The Detection of Periodic Reemergence Events of SARS-CoV-2 Delta Strain in Communities Dominated by Omicron.

Despite entering an endemic phase, SARS-CoV-2 remains a significant burden to public health across the global community. Wastewater sampling has consistently proven utility to understanding SARS-CoV-2 prevalence trends and genetic variation as it represents a less biased assessment of the corresponding communities. Here, we report that ongoing monitoring of SARS-CoV-2 genetic variation in samples obtained from the wastewatersheds of the city of Louisville in Jefferson county Kentucky has revealed the periodic reemergence of the Delta strain in the presence of the presumed dominant Omicron strain. Unlike previous SARS-CoV-2 waves/emergence events, the Delta reemergence events were geographically restricted in the community and failed to spread into other areas as determined by wastewater analyses. Moreover, the reemergence of the Delta strain did not correlate with vaccination rates as communities with lower relative vaccination have been, to date, not affected. Importantly, Delta reemergence events correlate with increased public health burdens, as indicated by increased daily case rates and mortality relative to non-Delta wastewatershed communities. While the underlying reasons for the reemergence of the Delta variant remain unclear, these data reaffirm the ongoing importance of wastewater genomic analyses towards understanding SARS-CoV-2 as it enters the endemic phase.

Combining Community Wastewater Genomic Surveillance with State Clinical Surveillance: A Framework for SARS-CoV-2 Public Health Practice.

This study aimed to develop a framework for combining community wastewater surveillance with state clinical surveillance for the confirmation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants within the community and to provide recommendations on how to expand on such research and apply the findings in public health responses. Wastewater samples were collected weekly from 17 geographically resolved locations in Louisville/Jefferson County, Kentucky (USA), from February 10 to December 13, 2021. Genomic surveillance and quantitative reverse transcription PCR (RT-qPCR) platforms were used to screen for SARS-CoV-2 in wastewater, and state clinical surveillance was used for confirmation. The study results highlighted an increased epidemiological value of combining community wastewater genomic surveillance and RT-qPCR with conventional case-auditing methods. The spatial scale and temporal frequency of wastewater sampling provided promising sensitivity and specificity for gaining public health screening insights about SARS-CoV-2 emergence, seeding, and spread in communities. Improved national surveillance systems are needed against future pathogens and variants, and wastewater-based genomic surveillance exhibits great potential when coupled with clinical testing. This paper presents evidence that complementary wastewater and clinical testing are cost-effectively enhanced when used in combination, as they provide a strong tool for a joint public health framework. Future pathogens of interest may be examined in either a targeted fashion or using a more global approach where all pathogens are monitored. This study has also provided novel insights developed from evidence-based public health practices.

Diversification of Potassium Currents in Excitable Cells via Kvß Proteins.

Excitable cells of the nervous and cardiovascular systems depend on an assortment of plasmalemmal potassium channels to control diverse cellular functions. Voltage-gated potassium (Kv) channels are central to the feedback control of membrane excitability in these processes due to their activation by depolarized membrane potentials permitting K+ efflux. Accordingly, Kv currents are differentially controlled not only by numerous cellular signaling paradigms that influence channel abundance and shape voltage sensitivity, but also by heteromeric configurations of channel complexes. In this context, we discuss the current knowledge related to how intracellular Kvß proteins interacting with pore complexes of Shaker-related Kv1 channels may establish a modifiable link between excitability and metabolic state. Past studies in heterologous systems have indicated roles for Kvß proteins in regulating channel stability, trafficking, subcellular targeting, and gating. More recent works identifying potential in vivo physiologic roles are considered in light of these earlier studies and key gaps in knowledge to be addressed by future research are described.

Rigor Me This: What Are the Basic Criteria for a Rigorous, Transparent, and Reproducible Scientific Study?

Scientific advancement is predicated upon the ability of a novel discovery to be independently reproduced and substantiated by others. Despite this inherent necessity, the research community is awash in published studies that cannot be replicated resulting in widespread confusion within the field and waning trust from the general public. In many cases, irreproducibility is the unavoidable consequence of a study that is conducted without the appropriate degree of rigor, typified by fundamental flaws in approach, design, execution, analysis, interpretation, and reporting. Combatting the irreproducibility pandemic in preclinical research is of urgent concern and is the primary responsibility of individual investigators, however there are important roles to be played by institutions, journals, government entities, and funding agencies as well. Herein, we provide an updated review of established rigor criteria pertaining to both in vitro and in vivo studies compiled from multiple sources across the research enterprise and present a practical checklist as a straightforward reference guide. It is our hope that this review may serve as an approachable resource for early career and experienced investigators alike, as they strive to improve all aspects of their scientific endeavors.

