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.

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.

Adverse effects of air pollution-derived fine particulate matter on cardiovascular homeostasis and disease.

Air pollution is a rapidly growing major health concern around the world. Atmospheric particulate matter that has a diameter of less than 2.5 µm (PM2.5) refers to an air pollutant composed of particles and chemical compounds that originate from various sources. While epidemiological studies have established the association between PM2.5 exposure and cardiovascular diseases, the precise cellular and molecular mechanisms by which PM2.5 promotes cardiovascular complications are yet to be fully elucidated. In this review, we summarize the various sources of PM2.5, its components, and the concentrations of ambient PM2.5 in various settings. We discuss the experimental findings to date that evaluate the potential adverse effects of PM2.5 on cardiovascular homeostasis and function, and the possible therapeutic options that may alleviate PM2.5-driven cardiovascular damage.

Endothelial-Myocardial Angiocrine Signaling in Heart Development.

Vascular endothelial cells are a multifunctional cell type with organotypic specificity in their function and structure. In this review, we discuss various subpopulations of endothelial cells in the mammalian heart, which spatiotemporally regulate critical cellular and molecular processes of heart development via unique sets of angiocrine signaling pathways. In particular, elucidation of intercellular communication among the functional cell types in the developing heart has recently been accelerated by the use of single-cell sequencing. Specifically, we overview the heterogeneic nature of cardiac endothelial cells and their contribution to heart tube and chamber formation, myocardial trabeculation and compaction, and endocardial cushion and valve formation via angiocrine pathways.

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.

Wnt Activation and Reduced Cell-Cell Contact Synergistically Induce Massive Expansion of Functional Human iPSC-Derived Cardiomyocytes.

Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro can expand CMs modestly (<5-fold). Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e., 100- to 250-fold) by glycogen synthase kinase-3ß (GSK-3ß) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show that GSK-3ß inhibition suppresses CM maturation, while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10 to 25 times greater expansion beyond GSK-3ß inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from yes-associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. In summary, we uncovered a molecular interplay that enables massive hiPSC-CM expansion for large-scale drug screening and tissue engineering applications.

Modeling Secondary Iron Overload Cardiomyopathy with Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Excessive iron accumulation in the heart causes iron overload cardiomyopathy (IOC), which initially presents as diastolic dysfunction and arrhythmia but progresses to systolic dysfunction and end-stage heart failure when left untreated. However, the mechanisms of iron-related cardiac injury and how iron accumulates in human cardiomyocytes are not well understood. Herein, using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we model IOC and screen for drugs to rescue the iron overload phenotypes. Human iPSC-CMs under excess iron exposure recapitulate early-stage IOC, including oxidative stress, arrhythmia, and contractile dysfunction. We find that iron-induced changes in calcium kinetics play a critical role in dysregulation of CM functions. We identify that ebselen, a selective divalent metal transporter 1 (DMT1) inhibitor and antioxidant, could prevent the observed iron overload phenotypes, supporting the role of DMT1 in iron uptake into the human myocardium. These results suggest that ebselen may be a potential preventive and therapeutic agent for treating patients with secondary iron overload.

High-throughput Preparation of DNA, RNA, and Protein from Cryopreserved Human iPSCs for Multi-omics Analysis.

We describe the procedure to isolate genomic DNA, RNA, and protein directly from cryopreserved induced pluripotent stem cell (iPSC) vials using commercially available solid-phase extraction kits, and we report the relationship between macromolecule yields and experimental and storage factors. Sufficient quantities of DNA, RNA, and protein are recoverable from as low as 1 million cryopreserved cells across 728 distinct iPSC lines suitable for whole-genome sequencing, RNA sequencing, and mass spectrometry experiments. Nucleic acids extracted from iPSC stocks cryopreserved up to 4 years maintain sufficient quantity and integrity for downstream analysis with minimal genomic DNA fragmentation. An expected positive correlation exists between cell count and DNA or RNA yield, with comparable yields recovered between cells across different cryostorage timespans. This article provides an effective way to simultaneously isolate iPSC biomolecules for multi-omics investigations. © 2020 Wiley Periodicals LLC. Basic Protocol 1: QIAshredder and AllPrep DNA/RNA/protein mini kit extraction and subsequent DNA quantification and quality analysis Basic Protocol 2: Broad-range RNA quantification and quality assay using QuBit 4 Fluorometer and associated kits.

