Miscarriage risk assessment: a bioinformatic approach to identifying candidate lethal genes and variants.

PURPOSE: Miscarriage, often resulting from a variety of genetic factors, is a common pregnancy outcome. Preconception genetic carrier screening (PGCS) identifies at-risk partners for newborn genetic disorders; however, PGCS panels currently lack miscarriage-related genes. In this study, we evaluated the potential impact of both known and candidate genes on prenatal lethality and the effectiveness of PGCS in diverse populations. METHODS: We analyzed 125,748 human exome sequences and mouse and human gene function databases. Our goals were to identify genes crucial for human fetal survival (lethal genes), to find variants not present in a homozygous state in healthy humans, and to estimate carrier rates of known and candidate lethal genes in various populations and ethnic groups. RESULTS: This study identified 138 genes in which heterozygous lethal variants are present in the general population with a frequency of 0.5% or greater. Screening for these 138 genes could identify 4.6% (in the Finnish population) to 39.8% (in the East Asian population) of couples at risk of miscarriage. This explains the cause of pregnancy loss in approximately 1.1-10% of cases affected by biallelic lethal variants. CONCLUSION: This study has identified a set of genes and variants potentially associated with lethality across different ethnic backgrounds. The variation of these genes across ethnic groups underscores the need for a comprehensive, pan-ethnic PGCS panel that includes genes related to miscarriage.

Reprogramming of cardiac phosphoproteome, proteome, and transcriptome confers resilience to chronic adenylyl cyclase-driven stress.

Our prior study (Tarasov et al., 2022) discovered that numerous adaptive mechanisms emerge in response to cardiac-specific overexpression of adenylyl cyclase type 8 (TGAC8) which included overexpression of a large number of proteins. Here, we conducted an unbiased phosphoproteomics analysis in order to determine the role of altered protein phosphorylation in the adaptive heart performance and protection profile of adult TGAC8 left ventricle (LV) at 3-4 months of age, and integrated the phosphoproteome with transcriptome and proteome. Based on differentially regulated phosphoproteins by genotype, numerous stress-response pathways within reprogrammed TGAC8 LV, including PKA, PI3K, and AMPK signaling pathways, predicted upstream regulators (e.g. PDPK1, PAK1, and PTK2B), and downstream functions (e.g. cell viability, protein quality control), and metabolism were enriched. In addition to PKA, numerous other kinases and phosphatases were hyper-phosphorylated in TGAC8 vs. WT. Hyper-phosphorylated transcriptional factors in TGAC8 were associated with increased mRNA transcription, immune responses, and metabolic pathways. Combination of the phosphoproteome with its proteome and with the previously published TGAC8 transcriptome enabled the elucidation of cardiac performance and adaptive protection profiles coordinately regulated at post-translational modification (PTM) (phosphorylation), translational, and transcriptional levels. Many stress-response signaling pathways, i.e., PI3K/AKT, ERK/MAPK, and ubiquitin labeling, were consistently enriched and activated in the TGAC8 LV at transcriptional, translational, and PTM levels. Thus, reprogramming of the cardiac phosphoproteome, proteome, and transcriptome confers resilience to chronic adenylyl cyclase-driven stress. We identified numerous pathways/function predictions via gene sets, phosphopeptides, and phosphoproteins, which may point to potential novel therapeutic targets to enhance heart adaptivity, maintaining heart performance while avoiding cardiac dysfunction.

Transcriptome of Left Ventricle and Sinoatrial Node in Young and Old C57 Mice.

Advancing age is the most important risk factor for cardiovascular diseases (CVDs). Two types of cells, within the heart pacemaker, sinoatrial node (SAN), and within the left ventricle (LV), control two crucial characteristics of heart function, heart beat rate and contraction strength. As age advances, the heart's structure becomes remodeled, and SAN and LV cell functions deteriorate, thus increasing the risk for CVDs. However, the different molecular features of age-associated changes in SAN and LV cells have never been compared in omics scale in the context of aging. We applied deep RNA sequencing to four groups of samples, young LV, old LV, young SAN and old SAN, followed by numerous bioinformatic analyses. In addition to profiling the differences in gene expression patterns between the two heart chambers (LV vs. SAN), we also identified the chamber-specific concordant or discordant age-associated changes in: (1) genes linked to energy production related to cardiomyocyte contraction, (2) genes related to post-transcriptional processing, (3) genes involved in KEGG longevity regulating pathway, (4) prolongevity and antilongevity genes recorded and curated in the GenAge database, and (5) CVD marker genes. Our bioinformatic analysis also predicted the regulation activities and mapped the expression of upstream regulators including transcription regulators and post-transcriptional regulator miRNAs. This comprehensive analysis promotes our understanding of regulation of heart functions and will enable discovery of gene-specific therapeutic targets of CVDs in advanced age.

