ATP-dependent citrate lyase Drives Left Ventricular Dysfunction by Metabolic Remodeling of the Heart.

Background: Metabolic remodeling is a hallmark of the failing heart. Oncometabolic stress during cancer increases the activity and abundance of the ATP-dependent citrate lyase (ACL, Acly ), which promotes histone acetylation and cardiac adaptation. ACL is critical for the de novo synthesis of lipids, but how these metabolic alterations contribute to cardiac structural and functional changes remains unclear. Methods: We utilized human heart tissue samples from healthy donor hearts and patients with hypertrophic cardiomyopathy. Further, we used CRISPR/Cas9 gene editing to inactivate Acly in cardiomyocytes of MyH6-Cas9 mice. In vivo, positron emission tomography and ex vivo stable isotope tracer labeling were used to quantify metabolic flux changes in response to the loss of ACL. We conducted a multi-omics analysis using RNA-sequencing and mass spectrometry-based metabolomics and proteomics. Experimental data were integrated into computational modeling using the metabolic network CardioNet to identify significantly dysregulated metabolic processes at a systems level. Results: Here, we show that in mice, ACL drives metabolic adaptation in the heart to sustain contractile function, histone acetylation, and lipid modulation. Notably, we show that loss of ACL increases glucose oxidation while maintaining fatty acid oxidation. Ex vivo isotope tracing experiments revealed a reduced efflux of glucose-derived citrate from the mitochondria into the cytosol, confirming that citrate is required for reductive metabolism in the heart. We demonstrate that YAP inactivation facilitates ACL deficiency. Computational flux analysis and integrative multi-omics analysis indicate that loss of ACL induces alternative isocitrate dehydrogenase 1 flux to compensate. Conclusions: This study mechanistically delineates how cardiac metabolism compensates for suppressed citrate metabolism in response to ACL loss and uncovers metabolic vulnerabilities in the heart.

A cost-effectiveness analysis of early detection and bundled treatment of postpartum hemorrhage alongside the E-MOTIVE trial.

Timely detection and treatment of postpartum hemorrhage (PPH) are crucial to prevent complications or death. A calibrated blood-collection drape can help provide objective, accurate and early diagnosis of PPH, and a treatment bundle can address delays or inconsistencies in the use of effective interventions. Here we conducted an economic evaluation alongside the E-MOTIVE trial, an international, parallel cluster-randomized trial with a baseline control phase involving 210,132 women undergoing vaginal delivery across 78 secondary-level hospitals in Kenya, Nigeria, South Africa and Tanzania. We aimed to assess the cost-effectiveness of the E-MOTIVE intervention, which included a calibrated blood-collection drape for early detection of PPH and a bundle of first-response treatments (uterine massage, oxytocic drugs, tranexamic acid, intravenous fluids, examination and escalation), compared with usual care. We used multilevel modeling to estimate incremental cost-effectiveness ratios from the perspective of the public healthcare system for outcomes of cost per severe PPH (blood loss ≥1,000 ml) avoided and cost per disability-adjusted life-year averted. Our findings suggest that the use of a calibrated blood-collection drape for early detection of PPH and bundled first-response treatment is cost-effective and should be perceived by decision-makers as a worthwhile use of healthcare budgets. ClinicalTrials.gov identifier: NCT04341662 .

SETD3 is a mechanosensitive enzyme that methylates actin on His-73 to regulate mitochondrial dynamics and function.

Mitochondria, which act as sensors of metabolic homeostasis and metabolite signaling, form a dynamic intracellular network of continuously changing shape, size, and localization to respond to localized cellular energy demands. Mitochondrial dynamics and function depend on interactions with the F-actin cytoskeleton that are poorly understood. Here, we show that SET domain protein 3 (SETD3), a recently described actin histidine methyltransferase, directly methylates actin Histidine-73 and enhances F-actin polymerization on mitochondria. SETD3 is a mechano-sensitive enzyme which is localized on the outer mitochondrial membrane and promotes actin polymerization around mitochondrias. SETD3 loss of function leads to diminished F-actin around mitochondria and a decrease in mitochondrial branch length, branch number, and mitochondrial movement. Our functional analysis revealed that SETD3 is required for oxidative phosphorylation and mitochondrial complex I assembly, and function. Our data further indicate that SETD3 regulates F-actin formation around mitochondria and is essential for maintaining mitochondrial morphology, movement, and function. Finally, we discovered that SETD3 levels are regulated by ECM stiffness and regulate mitochondrial shape in response to changes in ECM stiffness. These findings provide new insight into the mechanism for F-actin polymerization around mitochondria.