Single-cell RNA sequencing in cardiovascular development, disease and medicine.

Advances in single-cell RNA sequencing (scRNA-seq) technologies in the past 10 years have had a transformative effect on biomedical research, enabling the profiling and analysis of the transcriptomes of single cells at unprecedented resolution and throughput. Specifically, scRNA-seq has facilitated the identification of novel or rare cell types, the analysis of single-cell trajectory construction and stem or progenitor cell differentiation, and the comparison of healthy and disease-related tissues at single-cell resolution. These applications have been critical in advances in cardiovascular research in the past decade as evidenced by the generation of cell atlases of mammalian heart and blood vessels and the elucidation of mechanisms involved in cardiovascular development and stem or progenitor cell differentiation. In this Review, we summarize the currently available scRNA-seq technologies and analytical tools and discuss the latest findings using scRNA-seq that have substantially improved our knowledge on the development of the cardiovascular system and the mechanisms underlying cardiovascular diseases. Furthermore, we examine emerging strategies that integrate multimodal single-cell platforms, focusing on future applications in cardiovascular precision medicine that use single-cell omics approaches to characterize cell-specific responses to drugs or environmental stimuli and to develop effective patient-specific therapeutics.

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.

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.

Atheroprotective roles of smooth muscle cell phenotypic modulation and the TCF21 disease gene as revealed by single-cell analysis.

In response to various stimuli, vascular smooth muscle cells (SMCs) can de-differentiate, proliferate and migrate in a process known as phenotypic modulation. However, the phenotype of modulated SMCs in vivo during atherosclerosis and the influence of this process on coronary artery disease (CAD) risk have not been clearly established. Using single-cell RNA sequencing, we comprehensively characterized the transcriptomic phenotype of modulated SMCs in vivo in atherosclerotic lesions of both mouse and human arteries and found that these cells transform into unique fibroblast-like cells, termed 'fibromyocytes', rather than into a classical macrophage phenotype. SMC-specific knockout of TCF21-a causal CAD gene-markedly inhibited SMC phenotypic modulation in mice, leading to the presence of fewer fibromyocytes within lesions as well as within the protective fibrous cap of the lesions. Moreover, TCF21 expression was strongly associated with SMC phenotypic modulation in diseased human coronary arteries, and higher levels of TCF21 expression were associated with decreased CAD risk in human CAD-relevant tissues. These results establish a protective role for both TCF21 and SMC phenotypic modulation in this disease.

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.

Single-Cell RNA Sequencing of Human Embryonic Stem Cell Differentiation Delineates Adverse Effects of Nicotine on Embryonic Development.

Nicotine, the main chemical constituent of tobacco, is highly detrimental to the developing fetus by increasing the risk of gestational complications and organ disorders. The effects of nicotine on human embryonic development and related mechanisms, however, remain poorly understood. Here, we performed single-cell RNA sequencing (scRNA-seq) of human embryonic stem cell (hESC)-derived embryoid body (EB) in the presence or absence of nicotine. Nicotine-induced lineage-specific responses and dysregulated cell-to-cell communication in EBs, shedding light on the adverse effects of nicotine on human embryonic development. In addition, nicotine reduced cell viability, increased reactive oxygen species (ROS), and altered cell cycling in EBs. Abnormal Ca2+ signaling was found in muscle cells upon nicotine exposure, as verified in hESC-derived cardiomyocytes. Consequently, our scRNA-seq data suggest direct adverse effects of nicotine on hESC differentiation at the single-cell level and offer a new method for evaluating drug and environmental toxicity on human embryonic development in utero.

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.