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BACKGROUND: DNA methylation acts as a mechanism of gene transcription regulation. It has recently gained attention as a possible therapeutic target in cardiac hypertrophy and heart failure. However, its exact role in cardiomyocytes remains controversial. Thus, we knocked out the main de novo DNA methyltransferase in cardiomyocytes, DNMT3A, in human induced pluripotent stem cells. Functional consequences of DNA methylation-deficiency under control and stress conditions were then assessed in human engineered heart tissue from knockout human induced pluripotent stem cell-derived cardiomyocytes. METHODS: DNMT3A was knocked out in human induced pluripotent stem cells by CRISPR/Cas9gene editing. Fibrin-based engineered heart tissue was generated from knockout and control human induced pluripotent stem cell-derived cardiomyocytes. Development and baseline contractility were analyzed by video-optical recording. Engineered heart tissue was subjected to different stress protocols, including serum starvation, serum variation, and restrictive feeding. Molecular, histological, and ultrastructural analyses were performed afterward. RESULTS: Knockout of DNMT3A in human cardiomyocytes had three main consequences for cardiomyocyte morphology and function: (1) Gene expression changes of contractile proteins such as higher atrial gene expression and lower MYH7/MYH6 ratio correlated with different contraction kinetics in knockout versus wild-type; (2) Aberrant activation of the glucose/lipid metabolism regulator peroxisome proliferator-activated receptor gamma was associated with accumulation of lipid vacuoles within knockout cardiomyocytes; (3) Hypoxia-inducible factor 1α protein instability was associated with impaired glucose metabolism and lower glycolytic enzyme expression, rendering knockout-engineered heart tissue sensitive to metabolic stress such as serum withdrawal and restrictive feeding. CONCLUSION: The results suggest an important role of DNA methylation in the normal homeostasis of cardiomyocytes and during cardiac stress, which could make it an interesting target for cardiac therapy.
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DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Epigenômica/métodos , Regulação da Expressão Gênica/genética , Miócitos Cardíacos/metabolismo , Engenharia Tecidual/métodos , Cardiomegalia/patologia , DNA Metiltransferase 3A , HumanosRESUMO
The discovery of RNA interference (RNAi) gave rise to the development of new nucleic acid-based technologies as powerful investigational tools and potential therapeutics. Mechanistic key details of RNAi in humans need to be deciphered yet, before such approaches take root in biomedicine and molecular therapy. We developed and validated an in silico-based model of siRNA-mediated RNAi in human cells in order to link in vitro-derived pre-steady state kinetic data with a quantitative and time-resolved understanding of RNAi on the cellular level. The observation that product release by Argonaute 2 is accelerated in the presence of an excess of target RNA in vitro inspired us to suggest an associative mechanism for the RNA slicer reaction where incoming target mRNAs actively promote dissociation of cleaved mRNA fragments. This novel associative model is compatible with high multiple turnover rates of RNAi-based gene silencing in living cells and accounts for target mRNA concentration-dependent enhancement of the RNAi machinery.
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Interferência de RNA , RNA Mensageiro/metabolismo , Proteínas Argonautas/metabolismo , Simulação por Computador , Células HeLa , Humanos , Cinética , Modelos Genéticos , RNA Interferente Pequeno/metabolismoRESUMO
The heterozygous mutation c.155G > T in GNB2 clinically leads to sinus bradycardia and sinus node dysfunction. Here, patient-specific skin fibroblasts of the mutation carrier were used for Sendai virus reprogramming into human induced-pluripotent stem cells (hiPSC). For generating the isogenic control cell line, a CRISPR/Cas9-mediated HDR-repair of the hiPSCs was carried out. Both generated cell lines (GNB2 SV5528, GNB2 K26) maintained a normal karyotype, cell morphology, pluripotency in immunofluoresence and RT-qPCR analysis. Both hiPSC-lines showed differentiation potential into all three germ layers. Differentiated cardiomyocytes of this isogenic set may pave the way for investigating pharmacological rescue strategies for sinus node dysfunction.