Evaluation of sex differences in the receipt of concomitant atrial fibrillation procedures during nonmitral cardiac surgery.

OBJECTIVE: Women are less likely to receive guideline-recommended cardiovascular care, but evaluation of sex-based disparities in cardiac surgical procedures is limited. Receipt of concomitant atrial fibrillation (AF) procedures during nonmitral cardiac surgery was compared by sex for patients with preoperative AF. METHODS: Patients with preoperative AF undergoing coronary artery bypass grafting and/or aortic valve replacement at any of the 33 hospitals in Michigan from 2014 to 2022 were included. Patients with prior cardiac surgery, transcatheter AF procedure, or emergency/salvage status were excluded. Hierarchical logistic regression identified predictors of concomitant AF procedures, account for hospital and surgeon as random effects. RESULTS: Of 5460 patients with preoperative AF undergoing nonmitral cardiac surgery, 24% (n = 1291) were women with a mean age of 71 years. Women were more likely to have paroxysmal (vs persistent) AF than men (80% vs 72%; P < .001) and had a higher mean predicted risk of mortality (5% vs 3%; P < .001). The unadjusted rate of concomitant AF procedure was 59% for women and 67% for men (P < .001). After risk adjustment, women had 26% lower adjusted odds of concomitant AF procedure than men (adjusted odds ratio, 0.74; 95% CI, 0.64-0.86; P < .001). Female sex was the risk factor associated with the lowest odds of concomitant AF procedure. CONCLUSIONS: Women are less likely to receive guideline recommended concomitant AF procedure during nonmitral surgery. Identification of barriers to concomitant AF procedure in women may improve treatment of AF.

SRF Expression in Excess Permits a Dual Contractile-Proliferative Phenotype of Airway Smooth Muscle.

The transcription factors (TFs) myocardin (MyoCD) and ETS Like-1 protein (Elk-1) competitively bind to serum response factor (SRF) and control myogenic- and mitogenic-related gene expression in smooth muscle, respectively. Their functions are therefore mutually inhibitory, which result in a contractile versus proliferative phenotype dichotomy. Airway smooth muscle cell (ASMC) phenotype alterations occur in various inflammatory airway diseases, promoting pathological remodelling and contributing to airflow obstruction. We characterized MyoCD and Elk-1 interactions and their roles in phenotype determination in human ASMCs. MyoCD overexpression in ASMCs increased smooth muscle gene expression, force generation, and partially restored the loss of smooth muscle protein associated with prolonged culturing, while inhibiting Elk-1 transcriptional activities and proliferation induced by epidermal growth factor (EGF). However, MyoCD overexpression failed to suppress these responses induced by fetal bovine serum (FBS) as FBS also upregulated SRF expression to a degree that allowed unopposed function of both TFs. Inhibition of the RhoA pathway reversed said SRF changes, allowing inhibition of Elk-1 by MyoCD overexpression and suppressing FBS-mediated contractile protein gene upregulation. Our study confirmed that MyoCD in increased abundance can competitively inhibit Elk-1 function. However, SRF upregulation permits a dual contractile-proliferative ASMC phenotype, anticipated to exacerbate pathological alterations, whereas therapies targeting SRF may inhibit both pathological ASMC proliferation and contractile protein gene expression.

Spatiotemporal distribution and bionomics of Anopheles stephensi in different eco-epidemiological settings in Ethiopia.

