Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration.

Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the ß-myosin heavy chain (MYH7) can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant MYH7 G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions. We developed a collaborative pipeline to characterize myosin function across protein, myofibril, cell, and tissue levels to determine the multiscale effects on structure-function of the contractile apparatus and its implications for gene regulation and metabolic state. The G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 33%, resulting in more myosin heads available for contraction. Myofibrils from gene-edited MYH7WT/G256E human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibited greater and faster tension development. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. We demonstrated consistent hypercontractile myosin function as a primary consequence of the MYH7 G256E mutation across scales, highlighting the pathogenicity of this gene variant. Single-cell transcriptomic and metabolic profiling demonstrated upregulated mitochondrial genes and increased mitochondrial respiration, indicating early bioenergetic alterations. This work highlights the benefit of our multiscale platform to systematically evaluate the pathogenicity of gene variants at the protein and contractile organelle level and their early consequences on cellular and tissue function. We believe this platform can help elucidate the genotype-phenotype relationships underlying other genetic cardiovascular diseases.

Patient and Disease-Specific Induced Pluripotent Stem Cells for Discovery of Personalized Cardiovascular Drugs and Therapeutics.

Human induced pluripotent stem cells (iPSCs) have emerged as an effective platform for regenerative therapy, disease modeling, and drug discovery. iPSCs allow for the production of limitless supply of patient-specific somatic cells that enable advancement in cardiovascular precision medicine. Over the past decade, researchers have developed protocols to differentiate iPSCs to multiple cardiovascular lineages, as well as to enhance the maturity and functionality of these cells. Despite significant advances, drug therapy and discovery for cardiovascular disease have lagged behind other fields such as oncology. We speculate that this paucity of drug discovery is due to a previous lack of efficient, reproducible, and translational model systems. Notably, existing drug discovery and testing platforms rely on animal studies and clinical trials, but investigations in animal models have inherent limitations due to interspecies differences. Moreover, clinical trials are inherently flawed by assuming that all individuals with a disease will respond identically to a therapy, ignoring the genetic and epigenomic variations that define our individuality. With ever-improving differentiation and phenotyping methods, patient-specific iPSC-derived cardiovascular cells allow unprecedented opportunities to discover new drug targets and screen compounds for cardiovascular disease. Imbued with the genetic information of an individual, iPSCs will vastly improve our ability to test drugs efficiently, as well as tailor and titrate drug therapy for each patient.

Endocardial/endothelial angiocrines regulate cardiomyocyte development and maturation and induce features of ventricular non-compaction.

AIMS: Non-compaction cardiomyopathy is a devastating genetic disease caused by insufficient consolidation of ventricular wall muscle that can result in inadequate cardiac performance. Despite being the third most common cardiomyopathy, the mechanisms underlying the disease, including the cell types involved, are poorly understood. We have previously shown that endothelial cell-specific deletion of the chromatin remodeller gene Ino80 results in defective coronary vessel development that leads to ventricular non-compaction in embryonic mouse hearts. We aimed to identify candidate angiocrines expressed by endocardial and endothelial cells (ECs) in wildtype and LVNC conditions in Tie2Cre;Ino80fl/fltransgenic embryonic mouse hearts, and test the effect of these candidates on cardiomyocyte proliferation and maturation. METHODS AND RESULTS: We used single-cell RNA-sequencing to characterize endothelial and endocardial defects in Ino80-deficient hearts. We observed a pathological endocardial cell population in the non-compacted hearts and identified multiple dysregulated angiocrine factors that dramatically affected cardiomyocyte behaviour. We identified Col15a1 as a coronary vessel-secreted angiocrine factor, downregulated by Ino80-deficiency, that functioned to promote cardiomyocyte proliferation. Furthermore, mutant endocardial and endothelial cells up-regulated expression of secreted factors, such as Tgfbi, Igfbp3, Isg15, and Adm, which decreased cardiomyocyte proliferation and increased maturation. CONCLUSIONS: These findings support a model where coronary endothelial cells normally promote myocardial compaction through secreted factors, but that endocardial and endothelial cells can secrete factors that contribute to non-compaction under pathological conditions.

