Serine protease inhibitor, SerpinA3n regulates cardiac remodeling after myocardial infarction.

Following myocardial infarction, the heart repairs itself via a fibrotic repair response. The degree of fibrosis is determined by the balance between deposition of extracellular matrix by activated fibroblasts and breakdown of nascent scar tissue by proteases that are secreted predominantly by inflammatory cells. Excessive proteolytic activity and matrix turnover has been observed in human heart failure and protease inhibitors in the injured heart regulate matrix breakdown. Serine protease inhibitors (Serpins) represent the largest and the most functionally diverse family of evolutionary conserved protease inhibitors and levels of the specific Serpin, SerpinA3, have been strongly associated with clinical outcomes in human myocardial infarction as well as non-ischemic cardiomyopathies. Yet, the role of Serpins in regulating cardiac remodeling is poorly understood. We observed the robust expression of Serpins in the infarcted murine heart and demonstrate that genetic deletion of SerpinA3n (mouse homolog of SerpinA3) leads to increased activity of substrate proteases, poorly compacted matrix and significantly worse post infarct cardiac function. Single cell transcriptomics complemented with histology in SerpinA3n deficient animals, demonstrated increased inflammation, adverse myocyte hypertrophy and expression of pro-hypertrophic genes. Proteomic analysis of scar tissue demonstrated decreased cross linking of extracellular matrix peptides consistent with increased proteolysis in SerpinA3n deficient animals. Taken together these observations demonstrate a hitherto unappreciated causal role of Serpins in regulating matrix function and post infarct cardiac remodeling.

miR-378-mediated glycolytic metabolism enriches the Pax7Hi subpopulation of satellite cells.

Adult skeletal muscle stem cells, also known satellite cells (SCs), are a highly heterogeneous population and reside between the basal lamina and the muscle fiber sarcolemma. Myofibers function as an immediate niche to support SC self-renewal and activation during muscle growth and regeneration. Herein, we demonstrate that microRNA 378 (miR-378) regulates glycolytic metabolism in skeletal muscle fibers, as evidenced by analysis of myofiber-specific miR-378 transgenic mice (TG). Subsequently, we evaluate SC function and muscle regeneration using miR-378 TG mice. We demonstrate that miR-378 TG mice significantly attenuate muscle regeneration because of the delayed activation and differentiation of SCs. Furthermore, we show that the miR-378-mediated metabolic switch enriches Pax7Hi SCs, accounting for impaired muscle regeneration in miR-378 TG mice. Mechanistically, our data suggest that miR-378 targets the Akt1/FoxO1 pathway, which contributes the enrichment of Pax7Hi SCs in miR-378 TG mice. Together, our findings indicate that miR-378 is a target that links fiber metabolism to muscle stem cell heterogeneity and provide a genetic model to approve the metabolic niche role of myofibers in regulating muscle stem cell behavior and function.

Linc-RAM promotes muscle cell differentiation via regulating glycogen phosphorylase activity.

Long non-coding RNAs (lncRNAs) are important regulators of diverse biological processes, especially skeletal muscle cell differentiation. Most of the lncRNAs identified to date are localized in the nucleus and play regulatory roles in gene expression. The cytoplasmic lncRNAs are less well understood. We previously identified a long intergenic non-coding RNA (linc-RNA) activator of myogenesis (Linc-RAM) that directly binds MyoD in the nucleus to enhance muscle cell differentiation. Here, we report that a substantial fraction of Linc-RAM is localized in the cytoplasm of muscle cells. To explore the molecular functions of cytoplasmic Linc-RAM, we sought to identify Linc-RAM-binding proteins. We report here that Linc-RAM physically interacts with glycogen phosphorylase (PYGM) in the cytoplasm. Knockdown of PYGM significantly attenuates the function of Linc-RAM in promoting muscle cell differentiation. Loss-of-function and gain-of function assays demonstrated that PYGM enhances muscle cell differentiation in an enzymatic activity-dependent manner. Finally, we show that the interaction between Linc-RAM and PYGM positively regulates the enzymatic activity of PYGM in muscle cells. Collectively, our findings unveil a molecular mechanism through which cytoplasmic Linc-RAM contributes to muscle cell differentiation by regulating PYGM activity. Our findings establish that there is crosstalk between lncRNAs and cellular metabolism during myogenic cell differentiation.

Cardiomyocytes disrupt pyrimidine biosynthesis in nonmyocytes to regulate heart repair.

Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.

Acetoacetate promotes muscle cell proliferation via the miR-133b/SRF axis through the Mek-Erk-MEF2 pathway.

