Frataxin deficiency alters gene expression in Friedreich ataxia derived IPSC-neurons and cardiomyocytes.

BACKGROUND: Friedreich's ataxia (FRDA) is an autosomal recessive disease, whereby homozygous inheritance of an expanded GAA trinucleotide repeat expansion in the first intron of the FXN gene leads to transcriptional repression of the encoded protein frataxin. FRDA is a progressive neurodegenerative disorder, but the primary cause of death is heart disease which occurs in 60% of the patients. Several functions of frataxin have been proposed, but none of them fully explain why its deficiency causes the FRDA phenotypes nor why the most affected cell types are neurons and cardiomyocytes. METHODS: To investigate, we generated iPSC-derived neurons (iNs) and cardiomyocytes (iCMs) from an FRDA patient and upregulated FXN expression via lentivirus without altering genomic GAA repeats at the FXN locus. RESULTS: RNA-seq and differential gene expression enrichment analyses demonstrated that frataxin deficiency affected the expression of glycolytic pathway genes in neurons and extracellular matrix pathway genes in cardiomyocytes. Genes in these pathways were differentially expressed when compared to a control and restored to control levels when FRDA cells were supplemented with frataxin. CONCLUSIONS: These results offer novel insight into specific roles of frataxin deficiency pathogenesis in neurons and cardiomyocytes.

Corrigendum: Phenotypic variability in iPSC-induced cardiomyocytes and cardiac fibroblasts carrying diverse LMNA mutations.

[This corrects the article DOI: 10.3389/fphys.2021.778982.].

Generation of a heterozygous FLNC mutation-carrying human iPSC line, USFi002-A, for modeling dilated cardiomyopathy.

Dilated Cardiomyopathy (DCM) is one of the main causes of sudden cardiac death and heart failure and is the leading indication for cardiac transplantation worldwide. Mutations in different genes including TTN, MYH7, and LMNA, have been linked to the development of DCM. Here, we generated a human induced pluripotent stem cell (IPSC) line from a DCM patient with a familial history that carries a frameshift mutation in Filamin C (FLNC). The IPSCs show typical morphology of pluripotent cells, expression of pluripotency markers, normal karyotype, and in vitro capacity to differentiate into all three germ layers.

Generation of a Friedreich's Ataxia patient-derived iPSC line USFi001-A.

Friedreich's Ataxia (FA) is an autosomal recessive disorder with an incidence of 1 in 50,000 in Caucasians. Most cases are caused by a biallelic GAA expansion in the first intron of the Frataxin (FXN) gene. FA is a neurodegenerative disease, but the leading cause of death is hypertrophic cardiomyopathy (HCM) that develops in 60% of the patients. We generated an induced pluripotent stem cell (iPSC) line from an FA patient with a homozygous GAA expansion in intron 1 of the FXN gene. The IPSCs display pluripotent cell morphology, expression of pluripotency markers, normal karyotype, and the capability to differentiate into all three germ layers.

Establishment of an arrhythmogenic right ventricular cardiomyopathy derived iPSC cell line (USFi004-A) carrying a heterozygous mutation in PKP2 (c.1799delA).

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an autosomal dominant inherited disease, with variable penetrance and expressivity. Currently, more than 14 different genetic loci have been reported for ARVC, the majority being desmosomal genes like Plakophilin-2 (PKP2). Here, we generated an iPSC cell line bearing a pathogenic heterozygous mutation in PKP2 (c.1799delA) from a patient affected by ARVC. Peripheral blood mononuclear cells were reprogrammed by Sendai virus vectors encoding KOS, KLF4, and c-MYC. Derived iPSCs expressed pluripotent markers, showed intact karyotype and demonstrated the ability to differentiate into three germ layers.

Generation of an iPSC cell line (USFi003-A) from a patient with dilated cardiomyopathy carrying a heterozygous mutation in LMNA (p.R541C).

Mutations in the gene that encodes the nuclear envelope proteins lamin A/C (LMNA) are considered to be a prominent cause of Dilated cardiomyopathy (DCM), a leading cause of heart failure and a prevalent indication for heart transplant. Here we described the generation of induced pluripotent stem cells (iPSCs) from a 53-year-old female with DCM plus progressive conduction disease who carry a heterozygous mutation in LMNA (c.1621C > T, p.R541C). PBMCs isolated from the patient were reprogrammed with Yamanaka factors KOS, KLF4, and c-MYC by the non-integrating sendai virus vector system. The obtained iPSC lines demonstrated normal karyotype and pluripotent identity.

