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Forests around the world are experiencing changes due to climate variability and human land use. How these changes interact and influence the vulnerability of forests are not well understood. In the eastern United States, well-documented anthropogenic disturbances and land-use decisions, such as logging and fire suppression, have influenced forest species assemblages, leading to a demographic shift from forests dominated by xeric species to those dominated by mesic species. Contemporarily, the climate has changed and is expected to continue to warm and produce higher evaporative demand, imposing stronger drought stress on forest communities. Here, we use an extensive network of tree-ring records from common hardwood species across ~100 sites and ~1300 trees in the eastern United States to examine the magnitude of growth response to both wet and dry climate extremes. We find that growth reductions during drought exceed the positive growth response to pluvials. Mesic species such as Liriodendron tulipifera and Acer saccharum, which are becoming more dominant, are more sensitive to drought than more xeric species, such as oaks (Quercus) and hickory (Carya), especially at moderate and extreme drought intensities. Although more extreme droughts produce a larger annual growth reduction, mild droughts resulted in the largest cumulative growth decreases due to their higher frequency. When using global climate model projections, all scenarios show drought frequency increasing substantially (3-9 times more likely) by 2100. Thus, the ongoing demographic shift toward more mesic species in the eastern United States combined with drier conditions results in larger drought-induced growth declines, suggesting that drought will have an even larger impact on aboveground carbon uptake in the future in the eastern United States., (© 2024 The Author(s). Global Change Biology published by John Wiley & Sons Ltd.)

The field of regenerative medicine has witnessed remarkable advancements with the emergence of induced pluripotent stem cells (iPSCs) derived from a variety of sources. Among these, urine-derived induced pluripotent stem cells (u-iPSCs) have garnered substantial attention due to their non-invasive and patient-friendly acquisition method. This review manuscript delves into the potential and application of u-iPSCs in advancing precision medicine, particularly in the realms of drug testing, disease modeling, and cell therapy. U-iPSCs are generated through the reprogramming of somatic cells found in urine samples, offering a unique and renewable source of patient-specific pluripotent cells. Their utility in drug testing has revolutionized the pharmaceutical industry by providing personalized platforms for drug screening, toxicity assessment, and efficacy evaluation. The availability of u-iPSCs with diverse genetic backgrounds facilitates the development of tailored therapeutic approaches, minimizing adverse effects and optimizing treatment outcomes. Furthermore, u-iPSCs have demonstrated remarkable efficacy in disease modeling, allowing researchers to recapitulate patient-specific pathologies in vitro. This not only enhances our understanding of disease mechanisms but also serves as a valuable tool for drug discovery and development. In addition, u-iPSC-based disease models offer a platform for studying rare and genetically complex diseases, often underserved by traditional research methods. The versatility of u-iPSCs extends to cell therapy applications, where they hold immense promise for regenerative medicine. Their potential to differentiate into various cell types, including neurons, cardiomyocytes, and hepatocytes, enables the development of patient-specific cell replacement therapies. This personalized approach can revolutionize the treatment of degenerative diseases, organ failure, and tissue damage by minimizing immune rejection and optimizing therapeutic outcomes. However, several challenges and considerations, such as standardization of reprogramming protocols, genomic stability, and scalability, must be addressed to fully exploit u-iPSCs' potential in precision medicine. In conclusion, this review underscores the transformative impact of u-iPSCs on advancing precision medicine and highlights the future prospects and challenges in harnessing this innovative technology for improved healthcare outcomes., (© 2024. The Author(s).)

Objective: In this study we aimed to determine the impact of human urine derived stem cells (USC) and genetically modified USC that were designed to overexpress myogenic growth factor IGF1 (USC IGF ), on the regenerative capacity of cardiotoxin (CTX)-injured murine skeletal muscle., Methods: We overexpressed IGF1 in USC and investigated the alterations in myogenic capacity and regenerative function in cardiotoxin-injured muscle tissues., Results: Compared with USC alone, USC IGF1 activated the IGF1-Akt-mTOR signaling pathway, significantly improved myogenic differentiation capacity in vitro, and enhanced the secretion of myogenic growth factors and cytokines. In addition, IGF1 overexpression increased the ability of USC to fuse with skeletal myocytes to form myotubes, regulated the pro-regenerative immune response and inflammatory cytokines, and increased myogenesis in an in vivo model of skeletal muscle injury., Conclusion: Overall, USC genetically modified to overexpress IGF1 significantly enhanced skeletal muscle regeneration by regulating myogenic differentiation, paracrine effects, and cell fusion, as well as by modulating immune responses in injured skeletal muscles in vivo. This study provides a novel perspective for evaluating the myogenic function of USC as a nonmyogenic cell source in skeletal myogenesis. The combination of USC and IGF1 expression has the potential to provide a novel efficient therapy for skeletal muscle injury and associated muscular defects in patients with urinary incontinence., Competing Interests: None., (AJCEU Copyright © 2024.)

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) possess tremendous potential for basic research and translational application. However, these cells structurally and functionally resemble fetal cardiomyocytes, which is a major limitation of these cells. Microgravity can significantly alter cell behavior and function. Here we investigated the effect of simulated microgravity on hiPSC-CM maturation. Following culture under simulated microgravity in a random positioning machine for 7 days, 3D hiPSC-CMs had increased mitochondrial content as detected by a mitochondrial protein and mitochondrial DNA to nuclear DNA ratio. The cells also had increased mitochondrial membrane potential. Consistently, simulated microgravity increased mitochondrial respiration in 3D hiPSC-CMs, as indicated by higher levels of maximal respiration and ATP content, suggesting improved metabolic maturation in simulated microgravity cultures compared with cultures under normal gravity. Cells from simulated microgravity cultures also had improved Ca 2+ transient parameters, a functional characteristic of more mature cardiomyocytes. In addition, these cells had improved structural properties associated with more mature cardiomyocytes, including increased sarcomere length, z-disc length, nuclear diameter, and nuclear eccentricity. These findings indicate that microgravity enhances the maturation of hiPSC-CMs at the structural, metabolic, and functional levels., (© 2024. The Author(s).)

Background: Cardiac pathological outcome of metabolic remodeling is difficult to model using cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) due to low metabolic maturation., Methods: hiPSC-CM spheres were treated with AMP-activated protein kinase (AMPK) activators and examined for hiPSC-CM maturation features, molecular changes and the response to pathological stimuli., Results: Treatment of hiPSC-CMs with AMPK activators increased ATP content, mitochondrial membrane potential and content, mitochondrial DNA, mitochondrial function and fatty acid uptake, indicating increased metabolic maturation. Conversely, the knockdown of AMPK inhibited mitochondrial maturation of hiPSC-CMs. In addition, AMPK activator-treated hiPSC-CMs had improved structural development and functional features-including enhanced Ca 2+ transient kinetics and increased contraction. Transcriptomic, proteomic and metabolomic profiling identified differential levels of expression of genes, proteins and metabolites associated with a molecular signature of mature cardiomyocytes in AMPK activator-treated hiPSC-CMs. In response to pathological stimuli, AMPK activator-treated hiPSC-CMs had increased glycolysis, and other pathological outcomes compared to untreated cells., Conclusion: AMPK activator-treated cardiac spheres could serve as a valuable model to gain novel insights into cardiac diseases., (© 2023. The Author(s).)

