Pannexin 1 Channels Control Cardiomyocyte Metabolism and Neutrophil Recruitment During Non-Ischemic Heart Failure.

Pannexin 1 (PANX1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation, blood pressure regulation, and myocardial infarction. However, a possible role of PANX1 in cardiomyocytes in the progression of heart failure has not yet been investigated. We generated a novel mouse line with constitutive deletion of PANX1 in cardiomyocytes (Panx1 MyHC6 ). PANX1 deletion in cardiomyocytes had no effect on unstressed heart function but increased the glycolytic metabolism both in vivo and in vitro . In vitro , treatment of H9c2 cardiomyocytes with isoproterenol led to PANX1-dependent release of ATP and Yo-Pro-1 uptake, as assessed by pharmacological blockade with spironolactone and siRNA-mediated knock-down of PANX1. To investigate non-ischemic heart failure and the preceding cardiac hypertrophy we administered isoproterenol, and we demonstrate that Panx1 MyHC6 mice were protected from systolic and diastolic left ventricle volume increases and cardiomyocyte hypertrophy. Moreover, we found that Panx1 MyHC6 mice showed decreased isoproterenol-induced recruitment of immune cells (CD45 + ), particularly neutrophils (CD11b + , Ly6g + ), to the myocardium. Together these data demonstrate that PANX1 deficiency in cardiomyocytes impacts glycolytic metabolism and protects against cardiac hypertrophy in non-ischemic heart failure at least in part by reducing immune cell recruitment. Our study implies PANX1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in heart failure patients.

Cell cycle specific, differentially tagged ribosomal proteins to measure phase specific transcriptomes from asynchronously cycling cells.

Asynchronously cycling cells pose a challenge to the accurate characterization of phase-specific gene expression. Current strategies, including RNAseq, survey the steady state gene expression across the cell cycle and are inherently limited by their inability to resolve dynamic gene regulatory networks. Single cell RNAseq (scRNAseq) can identify different cell cycle transcriptomes if enough cycling cells are present, however some cells are not amenable to scRNAseq. Therefore, we merged two powerful strategies, the CDT1 and GMNN degrons used in Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) cell cycle sensors and the ribosomal protein epitope tagging used in RiboTrap/Tag technologies to isolate cell cycle phase-specific mRNA for sequencing. The resulting cell cycle dependent, tagged ribosomal proteins (ccTaggedRP) were differentially expressed during the cell cycle, had similar subcellular locations as endogenous ribosomal proteins, incorporated into ribosomes and polysomes, and facilitated the recovery of cell cycle phase-specific RNA for sequencing. ccTaggedRP has broad applications to investigate phase-specific gene expression in complex cell populations.

Comparison of Blood-Based Shotgun and Targeted Metagenomic Sequencing for Microbiological Diagnosis of Infective Endocarditis.

Background: Shotgun and targeted metagenomic sequencing have been shown in separate studies to be potentially useful for culture-free pathogen identification in blood and/or plasma of patients with infective endocarditis (IE). However, the 2 approaches have not been directly compared. The aim of this study was to compare shotgun metagenomic sequencing with targeted metagenomic sequencing (tMGS) for organism identification in blood or plasma of patients with IE. Methods: Patients with possible or definite IE were prospectively enrolled from October 2020 to July 2021. Shotgun metagenomic sequencing was performed with the Karius test, which uses microbial cell-free DNA (mcfDNA) sequencing to detect, identify, and quantitate DNA-based pathogens in plasma. tMGS was performed using a 16S ribosomal RNA (rRNA) polymerase chain reaction assay targeting the V1 to V3 regions of the 16S rRNA gene. Results were compared using the McNemar test of paired proportions. Results: Samples from 34 patients were investigated. The Karius test was positive in 24/34 (71%), including 3/6 (50%) with blood culture-negative endocarditis (BCNE), which was not significantly different from the positivity rate of tMGS (P = .41). Results of the Karius test were concordant with tMGS in 75% of cases. The Karius test detected 2 cases of methicillin-resistant Staphylococcus aureus among the 7 S. aureus detections, in accordance with results of phenotypic susceptibility testing. The combination of blood cultures, the Karius test, and tMGS found a potential causative pathogen in 33/34 (97%), including 5/6 with BCNE. Conclusions: The Karius test and tMGS yielded comparable detection rates; however, beyond organism identification, the Karius test generated potentially useful antibiotic resistance data.