Functional testing for variant prioritization in a family with long QT syndrome.

Next-generation sequencing platforms are being increasingly applied in clinical genetic settings for evaluation of families with suspected heritable disease. These platforms potentially improve the diagnostic yield beyond that of disease-specific targeted gene panels, but also increase the number of rare or novel genetic variants that may confound precise diagnostics. Here, we describe a functional testing approach used to interpret the results of whole exome sequencing (WES) in a family presenting with syncope and sudden death. One individual had a prolonged QT interval on electrocardiogram (ECG) and carried a diagnosis of long QT syndrome (LQTS), but a second individual did not meet criteria for LQTS. Filtering WES results for uncommon variants with arrhythmia association identified four for further analyses. In silico analyses indicated that two of these variants, KCNH2 p.(Cys555Arg) and KCNQ1 p.(Arg293Cys), were likely to be causal in this family's LQTS. We subsequently performed functional characterization of these variants in a heterologous expression system. The expression of KCNQ1-Arg293Cys did not show a deleterious phenotype but KCNH2-Cys555Arg demonstrated a loss-of-function phenotype that was partially dominant. Our stepwise approach identified a precise genetic etiology in this family, which resulted in the establishment of a LQTS diagnosis in the second individual as well as an additional asymptomatic family member, enabling personalized clinical management. Given its ability to aid in the diagnosis, the application of functional characterization should be considered as a value adjunct to in silico analyses of WES.

Phenotypic Variability in iPSC-Induced Cardiomyocytes and Cardiac Fibroblasts Carrying Diverse LMNA Mutations.

Mutations in the LMNA gene (encoding lamin A/C) are a significant cause of familial arrhythmogenic cardiomyopathy. Although the penetrance is high, there is considerable phenotypic variability in disease onset, rate of progression, arrhythmias, and severity of myopathy. To begin to address whether this variability stems from specific LMNA mutation sites and types, we generated seven patient-specific induced pluripotent stem cell (iPSC) lines with various LMNA mutations. IPSC-derived cardiomyocytes (iCMs) and cardiac fibroblasts (iCFs) were differentiated from each line for phenotypic analyses. LMNA expression and extracellular signal-regulated kinase pathway activation were perturbed to differing degrees in both iCMs and iCFs from the different lines. Enhanced apoptosis was observed in iCMs but not in iCFs. Markedly diverse irregularities of nuclear membrane morphology were present in iCFs but not iCMs, while iCMs demonstrated variable sarcomere disarray. Heterogenous electrophysiological aberrations assayed by calcium indicator imaging and multi-electrode array suggest differing substrates for arrhythmia that were accompanied by variable ion channel gene expression in the iCMs. Coculture studies suggest enhancement of the LMNA mutation effects on electrophysiological function exerted by iCFs. This study supports the utility of patient-specific iPSC experimental platform in the exploration of mechanistic and phenotypic heterogeneity of different mutations within a cardiac disease-associated gene. The addition of genetically defined coculture of cardiac-constituent non-myocytes further expands the capabilities of this approach.

Synonymous nucleotide modification of the KCNH2 gene affects both mRNA characteristics and translation of the encoded hERG ion channel.

Synonymous nucleotide variation is increasingly recognized as a factor than can affect protein expression, but the underlying mechanisms are incompletely understood. Here, we investigated whether synonymous changes could affect expression of the potassium voltage-gated channel subfamily H member 2 (KCNH2) gene, encoding the human ether-a-go-go-related gene (hERG) ion channel, which is linked to hereditary cardiac arrhythmia. We examined a previously described synthetic version (hERG-codon modified (CM)) with synonymous substitutions designed to reduce GC content, rare codons, and mRNA secondary structure relative to the native construct (hERG-NT). hERG-CM exhibited lower protein expression than hERG-NT in HEK293T cells. We found that the steady-state abundance of hERG-NT mRNA was greater than hERG-CM because of an enhanced transcription rate and increased mRNA stability for hERG-NT. Translation of hERG-CM was independently reduced, contributing to the overall greater synthesis of hERG-NT channel protein. This was partially offset, however, by a higher aggregation of a newly synthesized hERG-NT channel, resulting in nonfunctional protein. Regional mRNA analyses of chimeras of hERG-NT and hERG-CM revealed that synonymous changes in the 5' segments of the coding region had the greatest influence on hERG synthesis at both the mRNA and protein levels. Taken together, these results indicate that synonymous nucleotide variations within the coding region, particularly in the 5' region of the hERG mRNA, can affect both transcription and translation. These findings support the notion that greater attention should be given to the effects of synonymous genetic variation when analyzing hERG DNA sequences in the study of hereditary cardiac disease.

