Multiscale drug screening for cardiac fibrosis identifies MD2 as a therapeutic target.

Cardiac fibrosis impairs cardiac function, but no effective clinical therapies exist. To address this unmet need, we employed a high-throughput screening for antifibrotic compounds using human induced pluripotent stem cell (iPSC)-derived cardiac fibroblasts (CFs). Counter-screening of the initial candidates using iPSC-derived cardiomyocytes and iPSC-derived endothelial cells excluded hits with cardiotoxicity. This screening process identified artesunate as the lead compound. Following profibrotic stimuli, artesunate inhibited proliferation, migration, and contraction in human primary CFs, reduced collagen deposition, and improved contractile function in 3D-engineered heart tissues. Artesunate also attenuated cardiac fibrosis and improved cardiac function in heart failure mouse models. Mechanistically, artesunate targeted myeloid differentiation factor 2 (MD2) and inhibited MD2/Toll-like receptor 4 (TLR4) signaling pathway, alleviating fibrotic gene expression in CFs. Our study leverages multiscale drug screening that integrates a human iPSC platform, tissue engineering, animal models, in silico simulations, and multiomics to identify MD2 as a therapeutic target for cardiac fibrosis.

Tachycardia-induced metabolic rewiring as a driver of contractile dysfunction.

Prolonged tachycardia-a risk factor for cardiovascular morbidity and mortality-can induce cardiomyopathy in the absence of structural disease in the heart. Here, by leveraging human patient data, a canine model of tachycardia and engineered heart tissue generated from human induced pluripotent stem cells, we show that metabolic rewiring during tachycardia drives contractile dysfunction by promoting tissue hypoxia, elevated glucose utilization and the suppression of oxidative phosphorylation. Mechanistically, a metabolic shift towards anaerobic glycolysis disrupts the redox balance of nicotinamide adenine dinucleotide (NAD), resulting in increased global protein acetylation (and in particular the acetylation of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), a molecular signature of heart failure. Restoration of NAD redox by NAD+ supplementation reduced sarcoplasmic/endoplasmic reticulum Ca2+-ATPase acetylation and accelerated the functional recovery of the engineered heart tissue after tachycardia. Understanding how metabolic rewiring drives tachycardia-induced cardiomyopathy opens up opportunities for therapeutic intervention.

Contractility and Calcium Transient Maturation in the Human iPSC-Derived Cardiac Microfibers.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are considered immature in the sarcomere organization, contractile machinery, calcium transient, and transcriptome profile, which prevent them from further applications in modeling and studying cardiac development and disease. To improve the maturity of hiPSC-CMs, here, we engineered the hiPSC-CMs into cardiac microfibers (iCMFs) by a stencil-based micropatterning method, which enables the hiPSC-CMs to be aligned in an end-to-end connection for prolonged culture on the hydrogel of physiological stiffness. A series of characterization approaches were performed to evaluate the maturation in iCMFs on both structural and functional levels, including immunohistochemistry, calcium transient, reverse-transcription quantitative PCR, cardiac contractility, and electrical pacing analysis. Our results demonstrate an improved cardiac maturation of hiPSC-CMs in iCMFs compared to micropatterned or random single hiPSC-CMs and hiPSC-CMs in a random cluster at the same cell number of iCMFs. We found an increased sarcomere length, better regularity and alignment of sarcomeres, enhanced contractility, matured calcium transient, and T-tubule formation and improved adherens junction and gap junction formation. The hiPSC-CMs in iCMFs showed a robust calcium cycling in response to the programmed and continuous electrical pacing from 0.5 to 7 Hz. Moreover, we generated the iCMFs with hiPSC-CMs with mutations in myosin-binding protein C (MYBPC3) to have a proof-of-concept of iCMFs in modeling cardiac hypertrophic phenotype. These findings suggest that the multipatterned iCMF connection of hiPSC-CMs boosts the cardiac maturation structurally and functionally, which will reveal the full potential of the application of hiPSC-CM models in disease modeling of cardiomyopathy and cardiac regenerative medicine.

Human induced pluripotent stem cells for studying mitochondrial diseases in the heart.