Diagnostic Yield of 16S Ribosomal Ribonucleic Acid Gene-Based Targeted Metagenomic Sequencing for Evaluation of Pleural Space Infection: A Prospective Study.

Objective: To better understand the microbial profile of complicated parapneumonic effusions and empyema, and to evaluate whether antimicrobial selection would differ if guided by targeted metagenomic sequencing (tMGS) vs conventional cultures (CCs) alone. Patients and Methods: We analyzed the pleural fluid of a cohort of 47 patients undergoing thoracentesis from January 1, 2017 to August 31, 2019, to characterize their microbial profile. All samples underwent 16S ribosomal ribonucleic acid gene polymerase chain reaction, followed by tMGS. Results: Pleural space infection was deemed clinically present in 20 of the 47 (43%) participants. Of those, n=7 (35%) had positive pleural fluid cultures and n=14 (70%) had positive tMGS results. The organisms identified by tMGS were concordant with CCs; however, tMGS detected additional bacterial species over CCs alone. Streptococcus and Staphylococcus species were the most common organisms identified, with Streptococcus intermedius/constellatus identified in 5 patients. Polymicrobial infections were found in 6 of the 20 patients, with anaerobes being the most common organisms identified in these cases. Conclusion: Streptococci and staphylococci were the most common organisms identified in infected pleural fluid. Anaerobes were common in polymicrobial infections. When compared with CCs, tMGS had higher sensitivity than CCs. Targeted metagenomic sequencing identified additional organisms, not identified by CCs, with associated potential management implications.

Conserved chamber-specific polyploidy maintains heart function in Drosophila.

Developmentally programmed polyploidy (whole-genome duplication) of cardiomyocytes is common across evolution. Functions of such polyploidy are essentially unknown. Here, in both Drosophila larvae and human organ donors, we reveal distinct polyploidy levels in cardiac organ chambers. In Drosophila, differential growth and cell cycle signal sensitivity leads the heart chamber to reach a higher ploidy/cell size relative to the aorta chamber. Cardiac ploidy-reduced animals exhibit reduced heart chamber size, stroke volume and cardiac output, and acceleration of circulating hemocytes. These Drosophila phenotypes mimic human cardiomyopathies. Our results identify productive and likely conserved roles for polyploidy in cardiac chambers and suggest that precise ploidy levels sculpt many developing tissues. These findings of productive cardiomyocyte polyploidy impact efforts to block developmental polyploidy to improve heart injury recovery.

16S rRNA Gene PCR/Sequencing of Heart Valves for Diagnosis of Infective Endocarditis in Routine Clinical Practice.

Sequencing is increasingly used for infective endocarditis (IE) diagnosis. Here, the performance of 16S rRNA gene PCR/sequencing of heart valves utilized in routine clinical practice was compared with conventional IE diagnostics. Subjects whose heart valves were sent to the clinical microbiology laboratory for 16S rRNA gene PCR/sequencing from August 2020 through February 2022 were studied. A PCR assay targeting V1 to V3 regions of the 16S rRNA gene was performed, followed by Sanger and/or next-generation sequencing (NGS) (using an Illumina MiSeq), or reported as negative, depending on an algorithm that included the PCR cycle threshold value. Fifty-four subjects, including 40 with IE, three with cured IE, and 11 with noninfective valvular disease, were studied. Thirty-one positive results, 11 from NGS and 20 from Sanger sequencing, were generated from analysis of 16S rRNA gene sequence(s). Positivity rates of blood cultures and 16S rRNA gene PCR/sequencing of valves were 55% and 75%, respectively (P = 0.06). In those with prior antibiotic exposure, positivity rates of blood cultures and 16S rRNA gene PCR/sequencing of valves were 11% and 76%, respectively (P < 0.001). Overall, 61% of blood culture-negative IE subjects had positive valve 16S rRNA gene PCR/sequencing results. 16S rRNA gene-based PCR/sequencing of heart valves is a useful diagnostic tool for pathogen identification in patients with blood culture-negative IE undergoing valve surgery in routine clinical practice.

The Microenvironment of the Pathogenesis of Cardiac Hypertrophy.