Express with caution: Epitope tags and cDNA variants effects on hERG channel trafficking, half-life and function.

INTRODUCTION: Genetic mutations in KCNH2, which encodes hERG, the alpha subunit of the potassium channel responsible for the IKr current, cause long QT syndrome (LQTS), an inherited cardiac arrhythmia disorder. Electrophysiology techniques are used to correlate genotype with molecular phenotype to determine which mutations identified in patients diagnosed with LQTS are disease causing, and which are benign. These investigations are usually done using heterologous expression in cell lines, and often, epitope fusion tags are used to enable isolation and identification of the protein of interest. METHODS AND RESULTS: Here, we demonstrate through electrophysiology techniques and immunohistochemistry, that both N-terminal and C-terminal myc fusion tags may perturb hERG protein channel expression and kinetics of the IKr current. We also characterize the impact of 2 previously reported inadvertent cDNA variants on hERG channel expression and half-life. CONCLUSION: Our results underscore the importance of careful characterization of the impact of epitope fusion tags and of confirming complete sequence accuracy prior to genotype-phenotype studies for ion channel proteins such as hERG.

Frizzled-4 C-terminus Distal to KTXXXW Motif is Essential for Normal Dishevelled Recruitment and Norrin-stimulated Activation of Lef/Tcf-dependent Transcriptional Activation.

The carboxy (C)-termini of G protein coupled receptors (GPCR) dictate essential functions. The KTXXXW motif C-terminus of Frizzleds (FZD) has been implicated in recruitment of Dishevelled (DVL). Through study of FZD4 and its associated ligand Norrin, we report that a minimum of three residues distal to the KTXXXW motif in the C-terminal tail of Frizzled-4 are essential for DVL recruitment and robust Lef/Tcf-dependent transcriptional activation in response to Norrin.

The Intracellular Loop 2 F328S Frizzled-4 Mutation Implicated in Familial Exudative Vitreoretinopathy Impairs Dishevelled Recruitment.

Familial exudative vitreoretinopathy (FEVR) is a disease state characterized by aberrant retinal angiogenesis. Norrin-induced activation of Frizzled-4 (Fz4) has a major role in regulating beta-catenin levels in the eye that, in turn, modulate the blood retina barrier (BRB). Here we gain insight on the basis of the pathology of a FEVR implicated F328S Fz4 mutant by study. The receptor exhibits a substantially reduced ability to activate Lef/Tcf-dependent transcription. This impaired activation correlates with a decreased ability to stabilize and recruit Dishevelled-2 (Dvl2) to the cell surface. Aromaticity at position 328 of the intracellular loop 2 (iloop2) is revealed similarly as a prerequisite for Dvl2 recruitment to the Fz4. This aromaticity at 328 enables normal Norrin-induced canonical activation. The corresponding position in iloop2 of other Frizzleds likely functions in Dvl recruitment.

Probing the interaction of SR141716A with the CB1 receptor.

SR141716A binds selectively to the brain cannabinoid (CB1) receptor and exhibits a potent inverse agonist/antagonist activity. Although SR141716A, also known as rimonabant, has been withdrawn from the market due to severe side effects, there remains interest in some of its many potential medical applications. Consequently, it is imperative to understand the mechanism by which SR141716A exerts its inverse agonist activity. As a result of using an approach combining mutagenesis and molecular dynamics simulations, we determined the binding mode of SR141716A. We found from the simulation of the CB1-SR141716A complex that SR141716A projects toward TM5 to interact tightly with the major binding pocket, replacing the coordinated water molecules, and secures the Trp-356(6.48) rotameric switch in the inactive state to promote the formation of an extensive water-mediated H-bonding network to the highly conserved SLAXAD and NPXXY motifs in TM2/TM7. We identify for the first time the involvement of the minor binding pocket formed by TM2/TM3/TM7 for SR141716A binding, which complements the major binding pocket formed by TM3/TM5/TM6. Simulation of the F174(2.61)A mutant CB1-SR141716A complex demonstrates the perturbation of TM2 that attenuates SR141716A binding indirectly. These results suggest SR141716A exerts inverse agonist activity through the stabilization of both TM2 and TM5, securing the Trp-356(6.48) rotameric switch and restraining it from activation.

Identification of essential cannabinoid-binding domains: structural insights into early dynamic events in receptor activation.

