scholarly journals Functional study of a N-terminal CPVT mutation RyR2R420Q in patient specific hiPSC-CMs model

EP Europace ◽  
2021 ◽  
Vol 23 (Supplement_3) ◽  
Author(s):  
L Yin ◽  
P Joanne ◽  
R Perrier ◽  
P Gerbaud ◽  
P Lechene ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Calcium Transient ◽  
Polymorphic Ventricular Tachycardia ◽  
Functional Study ◽  
Patient Specific ◽  
Control Group ◽  
Structural Abnormality ◽  
Loss Of Function ◽  
Adrenergic Stimulation ◽  
Gain Of Function

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ANR (Agence Nationale de la Rercherche) Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a lethal genetic arrhythmia that manifests by syncope or sudden death in children and young adults under stress conditions without obvious cardiac structural abnormality. A novel CPVT mutation located in the RyR2 N terminal portion has been identified in a Spanish family (RyR2R420Q). According to the studies of RyR2 function in HEK293 cell line, this mutation presented gain of function at low cytosolic intracellular Ca2+ concentration ([Ca2+]i) and loss of function at high [Ca2+]i. Moreover, KI mice heterozygous for this mutation presented bradycardia and sino-atrial node (SAN) dysfunction. Here we generated induced pluripotent stem cell (hiPSC) from two brothers (one with mutation, the other without mutation as control) of this family and differentiated them into cardiomyocytes (hiPSC-CM). In order to verify that the differentiated cells were well cardiomyocytes, we did immunofluorescence labelling to detect the α-actinin expression and found that around 90% cells were α-actinin positive in both groups of hiPS-CMs. Then the calcium transient was studied by confocal microscopy and the action potential (AP) by micro-electrode technique. The characteristics of spontaneous AP of mutated cells were mostly similar to that of control cells, but more mutated cells presented proarrhythmic behaviors under adrenergic stimulation. hiPSC-CM are immature cardiomyocytes and contract spontaneously. In order to be able to analyze [Ca2+]i transient characteristics, we paced the cells at a constant rate of 1 Hz by field stimulation through two Pt electrodes. Sarcoplasmic reticulum (SR) Ca2+ load was estimated by rapid caffeine (10 mM) application. hiPSC-CMs from the RyR2R420Q carrier presented smaller SR Ca2+ load than those from the control person, whereas their fractional release (the [Ca2+]i transient normalized by the amount of Ca2+ stored in the SR) was higher than that in control group, indicating a gain-of-function mutation.  Even if SR Ca2+ load was smaller in RyR2R420Q cells, they often presented proarrhythmogenic behavior such as Ca2+ waves. The fact was further enhanced during β-adrenergic stimulation, pointing to this model as a valuable tool to study the CPVT disease in human cells.

scholarly journals CPVT-Associated Mutation P.G357S-RYR2 Promotes a Gain of Function in Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes (iPS-CM)

2020 ◽  
Vol 118 (3) ◽  
pp. 255a
Author(s):  
David Carreras ◽  
Rebecca Martinez-Moreno ◽  
Elisabet Selga ◽  
Ramon Brugada ◽  
Fabiana S. Scornik ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Gain Of Function ◽  
Induced Pluripotent

scholarly journals Mutation-specific differences in arrhythmias and drug responses in CPVT patients: simultaneous patch clamp and video imaging of iPSC derived cardiomyocytes

2019 ◽  
Vol 47 (2) ◽  
pp. 1067-1077 ◽  
Author(s):  
R. P. Pölönen ◽  
H. Swan ◽  
K. Aalto-Setälä
Keyword(s):  
Disease Modeling ◽  
Induced Pluripotent Stem Cell ◽  
Polymorphic Ventricular Tachycardia ◽  
Patient Specific ◽  
Electrophysiological Properties ◽  
Electrophysiological Measurements ◽  
Skin Biopsies ◽  
Induced Pluripotent ◽  
Almost All ◽  
And Control

AbstractCatecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disease characterized by arrhythmias under adrenergic stress. Mutations in the cardiac ryanodine receptor (RYR2) are the leading cause for CPVT. We characterized electrophysiological properties of CPVT patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying different mutations in RYR2 and evaluated effects of carvedilol and flecainide on action potential (AP) and contractile properties of hiPSC-CMs. iPSC-CMs were generated from skin biopsies of CPVT patients carrying exon 3 deletion (E3D) and L4115F mutation in RYR2. APs and contractile movement were recorded simultaneously from the same hiPSC-CMs. Differences in AP properties of ventricular like CMs were seen in CPVT and control CMs: APD90 of both E3D (n = 20) and L4115F (n = 25) CPVT CMs was shorter than in control CMs (n = 15). E3D-CPVT CMs had shortest AP duration, lowest AP amplitude, upstroke velocity and more depolarized diastolic potential than controls. Adrenaline had positive and carvedilol and flecainide negative chronotropic effect in all hiPSC CMs. CPVT CMs had increased amount of delayed after depolarizations (DADs) and early after depolarizations (EADs) after adrenaline exposure. E3D CPVT CMs had the most DADs, EADs, and tachyarrhythmia. Discordant negatively coupled alternans was seen in L4115F CPVT CMs. Carvedilol cured almost all arrhythmias in L4115F CPVT CMs. Both drugs decreased contraction amplitude in all hiPSC CMs. E3D CPVT CMs have electrophysiological properties, which render them more prone to arrhythmias. iPSC-CMs provide a unique platform for disease modeling and drug screening for CPVT. Combining electrophysiological measurements, we can gain deeper insight into mechanisms of arrhythmias.

Abstract 15566: Induced Pluripotent Stem Cell-Derived Cardiomyocytes Recapitulate Clinically Observed Refractoriness to Therapeutic β-Blockade in a Patient-Specific Model of Catecholaminergic Polymorphic Ventricular Tachycardia

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Marcela K Preininger ◽  
Rajneesh Jha ◽  
Qingling Wu ◽  
Monalisa Singh ◽  
Joshua T Maxwell ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ventricular Tachycardia ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Polymorphic Ventricular Tachycardia ◽  
Patient Specific ◽  
Blocker Therapy ◽  
Β Blocker ◽  
Induced Pluripotent

Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome characterized by diastolic store overload-induced Ca2+ waves during β-adrenergic receptor (β-AR) stimulation. Mysteriously, β-blockers are ineffective at abolishing stress-induced ventricular arrhythmias in ~25% of patients. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from these CPVT patients offer an attractive system for investigating the phenomenon, but it remains unknown whether iPSC-CMs can recapitulate clinically observed patient-specific drug responses. Hypothesis: This study assessed the hypothesis that patient-specific refractoriness to β-blocker therapy can be observed in vitro using CPVT iPSC-CMs. Methods: We generated iPSC-CMs from a control individual and a CPVT patient insensitive to the widely prescribed β-blocker nadolol, but responsive to flecainide, and compared the efficacy of the two drugs in vitro in diminishing diastolic Ca2+ waves and restoring Ca2+ spark parameters during β-AR stimulation. Results: In CPVT hiPSC-CMs (n = 34), β-AR agonism elicited intense diastolic Ca2+ waves and potentiated unduly frequent, large, and prolonged Ca2+ sparks compared to control iPSC-CMs (n = 12). Pursuant to the patient’s in vivo responses, nadolol-treated CPVT iPSC-CMs (n = 27) demonstrated inadequate improvement of Ca2+ handling defects during β-AR stimulation relative to flecainide-treated CPVT iPSC-CMs (n = 25). Nadolol showed no significant effect on the frequency of diastolic Ca2+ waves, but reduced mean amplitude by 50% (p < 0.0001). In contrast, flecainide reduced both frequency and amplitude by 83% (p < 0.001) and 72% (p < 0.0001), respectively. During nadolol treatment, Ca2+ spark frequency, width, and duration remained significantly altered, while flecainide restored all Ca2+ spark parameters to baseline levels. Conclusions: Clinically observed recalcitrance to β-blocker therapy in individuals with CPVT can be modeled in vitro using patient-derived iPSC-CMs. Furthermore, the efficacy of other drugs such as flecainide can be comparatively evaluated, supporting the use of patient-specific iPSC-CMs as a clinically-relevant implement of precision medicine.