Pathological cardiac hypertrophy is a key risk factor for the development of heart failure and predisposes individuals to cardiac arrhythmia and sudden death. While physiological cardiac hypertrophy is adaptive, hypertrophy resulting from conditions comprising hypertension, aortic stenosis, or genetic mutations, such as hypertrophic cardiomyopathy, is maladaptive. Here, we highlight the essential role and reciprocal interactions involving both cardiomyocytes and non-myocardial cells in response to pathological conditions. Prolonged cardiovascular stress causes cardiomyocytes and non-myocardial cells to enter an activated state releasing numerous pro-hypertrophic, pro-fibrotic, and pro-inflammatory mediators such as vasoactive hormones, growth factors, and cytokines, i.e., commencing signaling events that collectively cause cardiac hypertrophy. Fibrotic remodeling is mediated by cardiac fibroblasts as the central players, but also endothelial cells and resident and infiltrating immune cells enhance these processes. Many of these hypertrophic mediators are now being integrated into computational models that provide system-level insights and will help to translate our knowledge into new pharmacological targets. This perspective article summarizes the last decades' advances in cardiac hypertrophy research and discusses the herein-involved complex myocardial microenvironment and signaling components.

Obesity-Induced Coronary Microvascular Disease Is Prevented by iNOS Deletion and Reversed by iNOS Inhibition.

Coronary microvascular disease (CMD) caused by obesity and diabetes is major contributor to heart failure with preserved ejection fraction; however, the mechanisms underlying CMD are not well understood. Using cardiac magnetic resonance applied to mice fed a high-fat, high-sucrose diet as a model of CMD, we elucidated the role of inducible nitric oxide synthase (iNOS) and 1400W, an iNOS antagonist, in CMD. Global iNOS deletion prevented CMD along with the associated oxidative stress and diastolic and subclinical systolic dysfunction. The 1400W treatment reversed established CMD and oxidative stress and preserved systolic/diastolic function in mice fed a high-fat, high-sucrose diet. Thus, iNOS may represent a therapeutic target for CMD.

Molecular and cellular evidence for the impact of a hypertrophic cardiomyopathy-associated RAF1 variant on the structure and function of contractile machinery in bioartificial cardiac tissues.

Noonan syndrome (NS), the most common among RASopathies, is caused by germline variants in genes encoding components of the RAS-MAPK pathway. Distinct variants, including the recurrent Ser257Leu substitution in RAF1, are associated with severe hypertrophic cardiomyopathy (HCM). Here, we investigated the elusive mechanistic link between NS-associated RAF1S257L and HCM using three-dimensional cardiac bodies and bioartificial cardiac tissues generated from patient-derived induced pluripotent stem cells (iPSCs) harboring the pathogenic RAF1 c.770 C > T missense change. We characterize the molecular, structural, and functional consequences of aberrant RAF1-associated signaling on the cardiac models. Ultrastructural assessment of the sarcomere revealed a shortening of the I-bands along the Z disc area in both iPSC-derived RAF1S257L cardiomyocytes and myocardial tissue biopsies. The aforementioned changes correlated with the isoform shift of titin from a longer (N2BA) to a shorter isoform (N2B) that also affected the active force generation and contractile tensions. The genotype-phenotype correlation was confirmed using cardiomyocyte progeny of an isogenic gene-corrected RAF1S257L-iPSC line and was mainly reversed by MEK inhibition. Collectively, our findings uncovered a direct link between a RASopathy gene variant and the abnormal sarcomere structure resulting in a cardiac dysfunction that remarkably recapitulates the human disease.

Ionic Accumulation as a Diagnostic Tool in Perovskite Solar Cells: Characterizing Band Alignment with Rapid Voltage Pulses.

Despite record-breaking devices, interfaces in perovskite solar cells are still poorly understood, inhibiting further progress. Their mixed ionic-electronic nature results in compositional variations at the interfaces, depending on the history of externally applied biases. This makes it difficult to measure the band energy alignment of charge extraction layers accurately. As a result, the field often resorts to a trial-and-error process to optimize these interfaces. Current approaches are typically carried out in a vacuum and on incomplete cells, hence values may not reflect those found in working devices. To address this, a pulsed measurement technique characterizing the electrostatic potential energy drop across the perovskite layer in a functioning device is developed. This method reconstructs the current-voltage (JV) curve for a range of stabilization biases, holding the ion distribution "static" during subsequent rapid voltage pulses. Two different regimes are observed: at low biases, the reconstructed JV curve is "s-shaped", whereas, at high biases, typical diode-shaped curves are returned. Using drift-diffusion simulations, it is demonstrated that the intersection of the two regimes reflects the band offsets at the interfaces. This approach effectively allows measurements of interfacial energy level alignment in a complete device under illumination and without the need for expensive vacuum equipment.