Circulating MicroRNA-122-5p Is Associated With a Lack of Improvement in Left Ventricular Function After Transcatheter Aortic Valve Replacement and Regulates Viability of Cardiomyocytes Through Extracellular Vesicles.

BACKGROUND: Transcatheter aortic valve replacement (TAVR) is a well-established treatment option for high- and intermediate-risk patients with severe symptomatic aortic valve stenosis. A majority of patients exhibit improvements in left ventricular ejection fraction (LVEF) after TAVR in response to TAVR-associated afterload reduction. However, a specific role for circulating microRNAs (miRNAs) in the improvement of cardiac function for patients after TAVR has not yet been investigated. Here, we profiled the differential expression of miRNAs in circulating extracellular vesicles (EVs) in patients after TAVR and, in particular, the novel role of circulating miR-122-5p in cardiomyocytes. METHODS: Circulating EV-associated miRNAs were investigated by use of an unbiased Taqman-based human miRNA array. Several EV miRNAs (miR-122-5p, miR-26a, miR-192, miR-483-5p, miR-720, miR-885-5p, and miR-1274) were significantly deregulated in patients with aortic valve stenosis at day 7 after TAVR compared with the preprocedural levels in patients without LVEF improvement. The higher levels of miR-122-5p were negatively correlated with LVEF improvement at both day 7 (r=-0.264 and P=0.015) and 6 months (r=-0.328 and P=0.0018) after TAVR. RESULTS: Using of patient-derived samples and a murine aortic valve stenosis model, we observed that the expression of miR-122-5p correlates negatively with cardiac function, which is associated with LVEF. Mice with graded wire injury-induced aortic valve stenosis demonstrated a higher level of miR-122-5p, which was related to cardiomyocyte dysfunction. Murine ex vivo experiments revealed that miR-122-5p is highly enriched in endothelial cells compared with cardiomyocytes. Coculture experiments, copy-number analysis, and fluorescence microscopy with Cy3-labeled miR-122-5p demonstrated that miR-122-5p can be shuttled through large EVs from endothelial cells into cardiomyocytes. Gain- and loss-of-function experiments suggested that EV-mediated shuttling of miR-122-5p increases the level of miR-122-5p in recipient cardiomyocytes. Mechanistically, mass spectrometry, miRNA pulldown, electrophoretic mobility shift assay, and RNA immunoprecipitation experiments confirmed that miR-122-5p interacts with the RNA-binding protein hnRNPU (heterogeneous nuclear ribonucleoprotein U) in a sequence-specific manner to encapsulate miR-122-5p into large EVs. On shuttling, miR-122-5p reduces the expression of the antiapoptotic gene BCL2 by binding to its 3' untranslated region to inhibit its translation, thereby decreasing the viability of target cardiomyocytes. CONCLUSIONS: Increased levels of circulating proapoptotic EV-incorporated miR-122-5p are associated with reduced LVEF after TAVR. EV shuttling of miR-122-5p regulates the viability and apoptosis of cardiomyocytes in a BCL2-dependent manner.

Pyridine nucleotide redox potential in coronary smooth muscle couples myocardial blood flow to cardiac metabolism.

Adequate oxygen delivery to the heart during stress is essential for sustaining cardiac function. Acute increases in myocardial oxygen demand evoke coronary vasodilation and enhance perfusion via functional upregulation of smooth muscle voltage-gated K+ (Kv) channels. Because this response is controlled by Kv1 accessory subunits (i.e., Kvß), which are NAD(P)(H)-dependent aldo-keto reductases, we tested the hypothesis that oxygen demand modifies arterial [NAD(H)]i, and that resultant cytosolic pyridine nucleotide redox state influences Kv1 activity. High-resolution imaging mass spectrometry and live-cell imaging reveal cardiac workload-dependent increases in NADH:NAD+ in intramyocardial arterial myocytes. Intracellular NAD(P)(H) redox ratios reflecting elevated oxygen demand potentiate native coronary Kv1 activity in a Kvß2-dependent manner. Ablation of Kvß2 catalysis suppresses redox-dependent increases in Kv1 activity, vasodilation, and the relationship between cardiac workload and myocardial blood flow. Collectively, this work suggests that the pyridine nucleotide sensitivity and enzymatic activity of Kvß2 controls coronary vasoreactivity and myocardial blood flow during metabolic stress.

