS-CM, which have provided novel insights into the mechanisms and pathophysiology of Brugada syndrome. This review provides an evaluation of the strengths and limitations of each of these model systems and summarizes the key mechanisms that have been identified to date.
Brugada syndrome predisposes to sudden death due to disruption of normal cardiac ion channel function, yet our understanding of the underlying cellular mechanisms is incomplete. Commonly used ...heterologous expression models lack many characteristics of native cardiomyocytes and, in particular, the individual genetic background of a patient. Patient-specific induced pluripotent stem (iPS) cell-derived cardiomyocytes (iPS-CM) may uncover cellular phenotypical characteristics not observed in heterologous models. Our objective was to determine the properties of the sodium current in iPS-CM with a mutation in SCN5A associated with Brugada syndrome.
Dermal fibroblasts from a Brugada syndrome patient with a mutation in SCN5A (c.1100G>A, leading to Nav1.5_p.R367H) were reprogrammed to iPS cells. Clones were characterized and differentiated to form beating clusters and sheets. Patient and control iPS-CM were structurally indistinguishable. Sodium current properties of patient and control iPS-CM were compared. These results were contrasted with those obtained in tsA201 cells heterologously expressing sodium channels with the same mutation.
Patient-derived iPS-CM showed a 33.1–45.5% reduction in INa density, a shift in both activation and inactivation voltage-dependence curves, and faster recovery from inactivation. Co-expression of wild-type and mutant channels in tsA201 cells did not compromise channel trafficking to the membrane, but resulted in a reduction of 49.8% in sodium current density without affecting any other parameters.
Cardiomyocytes derived from iPS cells from a Brugada syndrome patient with a mutation in SCN5A recapitulate the loss of function of sodium channel current associated with this syndrome; including pro-arrhythmic changes in channel function not detected using conventional heterologous expression systems.
•iPS-CM were generated from a Brugada Syndrome patient who carries an SNV in SCN5A.•Patient-specific iPS-CM show a loss of function of the sodium current.•Use of iPS-CM uncovers changes in INa properties not apparent in tsA201 cells.
The
gene encodes the α-subunit of the voltage-gated cardiac sodium channel (Na
1.5), a key player in cardiac action potential depolarization. Genetic variants in protein-coding regions of the human
...have been largely associated with inherited cardiac arrhythmias. Increasing evidence also suggests that aberrant expression of the
gene could increase susceptibility to arrhythmogenic diseases, but the mechanisms governing
expression are not yet well understood. To gain insights into the molecular basis of
gene regulation, we used rat gastrocnemius muscle four days following denervation, a process well known to stimulate
expression. Our results show that denervation of rat skeletal muscle induces the expression of the adult cardiac
isoform. RNA-seq experiments reveal that denervation leads to significant changes in the transcriptome, with
amongst the fifty top upregulated genes. Consistent with this increase in expression, ChIP-qPCR assays show enrichment of H3K27ac and H3K4me3 and binding of the transcription factor Gata4 near the
promoter region. Also, Gata4 mRNA levels are significantly induced upon denervation. Genome-wide analysis of H3K27ac by ChIP-seq suggest that a super enhancer recently described to regulate
in cardiac tissue is activated in response to denervation. Altogether, our experiments reveal that similar mechanisms regulate the expression of
in denervated muscle and cardiac tissue, suggesting a conserved pathway for
expression among striated muscles.
Brugada syndrome (BrS) is an inherited cardiac arrhythmogenic disease that predisposes patients to sudden cardiac death. It is associated with mutations in SCN5A, which encodes the cardiac sodium ...channel alpha subunit (Na
1.5). BrS-related mutations have incomplete penetrance and variable expressivity within families.
The purpose of this study was to determine the role of patient-specific genetic background on the cellular and clinical phenotype among carriers of Na
1.5_p.V1525M.