Single-Cell RNA Sequencing Unveils Unique Transcriptomic Signatures of Organ-Specific Endothelial Cells.

BACKGROUND: Endothelial cells (ECs) display considerable functional heterogeneity depending on the vessel and tissue in which they are located. Whereas these functional differences are presumably imprinted in the transcriptome, the pathways and networks that sustain EC heterogeneity have not been fully delineated. METHODS: To investigate the transcriptomic basis of EC specificity, we analyzed single-cell RNA sequencing data from tissue-specific mouse ECs generated by the Tabula Muris consortium. We used a number of bioinformatics tools to uncover markers and sources of EC heterogeneity from single-cell RNA sequencing data. RESULTS: We found a strong correlation between tissue-specific EC transcriptomic measurements generated by either single-cell RNA sequencing or bulk RNA sequencing, thus validating the approach. Using a graph-based clustering algorithm, we found that certain tissue-specific ECs cluster strongly by tissue (eg, liver, brain), whereas others (ie, adipose, heart) have considerable transcriptomic overlap with ECs from other tissues. We identified novel markers of tissue-specific ECs and signaling pathways that may be involved in maintaining their identity. Sex was a considerable source of heterogeneity in the endothelial transcriptome and we discovered Lars2 to be a gene that is highly enriched in ECs from male mice. We found that markers of heart and lung ECs in mice were conserved in human fetal heart and lung ECs. We identified potential angiocrine interactions between tissue-specific ECs and other cell types by analyzing ligand and receptor expression patterns. CONCLUSIONS: We used single-cell RNA sequencing data generated by the Tabula Muris consortium to uncover transcriptional networks that maintain tissue-specific EC identity and to identify novel angiocrine and functional relationships between tissue-specific ECs.

Generation of Quiescent Cardiac Fibroblasts From Human Induced Pluripotent Stem Cells for In Vitro Modeling of Cardiac Fibrosis.

RATIONALE: Activated fibroblasts are the major cell type that secretes excessive extracellular matrix in response to injury, contributing to pathological fibrosis and leading to organ failure. Effective anti-fibrotic therapeutic solutions, however, are not available due to the poorly defined characteristics and unavailability of tissue-specific fibroblasts. Recent advances in single-cell RNA-sequencing fill such gaps of knowledge by enabling delineation of the developmental trajectories and identification of regulatory pathways of tissue-specific fibroblasts among different organs. OBJECTIVE: This study aims to define the transcriptome profiles of tissue-specific fibroblasts using recently reported mouse single-cell RNA-sequencing atlas and to develop a robust chemically defined protocol to derive cardiac fibroblasts (CFs) from human induced pluripotent stem cells for in vitro modeling of cardiac fibrosis and drug screening. METHODS AND RESULTS: By analyzing the single-cell transcriptome profiles of fibroblasts from 10 selected mouse tissues, we identified distinct tissue-specific signature genes, including transcription factors that define the identities of fibroblasts in the heart, lungs, trachea, and bladder. We also determined that CFs in large are of the epicardial lineage. We thus developed a robust chemically defined protocol that generates CFs from human induced pluripotent stem cells. Functional studies confirmed that iPSC-derived CFs preserved a quiescent phenotype and highly resembled primary CFs at the transcriptional, cellular, and functional levels. We demonstrated that this cell-based platform is sensitive to both pro- and anti-fibrosis drugs. Finally, we showed that crosstalk between human induced pluripotent stem cell-derived cardiomyocytes and CFs via the atrial/brain natriuretic peptide-natriuretic peptide receptor-1 pathway is implicated in suppressing fibrogenesis. CONCLUSIONS: This study uncovers unique gene signatures that define tissue-specific identities of fibroblasts. The bona fide quiescent CFs derived from human induced pluripotent stem cells can serve as a faithful in vitro platform to better understand the underlying mechanisms of cardiac fibrosis and to screen anti-fibrotic drugs.

Transcriptomic Profiling of the Developing Cardiac Conduction System at Single-Cell Resolution.

