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Supplemental Digital Content is available in the text., Background: Cardiac hypertrophy is an independent risk factor for heart failure, a leading cause of morbidity and mortality globally. The calcineurin/NFAT (nuclear factor of activated T cells) pathway and the MAPK (mitogen-activated protein kinase)/Erk (extracellular signal-regulated kinase) pathway contribute to the pathogenesis of cardiac hypertrophy as an interdependent network of signaling cascades. How these pathways interact remains unclear and few direct targets responsible for the prohypertrophic role of NFAT have been described. Methods: By engineering cardiomyocyte-specific ETS2 (a member of the E26 transformation-specific sequence [ETS] domain family) knockout mice, we investigated the role of ETS2 in cardiac hypertrophy. Primary cardiomyocytes were used to evaluate ETS2 function in cell growth. Results: ETS2 is phosphorylated and activated by Erk1/2 on hypertrophic stimulation in both mouse (n=3) and human heart samples (n=8 to 19). Conditional deletion of ETS2 in mouse cardiomyocytes protects against pressure overload–induced cardiac hypertrophy (n=6 to 11). Silencing of ETS2 in the hearts of calcineurin transgenic mice significantly attenuates hypertrophic growth and contractile dysfunction (n=8). As a transcription factor, ETS2 is capable of binding to the promoters of hypertrophic marker genes, such as ANP, BNP, and Rcan1.4 (n=4). We report that ETS2 forms a complex with NFAT to stimulate transcriptional activity through increased NFAT binding to the promoters of at least 2 hypertrophy-stimulated genes: Rcan1.4 and microRNA-223 (=n4 to 6). Suppression of microRNA-223 in cardiomyocytes inhibits calcineurin-mediated cardiac hypertrophy (n=6), revealing microRNA-223 as a novel prohypertrophic target of the calcineurin/NFAT and Erk1/2-ETS2 pathways. Conclusions: Our findings point to a critical role for ETS2 in calcineurin/NFAT pathway-driven cardiac hypertrophy and unveil a previously unknown molecular connection between the Erk1/2 activation of ETS2 and expression of NFAT/ETS2 target genes.

In urban harbors there is a reliance on the “gray infrastructure” of armored bulkheads along shorelines. While this form of shoreline technology provides stability and eliminates erosion, it limits shoreline biodiversity and lacks aesthetic value. In this paper, a living shoreline concept, termed the Green Bulkhead, is described and demonstrated. This is a kind of artificial vertical wetland with plants grown in a porous plastic fabric that is draped over the surface of an existing bulkhead. The system is irrigated with water that is pumped from the harbor. Several alternate designs have been tested for different medium types, planting patterns and water pumping regimes. It is proposed that the green bulkhead system can provide limited treatment wetland services in an urban setting where cost-effective water quality management options are minimal. Testing found that high percentages of sediment were retained within the matrix of the different media. The system has aesthetic benefits by “greening” the harbor environment with wetland plants and, because of its vertical orientation, it can be managed adaptively for sea level rise. This work is a report of an on-going ecological engineering project with demonstrations along the Baltimore, Maryland Inner Harbor and the Anacostia Waterfront in Washington, DC.

Using the example of analysis of 40-year series of vector fields of ice drift in the Arctic Ocean, the features of calculating the parameters of the linear trend of vector processes are considered. The decomposition of acceleration vectors into orthogonal and collinear components relative to the mean vector makes it possible to estimate an increase or decrease in the average velocity and regularity of change in the angular velocity of direction of the mean vector. The results of the analysis confirm the increase in the velocity modulus in the field of the average drift vector in most of the Arctic Ocean at the same time, seasonal and regional features of the angular velocity variation in the field mean vector drift. It is noted that in the winter season, the linear trend explains more than half the variance in annual drift variability in the Fram Strait, on the southern periphery of the anticyclonic gyre in the Beaufort Sea, in the Kara Sea, and in the Baffin Sea.

Heart failure with preserved ejection fraction (HFpEF) is now the dominant form of heart failure and one for which no efficacious therapies exist. Obesity and lipid mishandling greatly contribute to HFpEF. However, molecular mechanism(s) governing metabolic alterations and perturbations in lipid homeostasis in HFpEF are largely unknown. Here, we report that cardiomyocyte steatosis in HFpEF is coupled with increases in the activity of the transcription factor FoxO1 (Forkhead box protein O1). FoxO1 depletion, as well as over-expression of the Xbp1s (spliced form of the X-box-binding protein 1) arm of the UPR (unfolded protein response) in cardiomyocytes each ameliorates the HFpEF phenotype in mice and reduces myocardial lipid accumulation. Mechanistically, forced expression of Xbp1s in cardiomyocytes triggers ubiquitination and proteasomal degradation of FoxO1 which occurs, in large part, through activation of the E3 ubiquitin ligase STUB1 (STIP1 homology and U-box-containing protein 1) a novel and direct transcriptional target of Xbp1s. Our findings uncover the Xbp1s-FoxO1 axis as a pivotal mechanism in the pathogenesis of cardiometabolic HFpEF and unveil previously unrecognized mechanisms whereby the UPR governs metabolic alterations in cardiomyocytes., Heart failure with preserved ejection fraction (HFpEF) is a global, major health issue for which no effective therapies are available. Here, the authors discover that the interplay between two transcription factors, Xbp1s and FoxO1, is critical for metabolic adaptation and lipid handling in HFpEF-stressed cardiomyocytes.