We studied sodium currents from patient-specific human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and heterologously transfected human embryonic kidney (HEK) tsA201 cells using the whole-cell patch-clamp technique. We determined gene and protein expression by quantitative polymerase chain reaction, RNA sequencing, and western blot and performed a genetic panel for arrhythmogenic diseases.
Our results showed a large reduction in I
density in hiPSC-CM derived from 2 V1525M single nucleotide variant (SNV) carriers compared with hiPSC-CM derived from a noncarrier, suggesting a dominant-negative effect of the Na
1.5_p.V1525M channel. I
was not affected in hiPSC-CMs derived from a V1525M SNV carrier who also carries the Na
1.5_p.H558R polymorphism. Heterozygous expression of V1525M in HEK-293T cells produced a loss of I
function, not observed when this variant was expressed together with H558R. In addition, the antiarrhythmic drug mexiletine rescued I
function in hiPSC-CM. SCN5A expression was increased in the V1525M carrier who also expresses Na
1.5_p.H558R.
Our results in patient-specific hiPSC-CM point to a dominant-negative effect of Na
1.5_p.V1525M, which can be reverted by the presence of Na
1.5_p.H558R. Overall, our data points to a role of patient-specific genetic background as a determinant for incomplete penetrance in BrS.
Tissue-specific cells differentiated from patient-derived human induced pluripotent stem cells (hiPSC) are a relevant cellular model to study several diseases. We obtained a hiPSC line from skin ...fibroblasts of a patient affected by familial atrial fibrillation by nucleofection of non-integrating episomal vectors. The resulting hiPSC line displays a normal karyotype, expresses pluripotency surface markers and pluripotency genes, and differentiates into cells of the 3 germ layers. Therefore, it represents a reliable model to study the disease in a physiologically relevant cellular environment.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a difficult-to-diagnose cause of sudden cardiac death (SCD). We identified a family of 1400 individuals with multiple cases of CPVT, ...including 36 SCDs during youth.
We sought to identify the genetic cause of CPVT in this family, to preventively treat and clinically characterize the mutation-positive individuals, and to functionally characterize the pathogenic mechanisms of the mutation.
Genetic testing was performed for 1404 relatives. Mutation-positive individuals were preventively treated with β-blockers and clinically characterized with a serial exercise treadmill test (ETT) and Holter monitoring. In vitro functional studies included caffeine sensitivity and store overload-induced calcium release activity of the mutant channel in HEK293 cells.
We identified the p.G357S_RyR2 mutation, in the cardiac ryanodine receptor, in 179 family members and in 6 SCD cases. No SCD was observed among treated mutation-positive individuals over a median follow-up of 37 months; however, 3 relatives who had refused genetic testing (confirmed mutation-positive individuals) experienced SCD. Holter monitoring did not provide relevant information for CPVT diagnosis. One single ETT was unable to detect complex cardiac arrhythmias in 72% of mutation-positive individuals, though the serial ETT improved the accuracy. Functional studies showed that the G357S mutation increased caffeine sensitivity and store overload-induced calcium release activity under conditions that mimic catecholaminergic stress.
Our study supports the use of genetic testing to identify individuals at risk of SCD to undertake prophylactic interventions. We also show that the pathogenic mechanisms of p.G357S_RyR2 appear to depend on β-adrenergic stimulation.
Patient-derived induced pluripotent stem cells (iPSC) are a valuable approach to model cardiovascular diseases. We nucleofected non-integrating episomal vectors in skin fibroblasts of three family ...members carrying a single nucleotide variant (SNV) in SCN5A, which encodes the cardiac-type sodium channel, and of a related healthy control. The SNV SCN5A_c.4573G > A had been previously identified in a Brugada Syndrome patient. The resulting iPS cell lines differentiate into cells of the 3 germ layers, display normal karyotypes and express pluripotency surface markers and genes. Thus, they are a reliable source to study the effect of the identified mutation in a physiologically relevant environment.