RATIONALE: The cardiac conduction system (CCS) consists of distinct components including the sinoatrial node, atrioventricular node, His bundle, bundle branches, and Purkinje fibers. Despite an essential role for the CCS in heart development and function, the CCS has remained challenging to interrogate because of inherent obstacles including small cell numbers, large cell-type heterogeneity, complex anatomy, and difficulty in isolation. Single-cell RNA-sequencing allows for genome-wide analysis of gene expression at single-cell resolution. OBJECTIVE: Assess the transcriptional landscape of the entire CCS at single-cell resolution by single-cell RNA-sequencing within the developing mouse heart. METHODS AND RESULTS: Wild-type, embryonic day 16.5 mouse hearts (n=6 per zone) were harvested and 3 zones of microdissection were isolated, including: Zone I-sinoatrial node region; Zone II-atrioventricular node/His region; and Zone III-bundle branch/Purkinje fiber region. Tissue was digested into single-cell suspensions, cells isolated, mRNA reverse transcribed, and barcoded before high-throughput sequencing and bioinformatics analyses. Single-cell RNA-sequencing was performed on over 22 000 cells, and all major cell types of the murine heart were successfully captured including bona fide clusters of cells consistent with each major component of the CCS. Unsupervised weighted gene coexpression network analysis led to the discovery of a host of novel CCS genes, a subset of which were validated using fluorescent in situ hybridization as well as whole-mount immunolabeling with volume imaging (iDISCO+) in 3 dimensions on intact mouse hearts. Further, subcluster analysis unveiled isolation of distinct CCS cell subtypes, including the clinically relevant but poorly characterized transitional cells that bridge the CCS and surrounding myocardium. CONCLUSIONS: Our study represents the first comprehensive assessment of the transcriptional profiles from the entire CCS at single-cell resolution and provides a characterization in the context of development and disease.

Large-Scale Single-Cell RNA-Seq Reveals Molecular Signatures of Heterogeneous Populations of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells.

RATIONALE: Human-induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) have risen as a useful tool in cardiovascular research, offering a wide gamut of translational and clinical applications. However, inefficiency of the currently available iPSC-EC differentiation protocol and underlying heterogeneity of derived iPSC-ECs remain as major limitations of iPSC-EC technology. OBJECTIVE: Here, we performed droplet-based single-cell RNA sequencing (scRNA-seq) of the human iPSCs after iPSC-EC differentiation. Droplet-based scRNA-seq enables analysis of thousands of cells in parallel, allowing comprehensive analysis of transcriptional heterogeneity. METHODS AND RESULTS: Bona fide iPSC-EC cluster was identified by scRNA-seq, which expressed high levels of endothelial-specific genes. iPSC-ECs, sorted by CD144 antibody-conjugated magnetic sorting, exhibited standard endothelial morphology and function including tube formation, response to inflammatory signals, and production of NO. Nonendothelial cell populations resulting from the differentiation protocol were identified, which included immature cardiomyocytes, hepatic-like cells, and vascular smooth muscle cells. Furthermore, scRNA-seq analysis of purified iPSC-ECs revealed transcriptional heterogeneity with 4 major subpopulations, marked by robust enrichment of CLDN5, APLNR, GJA5, and ESM1 genes, respectively. CONCLUSIONS: Massively parallel, droplet-based scRNA-seq allowed meticulous analysis of thousands of human iPSCs subjected to iPSC-EC differentiation. Results showed inefficiency of the differentiation technique, which can be improved with further studies based on identification of molecular signatures that inhibit expansion of nonendothelial cell types. Subtypes of bona fide human iPSC-ECs were also identified, allowing us to sort for iPSC-ECs with specific biological function and identity.

Ex vivo anti-microbial efficacy of various formaldehyde releasers against antibiotic resistant and antibiotic sensitive microorganisms involved in infectious keratitis.

