Perlecan (HSPG2) promotes structural, contractile, and metabolic development of human cardiomyocytes.

Perlecan (HSPG2), a heparan sulfate proteoglycan similar to agrin, is key for extracellular matrix (ECM) maturation and stabilization. Although crucial for cardiac development, its role remains elusive. We show that perlecan expression increases as cardiomyocytes mature in vivo and during human pluripotent stem cell differentiation to cardiomyocytes (hPSC-CMs). Perlecan-haploinsuffient hPSCs (HSPG2+/-) differentiate efficiently, but late-stage CMs have structural, contractile, metabolic, and ECM gene dysregulation. In keeping with this, late-stage HSPG2+/- hPSC-CMs have immature features, including reduced ⍺-actinin expression and increased glycolytic metabolism and proliferation. Moreover, perlecan-haploinsuffient engineered heart tissues have reduced tissue thickness and force generation. Conversely, hPSC-CMs grown on a perlecan-peptide substrate are enlarged and display increased nucleation, typical of hypertrophic growth. Together, perlecan appears to play the opposite role of agrin, promoting cellular maturation rather than hyperplasia and proliferation. Perlecan signaling is likely mediated via its binding to the dystroglycan complex. Targeting perlecan-dependent signaling may help reverse the phenotypic switch common to heart failure.

p16INK4A-dependent senescence in the bone marrow niche drives age-related metabolic changes of hematopoietic progenitors.

Rapid and effective leukocyte response to infection is a fundamental function of the bone marrow (BM). However, with increasing age, this response becomes impaired, resulting in an increased burden of infectious diseases. Here, we investigate how aging changes the metabolism and function of hematopoietic progenitor cells (HPCs) and the impact of the BM niche on this phenotype. We found that, in response to lipopolysaccharide-induced stress, HPC mitochondrial function is impaired, and there is a failure to upregulate the TCA cycle in progenitor populations in aged animals compared with young animals. Furthermore, aged mesenchymal stromal cells (MSCs) of the BM niche, but not HPCs, exhibit a senescent phenotype, and selective depletion of senescent cells from the BM niche, as well as treatment with the senolytic drug ABT-263, improves mitochondrial function of HPCs when stressed with lipopolysaccharide. In summary, age-related HPC metabolic dysfunction occurs indirectly as a "bystander phenomenon" in the aging BM niche and can be restored by targeting senescent MSCs.

Genome-wide association study of delay discounting in Heterogenous Stock rats.

Delay discounting refers to the behavioral tendency to devalue rewards as a function of their delay in receipt. Heightened delay discounting has been associated with substance use disorders, as well as multiple co-occurring psychopathologies. Genetic studies in humans and animal models have established that delay discounting is a heritable trait, but only a few specific genes have been associated with delay discounting. Here, we aimed to identify novel genetic loci associated with delay discounting through a genome-wide association study (GWAS) using Heterogenous Stock rats, a genetically diverse outbred population derived from eight inbred founder strains. We assessed delay discounting in 650 male and female rats using an adjusting amount procedure in which rats chose between smaller immediate sucrose rewards or a larger reward at variable delays. Preference switch points were calculated for each rat and both exponential and hyperbolic functions were fitted to these indifference points. Area under the curve (AUC) and the discounting parameter k of both functions were used as delay discounting measures. GWAS for AUC, exponential k, and indifference points for a short delay identified significant loci on chromosomes 20 and 14. The gene Slc35f1, which encodes a member of the solute carrier family of nucleoside sugar transporters, was the only gene within the chromosome 20 locus. That locus also contained an eQTL for Slc35f1, suggesting that heritable differences in the expression of that gene might be responsible for the association with behavior. The gene Adgrl3, which encodes a member of the latrophilin family of G-protein coupled receptors, was the only gene within the chromosome 14 locus. These findings implicate novel genes in delay discounting and highlight the need for further exploration.

Transcriptomic Analysis of Cardiomyocyte Extracellular Vesicles in Hypertrophic Cardiomyopathy Reveals Differential snoRNA Cargo.