Collagen type XIX regulates cardiac extracellular matrix structure and ventricular function.

The cardiac extracellular matrix plays essential roles in homeostasis and injury responses. Although the role of fibrillar collagens have been thoroughly documented, the functions of non-fibrillar collagen members remain underexplored. These include a distinct group of non-fibrillar collagens, termed, fibril-associated collagens with interrupted triple helices (FACITs). Recent reports of collagen type XIX (encoded by Col19a1) expression in adult heart and evidence of its enhanced expression in cardiac ischemia suggest important functions for this FACIT in cardiac ECM structure and function. Here, we examined the cellular source of collagen XIX in the adult murine heart and evaluated its involvement in ECM structure and ventricular function. Immunodetection of collagen XIX in fractionated cardiovascular cell lineages revealed fibroblasts and smooth muscle cells as the primary sources of collagen XIX in the heart. Based on echocardiographic and histologic analyses, Col19a1 null (Col19a1N/N) mice exhibited reduced systolic function, thinning of left ventricular walls, and increased cardiomyocyte cross-sectional areas-without gross changes in myocardial collagen content or basement membrane morphology. Col19a1N/N cardiac fibroblasts had augmented expression of several enzymes involved in the synthesis and stability of fibrillar collagens, including PLOD1 and LOX. Furthermore, second harmonic generation-imaged ECM derived from Col19a1N/N cardiac fibroblasts, and transmission electron micrographs of decellularized hearts from Col19a1N/N null animals, showed marked reductions in fibrillar collagen structural organization. Col19a1N/N mice also displayed enhanced phosphorylation of focal adhesion kinase (FAK), signifying de-repression of the FAK pathway-a critical mediator of cardiomyocyte hypertrophy. Collectively, we show that collagen XIX, which had a heretofore unknown role in the mammalian heart, participates in the regulation of cardiac structure and function-potentially through modulation of ECM fibrillar collagen structural organization. Further, these data suggest that this FACIT may modify ECM superstructure via acting at the level of the fibroblast to regulate their expression of collagen synthetic and stabilization enzymes.

Myocardial Blood Flow Control by Oxygen Sensing Vascular Kvß Proteins.

[Figure: see text].

Administration of cardiac mesenchymal cells modulates innate immunity in the acute phase of myocardial infarction in mice.

Although cardiac mesenchymal cell (CMC) therapy mitigates post-infarct cardiac dysfunction, the underlying mechanisms remain unidentified. It is acknowledged that donor cells are neither appreciably retained nor meaningfully contribute to tissue regeneration-suggesting a paracrine-mediated mechanism of action. As the immune system is inextricably linked to wound healing/remodeling in the ischemically injured heart, the reparative actions of CMCs may be attributed to their immunoregulatory properties. The current study evaluated the consequences of CMC administration on post myocardial infarction (MI) immune responses in vivo and paracrine-mediated immune cell function in vitro. CMC administration preferentially elicited the recruitment of cell types associated with innate immunity (e.g., monocytes/macrophages and neutrophils). CMC paracrine signaling assays revealed enhancement in innate immune cell chemoattraction, survival, and phagocytosis, and diminished pro-inflammatory immune cell activation; data that identifies and catalogues fundamental immunomodulatory properties of CMCs, which have broad implications regarding the mechanism of action of CMCs in cardiac repair.

Macrophage Long Non-Coding RNAs in Pathogenesis of Cardiovascular Disease.

Chronic inflammation is inextricably linked to cardiovascular disease (CVD). Macrophages themselves play important roles in atherosclerosis, as well as acute and chronic heart failure. Although the role of macrophages in CVD pathophysiology is well-recognized, little is known regarding the precise mechanisms influencing their function in these contexts. Long non-coding RNAs (lncRNAs) have emerged as significant regulators of macrophage function; as such, there is rising interest in understanding how these nucleic acids influence macrophage signaling, cell fate decisions, and activity in health and disease. In this review, we summarize current knowledge regarding lncRNAs in directing various aspects of macrophage function in CVD. These include foam cell formation, Toll-like receptor (TLR) and NF-kß signaling, and macrophage phenotype switching. This review will provide a comprehensive understanding concerning previous, ongoing, and future studies of lncRNAs in macrophage functions and their importance in CVD.

Functional characterization of long noncoding RNAs.

