Direct interrogation of context-dependent GPCR activity with a universal biosensor platform.

G protein-coupled receptors (GPCRs) are the largest family of druggable proteins encoded in the human genome, but progress in understanding and targeting them is hindered by the lack of tools to reliably measure their nuanced behavior in physiologically relevant contexts. Here, we developed a collection of compact ONE vector G-protein Optical (ONE-GO) biosensor constructs as a scalable platform that can be conveniently deployed to measure G-protein activation by virtually any GPCR with high fidelity even when expressed endogenously in primary cells. By characterizing dozens of GPCRs across many cell types like primary cardiovascular cells or neurons, we revealed insights into the molecular basis for G-protein coupling selectivity of GPCRs, pharmacogenomic profiles of anti-psychotics on naturally occurring GPCR variants, and G-protein subtype signaling bias by endogenous GPCRs depending on cell type or upon inducing disease-like states. In summary, this open-source platform makes the direct interrogation of context-dependent GPCR activity broadly accessible.

Integrative single-cell analysis of cardiogenesis identifies developmental trajectories and non-coding mutations in congenital heart disease.

To define the multi-cellular epigenomic and transcriptional landscape of cardiac cellular development, we generated single-cell chromatin accessibility maps of human fetal heart tissues. We identified eight major differentiation trajectories involving primary cardiac cell types, each associated with dynamic transcription factor (TF) activity signatures. We contrasted regulatory landscapes of iPSC-derived cardiac cell types and their in vivo counterparts, which enabled optimization of in vitro differentiation of epicardial cells. Further, we interpreted sequence based deep learning models of cell-type-resolved chromatin accessibility profiles to decipher underlying TF motif lexicons. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in congenital heart disease (CHD) cases vs. controls. In vitro studies in iPSCs validated the functional impact of identified variation on the predicted developmental cell types. This work thus defines the cell-type-resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements in CHD.

Cannabinoid receptor 1 antagonist genistein attenuates marijuana-induced vascular inflammation.

Epidemiological studies reveal that marijuana increases the risk of cardiovascular disease (CVD); however, little is known about the mechanism. Δ9-tetrahydrocannabinol (Δ9-THC), the psychoactive component of marijuana, binds to cannabinoid receptor 1 (CB1/CNR1) in the vasculature and is implicated in CVD. A UK Biobank analysis found that cannabis was an risk factor for CVD. We found that marijuana smoking activated inflammatory cytokines implicated in CVD. In silico virtual screening identified genistein, a soybean isoflavone, as a putative CB1 antagonist. Human-induced pluripotent stem cell-derived endothelial cells were used to model Δ9-THC-induced inflammation and oxidative stress via NF-κB signaling. Knockdown of the CB1 receptor with siRNA, CRISPR interference, and genistein attenuated the effects of Δ9-THC. In mice, genistein blocked Δ9-THC-induced endothelial dysfunction in wire myograph, reduced atherosclerotic plaque, and had minimal penetration of the central nervous system. Genistein is a CB1 antagonist that attenuates Δ9-THC-induced atherosclerosis.

A Tension-Based Model Distinguishes Hypertrophic versus Dilated Cardiomyopathy.

The heart either hypertrophies or dilates in response to familial mutations in genes encoding sarcomeric proteins, which are responsible for contraction and pumping. These mutations typically alter calcium-dependent tension generation within the sarcomeres, but how this translates into the spectrum of hypertrophic versus dilated cardiomyopathy is unknown. By generating a series of cardiac-specific mouse models that permit the systematic tuning of sarcomeric tension generation and calcium fluxing, we identify a significant relationship between the magnitude of tension developed over time and heart growth. When formulated into a computational model, the integral of myofilament tension development predicts hypertrophic and dilated cardiomyopathies in mice associated with essentially any sarcomeric gene mutations, but also accurately predicts human cardiac phenotypes from data generated in induced-pluripotent-stem-cell-derived myocytes from familial cardiomyopathy patients. This tension-based model also has the potential to inform pharmacologic treatment options in cardiomyopathy patients.

Leveraging physiology and artificial intelligence to deliver advancements in health care.