Abstract 16472: Deciphering a Mechanistic Link Between Enhanced Late Sodium Current and Triggered Atrial Fibrillation Using Patient-specific and Gene-corrected Induced Pluripotent Stem Cell-derived Atrial Cardiomyocytes

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Liang HONG ◽  
Olivia T Ly ◽  
Hanna Chen ◽  
Arvind Sridhar ◽  
Meihong Zhang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Atrial Fibrillation ◽  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Sodium Current ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Gain Of Function ◽  
Late Sodium Current ◽  
Induced Pluripotent

Introduction: Gain-of-function mutations in SCN5A, which encodes the cardiac sodium channel, have been linked with familial atrial fibrillation (AF). However, the mechanistic link between the late sodium current (I Na,L ) and triggered arrhythmia remains unclear. Hypothesis: To characterize the electrophysiological (EP) phenotype of gain-of-function AF-linked SCN5A mutations, elucidate the underlying cellular mechanisms using patient-specific and gene-corrected (GC) induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs). Methods: We generated iPSC-aCMs from two families carrying SCN5A mutations (E428K and N470K) and control subjects. Whole-cell patch clamp and multi-electrode arrays were recorded to assess the EP phenotypes of the atrial iPSC-CMs. We corrected the E428K iPSC-aCMs using CRISPR/Cas9 gene editing approach (isogenic control). Results: The SCN5A mutation lines displayed abnormal EP properties including increased beating frequency and irregularity with triggered beats characteristic of AF ( Fig. 1 ). E428K iPSC-aCMs displayed spontaneous arrhythmogenic activity with beat-to-beat irregularity ( Fig. 1 A-D ) with the prolonged APD ( Fig. 1 E-H ) associated with enhanced I Na,L ( Fig. 1 I-L ). In contrast, expression of SCN5A -E428K in heterologous expression system failed to show enhanced I Na,L . The gene-corrected E428K iPSC-aCMs normalized the aberrant EP phenotype. Gene expression profiling revealed differential expression of calcium and potassium channel homeostasis and nitric oxide mediated signal transduction which could result in EP remodeling of atrial CMs. Conclusions: Patient-specific and gene-corrected iPSC-aCMs exhibited striking ex-vivo EP phenotype of an AF-causing SCN5A gain-of-function mutation that produced minimal changes in-vitro . We established a mechanistic link between enhanced I Na,L , ion channel remodeling and nitric oxide signaling pathways, and triggered AF.

scholarly journals A Type 2 Ryanodine Receptor Variant in the Helical Domain 2 Associated with an Impairment of the Adrenergic Response

2021 ◽  
Vol 11 (6) ◽  
pp. 579
Author(s):  
Malorie Blancard ◽  
Zahia Touat-Hamici ◽  
Yuriana Aguilar-Sanchez ◽  
Liheng Yin ◽  
Guy Vaksmann ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Missense Variant ◽  
Polymorphic Ventricular Tachycardia ◽  
Loss Of Function ◽  
Adrenergic Stimulation ◽  
Normal Response ◽  
Atypical Form ◽  
Helical Domain ◽  
Adrenergic Response

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is triggered by exercise or acute emotion in patients with normal resting electrocardiogram. The major disease-causing gene is RYR2, encoding the cardiac ryanodine receptor (RyR2). We report a novel RYR2 variant, p.Asp3291Val, outside the four CPVT mutation hotspots, in three CPVT families with numerous sudden deaths. This missense variant was first identified in a four-generation family, where eight sudden cardiac deaths occurred before the age of 30 in the context of adrenergic stress. All affected subjects harbored at least one copy of the RYR2 variant. Three affected sisters were homozygous for the variant. The same variant was found in two additional CPVT families. It is located in the helical domain 2 and changes a negatively charged amino acid widely conserved through evolution. Functional analysis of D3291V channels revealed a normal response to cytosolic Ca2+, a markedly reduced luminal Ca2+ sensitivity and, more importantly, an absence of normal response to 8-bromo-cAMP and forskolin stimulation in both transfected HEK293 and HL-1 cells. Our data support that the D3291V-RyR2 is a loss-of-function RyR2 variant responsible for an atypical form of CPVT inducing a mild dysfunction in basal conditions but leading potentially to fatal events through its unresponsiveness to adrenergic stimulation.