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Sistemas CRISPR-Cas , Células-Tronco Pluripotentes Induzidas , Mutação , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Sistemas CRISPR-Cas/genética , Heterozigoto , Linhagem Celular , Diferenciação Celular , Síndrome do Nó Sinusal/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismoRESUMO
Current therapies for Fabry disease are based on reversing intracellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosomal dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease, remains unclear. In this study, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/Cas9-mediated α-galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified α-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.
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Doença de Fabry , Podócitos , Humanos , Podócitos/patologia , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Doença de Fabry/genética , Doença de Fabry/tratamento farmacológico , Doença de Fabry/patologia , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo , alfa-Galactosidase/uso terapêutico , Rim/metabolismo , Triexosilceramidas/metabolismo , Triexosilceramidas/farmacologia , Triexosilceramidas/uso terapêuticoRESUMO
Kidney organoids are a promising model to study kidney disease, but their use is constrained by limited knowledge of their functional protein expression profile. Here, we define the organoid proteome and transcriptome trajectories over culture duration and upon exposure to TNFα, a cytokine stressor. Older organoids increase deposition of extracellular matrix but decrease expression of glomerular proteins. Single cell transcriptome integration reveals that most proteome changes localize to podocytes, tubular and stromal cells. TNFα treatment of organoids results in 322 differentially expressed proteins, including cytokines and complement components. Transcript expression of these 322 proteins is significantly higher in individuals with poorer clinical outcomes in proteinuric kidney disease. Key TNFα-associated protein (C3 and VCAM1) expression is increased in both human tubular and organoid kidney cell populations, highlighting the potential for organoids to advance biomarker development. By integrating kidney organoid omic layers, incorporating a disease-relevant cytokine stressor and comparing with human data, we provide crucial evidence for the functional relevance of the kidney organoid model to human kidney disease.
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Nefropatias , Fator de Necrose Tumoral alfa , Humanos , Fator de Necrose Tumoral alfa/metabolismo , Proteoma/metabolismo , Rim , Nefropatias/genética , Nefropatias/metabolismo , Organoides/metabolismoRESUMO
Single nucleotide polymorphisms (SNPs) in microRNAs (miRNAs) or their target sites (miR-SNPs) within the 3'-UTR of mRNAs are increasingly thought to play a major role in pathological dysregulation of gene expression. Here, we studied the functional role of miR-SNPs on miRNA-mediated post-transcriptional regulation of gene expression. First, analyses were performed on a SNP located in the miR-155 target site within the 3'-UTR of the Angiotensin II type 1 receptor (AGTR1; rs5186, A > C) mRNA. Second, a SNP in the 3'-UTR of the muscle RAS oncogene homolog (MRAS; rs9818870, C > T) mRNA was studied which is located outside of binding sites of miR-195 and miR-135. Using these SNPs we investigated their effects on local RNA structure, on local structural accessibility and on functional miRNA binding, respectively. Systematic computational RNA folding analyses of the allelic mRNAs in either case predicted significant changes of local RNA structure in the vicinity of the cognate miRNA binding sites. Consistently, experimental in vitro probing of RNA showing differential cleavage patterns and reporter gene-based assays indicated functional differences of miRNA-mediated regulation of the two AGTR1 and MRAS alleles. In conclusion, we describe a novel model explaining the functional influence of 3'-UTR-located SNPs on miRNA-mediated control of gene expression via SNP-related changes of local RNA structure in non-coding regions of mRNA. This concept substantially extends the meaning of disease-related SNPs identified in non protein-coding transcribed sequences within or close to miRNA binding sites.