Hypertrophic cardiomyopathy (HCM) is characterized by increased left ventricular wall thickness that can lead to devastating conditions such as heart failure and sudden cardiac death. Despite extensive study, the mechanisms mediating many of the associated clinical manifestations remain unknown and human models are required. To address this, human-induced pluripotent stem cell (hiPSC) lines were generated from patients with a HCM-associated mutation (c.ACTC1G301A) and isogenic controls created by correcting the mutation using CRISPR/Cas9 gene editing technology. Cardiomyocytes (hiPSC-CMs) were differentiated from these hiPSCs and analyzed at baseline, and at increased contractile workload (2 Hz electrical stimulation). Released extracellular vesicles (EVs) were isolated and characterized after a 24-h culture period and transcriptomic analysis performed on both hiPSC-CMs and released EVs. Transcriptomic analysis of cellular mRNA showed the HCM mutation caused differential splicing within known HCM pathways, and disrupted metabolic pathways. Analysis at increasing contraction frequency showed further disruption of metabolic gene expression, with an additive effect in the HCM background. Intriguingly, we observed differences in snoRNA cargo within HCM released EVs that specifically altered when HCM hiPSC-CMs were subjected to increased workload. These snoRNAs were predicted to have roles in post-translational modifications and alternative splicing, processes differentially regulated in HCM. As such, the snoRNAs identified in this study may unveil mechanistic insight into unexplained HCM phenotypes and offer potential future use as HCM biomarkers or as targets in future RNA-targeting therapies.

Genome size influences adaptive plasticity of water loss, but not metabolic rates, in lungless salamanders.

Many expressions of phenotype, such as physiological performance, integrate multiple underlying traits to function. Linking component traits to adaptive physiology thus gives insight into mechanisms of selection acting on performance. Genome size (C-value) is a trait that influences physiology in multiple taxa by exerting a nucleotypic effect, constraining cell size and cellular physiology such that whole-organism mass-specific metabolism is reduced with increasing C-value. We tested for this mechanism of C-value function acting in lungless salamanders, plus an unexplored potential mechanism of C-value effects constraining water transport across the body surface to influence cutaneous water loss rates. We found no evidence for a nucleotypic effect on metabolic rates, but we demonstrate a relationship between C-value and water loss physiology. Under warmer experimental conditions, C-value was inversely correlated with water loss and positively correlated with resistance to water loss, which demonstrated adaptive plasticity at higher temperatures. We hypothesize that this pattern results from differences in cell size constraining diffusion and evaporation of water from the skin under warm conditions when cutaneous perfusion is reduced. Testing this hypothesis may confirm a previously unappreciated adaptive role for C-value variation in this group, and reveals the possibility that genome size influences physiological exchange across transport barriers more broadly.

Modelling Metabolic Shifts during Cardiomyocyte Differentiation, Iron Deficiency and Transferrin Rescue Using Human Pluripotent Stem Cells.

Cardiomyocytes rely on specialised metabolism to meet the high energy demand of the heart. During heart development, metabolism matures and shifts from the predominant utilisation of glycolysis and glutamine oxidation towards lactate and fatty acid oxidation. Iron deficiency (ID) leads to cellular metabolism perturbations. However, the exact alterations in substrate metabolism during ID are poorly defined. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), the present study investigated changes in major metabolic substrate utilisation in the context of ID or upon transferrin rescue. Typically, during hiPSC-CM differentiation, the greatest increase in total metabolic output and rate was seen in fatty acid metabolism. When ID was induced, hiPSC-CMs displayed increased reliance on glycolytic metabolism, and six TCA cycle, five amino acid, and four fatty acid substrates were significantly impaired. Transferrin rescue was able to improve TCA cycle substrate metabolism, but the amino acid and fatty acid metabolism remained perturbed. Replenishing iron stores partially reverses the adverse metabolic changes that occur during ID. Understanding the changes in metabolic substrate utilisation and their modification may provide potential for discovery of new biomarkers and therapeutic targets in cardiovascular diseases.

Experimental removal of introduced slider turtles offers new insight into competition with a native, threatened turtle.