The aim of the study was to identify the basic characteristics of ice drift in the south-western Kara Sea and to estimate the extreme drift speed of given probability, including its spatial variability and statistical correlation with the main drift-forming factors. In order to obtain the ice drift data, the numerical dynamic-thermodynamic model of ice cover evolution developed in AARI was used. Its basic specific feature is imitation of ice cover with the help of so-called markers (conventional ice floes). Using three variants of the model grid net (25, 12.5 and 5 km), the ice conditions in the Baidara Bay and the adjoining area in January-March 2018 were simulated. The analysis of the simulation results showed that the average ice drift (average ice transport) is directed from the Baidara Bay to the open sea, i.e. northward with slight deviations mostly to the west. A less detailed grid net results in a smoothed ice drift field, while an increase in the spatial resolution of the model increases the spatial contrasts of the ice drift. The maximum values of the extreme ice drift velocity expressed as “once per N years” occur in the northern part of the model area at the directions of the north-western quarter (up to 1.5-1.8 m/s “once per 10 years” - “once per 100 years”, respectively). The frequency of ice drift velocity exceeding 0.3 m/s is about 4-7 %, and that of ice drift velocity exceeding 0.6 m/s is not more than 1 %. At low drift velocity, the role of inertia is very high, but as the drift rate grows, the inertia contribution decreases noticeably. At increasing drift velocity, the statistical correlation between the ice drift (on the one hand) and the wind, current and sea level tilt (on the other hand) becomes evident. This effect is especially evident for the correlation “drift / wind”. The correlation “drift / ice pressure” depends on the drift speed more or less noticeably at low and high drift speeds, when unidirectional changes of the ice drift and ice pressure happen more often than the opposite ones. At the drift velocities within 0.15-0.60 m\s, the correlation between the ice drift and ice pressure is insignificant, i.e. the unidirectional and opposite changes of the ice drift and ice pressure are almost equally likely.

The mechanism and function of autophagy as a highly-conserved bulk degradation pathway are well studied, but the physiological role of autophagy remains poorly understood. We show that autophagy is involved in the adaptation of Saccharomyces cerevisiae to respiratory growth through its recycling of serine. On respiratory media, growth onset, mitochondrial initiator tRNA modification and mitochondrial protein expression are delayed in autophagy defective cells, suggesting that mitochondrial one-carbon metabolism is perturbed in these cells. The supplementation of serine, which is a key one-carbon metabolite, is able to restore mitochondrial protein expression and alleviate delayed respiratory growth. These results indicate that autophagy-derived serine feeds into mitochondrial one-carbon metabolism, supporting the initiation of mitochondrial protein synthesis and allowing rapid adaptation to respiratory growth., Autophagy is important during stress and development, but how the metabolites generated are used by the cell remains unclear. Here, the authors demonstrate that budding yeast require autophagy to provide serine for one-carbon metabolism during the switch from glycolytic to respiratory growth.

In May 2015, the old hummock located to the North of the Bennett island (the East Siberian Sea) was investigated using several methods, among which were water thermal drilling, tachometric and sonar surveys, underwater video recording, as well as techniques to determine the strength and physical properties of ice. It was found out that only a combination of different methods provides a way to correctly estimate the main morphometric characteristics of a large ice formation and to determine its volume and mass. Analysis of the internal structure of the hummock, obtained by the water thermal drilling with a record of the drilling rate on the logger, made it possible to reveal a composite character of the ice formation (the hummock consisted of two fragments – the large old one and the smaller first-year piece) and to estimate approximately its age (3–4 years). Comparison of the main morphometric characteristics of the old hummock with the average values of first-year hummocks, investigated in the same area and the time, showed that the old hummock had significantly greater geometric parameters: its volume and mass exceeded similar parameters of the average younger formation by factors 5.6 and 5.8, respectively. This significant difference allows suggestion that the reason is not the age but a composite structure of the old formation. The average thickness of the consolidated layer of the old hummock equal to 4.6 m is almost twice larger than similar parameter of a first-year hummock (2.33 m) while the average value of the thickness in the old part of the old hummock (5.22 m) is larger than that of a young one by the factor 2.2. Note also, that the old hummock is characterized by almost complete smoothness (impossible to separate individual blocks) and minimal porosity (1%) of its ice. The salinity and density of the ice composing the frontal part of the old hummock is much smaller than in first-year hummocks. The average density of ice in the old hummock, determined analytically from the buoyancy condition, was equal to 896 kg/m3, while the average density of ice in the first-year hummocks, determined from measurements, – 917 kg/m3.

Many biomolecules undergo liquid–liquid phase separation to form liquid-like condensates that mediate diverse cellular functions1,2. Autophagy is able to degrade such condensates using autophagosomes—double-membrane structures that are synthesized de novo at the pre-autophagosomal structure (PAS) in yeast3–5. Whereas Atg proteins that associate with the PAS have been characterized, the physicochemical and functional properties of the PAS remain unclear owing to its small size and fragility. Here we show that the PAS is in fact a liquid-like condensate of Atg proteins. The autophagy-initiating Atg1 complex undergoes phase separation to form liquid droplets in vitro, and point mutations or phosphorylation that inhibit phase separation impair PAS formation in vivo. In vitro experiments show that Atg1-complex droplets can be tethered to membranes via specific protein–protein interactions, explaining the vacuolar membrane localization of the PAS in vivo. We propose that phase separation has a critical, active role in autophagy, whereby it organizes the autophagy machinery at the PAS. The pre-autophagosomal structure in yeast is a liquid-like condensate of Atg proteins whose phase separation may have a critical, active role in autophagy.

Phase-separated droplets with liquid-like properties can be degraded by macroautophagy/autophagy, but the mechanism underlying this degradation is poorly understood. We have recently derived a physical model to investigate the interaction between autophagic membranes and such droplets, uncovering that intrinsic wetting interactions underlie droplet-membrane contacts. We found that the competition between droplet surface tension and the increasing tendency of growing membrane sheets to bend determines whether a droplet is completely engulfed or isolated in a piecemeal fashion, a process we term fluidophagy. Intriguingly, we found that another critical parameter of droplet-membrane interactions, the spontaneous curvature of the membrane, determines whether the droplet is degraded by autophagy or - counterintuitively - serves as a platform from which autophagic membranes expand into the cytosol. We also discovered that the interaction of membrane-associated LC3 with the LC3-interacting region (LIR) found in the autophagic cargo receptor protein SQSTM1/p62 and many other autophagy-related proteins influences the preferred bending directionality of forming autophagosomes in living cells. Our study provides a physical account of how droplet-membrane wetting underpins the structure and fate of forming autophagosomes.