Acetoacetate (AA) is an important ketone body that is used as an oxidative fuel to supply energy for the cellular activities of various tissues, including the brain and skeletal muscle. We recently revealed a new signaling role for AA by showing that it promotes muscle cell proliferation in vitro, enhances muscle regeneration in vivo, and ameliorates the dystrophic muscle phenotype of Mdx mice. In this study, we provide new molecular insight into this function of AA. We show that AA promotes C2C12 cell proliferation by transcriptionally upregulating the expression of muscle-specific miR-133b, which in turn stimulates muscle cell proliferation by targeting serum response factor. Furthermore, we show that the AA-induced upregulation of miR-133b is transcriptionally mediated by MEF2 via the Mek-Erk1/2 signaling pathway. Mechanistically, our findings provide further convincing evidence that AA acts as signaling metabolite to actively regulate various cellular activities in mammalian cells.

Metabolic reprogramming and epigenetic changes of vital organs in SARS-CoV-2-induced systemic toxicity.

Extrapulmonary manifestations of COVID-19 are associated with a much higher mortality rate than pulmonary manifestations. However, little is known about the pathogenesis of systemic complications of COVID-19. Here, we create a murine model of SARS-CoV-2-induced severe systemic toxicity and multiorgan involvement by expressing the human ACE2 transgene in multiple tissues via viral delivery, followed by systemic administration of SARS-CoV-2. The animals develop a profound phenotype within 7 days with severe weight loss, morbidity, and failure to thrive. We demonstrate that there is metabolic suppression of oxidative phosphorylation and the tricarboxylic acid (TCA) cycle in multiple organs with neutrophilia, lymphopenia, and splenic atrophy, mirroring human COVID-19 phenotypes. Animals had a significantly lower heart rate, and electron microscopy demonstrated myofibrillar disarray and myocardial edema, a common pathogenic cardiac phenotype in human COVID-19. We performed metabolomic profiling of peripheral blood and identified a panel of TCA cycle metabolites that served as biomarkers of depressed oxidative phosphorylation. Finally, we observed that SARS-CoV-2 induces epigenetic changes of DNA methylation, which affects expression of immune response genes and could, in part, contribute to COVID-19 pathogenesis. Our model suggests that SARS-CoV-2-induced metabolic reprogramming and epigenetic changes in internal organs could contribute to systemic toxicity and lethality in COVID-19.

Cardiac fibroblast proliferation rates and collagen expression mature early and are unaltered with advancing age.

Cardiac fibrosis is a pathophysiologic hallmark of the aging heart, but little is known about how fibroblast proliferation and transcriptional programs change throughout the life span of the organism. Using EdU pulse labeling, we demonstrated that more than 50% of cardiac fibroblasts were actively proliferating in the first day of postnatal life. However, by 4 weeks, only 10% of cardiac fibroblasts were proliferating. By early adulthood, the fraction of proliferating cardiac fibroblasts further decreased to approximately 2%, where it remained throughout the rest of the organism's life. We observed that maximal changes in cardiac fibroblast transcriptional programs and, in particular, collagen and ECM gene expression both in the heart and cardiac fibroblast were maximal in the newly born and juvenile animal and decreased with organismal aging. Examination of DNA methylation changes both in the heart and in cardiac fibroblasts did not demonstrate significant changes in differentially methylated regions between young and old mice. Our observations demonstrate that cardiac fibroblasts attain a stable proliferation rate and transcriptional program early in the life span of the organism and suggest that phenotypic changes in the aging heart are not directly attributable to changes in proliferation rate or altered collagen expression in cardiac fibroblasts.

Type V Collagen in Scar Tissue Regulates the Size of Scar after Heart Injury.

Scar tissue size following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors regulating scar size. We demonstrate that collagen V, a minor constituent of heart scars, regulates the size of heart scars after ischemic injury. Depletion of collagen V led to a paradoxical increase in post-infarction scar size with worsening of heart function. A systems genetics approach across 100 in-bred strains of mice demonstrated that collagen V is a critical driver of postinjury heart function. We show that collagen V deficiency alters the mechanical properties of scar tissue, and altered reciprocal feedback between matrix and cells induces expression of mechanosensitive integrins that drive fibroblast activation and increase scar size. Cilengitide, an inhibitor of specific integrins, rescues the phenotype of increased post-injury scarring in collagen-V-deficient mice. These observations demonstrate that collagen V regulates scar size in an integrin-dependent manner.

miR-127 enhances myogenic cell differentiation by targeting S1PR3.