Conserved Chamber-Specific Polyploidy Maintains Heart Function in Drosophila.

Developmentally programmed polyploidy (whole-genome-duplication) of cardiomyocytes is common across evolution. Functions of such polyploidy are essentially unknown. Here, we reveal roles for precise polyploidy levels in cardiac tissue. We highlight a conserved asymmetry in polyploidy level between cardiac chambers in Drosophila larvae and humans. In Drosophila , differential Insulin Receptor (InR) sensitivity leads the heart chamber to reach a higher ploidy/cell size relative to the aorta chamber. Cardiac ploidy-reduced animals exhibit reduced heart chamber size, stroke volume, cardiac output, and acceleration of circulating hemocytes. These Drosophila phenotypes mimic systemic human heart failure. Using human donor hearts, we reveal asymmetry in nuclear volume (ploidy) and insulin signaling between the left ventricle and atrium. Our results identify productive and likely conserved roles for polyploidy in cardiac chambers and suggest precise ploidy levels sculpt many developing tissues. These findings of productive cardiomyocyte polyploidy impact efforts to block developmental polyploidy to improve heart injury recovery.

Opposing effects of genetic variation in MTCH2 for obesity versus heart failure.

Genetic variation in genes regulating metabolism may be advantageous in some settings but not others. The non-failing adult heart relies heavily on fatty acids as a fuel substrate and source of ATP. In contrast, the failing heart favors glucose as a fuel source. A bootstrap analysis for genes with deviant allele frequencies in cardiomyopathy cases versus controls identified the MTCH2 gene as having unusual variation. MTCH2 encodes an outer mitochondrial membrane protein, and prior genome-wide studies associated MTCH2 variants with body mass index, consistent with its role in metabolism. We identified the referent allele of rs1064608 (p.Pro290) as being overrepresented in cardiomyopathy cases compared to controls, and linkage disequilibrium analysis associated this variant with the MTCH2 cis eQTL rs10838738 and lower MTCH2 expression. To evaluate MTCH2, we knocked down Mtch in Drosophila heart tubes which produced a dilated and poorly functioning heart tube, reduced adiposity and shortened life span. Cardiac Mtch mutants generated more lactate at baseline, and they displayed impaired oxygen consumption in the presence of glucose but not palmitate. Treatment of cardiac Mtch mutants with dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, reduced lactate and rescued lifespan. Deletion of MTCH2 in human cells similarly impaired oxygen consumption in the presence of glucose but not fatty acids. These data support a model in which MTCH2 reduction may be favorable when fatty acids are the major fuel source, favoring lean body mass. However, in settings like heart failure, where the heart shifts toward using more glucose, reduction of MTCH2 is maladaptive.

Targeted Versus Shotgun Metagenomic Sequencing-based Detection of Microorganisms in Sonicate Fluid for Periprosthetic Joint Infection Diagnosis.

BACKGROUND: Next-generation sequencing (NGS) is increasingly used for periprosthetic joint infection (PJI) diagnosis, but its clinical utility is poorly defined. Shotgun metagenomic sequencing (sNGS) has been reported to identify PJI pathogens undetected by culture in sonicate fluid. However, sNGS is complex and costly. Here, 16S ribosomal RNA (rRNA) gene-based targeted metagenomic sequencing (tNGS) was compared to sNGS of sonicate fluid for microbial detection and identification in patients with total hip arthroplasty (THA) and total knee arthroplasty (TKA) failure. METHODS: A convenience sample of sonicate fluids derived from patients who had undergone THA or TKA removal, enriched with culture negative PJI cases, was tested. Samples had been previously tested by sNGS. For tNGS, samples were extracted, amplified by polymerase chain reaction targeting the V1 to V3 regions of the 16S rRNA gene, and sequenced on an Illumina MiSeq. RESULTS: A total of 395 sonicate fluids, including 208 from subjects with PJI, were studied. Compared with sonicate fluid culture, tNGS had higher positive percent agreement (72.1 vs 52.9%, P < .001), detecting potential pathogens in 48.0% of culture-negative PJIs. There was no difference between the positive percent agreement of tNGS (72.1%) and sNGS (73.1%, P = .83). CONCLUSIONS: 16S rRNA gene-based tNGS is a potential diagnostic tool for PJI pathogen identification in sonicate fluid from failed THAs and TKAs in culture-negative cases, with similar performance characteristics to sNGS.