Enhanced Myocardial Adenylyl Cyclase Activity Alters Heart-Brain Communication.

BACKGROUND: The central nervous system's influence on cardiac function is well described; however, direct evidence for signaling from heart to brain remains sparse. Mice with cardiac-selective overexpression of adenylyl cyclase type 8 (TGAC8) display elevated heart rate/contractility and altered neuroautonomic surveillance. OBJECTIVES: In this study the authors tested whether elevated adenylyl cyclase type 8-dependent signaling at the cardiac cell level affects brain activity and behavior. METHODS: A telemetry system was used to record electrocardiogram (ECG) and electroencephalogram (EEG) in TGAC8 and wild-type mice simultaneously. The Granger causality statistical approach evaluated variations in the ECG/EEG relationship. Mouse behavior was assessed via elevated plus maze, open field, light-dark box, and fear conditioning tests. Transcriptomic and proteomic analyses were performed on brain tissue lysates. RESULTS: Behavioral testing revealed increased locomotor activity in TGAC8 that included a greater total distance traveled (+43%; P < 0.01), a higher average speed (+38%; P < 0.01), and a reduced freezing time (-45%; P < 0.01). Dual-lead telemetry recording confirmed a persistent heart rate elevation with a corresponding reduction in ECG-R-waves interval variability and revealed increased EEG-gamma activity in TGAC8 vs wild-type. Bioinformatic assessment of hippocampal tissue indicated upregulation of dopamine 5, gamma-aminobutyric acid A, and metabotropic glutamate 1/5 receptors, major players in gamma activity generation. Granger causality analyses of ECG and EEG recordings showed a marked increase in informational flow between the TGAC8 heart and brain. CONCLUSIONS: Perturbed signals arising from the heart cause changes in brain activity, altering mouse behavior. More specifically, the brain interprets augmented myocardial humoral/functional output as a "sustained exercise-like" situation and responds by activating central nervous system output controlling locomotion.

Preconception Genetic Carrier Screening for Miscarriage Risk Assessment: A Bioinformatic Approach to Identifying Candidate Lethal Genes and Variants.

Purpose: Miscarriage, due to genetically heterogeneous etiology, is a common outcome of pregnancy. Preconception genetic carrier screening (PGCS) identifies at-risk partners for newborn genetic disorders; however, PGCS panels currently lack miscarriage-related genes. Here we assessed the theoretical impact of known and candidate genes on prenatal lethality and the PGCS among diverse populations. Methods: Human exome sequencing and mouse gene function databases were analyzed to define genes essential for human fetal survival (lethal genes), identify variants that are absent in a homozygous state in healthy human population, and to estimate carrier rates for known and candidate lethal genes. Results: Among 138 genes, potential lethal variants are present in the general population with a frequency of 0.5% or greater. Preconception screening for these 138 genes would identify from 4.6% (Finnish population) to 39.8% (East Asian population) of couples that are at-risk for miscarriage, explaining a cause for pregnancy loss for ∼1.1-10% of conceptions affected by biallelic lethal variants. Conclusion: This study identified a set of genes and variants potentially associated with lethality across different ethnic backgrounds. The diversity of these genes amongst the various ethnic groups highlights the importance of designing a pan-ethnic PGCS panel comprising miscarriage-related genes.

Characterization of diverse populations of sinoatrial node cells and their proliferation potential at single nucleus resolution.