Increasing tropical cyclone (TC) pressure on temperate forests is inevitable under the recent global increase of the intensity and poleward migration of TCs. However, the long-term effects of TCs on large-scale structure and diversity of temperate forests remain unclear. Here, we aim to ascertain the legacy of TCs on forest structure and tree species richness by using structural equation models that consider several environmental gradients and use an extensive dataset containing >140,000 plots with >3 million trees from natural temperate forests across eastern United States impacted by TCs. We found that high TC activity (a combination of TC frequency and intensity) leads to a decrease in maximum tree sizes (height and diameter), an increase in tree density and basal area, and a decline in the number of tree species and recruits. We identified TC activity as the strongest predictor of forest structure and species richness in xeric (dry) forests, while it had a weaker impact on hydric (wet) forests. We highlight the sensitivity of forest structure and tree species richness to impacts of likely further increase of TC activity in interaction with climate extremes, especially drought. Our results show that increased TC activity leads to the homogenization of forest structure and reduced tree species richness in U.S. temperate forests. These findings suggest that further declines in tree species richness may be expected because of the projected increase of future levels of TC activity., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

As the climate changes, warmer spring temperatures are causing earlier leaf-out 1-3 and commencement of CO 2 uptake 1,3 in temperate deciduous forests, resulting in a tendency towards increased growing season length 3 and annual CO 2 uptake 1,3-7 . However, less is known about how spring temperatures affect tree stem growth 8,9 , which sequesters carbon in wood that has a long residence time in the ecosystem 10,11 . Here we show that warmer spring temperatures shifted stem diameter growth of deciduous trees earlier but had no consistent effect on peak growing season length, maximum growth rates, or annual growth, using dendrometer band measurements from 440 trees across two forests. The latter finding was confirmed on the centennial scale by 207 tree-ring chronologies from 108 forests across eastern North America, where annual ring width was far more sensitive to temperatures during the peak growing season than in the spring. These findings imply that any extra CO 2 uptake in years with warmer spring temperatures 4,5 does not significantly contribute to increased sequestration in long-lived woody stem biomass. Rather, contradicting projections from global carbon cycle models 1,12 , our empirical results imply that warming spring temperatures are unlikely to increase woody productivity enough to strengthen the long-term CO 2 sink of temperate deciduous forests., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Uncertainties surrounding tree carbon allocation to growth are a major limitation to projections of forest carbon sequestration and response to climate change. The prevalence and extent to which carbon assimilation (source) or cambial activity (sink) mediate wood production are fundamentally important and remain elusive. We quantified source-sink relations across biomes by combining eddy-covariance gross primary production with extensive on-site and regional tree ring observations. We found widespread temporal decoupling between carbon assimilation and tree growth, underpinned by contrasting climatic sensitivities of these two processes. Substantial differences in assimilation-growth decoupling between angiosperms and gymnosperms were determined, as well as stronger decoupling with canopy closure, aridity, and decreasing temperatures. Our results reveal pervasive sink control over tree growth that is likely to be increasingly prominent under global climate change.

Nearly 1 in every 100 children born have a congenital heart defect. Many of these defects primarily affect the right heart causing pressure overload of the right ventricle (RV). The RV maintains function by adapting to the increased pressure; however, many of these adaptations eventually lead to RV hypertrophy and failure. In this study, we aim to identify the cellular and molecular mechanisms of these adaptions. We utilized a surgical animal model of pulmonary artery banding (PAB) in juvenile rats that has been shown to accurately recapitulate the physiology of right ventricular pressure overload in young hearts. Using this model, we examined changes in cardiac myocyte protein expression as a result of pressure overload with mass spectrometry 4 weeks post-banding. We found pressure overload of the RV induced significant downregulation of cardiac myosin light chain kinase (cMLCK). Single myocyte calcium and contractility recordings showed impaired contraction and relaxation in PAB RV myocytes, consistent with the loss of cMLCK. In the PAB myocytes, calcium transients were of smaller amplitude and decayed at a slower rate compared to controls. We also identified miR-200c, which has been shown to regulate cMLCK expression, as upregulated in the RV in response to pressure overload. These results indicate the loss of cMLCK is a critical maladaptation of the RV to pressure overload and represents a novel target for therapeutic approaches to treat RV hypertrophy and failure associated with congenital heart defects., (© 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Pediatric patients with congenital heart defects (CHD) often present with heart failure from increased load on the right ventricle (RV) due to both surgical methods to treat CHD and the disease itself. Patients with RV failure often require transplantation, which is limited due to lack of donor availability and rejection. Previous studies investigating the development and in vitro assessment of a bioprinted cardiac patch composed of cardiac extracellular matrix (cECM) and human c-kit + progenitor cells (hCPCs) showed that the construct has promise in treating cardiac dysfunction. The current study investigates in vivo cardiac outcomes of patch implantation in a rat model of RV failure. Patch parameters including cECM-inclusion and hCPC-inclusion are investigated. Assessments include hCPC retention, RV function, and tissue remodeling (vascularization, hypertrophy, and fibrosis). Animal model evaluation shows that both cell-free and neonatal hCPC-laden cECM-gelatin methacrylate (GelMA) patches improve RV function and tissue remodeling compared to other patch groups and controls. Inclusion of cECM is the most influential parameter driving therapeutic improvements, with or without cell inclusion. This study paves the way for clinical translation in treating pediatric heart failure using bioprinted GelMA-cECM and hCPC-GelMA-cECM patches.

Tree rings provide an invaluable long-term record for understanding how climate and other drivers shape tree growth and forest productivity. However, conventional tree-ring analysis methods were not designed to simultaneously test effects of climate, tree size, and other drivers on individual growth. This has limited the potential to test ecologically relevant hypotheses on tree growth sensitivity to environmental drivers and their interactions with tree size. Here, we develop and apply a new method to simultaneously model nonlinear effects of primary climate drivers, reconstructed tree diameter at breast height (DBH), and calendar year in generalized least squares models that account for the temporal autocorrelation inherent to each individual tree's growth. We analyze data from 3811 trees representing 40 species at 10 globally distributed sites, showing that precipitation, temperature, DBH, and calendar year have additively, and often interactively, influenced annual growth over the past 120 years. Growth responses were predominantly positive to precipitation (usually over ≥3-month seasonal windows) and negative to temperature (usually maximum temperature, over ≤3-month seasonal windows), with concave-down responses in 63% of relationships. Climate sensitivity commonly varied with DBH (45% of cases tested), with larger trees usually more sensitive. Trends in ring width at small DBH were linked to the light environment under which trees established, but basal area or biomass increments consistently reached maxima at intermediate DBH. Accounting for climate and DBH, growth rate declined over time for 92% of species in secondary or disturbed stands, whereas growth trends were mixed in older forests. These trends were largely attributable to stand dynamics as cohorts and stands age, which remain challenging to disentangle from global change drivers. By providing a parsimonious approach for characterizing multiple interacting drivers of tree growth, our method reveals a more complete picture of the factors influencing growth than has previously been possible., (© 2021 The Authors. Global Change Biology published by John Wiley & Sons Ltd. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Background Anticancer therapies have significantly improved patient outcomes; however, cardiac side effects from cancer therapies remain a significant challenge. Cardiotoxicity following treatment with proteasome inhibitors such as carfilzomib is known in clinical settings, but the underlying mechanisms have not been fully elucidated. Methods and Results Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a cell model for drug-induced cytotoxicity in combination with traction force microscopy, functional assessments, high-throughput imaging, and comprehensive omic analyses, we examined the molecular mechanisms involved in structural and functional alterations induced by carfilzomib in hiPSC-CMs. Following the treatment of hiPSC-CMs with carfilzomib at 0.01 to 10 µmol/L, we observed a concentration-dependent increase in carfilzomib-induced toxicity and corresponding morphological, structural, and functional changes. Carfilzomib treatment reduced mitochondrial membrane potential, ATP production, and mitochondrial oxidative respiration and increased mitochondrial oxidative stress. In addition, carfilzomib treatment affected contractility of hiPSC-CMs in 3-dimensional microtissues. At a single cell level, carfilzomib treatment impaired Ca 2+ transients and reduced integrin-mediated traction forces as detected by piconewton tension sensors. Transcriptomic and proteomic analyses revealed that carfilzomib treatment downregulated the expression of genes involved in extracellular matrices, integrin complex, and cardiac contraction, and upregulated stress responsive proteins including heat shock proteins. Conclusions Carfilzomib treatment causes deleterious changes in cellular and functional characteristics of hiPSC-CMs. Insights into these changes could be gained from the changes in the expression of genes and proteins identified from our omic analyses.