Mitochondrial dysfunction is known to contribute to a range of diseases, and primary mitochondrial defects strongly impact high-energy organs such as the heart. Platforms for high-throughput and human-relevant assessment of mitochondrial diseases are currently lacking, hindering the development of targeted therapies. In the past decade, human-induced pluripotent stem cells (iPSCs) have become a promising technology for drug discovery in basic and clinical research. In particular, human iPSC-derived cardiomyocytes (iPSC-CMs) offer a unique tool to study a wide range of mitochondrial functions and possess the potential to become a key translational asset for mitochondrial drug development. This review summarizes mitochondrial functions and recent therapeutic discoveries, advancements and limitations of using iPSC-CMs to study mitochondrial diseases of the heart with an emphasis on cardiac applications.

Alpine Treeline Dynamics and the Special Exposure Effect in the Hengduan Mountains.

Alpine treeline is highly sensitive to climate change, but there remains a lack of research on the spatiotemporal heterogeneity of treeline and their relationships with climate change at the landscape scale. We extracted positions of alpine treeline from high-resolution Google Earth images from three periods (2000, 2010, and 2020) and analyzed the elevation patterns and dynamics of treeline positions in the Hengduan Mountains. Based on the treeline positions in 2020, a buffer zone of 300 m is established as the treeline transition zone, and the changing trend of the fraction vegetation cover (FVC) from 2000 to 2020 and its relationship with climate are also analyzed. Due to the special geographical and climatic environment, the treeline in the Hengduan Mountains area is high in the middle but lower in the surrounding areas. We found that over the past 20 years, the treeline position did not change significantly but that the FVC increased in 80.3% of the treeline areas. The increase in FVC was related to the decrease in precipitation in the growing season. The results also revealed a special exposure effect on the alpine treeline in the Hengduan Mountains. Because of the lower treeline, isotherm position caused by the monsoon climate, the treeline position on south-facing slopes is lower than that on slopes with other exposures. Our results confirmed that the pattern and dynamics of the alpine treeline are driven by the regional monsoon climate regime.

Herbivore-Mediated Selection on Floral Display Covaries Nonlinearly With Plant-Antagonistic Interaction Intensity Among Primrose Populations.

Quantifying the relations between plant-antagonistic interactions and natural selection among populations is important for predicting how spatial variation in ecological interactions drive adaptive differentiation. Here, we investigate the relations between the opportunity for selection, herbivore-mediated selection, and the intensity of plant-herbivore interaction among 11 populations of the insect-pollinated plant Primula florindae over 2 years. We experimentally quantified herbivore-mediated directional selection on three floral traits (two display and one phenological) within populations and found evidence for herbivore-mediated selection for a later flowering start date and a greater number of flowers per plant. The opportunity for selection and strength of herbivore-mediated selection on number of flowers varied nonlinearly with the intensity of herbivory among populations. These parameters increased and then decreased with increasing intensity of plant-herbivore interactions, defined as an increase in the ratio of herbivore-damaged flowers per individual. Our results provide novel insights into how plant-antagonistic interactions can shape spatial variation in selection on floral traits and contribute toward understanding the mechanistic basis of geographic variation in angiosperm flowers.

Protocol to measure contraction, calcium, and action potential in human-induced pluripotent stem cell-derived cardiomyocytes.

Multiple strategies have been developed to efficiently differentiate human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we describe a protocol for measuring three key functional parameters of hiPSC-CMs, including contractile function, calcium (Ca2+) handling, and action potential. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021).

Microclimate feedbacks sustain power law clustering of encroaching coastal woody vegetation.

The spatial pattern of vegetation patchiness may follow universal characteristic rules when the system is close to critical transitions between alternative states, which improves the anticipation of ecosystem-level state changes which are currently difficult to detect in real systems. However, the spatial patterning of vegetation patches in temperature-driven ecosystems have not been investigated yet. Here, using high-resolution imagery from 1972 to 2013 and a stochastic cellular automata model, we show that in a North American coastal ecosystem where woody plant encroachment has been happening, the size distribution of woody patches follows a power law when the system approaches a critical transition, which is sustained by the local positive feedbacks between vegetation and the surrounding microclimate. Therefore, the observed power law distribution of woody vegetation patchiness may be suggestive of critical transitions associated with temperature-driven woody plant encroachment in coastal and potentially other ecosystems.

Human Induced Pluripotent Stem Cells as a Screening Platform for Drug-Induced Vascular Toxicity.