Rationale: Reperfusion injury accounts for up to half of myocardial infarct size, and meaningful clinical therapies targeting it do not exist. We have reported previously that autophagy is reduced during reperfusion and that HDAC (histone deacetylase) inhibition enhances cardiomyocyte autophagy and blunts ischemia/reperfusion (I/R) injury when administered at the time of reperfusion. However, whether inducing autophagy per se, as opposed to other effects triggered by HDAC inhibition, is sufficient to protect against reperfusion injury is not clear. Objective: We set out to test whether augmentation of autophagy using a specific autophagy-inducing peptide, TB (Tat-Beclin), protects the myocardium through reduction of reactive oxygen species (ROS) during reperfusion injury. Methods and Results: Eight- to 12-week-old, WT (wild type) C57BL6 mice and tamoxifen-inducible cardiomyocyte-specific ATG7 KO (ATG7 knockout) mice (to test the dependency on autophagy) were randomized into 2 groups: exposed to a control TS (Tat-scrambled) peptide or a TB peptide. Each group was subjected to I/R surgery (45-minute coronary ligation, 24-hour reperfusion). Infarct size, systolic function, autophagic flux, and ROS were assayed. Cultured neonatal rat ventricular myocytes were exposed to TB during simulated I/R injury. ATG7 knockdown by small interfering RNA in neonatal rat ventricular myocytes was used to evaluate the role of autophagy. TB treatment at reperfusion reduced infarct size by 20% (absolute reduction; 50% relative reduction) and improved contractile function. Improvement correlated with increased autophagic flux in the border zone with less oxidative stress. ATG7 KO mice did not manifest TB-promoted cardioprotection during I/R. In neonatal rat ventricular myocytes subjected to I/R, TB reduced cell death by 41% and reduced I/R-induced ROS generation. Conversely, ATG7 knockdown in neonatal rat ventricular myocytes abolished these beneficial effects of TB on cell death and ROS reduction. Conclusions: Induction of autophagy at the time of reperfusion is sufficient to mitigate myocardial reperfusion injury by reducing ROS and cell death. Maintenance of appropriate autophagic flux may emerge as a viable clinical therapy to reduce reperfusion injury in acute myocardial infarction.

Background Energy metabolism and substrate selection are key aspects of correct myocardial mechanical function. Myocardial preference for oxidizable substrates changes in both hypertrophy and in overt failure. Previous work has shown that glucose oxidation is upregulated in overpressure hypertrophy, but its fate in overt failure is less clear. Anaplerotic flux of pyruvate into the tricarboxylic acid cycle (TCA) has been posited as a secondary fate of glycolysis, aside from pyruvate oxidation or lactate production. Methods and results A model of heart failure that emulates both valvular and hypertensive heart disease, the severe transaortic constriction (sTAC) mouse, was assayed for changes in substrate preference using metabolomic and carbon-13 flux measurements. Quantitative measures of O2 consumption in the Langendorff perfused mouse heart were paired with 13C isotopomer analysis to assess TCA cycle turnover. Since the heart accommodates oxidation of all physiological energy sources, the utilization of carbohydrates, fatty acids, and ketones were measured simultaneously using a triple-tracer NMR method. The fractional contribution of glucose to acetyl-CoA production was upregulated in heart failure, while other sources were not significantly different. A model that includes both pyruvate carboxylation and anaplerosis through succinyl-CoA produced superior fits to the data compared to a model using only pyruvate carboxylation. In the sTAC heart, anaplerosis through succinyl-CoA is elevated, while pyruvate carboxylation was not. Metabolomic data showed depleted TCA cycle intermediate pool sizes versus the control, in agreement with previous results. Conclusion In the sTAC heart failure model, the glucose contribution to acetyl-CoA production was significantly higher, with compensatory changes in fatty acid and ketone oxidation not reaching a significant level. Anaplerosis through succinyl-CoA is also upregulated, and is likely used to preserve TCA cycle intermediate pool sizes. The triple tracer method used here is new, and can be used to assess sources of acetyl-CoA production in any oxidative tissue.

Heart failure with preserved ejection fraction (HFpEF) is a common syndrome with high morbidity and mortality for which there are no evidence-based therapies. Here we report that concomitant metabolic and hypertensive stress in mice—elicited by a combination of high-fat diet and inhibition of constitutive nitric oxide synthase using Nω-nitro-l-arginine methyl ester (l-NAME)—recapitulates the numerous systemic and cardiovascular features of HFpEF in humans. Expression of one of the unfolded protein response effectors, the spliced form of X-box-binding protein 1 (XBP1s), was reduced in the myocardium of our rodent model and in humans with HFpEF. Mechanistically, the decrease in XBP1s resulted from increased activity of inducible nitric oxide synthase (iNOS) and S-nitrosylation of the endonuclease inositol-requiring protein 1α (IRE1α), culminating in defective XBP1 splicing. Pharmacological or genetic suppression of iNOS, or cardiomyocyte-restricted overexpression of XBP1s, each ameliorated the HFpEF phenotype. We report that iNOS-driven dysregulation of the IRE1α–XBP1 pathway is a crucial mechanism of cardiomyocyte dysfunction in HFpEF. iNOS-driven dysregulation of the IRE1α–XBP1 pathway leads to cardiomyocyte dysfunction in mice and recapitulates the systemic and cardiovascular features of human heart failure with preserved ejection fraction.

Protein-rich droplets, such as stress granules, P-bodies, and the nucleolus, perform diverse and specialized cellular functions. Recent evidence has shown the droplets, which are also known as biomolecular condensates or membrane-less compartments, form by phase separation. Many droplets also contact membrane-bound organelles, thereby functioning in development, intracellular degradation, and organization. These underappreciated interactions have major implications for our fundamental understanding of cells. Starting with a brief introduction to wetting phenomena, we summarize recent progress in the emerging field of droplet–membrane contact. We describe the physical mechanism of droplet–membrane interactions, discuss how these interactions remodel droplets and membranes, and introduce "membrane scaffolding" by liquids as a novel reshaping mechanism, thereby demonstrating that droplet–membrane interactions are elastic wetting phenomena. “Membrane-less” and “membrane-bound” condensates likely represent distinct wetting states that together link phase separation with mechanosensitivity and explain key structures observed during embryogenesis, during autophagy, and at synapses. We therefore contend that droplet wetting on membranes provides a robust and intricate means of intracellular organization.