Myocardial infarction is one of the largest contributors to cardiovascular disease and reduces the ability of the heart to pump blood. One promising therapeutic approach to address the diminished function is the use of cardiac patches composed of biomaterial substrates and cardiac cells. These patches can be enhanced with the application of an auxetic design, which has a negative Poisson's ratio and can be modified to suit the mechanics of the infarct and surrounding cardiac tissue. Here, we examined multiple auxetic models (orthogonal missing rib and re-entrant honeycomb in two orientations) with tunable mechanical properties as a cardiac patch substrate. Further, we demonstrated that 3D printing based auxetic cardiac patches of varying thicknesses (0.2, 0.4, and 0.6 mm) composed of polycaprolactone and gelatin methacrylate can support induced pluripotent stem cell-derived cardiomyocyte function for 14-day culture. Taken together, this work shows the potential of cellularized auxetic cardiac patches as a suitable tissue engineering approach to treating cardiovascular disease.

Growth differentiation factor 11 (GDF11) is a member of the TGF-β protein family that has been implicated in the development of cardiac hypertrophy. While some studies have suggested that systemic GDF11 protects against cardiomyocyte enlargement and left ventricular wall thickening, there remains uncertainty about the true impact of GDF11 and whether its purported effects are actually attributable to its homolog myostatin. This study was conducted to resolve the statistical and genetic relationships among GDF11, myostatin, and cardiac hypertrophy in a mouse model of human genetics, the Diversity Outbred (DO) stock. In the DO population, serum GDF11 concentrations positively correlated with cardiomyocyte cross-sectional area, while circulating myostatin levels were negatively correlated with body weight, heart weight, and left ventricular wall thickness and mass. Genetic analyses revealed that serum GDF11 concentrations are modestly heritable (0.23) and identified a suggestive peak on murine chromosome 3 in close proximity to the gene Hey1, a transcriptional repressor. Bioinformatic analyses located putative binding sites for the HEY1 protein upstream of the Gdf11 gene in the mouse and human genomes. In contrast, serum myostatin concentrations were more heritable (0.57) than GDF11 concentrations, and mapping identified a significant locus near the gene FoxO1, which has binding motifs within the promoter regions of human and mouse myostatin genes. Together, these findings more precisely define the independent cardiovascular effects of GDF11 and myostatin, as well as their distinct regulatory pathways. Hey1 is a compelling candidate for the regulation of GDF11 and will be further evaluated in future studies., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

The impacts of inland flooding caused by tropical cyclones (TCs), including loss of life, infrastructure disruption, and alteration of natural landscapes, have increased over recent decades. While these impacts are well documented, changes in TC precipitation extremes-the proximate cause of such inland flooding-have been more difficult to detect. Here, we present a latewood tree-ring-based record of seasonal (June 1 through October 15) TC precipitation sums (ΣTCP) from the region in North America that receives the most ΣTCP: coastal North and South Carolina. Our 319-y-long ΣTCP reconstruction reveals that ΣTCP extremes (≥0.95 quantile) have increased by 2 to 4 mm/decade since 1700 CE, with most of the increase occurring in the last 60 y. Consistent with the hypothesis that TCs are moving slower under anthropogenic climate change, we show that seasonal ΣTCP along the US East Coast are positively related to seasonal average TC duration and TC translation speed., Competing Interests: The authors declare no competing interest.

Proinflammatory molecule tumor necrosis factor alpha (TNF-α) is predominantly elevated in cytokine storm as well as worsening cardiac function. Here we model the molecular and functional effects of TNF-α in cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSC). We found that treatment of hiPSC-CMs with TNF-α increased reactive oxygen species (ROS) and caspase 3/7 activity and caused cell death and apoptosis. TNF-α treatment also resulted in dysregulation of cardiomyocyte function with respect to the increased abnormal calcium handling, calcium wave propagation between cells and excitation-contraction coupling. We also uncovered significant changes in gene expression and protein localization caused by TNF-α treatment. Notably, TNF-α treatment altered the expression of ion channels, dysregulated cadherins, and affected the localization of gap-junction protein connexin-43. In addition, TNF-α treatment up-regulated IL-32 (a human specific cytokine, not present in rodents and an inducer of TNF-α) and IL-34 and down-regulated glutamate receptors and cardiomyocyte contractile proteins. These findings provide insights into the molecular and functional consequences from the exposure of human cardiomyocytes to TNF-α. Our study provides a model to incorporate inflammatory factors into hiPSC-CM-based studies to evaluate mechanistic aspects of heart disease., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Treatment-induced cardiotoxicity is a leading noncancer-related cause of acute and late onset morbidity and mortality in cancer patients on antineoplastic drugs such as melphalan-increasing clinical case reports have documented that it could induce cardiotoxicity including severe arrhythmias and heart failure. As the mechanism by which melphalan impairs cardiac cells remains poorly understood, here, we aimed to use cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) to investigate the cellular and molecular mechanisms of melphalan-induced cardiotoxicity., Methods: hiPSC-CMs were generated and treated with clinically relevant doses of melphalan. To characterize melphalan-induced cardiotoxicity, cell viability and apoptosis were quantified at various treatment durations. Ca 2+ transient and contractility analyses were used to examine the alterations of hiPSC-CM function. Proteomic analysis, reactive oxygen species detection, and RNA-Sequencing were conducted to investigate underlying mechanisms., Results: Melphalan treatment of hiPSC-CMs induced oxidative stress, caused Ca 2+ handling defects and dysfunctional contractility, altered global transcriptomic and proteomic profiles, and resulted in apoptosis and cell death. The antioxidant N-acetyl-L-cysteine attenuated these genomic, cellular, and functional alterations. In addition, several other signaling pathways including the p53 and transforming growth factor-β signaling pathways were also implicated in melphalan-induced cardiotoxicity according to the proteomic and transcriptomic analyses., Conclusions: Melphalan induces cardiotoxicity through the oxidative stress pathway. This study provides a unique resource of the global transcriptomic and proteomic datasets for melphalan-induced cardiotoxicity and can potentially open up new clinical mechanism-based targets to prevent and treat melphalan-induced cardiotoxicity.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide an excellent platform for potential clinical and research applications. Identifying abnormal Ca 2+ transients is crucial for evaluating cardiomyocyte function that requires labor-intensive manual effort. Therefore, we develop an analytical pipeline for automatic assessment of Ca 2+ transient abnormality, by employing advanced machine learning methods together with an Analytical Algorithm. First, we adapt an existing Analytical Algorithm to identify Ca 2+ transient peaks and determine peak abnormality based on quantified peak characteristics. Second, we train a peak-level Support Vector Machine (SVM) classifier by using human-expert assessment of peak abnormality as outcome and profiled peak variables as predictive features. Third, we train another cell-level SVM classifier by using human-expert assessment of cell abnormality as outcome and quantified cell-level variables as predictive features. This cell-level SVM classifier can be used to assess additional Ca 2+ transient signals. By applying this pipeline to our Ca 2+ transient data, we trained a cell-level SVM classifier using 200 cells as training data, then tested its accuracy in an independent dataset of 54 cells. As a result, we obtained 88% training accuracy and 87% test accuracy. Further, we provide a free R package to implement our pipeline for high-throughput CM Ca 2+ analysis.