MicroRNAs (miRNAs) have recently been implicated in muscle stem cell function. miR-127 is known to be predominantly expressed in skeletal muscle, but its roles in myogenic differentiation and muscle regeneration are unknown. Here, we show that miR-127 is upregulated during C2C12 and satellite cell (SC) differentiation and, by establishing C2C12 cells stably expressing miR-127, demonstrate that overexpression of miR-127 in C2C12 cells enhances myogenic cell differentiation. To investigate the function of miR-127 during muscle development and regeneration in vivo, we generated miR-127 transgenic mice. These mice exhibited remarkably accelerated muscle regeneration compared with wild-type mice by promoting SC differentiation. Mechanistically, we demonstrated that the gene encoding sphingosine-1-phosphate receptor 3 (S1PR3), a G-protein-coupled receptor for sphingosine-1-phosphate, is a target of miR-127 required for its function in promoting myogenic cell differentiation. Importantly, overexpression of miR-127 in muscular dystrophy model mdx mice considerably ameliorated the disease phenotype. Thus, our findings suggest that miR-127 may serve as a potential therapeutic target for the treatment of skeletal muscle disease in humans.

miR-431 is involved in regulating cochlear function by targeting Eya4.

To understand the relationship between microRNAs and hearing loss and help clarify the causes of hereditary deafness, we studied the functions of miR-431 in cochleae. We first investigated the spatial-temporal expression profiles of miR-431 in spiral ganglion neurons (SGNs) in cochleae using real-time PCR and miRNA in situ hybridization. These studies showed that expression of miR-431 was high in SGNs in the cochleae of newborn mice, and decreased as development progressed. To test the functional effects of miR-431, we established miR-431 overexpressing transgenic (Tg) mice. Surface preparations of the cochlear basilar membrane and cochlear sections revealed no major structural differences between Tg and wild-type (Wt) mice. However, a comparison of auditory brain stem responses (ABRs) in Tg and Wt mice showed that ABR thresholds were significantly higher in Tg mice than in Wt mice. Notably, the density of SGNs was significantly lower in Tg mice than in Wt mice. We also found that the proportion of mature SGNs in cultures of primary SGNs from Tg cochleae was lower and their axons were shorter. A bioinformatics analysis predicted that the mRNA target of miR-431 was Eya4, a finding confirmed by luciferase reporter assays and western blotting. Importantly, overexpression of miR-431 in cochleae of Tg mice inhibited the translation of Eya4 mRNA, leading to a deficiency of EYA4. Thus, excessive amounts of miR-431 in cochleae of Tg mice may be the cause of sparse SGNs, which in turn could be responsible for hearing loss.

Overexpression of Both ERG11 and ABC2 Genes Might Be Responsible for Itraconazole Resistance in Clinical Isolates of Candida krusei.

OBJECTIVE: To study the main molecular mechanisms responsible for itraconazole resistance in clinical isolates of Candida krusei. METHODS: The 14α-demethylases encoded by ERG11 gene in the 16 C.krusei clinical isolates were amplified by polymerase chain reaction (PCR), and their nucleotide sequences were determined to detect point mutations. Meanwhile, ERG11 and efflux transporters (ABC1 and ABC2) genes were determined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) for their expression in itraconazole-resistant (R), itraconazole-susceptible dose dependent (SDD) and itraconazole-susceptible (S) C.krusei at the mRNA level. RESULTS: We found 7-point mutations in ERG11 gene of all the C.krusei clinical isolates, including 6 synonymous mutations and 1 missense mutation (C44T). However, the missense mutation was found in the three groups. The mRNA levels of ERG11 gene in itraconazole-resistant isolates showed higher expression compared with itraconazole-susceptible dose dependent and itraconazole-susceptible ones (P = 0.015 and P = 0.002 respectively). ABC2 gene mRNA levels in itraconazole-resistant group was significantly higher than the other two groups, and the levels of their expression in the isolates appeared to increase with the decrease of susceptibility to itraconazole (P = 0.007 in SDD compared with S, P = 0.016 in SDD with R, and P<0.001 in S with R respectively). While ABC1 gene presented lower expression in itraconazole resistant strains. However, the mRNA levels of ERG11, ABC1 and ABC2 in a C.krusei (CK10) resistant to both itraconazole and voriconazole were expressed highest in all the itraconazole-resistant isolates. CONCLUSIONS: There are ERG11 gene polymorphisms in clinical isolates of C.krusei. ERG11 gene mutations may not be involved in the development of itraconazole resistance in C.krusei. ERG11 and ABC2 overexpression might be responsible for the acquired itraconazole resistance of these clinical isolates.