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Regiões 3' não Traduzidas , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Interferência de RNA , RNA Mensageiro/genética , Sequência de Bases , Sítios de Ligação , Doenças Cardiovasculares/genética , Células HeLa , Humanos , MicroRNAs/metabolismo , MicroRNAs/fisiologia , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Termodinâmica , Proteínas ras/genéticaRESUMO
PURPOSE: Dilated cardiomyopathy (DCM) is a primary disorder of the cardiac muscle, characterised by dilatation of the left ventricle and contractile dysfunction. About 50% of DCM cases can be attributed to monogenic causes, whereas the aetiology in the remaining patients remains unexplained. METHODS: We report a family with two brothers affected by severe DCM with onset in the adolescent period. Using exome sequencing, we identified a homozygous premature termination variant in the MYZAP gene in both affected sibs. MYZAP encodes for myocardial zonula adherens protein - a conserved cardiac protein in the intercalated disc structure of cardiomyocytes. RESULTS: The effect of the variant was demonstrated by light and electron microscopy of the heart muscle and immunohistochemical and Western blot analysis of MYZAP protein in the heart tissue of the proband. Functional characterization using patient-derived induced pluripotent stem cell cardiomyocytes revealed significantly lower force and longer time to peak contraction and relaxation consistent with severe contractile dysfunction. CONCLUSION: We provide independent support for the role of biallelic loss-of-function MYZAP variants in dilated cardiomyopathy. This report extends the spectrum of cardiac disease associated with dysfunction of cardiac intercalated disc junction and sheds light on the mechanisms leading to DCM.
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MYBPC3 is the most frequently affected gene in hypertrophic cardiomyopathy (HCM), which is an autosomal-dominant cardiac disease caused by mutations in sarcomeric proteins. Bi-allelic truncating MYBPC3 mutations are associated with severe forms of neonatal cardiomyopathy. We reprogrammed skin fibroblasts from a HCM patient carrying a heterozygous MYBPC3 truncating mutation into human induced pluripotent stem cells (iPSC) and used CRISPR/Cas9 to generate bi-allelic MYBPC3 truncating mutation and isogenic control hiPSC lines. All lines expressed pluripotency markers, had normal karyotype and differentiated into endoderm, ectoderm and cardiomyocytes in vitro. This set of three lines provides a useful tool to study HCM pathomechanisms.
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Cardiomiopatia Hipertrófica , Células-Tronco Pluripotentes Induzidas , Alelos , Cardiomiopatia Hipertrófica/genética , Heterozigoto , Humanos , Mutação , Miócitos CardíacosRESUMO
The phospholamban (PLN) p.Arg14del mutation causes dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. Patient dermal fibroblasts were reprogrammed to hiPSC, isogenic controls were established by CRISPR/Cas9, and cardiomyocytes were differentiated. Mutant cardiomyocytes revealed significantly prolonged Ca2+ transient decay time, Ca2+ -load dependent irregular beating pattern, and lower force. Proteomic analysis revealed less endoplasmic reticulum (ER) and ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the ER and large lipid droplets in close association with mitochondria. Follow-up experiments confirmed impairment of the ER/mitochondria compartment. PLN p.Arg14del end-stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison with ischemic heart failure and non-failing donor heart samples. Transduction of PLN p.Arg14del EHTs with the Ca2+ -binding proteins GCaMP6f or parvalbumin improved the disease phenotype. This study identified impairment of the ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca2+ -scavenging, suggesting impaired local Ca2+ cycling as an important disease culprit.
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Transplante de Coração , Miócitos Cardíacos , Proteínas de Ligação ao Cálcio/metabolismo , Retículo Endoplasmático , Humanos , Mitocôndrias , Mutação , Miócitos Cardíacos/metabolismo , Proteômica , Doadores de TecidosRESUMO
Direct reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) requires a resetting of the epigenome in order to facilitate a cell fate transition. Previous studies have shown that epigenetic modifying enzymes play a central role in controlling induced pluripotency and the generation of iPSC. Here we show that RNF40, a histone H2B lysine 120 E3 ubiquitin-protein ligase, is specifically required for early reprogramming during induced pluripotency. Loss of RNF40-mediated H2B monoubiquitination (H2Bub1) impaired early gene activation in reprogramming. We further show that RNF40 contributes to tissue-specific gene suppression via indirect effects by controlling the expression of the polycomb repressive complex-2 histone methyltransferase component EZH2, as well as through more direct effects by promoting the resolution of H3K4me3/H3K27me3 bivalency on H2Bub1-occupied pluripotency genes. Thus, we identify RNF40 as a central epigenetic mediator of cell state transition with distinct functions in resetting somatic cell state to pluripotency.