Introduction: Heart failure with preserved ejection fraction (HFpEF) is now the dominant form of heart failure (HF). Limited insight into underlying mechanisms has culminated in the longstanding absence of evidence-based therapies capable of mitigating the substantial morbidity and mortality associated with the syndrome. Existing clinical and epidemiological evidence suggests that excessive body fat and lipid mishandling contribute to HFpEF. However, molecular mechanism(s) governing metabolic alterations and perturbations in lipid homeostasis in HFpEF are unknown. We recently developed a novel, clinically relevant, murine model of HFpEF, uncovering suppression of the Xbp1s (spliced form of the X-box-binding protein 1) arm of the UPR (unfolded protein response) signaling pathway as a critical driver of HFpEF pathogenesis. Objectives: To define and manipulate mechanisms downstream of Xbp1s in HFpEF and decipher its cardioprotective actions. Methods and Results: In the myocardium of experimental HFpEF, we detected cardiomyocyte steatosis coupled with increases in the abundance and activity of FoxO1 (Forkhead box protein O1), a conserved transcription factor involved in cell metabolism. FoxO1 depletion, as well as Xbp1s over-expression, in cardiomyocytes each ameliorated the HFpEF phenotype and reduced myocardial lipid accumulation. Strikingly, forced expression of Xbp1s in cardiomyocytes triggered proteasomal degradation of FoxO1. Furthermore, we discovered that FoxO1 is ubiquitinated upon Xbp1s over-expression, and Xbp1s-induced proteasomal degradation of FoxO1 occurs, in large part, through activation of the E3 ubiquitin ligase STUB1 (STIP1 homology and U-Box-containing protein 1), a protein we identified as a novel and direct transcriptional target of Xbp1s. Conclusions: Our findings uncover the Xbp1s-FoxO1 axis as a pivotal mechanism in the pathogenesis of HFpEF and unveil previously unrecognized mechanisms whereby the UPR governs metabolic alterations in cardiomyocytes.

Background: BET (bromodomain and extraterminal) epigenetic reader proteins, in particular BRD4 (bromodomain-containing protein 4), have emerged as potential therapeutic targets in a number of pathological conditions, including cancer and cardiovascular disease. Small-molecule BET protein inhibitors such as JQ1 have demonstrated efficacy in reversing cardiac hypertrophy and heart failure in preclinical models. Yet, genetic studies elucidating the biology of BET proteins in the heart have not been conducted to validate pharmacological findings and to unveil potential pharmacological side effects. Methods: By engineering a cardiomyocyte-specific BRD4 knockout mouse, we investigated the role of BRD4 in cardiac pathophysiology. We performed functional, transcriptomic, and mitochondrial analyses to evaluate BRD4 function in developing and mature hearts. Results: Unlike pharmacological inhibition, loss of BRD4 protein triggered progressive declines in myocardial function, culminating in dilated cardiomyopathy. Transcriptome analysis of BRD4 knockout mouse heart tissue identified early and specific disruption of genes essential to mitochondrial energy production and homeostasis. Functional analysis of isolated mitochondria from these hearts confirmed that BRD4 ablation triggered significant changes in mitochondrial electron transport chain protein expression and activity. Computational analysis identified candidate transcription factors participating in the BRD4-regulated transcriptome. In particular, estrogen-related receptor α, a key nuclear receptor in metabolic gene regulation, was enriched in promoters of BRD4-regulated mitochondrial genes. Conclusions: In aggregate, we describe a previously unrecognized role for BRD4 in regulating cardiomyocyte mitochondrial homeostasis, observing that its function is indispensable to the maintenance of normal cardiac function.

This paper presents field results and analysis of iceberg towing experiments performed in the Barents and Kara Sea in 2016-2017. For the first time ever in the industry, this research was based on a complex study of the icebergs origin and properties. For that purpose prior to the trials outlet glaciers of Novaya Zemlya, Franz Josef Land and Severnaya Zemlya were examined with airborne radar that allowed to measure their thickness and to build 3D models. Satellite remote sensing data were used to derive glacier fronts position, ice surface flow velocity, changes of glacier margins, and parameters of iceberg distribution. The data on the flow rates of the main glaciers were compared with the satellite beacons equipped with GPS (ARGOS) installed on several glaciers. Empirical relationships were derived to determine the mass and geometry of icebergs based on instrumental measurements and airborne data in the Barents and Kara seas. All this information was used to estimate iceberg towing possibilities in different conditions, analyze obtained data and deeper understand the process. Influence of oscillations during the towing process was studied. For a wide range of towing speeds, drag force coefficients were determined for icebergs of various sizes and shapes. Experimental iceberg towing operations performed in ice fields during the early stages of ice formation are described. These works were conducted in October 2017 in the Kara sea. Limitations of iceberg towing under different ice conditions are determined and discussed. Technological features of towing operations within negative air temperatures and the presence of sea ice are also highlighted.

Forkhead box O (FoxO) proteins and thyroid hormone (TH) have well established roles in cardiovascular morphogenesis and remodeling. However, specific role(s) of individual FoxO family members in stress-induced growth and remodeling of cardiomyocytes remains unknown. Here, we report that FoxO1, but not FoxO3, activity is essential for reciprocal regulation of types II and III iodothyronine deiodinases (Dio2 and Dio3, respectively), key enzymes involved in intracellular TH metabolism. We further show that Dio2 is a direct transcriptional target of FoxO1, and the FoxO1–Dio2 axis governs TH-induced hypertrophic growth of neonatal cardiomyocytes in vitro and in vivo. Utilizing transverse aortic constriction as a model of hemodynamic stress in wild-type and cardiomyocyte-restricted FoxO1 knockout mice, we unveil an essential role for the FoxO1–Dio2 axis in afterload-induced pathological cardiac remodeling and activation of TRα1. These findings demonstrate a previously unrecognized FoxO1–Dio2 signaling axis in stress-induced cardiomyocyte growth and remodeling and intracellular TH homeostasis., Disease stress-induced cardiac hypertrophy is a major mechanism of pathological cardiac remodeling. Here, the authors unveil a previously unrecognized role of a FoxO1–Dio2 signaling axis in maladaptive, afterload-induced cardiac hypertrophy and intracellular thyroid hormone homeostasis.