BACKGROUND: Malaria is a major public health concern in Ethiopia, and its incidence could worsen with the spread of the invasive mosquito species Anopheles stephensi in the country. This study aimed to provide updates on the distribution of An. stephensi and likely household exposure in Ethiopia. METHODS: Entomological surveillance was performed in 26 urban settings in Ethiopia from 2021 to 2023. A kilometer-by-kilometer quadrant was established per town, and approximately 20 structures per quadrant were surveyed every 3 months. Additional extensive sampling was conducted in 50 randomly selected structures in four urban centers in 2022 and 2023 to assess households' exposure to An. stephensi. Prokopack aspirators and CDC light traps were used to collect adult mosquitoes, and standard dippers were used to collect immature stages. The collected mosquitoes were identified to species level by morphological keys and molecular methods. PCR assays were used to assess Plasmodium infection and mosquito blood meal source. RESULTS: Catches of adult An. stephensi were generally low (mean: 0.15 per trap), with eight positive sites among the 26 surveyed. This mosquito species was reported for the first time in Assosa, western Ethiopia. Anopheles stephensi was the predominant species in four of the eight positive sites, accounting for 75-100% relative abundance of the adult Anopheles catches. Household-level exposure, defined as the percentage of households with a peridomestic presence of An. stephensi, ranged from 18% in Metehara to 30% in Danan. Anopheles arabiensis was the predominant species in 20 of the 26 sites, accounting for 42.9-100% of the Anopheles catches. Bovine blood index, ovine blood index and human blood index values were 69.2%, 32.3% and 24.6%, respectively, for An. stephensi, and 65.4%, 46.7% and 35.8%, respectively, for An. arabiensis. None of the 197 An. stephensi mosquitoes assayed tested positive for Plasmodium sporozoite, while of the 1434 An. arabiensis mosquitoes assayed, 62 were positive for Plasmodium (10 for P. falciparum and 52 for P. vivax). CONCLUSIONS: This study shows that the geographical range of An. stephensi has expanded to western Ethiopia. Strongly zoophagic behavior coupled with low adult catches might explain the absence of Plasmodium infection. The level of household exposure to An. stephensi in this study varied across positive sites. Further research is needed to better understand the bionomics and contribution of An. stephensi to malaria transmission.

YAP induces a neonatal-like pro-renewal niche in the adult heart.

After myocardial infarction (MI), mammalian hearts do not regenerate, and the microenvironment is disrupted. Hippo signaling loss of function with activation of transcriptional co-factor YAP induces heart renewal and rebuilds the post-MI microenvironment. In this study, we investigated adult renewal-competent mouse hearts expressing an active version of YAP, called YAP5SA, in cardiomyocytes (CMs). Spatial transcriptomics and single-cell RNA sequencing revealed a conserved, renewal-competent CM cell state called adult (a)CM2 with high YAP activity. aCM2 co-localized with cardiac fibroblasts (CFs) expressing complement pathway component C3 and macrophages (MPs) expressing C3ar1 receptor to form a cellular triad in YAP5SA hearts and renewal-competent neonatal hearts. Although aCM2 was detected in adult mouse and human hearts, the cellular triad failed to co-localize in these non-renewing hearts. C3 and C3ar1 loss-of-function experiments indicated that C3a signaling between MPs and CFs was required to assemble the pro-renewal aCM2, C3+ CF and C3ar1+ MP cellular triad.

Activated fibroblasts drive cellular interactions in end-stage pediatric hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a relatively rare but debilitating diagnosis in the pediatric population and patients with end-stage HCM require heart transplantation. In this study, we performed single-nucleus RNA sequencing on pediatric HCM and control myocardium. We identified distinct underling cellular processes in pediatric, end-stage HCM in cardiomyocytes, fibroblasts, endothelial cells, and myeloid cells, compared to controls. Pediatric HCM was enriched in cardiomyocytes exhibiting "stressed" myocardium gene signatures and underlying pathways associated with cardiac hypertrophy. Cardiac fibroblasts exhibited clear activation signatures and heightened downstream processes associated with fibrosis, more so than adult counterparts. There was notable depletion of tissue-resident macrophages, and increased vascular remodeling in endothelial cells. Our analysis provides the first single nuclei analysis focused on end-stage pediatric HCM.