Nearly 1 in every 120 children born has a congenital heart defect. Although surgical therapy has improved survival, many of these children go on to develop right ventricular heart failure (RVHF). The emergence of cardiovascular regenerative medicine as a potential therapeutic strategy for pediatric HF has provided new avenues for treatment with a focus on repairing or regenerating the diseased myocardium to restore cardiac function. Although primarily tried using adult cells and adult disease models, stem cell therapy is relatively untested in the pediatric population. Here, we investigate the ability of electrical stimulation (ES) to enhance the retention and therapeutic function of pediatric cardiac-derived c-kit + progenitor cells (CPCs) in an animal model of RVHF. Human CPCs isolated from pediatric patients were exposed to chronic ES and implanted into the RV myocardium of rats. Cardiac function and cellular retention analysis showed electrically stimulated CPCs (ES-CPCs) were retained in the heart at a significantly higher level and longer time than control CPCs and also significantly improved right ventricular functional parameters. ES also induced upregulation of extracellular matrix and adhesion genes and increased in vitro survival and adhesion of cells. Specifically, upregulation of β1 and β5 integrins contributed to the increased retention of ES-CPCs. Lastly, we show that ES induces CPCs to release higher levels of pro-reparative factors in vitro. These findings suggest that ES can be used to increase the retention, survival, and therapeutic effect of human c-kit + progenitor cells and can have implications on a variety of cell-based therapies. Stem Cells 2019;37:1528-1541., (© 2019 The Authors. Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press 2019.)

Congenital heart disease can lead to severe right ventricular heart failure (RVHF). We have shown that aggregated c-kit + progenitor cells (CPCs) can improve RVHF repair, likely due to exosome-mediated effects. Here, we demonstrate that miRNA content from monolayer (2D) and aggregated (3D) CPC exosomes can be related to in vitro angiogenesis and antifibrosis responses using partial least squares regression (PLSR). PLSR reduced the dimensionality of the data set to the top 40 miRNAs with the highest weighted coefficients for the in vitro biological responses. Target pathway analysis of these top 40 miRNAs demonstrated significant fit to cardiac angiogenesis and fibrosis pathways. Although the model was trained on in vitro data, we demonstrate that the model can predict angiogenesis and fibrosis responses to exosome treatment in vivo with a strong correlation with published in vivo responses. These studies demonstrate that PLSR modeling of exosome miRNA content has the potential to inform preclinical trials and predict new promising CPC therapies. Stem Cells Translational Medicine 2019;8:1212-1221., (© 2019 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Severe drought can cause lagged effects on tree physiology that negatively impact forest functioning for years. These "drought legacy effects" have been widely documented in tree-ring records and could have important implications for our understanding of broader scale forest carbon cycling. However, legacy effects in tree-ring increments may be decoupled from ecosystem fluxes due to (a) postdrought alterations in carbon allocation patterns; (b) temporal asynchrony between radial growth and carbon uptake; and (c) dendrochronological sampling biases. In order to link legacy effects from tree rings to whole forests, we leveraged a rich dataset from a Midwestern US forest that was severely impacted by a drought in 2012. At this site, we compiled tree-ring records, leaf-level gas exchange, eddy flux measurements, dendrometer band data, and satellite remote sensing estimates of greenness and leaf area before, during, and after the 2012 drought. After accounting for the relative abundance of tree species in the stand, we estimate that legacy effects led to ~10% reductions in tree-ring width increments in the year following the severe drought. Despite this stand-scale reduction in radial growth, we found that leaf-level photosynthesis, gross primary productivity (GPP), and vegetation greenness were not suppressed in the year following the 2012 drought. Neither temporal asynchrony between radial growth and carbon uptake nor sampling biases could explain our observations of legacy effects in tree rings but not in GPP. Instead, elevated leaf-level photosynthesis co-occurred with reduced leaf area in early 2013, indicating that resources may have been allocated away from radial growth in conjunction with postdrought upregulation of photosynthesis and repair of canopy damage. Collectively, our results indicate that tree-ring legacy effects were not observed in other canopy processes, and that postdrought canopy allocation could be an important mechanism that decouples tree-ring signals from GPP., (© 2019 John Wiley & Sons Ltd.)

Alcohol use prior to and during pregnancy remains a significant societal problem and can lead to developmental fetal abnormalities including compromised myocardia function and increased risk for heart disease later in life. Alcohol-induced cardiac toxicity has traditionally been studied in animal-based models. These models have limitations due to physiological differences from human cardiomyocytes (CMs) and are also not suitable for high-throughput screening. We hypothesized that human-induced pluripotent stem cell-derived CMs (hiPSC-CMs) could serve as a useful tool to study alcohol-induced cardiac defects and/or toxicity. In this study, hiPSC-CMs were treated with ethanol at doses corresponding to the clinically relevant levels of alcohol intoxication. hiPSC-CMs exposed to ethanol showed a dose-dependent increase in cellular damage and decrease in cell viability, corresponding to increased production of reactive oxygen species. Furthermore, ethanol exposure also generated dose-dependent increased irregular Ca2+ transients and contractility in hiPSC-CMs. RNA-seq analysis showed significant alteration in genes belonging to the potassium voltage-gated channel family or solute carrier family, partially explaining the irregular Ca2+ transients and contractility in ethanol-treated hiPSC-CMs. RNA-seq also showed significant upregulation in the expression of genes associated with collagen and extracellular matrix modeling, and downregulation of genes involved in cardiovascular system development and actin filament-based process. These results suggest that hiPSC-CMs can be a novel and physiologically relevant system for the study of alcohol-induced cardiac toxicity., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Rationale: Congenital heart disease can lead to life-threatening right ventricular (RV) heart failure. Results from clinical trials support expanding cardiac progenitor cell (CPC) based therapies. However, our recent data show that CPCs lose function as they age, starting as early as 1 year., Objective: To determine whether the aggregation of child (1-5-year-old) CPCs into scaffold-free spheres can improve differentiation by enhancing Notch signaling, a known regulator of CPC fate. We hypothesized that aggregated (3-dimensional [3D]) CPCs will repair RV heart failure better than monolayer (2-dimensional [2D]) CPCs., Methods and Results: Spheres were produced with 1500 CPCs each using a microwell array. CPC aggregation significantly increased gene expression of Notch1 compared with 2D CPCs, accompanied by significant upregulation of cardiogenic transcription factors (GATA4, HAND1, MEF2C, NKX2.5, and TBX5) and endothelial markers (CD31, FLK1, FLT1, VWF). Blocking Notch receptor activation with the γ-secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) diminished these effects. To evaluate the therapeutic improvements of CPC aggregation, RV heart failure was induced in athymic rats by pulmonary artery banding, and cells were implanted into the RV free wall. Echocardiographic measurements 28 days postimplantation showed significantly improved RV function with 3D compared with 2D CPCs. Tracking implanted CPCs via DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide)-labeling showed improved retention of 3D CPCs. Transducing 3D CPCs with Notch1-shRNA (short hairpin RNA) did not reduce retention, but significantly reduced RV functional improvements. Histological analyses showed 3D treatment reduced RV fibrosis and increased angiogenesis. Although 3D CPCs formed CD31 + vessel-like cells in vivo, these effects are more likely because of improved 3D CPC exosome function compared with 2D CPC exosomes., Conclusions: Spherical aggregation improves child CPC function in a Notch-dependent manner. The strong reparative ability of CPC spheres warrants further investigation as a treatment for pediatric heart failure, especially in older children where reparative ability may be reduced.