BACKGROUND: Corneal infections with antibiotic-resistant microorganisms are an increasingly difficult management challenge and chemically or photochemically cross-linking the cornea for therapy presents a unique approach to managing such infections since both direct microbial pathogens killing and matrix stabilization can occur simultaneously. The present study was undertaken in order to compare the anti-microbial efficacy, in vitro, of 5 candidate cross-linking solutions against 5 different microbial pathogens with relevance to infectious keratitis. METHODS: In vitro bactericidal efficacy studies were carried out using 5 different FARs [diazolidinyl urea (DAU), 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione (DMDM), sodium hydroxymethylglycinate (SMG), 2-(hydroxymethyl)-2-nitro-1,3-propanediol (NT = nitrotriol), 2-nitro-1-propanol (NP)] against 5 different microbial pathogens including two antibiotic-resistant species [methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Pseudomonas aeruginosa (PA), and Candida albicans (CA)]. Standard in vitro antimicrobial testing methods were used. RESULTS: The results for MSSA were similar to those for MRSA. DAU, DMDM, and SMG all showed effectiveness with greater effects generally observed with longer incubation times and higher concentrations. Against MRSA, 40 mM SMG at 120 min showed a > 95% kill rate, p < 0.02. Against VRE, 40 mM DAU for 120 min showed a > 94% kill rate, p < 0.001. All FARs showed bactericidal effect against Pseudomonas aeruginosa, making PA the most susceptible of the strains tested. Candida showed relative resistance to these compounds, requiring high concentrations (100 mM) to achieve kill rates greater than 50%. CONCLUSION: Our results show that each FAR compound has different effects against different cultures. Our antimicrobial armamentarium could potentially be broadened by DAU, DMDM, SMG and other FARs for antibiotic-resistant keratitis. Further testing in live animal models are indicated.

Marked Vascular Dysfunction in a Case of Peripartum Cardiomyopathy.

Peripartum cardiomyopathy (PPCM) is a rare form of congestive heart failure characterized by left ventricular dysfunction that develops towards the end of pregnancy or during the early postpartum phase. Even though the majority of PPCM patients show partial or complete recovery of their heart functions, the mortality rate of PPCM remains high. Previous research has suggested that vascular dysfunction triggered by late-gestational hormones and potent anti-angiogenic factors play key roles in the pathogenesis of PPCM; however, the exact mechanisms remain elusive due to limited patient tissues for characterization. Here, we report a case of PPCM where the coronary vessels from the patient's explanted heart showed marked vascular dysfunction with impaired nitric oxide response. Importantly, these vessels exhibited deficient adenosine-mediated vasorelaxation when subjected to myograph studies, suggesting impaired Kv7 ion channels. Results from this work may lead to new therapeutic strategies for improving Kv7 function in PPCM patients.

Quantification of Cerenkov Luminescence Imaging (CLI) Comparable With 3-D PET Standard Measurements.

Cerenkov luminescence imaging (CLI) is commonly performed using two-dimensional (2-D) conventional optical imaging systems for its cost-effective solution. However, quantification of CLI comparable to conventional three-dimensional positron emission tomography (PET) is challenging using these systems due to both the high attenuation of Cerenkov radiation (CR) on mouse tissue and nonexisting depth resolution of CLI using 2-D imaging systems (2-D CLI). In this study, we developed a model that estimates effective tissue attenuation coefficient and corrects the tissue attenuation of CLI signal intensity independent of tissue depth and size. To evaluate this model, we used several thin slices of ham as a phantom and placed a radionuclide (89Zr and 64Cu) inside the phantom at different tissue depths and sizes (2, 7, and 12 mm). We performed 2-D CLI and MicroPET/CT (Combined small animal PET and Computed Tomography (CT)) imaging of the phantom and in vivo mouse model after administration of 89Zr tracer. Estimates of the effective tissue attenuation coefficient (µeff) for 89Zr and 64Cu were ∼2.4 and ∼2.6 cm-1, respectively. The computed unit conversion factor to %ID/g from 2-D CLI signal was 2.74 × 10-3 µCi/radiance estimated from phantom study. After applying tissue attenuation correction and unit conversion to the in vivo animal study, an average quantification difference of 10% for spleen and 35% for liver was obtained compared to PET measurements. The proposed model provides comparable quantification accuracy to standard PET system independent of deep tissue CLI signal attenuation.