MicroRNA-431 accelerates muscle regeneration and ameliorates muscular dystrophy by targeting Pax7 in mice.

Skeletal muscle stem cells, called satellite cells, are a quiescent heterogeneous population. Their heterogeneity is influenced by Pax7, a well-defined transcriptional regulator of satellite cell functions that defines two subpopulations: Pax7(Hi) and Pax7(Lo). However, the mechanisms by which these subpopulations are established and maintained during myogenesis are not completely understood. Here we show that miR-431, which is predominantly expressed in the skeletal muscle, mediates satellite cell heterogeneity by fine-tuning Pax7 levels during muscle development and regeneration. In miR-431 transgenic mice, the Pax7(Lo) subpopulation is enriched, enhances myogenic differentiation and accelerates muscle regeneration. Notably, miR-431 attenuates the muscular dystrophic phenotype in mdx mice and may be a potential therapeutic target in muscular diseases. miR-431 transgenic mice are a unique genetic model for investigating the cellular features and biological functions of Pax7(Lo) satellite cells during muscle development and regeneration.

Myostatin regulates miR-431 expression via the Ras-Mek-Erk signaling pathway.

MicroRNAs (miRNAs) play critical regulatory roles in controlling myogenic development both in vitro and in vivo; however, the molecular mechanisms underlying transcriptional regulation of miRNA genes in skeletal muscle cells are largely unknown. Here, using a microarray hybridization approach, we identified myostatin-regulated miRNA genes in skeletal muscle tissues by systematically searching miRNAs that are differentially expressed between wild-type and myostatin-null mice during development. We found that 116 miRNA genes were differentially expressed in muscles between these mice across different developmental stages. We further characterized myostatin-regulated miR-431 was upregulated in skeletal muscle tissues of myostatin-null mice. In functional studies, we found that overexpression of miR-431 in C2C12 myoblast cells attenuated myostatin-induced suppression of myogenic differentiation. Mechanistic studies further demonstrated that myostatin acted through the Ras-Mek-Erk signaling pathway to transcriptionally regulate miR-431 expression C2C12 cells. Our findings provide new insight into the mechanisms underlying transcriptional regulation of miRNA genes by myostatin during skeletal muscle development.

Accelerated regeneration of the skeletal muscle in RNF13-knockout mice is mediated by macrophage-secreted IL-4/IL-6.

RING finger protein 13 (RNF13) is a newly identified E3 ligase reported to be functionally significant in the regulation of cancer development, muscle cell growth, and neuronal development. In this study, the function of RNF13 in cardiotoxin-induced skeletal muscle regeneration was investigated using RNF13-knockout mice. RNF13(-/-) mice exhibited enhanced muscle regeneration-characterized by accelerated satellite cell proliferation-compared with wild-type mice. The expression of RNF13 was remarkably induced in macrophages rather than in the satellite cells of wild-type mice at the very early stage of muscle damage. This result indicated that inflammatory cells are important in RNF13-mediated satellite cell functions. The cytokine levels in skeletal muscles were further analyzed and showed that RNF13(-/-) mice produced greater amounts of various cytokines than wild-type mice. Among these, IL-4 and IL-6 levels significantly increased in RNF13(-/-) mice. The accelerated muscle regeneration phenotype was abrogated by inhibiting IL-4/IL-6 action in RNF13(-/-) mice with blocking antibodies. These results indicate that RNF13 deficiency promotes skeletal muscle regeneration via the effects on satellite cell niche mediated by IL-4 and IL-6.

Identification of long non-protein coding RNAs in chicken skeletal muscle using next generation sequencing.

Vertebrate genomes encode thousands of non-coding RNAs including short non-coding RNAs (such as microRNAs) and long non-coding RNAs (lncRNAs). Chicken (Gallus gallus) is an important model organism for developmental biology, and the recently assembled genome sequences for chicken will facilitate the understanding of the functional roles of non-coding RNA genes during development. The present study concerns the first systematic identification of lncRNAs using RNA-Seq to sample the transcriptome during chicken muscle development. A computational approach was used to identify 281 new intergenic lncRNAs in the chicken genome. Novel lncRNAs in general are less conserved than protein-coding genes and slightly more conserved than random non-coding sequences. The present study has provided an initial chicken lncRNA catalog and greatly increased the number of chicken ncRNAs in the non-protein coding RNA database. Furthermore, the computational pipeline presented in the current work will be useful for characterizing lncRNAs obtained from deep sequencing data.