Artificial intelligence in health care has experienced remarkable innovation and progress in the last decade. Significant advancements can be attributed to the utilization of artificial intelligence to transform physiology data to advance health care. In this review, we explore how past work has shaped the field and defined future challenges and directions. In particular, we focus on three areas of development. First, we give an overview of artificial intelligence, with special attention to the most relevant artificial intelligence models. We then detail how physiology data have been harnessed by artificial intelligence to advance the main areas of health care: automating existing health care tasks, increasing access to care, and augmenting health care capabilities. Finally, we discuss emerging concerns surrounding the use of individual physiology data and detail an increasingly important consideration for the field, namely the challenges of deploying artificial intelligence models to achieve meaningful clinical impact.

Dissecting human population variation in single-cell responses to SARS-CoV-2.

Humans display substantial interindividual clinical variability after SARS-CoV-2 infection1-3, the genetic and immunological basis of which has begun to be deciphered4. However, the extent and drivers of population differences in immune responses to SARS-CoV-2 remain unclear. Here we report single-cell RNA-sequencing data for peripheral blood mononuclear cells-from 222 healthy donors of diverse ancestries-that were stimulated with SARS-CoV-2 or influenza A virus. We show that SARS-CoV-2 induces weaker, but more heterogeneous, interferon-stimulated gene activity compared with influenza A virus, and a unique pro-inflammatory signature in myeloid cells. Transcriptional responses to viruses display marked population differences, primarily driven by changes in cell abundance including increased lymphoid differentiation associated with latent cytomegalovirus infection. Expression quantitative trait loci and mediation analyses reveal a broad effect of cell composition on population disparities in immune responses, with genetic variants exerting a strong effect on specific loci. Furthermore, we show that natural selection has increased population differences in immune responses, particularly for variants associated with SARS-CoV-2 response in East Asians, and document the cellular and molecular mechanisms by which Neanderthal introgression has altered immune functions, such as the response of myeloid cells to viruses. Finally, colocalization and transcriptome-wide association analyses reveal an overlap between the genetic basis of immune responses to SARS-CoV-2 and COVID-19 severity, providing insights into the factors contributing to current disparities in COVID-19 risk.

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.

Adverse Impact of Cannabis on Human Health.

Cannabis, the most commonly used recreational drug, is illicit in many areas of the world. With increasing decriminalization and legalization, cannabis use is increasing in the United States and other countries. The adverse effects of cannabis are unclear because its status as a Schedule 1 drug in the United States restricts research. Despite a paucity of data, cannabis is commonly perceived as a benign or even beneficial drug. However, recent studies show that cannabis has adverse cardiovascular and pulmonary effects and is linked with malignancy. Moreover, case reports have shown an association between cannabis use and neuropsychiatric disorders. With growing availability, cannabis misuse by minors has led to increasing incidences of overdose and toxicity. Though difficult to detect, cannabis intoxication may be linked to impaired driving and motor vehicle accidents. Overall, cannabis use is on the rise, and adverse effects are becoming apparent in clinical data sets.

Pericytes contribute to pulmonary vascular remodeling via HIF2α signaling.

Vascular remodeling is the process of structural alteration and cell rearrangement of blood vessels in response to injury and is the cause of many of the world's most afflicted cardiovascular conditions, including pulmonary arterial hypertension (PAH). Many studies have focused on the effects of vascular endothelial cells and smooth muscle cells (SMCs) during vascular remodeling, but pericytes, an indispensable cell population residing largely in capillaries, are ignored in this maladaptive process. Here, we report that hypoxia-inducible factor 2α (HIF2α) expression is increased in the lung tissues of PAH patients, and HIF2α overexpressed pericytes result in greater contractility and an impaired endothelial-pericyte interaction. Using single-cell RNAseq and hypoxia-induced pulmonary hypertension (PH) models, we show that HIF2α is a major molecular regulator for the transformation of pericytes into SMC-like cells. Pericyte-selective HIF2α overexpression in mice exacerbates PH and right ventricular hypertrophy. Temporal cellular lineage tracing shows that HIF2α overexpressing reporter NG2+ cells (pericyte-selective) relocate from capillaries to arterioles and co-express SMA. This novel insight into the crucial role of NG2+ pericytes in pulmonary vascular remodeling via HIF2α signaling suggests a potential drug target for PH.