Atherosclerotic Plaque Tissue: Noninvasive Quantitative Assessment of Characteristics with Software-aided Measurements from Conventional CT Angiography.

Purpose To (a) evaluate whether plaque tissue characteristics determined with conventional computed tomographic (CT) angiography could be quantitated at higher levels of accuracy by using image processing algorithms that take characteristics of the image formation process coupled with biologic insights on tissue distributions into account by comparing in vivo results and ex vivo histologic findings and (b) assess reader variability. Materials and Methods Thirty-one consecutive patients aged 43-85 years (average age, 64 years) known to have or suspected of having atherosclerosis who underwent CT angiography and were referred for endarterectomy were enrolled. Surgical specimens were evaluated with histopathologic examination to serve as standard of reference. Two readers used lumen boundary to determine scanner blur and then optimized component densities and subvoxel boundaries to best fit the observed image by using semiautomatic software. The accuracy of the resulting in vivo quantitation of calcification, lipid-rich necrotic core (LRNC), and matrix was assessed with statistical estimates of bias and linearity relative to ex vivo histologic findings. Reader variability was assessed with statistical estimates of repeatability and reproducibility. Results A total of 239 cross sections obtained with CT angiography and histologic examination were matched. Performance on held-out data showed low levels of bias and high Pearson correlation coefficients for calcification (-0.096 mm2 and 0.973, respectively), LRNC (1.26 mm2 and 0.856), and matrix (-2.44 mm2 and 0.885). Intrareader variability was low (repeatability coefficient ranged from 1.50 mm2 to 1.83 mm2 among tissue characteristics), as was interreader variability (reproducibility coefficient ranged from 2.09 mm2 to 4.43 mm2). Conclusion There was high correlation and low bias between the in vivo software image analysis and ex vivo histopathologic quantitative measures of atherosclerotic plaque tissue characteristics, as well as low reader variability. Software algorithms can mitigate the blurring and partial volume effects of routine CT angiography acquisitions to produce accurate quantification to enhance current clinical practice. Clinical trial registration no. NCT02143102 © RSNA, 2017 Online supplemental material is available for this article. An earlier incorrect version of this article appeared online. This article was corrected on September 15, 2017.

Wnt10b Gain-of-Function Improves Cardiac Repair by Arteriole Formation and Attenuation of Fibrosis.

RATIONALE: Myocardial infarction causes irreversible tissue damage, leading to heart failure. We recently discovered that canonical Wnt signaling and the Wnt10b ligand are strongly induced in mouse hearts after infarction. Wnt10b regulates cell fate in various organs, but its role in the heart is unknown. OBJECTIVE: To investigate the effect of Wnt10b gain-of-function on cardiac repair mechanisms and to assess its potential to improve ventricular function after injury. METHODS AND RESULTS: Histological and molecular analyses showed that Wnt10b is expressed in cardiomyocytes and localized in the intercalated discs of mouse and human hearts. After coronary artery ligation or cryoinjury in mice, Wnt10b is strongly and transiently induced in peri-infarct cardiomyocytes during granulation tissue formation. To determine the effect of Wnt10b on neovascularization and fibrosis, we generated a mouse line to increase endogenous Wnt10b levels in cardiomyocytes. We found that gain of Wnt10b function orchestrated a recovery phenotype characterized by robust neovascularization of the injury zone, less myofibroblasts, reduced scar size, and improved ventricular function compared with wild-type mice. Wnt10b stimulated expression of vascular endothelial growth factor receptor 2 in endothelial cells and angiopoietin-1 in vascular smooth muscle cells through nuclear factor-κB activation. These effects coordinated endothelial growth and smooth muscle cell recruitment, promoting robust formation of large, coronary-like blood vessels. CONCLUSION: Wnt10b gain-of-function coordinates arterial formation and attenuates fibrosis in cardiac tissue after injury. Because generation of mature blood vessels is necessary for efficient perfusion, our findings could lead to novel strategies to optimize the inherent repair capacity of the heart and prevent the onset of heart failure.