scholarly journals Functional Characterization of KCNMA1 mutation associated with dyskinesia, seizure, developmental delay, and cerebellar atrophy

2021 ◽  
Author(s):  
Emrah Yucesan ◽  
Beyza Goncu ◽  
Cemil Ozgul ◽  
Arda Kebapci ◽  
Ayca Dilruba Aslanger ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Cerebellar Atrophy ◽  
Bk Channels ◽  
Bk Channel ◽  
Functional Study ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Α Subunit ◽  
Truncating Mutation

Abstract KCNMA1 located on chromosome 10q22.3, encodes the pore-forming α subunit of the “Big K+” (BK) large conductance calcium and voltage-activated K + channel. BK channels are widely distributed across tissues, including both excitable and non excitable cells. Numerous evidence suggests the functional BK channel alterations produced by different KCNMA1 alleles may associate with different symptoms, such as paroxysmal non kinesigenic dyskinesia with gain of function and ataxia with loss of function. Functional classifications revealed two major patterns, gain of function and loss of function effects on channel properties in different cell lines. In the literature, two mutations have been shown to confer gain of function properties to BK channels: D434G and N995S. On the other hand 10 mutations have been classified as loss of function (S351Y,G354S, G356R, G375R, C413Y/N449fs, I663V, P805L, and D984N) or putative loss of function (premature truncation mutations: Y676Lfs*7 and Arg458Ter). In this study, we report the functional characterization of a variant which was previously reported the whole exome sequencing revealed bi-allelic nonsense variation (NM_001161352.1 (ENST00000286628.8):c.1372C > T; Arg458*) of the cytoplasmic domain of calcium-activated potassium channel subunit alpha-1 protein. To detect functional consequences of the variation immunostaining and electrophysiological studies were conducted. In this study, we conducted patch-clamp recordings on WT and R458X mutant cells. We found the gain of function effect for the mutation. This is the first functional study observing an increased current in the KCNMA1 gene resulting from a truncating mutation

Abstract 251: Modeling LMNA Associated Cardiomyopathy Using Patient-Specific Human Induced Pluripotent Stem Cells

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Hananeh Fonoudi ◽  
Jane Wilcox ◽  
Paul W Burridge
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Clinical Symptoms ◽  
Field Potential ◽  
Calcium Transient ◽  
Patient Specific ◽  
Control Group ◽  
Induced Pluripotent

Mutations in LMNA are the most prevalent cause of dilated cardiomyopathy (DCM), accounting for up to 5-10% all the familial DCM. LMNA encodes the lamin A/C proteins which form filamentous structures that underline nuclear envelop. Sudden cardiac death and arrhythmia are common in patients with LMNA mutations. Despite recent advancement in the field, still the exact mechanism that link the mutation in LMNA to the formation of DCM and arrythmia is still largely unknown. In this study, we have generated an in vitro model of LMNA associated DCM using patient specific human induced pluripotent stem cells (hiPSCs). A family with pathogenic deletion in LMNA gene (c. 1142-1157 + 1del17) and history of DCM were selected. hiPSCs were generated from 4 affected individuals in the family and 5 healthy individuals. hiPSCs were then directly differentiated into cardiomyocytes and assessed at day 30 post differentiation. Cardiomyocytes derived from LMNA variant patients showed significantly higher level of nuclear deformation compared to control group. Moreover, after 2 days of mechanical stress cardiomyocytes derived from LMNA variant patients showed significantly higher level of nuclear dysmorphism while the control group were not affected. Field potential analysis of cardiomyocytes derived from LMNA patients compared to controls using multielectrode array revealed significantly higher beat rate irregularity in LMNA variant group which was consistent with the clinical symptoms of the patients. Furthermore, calcium transient of the cardiomyocytes derived from LMNA variant patients were significantly different from control group. Finally, patch clamp analysis also proved our previous findings and showed electrophysiological abnormalities in patients’ cells. In summary our finding thus far shows significant electrophysiological differences between cardiomyocytes derived from LMNA variant patients and control group which could help to unravel the cellular mechanism underlying formation of arrhythmia in LMNA variant patients.

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