Caution! Static Supercell Calculations of Defect Migration in Higher Symmetry ABX3 Perovskite Halides May Be Unreliable: A Case Study of Methylammonium Lead Iodide.

Activation energies of defect migration in ABX3 perovskite halides are widely obtained through static supercell calculations with the nudged-elastic-band method. Taking methylammonium lead iodide (CH3NH3PbI3, MAPbI3) as an example, we demonstrate that such calculations are unreliable for the higher symmetry structures adopted by the material at temperatures relevant to device operation (tetragonal and cubic MAPbI3) because, in addition to ion relaxation around the point defects, local structural modifications characteristic of the ground-state (orthorhombic) structure occur. In this way, we offer a simple explanation of why calculated activation energies of defect migration in MAPbI3 suffer from surprisingly large scatter. We propose a robust test to determine whether static supercell calculations of point-defect processes in ABX3 perovskite systems are reliable.

Clinical Use of a 16S Ribosomal RNA Gene-Based Sanger and/or Next Generation Sequencing Assay to Test Preoperative Synovial Fluid for Periprosthetic Joint Infection Diagnosis.

Preoperative pathogen identification in patients with periprosthetic joint infection (PJI) is typically limited to synovial fluid culture. Whether sequencing-based approaches are of potential use in identification of pathogens in PJI, and if so which approach is ideal, is incompletely defined. The objective of the study was to analyze the accuracy of a 16S rRNA (rRNA) gene-based PCR followed by Sanger sequencing and/or targeted metagenomic sequencing approach (tMGS) performed on synovial fluid for PJI diagnosis. A retrospective study was conducted, analyzing synovial fluids tested between August 2020 and May 2021 at a single center. Subjects with hip, knee, shoulder, and elbow arthroplasties who had synovial fluid aspirated and clinically subjected to sequence-based testing and conventional culture were studied. A total of 154 subjects were included in the study; 118 had noninfectious arthroplasty failure (NIAF), while 36 had PJI. Clinical sensitivity and specificity for diagnosis of PJI were 69% and 100%, respectively, for the sequencing-based approach and 72% and 100%, respectively, for conventional culture (P = 0.74). The combination of both tests was more sensitive (83%) than culture alone (P = 0.04). Results of sequencing-based testing led to changes in treatment in four of 36 (11%) PJI subjects. Microbial identification was achieved using Sanger and next generation sequencing in 19 and 6 subjects, respectively. When combined with culture, the described 16S rRNA gene sequencing-based approach increased sensitivity compared to culture alone, suggesting its potential use in the diagnosis of PJI when synovial fluid culture is negative. IMPORTANCE Periprosthetic joint infection (PJI) is a dreadful complication of joint replacement. Noninvasive identification of infectious pathogens has been traditionnally limited to culture-based testing of synovial fluid which has poor sensitivity. Sanger and Next-generation sequencing (NGS) may be used for synovial fluid testing in PJI, but experience in routine practice is sparse. We used a targeted metagenomic sequencing approach for routine testing of synovial fluid involving NGS when Sanger sequencing had failed or was likely to fail. The objective of this study was to analyze the approach's performance for diagnosis of PJI in comparison to culture for testing synovial fluid. Overall, the sequencing-based approach was not superior to culture for diagnosis of PJI, but yielded positive results in some culture-negative samples.

Comparison of the BioFire Joint Infection Panel to 16S Ribosomal RNA Gene-Based Targeted Metagenomic Sequencing for Testing Synovial Fluid from Patients with Knee Arthroplasty Failure.