Species-specific responses of plant intrinsic water-use efficiency (iWUE) to multiple environmental drivers associated with climate change, including soil moisture (θ), vapor pressure deficit (D), and atmospheric CO 2 concentration (c a ), are poorly understood. We assessed how the iWUE and growth of several species of deciduous trees that span a gradient of isohydric to anisohydric water-use strategies respond to key environmental drivers (θ, D and c a ). iWUE was calculated for individual tree species using leaf-level gas exchange and tree-ring δ 13 C in wood measurements, and for the whole forest using the eddy covariance method. The iWUE of the isohydric species was generally more sensitive to environmental change than the anisohydric species was, and increased significantly with rising D during the periods of water stress. At longer timescales, the influence of c a was pronounced for isohydric tulip poplar but not for others. Trees' physiological responses to changing environmental drivers can be interpreted differently depending on the observational scale. Care should be also taken in interpreting observed or modeled trends in iWUE that do not explicitly account for the influence of D., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

The mitochondrial Ca2+ uniporter (MCU) complex mediates acute mitochondrial Ca2+ influx. In skeletal muscle, MCU links Ca2+ signaling to energy production by directly enhancing the activity of key metabolic enzymes in the mitochondria. Here, we examined the role of MCU in skeletal muscle development and metabolic function by generating mouse models for the targeted deletion of Mcu in embryonic, postnatal, and adult skeletal muscle. Loss of Mcu did not affect muscle growth and maturation or otherwise cause pathology. Skeletal muscle-specific deletion of Mcu in mice also did not affect myofiber intracellular Ca2+ handling, but it did inhibit acute mitochondrial Ca2+ influx and mitochondrial respiration stimulated by Ca2+, resulting in reduced acute exercise performance in mice. However, loss of Mcu also resulted in enhanced muscle performance under conditions of fatigue, with a preferential shift toward fatty acid metabolism, resulting in reduced body fat with aging. Together, these results demonstrate that MCU-mediated mitochondrial Ca2+ regulation underlies skeletal muscle fuel selection at baseline and under enhanced physiological demands, which affects total homeostatic metabolism.

Engineering cardiac tissue that accurately recapitulates adult myocardium is critical for advancing disease modeling, drug screening, and regenerative medicine. Ronaldson-Bouchard et al. report a new strategy for generating cardiac tissues from stem-cell-derived cardiomyocytes that reach a maturation level closer to human adult cardiac structure and function., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Ca 2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca 2+ from mitochondria is important for determining the role of mitochondrial Ca 2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca 2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca 2+ uptake using the Ca 2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca 2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca 2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca 2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca 2+ in permeabilized cells using the Ca 2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca 2+ . Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca 2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca 2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Key Points: In atrial myocytes excitation-contraction coupling is strikingly different from ventricle because atrial myocytes lack a transverse tubule membrane system: Ca 2+ release starts in the cell periphery and propagates towards the cell centre by Ca 2+ -induced Ca 2+ release from the sarcoplasmic reticulum (SR) Ca 2+ store. The cytosolic Ca 2+ sensitivity of the ryanodine receptor (RyRs) Ca 2+ release channel is low and it is unclear how Ca 2+ release can be activated in the interior of atrial cells. Simultaneous confocal imaging of cytosolic and intra-SR calcium revealed a transient elevation of store Ca 2+ that we termed 'Ca 2+ sensitization signal'. We propose a novel paradigm of atrial ECC that is based on tandem activation of the RyRs by cytosolic and luminal Ca 2+ through a 'fire-diffuse-uptake-fire' (or FDUF) mechanism: Ca 2+ uptake by SR Ca 2+ pumps at the propagation front elevates Ca 2+ inside the SR locally, leading to luminal RyR sensitization and lowering of the cytosolic Ca 2+ activation threshold., Abstract: In atrial myocytes Ca 2+ release during excitation-contraction coupling (ECC) is strikingly different from ventricular myocytes. In many species atrial myocytes lack a transverse tubule system, dividing the sarcoplasmic reticulum (SR) Ca 2+ store into the peripheral subsarcolemmnal junctional (j-SR) and the much more abundant central non-junctional (nj-SR) SR. Action potential (AP)-induced Ca 2+ entry activates Ca 2+ -induced Ca 2+ release (CICR) from j-SR ryanodine receptor (RyR) Ca 2+ release channels. Peripheral elevation of [Ca 2+ ] i initiates CICR from nj-SR and sustains propagation of CICR to the cell centre. Simultaneous confocal measurements of cytosolic ([Ca 2+ ] i ; with the fluorescent Ca 2+ indicator rhod-2) and intra-SR ([Ca 2+ ] SR ; fluo-5N) Ca 2+ in rabbit atrial myocytes revealed that Ca 2+ release from j-SR resulted in a cytosolic Ca 2+ transient of higher amplitude compared to release from nj-SR; however, the degree of depletion of j-SR [Ca 2+ ] SR was smaller than nj-SR [Ca 2+ ] SR . Similarly, Ca 2+ signals from individual release sites of the j-SR showed a larger cytosolic amplitude (Ca 2+ sparks) but smaller depletion (Ca 2+ blinks) than release from nj-SR. During AP-induced Ca 2+ release the rise of [Ca 2+ ] i detected at individual release sites of the nj-SR preceded the depletion of [Ca 2+ ] SR , and during this latency period a transient elevation of [Ca 2+ ] SR occurred. We propose that Ca 2+ release from nj-SR is activated by cytosolic and luminal Ca 2+ (tandem RyR activation) via a novel 'fire-diffuse-uptake-fire' (FDUF) mechanism. This novel paradigm of atrial ECC predicts that Ca 2+ uptake by sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA) at the propagation front elevates local [Ca 2+ ] SR , leading to luminal RyR sensitization and lowering of the activation threshold for cytosolic CICR., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Rationale: Studies have demonstrated that exosomes can repair cardiac tissue post-myocardial infarction and recapitulate the benefits of cellular therapy., Objective: We evaluated the role of donor age and hypoxia of human pediatric cardiac progenitor cell (CPC)-derived exosomes in a rat model of ischemia-reperfusion injury., Methods and Results: Human CPCs from the right atrial appendages from children of different ages undergoing cardiac surgery for congenital heart defects were isolated and cultured under hypoxic or normoxic conditions. Exosomes were isolated from the culture-conditioned media and delivered to athymic rats after ischemia-reperfusion injury. Echocardiography at day 3 post-myocardial infarction suggested statistically improved function in neonatal hypoxic and neonatal normoxic groups compared with saline-treated controls. At 28 days post-myocardial infarction, exosomes derived from neonatal normoxia, neonatal hypoxia, infant hypoxia, and child hypoxia significantly improved cardiac function compared with those from saline-treated controls. Staining showed decreased fibrosis and improved angiogenesis in hypoxic groups compared with controls. Finally, using sequencing data, a computational model was generated to link microRNA levels to specific outcomes., Conclusions: CPC exosomes derived from neonates improved cardiac function independent of culture oxygen levels, whereas CPC exosomes from older children were not reparative unless subjected to hypoxic conditions. Cardiac functional improvements were associated with increased angiogenesis, reduced fibrosis, and improved hypertrophy, resulting in improved cardiac function; however, mechanisms for normoxic neonatal CPC exosomes improved function independent of those mechanisms. This is the first study of its kind demonstrating that donor age and oxygen content in the microenvironment significantly alter the efficacy of human CPC-derived exosomes., (© 2016 American Heart Association, Inc.)