The Macrophage Landscape Across the Lifespan of a Human Cardiac Allograft.

BACKGROUND: Much of our knowledge of organ rejection after transplantation is derived from rodent models. METHODS: We used single-nucleus RNA sequencing to investigate the inflammatory myocardial microenvironment in human pediatric cardiac allografts at different stages after transplantation. We distinguished donor- from recipient-derived cells using naturally occurring genetic variants embedded in single-nucleus RNA sequencing data. RESULTS: Donor-derived tissue resident macrophages, which accompany the allograft into the recipient, are lost over time after transplantation. In contrast, monocyte-derived macrophages from the recipient populate the heart within days after transplantation and form 2 macrophage populations: recipient MP1 and recipient MP2. Recipient MP2s have cell signatures similar to donor-derived resident macrophages; however, they lack signatures of pro-reparative phagocytic activity typical of donor-derived resident macrophages and instead express profibrotic genes. In contrast, recipient MP1s express genes consistent with hallmarks of cellular rejection. Our data suggest that recipient MP1s activate a subset of natural killer cells, turning them into a cytotoxic cell population through feed-forward signaling between recipient MP1s and natural killer cells. CONCLUSIONS: Our findings reveal an imbalance of donor-derived and recipient-derived macrophages in the pediatric cardiac allograft that contributes to allograft failure.

Induction of a memory-like CD4+ T-cell phenotype by airway smooth muscle cells.

In asthma, CD4+ T-cell interaction with airway smooth muscle (ASM) may enhance its contractile properties and promote its proliferation. However, less is known about the effects of this interaction on T cells. To explore the consequences of interaction of CD4+ T cells with ASM we placed the cells in co-culture and analyzed the phenotypic and functional changes in the T cells. Effector status as well as cytokine expression was assessed by flow cytometry. An increase in CD45RA-CD45RO+ memory T cells was observed after co-culture; however, these cells were not more responsive to CD3/28 restimulation. A reduction in mitochondrial coupling and an increase in the production of mitochondrial reactive oxygen species by CD4+ T cells post-restimulation suggested altered mitochondrial metabolism after co-culture. RNA sequencing analysis of the T cells revealed characteristic downregulation of effector T-cell-associated genes, but a lack of upregulation of memory T-cell-associated genes. The results of this study demonstrate that ASM cells can induce a phenotypic shift in CD4+ T cells into memory-like T cells but with reduced capacity for activation.

Surveillance for Selected Pathogens and Parasites of Northern Bobwhite (Colinus virginianus) from Western Oklahoma, USA, 2018-20.

The Northern Bobwhite (Colinus virginianus) has been undergoing a range-wide population decline. Potential causes for declines across its historic range have been investigated for decades and include habitat loss and fragmentation and a variety of parasitic and infectious diseases. Although there have been studies on bobwhite ecology in Oklahoma, USA, relatively little is known about parasites and pathogens in the region. We evaluated the health of free-ranging bobwhites from nine sites in western Oklahoma. From 2018 to 2020, 206 bobwhites were evaluated for gross and microscopic lesions and tested for selected pathogens. In general, bobwhites were in good nutritional condition with ample muscle mass and fat stores. No significant gross lesions were observed in any bobwhite and no significant histologic lesions were detected in a subset. There was no evidence of infection with or exposure to reticuloendotheliosis virus, West Nile virus, respiratory Mycoplasmataceae species, Pasteurella multocida, intestinal Eimeria spp., or oral Trichomonas spp. Several pathogens of potential concern were detected, including avian adenovirus (8.6%), Toxoplasma gondii (2.3%), and haemosporidians (a Haemoproteus sp. (1.5%), Leucocytozoon schoutedeni (1.5%), and Plasmodium homopolare haplotype 2 [lineage LAIRI01; 3.6%]). Physaloptera sp. (12%) and Sarcocystis sp. (1%) were detected in the breast muscle. Low intraspecific genetic diversity was noted for Physaloptera sp., and sequences were most similar to Physaloptera sequences from bobwhites and grasshoppers (Orthoptera) in Texas. Low intensities of chewing lice, chiggers, and ticks were observed. A subset of bobwhites had evidence of exposure to selected toxicants and heavy metals; a small number had low levels of iron, manganese, zinc, molybdenum, and copper, which were not considered diagnostically relevant. In general, bobwhites from western Oklahoma appeared to be in good health with a low diversity of pathogens detected, but future work is needed to understand potentially changing disease risks for this population.