The red-eared slider turtle (Trachemys scripta elegans; RES) is often considered one of the world's most invasive species. Results from laboratory and mesocosm experiments suggest that introduced RES outcompete native turtles for key ecological resources, but such experiments can overestimate the strength of competition. We report on the first field experiment with a wild turtle community, involving introduced RES and a declining native species of conservation concern, the western pond turtle (Emys marmorata; WPT). Using a before/after experimental design, we show that after removing most of an introduced RES population, the remaining RES dramatically shifted their spatial basking distribution in a manner consistent with strong intraspecific competition. WPT also altered their spatial basking distribution after the RES removal, but in ways inconsistent with strong interspecific competition. However, we documented reduced levels of WPT basking post-removal, which may reflect a behavioral shift attributable to the lower density of the turtle community. WPT body condition also increased after we removed RES, consistent with either indirect or direct competition between WPT and RES and providing the first evidence that RES can compete with a native turtle in the wild. We conclude that the negative impacts on WPT basking by RES in natural contexts are more limited than suggested by experiments with captive turtles, although wild WPT do appear to compete for food with introduced RES. Our results highlight the importance of manipulative field experiments when studying biological invasions, and the potential value of RES removal as a management strategy for WPT.

Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour.

Background: Diseases such as hypertrophic cardiomyopathy (HCM) can lead to severe outcomes including sudden death. The generation of human induced pluripotent stem cell (hiPSC) reporter lines can be useful for disease modelling and drug screening by providing physiologically relevant in vitro models of disease. The AAVS1 locus is cited as a safe harbour that is permissive for stable transgene expression, and hence is favoured for creating gene targeted reporter lines. Methods: We generated hiPSC reporters using a plasmid-based CRISPR/Cas9 nickase strategy. The first intron of PPP1R12C, the AAVS1 locus, was targeted with constructs expressing a genetically encoded calcium indicator (R-GECO1.0) or HOXA9-T2A-mScarlet reporter under the control of a pCAG or inducible pTRE promoter, respectively. Transgene expression was compared between clones before, during and/or after directed differentiation to mesodermal lineages. Results: Successful targeting to AAVS1 was confirmed by PCR and sequencing. Of 24 hiPSC clones targeted with pCAG-R-GECO1.0, only 20 expressed the transgene and in these, the percentage of positive cells ranged from 0% to 99.5%. Differentiation of a subset of clones produced cardiomyocytes, wherein the percentage of cells positive for R-GECO1.0 ranged from 2.1% to 93.1%. In the highest expressing R-GECO1.0 clones, transgene silencing occurred during cardiomyocyte differentiation causing a decrease in expression from 98.93% to 1.3%. In HOXA9-T2A-mScarlet hiPSC reporter lines directed towards mesoderm lineages, doxycycline induced a peak in transgene expression after two days but this reduced by up to ten-thousand-fold over the next 8-10 days. Nevertheless, for R-GECO1.0 lines differentiated into cardiomyocytes, transgene expression was rescued by continuous puromycin drug selection, which allowed the Ca 2+ responses associated with HCM to be investigated in vitro using single cell analysis. Conclusions: Targeted knock-ins to AAVS1 can be used to create reporter lines but variability between clones and transgene silencing requires careful attention by researchers seeking robust reporter gene expression.

Mechanistic Insights into the Cholesterol-dependent Binding of Perfringolysin O-based Probes and Cell Membranes.

Cholesterol distribution in the cell is maintained by both vesicular and non-vesicular sterol transport. Non-vesicular transport is mediated by the interaction of membrane-embedded cholesterol and water-soluble proteins. Small changes to the lipid composition of the membrane that do not change the total cholesterol content, can significantly affect how cholesterol interacts with other molecules at the surface of the membrane. The cholesterol-dependent cytolysin Perfringolysin O (PFO) constitutes a powerful tool to detect cholesterol in membranes, and the use of PFO-based probes has flourished in recent years. By using a non-lytic PFO derivative, we showed that the sensitivity of the probes for cholesterol can be tuned by modifications introduced directly in the membrane-interacting loops and/or by modifying residues away from the membrane-interacting domain. Through the use of these biosensors on live RAW 264.7 cells, we found that changes in the overall cholesterol content have a limited effect on the average cholesterol accessibility at the surface of the membrane. We showed that these exquisite biosensors report on changes in cholesterol reactivity at the membrane surface independently of the overall cholesterol content in the membrane.

Asymmetric Introgression in a Spotted Salamander Hybrid Zone.