Median nerve penetration by a persistent median artery and vein mimicking carpal tunnel syndrome.

INTRODUCTION: Carpal tunnel syndrome (CTS) is a common clinical syndrome seen in the outpatient setting that is easily confirmed by electrodiagnostic testing. METHODS: We describe the case of a patient who presented with the classic symptoms and neurological examination for CTS, but had a normal nerve conduction study and electromyogram. RESULTS: Neuromuscular ultrasound of the median nerve on the symptomatic side revealed penetration of the nerve by a persistent median artery and vein in the mid-forearm, with a positive sonographic Tinel sign over this spot. This finding is an anatomical variation that has been described sparingly in the literature, mostly in cadavers. It has not been reported previously to be a mimic of CTS. CONCLUSIONS: This case demonstrates the diagnostic utility of neuromuscular ultrasound and the importance of considering an anatomical variation involving the median nerve in the differential diagnosis of CTS.

ISA-TAB-Nano: a specification for sharing nanomaterial research data in spreadsheet-based format.

BACKGROUND AND MOTIVATION: The high-throughput genomics communities have been successfully using standardized spreadsheet-based formats to capture and share data within labs and among public repositories. The nanomedicine community has yet to adopt similar standards to share the diverse and multi-dimensional types of data (including metadata) pertaining to the description and characterization of nanomaterials. Owing to the lack of standardization in representing and sharing nanomaterial data, most of the data currently shared via publications and data resources are incomplete, poorly-integrated, and not suitable for meaningful interpretation and re-use of the data. Specifically, in its current state, data cannot be effectively utilized for the development of predictive models that will inform the rational design of nanomaterials. RESULTS: We have developed a specification called ISA-TAB-Nano, which comprises four spreadsheet-based file formats for representing and integrating various types of nanomaterial data. Three file formats (Investigation, Study, and Assay files) have been adapted from the established ISA-TAB specification; while the Material file format was developed de novo to more readily describe the complexity of nanomaterials and associated small molecules. In this paper, we have discussed the main features of each file format and how to use them for sharing nanomaterial descriptions and assay metadata. CONCLUSION: The ISA-TAB-Nano file formats provide a general and flexible framework to record and integrate nanomaterial descriptions, assay data (metadata and endpoint measurements) and protocol information. Like ISA-TAB, ISA-TAB-Nano supports the use of ontology terms to promote standardized descriptions and to facilitate search and integration of the data. The ISA-TAB-Nano specification has been submitted as an ASTM work item to obtain community feedback and to provide a nanotechnology data-sharing standard for public development and adoption.

A novel knowledge representation framework for the statistical validation of quantitative imaging biomarkers.

Quantitative imaging biomarkers are of particular interest in drug development for their potential to accelerate the drug development pipeline. The lack of consensus methods and carefully characterized performance hampers the widespread availability of these quantitative measures. A framework to support collaborative work on quantitative imaging biomarkers would entail advanced statistical techniques, the development of controlled vocabularies, and a service-oriented architecture for processing large image archives. Until now, this framework has not been developed. With the availability of tools for automatic ontology-based annotation of datasets, coupled with image archives, and a means for batch selection and processing of image and clinical data, imaging will go through a similar increase in capability analogous to what advanced genetic profiling techniques have brought to molecular biology. We report on our current progress on developing an informatics infrastructure to store, query, and retrieve imaging biomarker data across a wide range of resources in a semantically meaningful way that facilitates the collaborative development and validation of potential imaging biomarkers by many stakeholders. Specifically, we describe the semantic components of our system, QI-Bench, that are used to specify and support experimental activities for statistical validation in quantitative imaging.

Quantitative imaging biomarker ontology (QIBO) for knowledge representation of biomedical imaging biomarkers.