Evaluation of potential vascular injury is an essential part of the safety study during pharmaceutical development. Vascular liability issues are important causes of drug termination during preclinical investigations. Currently, preclinical assessment of vascular toxicity primarily relies on the use of animal models. However, accumulating evidence indicates a significant discrepancy between animal toxicity and human toxicity, casting doubt on the clinical relevance of animal models for such safety studies. While the causes of this discrepancy are expected to be multifactorial, species differences are likely a key factor. Consequently, a human-based model is a desirable solution to this problem, which has been made possible by the advent of human induced pluripotent stem cells (iPSCs). In particular, recent advances in the field now allow the efficient generation of a variety of vascular cells (e.g., endothelial cells, smooth muscle cells, and pericytes) from iPSCs. Using these cells, different vascular models have been established, ranging from simple 2D cultures to highly sophisticated vascular organoids and microfluidic devices. Toxicity testing using these models can recapitulate key aspects of vascular pathology on molecular (e.g., secretion of proinflammatory cytokines), cellular (e.g., cell apoptosis), and in some cases, tissue (e.g., endothelium barrier dysfunction) levels. These encouraging data provide the rationale for continuing efforts in the exploration, optimization, and validation of the iPSC technology in vascular toxicology.

Transcriptome analysis of non human primate-induced pluripotent stem cell-derived cardiomyocytes in 2D monolayer culture vs. 3D engineered heart tissue.

AIMS: Stem cell therapy has shown promise for treating myocardial infarction via re-muscularization and paracrine signalling in both small and large animals. Non-human primates (NHPs), such as rhesus macaques (Macaca mulatta), are primarily utilized in preclinical trials due to their similarity to humans, both genetically and physiologically. Currently, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are delivered into the infarcted myocardium by either direct cell injection or an engineered tissue patch. Although both approaches have advantages in terms of sample preparation, cell-host interaction, and engraftment, how the iPSC-CMs respond to ischaemic conditions in the infarcted heart under these two different delivery approaches remains unclear. Here, we aim to gain a better understanding of the effects of hypoxia on iPSC-CMs at the transcriptome level. METHODS AND RESULTS: NHP iPSC-CMs in both monolayer culture (2D) and engineered heart tissue (EHT) (3D) format were exposed to hypoxic conditions to serve as surrogates of direct cell injection and tissue implantation in vivo, respectively. Outcomes were compared at the transcriptome level. We found the 3D EHT model was more sensitive to ischaemic conditions and similar to the native in vivo myocardium in terms of cell-extracellular matrix/cell-cell interactions, energy metabolism, and paracrine signalling. CONCLUSION: By exposing NHP iPSC-CMs to different culture conditions, transcriptome profiling improves our understanding of the mechanism of ischaemic injury.

Dimensionality reduction of complex dynamical systems.

One of the outstanding problems in complexity science and engineering is the study of high-dimensional networked systems and of their susceptibility to transitions to undesired states as a result of changes in external drivers or in the structural properties. Because of the incredibly large number of parameters controlling the state of such complex systems and the heterogeneity of its components, the study of their dynamics is extremely difficult. Here we propose an analytical framework for collapsing complex N-dimensional networked systems into an S+1-dimensional manifold as a function of S effective control parameters with S << N. We test our approach on a variety of real-world complex problems showing how this new framework can approximate the system's response to changes and correctly identify the regions in the parameter space corresponding to the system's transitions. Our work offers an analytical method to evaluate optimal strategies in the design or management of networked systems.

Critical slowing down associated with critical transition and risk of collapse in crypto-currency.

The year 2017 saw the rise and fall of the crypto-currency market, followed by high variability in the price of all crypto-currencies. In this work, we study the abrupt transition in crypto-currency residuals, which is associated with the critical transition (the phenomenon of critical slowing down) or the stochastic transition phenomena. We find that, regardless of the specific crypto-currency or rolling window size, the autocorrelation always fluctuates around a high value, while the standard deviation increases monotonically. Therefore, while the autocorrelation does not display the signals of critical slowing down, the standard deviation can be used to anticipate critical or stochastic transitions. In particular, we have detected two sudden jumps in the standard deviation, in the second quarter of 2017 and at the beginning of 2018, which could have served as the early warning signals of two major price collapses that have happened in the following periods. We finally propose a mean-field phenomenological model for the price of crypto-currency to show how the use of the standard deviation of the residuals is a better leading indicator of the collapse in price than the time-series' autocorrelation. Our findings represent a first step towards a better diagnostic of the risk of critical transition in the price and/or volume of crypto-currencies.

Universal Cointegration and Its Applications.