Before the establishment of reproductive isolation, deeply diverged intraspecific lineages can experience complex genetic and behavioral interactions as they come into secondary contact. Divergent selective and demographic processes mediate gene flow among lineages, resulting in hybrid zones with complex biogeographic structure. Discordance in the biogeographic patterns of autosomal and maternally inherited loci provides a useful window to infer the processes mediating admixture and introgression across hybrid zones. Here, we sampled 489 genotypes across a hybrid zone between 2 phylogeographic lineages of the spotted salamander, Ambystoma maculatum, and characterize discordant patterns of nuclear and mitochondrial introgression across the contact boundary. Our results indicate asymmetric introgression of nuclear DNA beyond the contact boundary from the western to eastern lineage, with introgression of eastern mitochondrial DNA into the western lineage. We discuss alternative mechanisms for this pattern and attribute this result to neutral patterns of population expansion of the western lineage into the east in combination with female mate choice for larger-bodied western males. Our results underscore the complexity of interacting mechanisms that give rise to reproductive asymmetries in the earliest stages of the speciation process.

Perfringolysin O structure and mechanism of pore formation as a paradigm for cholesterol-dependent cytolysins.

Cholesterol-dependent cytolysins (CDCs) constitute a family of pore forming toxins secreted by Gram-positive bacteria. These toxins form transmembrane pores by inserting a large ß-barrel into cholesterol-containing membrane bilayers. Binding of water-soluble CDCs to the membrane triggers the formation of oligomers containing 35-50 monomers. The coordinated insertion of more than seventy ß-hairpins into the membrane requires multiple structural conformational changes. Perfringolysin O (PFO), secreted by Clostridium perfringens, has become the prototype for the CDCs. In this chapter, we will describe current knowledge on the mechanism of PFO cytolysis, with special focus on cholesterol recognition, oligomerization, and the conformational changes involved in pore formation.

Modifications in perfringolysin O domain 4 alter the cholesterol concentration threshold required for binding.

Changes in the cholesterol content of cell membranes affect many physiological and pathological events, including the formation of arterial plaques, the entry of virus into cells, and receptor organization. Measuring the trafficking and distribution of cholesterol is essential to understanding how cells regulate sterol levels in membranes. Perfringolysin O (PFO) is a cytolysin secreted by Clostridium perfringens that requires cholesterol in the target membrane for binding. The specificity of PFO for high levels of cholesterol makes the toxin an attractive tool for studying the distribution and trafficking of cholesterol in cells. However, the use of the native toxin is limited given that binding is triggered only above a determined cholesterol concentration. To this end, we have identified mutations in PFO that altered the threshold for how much cholesterol is required to trigger binding. The cholesterol threshold among different PFO derivatives varied up to 10 mol % sterol, and these variations were not dependent on the lipid composition of the membrane. We characterized the binding of these PFO derivatives on murine macrophage-like cells whose cholesterol content was reduced or augmented. Our findings revealed that engineered PFO derivatives differentially associated with these cells in response to changes in cholesterol levels in the plasma membrane.

The cholesterol-dependent cytolysin family of gram-positive bacterial toxins.

The cholesterol-dependent cytolysins (CDCs) are a family of beta-barrel pore-forming toxins secreted by Gram-positive bacteria. These toxins are produced as water-soluble monomeric proteins that after binding to the target cell oligomerize on the membrane surface forming a ring-like pre-pore complex, and finally insert a large beta-barrel into the membrane (about 250 A in diameter). Formation of such a large transmembrane structure requires multiple and coordinated conformational changes. The presence of cholesterol in the target membrane is absolutely required for pore-formation, and therefore it was long thought that cholesterol was the cellular receptor for these toxins. However, not all the CDCs require cholesterol for binding. Intermedilysin, secreted by Streptoccocus intermedius only binds to membranes containing a protein receptor, but forms pores only if the membrane contains sufficient cholesterol. In contrast, perfringolysin O, secreted by Clostridium perfringens, only binds to membranes containing substantial amounts of cholesterol. The mechanisms by which cholesterol regulates the cytolytic activity of the CDCs are not understood at the molecular level. The C-terminus of perfringolysin O is involved in cholesterol recognition, and changes in the conformation of the loops located at the distal tip of this domain affect the toxin-membrane interactions. At the same time, the distribution of cholesterol in the membrane can modulate toxin binding. Recent studies support the concept that there is a dynamic interplay between the cholesterol-binding domain of the CDCs and the excess of cholesterol molecules in the target membrane.