The activation of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle coordinates systemic metabolic responses to exercise1. Autophagy—a lysosomal degradation pathway that maintains cellular homeostasis2—is upregulated during exercise, and a core autophagy protein, beclin 1, is required for AMPK activation in skeletal muscle3. Here we describe a role for the innate immune-sensing molecule Toll-like receptor 9 (TLR9)4, and its interaction with beclin 1, in exercise-induced activation of AMPK in skeletal muscle. Mice that lack TLR9 are deficient in both exercise-induced activation of AMPK and plasma membrane localization of the GLUT4 glucose transporter in skeletal muscle, but are not deficient in autophagy. TLR9 binds beclin 1, and this interaction is increased by energy stress (glucose starvation and endurance exercise) and decreased by a BCL2 mutation3,5 that blocks the disruption of BCL2–beclin 1 binding. TLR9 regulates the assembly of the endolysosomal phosphatidylinositol 3-kinase complex (PI3KC3-C2)—which contains beclin 1 and UVRAG—in skeletal muscle during exercise, and knockout of beclin 1 or UVRAG inhibits the cellular AMPK activation induced by glucose starvation. Moreover, TLR9 functions in a muscle-autonomous fashion in ex vivo contraction-induced AMPK activation, glucose uptake and beclin 1–UVRAG complex assembly. These findings reveal a heretofore undescribed role for a Toll-like receptor in skeletal-muscle AMPK activation and glucose metabolism during exercise, as well as unexpected crosstalk between this innate immune sensor and autophagy proteins. In mice, the interaction of the innate immune sensor TLR9 with beclin 1 is shown to have a role in glucose metabolism and AMPK activation in skeletal muscle during exercise.

Background: Myocardium irreversibly injured by ischemic stress must be efficiently repaired to maintain tissue integrity and contractile performance. Macrophages play critical roles in this process. These cells transform across a spectrum of phenotypes to accomplish diverse functions ranging from mediating the initial inflammatory responses that clear damaged tissue to subsequent reparative functions that help rebuild replacement tissue. Although macrophage transformation is crucial to myocardial repair, events governing this transformation are poorly understood. Methods: Here, we set out to determine whether innate immune responses triggered by cytoplasmic DNA play a role. Results: We report that ischemic myocardial injury, along with the resulting release of nucleic acids, activates the recently described cyclic GMP-AMP synthase–stimulator of interferon genes pathway. Animals lacking cyclic GMP-AMP synthase display significantly improved early survival after myocardial infarction and diminished pathological remodeling, including ventricular rupture, enhanced angiogenesis, and preserved ventricular contractile function. Furthermore, cyclic GMP-AMP synthase loss of function abolishes the induction of key inflammatory programs such as inducible nitric oxide synthase and promotes the transformation of macrophages to a reparative phenotype, which results in enhanced repair and improved hemodynamic performance. Conclusions: These results reveal, for the first time, that the cytosolic DNA receptor cyclic GMP-AMP synthase functions during cardiac ischemia as a pattern recognition receptor in the sterile immune response. Furthermore, we report that this pathway governs macrophage transformation, thereby regulating postinjury cardiac repair. Because modulators of this pathway are currently in clinical use, our findings raise the prospect of new treatment options to combat ischemic heart disease and its progression to heart failure.

Rationale: Restoration of coronary artery blood flow is the most effective means of ameliorating myocardial damage triggered by ischemic heart disease. However, coronary reperfusion elicits an increment of additional injury to the myocardium. Accumulating evidence indicates that the unfolded protein response (UPR) in cardiomyocytes is activated by ischemia/reperfusion (I/R) injury. Xbp1s (spliced X-box binding protein 1), the most highly conserved branch of the unfolded protein response, is protective in response to cardiac I/R injury. GRP78 (78 kDa glucose-regulated protein), a master regulator of the UPR and an Xbp1s target, is upregulated after I/R. However, its role in the protective response of Xbp1s during I/R remains largely undefined. Objective: To elucidate the role of GRP78 in the cardiomyocyte response to I/R using both in vitro and in vivo approaches. Methods and Results: Simulated I/R injury to cultured neonatal rat ventricular myocytes induced apoptotic cell death and strong activation of the UPR and GRP78. Overexpression of GRP78 in neonatal rat ventricular myocytes significantly protected myocytes from I/R-induced cell death. Furthermore, cardiomyocyte-specific overexpression of GRP78 ameliorated I/R damage to the heart in vivo. Exploration of underlying mechanisms revealed that GRP78 mitigates cellular damage by suppressing the accumulation of reactive oxygen species. We go on to show that the GRP78-mediated cytoprotective response involves plasma membrane translocation of GRP78 and interaction with PI3 kinase, culminating in stimulation of Akt. This response is required as inhibition of the Akt pathway significantly blunted the antioxidant activity and cardioprotective effects of GRP78. Conclusions: I/R induction of GRP78 in cardiomyocytes stimulates Akt signaling and protects against oxidative stress, which together protect cells from I/R damage.

In hospitals, the nursing staff is often confronted with the problem of the preparation and administration of drugs for their pediatric patients because of the lack of indication, pediatric dosage, and appropriate galenic form. The goal of this study was to give an overview of the nurses’ preparation habits in pediatric units and highlight their daily problems. This single-center prospective study was conducted through an observation of the nursing staff during the drug preparation process in medicine, surgery and intensive care units. We included 91 patients (55 boys and 36 girls), with an average age of 6.3 years (youngest child, 10 days old; oldest child, 18 years old). We observed a mean 2.16 drug preparations per patient [1–5]. We collected 197 observation reports regarding 66 injectable drugs and 131 oral drugs (71 liquid forms and 60 solid forms). The majority of these reports concerned central nervous system drugs (63/197), metabolism and digestive system drugs (50/197), and anti-infective drugs (46/197). The study highlights the nurses’ difficulties: modification of the solid galenic forms, lack of knowledge on oral liquid form preservation or reconstitution methods, withdrawal of small volumes, and vague and noncompliant labeling. This study led to the creation of a specific working group for pediatrics. This multidisciplinary team meets on a regular basis to work toward improving the current habits to both simplify and secure drug administration to hospitalized children.