The diagnosis of periprosthetic joint infection (PJI) is challenging, often requiring multiple clinical specimens and diagnostic techniques, some with prolonged result turnaround times. Here, the diagnostic performance of the Investigational Use Only (IUO) BioFire Joint Infection (JI) Panel was compared to 16S rRNA gene-based targeted metagenomic sequencing (tMGS) applied to synovial fluid for PJI diagnosis. Sixty synovial fluid samples from knee arthroplasty failure archived at -80°C were tested. Infectious Diseases Society of America (IDSA) diagnostic criteria were used to classify PJI. For culture-positive PJI with pathogens targeted by the JI panel, JI panel sensitivity was 91% (21/23; 95% confidence interval [CI], 73 to 98%), and tMGS sensitivity was 96% (23/24; 95% CI, 80 to 99%) (P = 0.56). Overall sensitivities of the JI panel and tMGS for PJI diagnosis were 56% (24/43; 95% CI, 41 to 70%) and 93% (41/44; 95% CI, 82 to 98%), respectively (P < 0.001). JI panel and tMGS overall specificities were 100% (16/16; 95% CI, 81 to 100%) and 94% (15/16; 95% CI, 72 to 99%), respectively. While the clinical sensitivity of the JI panel was excellent for on-panel microorganisms, overall sensitivity for PJI diagnosis was low due to the absence of Staphylococcus epidermidis, a common causative pathogen of PJI, on the panel. A PJI diagnostic algorithm for the use of both molecular tests is proposed.

Pathogen Detection in Infective Endocarditis Using Targeted Metagenomics on Whole Blood and Plasma: a Prospective Pilot Study.

Initial microbiologic diagnosis of infective endocarditis (IE) relies on blood cultures and Bartonella and Coxiella burnetii serology. Small case series and one prospective study have preliminarily reported application of metagenomic sequencing on blood or plasma for IE diagnosis. Here, results of a prospective pilot study evaluating targeted metagenomic sequencing (tMGS) for blood-based early pathogen detection and identification in IE are reported. Subjects diagnosed with possible or definite IE at a single institution were prospectively enrolled with informed consent from October 2020 to July 2021. Blood was drawn and separated into whole blood and plasma. Both specimen types were subjected to nucleic acid extraction and PCR targeting the V1-V3 region of the 16S ribosomal RNA gene, followed by next-generation sequencing on an Illumina MiSeqTM platform. 35 subjects, 28 (80%) with definite and 7 (20%) with possible IE were enrolled, including 6 (17%) with blood culture-negative endocarditis (BCNE). Overall, 20 whole blood (59%) and 16 plasma (47%) samples tested positive (P = 0.47). When results of whole blood and plasma testing were combined, a positive tMGS result was found in 23 subjects (66%). tMGS identified a potential pathogen in 5 of 6 culture-negative IE cases. Although further study is needed, the results of this pilot study suggest that blood-based tMGS may provide pathogen identification in subjects with IE, including in culture-negative cases.

Accelerated fatty acid composition MRI of epicardial adipose tissue: Development and application to eplerenone treatment in a mouse model of obesity-induced coronary microvascular disease.

PURPOSE: To develop an accelerated MRI method to quantify the epicardial adipose tissue (EAT) fatty acid composition (FAC) and test the hypothesis that eplerenone (EPL) shifts the EAT FAC toward unsaturation in obese mice. METHODS: Undersampled multi-echo gradient echo imaging employing a dictionary-based compressed-sensing reconstruction and iterative decomposition with echo asymmetry and least-squares-based mapping (IDEAL) was developed, validated, and used to study EAT in obese mice scanned at 7T. Fully sampled and rate 2, 2.5, 3, and 3.5 undersampled image data were acquired, reconstructed, and assessed using RMSE and structural similarity (SSIM). Two groups of mice were studied: untreated (control, n = 10) and EPL-treated (n = 10) mice fed a high-fat high-sucrose diet. MRI included imaging of EAT FAC, EAT volume, and myocardial perfusion reserve. RESULTS: Rate 3 acceleration provided RMSE <5% and structural similarity >0.85 for FAC MRI. After 6 weeks of diet, EPL-treated compared to untreated mice had a reduced EAT saturated fatty acid fraction (0.27 ± 0.09 vs. 0.39 ± 0.07, P < 0.05) and increased EAT unsaturation degree (4.37 ± 0.32 vs. 3.69 ± 0.58, P < 0.05). Also, EAT volume in EPL-treated compared to untreated mice was reduced (8.1 ± 0.6 mg vs. 11.4 ± 0.7 mg, P < 0.01), and myocardial perfusion reserve was improved (1.83 ± 0.15 vs. 1.61 ± 0.17, P < 0.05). CONCLUSION: Rate 3 accelerated FAC MRI enabled accurate quantification of EAT FAC in mice. EPL treatment shifted the EAT FAC toward increased unsaturation and was associated with improvement of coronary microvascular function.