Cointegration focuses on whether the long-term linear relationship between two or more time series is stationary even if this linear relationship does not exist or is not strong for the short term. Identifying the potential cointegration is important for economics, ecology, meteorology, neuroscience, and much more. Classic methods only considered or restricted in cointegration where the order of integration of all time series is 1. We introduce a method based on searching the vector to minimize the absolute correlation of convergent cross-mapping that can explore the universal cointegration and its extent. The proposed method can be applied to time series whose order of integration is not 1, cases that are not covered by classic cointegration. The proposed method is first illustrated and validated through time series generated by mathematical models in which the underlying relationships are known and then applied to three real-world examples.

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.

Brain controllability: Not a slam dunk yet.

In our recent article [1] published in this journal we provide quantitative evidence to show that there are warnings and caveats in the way Gu and collaborators [2] define controllability of brain networks and measure the contribution of each of its nodes. The comment by Pasqualetti et al. [3] confirms the need to go beyond the methodology and approach presented in Gu et al.'s original work. In fact, they recognize that "the source of confusion is due to the fact that assessing controllability via numerical analysis typically leads to ill-conditioned problems, and thus often generates results that are difficult to interpret". This is indeed the first warning we discussed in [1]: our work was not meant to prove that brain networks are not controllable from one node, rather we wished to highlight that the one node controllability framework and all consequent results were not properly justified based on the methodology presented in Gu et al. [2]. We used in our work the same method of Gu et al. not because we believe it is the best methodology, but because we extensively investigated it with the aim of replicating, testing, and extending their results. The warning and caveats we have proposed are the results of this investigation. Indeed, on the basis of our controllability analyses of multiple human brain networks datasets, we concluded: "The λmin(WK) are statistically compatible with zero and thus the associated controllability Gramian cannot be inverted1. These results show that it is not possible to infer one node controllability of the brain numerically". Hence both groups agree that one node controllability cannot be inferred numerically.

Reconciling cooperation, biodiversity and stability in complex ecological communities.

Empirical evidences show that ecosystems with high biodiversity can persist in time even in the presence of few types of resources and are more stable than low biodiverse communities. This evidence is contrasted by the conventional mathematical modeling, which predicts that the presence of many species and/or cooperative interactions are detrimental for ecological stability and persistence. Here we propose a modelling framework for population dynamics, which also include indirect cooperative interactions mediated by other species (e.g. habitat modification). We show that in the large system size limit, any number of species can coexist and stability increases as the number of species grows, if mediated cooperation is present, even in presence of exploitative or harmful interactions (e.g. antibiotics). Our theoretical approach thus shows that appropriate models of mediated cooperation naturally lead to a solution of the long-standing question about complexity-stability paradox and on how highly biodiverse communities can coexist.

Temporal Impact of Substrate Anisotropy on Differentiating Cardiomyocyte Alignment and Functionality.

Anisotropic biomaterials can affect cell function by driving cell alignment, which is critical for cardiac engineered tissues. Recent work, however, has shown that pluripotent stem cell-derived cardiomyocytes may self-align over long periods of time. To determine how the degree of biomaterial substrate anisotropy impacts differentiating cardiomyocyte structure and function, we differentiated mouse embryonic stem cells to cardiomyocytes on nonaligned, semialigned, and aligned fibrous substrates and evaluated cell alignment, contractile displacement, and calcium transient synchronicity over time. Although cardiomyocyte gene expression was not affected by fiber alignment, we observed gradient- and threshold-based differences in cardiomyocyte alignment and function. Cardiomyocyte alignment increased with the degree of fiber alignment in a gradient-based manner at early time points and in a threshold-based manner at later time points. Calcium transient synchronization tightly followed cardiomyocyte alignment behavior, allowing highly anisotropic biomaterials to drive calcium transient synchronization within 8 days, while such synchronized cardiomyocyte behavior required 20 days of culture on nonaligned biomaterials. In contrast, cardiomyocyte contractile displacement had no directional preference on day 8 yet became anisotropic in the direction of fiber alignment on aligned fibers by day 20. Biomaterial anisotropy impact on differentiating cardiomyocyte structure and function is temporally dependent. Impact Statement This work demonstrates that biomaterial anisotropy can quickly drive desired pluripotent stem cell-derived cardiomyocyte structure and function. Such an understanding of matrix anisotropy's time-dependent influence on stem cell-derived cardiomyocyte function will have future applications in the development of cardiac cell therapies and in vitro cardiac tissues for drug testing. Furthermore, our work has broader implications concerning biomaterial anisotropy effects on other cell types in which function relies on alignment, such as myocytes and neurons.