PURPOSE OF REVIEW: Mounting evidence suggests that long noncoding RNAs (lncRNAs) are essential regulators of gene expression. Although few lncRNAs have been the subject of detailed molecular and functional characterization, it is believed that lncRNAs play an important role in tissue homeostasis and development. In fact, gene expression profiling studies reveal lncRNAs are developmentally regulated in a tissue-type and cell-type specific manner. Such findings have brought significant attention to their potential contribution to disease cause. The current review summarizes recent studies of lncRNAs in the heart. RECENT FINDINGS: lncRNA discovery has largely been driven by the implementation of next generation sequencing technologies. To date, such technologies have contributed to the identification of tens of thousands of distinct lncRNAs in humans -- accounting for a large majority of all RNA sequences transcribed across the human genome. Although the functions of these lncRNAs remain largely unknown, gain-of-function and loss-of-function studies (in vivo and in vitro) have uncovered a number of mechanisms by which lncRNAs regulate gene expression and protein function. Such mechanisms have been stratified according to three major functional categories: RNA sponges (RNA-mediated sequestration of free miRNAs; e.g. H19, MEG3, and MALAT1); transcription-modulating lncRNAs (RNA influences regulatory factor recruitment by binding to histone modifiers or transcription factors; e.g. CAIF, MANTIS, and NEAT1); and translation-modulating lncRNAs (RNA modifies protein function via directly interacting with a protein itself or binding partners; e.g. Airn, CCRR, and ZFAS1). SUMMARY: Recent studies strongly suggest that lncRNAs function via binding to macromolecules (e.g. genomic DNA, miRNAs, or proteins). Thus, lncRNAs constitute an additional mode by which cells regulate gene expression.

Clinical value of non-coding RNAs in cardiovascular, pulmonary, and muscle diseases.

Although a majority of the mammalian genome is transcribed to RNA, mounting evidence indicates that only a minor proportion of these transcriptional products are actually translated into proteins. Since the discovery of the first non-coding RNA (ncRNA) in the 1980s, the field has gone on to recognize ncRNAs as important molecular regulators of RNA activity and protein function, knowledge of which has stimulated the expansion of a scientific field that quests to understand the role of ncRNAs in cellular physiology, tissue homeostasis, and human disease. Although our knowledge of these molecules has significantly improved over the years, we have limited understanding of their precise functions, protein interacting partners, and tissue-specific activities. Adding to this complexity, it remains unknown exactly how many ncRNAs there are in existence. The increased use of high-throughput transcriptomics techniques has rapidly expanded the list of ncRNAs, which now includes classical ncRNAs (e.g., ribosomal RNAs and transfer RNAs), microRNAs, and long ncRNAs. In addition, splicing by-products of protein-coding genes and ncRNAs, so-called circular RNAs, are now being investigated. Because there is substantial heterogeneity in the functions of ncRNAs, we have summarized the present state of knowledge regarding the functions of ncRNAs in heart, lungs, and skeletal muscle. This review highlights the pathophysiologic relevance of these ncRNAs in the context of human cardiovascular, pulmonary, and muscle diseases.

Research and therapy with induced pluripotent stem cells (iPSCs): social, legal, and ethical considerations.

Induced pluripotent stem cells (iPSCs) can self-renew indefinitely in culture and differentiate into all specialized cell types including gametes. iPSCs do not exist naturally and are instead generated ("induced" or "reprogrammed") in culture from somatic cells through ectopic co-expression of defined pluripotency factors. Since they can be generated from any healthy person or patient, iPSCs are considered as a valuable resource for regenerative medicine to replace diseased or damaged tissues. In addition, reprogramming technology has provided a powerful tool to study mechanisms of cell fate decisions and to model human diseases, thereby substantially potentiating the possibility to (i) discover new drugs in screening formats and (ii) treat life-threatening diseases through cell therapy-based strategies. However, various legal and ethical barriers arise when aiming to exploit the full potential of iPSCs to minimize abuse or unauthorized utilization. In this review, we discuss bioethical, legal, and societal concerns associated with research and therapy using iPSCs. Furthermore, we present key questions and suggestions for stem cell scientists, legal authorities, and social activists investigating and working in this field.

Pro-Angiogenic Actions of CMC-Derived Extracellular Vesicles Rely on Selective Packaging of Angiopoietin 1 and 2, but Not FGF-2 and VEGF.

