Disease modeling hypertrophic cardiomyopathy using CRISPR/Cas9 genome editing technology in human pluripotent stem cell-derived cardiomyocytes

Hypertrophic cardiomyopathy (HCM) is a prevalent genetic cardiovascular disease affecting 1:500 individuals whose cardiac function is deteriorated due to thickening of the left ventricle of the heart, mostly owing to mutations in sarcomeric genes. Modeling HCM in vitro using human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) offers promise to further investigate the disease mechanisms, towards the development of effective drugs. Herein, nickase CRISPR/Cas9 genome editing technology was harnessed to introduce the R453C pathological mutation in the MYH7 sarcomeric gene, in three healthy hPSC lines. Monoclonal hPSC lines generated displayed the mutation in one or both alleles, as confirmed by PCR-genotyping and Sanger sequencing. A monolayer cardiac differentiation protocol was applied to the generated hPSC lines, resulting in >90% cardiomyocyte purities, and expression of mutant allele(s) of the MYH7 gene was analysed by RT-PCR. High-content imaging analysis showed that mutant hPSC-CMs displayed higher expression of hypertrophic marker Brain Natriuretic Peptide (BNP), in comparison to isogenic controls. BNP expression was maximised by treatment with hypertrophic inducer Endothelin-1 and rescued by its antagonist Bosentan. Flow cytometry analysis revealed a mild increase in cell volume of mutant cardiomyocytes relative to their wild-type controls. Functional evaluation of gene-edited lines exposed higher mitochondrial respiration rates relative to the isogenic controls, with the same mitochondrial content, resulting in a trend towards oxidative stress. Further genome engineering to incorporate a calcium indicator in R453C-MYH7 lines enabled confocal line analysis of calcium transients. MYH7-mutant hPSC-CMs exhibited higher frequency of irregular events in comparison to the healthy control, faster calcium kinetics, and higher resting cytosolic calcium concentration. Integration of hPSC-CMs in Engineered Heart Tissues (EHTs) and subsequent analysis of contractile force showed that mutant lines had a hypo-contractile and negative clinotropic phenotype relative to their isogenic controls. Moreover, R453C-MYH7 hEHTs showed a more pronounced negative force-frequency relationship in comparison with the healthy lines. These phenotypes were not rescued by treatment with cardiac myosin activator Omecamtiv Mecarbil, suggesting that targeting other mechanisms indirectly related with the contractile apparatus may be a preferred route to attenuate the observed pathological changes. Finally, transcriptomic analysis of gene-edited lines showed up-regulation of genes associated with fetal gene program, hypertrophy, fibrosis, apoptosis and autophagy, indicating potential molecular mechanisms associated with the observed phenotypes and HCM progression. Overall, hPSC-CMs bearing the R453C-MYH7 mutation exhibit the main molecular and functional hallmarks of HCM, providing a physiologically-relevant platform that enables further dissection of disease mechanisms and promotes pharmacological intervention

High-Throughput Phenotyping Toolkit for Characterizing Cellular Models of Hypertrophic Cardiomyopathy in Vitro

Hypertrophic cardiomyopathy (HCM) is a prevalent and complex cardiovascular disease characterised by multifarious hallmarks, a heterogeneous set of clinical manifestations, and several molecular mechanisms. Various disease models have been developed to study this condition, but they often show contradictory results, due to technical constraints and/or model limitations. Therefore, new tools are needed to better investigate pathological features in an unbiased and technically refined approach, towards improving understanding of disease progression. Herein, we describe three simple protocols to phenotype cellular models of HCM in vitro, in a high-throughput manner where technical artefacts are minimized. These are aimed at investigating: (1) Hypertrophy, by measuring cell volume by flow cytometry; (2) HCM molecular features, through the analysis of a hypertrophic marker, multinucleation, and sarcomeric disarray by high-content imaging; and (3) mitochondrial respiration and content via the Seahorse™ platform. Collectively, these protocols comprise straightforward tools to evaluate molecular and functional parameters of HCM phenotypes in cardiomyocytes in vitro. These facilitate greater understanding of HCM and high-throughput drug screening approaches and are accessible to all researchers of cardiac disease modelling. Whilst HCM is used as an exemplar, the approaches described are applicable to other cellular models where the investigation of identical biological changes is paramount