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Células-Tronco Pluripotentes Induzidas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Diferenciação Celular/fisiologia , Reprogramação Celular/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Processamento de Proteína Pós-Traducional , Ubiquitina-Proteína Ligases/genéticaRESUMO
The reproducibility of stem cell research relies on the constant availability of quality-controlled cells. As the quality of human induced pluripotent stem cells (hiPSCs) can deteriorate in the course of a few passages, cell banking is key to achieve consistent results and low batch-to-batch variation. Here, we provide a cost-efficient route to generate master and working cell banks for basic research projects. In addition, we describe minimal protocols for quality assurance including tests for sterility, viability, pluripotency, and genetic integrity. © 2020 The Authors. Basic Protocol 1: Expansion of hiPSCs Basic Protocol 2: Cell banking of hiPSCs Support Protocol 1: Pluripotency assessment by flow cytometry Support Protocol 2: Thawing control: Viability and sterility Support Protocol 3: Potency, viral clearance, and pluripotency: Spontaneous differentiation and qRT-PCR Support Protocol 4: Identity: Short tandem repeat analysis.
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Criopreservação/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes/citologia , Linhagem Celular , Humanos , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease accompanied by structural and contractile alterations. We identified a rare c.740C>T (p.T247M) mutation in ACTN2, encoding α-actinin 2 in a HCM patient, who presented with left ventricular hypertrophy, outflow tract obstruction, and atrial fibrillation. We generated patient-derived human-induced pluripotent stem cells (hiPSCs) and show that hiPSC-derived cardiomyocytes and engineered heart tissues recapitulated several hallmarks of HCM, such as hypertrophy, myofibrillar disarray, hypercontractility, impaired relaxation, and higher myofilament Ca2+ sensitivity, and also prolonged action potential duration and enhanced L-type Ca2+ current. The L-type Ca2+ channel blocker diltiazem reduced force amplitude, relaxation, and action potential duration to a greater extent in HCM than in isogenic control. We translated our findings to patient care and showed that diltiazem application ameliorated the prolonged QTc interval in HCM-affected son and sister of the index patient. These data provide evidence for this ACTN2 mutation to be disease-causing in cardiomyocytes, guiding clinical therapy in this HCM family. This study may serve as a proof-of-principle for the use of hiPSC for personalized treatment of cardiomyopathies.
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Actinina/genética , Cardiomiopatia Hipertrófica/genética , Animais , Modelos Animais de Doenças , Humanos , Síndrome do QT Longo/genética , Mutação , Medicina de PrecisãoRESUMO
The enzymatic activities of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are strictly correlated with the dimeric forms of this vital retroviral enzyme. Accordingly, the development of inhibitors targeting the dimerization of RT represents a promising alternative antiviral strategy. Based on mutational studies, we applied a structure-based ligand design approach generating pharmacophoric models of the large subunit connection subdomain to possibly identify small molecules from the ASINEX database, which might interfere with the RT subunit interaction. Docking studies of the selected compounds identified several candidates, which were initially tested in an in vitro subunit association assay. One of these molecules (MAS0) strongly reduced the association of the two RT subunits p51 and p66. Most notably, the compound simultaneously inhibited both the polymerase as well as the RNase H activity of the retroviral enzyme, following preincubation with t(1/2) of about 2 h, indicative of a slow isomerization step. This step most probably represents a shift of the RT dimer equilibrium from an active to an inactive conformation. Taken together, to the best of our knowledge, this study represents the first successful rational screen for a small molecule HIV RT dimerization inhibitor, which may serve as attractive hit compound for the development of novel therapeutic agents.
