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 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

Materials for stem cell factories of the future

The materials community is now identifying polymeric substrates that could permit translation of human pluripotent stem cells (hPSCs) from lab-based research to industrial scale biomedicine. Well defined materials are required to allow cell banking and to provide the raw material for reproducible differentiation into lineages for large scale drug screening programs and clinical use, wherein >1 billion cells for each patient are needed to replace losses during heart attack, multiple sclerosis and diabetes. Producing this number of cells for one patient is challenging and a rethink is needed to scalable technology with the potential to meet the needs of millions of patients a year. Here we consider the role of materials discovery, an emerging area of materials chemistry that is in a large part driven by the challenges posed by biologists to materials scientists1-4

Isogenic Pairs of hiPSC-CMs with Hypertrophic Cardiomyopathy/LVNC-Associated ACTC1 E99K Mutation Unveil Differential Functional Deficits

Hypertrophic cardiomyopathy (HCM) is a primary disorder of contractility in heart muscle. To gain mechanistic insight and guide pharmacological rescue, this study models HCM using isogenic pairs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the E99K-ACTC1 cardiac actin mutation. In both 3D engineered heart tissues and 2D monolayers, arrhythmogenesis was evident in all E99K-ACTC1 hiPSC-CMs. Aberrant phenotypes were most common in hiPSC-CMs produced from the heterozygote father. Unexpectedly, pathological phenotypes were less evident in E99K-expressing hiPSC-CMs from the two sons. Mechanistic insight from Ca2+ handling expression studies prompted pharmacological rescue experiments, wherein dual dantroline/ranolazine treatment was most effective. Our data are consistent with E99K mutant protein being a central cause of HCM but the three-way interaction between the primary genetic lesion, background (epi)genetics, and donor patient age may influence the pathogenic phenotype. This illustrates the value of isogenic hiPSC-CMs in genotype-phenotype correlations

Simplified footprint-free Cas9/CRISPR editing of cardiac-associated genes in human pluripotent stem cells

Modelling disease with hPSCs is hindered because the impact on cell phenotype from genetic variability between individuals can be greater than from the pathogenic mutation. While ‘footprint-free’ Cas9/CRISPR editing solves this issue, existing approaches are inefficient or lengthy. Here, a simplified PiggyBac strategy shortened hPSC editing by 2 weeks and required one round of clonal expansion and genotyping rather than two, with similar efficiencies to the longer conventional process. Success was shown across 4 cardiac-associated loci (ADRB2, GRK5, RYR2, ACTC1) by genomic cleavage and editing efficiencies of 8-93% and 8-67%, respectively, including mono- and/or bi-allelic events. Pluripotency was retained, as was differentiation into high purity cardiomyocytes (CMs; 88-99%). Using the GRK5 isogenic lines as an exemplar, chronic stimulation with the beta-adrenoceptor agonist, isoprenaline, reduced beat rate in hPSC-CMs expressing GRK5-Q41 but not GRK5-L41; this was reversed by the beta-blocker, propranolol. This shortened, footprint-free approach will be useful for mechanistic studies

p16INK4A-dependent senescence in the bone marrow niche drives age-related metabolic changes of hematopoietic progenitors

Rapid and effective leukocyte response to infection is a fundamental function of the bone marrow (BM). However, with increasing age, this response becomes impaired, resulting in an increased burden of infectious diseases. Here, we investigate how aging changes the metabolism and function of hematopoietic progenitor cells (HPCs) and the impact of the BM niche on this phenotype. We found that, in response to lipopolysaccharide-induced stress, HPC mitochondrial function is impaired, and there is a failure to upregulate the TCA cycle in progenitor populations in aged animals compared with young animals. Furthermore, aged mesenchymal stromal cells (MSCs) of the BM niche, but not HPCs, exhibit a senescent phenotype, and selective depletion of senescent cells from the BM niche, as well as treatment with the senolytic drug ABT-263, improves mitochondrial function of HPCs when stressed with lipopolysaccharide. In summary, age-related HPC metabolic dysfunction occurs indirectly as a “bystander phenomenon” in the aging BM niche and can be restored by targeting senescent MSCs

CRISPR/Cas9 editing in human pluripotent stem cell-cardiomyocytes highlights arrhythmias, hypocontractility, and energy depletion as potential therapeutic targets for hypertrophic cardiomyopathy