The lysine-specific histone demethylase 1A (LSD1) also known as lysine (K)-specific demethylase 1A (KDM1A) is a central epigenetic regulator of metabolic reprogramming in obesity-associated diseases, ...neurological disorders, and cancer. Here, we evaluated the ability of oleacein, a biophenol secoiridoid naturally present in extra virgin olive oil (EVOO), to target LSD1. Molecular docking and dynamic simulation approaches revealed that oleacein could target the binding site of the LSD1 cofactor flavin adenosine dinucleotide with high affinity and at low concentrations. At higher concentrations, oleacein was predicted to target the interaction of LSD1 with histone H3 and the LSD1 co-repressor (RCOR1/CoREST), likely disturbing the anchorage of LSD1 to chromatin. AlphaScreen-based in vitro assays confirmed the ability of oleacein to act as a direct inhibitor of recombinant LSD1, with an IC
as low as 2.5 μmol/L. Further, oleacein fully suppressed the expression of the transcription factor SOX2 (SEX determining Region Y-box 2) in cancer stem-like and induced pluripotent stem (iPS) cells, which specifically occurs under the control of an LSD1-targeted distal enhancer. Conversely, oleacein failed to modify ectopic SOX2 overexpression driven by a constitutive promoter. Overall, our findings provide the first evidence that EVOO contains a naturally occurring phenolic inhibitor of LSD1, and support the use of oleacein as a template to design new secoiridoid-based LSD1 inhibitors.
Brugada Syndrome (BrS) is a familial disease associated with sudden cardiac death. A 20%-25% of BrS patients carry genetic defects that cause loss-of-function of the voltage-gated cardiac sodium ...channel. Thus, 70%-75% of patients remain without a genetic diagnosis. In this work, we identified a novel missense mutation (p.Asp211Gly) in the sodium beta2 subunit encoded by SCN2B, in a woman diagnosed with BrS. We studied the sodium current (INa) from cells coexpressing Nav1.5 and wild-type (beta2WT) or mutant (beta2D211G) beta2 subunits. Our electrophysiological analysis showed a 39.4% reduction in INa density when Nav1.5 was coexpressed with the beta2D211G. Single channel analysis showed that the mutation did not affect the Nav1.5 unitary channel conductance. Instead, protein membrane detection experiments suggested that beta2D211G decreases Nav1.5 cell surface expression. The effect of the mutant beta2 subunit on the INa strongly suggests that SCN2B is a new candidate gene associated with BrS.
ABSTRACT
Brugada Syndrome (BrS) is a familial disease associated with sudden cardiac death. A 20%–25% of BrS patients carry genetic defects that cause loss‐of‐function of the voltage‐gated cardiac ...sodium channel. Thus, 70%–75% of patients remain without a genetic diagnosis. In this work, we identified a novel missense mutation (p.Asp211Gly) in the sodium β2 subunit encoded by SCN2B, in a woman diagnosed with BrS. We studied the sodium current (INa) from cells coexpressing Nav1.5 and wild‐type (β2WT) or mutant (β2D211G) β2 subunits. Our electrophysiological analysis showed a 39.4% reduction in INa density when Nav1.5 was coexpressed with the β2D211G. Single channel analysis showed that the mutation did not affect the Nav1.5 unitary channel conductance. Instead, protein membrane detection experiments suggested that β2D211G decreases Nav1.5 cell surface expression. The effect of the mutant β2 subunit on the INa strongly suggests that SCN2B is a new candidate gene associated with BrS.
Brugada Syndrome (BrS) is a familial disease associated mainly to a loss‐of‐function of the cardiac sodium channel. In this work, we identified a novel missense mutation (p.Asp211Gly) in the sodium β2 subunit encoded by SCN2B, in a woman diagnosed with BrS. Our electrophysiological analysis showed that the β2D211G provoked a reduction in INa density by decreasing Nav1.5 cell surface expression. These results strongly suggest that SCN2B is a new candidate gene for BrS.
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