TRF2 rescues telomere attrition and prolongs cell survival in Duchenne muscular dystrophy cardiomyocytes derived from human iPSCs.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of dystrophin. Heart failure, driven by cardiomyocyte death, fibrosis, and the development of dilated cardiomyopathy, is the leading cause of death in DMD patients. Current treatments decrease the mechanical load on the heart but do not address the root cause of dilated cardiomyopathy: cardiomyocyte death. Previously, we showed that telomere shortening is a hallmark of DMD cardiomyocytes. Here, we test whether prevention of telomere attrition is possible in cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPSC-CMs) and if preventing telomere shortening impacts cardiomyocyte function. We observe reduced cell size, nuclear size, and sarcomere density in DMD iPSC-CMs compared with healthy isogenic controls. We find that expression of just one telomere-binding protein, telomeric repeat-binding factor 2 (TRF2), a core component of the shelterin complex, prevents telomere attrition and rescues deficiencies in cell size as well as sarcomere density. We employ a bioengineered platform to micropattern cardiomyocytes for calcium imaging and perform Southern blots of telomere restriction fragments, the gold standard for telomere length assessments. Importantly, preservation of telomere lengths in DMD cardiomyocytes improves their viability. These data provide evidence that preventing telomere attrition ameliorates deficits in cell morphology, activation of the DNA damage response, and premature cell death, suggesting that TRF2 is a key player in DMD-associated cardiac failure.

Addressing Structural Racism Through Public Policy Advocacy: A Policy Statement From the American Heart Association.

During the COVID-19 pandemic, the American Heart Association created a new 2024 Impact Goal with health equity at its core, in recognition of the increasing health disparities in our country and the overwhelming evidence of the damaging effect of structural racism on cardiovascular and stroke health. Concurrent with the announcement of the new Impact Goal was the release of an American Heart Association presidential advisory on structural racism, recognizing racism as a fundamental driver of health disparities and directing the American Heart Association to advance antiracist strategies regarding science, business operations, leadership, quality improvement, and advocacy. This policy statement builds on the call to action put forth in our presidential advisory, discussing specific opportunities to leverage public policy in promoting overall well-being and rectifying those long-standing structural barriers that impede the progress that we need and seek for the health of all communities. Although this policy statement discusses difficult aspects of our past, it is meant to provide a forward-looking blueprint that can be embraced by a broad spectrum of stakeholders who share the association's commitment to addressing structural racism and realizing true health equity.

The American Heart Association at 100: A Century of Scientific Progress and the Future of Cardiovascular Science: A Presidential Advisory From the American Heart Association.

In 1924, the founders of the American Heart Association (AHA) envisioned an international society focused on the heart and aimed at facilitating research, disseminating information, increasing public awareness, and developing public health policy related to heart disease. This presidential advisory provides a comprehensive review of the past century of cardiovascular and stroke science, with a focus on the AHA's contributions, as well as informed speculation about the future of cardiovascular science into the next century of the organization's history. The AHA is a leader in fundamental, translational, clinical, and population science, and it promotes the concept of the "learning health system," in which a continuous cycle of evidence-based practice leads to practice-based evidence, permitting an iterative refinement in clinical evidence and care. This advisory presents the AHA's journey over the past century from instituting professional membership to establishing extraordinary research funding programs; translating evidence to practice through clinical practice guidelines; affecting systems of care through quality programs, certification, and implementation; leading important advocacy efforts at the federal, state and local levels; and building global coalitions around cardiovascular and stroke science and public health. Recognizing an exciting potential future for science and medicine, the advisory offers a vision for even greater impact for the AHA's second century in its continued mission to be a relentless force for longer, healthier lives.

Uterus Transplantation: The Translational Evolution of a Clinical Breakthrough.