French preparation guidelines state that pharmacy staff who manipulate cytotoxic drugs have to follow specific training. In order to assess the pharmaceutical assistants’ skills and knowledge, we developed a “Cytotoxic Preparation Centralized Unit (CPCU) of errors,” derived from the Canadian concept of “Chamber of horrors.” A table listing 20 mistakes to track down was created and each pharmaceutical assistant spent 20 min in the “CPCU of errors” with the pharmacist, who wrote down the spotted mistakes in real time. Among the 21 trained pharmaceutical assistants, 15 were evaluated. On average, 11.9 mistakes on 20 were detected. The lowest score was 7 spotted errors on 20 and the highest was 16 on 20. Those results should be qualified depending on pharmaceutical assistants’ years of experience in the preparation of chemotherapy. Those results may be explained by the way the role-playing was conducted. The simulation was not conducted during an actual preparation using the usual equipment. One of the major obstacles was the difficulty to clear some time for this project because its realization required a full-time pharmacist and the referring pharmaceutical assistant in addition to the evaluated pharmaceutical assistants. Overall, the staff feedback was positive and the role-playing led to a reminder of theoretical knowledge and the good use of some devices. It would be interesting to develop this type of project through a regional oncology network to create a medium that can be used by other hospitals.

Hypertension is one of the most important risk factors of heart failure. In response to high blood pressure, the left ventricle manifests hypertrophic growth to ameliorate wall stress, which may progress into decompensation and trigger pathological cardiac remodeling. Despite the clinical importance, the temporal dynamics of pathological cardiac growth remain elusive. Here, we took advantage of the puromycin labeling approach to measure the relative rates of protein synthesis as a way to delineate the temporal regulation of cardiac hypertrophic growth. We first identified the optimal treatment conditions for puromycin in neonatal rat ventricular myocyte culture. We went on to demonstrate that myocyte growth reached its peak rate after 8–10 h of growth stimulation. At the in vivo level, with the use of an acute surgical model of pressure-overload stress, we observed the maximal growth rate to occur at day 7 after surgery. Moreover, RNA sequencing analysis supports that the most profound transcriptomic changes occur during the early phase of hypertrophic growth. Our results therefore suggest that cardiac myocytes mount an immediate growth response in reply to pressure overload followed by a gradual return to basal levels of protein synthesis, highlighting the temporal dynamics of pathological cardiac hypertrophic growth. NEW & NOTEWORTHY We determined the optimal conditions of puromycin incorporation in cardiac myocyte culture. We took advantage of this approach to identify the growth dynamics of cardiac myocytes in vitro. We went further to discover the protein synthesis rate in vivo, which provides novel insights about cardiac temporal growth dynamics in response to pressure overload.

During the past decade, epigenetic control of cardiac remodeling has attracted attention as a major mechanism contributing to heart failure. The BET family of bromodomain proteins (BRD) functions as “readers” of acetylated chromatin. Pharmacological inhibition of BRD proteins prevents cardiac hypertrophy and heart failure in preclinical models, yet mechanistic studies elucidating the biology of BRD proteins in the heart are lacking. Here, we investigated the role(s) of BRD4 in cardiac physiology and pathophysiology. By engineering a cardiomyocyte-specific BRD4 knockout mouse, we discovered that loss of BRD4 triggered progressive decline in ventricular contractile function, culminating in dilated cardiomyopathy in both post-natal and adult hearts. To identify early transcriptomic changes in BRD4-ablated heart, we conducted a global transcriptome analysis in KO hearts prior to the onset of ventricular dysfunction. RNA sequencing analysis of BRD4 KO hearts revealed disruption of genes essential to mitochondrial energy production and homeostasis, as well as key cardiac sarcomeric components. Computational analysis identified key transcription factors involved in BRD4-mediated gene regulation, including MEF2 complex, ERR1, SRF, and Nkx2.5. By studying isolated cardiomyocytes maintained in primary culture, we confirmed that disruption of the candidate TF complexes by BRD4 ablation altered mitochondrial morphology and provoked progressive contractile dysfunction. In aggregate, we describe for the first time a critical role of BRD4 in regulating key cardiomyocyte gene networks, in particular genes involved in mitochondrial homeostasis and contractility, to maintain cardiac function. Moreover, our data suggest that BRD4 is a novel regulator of epigenetic events in dilated cardiomyopathy. Elucidating these novel roles of BRD4 in cardiomyocyte remodeling will provide critical insights into the epigenetic control of stress-related cardiac events with potential clinical relevance.

Background: The unfolded protein response plays versatile roles in physiology and pathophysiology. Its connection to cell growth, however, remains elusive. Here, we sought to define the role of unfolded protein response in the regulation of cardiomyocyte growth in the heart. Methods: We used both gain- and loss-of-function approaches to genetically manipulate XBP1s (spliced X-box binding protein 1), the most conserved signaling branch of the unfolded protein response, in the heart. In addition, primary cardiomyocyte culture was used to address the role of XBP1s in cell growth in a cell-autonomous manner. Results: We found that XBP1s expression is reduced in both human and rodent cardiac tissues under heart failure. Furthermore, deficiency of XBP1s leads to decompensation and exacerbation of heart failure progression under pressure overload. On the other hand, cardiac-restricted overexpression of XBP1s prevents the development of cardiac dysfunction. Mechanistically, we found that XBP1s stimulates adaptive cardiac growth through activation of the mechanistic target of rapamycin signaling, which is mediated via FKBP11 (FK506-binding protein 11), a novel transcriptional target of XBP1s. Moreover, silencing of FKBP11 significantly diminishes XBP1s-induced mechanistic target of rapamycin activation and adaptive cell growth. Conclusions: Our results reveal a critical role of the XBP1s–FKBP11–mechanistic target of rapamycin axis in coupling of the unfolded protein response and cardiac cell growth regulation.