scGIST: gene panel design for spatial transcriptomics with prioritized gene sets.

A critical challenge of single-cell spatial transcriptomics (sc-ST) technologies is their panel size. Being based on fluorescence in situ hybridization, they are typically limited to panels of about a thousand genes. This constrains researchers to build panels from only the marker genes of different cell types and forgo other genes of interest, e.g., genes encoding ligand-receptor complexes or those in specific pathways. We propose scGIST, a constrained feature selection tool that designs sc-ST panels prioritizing user-specified genes without compromising cell type detection accuracy. We demonstrate scGIST's efficacy in diverse use cases, highlighting it as a valuable addition to sc-ST's algorithmic toolbox.

Buffering Mitigates Chondrocyte Oxidative Stress, Metabolic Dysfunction, and Death Induced by Normal Saline: Formulation of a Novel Arthroscopic Irrigant.

For decades, surgeons have utilized 0.9% normal saline (NS) for joint irrigation to improve visualization during arthroscopic procedures. This continues despite mounting evidence that NS exposure impairs chondrocyte metabolism and compromises articular cartilage function. We hypothesized that chondrocyte oxidative stress induced by low pH is the dominant factor driving NS toxicity, and that buffering NS to increase its pH would mitigate these effects. Effects on chondrocyte viability, reactive oxygen species (ROS) production, and overall metabolic function were assessed. Even brief exposure to NS caused cell death, ROS overproduction, and disruption of glycolysis, pentose phosphate, and tricarboxylic acid (TCA) cycle pathways. NS also stimulated ROS overproduction in synovial cells that could adversely alter the synovial function and subsequently the entire joint health. Buffering NS with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) significantly increased chondrocyte viability, reduced ROS production, and returned metabolite levels to near control levels while also reducing ROS production in synovial cells. These results confirm that chondrocytes and synoviocytes are vulnerable to insult from the acidic pH of NS and demonstrate that adding a buffering agent to NS averts many of its most harmful effects.

Single nucleus transcriptome of a "Super RV" shows increased insulin and angiogenesis signaling.

The right ventricle (RV) is one of the four pumping chambers of the heart, pumping blood to the lungs. In severe forms of congenital heart disease and pulmonary hypertension, the RV is made to pump into the systemic circulation. Such systemic RVs typically display early failure due to pressure overload. In rare cases a systemic RV persists into later decades of life - colloquially called a 'Super RV'. Here we present the single-nucleus transcriptome of a systemic RV from a 60-year-old with congenitally corrected transposition of great arteries (ccTGA). Our data shows two specific signaling pathways enriched in the ccTGA RV myocardium. First, we show increased insulin like growth factor (IGF1) signaling within the systemic RV myocardium: there is increased expression of the main receptor IGFR1 within the cardiomyocytes, and IGF1 ligands within the cardiofibroblasts and macrophages. Second, we find increased VEGF and Wnt9 ligand expression in cardiomyocytes and increased VEGF1R and Wnt9 receptors in endothelial cells, which are implicated in angiogenesis. We show that increased insulin and angiogenesis signaling are potentially beneficial RV adaptations to increased pressure overload. This study of an adult systemic RV provides an important framework for understanding RV remodeling to systemic pressures in congenital heart disease and pulmonary hypertension.

Increased iron uptake by splenic hematopoietic stem cells promotes TET2-dependent erythroid regeneration.