The classical cannabinoid agonist HU210, a structural analog of (-)-Δ(9)-tetrahydrocannabinol, binds to brain cannabinoid (CB1) receptors and activates signal transduction pathways. To date, an exact molecular description of the CB1 receptor is not yet available. Utilizing the minor binding pocket of the CB1 receptor as the primary ligand interaction site, we explored HU210 binding using lipid bilayer molecular dynamics (MD) simulations. Among the potential ligand contact residues, we identified residues Phe-174(2.61), Phe-177(2.64), Leu-193(3.29), and Met-363(6.55) as being critical for HU210 binding by mutational analysis. Using these residues to guide the simulations, we determined essential cannabinoid-binding domains in the CB1 receptor, including the highly sought after hydrophobic pocket important for the binding of the C3 alkyl chain of classical and nonclassical cannabinoids. Analyzing the simulations of the HU210-CB1 receptor complex, the CP55940-CB1 receptor complex, and the (-)-Δ(9)-tetrahydrocannabinol-CB1 receptor complex, we found that the positioning of the C3 alkyl chain and the aromatic stacking between Trp-356(6.48) and Trp-279(5.43) is crucial for the Trp-356(6.48) rotamer change toward receptor activation through the rigid-body movement of H6. The functional data for the mutant receptors demonstrated reductions in potency for G protein activation similar to the reductions seen in ligand binding affinity for HU210.

Ligand Binding Sensitivity of the Extracellular Loop Two of the Cannabinoid Receptor 1.

The cannabinoid receptor one (CB1) is a class A G-protein-coupled receptor thought to bind ligands primarily within its helical bundle. Evidence suggests, however, that the extracellular domain may also play a role. We have previously shown that the C-terminus of the extracellular loop 2 of CB1 is important in binding some compounds; receptors with mutations in this region (F268W, P269A, H270A, and I271A) bound some agonists with severely reduced affinity relative to the wild-type receptor. In the present work, we examine the impact of these mutations on binding a chemically diverse set of ligands. The receptors, F268W and I271A, exhibited a greater sensitivity to binding the inverse agonists/antagonists SLV319, AVE1625, NESS0327 relative to P269A and H270A, suggesting that the Pro and His are not involved in binding those compounds. In contrast, binding of the agonists, BAY593074 and WIN55212-2, was diminished in all four receptors, suggesting the conformational unit contributed by all four residues is important. A more marked loss in binding was observed for agonists of the nonclassical (CP55940) and classical (HU-210, JWH061, JWH179) cannabinoid classes and for a silent antagonist derivative (O-2050), pointing to the critical nature of this region for binding both the bicyclic/tricyclic core and the alkyl chain of these derivatives. However, moving the location of the alkyl chain on a series of pyrazole analogues shows it can be better accommodated in certain locations (O-1255) than others (O-1302, O-1690) and underscores the involvement of residues F268 and I271.

Dual role of the second extracellular loop of the cannabinoid receptor 1: ligand binding and receptor localization.

The seven transmembrane alpha-helices of G protein-coupled receptors (GPCRs) are the hallmark of this superfamily. Intrahelical interactions are critical to receptor assembly and, for the GPCR subclass that binds small molecules, ligand binding. Most research has focused on identifying the ligand binding pocket within the helical bundle, whereas the role of the extracellular loops remains undefined. Molecular modeling of the cannabinoid receptor 1 (CB1) extracellular loop 2 (EC2), however, suggests that EC2 is poised for key interactions. To test this possibility, we employed alanine scanning mutagenesis of CB1 EC2 and identified two distinct regions critical for ligand binding, G protein coupling activity, and receptor trafficking. Receptors with mutations in the N terminus of EC2 (W255A, N256A) were retained in the endoplasmic reticulum and did not bind the agonist (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol (CP55940) or the inverse agonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(SR141716A). In contrast, the C terminus of EC2 differentiates agonist and inverse agonist; the P269A, H270A, and I271A receptors exhibited diminished binding for several agonists but bound inverse agonists SR141716A, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), and 4-[6-methoxy-2-(4-methoxyphenyl)benzofuran-3-carbonyl]benzonitrile (LY320135) with wild-type receptor affinity. The F268A receptor involving substitution in the Cys-X-X-X-Ar motif, displayed both impaired localization and ligand binding. Other amino acid substitutions at position 268 revealed that highly hydrophobic residues are required to accomplish both functions. It is noteworthy that a F268W receptor was trafficked to the cell surface yet displayed differential binding preference for inverse agonists comparable with the P269A, H270A, and I271A receptors. The findings are consistent with a dual role for EC2 in stabilizing receptor assembly and in ligand binding.