We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates β2-adrenergic receptor (β2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates β2-AR stimulation of cPLA2. We therefore determined the effects of zinterol (0.1 μM; zint-β2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 μM) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 μM GW5074), phospholipase C (PLC; 0.5 μM edelfosine), PKC (4 μM chelerythrine) or IP3 receptor (IP3R) signaling (2 μM 2-APB) significantly inhibited zint-β2-AR stimulation of ICa,L in-PKA but not +PKA myocytes. Western blots showed that zint-β2-AR stimulation increased ERK1/2 phosphorylation in-PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKCα, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-β2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-βgal. In +LMN myocytes, zint-β2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A/S515A mutant compared to control +LMN myocytes infected with Adv-βgal. In-PKA myocytes depletion of intracellular Ca2+ stores by 5 μM thapsigargin failed to inhibit zint-β2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-β-cyclodextrin inhibited zint-β2-AR stimulation of ICa,L in-PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows β2-AR stimulation to act via PKCα/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the remodeling of β-AR signaling in failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity., Competing Interests: The authors have declared that no competing interests exist.

Although β-blockers can be used to eliminate stress-induced ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), this treatment is unsuccessful in ∼25% of cases. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from these patients have potential for use in investigating the phenomenon, but it remains unknown whether they can recapitulate patient-specific drug responses to β-blockers. This study assessed whether the inadequacy of β-blocker therapy in an individual can be observed in vitro using patient-derived CPVT iPSC-CMs. An individual with CPVT harboring a novel mutation in the type 2 cardiac ryanodine receptor (RyR2) was identified whose persistent ventricular arrhythmias during β-blockade with nadolol were abolished during flecainide treatment. iPSC-CMs generated from this patient and two control individuals expressed comparable levels of excitation-contraction genes, but assessment of the sarcoplasmic reticulum Ca(2+) leak and load relationship revealed intracellular Ca(2+) homeostasis was altered in the CPVT iPSC-CMs. β-adrenergic stimulation potentiated spontaneous Ca(2+) waves and unduly frequent, large and prolonged Ca(2+) sparks in CPVT compared with control iPSC-CMs, validating the disease phenotype. Pursuant to the patient's in vivo responses, nadolol treatment during β-adrenergic stimulation achieved negligible reduction of Ca(2+) wave frequency and failed to rescue Ca(2+) spark defects in CPVT iPSC-CMs. In contrast, flecainide reduced both frequency and amplitude of Ca(2+) waves and restored the frequency, width and duration of Ca(2+) sparks to baseline levels. By recapitulating the improved response of an individual with CPVT to flecainide compared with β-blocker therapy in vitro, these data provide new evidence that iPSC-CMs can capture basic components of patient-specific drug responses., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

In this study, we used deoxyribozyme (DNAzyme) functionalized gold nanoparticles (AuNPs) to catalytically silence tumor necrosis factor-α (TNF-α) in vivo as a potential therapeutic for myocardial infarction (MI). Using primary macrophages as a model, we demonstrated 50% knockdown of TNF-α, which was not attainable using Lipofectamine-based approaches. Local injection of DNAzyme conjugated to gold particles (AuNPs) in the rat myocardium yielded TNF-α knockdown efficiencies of 50%, which resulted in significant anti-inflammatory effects and improvement in acute cardiac function following MI. Our results represent the first example showing the use of DNAzyme AuNP conjugates in vivo for viable delivery and gene regulation. This is significant as TNF-α is a multibillion dollar drug target implicated in many inflammatory-mediated disorders, thus underscoring the potential impact of DNAzyme-conjugated AuNPs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Uremic cardiomyopathy is responsible for high morbidity and mortality rates among patients with chronic kidney disease (CKD), but the underlying mechanisms contributing to this complex phenotype are incompletely understood. Myocardial deformation analyses (ventricular strain) of patients with mild CKD have recently been reported to predict adverse clinical outcome. We aimed to determine if early myocardial dysfunction in a mouse model of CKD could be detected using ventricular strain analyses. CKD was induced in 5-week-old male 129X1/SvJ mice through partial nephrectomy (5/6Nx) with age-matched mice undergoing bilateral sham surgeries serving as controls. Serial transthoracic echocardiography was performed over 16 weeks following induction of CKD. Invasive hemodynamic measurements were performed at 8 weeks. Gene expression and histology was performed on hearts at 8 and 16 weeks. CKD mice developed decreased longitudinal strain (-25 ± 4.2% vs. -29 ± 2.3%; P = 0.01) and diastolic dysfunction (E/A ratio 1.2 ± 0.15 vs. 1.9 ± 0.18; P < 0.001) compared to controls as early as 2 weeks following 5/6Nx. In contrast, ventricular hypertrophy was not apparent until 4 weeks. Hearts from CKD mice developed progressive fibrosis at 8 and 16 weeks with gene signatures suggestive of evolving heart failure with elevated expression of natriuretic peptides. Uremic cardiomyopathy in this model is characterized by early myocardial dysfunction which preceded observable changes in ventricular geometry. The model ultimately resulted in myocardial fibrosis and increased expression of natriuretic peptides suggestive of progressive heart failure., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Cardiac progenitor cells (CPCs) have rapidly advanced to clinical trials, yet little is known regarding their interaction with the microenvironment. Signaling cues present in the microenvironment change with development and disease. This work aims to assess the influence of two distinct signaling moieties on CPCs: cyclic biaxial strain and extracellular matrix. We evaluate four endpoints for improving CPC therapy: paracrine signaling, proliferation, connexin43 expression, and alignment. Vascular endothelial growth factor A (about 900 pg/mL) was secreted by CPCs cultured on fibronectin and collagen I. The application of mechanical strain increased vascular endothelial growth factor A secretion 2-4-fold for CPCs cultured on poly-L-lysine, laminin, or a naturally derived cardiac extracellular matrix. CPC proliferation was at least 25% higher on fibronectin than that on other matrices, especially for lower strain magnitudes. At 5% strain, connexin43 expression was highest on fibronectin. With increasing strain magnitude, connexin43 expression decreased by as much as 60% in CPCs cultured on collagen I and a naturally derived cardiac extracellular matrix. Cyclic mechanical strain induced the strongest CPC alignment when cultured on fibronectin or collagen I. This study demonstrates that culturing CPCs on fibronectin with 5% strain magnitude is optimal for their vascular endothelial growth factor A secretion, proliferation, connexin43 expression, and alignment.

For nearly a century, the heart was viewed as a terminally differentiated organ until the discovery of a resident population of cardiac stem cells known as cardiac progenitor cells (CPCs). It has been shown that the regenerative capacity of CPCs can be enhanced by ex vivo modification. Preconditioning CPCs could provide drastic improvements in cardiac structure and function; however, a systematic approach to determining a mechanistic basis for these modifications founded on the physiology of CPCs is lacking. We have identified a novel property of CPCs to respond to electrical stimulation by initiating intracellular Ca 2+ oscillations. We used confocal microscopy and intracellular calcium imaging to determine the spatiotemporal properties of the Ca 2+ signal and the key proteins involved in this process using pharmacological inhibition and confocal Ca 2+ imaging. Our results provide valuable insights into mechanisms to enhance the therapeutic potential in stem cells and further our understanding of human CPC physiology.