A systematic analysis of the skeletal muscle miRNA transcriptome of chicken varieties with divergent skeletal muscle growth identifies novel miRNAs and differentially expressed miRNAs.

BACKGROUND: Functional studies have demonstrated that microRNAs (miRNAs or miRs) play critical roles in a wide spectrum of biological processes including development and disease pathogenesis. To investigate the functional roles that miRNAs play during chicken skeletal muscle development, the miRNA transcriptomes of skeletal muscles from broiler and layer chickens were profiled using Solexa deep sequencing. RESULTS: Some miRNAs have multiple isoforms and several miRNAs* are present at higher levels than their corresponding miRNAs. Thirty three novel and 189 known chicken miRNAs were identified using computational approaches. Subsequent miRNA transcriptome comparisons and real-time PCR validation experiments revealed 17 miRNAs that were differentially expressed between broilers and layers, and a number of targets of these miRNAs have been implicated in myogenesis regulation. Using integrative miRNA target-prediction and network-analysis approaches an interaction network of differentially expressed and muscle-related miRNAs and their putative targets was constructed, and miRNAs that could contribute to the divergent muscle growth of broiler and layer chickens by targeting the ACVR2B gene were identified, which can causes dramatic increases in muscle mass. CONCLUSIONS: The present study provides the first transcriptome profiling-based evaluation of miRNA function during skeletal muscle development in chicken. Systematic predictions aided the identification of potential miRNAs and their targets, which could contribute to divergent muscle growth in broiler and layer chickens. Furthermore, these predictions generated information that can be utilized in further research investigating the involvement of interaction networks, containing miRNAs and their targets, in the regulation of muscle development.

Systematic identification and evolutionary features of rhesus monkey small nucleolar RNAs.

BACKGROUND: Recent studies have demonstrated that non-protein-coding RNAs (npcRNAs/ncRNAs) play important roles during eukaryotic development, species evolution, and in the etiology of disease. Rhesus macaques are the most widely used primate model in both biomedical research and primate evolutionary studies. However, most reports on these animals focus on the functional roles of protein-coding sequences, whereas very little is known about macaque ncRNAs. RESULTS: In the present study, we performed the first systematic profiling of intermediate-size ncRNAs (50 to 500 nt) from the rhesus monkey by constructing a cDNA library. We identified 117 rhesus monkey ncRNAs, including 80 small nucleolar RNAs (snoRNAs), 29 other types of known RNAs (snRNAs, Y RNA, and others), and eight unclassified ncRNAs. Comparative genomic analysis and northern blot hybridizations demonstrated that some snoRNAs were lineage- or species-specific. Paralogous sequences were found for most rhesus monkey snoRNAs, the expression of which might be attributable to extensive duplication within the rhesus monkey genome. Further investigation of snoRNA flanking sequences showed that some rhesus monkey snoRNAs are retrogenes derived from L1-mediated integration. Finally, phylogenetic analysis demonstrated that birds and primates share some snoRNAs and host genes thereof, suggesting that both the relevant host genes and the snoRNAs contained therein may be inherited from a common ancestor. However, some rhesus monkey snoRNAs hosted by non-ribosome-related genes appeared after the evolutionary divergence between birds and mammals. CONCLUSIONS: We provide the first experimentally-derived catalog of rhesus monkey ncRNAs and uncover some interesting genomic and evolutionary features. These findings provide important information for future functional characterization of snoRNAs during primate evolution.

Systematic identification and characterization of chicken (Gallus gallus) ncRNAs.

Recent studies have demonstrated that non-coding RNAs (ncRNAs) play important roles during development and evolution. Chicken, the first genome-sequenced non-mammalian amniote, possesses unique features for developmental and evolutionary studies. However, apart from microRNAs, information on chicken ncRNAs has mainly been obtained from computational predictions without experimental validation. In the present study, we performed a systematic identification of intermediate size ncRNAs (50-500 nt) by ncRNA library construction and identified 125 chicken ncRNAs. Importantly, through the bioinformatics and expression analysis, we found the chicken ncRNAs has several novel features: (i) comparative genomic analysis against 18 sequenced vertebrate genomes revealed that the majority of the newly identified ncRNA candidates is not conserved and most are potentially bird/chicken specific, suggesting that ncRNAs play roles in lineage/species specification during evolution. (ii) The expression pattern analysis of intronic snoRNAs and their host genes suggested the coordinated expression between snoRNAs and their host genes. (iii) Several spatio-temporal specific expression patterns suggest involvement of ncRNAs in tissue development. Together, these findings provide new clues for future functional study of ncRNAs during development and evolution.