A widening array of novel imaging biomarkers is being developed using ever more powerful clinical and preclinical imaging modalities. These biomarkers have demonstrated effectiveness in quantifying biological processes as they occur in vivo and in the early prediction of therapeutic outcomes. However, quantitative imaging biomarker data and knowledge are not standardized, representing a critical barrier to accumulating medical knowledge based on quantitative imaging data. We use an ontology to represent, integrate, and harmonize heterogeneous knowledge across the domain of imaging biomarkers. This advances the goal of developing applications to (1) improve precision and recall of storage and retrieval of quantitative imaging-related data using standardized terminology; (2) streamline the discovery and development of novel imaging biomarkers by normalizing knowledge across heterogeneous resources; (3) effectively annotate imaging experiments thus aiding comprehension, re-use, and reproducibility; and (4) provide validation frameworks through rigorous specification as a basis for testable hypotheses and compliance tests. We have developed the Quantitative Imaging Biomarker Ontology (QIBO), which currently consists of 488 terms spanning the following upper classes: experimental subject, biological intervention, imaging agent, imaging instrument, image post-processing algorithm, biological target, indicated biology, and biomarker application. We have demonstrated that QIBO can be used to annotate imaging experiments with standardized terms in the ontology and to generate hypotheses for novel imaging biomarker-disease associations. Our results established the utility of QIBO in enabling integrated analysis of quantitative imaging data.

Scleral growth stunting via sub-Tenon injection of cross-linking solutions in live rabbits.

BACKGROUND: Scleral cross-linking is a potential method to inhibit axial elongation of the eye, preventing the progression of pathological myopia. Formaldehyde releasers, which are common preservatives found in cosmetics and ophthalmic solutions, have been shown to be not only effective in cross-linking corneal collagen in vitro and in vivo, but also have minimal toxicity effects on the eye. The present study aims to evaluate the efficacy of scleral cross-linking using sodium hydroxymethylglycinate (SMG) to inhibit eye growth using an in vivo rabbit model. METHODS: A cross-linking solution containing 40 mM SMG was delivered to the sub-Tenon's space behind the equator. The application regimen included a two-quadrant injection performed five times over 2 weeks on New Zealand White rabbits (n=5, group 1), and one-time injection followed for up to 5 days on Dutch-Belted rabbits (n=6, group 2). Group 1 was monitored serially for axial length changes using B-scan ultrasound for 5-6 weeks. Group 2 was injected with a higher viscosity solution formulation. Both groups were evaluated for thermal denaturation temperature changes of the sclera postmortem. RESULTS: Axial growth was limited by 10%-20% following SMG treatment as compared with the untreated eye. Thermal denaturation analysis showed increased heat resistance of the treated eyes in the areas of injection. Overall, the SMG treatment inhibited eye growth with few side effects from the injections. CONCLUSIONS: Cross-linking solutions delivered via sub-Tenon injection provide a potential method for limiting axial length growth in progressive myopia and could be used as a potential treatment for myopia.

Age-related enhanced degeneration of bioprosthetic valves due to leaflet calcification, tissue crosslinking, and structural changes.

AIMS: Bioprosthetic heart valves (BHVs), made from glutaraldehyde-fixed heterograft materials, are subject to more rapid structural valve degeneration (SVD) in paediatric and young adult patients. Differences in blood biochemistries and propensity for disease accelerate SVD in these patients, which results in multiple re-operations with compounding risks. The goal of this study is to investigate the mechanisms of BHV biomaterial degeneration and present models for studying SVD in young patients and juvenile animal models. METHODS AND RESULTS: We studied SVD in clinical BHV explants from paediatric and young adult patients, juvenile sheep implantation model, rat subcutaneous implants, and an ex vivo serum incubation model. BHV biomaterials were analysed for calcification, collagen microstructure (alignment and crimp), and crosslinking density. Serum markers of calcification and tissue crosslinking were compared between young and adult subjects. We demonstrated that immature subjects were more susceptible to calcification, microstructural changes, and advanced glycation end products formation. In vivo and ex vivo studies comparing immature and mature subjects mirrored SVD in clinical observations. The interaction between host serum and BHV biomaterials leads to significant structural and biochemical changes which impact their functions. CONCLUSIONS: There is an increased risk for accelerated SVD in younger subjects, both experimental animals and patients. Increased calcification, altered collagen microstructure with loss of alignment and increased crimp periods, and increased crosslinking are three main characteristics in BHV explants from young subjects leading to SVD. Together, our studies establish a basis for assessing the increased susceptibility of BHV biomaterials to accelerated SVD in young patients.