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Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/química , HIV-1/enzimologia , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Cristalografia por Raios X , Dimerização , Desenho de Fármacos , Estabilidade Enzimática/efeitos dos fármacos , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , Ligantes , Mutação , Ligação Proteica/efeitos dos fármacos , Ribonuclease H do Vírus da Imunodeficiência Humana/genética , Ribonuclease H do Vírus da Imunodeficiência Humana/metabolismo , Especificidade por SubstratoRESUMO
Aptamers targeting reverse transcriptase (RT) from HIV-1 inhibit viral replication in vitro, presumably by competing with binding of the primer/template complex. This site is not targeted by the currently available small-molecule anti-HIV-1 RT inhibitors. We have identified SY-3E4, a small-molecule inhibitor of HIV-1 RT, by applying a screening assay that utilizes a reporter-ribozyme regulated by the anti-HIV-1 RT aptamer. SY-3E4 displaces the aptamer from the protein, selectively inhibits DNA-dependent, but not RNA-dependent, polymerase activity, and inhibits the replication of both the wild-type virus and a multidrug-resistant strain. Analysis of available structural data of HIV-1 and HIV-2 RTs rationalizes many of the observed characteristics of the inhibitory profiles of SY-3E4 and the aptamer and suggests a previously not considered region in these RTs as a target for antiviral therapy. Our study reveals unexplored ways for rapidly identifying alternative small-molecule target sites in proteins and illustrates strategies for overcoming resistance-conferring mutations with small molecules.
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Aptâmeros de Nucleotídeos , Farmacorresistência Viral , Linhagem Celular , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , HIV-1/enzimologia , HIV-1/fisiologia , Humanos , Modelos Moleculares , Replicação ViralRESUMO
Cell-penetrating peptides (CPPs) have evolved as promising new tools to deliver nucleic acids into cells. So far, the majority of these delivery systems require a covalent linkage between carrier and cargo. To exploit the higher flexibility of a non-covalent strategy, we focused on the characterisation of a novel carrier peptide termed MPGalpha, which spontaneously forms complexes with nucleic acids. Using a luciferase-targeted small interfering RNA (siRNA) as cargo, we optimised the conditions for MPGalpha-mediated transfection of mammalian cells. In this system, reporter gene activity could be inhibited up to 90% with an IC50 value in the sub-nanomolar range. As a key issue, we addressed the cellular uptake mechanism of MPGalpha/siRNA complexes applying various approaches. First, transfection of HeLa cells with MPGalpha/siRNA complexes in the presence of several inhibitors of endocytosis showed a significant reduction of the RNA interference (RNAi) effect. Second, confocal laser microscopy revealed a punctual intracellular pattern rather than a diffuse distribution of fluorescently labelled RNA-cargo. These data provide strong evidence of an endocytotic pathway contributing significantly to the uptake of MPGalpha/siRNA complexes. Finally, we quantified the intracellular number of siRNA molecules after MPGalpha-mediated transfection. The amount of siRNA required to induce half maximal RNAi was 10 000 molecules per cell. Together, the combination of methods provided allows for a detailed side by side quantitative analysis of cargo internalisation and related biological effects. Thus, the overall efficiency of a given delivery technique as well as the mechanism of uptake can be assessed.
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Peptídeos/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transfecção , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Endocitose , Genes Reporter , Células HeLa , HumanosRESUMO
Short oligonucleotides below 8-10 nt in length adopt relatively simple structures. Accordingly, they represent interesting and so far unexplored lead compounds as molecular tools and, potentially, for drug development as a rational improvement of efficacy seem to be less complex than for other classes of longer oligomeric nucleic acid. As a 'proof of concept', we describe the highly specific binding of the hexanucleotide UCGUGU (Hex-S3) to human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) as a model target. Ultraviolet (UV) cross-linking studies and competition experiments with primer/template substrates and a RT-directed aptamer suggest site-specific binding of Hex-S3 to the large subunit (p66) of the viral enzyme. The affinity of 5.3 muM is related to hexanucleotide-specific suppression of HIV-1 replication in human cells by up to three orders of magnitude indicating that Hex-S3 exerts specific and biologically relevant activity. Experimental evidence described here further suggests a systematic hexamer array-based search for new tools for molecular biology and novel lead compounds in nucleic acid-based drug development.