Inhibition of DYRK1a Enhances Cardiomyocyte Cycling After Myocardial Infarction.

BACKGROUND: DYRK1a (dual-specificity tyrosine phosphorylation-regulated kinase 1a) contributes to the control of cycling cells, including cardiomyocytes. However, the effects of inhibition of DYRK1a on cardiac function and cycling cardiomyocytes after myocardial infarction (MI) remain unknown. METHODS: We investigated the impacts of pharmacological inhibition and conditional genetic ablation of DYRK1a on endogenous cardiomyocyte cycling and left ventricular systolic function in ischemia-reperfusion (I/R) MI using αMHC-MerDreMer-Ki67p-RoxedCre::Rox-Lox-tdTomato-eGFP (RLTG) (denoted αDKRC::RLTG) and αMHC-Cre::Fucci2aR::DYRK1aflox/flox mice. RESULTS: We observed that harmine, an inhibitor of DYRK1a, improved left ventricular ejection fraction (39.5±1.6% and 29.1±1.6%, harmine versus placebo, respectively), 2 weeks after I/R MI. Harmine also increased cardiomyocyte cycling after I/R MI in αDKRC::RLTG mice, 10.8±1.5 versus 24.3±2.6 enhanced Green Fluorescent Protein (eGFP)+ cardiomyocytes, placebo versus harmine, respectively, P=1.0×10-3. The effects of harmine on left ventricular ejection fraction were attenuated in αDKRC::DTA mice that expressed an inducible diphtheria toxin in adult cycling cardiomyocytes. The conditional cardiomyocyte-specific genetic ablation of DYRK1a in αMHC-Cre::Fucci2aR::DYRK1aflox/flox (denoted DYRK1a k/o) mice caused cardiomyocyte hyperplasia at baseline (210±28 versus 126±5 cardiomyocytes per 40× field, DYRK1a k/o versus controls, respectively, P=1.7×10-2) without changes in cardiac function compared with controls, or compensatory changes in the expression of other DYRK isoforms. After I/R MI, DYRK1a k/o mice had improved left ventricular function (left ventricular ejection fraction 41.8±2.2% and 26.4±0.8%, DYRK1a k/o versus control, respectively, P=3.7×10-2). RNAseq of cardiomyocytes isolated from αMHC-Cre::Fucci2aR::DYRK1aflox/flox and αMHC-Cre::Fucci2aR mice after I/R MI or Sham surgeries identified enrichment in mitotic cell cycle genes in αMHC-Cre::Fucci2aR::DYRK1aflox/flox compared with αMHC-Cre::Fucci2aR. CONCLUSIONS: The pharmacological inhibition or cardiomyocyte-specific ablation of DYRK1a caused baseline hyperplasia and improved cardiac function after I/R MI, with an increase in cell cycle gene expression, suggesting the inhibition of DYRK1a may serve as a therapeutic target to treat MI.

Targeted Metagenomic Sequencing-based Approach Applied to 2146 Tissue and Body Fluid Samples in Routine Clinical Practice.

BACKGROUND: The yield of next-generation sequencing (NGS) added to a Sanger sequencing-based 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR) assay was evaluated in clinical practice for diagnosis of bacterial infection. METHODS: PCR targeting the V1 to V3 regions of the 16S rRNA gene was performed, with amplified DNA submitted to Sanger sequencing and/or NGS (Illumina MiSeq) or reported as negative, depending on the cycle threshold value. A total of 2146 normally sterile tissues or body fluids were tested between August 2020 and March 2021. Clinical sensitivity was assessed in 579 patients from whom clinical data were available. RESULTS: Compared with Sanger sequencing alone (400 positive tests), positivity increased by 87% by adding NGS (347 added positive tests). Clinical sensitivity of the assay that incorporated NGS was 53%, which was higher than culture (42%, P < .001), with an impact on clinical decision-making in 14% of infected cases. Clinical sensitivity in the subgroup that received antibiotics at sampling was 41% for culture and 63% for the sequencing assay (P < .001). CONCLUSIONS: Adding NGS to Sanger sequencing of the PCR-amplified 16S rRNA gene substantially improved test positivity. In the patient population studied, the assay was more sensitive than culture, especially in patients who had received antibiotic therapy.