Mechanotransduction in cardiac stem cells : role of YAP/TAZ in the cellular response to the microenvironment

Trabalho de investigação desenvolvido na Universidade do Porto. Instituto de Ciências Biomédicas Abel Salazar, e no National Institute for Materials Science(Tsukuba, Japan)Tese de mestrado integrado. Bioengenharia (Biotecnologia Molecular). Faculdade de Engenharia. Universidade do Porto. 201

Transfection of hPSC-Cardiomyocytes Using Viafect™ Transfection Reagent

Twenty years since their first derivation, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have shown promise in disease modelling research, while their potential for cardiac repair is being investigated. However, low transfection efficiency is a barrier to wider realisation of the potential this model system has to offer. We endeavoured to produce a protocol for improved transfection of hPSC-CMs using the ViafectTM reagent by Promega. Through optimisation of four essential parameters: (i) serum supplementation, (ii) time between replating and transfection, (iii) reagent to DNA ratio and (iv) cell density, we were able to successfully transfect hPSC-CMs to ~95% efficiencies. Transfected hPSC-CMs retained high purity and structural integrity despite a mild reduction in viability, and preserved compatibility with phenotyping assays of hypertrophy. This protocol greatly adds value to the field by overcoming limited transfection efficiencies of hPSC-CMs in a simple and quick approach that ensures sustained expression of transfected genes for at least 14 days, opening new opportunities in mechanistic discovery for cardiac-related disease

Modeling hypertrophic cardiomyopathy: Mechanistic insights and pharmacological intervention

Hypertrophic cardiomyopathy (HCM) is a prevalent and complex cardiovascular disease where cardiac dysfunction often associates with mutations in sarcomeric genes. Various models based on tissue explants, isolated cardiomyocytes, skinned myofibrils, and purified actin/myosin preparations have uncovered disease hallmarks, enabling the development of putative therapeutics, with some reaching clinical trials. Newly developed human pluripotent stem cell (hPSC)-based models could be complementary by overcoming some of the inconsistencies of earlier systems, whilst challenging and/or clarifying previous findings. In this article we compare recent progress in unveiling multiple HCM mechanisms in different models, highlighting similarities and discrepancies. We explore how insight is facilitating the design of new HCM therapeutics, including those that regulate metabolism, contraction and heart rhythm, providing a future perspective for treatment of HCM

Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour

Background: Diseases such as hypertrophic cardiomyopathy (HCM) can lead to severe outcomes including sudden death. The generation of human induced pluripotent stem cell (hiPSC) reporter lines can be useful for disease modelling and drug screening by providing physiologically relevant in vitro models of disease. The AAVS1 locus is cited as a safe harbour that is permissive for stable transgene expression, and hence is favoured for creating gene targeted reporter lines. Methods: We generated hiPSC reporters using a plasmid-based CRISPR/Cas9 nickase strategy. The first intron of PPP1R12C, the AAVS1 locus, was targeted with constructs expressing a genetically encoded calcium indicator (R-GECO1.0) or HOXA9-T2A-mScarlet reporter under the control of a pCAG or inducible pTRE promoter, respectively. Transgene expression was compared between clones before, during and/or after directed differentiation to mesodermal lineages. Results: Successful targeting to AAVS1 was confirmed by PCR and sequencing. Of 24 hiPSC clones targeted with pCAG-R-GECO1.0, only 20 expressed the transgene and in these, the percentage of positive cells ranged from 0% to 99.5%. Differentiation of a subset of clones produced cardiomyocytes, wherein the percentage of cells positive for R-GECO1.0 ranged from 2.1% to 93.1%. In the highest expressing R-GECO1.0 clones, transgene silencing occurred during cardiomyocyte differentiation causing a decrease in expression from 98.93% to 1.3%. In HOXA9-T2A-mScarlet hiPSC reporter lines directed towards mesoderm lineages, doxycycline induced a peak in transgene expression after two days but this reduced by up to ten-thousand-fold over the next 8-10 days. Nevertheless, for R-GECO1.0 lines differentiated into cardiomyocytes, transgene expression was rescued by continuous puromycin drug selection, which allowed the Ca 2+ responses associated with HCM to be investigated in vitro using single cell analysis. Conclusions: Targeted knock-ins to AAVS1 can be used to create reporter lines but variability between clones and transgene silencing requires careful attention by researchers seeking robust reporter gene expression