Women suffering from absolute uterine factor infertility (AUFI), due to either lack of a uterus or one unable to sustain neonatal viability, presented as one of the last frontiers in conquering infertility. Following systematic animal research for over a decade, uterus transplantation was tested as a treatment for AUFI in 2012, which culminated in the first human live birth in 2014. The development of uterus transplantation from mouse to human has followed both the Moore Criteria for introduction of a surgical innovation and the IDEAL concept for evaluation of a novel major surgical procedure. In this article we review the important pre-clinical animal and human studies that paved the way for the successful introduction of human uterus transplantation a decade ago. We discuss this in the context of the Moore Criteria and describe the different procedures of preparation, surgeries, post-operative monitoring, and use of assisted reproduction in human uterus transplantation. We review the world-wide activities and associated results in the context of the IDEAL concept for evaluation of surgical innovation and appraise the ethical considerations relevant to uterus transplantation. We conclude that rigorous application of the Moore Criteria and strict alignment with the IDEAL concept has resulted in the establishment of uterus transplantation as a novel, safe and effective infertility therapy that is now being used worldwide for the treatment of women suffering from AUFI.

Challenges and opportunities for the next generation of cardiovascular tissue engineering.

Engineered cardiac tissues derived from human induced pluripotent stem cells offer unique opportunities for patient-specific disease modeling, drug discovery and cardiac repair. Since the first engineered hearts were introduced over two decades ago, human induced pluripotent stem cell-based three-dimensional cardiac organoids and heart-on-a-chip systems have now become mainstays in basic cardiovascular research as valuable platforms for investigating fundamental human pathophysiology and development. However, major obstacles remain to be addressed before the field can truly advance toward commercial and clinical translation. Here we provide a snapshot of the state-of-the-art methods in cardiac tissue engineering, with a focus on in vitro models of the human heart. Looking ahead, we discuss major challenges and opportunities in the field and suggest strategies for enabling broad acceptance of engineered cardiac tissues as models of cardiac pathophysiology and testbeds for the development of therapies.

ADMET-AI: A machine learning ADMET platform for evaluation of large-scale chemical libraries.

MOTIVATION: The emergence of large chemical repositories and combinatorial chemical spaces, coupled with high-throughput docking and generative AI, have greatly expanded the chemical diversity of small molecules for drug discovery. Selecting compounds for experimental validation requires filtering these molecules based on favourable druglike properties, such as Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET). RESULTS: We developed ADMET-AI, a machine learning platform that provides fast and accurate ADMET predictions both as a website and as a Python package. ADMET-AI has the highest average rank on the TDC ADMET Leaderboard, and it is currently the fastest web-based ADMET predictor, with a 45% reduction in time compared to the next fastest public ADMET web server. ADMET-AI can also be run locally with predictions for one million molecules taking just 3.1 hours. AVAILABILITY: The ADMET-AI platform is freely available both as a web server at admet.ai.greenstonebio.com and as an open-source Python package for local batch prediction at github.com/swansonk14/admet_ai (also archived on Zenodo at doi.org/10.5281/zenodo.10372930). All data and models are archived on Zenodo at doi.org/10.5281/zenodo.10372418. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Impaired Human Cardiac Cell Development due to NOTCH1 Deficiency.

BACKGROUND: NOTCH1 pathogenic variants are implicated in multiple types of congenital heart defects including hypoplastic left heart syndrome, where the left ventricle is underdeveloped. It is unknown how NOTCH1 regulates human cardiac cell lineage determination and cardiomyocyte proliferation. In addition, mechanisms by which NOTCH1 pathogenic variants lead to ventricular hypoplasia in hypoplastic left heart syndrome remain elusive. METHODS: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 genome editing was utilized to delete NOTCH1 in human induced pluripotent stem cells. Cardiac differentiation was carried out by sequential modulation of WNT signaling, and NOTCH1 knockout and wild-type differentiating cells were collected at day 0, 2, 5, 10, 14, and 30 for single-cell RNA-seq. RESULTS: Human NOTCH1 knockout induced pluripotent stem cells are able to generate functional cardiomyocytes and endothelial cells, suggesting that NOTCH1 is not required for mesoderm differentiation and cardiovascular development in vitro. However, disruption of NOTCH1 blocks human ventricular-like cardiomyocyte differentiation but promotes atrial-like cardiomyocyte generation through shortening the action potential duration. NOTCH1 deficiency leads to defective proliferation of early human cardiomyocytes, and transcriptomic analysis indicates that pathways involved in cell cycle progression and mitosis are downregulated in NOTCH1 knockout cardiomyocytes. Single-cell transcriptomic analysis reveals abnormal cell lineage determination of cardiac mesoderm, which is manifested by the biased differentiation toward epicardial and second heart field progenitors at the expense of first heart field progenitors in NOTCH1 knockout cell populations. CONCLUSIONS: NOTCH1 is essential for human ventricular-like cardiomyocyte differentiation and proliferation through balancing cell fate determination of cardiac mesoderm and modulating cell cycle progression. Because first heart field progenitors primarily contribute to the left ventricle, we speculate that pathogenic NOTCH1 variants lead to biased differentiation of first heart field progenitors, blocked ventricular-like cardiomyocyte differentiation, and defective cardiomyocyte proliferation, which collaboratively contribute to left ventricular hypoplasia in hypoplastic left heart syndrome.