While the fundamental mechanism by which cardiac cell therapy mitigates ventricular dysfunction in the post ischemic heart remains poorly defined, donor cell paracrine signaling is presumed to be a chief contributor to the afforded benefits. Of the many bioactive molecules secreted by transplanted cells, extracellular vesicles (EVs) and their proteinaceous, nucleic acid, and lipid rich contents, comprise a heterogeneous assortment of prospective cardiotrophic factors-whose involvement in the activation of endogenous cardiac repair mechanism(s), including reducing fibrosis and promoting angiogenesis, have yet to be fully explained. In the current study we aimed to interrogate potential mechanisms by which cardiac mesenchymal stromal cell (CMC)-derived EVs contribute to the CMC pro-angiogenic paracrine signaling capacity in vitro. Vesicular transmission and biological activity of human CMC-derived EVs was evaluated in in vitro assays for human umbilical vein endothelial cell (HUVEC) function, including EV uptake, cell survival, migration, tube formation, and intracellular pathway activation. HUVECs incubated with EVs exhibited augmented cell migration, tube formation, and survival under peroxide exposure; findings which paralleled enhanced activation of the archetypal pro-survival/pro-angiogenic pathways, STAT3 and PI3K-AKT. Cytokine array analyses revealed preferential enrichment of a subset of prototypical angiogenic factors, Ang-1 and Ang-2, in CMC EVs. Interestingly, pharmacologic inhibition of Tie2 in HUVECs, the cognate receptors of angiopoietins, efficiently attenuated CMC-EV-induced HUVEC migration. Further, in additional assays a Tie2 kinase inhibitor exhibited specificity to inhibit Ang-1-, but not Ang-2-, induced HUVEC migration. Overall, these findings suggest that the pro-angiogenic activities of CMC EVs are principally mediated by Ang-1-Tie2 signaling.

Epigenetically modified cardiac mesenchymal stromal cells limit myocardial fibrosis and promote functional recovery in a model of chronic ischemic cardiomyopathy.

Preclinical investigations support the concept that donor cells more oriented towards a cardiovascular phenotype favor repair. In light of this philosophy, we previously identified HDAC1 as a mediator of cardiac mesenchymal cell (CMC) cardiomyogenic lineage commitment and paracrine signaling potency in vitro-suggesting HDAC1 as a potential therapeutically exploitable target to enhance CMC cardiac reparative capacity. In the current study, we examined the effects of pharmacologic HDAC1 inhibition, using the benzamide class 1 isoform-selective HDAC inhibitor entinostat (MS-275), on CMC cardiomyogenic lineage commitment and CMC-mediated myocardial repair in vivo. Human CMCs pre-treated with entinostat or DMSO diluent control were delivered intramyocardially in an athymic nude rat model of chronic ischemic cardiomyopathy 30 days after a reperfused myocardial infarction. Indices of cardiac function were assessed by echocardiography and left ventricular (LV) Millar conductance catheterization 35 days after treatment. Compared with naïve CMCs, entinostat-treated CMCs exhibited heightened capacity for myocyte-like differentiation in vitro and superior ability to attenuate LV remodeling and systolic dysfunction in vivo. The improvement in CMC therapeutic efficacy observed with entinostat pre-treatment was not associated with enhanced donor cell engraftment, cardiomyogenesis, or vasculogenesis, but instead with more efficient inhibition of myocardial fibrosis and greater increase in myocyte size. These results suggest that HDAC inhibition enhances the reparative capacity of CMCs, likely via a paracrine mechanism that improves ventricular compliance and contraction and augments myocyte growth and function.

Airn Regulates Igf2bp2 Translation in Cardiomyocytes.

RATIONALE: Increasing evidence indicates the presence of lncRNAs in various cell types. Airn is an imprinting gene transcribed from the paternal chromosome. It is in antisense orientation to the imprinted, but maternally derived, Igf2r gene, on which Airn exerts its regulation in cis. Although Airn is highly expressed in the heart, functions aside from imprinting remain unknown. OBJECTIVE: Here, we studied the functions of Airn in the heart, especially cardiomyocytes. METHODS AND RESULTS: Silencing of Airn via siRNAs augmented cell death, vulnerability to cellular stress, and reduced cell migration. To find the cause of such phenotypes, the potential binding partners of Airn were identified via RNA pull-down followed by mass spectrometry, which indicated Igf2bp2 (insulin-like growth factor 2 mRNA-binding protein 2) and Rpa1 (replication protein A1) as potential binding partners. Further experiments showed that Airn binds to Igf2bp2 to control the translation of several genes. Moreover, silencing of Airn caused less binding of Igf2bp2 to other mRNAs and reduced translation of Igf2bp2 protein. CONCLUSIONS: Our study uncovers a new function of Airn and demonstrates that Airn is important for the physiology of cardiomyocytes.