Mitochondrial DNA: Hotspot for potential gene modifiers regulating hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a prevalent and untreatable cardiovascular disease with a highly complex clinical and genetic causation. HCM patients bearing similar sarcomeric mutations display variable clinical outcomes, implying the involvement of gene modifiers that regulate disease progression. As individuals exhibiting mutations in mitochondrial DNA (mtDNA) present cardiac phenotypes, the mitochondrial genome is a promising candidate to harbor gene modifiers of HCM. Herein, we sequenced the mtDNA of isogenic pluripotent stem cell-cardiomyocyte models of HCM focusing on two sarcomeric mutations. This approach was extended to unrelated patient families totaling 52 cell lines. By correlating cellular and clinical phenotypes with mtDNA sequencing, potentially HCM-protective or -aggravator mtDNA variants were identified. These novel mutations were mostly located in the non-coding control region of the mtDNA and did not overlap with those of other mitochondrial diseases. Analysis of unrelated patients highlighted family-specific mtDNA variants, while others were common in particular population haplogroups. Further validation of mtDNA variants as gene modifiers is warranted but limited by the technically challenging methods of editing the mitochondrial genome. Future molecular characterization of these mtDNA variants in the context of HCM may identify novel treatments and facilitate genetic screening in cardiomyopathy patients towards more efficient treatment options

Isogenic models of hypertrophic cardiomyopathy unveil differential phenotypes and mechanism-driven therapeutics

Background: Hypertrophic cardiomyopathy (HCM) is a prevalent and complex cardiovascular condition. Despite being strongly associated with genetic alterations, wide variation of disease penetrance, expressivity and hallmarks of progression complicate treatment. We aimed to characterize different human isogenic cellular models of HCM bearing patient-relevant mutations to clarify genetic causation and disease mechanisms, hence facilitating the development of effective therapeutics. Methods: We directly compared the p.β-MHC-R453C and p.ACTC1-E99K HCM-associated mutations in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and their healthy isogenic counterparts, generated using CRISPR/Cas9 genome editing technology. By harnessing several state-of-the-art HCM phenotyping techniques, these mutations were investigated to identify similarities and differences in disease progression and hypertrophic signaling pathways, towards establishing potential targets for pharmacological treatment. CRISPR/Cas9 knock-in of the genetically-encoded calcium indicator R-GECO1.0 to the AAVS1 locus into these disease models resulted in calcium reporter lines. Results: Confocal line scan analysis identified calcium transient arrhythmias and intracellular calcium overload in both models. The use of optogenetics and 2D/3D contractility assays revealed opposing phenotypes in the two mutations. Gene expression analysis highlighted upregulation of CALM1, CASQ2 and CAMK2D, and downregulation of IRF8 in p.β-MHC-R453C mutants, whereas the opposite changes were detected in p.ACTC1-E99K mutants. Contrasting profiles of nuclear translocation of NFATc1 and MEF2 between the two HCM models suggest differential hypertrophic signaling pathway activation. Calcium transient abnormalities were rescued with combination of dantrolene and ranolazine, whilst mavacamten reduced the hyper-contractile phenotype of p.ACTC1-E99K hiPSC-CMs. Conclusions: Our data show that hypercontractility and molecular signaling within HCM are not uniform between different gene mutations, suggesting that a ‘one-size fits all’ treatment underestimates the complexity of the disease. Understanding where the similarities (arrhythmogenesis, bioenergetics) and differences (contractility, molecular profile) lie will allow development of therapeutics that are directed towards common mechanisms or tailored to each disease variant, hence providing effective patient-specific therapy

Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour

Background: Diseases such as hypertrophic cardiomyopathy (HCM) can lead to severe outcomes including sudden death. The generation of human induced pluripotent stem cell (hiPSC) reporter lines can be useful for disease modelling and drug screening by providing physiologically relevant in vitro models of disease. The AAVS1 locus is cited as a safe harbour that is permissive for stable transgene expression, and hence is favoured for creating gene targeted reporter lines.Methods: We generated hiPSC reporters using a plasmid-based CRISPR/Cas9 nickase strategy. The first intron of PPP1R12C, the AAVS1 locus, was targeted with constructs expressing a genetically encoded calcium indicator (R-GECO1.0) or HOXA9-T2A-mScarlet reporter under the control of a pCAG or inducible pTRE promoter, respectively. Transgene expression was compared between clones before, during and/or after directed differentiation to mesodermal lineages.Results: Successful targeting to AAVS1 was confirmed by PCR and sequencing. Of 24 hiPSC clones targeted with pCAG-R-GECO1.0, only 20 expressed the transgene and in these, the percentage of positive cells ranged from 0% to 99.5%. Differentiation of a subset of clones produced cardiomyocytes, wherein the percentage of cells positive for R-GECO1.0 ranged from 2.1% to 93.1%. In the highest expressing R-GECO1.0 clones, transgene silencing occurred during cardiomyocyte differentiation causing a decrease in expression from 98.93% to 1.3%. In HOXA9-T2A-mScarlet hiPSC reporter lines directed towards mesoderm lineages, doxycycline induced a peak in transgene expression after two days but this reduced by up to ten-thousand-fold over the next 8-10 days. Nevertheless, for R-GECO1.0 lines differentiated into cardiomyocytes, transgene expression was rescued by continuous puromycin drug selection, which allowed the Ca2+ responses associated with HCM to be investigated in vitro using single cell analysis.Conclusions: Targeted knock-ins to AAVS1 can be used to create reporter lines but variability between clones and transgene silencing requires careful attention by researchers seeking robust reporter gene expression

Development of a 3D tissue-engineered skeletal muscle and bone co‐culture system

In vitro three‐dimensional (3D) tissue engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer culture, and are less ethically questionable than animal models. However, to create systems with even greater relevance, multiple integrated tissue systems should be recreated in vitro. In the present study, the effects and conditions most suitable for the co‐culture of TE skeletal muscle and bone were investigated. High‐glucose Dulbecco's Modified Eagle Medium (HG‐DMEM) supplemented with 20% foetal bovine serum (FBS) followed by HG‐DMEM with 2% horse serum was found to enable proliferation of both C2C12 muscle precursor cells and TE85 human osteosarcoma cells, fusion of C2C12s into myotubes, as well as an up‐regulation of RUNX2/CBFa1 in TE85s. Myotube formation was also evident within indirect contact monolayer cultures. Finally, in 3D co‐cultures, TE85 collagen/hydroxyapatite constructs had significantly greater expression of RUNX2/CBFa1 and osteocalcin/BGLAP in the presence of collagen‐based C2C12 skeletal muscle constructs; however, fusion within these constructs appeared reduced. This work demonstrates the first report of the simultaneous co‐culture and differentiation of 3D TE skeletal muscle and bone, and represents a significant step towards a full in vitro 3D musculoskeletal junction model