Background: Each heartbeat is initiated in the sinoatrial node (SAN), and although a recent study (GSE130710) using single nucleus RNA-seq had discovered different populations of cell types within SAN tissue, the distinct potential functions of these cell types have not been delineated. Methods: To infer some special potential functions of different SAN cell clusters, we applied principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE) and uniform manifold approximation and projection (UMAP) to the GSE130710 dataset to reduce dimensions, followed by Pseudotime trajectory and AUCell analyses, ANOVA and Hurdle statistical models, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichments to determine functional potential of cell types. Nuclear EdU immuno-labeling of SAN tissue confirmed cell type proliferation. Findings: We identified elements of a coupled clock system known to drive SAN cell pacemaking within the GSE130710 sinus node myocyte cluster, which, surprisingly, manifested signals of suppressed fatty acid and nitrogen metabolism and reduced immune gene expression. Proliferation signaling was enriched in endocardial, epicardial, epithelial cells, and macrophages, in which, fatty acid and nitrogen metabolic signals were also suppressed, but immune signaling was enhanced. EdU labeling was rare in pacemaker cells but was robust in interstitial cells. Interpretation: Pacemaker cells that initiate each heartbeat manifest suppressed fatty acid and nitrogen metabolism and limited immune signaling and proliferation potential. In contrast, other populations of SAN cells not directly involved in the initiation of heartbeats, manifest robust proliferation and immune potential, likely to ensure an environment required to sustain healthy SAN tissue pacemaker function.

A remarkable adaptive paradigm of heart performance and protection emerges in response to marked cardiac-specific overexpression of ADCY8.

Adult (3 month) mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt to an increased cAMP-induced cardiac workload (~30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here, we show classical cardiac hypertrophy markers were absent in TGAC8, and that total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TGAC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes, and a network of small interstitial proliferative non-cardiac myocytes compared to wild type (WT) littermates; Protein synthesis, proteosome activity, and autophagy were enhanced in TGAC8 vs WT, and Nrf-2, Hsp90α, and ACC2 protein levels were increased. Despite increased energy demands in vivo LV ATP and phosphocreatine levels in TGAC8 did not differ from WT. Unbiased omics analyses identified more than 2,000 transcripts and proteins, comprising a broad array of biological processes across multiple cellular compartments, which differed by genotype; compared to WT, in TGAC8 there was a shift from fatty acid oxidation to aerobic glycolysis in the context of increased utilization of the pentose phosphate shunt and nucleotide synthesis. Thus, marked overexpression of AC8 engages complex, coordinate adaptation "circuity" that has evolved in mammalian cells to defend against stress that threatens health or life (elements of which have already been shown to be central to cardiac ischemic pre-conditioning and exercise endurance cardiac conditioning) that may be of biological significance to allow for proper healing in disease states such as infarction or failure of the heart.

Proteomic Landscape and Deduced Functions of the Cardiac 14-3-3 Protein Interactome.

Rationale: The 14-3-3 protein family is known to interact with many proteins in non-cardiac cell types to regulate multiple signaling pathways, particularly those relating to energy and protein homeostasis; and the 14-3-3 network is a therapeutic target of critical metabolic and proteostatic signaling in cancer and neurological diseases. Although the heart is critically sensitive to nutrient and energy alterations, and multiple signaling pathways coordinate to maintain the cardiac cell homeostasis, neither the structure of cardiac 14-3-3 protein interactome, nor potential functional roles of 14-3-3 protein-protein interactions (PPIs) in heart has been explored. Objective: To establish the comprehensive landscape and characterize the functional role of cardiac 14-3-3 PPIs. Methods and Results: We evaluated both RNA expression and protein abundance of 14-3-3 isoforms in mouse heart, followed by co-immunoprecipitation of 14-3-3 proteins and mass spectrometry in left ventricle. We identified 52 proteins comprising the cardiac 14-3-3 interactome. Multiple bioinformatic analyses indicated that more than half of the proteins bound to 14-3-3 are related to mitochondria; and the deduced functions of the mitochondrial 14-3-3 network are to regulate cardiac ATP production via interactions with mitochondrial inner membrane proteins, especially those in mitochondrial complex I. Binding to ribosomal proteins, 14-3-3 proteins likely coordinate protein synthesis and protein quality control. Localizations of 14-3-3 proteins to mitochondria and ribosome were validated via immunofluorescence assays. The deduced function of cardiac 14-3-3 PPIs is to regulate cardiac metabolic homeostasis and proteostasis. Conclusions: Thus, the cardiac 14-3-3 interactome may be a potential therapeutic target in cardiovascular metabolic and proteostatic disease states, as it already is in cancer therapy.