Aims: To evaluate the ability of N-acetylglucosamine (GlcNAc) decorated nanoparticles and their cargo to modulate calcium handling in failing cardiac myocytes (CMs)., Materials & Methods: Primary CMs isolated from normal and failing hearts were treated with GlcNAc nanoparticles in order to assess the ability of the nanoparticles and their cargo to correct dysfunctional calcium handling in failing myocytes., Results & Conclusion: GlcNAc particles reduced aberrant calcium release in failing CMs and restored sarcomere function. Additionally, encapsulation of a small calcium-modulating protein, S100A1, in GlcNAc nanoparticles also showed improved calcium regulation. Thus, the development of our bioactive nanoparticle allows for a 'two-hit' treatment, by which the cargo and also the nanoparticle itself can modulate intracellular protein activity., Competing Interests: Financial & competing interests disclosure This publication has been funded in whole or in part with the federal funds from the National Heart, Lung and Blood Institute, NIH, Department of Health and Human Services, under Contract No. HHSN268201000043C, as well as grant HL090601 to ME Davis and American Heart Association grant 11GRNT7490000 to MB Wagner. The authors also wish to acknowledge the support of the Children's Miracle Network gift to Children's Healthcare of Atlanta, funding from the Center for Pediatric Nanomedicine at Georgia Institute of Technology and Children's Healthcare of Atlanta. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Key Points: Impaired calcium (Ca(2+)) signalling is the main contributor to depressed ventricular contractile function and occurrence of arrhythmia in heart failure (HF). Here we report that in atrial cells of a rabbit HF model, Ca(2+) signalling is enhanced and we identified the underlying cellular mechanisms. Enhanced Ca(2+) transients (CaTs) are due to upregulation of inositol-1,4,5-trisphosphate receptor induced Ca(2+) release (IICR) and decreased mitochondrial Ca(2+) sequestration. Enhanced IICR, however, together with an increased activity of the sodium-calcium exchange mechanism, also facilitates spontaneous Ca(2+) release in form of arrhythmogenic Ca(2+) waves and spontaneous action potentials, thus enhancing the arrhythmogenic potential of atrial cells. Our data show that enhanced Ca(2+) signalling in HF provides atrial cells with a mechanism to improve ventricular filling and to maintain cardiac output, but also increases the susceptibility to develop atrial arrhythmias facilitated by spontaneous Ca(2+) release., Abstract: We studied excitation-contraction coupling (ECC) and inositol-1,4,5-triphosphate (IP3)-dependent Ca(2+) release in normal and heart failure (HF) rabbit atrial cells. Left ventricular HF was induced by combined volume and pressure overload. In HF atrial myocytes diastolic [Ca(2+)]i was increased, action potential (AP)-induced Ca(2+) transients (CaTs) were larger in amplitude, primarily due to enhanced Ca(2+) release from central non-junctional sarcoplasmic reticulum (SR) and centripetal propagation of activation was accelerated, whereas HF ventricular CaTs were depressed. The larger CaTs were due to enhanced IP3 receptor-induced Ca(2+) release (IICR) and reduced mitochondrial Ca(2+) buffering, consistent with a reduced mitochondrial density and Ca(2+) uptake capacity in HF. Elementary IP3 receptor-mediated Ca(2+) release events (Ca(2+) puffs) were more frequent in HF atrial myoctes and were detected more often in central regions of the non-junctional SR compared to normal cells. HF cells had an overall higher frequency of spontaneous Ca(2+) waves and a larger fraction of waves (termed arrhythmogenic Ca(2+) waves) triggered APs and global CaTs. The higher propensity of arrhythmogenic Ca(2+) waves resulted from the combined action of enhanced IICR and increased activity of sarcolemmal Na(+)-Ca(2+) exchange depolarizing the cell membrane. In conclusion, the data support the hypothesis that in atrial myocytes from hearts with left ventricular failure, enhanced CaTs during ECC exert positive inotropic effects on atrial contractility which facilitates ventricular filling and contributes to maintaining cardiac output. However, HF atrial cells were also more susceptible to developing arrhythmogenic Ca(2+) waves which might form the substrate for atrial rhythm disorders frequently encountered in HF., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

Rationale: Myocardial infarction is a leading cause of death in developed nations, and there remains a need for cardiac therapeutic systems that mitigate tissue damage. Cardiac progenitor cells (CPCs) and other stem cell types are attractive candidates for treatment of myocardial infarction; however, the benefit of these cells may be as a result of paracrine effects., Objective: We tested the hypothesis that CPCs secrete proregenerative exosomes in response to hypoxic conditions., Methods and Results: The angiogenic and antifibrotic potential of secreted exosomes on cardiac endothelial cells and cardiac fibroblasts were assessed. We found that CPC exosomes secreted in response to hypoxia enhanced tube formation of endothelial cells and decreased profibrotic gene expression in TGF-β-stimulated fibroblasts, indicating that these exosomes possess therapeutic potential. Microarray analysis of exosomes secreted by hypoxic CPCs identified 11 miRNAs that were upregulated compared with exosomes secreted by CPCs grown under normoxic conditions. Principle component analysis was performed to identify miRNAs that were coregulated in response to distinct exosome-generating conditions. To investigate the cue-signal-response relationships of these miRNA clusters with a physiological outcome of tube formation or fibrotic gene expression, partial least squares regression analysis was applied. The importance of each up- or downregulated miRNA on physiological outcomes was determined. Finally, to validate the model, we delivered exosomes after ischemia-reperfusion injury. Exosomes from hypoxic CPCs improved cardiac function and reduced fibrosis., Conclusions: These data provide a foundation for subsequent research of the use of exosomal miRNA and systems biology as therapeutic strategies for the damaged heart., (© 2014 American Heart Association, Inc.)

In rabbit atrial myocytes Ca signaling has unique features due to the lack of transverse (t) tubules, the spatial arrangement of mitochondria and the contribution of inositol-1,4,5-trisphosphate (IP3) receptor-induced Ca release (IICR). During excitation-contraction coupling action potential-induced elevation of cytosolic [Ca] originates in the cell periphery from Ca released from the junctional sarcoplasmic reticulum (j-SR) and then propagates by Ca-induced Ca release from non-junctional (nj-) SR toward the cell center. The subsarcolemmal region between j-SR and the first array of nj-SR Ca release sites is devoid of mitochondria which results in a rapid propagation of activation through this domain, whereas the subsequent propagation through the nj-SR network occurs at a velocity typical for a propagating Ca wave. Inhibition of mitochondrial Ca uptake with the Ca uniporter blocker Ru360 accelerates propagation and increases the amplitude of Ca transients (CaTs) originating from nj-SR. Elevation of cytosolic IP3 levels by rapid photolysis of caged IP3 has profound effects on the magnitude of subcellular CaTs with increased Ca release from nj-SR and enhanced CaTs in the nuclear compartment. IP3 uncaging restricted to the nucleus elicites 'mini'-Ca waves that remain confined to this compartment. Elementary IICR events (Ca puffs) preferentially originate in the nucleus in close physical association with membrane structures of the nuclear envelope and the nucleoplasmic reticulum. The data suggest that in atrial myocytes the nucleus is an autonomous Ca signaling domain where Ca dynamics are primarily governed by IICR.

Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca(2+)-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr308), endothelial NO synthase (eNOS) (Ser1177), and ERK1/2 (Thr202/Tyr204). ERK1/2 phosphorylation, but not Akt and eNOS phosphorylation, was suppressed by inhibition of MEK1/2. Increased Akt phosphorylation resulted in increased Akt kinase activity and was mediated by corticotropin-releasing factor 2 (CRF2) receptors (astressin-2B sensitive). Inhibition of phosphatidylinositol 3-kinase (PI3K) diminished both Akt as well as eNOS phosphorylation mediated by Ucn2. Inhibition of protein kinase A (PKA) reduced Ucn2-induced phosphorylation of eNOS but did not affect the increase in phosphorylation of Akt. Conversely, direct receptor-independent elevation of cAMP via forskolin increased phosphorylation of eNOS but not of Akt. Ucn2 increased intracellular NO concentration ([NO]i), [cGMP], [cAMP], and cell shortening. Inhibition of eNOS suppressed the increases in [NO]i and cell shortening. When both PI3K-Akt and cAMP-PKA signaling were inhibited, the Ucn2-induced increases in [NO]i and cell shortening were attenuated. Thus, in rabbit ventricular myocytes, Ucn2 causes activation of cAMP-PKA, PI3K-Akt, and MEK1/2-ERK1/2 signaling. The MEK1/2-ERK1/2 pathway is not required for stimulation of NO signaling in these cells. The other two pathways, cAMP-PKA and PI3K-Akt, converge on eNOS phosphorylation at Ser1177 and result in pronounced and sustained cellular NO production with subsequent stimulation of cGMP signaling., (Copyright © 2014 the American Physiological Society.)

The functional role of inositol 1,4,5-trisphosphate (InsP3) signaling in cardiomyocytes is not entirely understood but it was linked to an increased propensity for triggered activity. The aim of this study was to determine how InsP3 receptors can translate Ca(2+) release into a depolarization of the plasma membrane and consequently arrhythmic activity. We used embryonic stem cell-derived cardiomyocytes (ESdCs) as a model system since their spontaneous electrical activity depends on InsP3-mediated Ca(2+) release. [InsP3]i was monitored with the FRET-based InsP3-biosensor FIRE-1 (Fluorescent InsP3 Responsive Element) and heterogeneity in sub-cellular [InsP3]i was achieved by targeted expression of FIRE-1 in the nucleus (FIRE-1nuc) or expression of InsP3 5-phosphatase (m43) localized to the plasma membrane. Spontaneous activity of ESdCs was monitored simultaneously as cytosolic Ca(2+) transients (Fluo-4/AM) and action potentials (current clamp). During diastole, the diastolic depolarization was paralleled by an increase of [Ca(2+)]i and spontaneous activity was modulated by [InsP3]i. A 3.7% and 1.7% increase of FIRE-1 FRET ratio and 3.0 and 1.5 fold increase in beating frequency was recorded upon stimulation with endothelin-1 (ET-1, 100 nmol/L) or phenylephrine (PE, 10 µmol/L), respectively. Buffering of InsP3 by FIRE-1nuc had no effect on the basal frequency while attenuation of InsP3 signaling throughout the cell (FIRE-1), or at the plasma membrane (m43) resulted in a 53.7% and 54.0% decrease in beating frequency. In m43 expressing cells the response to ET-1 was completely suppressed. Ca(2+) released from InsP3Rs is more effective than Ca(2+) released from RyRs to enhance INCX. The results support the hypothesis that in ESdCs InsP3Rs form a functional signaling domain with NCX that translates Ca(2+) release efficiently into a depolarization of the membrane potential.

β-adrenergic stimulation of cardiac myocytes enhances intracellular calcium cycling, which frequently associates with pro-arrhythmic Ca waves. The threshold level of free calcium in the sarcoplasmic reticulum ([Ca]SR) where waves initiate is increased during β-adrenergic stimulation. ( 1) Here, we measured [Ca]SR directly to monitor the [Ca]SR level at which spontaneous Ca waves terminated (termination threshold) during β-adrenergic stimulation. Compared with control conditions, application of the β-adrenergic receptor agonist isoproterenol (ISO; 1 μM) resulted in an increase in basal [Ca]SR and an increase in the [Ca]SR at which spontaneous Ca waves terminated. When [Ca]SR was experimentally matched, ISO stimulation resulted in a decrease in the depletion amplitude of the waves. Sensitization of ryanodine receptor SR Ca release channels to Ca with a low dose of caffeine in the presence of ISO was able to decrease the termination threshold compared with control conditions. Therefore, the Ca wave termination level may represent an important mode of altering Ca depletion from the SR and reducing the arrhythmogenic potential during β-adrenergic stimulation.

A historically unprecedented mountain pine beetle (MPB) outbreak affected western Montana during the past decade. We examined radial growth rates (AD 1860-2007/8) of co-occurring mature healthy and MPB-infected ponderosa pine trees collected at two sites (Cabin Gulch and Kitchen Gulch) in western Montana and: (1) compared basal area increment (BAI) values within populations and between sites; (2) used carbon isotope analysis to calculate intrinsic water-use efficiency (iWUE) at Cabin Gulch; and (3) compared climate-growth responses using a suite of monthly climatic variables. BAI values within populations and between sites were similar until the last 20-30 years, at which point the visually healthy populations had consistently higher BAI values (22-34%) than the MPB-infected trees. These results suggest that growth rates two-three decades prior to the current outbreak diverged between our selected populations, with the slower-growing trees being more vulnerable to beetle infestation. Both samples from Cabin Gulch experienced upward trends in iWUE, with significant regime shifts toward higher iWUE beginning in 1955-59 for the visually healthy trees and 1960-64 for the MPB-infected trees. Drought tolerance also varied between the two populations with the visually healthy trees having higher growth rates than MPB-infected trees prior to infection during a multi-decadal period of drying summertime conditions. Intrinsic water-use efficiency significantly increased for both populations during the past 150 years, but there were no significant differences between the visually healthy and MPB-infected chronologies.

β-Adrenergic signalling induces positive inotropic effects on the heart that associate with pro-arrhythmic spontaneous Ca(2+) waves. A threshold level of sarcoplasmic reticulum (SR) Ca(2+) ([Ca(2+)](SR)) is necessary to trigger Ca(2+) waves, and whether the increased incidence of Ca(2+) waves during β-adrenergic stimulation is due to an alteration in this threshold remains controversial. Using the low-affinity Ca(2+) indicator fluo-5N entrapped within the SR of rabbit ventricular myocytes, we addressed this controversy by directly monitoring [Ca(2+)](SR) and Ca(2+) waves during β-adrenergic stimulation. Electrical pacing in elevated extracellular Ca(2+) ([Ca(2+)](o) = 7 mM) was used to increase [Ca(2+)](SR) to the threshold where Ca(2+) waves were consistently observed. The β-adrenergic agonist isoproterenol (ISO; 1 μM) increased [Ca(2+)](SR) well above the control threshold and consistently triggered Ca(2+) waves. However, when [Ca(2+)](SR) was subsequently lowered in the presence of ISO (by lowering [Ca(2+)](o) to 1 mM and partially inhibiting sarcoplasmic/endoplasmic reticulum calcium ATPase with cyclopiazonic acid or thapsigargin), Ca(2+) waves ceased to occur at a [Ca(2+)](SR) that was higher than the control threshold. Furthermore, for a set [Ca(2+)](SR) level the refractoriness of wave occurrence (Ca(2+) wave latency) was prolonged during β-adrenergic stimulation, and was highly dependent on the extent that [Ca](SR) exceeded the wave threshold. These data show that acute β-adrenergic stimulation increases the [Ca(2+)](SR) threshold for Ca(2+) waves, and therefore the primary cause of Ca(2+) waves is the robust increase in [Ca(2+)](SR) above this higher threshold level. Elevation of the [Ca(2+)](SR) wave threshold and prolongation of wave latency represent potentially protective mechanisms against pro-arrhythmogenic Ca(2+) release during β-adrenergic stimulation.