Background: The primary cilium is a singular cellular structure that extends from the surface of many cell types and plays crucial roles in vertebrate development, including that of the heart. Whereas ciliated cells have been described in developing heart, a role for primary cilia in adult heart has not been reported. This, coupled with the fact that mutations in genes coding for multiple ciliary proteins underlie polycystic kidney disease, a disorder with numerous cardiovascular manifestations, prompted us to identify cells in adult heart harboring a primary cilium and to determine whether primary cilia play a role in disease-related remodeling. Methods: Histological analysis of cardiac tissues from C57BL/6 mouse embryos, neonatal mice, and adult mice was performed to evaluate for primary cilia. Three injury models (apical resection, ischemia/reperfusion, and myocardial infarction) were used to identify the location and cell type of ciliated cells with the use of antibodies specific for cilia (acetylated tubulin, γ-tubulin, polycystin [PC] 1, PC2, and KIF3A), fibroblasts (vimentin, α-smooth muscle actin, and fibroblast-specific protein-1), and cardiomyocytes (α-actinin and troponin I). A similar approach was used to assess for primary cilia in infarcted human myocardial tissue. We studied mice silenced exclusively in myofibroblasts for PC1 and evaluated the role of PC1 in fibrogenesis in adult rat fibroblasts and myofibroblasts. Results: We identified primary cilia in mouse, rat, and human heart, specifically and exclusively in cardiac fibroblasts. Ciliated fibroblasts are enriched in areas of myocardial injury. Transforming growth factor β-1 signaling and SMAD3 activation were impaired in fibroblasts depleted of the primary cilium. Extracellular matrix protein levels and contractile function were also impaired. In vivo, depletion of PC1 in activated fibroblasts after myocardial infarction impaired the remodeling response. Conclusions: Fibroblasts in the neonatal and adult heart harbor a primary cilium. This organelle and its requisite signaling protein, PC1, are required for critical elements of fibrogenesis, including transforming growth factor β-1–SMAD3 activation, production of extracellular matrix proteins, and cell contractility. Together, these findings point to a pivotal role of this organelle, and PC1, in disease-related pathological cardiac remodeling and suggest that some of the cardiovascular manifestations of autosomal dominant polycystic kidney disease derive directly from myocardium-autonomous abnormalities.

The hexosamine biosynthetic pathway (HBP) plays critical roles in nutrient sensing, stress response, and cell growth. However, its contribution to cardiac hypertrophic growth and heart failure remains incompletely understood. Here, we show that the HBP is induced in cardiomyocytes during hypertrophic growth. Overexpression of Gfat1 (glutamine:fructose-6-phosphate amidotransferase 1), the rate-limiting enzyme of HBP, promotes cardiomyocyte growth. On the other hand, Gfat1 inhibition significantly blunts phenylephrine-induced hypertrophic growth in cultured cardiomyocytes. Moreover, cardiac-specific overexpression of Gfat1 exacerbates pressure overload-induced cardiac hypertrophy, fibrosis, and cardiac dysfunction. Conversely, deletion of Gfat1 in cardiomyocytes attenuates pathological cardiac remodeling in response to pressure overload. Mechanistically, persistent upregulation of the HBP triggers decompensated hypertrophy through activation of mTOR while Gfat1 deficiency shows cardioprotection and a concomitant decrease in mTOR activity. Taken together, our results reveal that chronic upregulation of the HBP under hemodynamic stress induces pathological cardiac hypertrophy and heart failure through persistent activation of mTOR., Metabolic remodeling plays an important role in pathological cardiac hypertrophy. Here, the authors show that hexosamine biosynthetic pathway is elevated in the heart by pressure overload, which contributes to heart failure by persistent activation of mTOR.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20648-5

Background— The clinical use of doxorubicin is limited by cardiotoxicity. Histopathological changes include interstitial myocardial fibrosis and the appearance of vacuolated cardiomyocytes. Whereas dysregulation of autophagy in the myocardium has been implicated in a variety of cardiovascular diseases, the role of autophagy in doxorubicin cardiomyopathy remains poorly defined. Methods and Results— Most models of doxorubicin cardiotoxicity involve intraperitoneal injection of high-dose drug, which elicits lethargy, anorexia, weight loss, and peritoneal fibrosis, all of which confound the interpretation of autophagy. Given this, we first established a model that provokes modest and progressive cardiotoxicity without constitutional symptoms, reminiscent of the effects seen in patients. We report that doxorubicin blocks cardiomyocyte autophagic flux in vivo and in cardiomyocytes in culture. This block was accompanied by robust accumulation of undegraded autolysosomes. We go on to localize the site of block as a defect in lysosome acidification. To test the functional relevance of doxorubicin-triggered autolysosome accumulation, we studied animals with diminished autophagic activity resulting from haploinsufficiency for Beclin 1 . Beclin 1 +/− mice exposed to doxorubicin were protected in terms of structural and functional changes within the myocardium. Conversely, animals overexpressing Beclin 1 manifested an amplified cardiotoxic response. Conclusions— Doxorubicin blocks autophagic flux in cardiomyocytes by impairing lysosome acidification and lysosomal function. Reducing autophagy initiation protects against doxorubicin cardiotoxicity.

Multiwalled carbon nanotubes (MWCNT) have been shown to induce lung fibrosis in animal models, however the underlying molecular factors/mechanisms are still unclear. In this study, we investigated the effects of lipopolysaccharide (LPS), MWCNT, and the combination of LPS and MWCNT on the expression of matrix metalloproteinase-9 and metalloproteinase-12 (MMP-9, MMP-12), collagen 3A1 (Col3A1), and transforming growth factor beta (TGFβ) in alveolar epithelial A549 cells. MMPs are proteinases that degrade extracellular matrix and play a role in lung fibrosis. A549 cells were exposed to LPS (1 ng/mL), MWCNT (20 μg/mL), and the combination and analyzed for paracellular permeability, TGFβ, Col3A1, MMP-9, MMP-12, NF-κB activation, and cell migration by real-time PCR and immunofluorescence. LPS, the combination of LPS and MWCNT, and MWCNT only at the highest tested dose induced blue dextran extravasation. LPS and MWCNT increased the expression of TGFβ and its downstream target gene Col3A, and MMP-9 and MMP-12 mRNA. MWCNT potently induced cell migration toward wound healing, whereas LPS slightly induced cell migration. Both, LPS and MWCNT, induced NF-κB nuclear translocation. Our results indicate that MWCNT activated alveolar epithelial cells to promote fibrogenesis, and that LPS differentially primes molecular factors involved in lung remodeling. These findings suggest a role of alveolar epithelial cells in fibrogenesis and also may aid in the design and development of tests for screening of fibrogenic agents. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 445-455, 2017.