Multi-scale models reveal hypertrophic cardiomyopathy MYH7 G256E mutation drives hypercontractility and elevated mitochondrial respiration.

Rationale: Over 200 mutations in the sarcomeric protein ß-myosin heavy chain (MYH7) have been linked to hypertrophic cardiomyopathy (HCM). However, different mutations in MYH7 lead to variable penetrance and clinical severity, and alter myosin function to varying degrees, making it difficult to determine genotype-phenotype relationships, especially when caused by rare gene variants such as the G256E mutation. Objective: This study aims to determine the effects of low penetrant MYH7 G256E mutation on myosin function. We hypothesize that the G256E mutation would alter myosin function, precipitating compensatory responses in cellular functions. Methods: We developed a collaborative pipeline to characterize myosin function at multiple scales (protein to myofibril to cell to tissue). We also used our previously published data on other mutations to compare the degree to which myosin function was altered. Results: At the protein level, the G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 50.9%, suggesting more myosins available for contraction. Myofibrils isolated from hiPSC-CMs CRISPR-edited with G256E (MYH7 WT/G256E ) generated greater tension, had faster tension development and slower early phase relaxation, suggesting altered myosin-actin crossbridge cycling kinetics. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. Single-cell transcriptomic and metabolic profiling demonstrated upregulation of mitochondrial genes and increased mitochondrial respiration, suggesting altered bioenergetics as an early feature of HCM. Conclusions: MYH7 G256E mutation causes structural instability in the transducer region, leading to hypercontractility across scales, perhaps from increased myosin recruitment and altered crossbridge cycling. Hypercontractile function of the mutant myosin was accompanied by increased mitochondrial respiration, while cellular hypertrophy was modest in the physiological stiffness environment. We believe that this multi-scale platform will be useful to elucidate genotype-phenotype relationships underlying other genetic cardiovascular diseases.

SGLT2 inhibitor ameliorates endothelial dysfunction associated with the common ALDH2 alcohol flushing variant.

The common aldehyde dehydrogenase 2 (ALDH2) alcohol flushing variant known as ALDH2*2 affects ∼8% of the world's population. Even in heterozygous carriers, this missense variant leads to a severe loss of ALDH2 enzymatic activity and has been linked to an increased risk of coronary artery disease (CAD). Endothelial cell (EC) dysfunction plays a determining role in all stages of CAD pathogenesis, including early-onset CAD. However, the contribution of ALDH2*2 to EC dysfunction and its relation to CAD are not fully understood. In a large genome-wide association study (GWAS) from Biobank Japan, ALDH2*2 was found to be one of the strongest single-nucleotide polymorphisms associated with CAD. Clinical assessment of endothelial function showed that human participants carrying ALDH2*2 exhibited impaired vasodilation after light alcohol drinking. Using human induced pluripotent stem cell-derived ECs (iPSC-ECs) and CRISPR-Cas9-corrected ALDH2*2 iPSC-ECs, we modeled ALDH2*2-induced EC dysfunction in vitro, demonstrating an increase in oxidative stress and inflammatory markers and a decrease in nitric oxide (NO) production and tube formation capacity, which was further exacerbated by ethanol exposure. We subsequently found that sodium-glucose cotransporter 2 inhibitors (SGLT2i) such as empagliflozin mitigated ALDH2*2-associated EC dysfunction. Studies in ALDH2*2 knock-in mice further demonstrated that empagliflozin attenuated ALDH2*2-mediated vascular dysfunction in vivo. Mechanistically, empagliflozin inhibited Na+/H+-exchanger 1 (NHE-1) and activated AKT kinase and endothelial NO synthase (eNOS) pathways to ameliorate ALDH2*2-induced EC dysfunction. Together, our results suggest that ALDH2*2 induces EC dysfunction and that SGLT2i may potentially be used as a preventative measure against CAD for ALDH2*2 carriers.