SIRT6 in Vascular Diseases, from Bench to Bedside.

Aging is a key risk factor for angiogenic dysfunction and cardiovascular diseases, including heart failure, hypertension, atherosclerosis, diabetes, and stroke. Members of the NAD+-dependent class III histone deacetylase family, sirtuins, are conserved regulators of aging and cardiovascular and cerebrovascular diseases. The sirtuin SIRT6 is predominantly located in the nucleus and shows deacetylase activity for acetylated histone 3 lysine 56 and lysine 9 as well as for some non-histone proteins. Over the past decade, experimental analyses in rodents and non-human primates have demonstrated the critical role of SIRT6 in extending lifespan. Recent studies highlighted the pleiotropic protective actions of SIRT6 in angiogenesis and cardiovascular diseases, including atherosclerosis, hypertension, heart failure, and stroke. Mechanistically, SIRT6 participates in vascular diseases via epigenetic regulation of endothelial cells, vascular smooth muscle cells, and immune cells. Importantly, SIRT6 activators (e.g., MDL-800/MDL-811) have provided therapeutic value for treating age-related vascular disorders. Here, we summarized the roles of sirtuins in cardiovascular diseases; reviewed recent advances in the understanding of SIRT6 in vascular biology, cardiovascular aging, and diseases; highlighted its therapeutic potential; and discussed future perspectives.

Diurnal oscillations of endogenous H2O2 sustained by p66Shc regulate circadian clocks.

Redox balance, an essential feature of healthy physiological steady states, is regulated by circadian clocks, but whether or how endogenous redox signalling conversely regulates clockworks in mammals remains unknown. Here, we report circadian rhythms in the levels of endogenous H2O2 in mammalian cells and mouse livers. Using an unbiased method to screen for H2O2-sensitive transcription factors, we discovered that rhythmic redox control of CLOCK directly by endogenous H2O2 oscillations is required for proper intracellular clock function. Importantly, perturbations in the rhythm of H2O2 levels induced by the loss of p66Shc, which oscillates rhythmically in the liver and suprachiasmatic nucleus (SCN) of mice, disturb the rhythmic redox control of CLOCK function, reprogram hepatic transcriptome oscillations, lengthen the circadian period in mice and modulate light-induced clock resetting. Our findings suggest that redox signalling rhythms are intrinsically coupled to the circadian system through reversible oxidative modification of CLOCK and constitute essential mechanistic timekeeping components in mammals.

Caloric Restriction Induces MicroRNAs to Improve Mitochondrial Proteostasis.

Both caloric restriction (CR) and mitochondrial proteostasis are linked to longevity, but how CR maintains mitochondrial proteostasis in mammals remains elusive. MicroRNAs (miRNAs) are well known for gene silencing in cytoplasm and have recently been identified in mitochondria, but knowledge regarding their influence on mitochondrial function is limited. Here, we report that CR increases miRNAs, which are required for the CR-induced activation of mitochondrial translation, in mouse liver. The ablation of miR-122, the most abundant miRNA induced by CR, or the retardation of miRNA biogenesis via Drosha knockdown significantly reduces the CR-induced activation of mitochondrial translation. Importantly, CR-induced miRNAs cause the overproduction of mtDNA-encoded proteins, which induces the mitochondrial unfolded protein response (UPRmt), and consequently improves mitochondrial proteostasis and function. These findings establish a physiological role of miRNA-enhanced mitochondrial function during CR and reveal miRNAs as critical mediators of CR in inducing UPRmt to improve mitochondrial proteostasis.

[Study on the effect of GDG on secondary structure of plasminogen and plasminogen activators by circular dichroism].

To investigate the effect of GDG on the secondary structure of plasminogen and plasminogen activators, circular dichroism and SDS-PAGD were applied. The results show that the secondary structures of prourokinase and streptokinase were changed by GDG. The amount of alpha-helix, beta-sheet, beta-turn and random coil of fibrinolytic factors relates with the different concentrations of GDG in the study. GDG has no effects on the secondary structure of plasmin and plasminogen. GDG enhancing the interactivation of plasminogen and prourokinase was indicated by SDS-PAGE. It was found that GDG significantly affected the activity of prourokinase and streptokinase, and increased intrinsic fluorescence of prourokinase.