Resume Introduction/objectif Dans les essais cliniques, les promoteurs sont tenus de mettre gratuitement a disposition des investigateurs aussi bien le medicament a l’etude que les traitements de reference utilises dans l’essai. Materiel et methode Nous avons recense sur l’annee 2011 les essais en cours dans notre etablissement et determine si les medicaments etaient fournis ou non par le promoteur. Les medicaments non fournis ont ensuite ete identifies, ainsi que les essais dans lesquels ils etaient utilises et le nombre de dispensations intervenues durant l’annee afin de chiffrer le montant impute a l’hopital et l’Assurance maladie. Resultats et discussion Sur les 269 essais etudies, 388 898 € de medicaments experimentaux ont ete pris en charge par le systeme de sante (CHU : 46 400 € ; Assurance maladie : 342 498 €). Le service d’hematologie a genere la quasi-totalite des couts recenses tous types de promoteurs confondus, representant 364 447 € soit 93 %, dont 241 472 € proviennent d’essais promus par des societes savantes. D’autres etudes menees dans notre etablissement ont montre que l’activite de gestion des essais cliniques genere des benefices pour l’hopital. Conclusion Si le gain pour l’avancee de la recherche biomedicale est incontestable, en revanche, l’economie realisee s’en trouve grevee par des depenses qui devraient, dans la plupart des cas, etre supportees par le promoteur.

SUMMARY The heart manifests hypertrophic growth in response to high blood pressure, which may decompensate and progress to heart failure under persistent stress. Metabolic remodeling is an early event in this process. However, its role remains to be fully characterized. Here, we show that lactate dehydrogenase A (LDHA), a critical glycolytic enzyme, is elevated in the heart in response to hemodynamic stress. Cardiomyocyte-restricted deletion of LDHA leads to defective cardiac hypertrophic growth and heart failure by pressure overload. Silencing of LDHA in cultured cardiomyocytes suppresses cell growth from pro-hypertrophic stimulation in vitro, while overexpression of LDHA is sufficient to drive cardiomyocyte growth. Furthermore, we find that lactate is capable of rescuing the growth defect from LDHA knockdown. Mechanistically, lactate stabilizes NDRG3 (N-myc downregulated gene family 3) and stimulates ERK (extracellular signal-regulated kinase). Our results together suggest that the LDHA/NDRG3 axis may play a critical role in adaptive cardiomyocyte growth in response to hemodynamic stress., In Brief Dai et al. find that LDHA is significantly increased in the heart under hemodynamic stress, and cardiomyocyte-specific deletion of LDHA leads to severe cardiac dysfunction in response to pressure overload. LDHA may govern adaptive growth through elevation of NDRG3 and activation of ERK., Graphical Abstract

Algal turf scrubbers (ATS) and anaerobic digesters (AD), when combined, remove nutrients from eutrophic water while producing renewable energy through biogas production. Algal biomass was produced from an ATS system that used tipping buckets to introduce riverine water in pulses onto a flow-way with a mesh screen installed to facilitate algal attachment. This study was the first to determine methane (CH4) production from AD of a micro- and macro-algae polyculture, harvested from an ATS, using lab-scale reactors under batch and semi-continuous feeding regimes. The lab-scale study was followed by a pilot-scale (7 m3) ATS-AD study, the largest scale study of biogas production from ATS-derived algae. Batch digestion of ‘wet’ algae (93% moisture) produced 158 ± 13 L CH4/kg VS, which was higher than algae with a lower moisture content (≤62% moisture). Continuing use of ‘wet’ algae as the substrate, the 2 L semi-continuously-fed lab system yielded a maximum CH4 production value of 144 ± 7 L CH4/kg VS, with 60%–62% CH4 in the biogas. The 7 m3 pilot-scale system produced 107 ± 15 L CH4/kg VS, with 66.4% CH4 in the biogas. Scale-up calculations showed that a 1 ha ATS system could capture 8.73 kg N, 1.18 kg P, and 52.5 kg C/ha-d, with the resulting algae being used in AD to produce biogas for direct heating (2.8*108 J) or to power a 1.13 kW generator. While the total energy output from the 1 ha ATS-AD system is relatively low, energy production from the AD system is a supplemental benefit to an ATS system that captures nutrients from eutrophic water. This ATS-AD system could contribute to the overall Chesapeake Bay cleanup, especially in nutrient hot-spots, like the Baltimore Harbor, where high nutrient concentrations support high algal productivity, which would provide substrate for on-site energy production from AD.

Objective In humans, loss-of-function mutations in the gene encoding pregnancy-associated pregnancy protein-A2 cause short stature and slightly reduced bone density. The goal of this study was to determine the effects of Pappa2 deletion on bone in mice. Design Pappa2 deletion mice and littermate controls were culled at 10, 19 or 30 weeks of age and femurs were analysed by micro-computed tomography. Serum markers of bone turnover and insulin-like growth factor binding protein 5 (IGFBP-5), a proteolytic target of PAPP-A2, were measured by ELISA. Results At 10 and 19 weeks of age, Pappa2 deletion mice had slightly reduced trabecular parameters, but by 19 weeks of age, female deletion mice had increased cortical tissue mineral density, and this trait was increased by a small amount in deletion mice of both sexes at 30 weeks. Cortical area fraction was increased in Pappa2 deletion mice at all ages. Deletion of Pappa2 increased circulating IGFBP-5 levels and reduced markers of bone turnover (PINP and TRACP 5b). Conclusions PAPP-A2 contributes to the regulation of bone structure and mass in mice, likely through control of IGFBP-5 levels. The net effect of changes in bone formation and resorption depend on sex and age, and differ between trabecular and cortical bone.