Aims: Sarcomeric gene mutations frequently underlie hypertrophic cardiomyopathy (HCM), a prevalent and complex condition leading to left ventricle thickening and heart dysfunction. We evaluated isogenic genome-edited human pluripotent stem cell-cardiomyocytes (hPSC-CM) for their validity to model, and add clarity to, HCM. Methods and results: CRISPR/Cas9 editing produced 11 variants of the HCM-causing mutation c.C9123T-MYH7 [(p.R453C-?-myosin heavy chain (MHC)] in 3 independent hPSC lines. Isogenic sets were differentiated to hPSC-CMs for high-throughput, non-subjective molecular and functional assessment using 12 approaches in 2D monolayers and/or 3D engineered heart tissues. Although immature, edited hPSC-CMs exhibited the main hallmarks of HCM (hypertrophy, multi-nucleation, hypertrophic marker expression, sarcomeric disarray). Functional evaluation supported the energy depletion model due to higher metabolic respiration activity, accompanied by abnormalities in calcium handling, arrhythmias, and contraction force. Partial phenotypic rescue was achieved with ranolazine but not omecamtiv mecarbil, while RNAseq highlighted potentially novel molecular targets. Conclusion: Our holistic and comprehensive approach showed that energy depletion affected core cardiomyocyte functionality. The engineered R453C-?MHC-mutation triggered compensatory responses in hPSC-CMs, causing increased ATP production and ?MHC to energy-efficient ?MHC switching. We showed that pharmacological rescue of arrhythmias was possible, while MHY7: MYH6 and mutant: wild-type MYH7 ratios may be diagnostic, and previously undescribed lncRNAs and gene modifiers are suggestive of new mechanisms

The PHENIX Experiment at RHIC

The physics emphases of the PHENIX collaboration and the design and current status of the PHENIX detector are discussed. The plan of the collaboration for making the most effective use of the available luminosity in the first years of RHIC operation is also presented.Comment: 5 pages, 1 figure. Further details of the PHENIX physics program available at http://www.rhic.bnl.gov/phenix

Search for Higgs Bosons in e+e- Collisions at 183 GeV

The data collected by the OPAL experiment at sqrts=183 GeV were used to search for Higgs bosons which are predicted by the Standard Model and various extensions, such as general models with two Higgs field doublets and the Minimal Supersymmetric Standard Model (MSSM). The data correspond to an integrated luminosity of approximately 54pb-1. None of the searches for neutral and charged Higgs bosons have revealed an excess of events beyond the expected background. This negative outcome, in combination with similar results from searches at lower energies, leads to new limits for the Higgs boson masses and other model parameters. In particular, the 95% confidence level lower limit for the mass of the Standard Model Higgs boson is 88.3 GeV. Charged Higgs bosons can be excluded for masses up to 59.5 GeV. In the MSSM, mh > 70.5 GeV and mA > 72.0 GeV are obtained for tan{beta}>1, no and maximal scalar top mixing and soft SUSY-breaking masses of 1 TeV. The range 0.8 < tanb < 1.9 is excluded for minimal scalar top mixing and m{top} < 175 GeV. More general scans of the MSSM parameter space are also considered.Comment: 49 pages. LaTeX, including 33 eps figures, submitted to European Physical Journal

A Measurement of the Product Branching Ratio f(b->Lambda_b).BR(Lambda_b->Lambda X) in Z0 Decays

The product branching ratio, f(b->Lambda_b).BR(Lambda_b->Lambda X), where Lambda_b denotes any weakly-decaying b-baryon, has been measured using the OPAL detector at LEP. Lambda_b are selected by the presence of energetic Lambda particles in bottom events tagged by the presence of displaced secondary vertices. A fit to the momenta of the Lambda particles separates signal from B meson and fragmentation backgrounds. The measured product branching ratio is f(b->Lambda_b).BR(Lambda_b->Lambda X) = (2.67+-0.38(stat)+0.67-0.60(sys))% Combined with a previous OPAL measurement, one obtains f(b->Lambda_b).BR(Lambda_b->Lambda X) = (3.50+-0.32(stat)+-0.35(sys))%.Comment: 16 pages, LaTeX, 3 eps figs included, submitted to the European Physical Journal