Hematopoietic stem cells (HSCs) are capable of regenerating the blood system, but the instructive cues that direct HSCs to regenerate particular lineages lost to the injury remain elusive. Here, we show that iron is increasingly taken up by HSCs during anemia and induces erythroid gene expression and regeneration in a Tet2-dependent manner. Lineage tracing of HSCs reveals that HSCs respond to hemolytic anemia by increasing erythroid output. The number of HSCs in the spleen, but not bone marrow, increases upon anemia and these HSCs exhibit enhanced proliferation, erythroid differentiation, iron uptake, and TET2 protein expression. Increased iron in HSCs promotes DNA demethylation and expression of erythroid genes. Suppressing iron uptake or TET2 expression impairs erythroid genes expression and erythroid differentiation of HSCs; iron supplementation, however, augments these processes. These results establish that the physiological level of iron taken up by HSCs has an instructive role in promoting erythroid-biased differentiation of HSCs.

CD4+ T cell-derived IFN-γ and LIGHT synergistically upregulate chemokine production from airway smooth muscle cells.

Airway smooth muscle (ASM) remodeling in asthmatic airways may contribute to persistent airflow limitation and airway hyperresponsiveness. CD4+ T cells infiltrate the ASM layer where they may induce a proliferative and secretory ASM cell phenotype. We studied the interaction between activated CD4+ T cells and ASM cells in co-culture in vitro and investigated the effects of CD4+ T cells on chemokine production by ASM cells. CD4+ T cells induced marked upregulation of C-X-C motif chemokine ligands (CXCL) 9, 10, and 11 in ASM cells. Blockade of the IFN-γ receptor on ASM cells prevented this upregulation. Furthermore, T cell-derived IFN-γ and LIGHT (lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes) synergize in a dose-dependent manner to coordinately enhance CXCL9, 10, and 11 expression. The synergistic property of LIGHT was mediated exclusively through the lymphotoxin-ß receptor (LTBR), but not herpes virus entry mediator (HVEM). Disruption of LTBR signaling in ASM cells reduced CXCL9, 10, and 11 production and ASM cell-mediated CD4+ T cell chemotaxis. We conclude that the LIGHT-LTBR signaling axis acts together with IFN-γ to regulate chemokines that mediate lymphocyte infiltration in asthmatics.

Implementation of clopidogrel pharmacogenetic clinical decision support for a preemptive return of results program.

PURPOSE: To describe our experiences implementing and iterating CYP2C19 genotype-guided clopidogrel pharmacogenetic clinical decision support (CDS) tools over time in the setting of a large health system-wide, preemptive pharmacogenomics program. SUMMARY: Clopidogrel-treated patients who are genetically predicted cytochrome P450 isozyme 2C19 (CYP2C19) intermediate or poor metabolizers have an increased risk of atherothrombotic events, some of which can be life-threatening. The Clinical Pharmacogenetics Implementation Consortium provides guidance for the use of clopidogrel based on CYP2C19 genotype in patients with cardiovascular and cerebrovascular diseases. Our multidisciplinary team implemented an automated, interruptive alert that fires when clopidogrel is ordered or refilled for biobank participants with structured CYP2C19 intermediate or poor metabolizer genomic indicators in the electronic health record. The implementation began with a narrow cardiovascular indication and setting and was then scaled in 4 primary dimensions: (1) clinical indication; (2) availability across health-system locations; (3) care venue (e.g., inpatient vs outpatient); and (4) provider groups (eg, cardiology and neurology). We iterated our approach over time based on evolving clinical evidence and proactive strategies to optimize CDS maintenance and sustainability. A key facilitator of expansion was socialization of the broader pharmacogenomics initiative among our academic medical center community, accompanied by clinician acceptance of pharmacogenetic alerts in practice. CONCLUSION: A multidisciplinary collaboration is recommended to facilitate the use of CYP2C19 genotype-guided antiplatelet therapy in patients with cardiovascular and cerebrovascular diseases. Evolving clopidogrel pharmacogenetic evidence necessitates thoughtful iteration of implementation efforts and strategies to optimize long-term maintenance and sustainability.