Robust expansion of phylogeny for fast-growing genome sequence data.

Massive sequencing of SARS-CoV-2 genomes has urged novel methods that employ existing phylogenies to add new samples efficiently instead of de novo inference. 'TIPars' was developed for such challenge integrating parsimony analysis with pre-computed ancestral sequences. It took about 21 seconds to insert 100 SARS-CoV-2 genomes into a 100k-taxa reference tree using 1.4 gigabytes. Benchmarking on four datasets, TIPars achieved the highest accuracy for phylogenies of moderately similar sequences. For highly similar and divergent scenarios, fully parsimony-based and likelihood-based phylogenetic placement methods performed the best respectively while TIPars was the second best. TIPars accomplished efficient and accurate expansion of phylogenies of both similar and divergent sequences, which would have broad biological applications beyond SARS-CoV-2. TIPars is accessible from https://tipars.hku.hk/ and source codes are available at https://github.com/id-bioinfo/TIPars.

Activation of PDGF pathway links LMNA mutation to dilated cardiomyopathy.

Lamin A/C (LMNA) is one of the most frequently mutated genes associated with dilated cardiomyopathy (DCM). DCM related to mutations in LMNA is a common inherited cardiomyopathy that is associated with systolic dysfunction and cardiac arrhythmias. Here we modelled the LMNA-related DCM in vitro using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Electrophysiological studies showed that the mutant iPSC-CMs displayed aberrant calcium homeostasis that led to arrhythmias at the single-cell level. Mechanistically, we show that the platelet-derived growth factor (PDGF) signalling pathway is activated in mutant iPSC-CMs compared to isogenic control iPSC-CMs. Conversely, pharmacological and molecular inhibition of the PDGF signalling pathway ameliorated the arrhythmic phenotypes of mutant iPSC-CMs in vitro. Taken together, our findings suggest that the activation of the PDGF pathway contributes to the pathogenesis of LMNA-related DCM and point to PDGF receptor-ß (PDGFRB) as a potential therapeutic target.

The role of metabolism in directed differentiation versus trans-differentiation of cardiomyocytes.

The advent of induced pluripotent stem cells (iPSCs) and identification of transcription factors for cardiac reprogramming have raised hope to cure heart disease, the leading cause of death in the world. Our knowledge in heart development and molecular barriers of cardiac reprogramming is advancing, but many hurdles are yet to be overcome for clinical translation. Importantly, we lack a full understanding of molecular mechanisms governing cell fate conversion toward cardiomyocytes. In this review, we will discuss the role of metabolism in directed differentiation versus trans-differentiation of cardiomyocytes. Cardiomyocytes exhibit a unique metabolic feature distinct from PSCs and cardiac fibroblasts, and there are multiple overlapping molecular mechanisms underlying metabolic reprogramming during cardiomyogenesis. We will discuss key metabolic changes occurring during cardiomyocytes differentiation from PSCs and cardiac fibroblasts, and the potential role of metabolic reprogramming in the enhancement strategies for cardiomyogenesis. Only when such details are discovered will more effective strategies to enhance the de novo production of cardiomyocytes be possible.