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Fármacos Anti-HIV/química , Transcriptase Reversa do HIV/antagonistas & inibidores , Oligorribonucleotídeos/química , Sítios de Ligação , Linhagem Celular , Desenho de Fármacos , Inibidores Enzimáticos/química , Transcriptase Reversa do HIV/química , HIV-1/fisiologia , Humanos , Oligodesoxirribonucleotídeos/química , Análise de Sequência com Séries de Oligonucleotídeos , Transfecção , Replicação ViralRESUMO
Despite the fact that non-viral nucleic acid delivery systems are generally considered to be less efficient than viral vectors, they have gained much interest in recent years due to their superior safety profile compared to their viral counterpart. Among these synthetic vectors are cationic polymers, branched dendrimers, cationic liposomes and cell-penetrating peptides (CPPs). The latter represent an assortment of fairly unrelated sequences essentially characterised by a high content of basic amino acids and a length of 10-30 residues. CPPs are capable of mediating the cellular uptake of hydrophilic macromolecules like peptides and nucleic acids (e.g. siRNAs, aptamers and antisense-oligonucleotides), which are internalised by cells at a very low rate when applied alone. Up to now, numerous sequences have been reported to show cell-penetrating properties and many of them have been used to successfully transport a variety of different cargos into mammalian cells. In recent years, it has become apparent that endocytosis is a major route of internalisation even though the mechanisms underlying the cellular translocation of CPPs are poorly understood and still subject to controversial discussions. In this review, we will summarise the latest developments in peptide-based cellular delivery of nucleic acid cargos. We will discuss different mechanisms of entry, the intracellular fate of the cargo, correlation studies of uptake versus biological activity of the cargo as well as technical problems and pitfalls.
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Gene therapy is a promising option for severe forms of genetic diseases. We previously provided evidence for the feasibility of trans-splicing, exon skipping, and gene replacement in a mouse model of hypertrophic cardiomyopathy (HCM) carrying a mutation in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C). Here we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from an HCM patient carrying a heterozygous c.1358-1359insC MYBPC3 mutation and from a healthy donor. HCM hiPSC-CMs exhibited â¼50% lower MYBPC3 mRNA and cMyBP-C protein levels than control, no truncated cMyBP-C, larger cell size, and altered gene expression, thus reproducing human HCM features. We evaluated RNA trans-splicing and gene replacement after transducing hiPSC-CMs with adeno-associated virus. trans-splicing with 5' or 3' pre-trans-splicing molecules represented â¼1% of total MYBPC3 transcripts in healthy hiPSC-CMs. In contrast, gene replacement with the full-length MYBPC3 cDNA resulted in â¼2.5-fold higher MYBPC3 mRNA levels in HCM and control hiPSC-CMs. This restored the cMyBP-C level to 81% of the control level, suppressed hypertrophy, and partially restored gene expression to control level in HCM cells. This study provides evidence for (1) the feasibility of trans-splicing, although with low efficiency, and (2) efficient gene replacement in hiPSC-CMs with a MYBPC3 mutation.
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Since the advent of the generation of human induced pluripotent stem cells (hiPSCs), numerous protocols have been developed to differentiate hiPSCs into cardiomyocytes and then subsequently assess their ability to recapitulate the properties of adult human cardiomyocytes. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) are often assessed in single-cell assays. A shortcoming of these assays is the limited ability to characterize the physiological parameters of cardiomyocytes, such as contractile force, due to random orientations. This protocol describes the differentiation of cardiomyocytes from hiPSCs, which occurs within 14 d. After casting, cardiomyocytes undergo 3D assembly. This produces fibrin-based engineered heart tissues (EHTs)-in a strip format-that generate force under auxotonic stretch conditions. 10-15 d after casting, the EHTs can be used for contractility measurements. This protocol describes parallel expansion of hiPSCs; standardized generation of defined embryoid bodies, growth factor and small-molecule-based cardiac differentiation; and standardized generation of EHTs. To carry out the protocol, experience in advanced cell culture techniques is required.