Prostaglandin E2 mediates the late phase of ischemic preconditioning in the heart via its receptor subtype EP4.

Ischemic preconditioning (IPC) describes a phenomenon wherein brief ischemia of the heart induces a potent cardioprotective mechanism against succeeding ischemic insult. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostanoid biosynthesis, is upregulated in the ischemic heart and contributes to IPC. Prostaglandin E2 (PGE2) protects the heart from ischemia-reperfusion (I/R) injury via its receptor subtype EP4. We sought to clarify the role of the PGE2/EP4 system in the late phase of IPC. Mice were subjected to four IPC treatment cycles, consisting of 5 min of occlusion of the left anterior descending coronary artery (LAD). We found that COX-2 mRNA was significantly upregulated in wild-type hearts at 6 h after IPC treatment. Cardiac PGE2 levels at 24 h after IPC treatment were significantly increased in both wild-type mice and mice lacking EP4 (EP4-/-). At 24 h after IPC treatment, I/R injury was induced by 30 min of LAD occlusion followed by 2 h of reperfusion and the cardiac infarct size was determined. The infarct size was significantly reduced by IPC treatment in wild-type mice; a reduction was not observed in EP4-/- mice. AE1-329, an EP4 agonist, significantly reduced infarct size and significantly ameliorated deterioration of cardiac function in wild-type mice subjected to I/R without IPC treatment. Furthermore, AE1-329 significantly enhanced the I/R-induced activation of Akt, a pro-survival kinase. We demonstrated that the PGE2/EP4 system in the heart plays a critical role in the late phase of IPC, partly by augmenting Akt-mediated signaling. These findings clarify the mechanism of IPC and may contribute to the development of therapeutic strategies for ischemic heart disease.

Performance of using opposing homozygotes for paternity testing in Japanese Black cattle.

Genome-wide single nucleotide polymorphism (SNP) markers in Japanese Black cattle enable genomic prediction and verifying parent-offspring relationships. We assessed the performance of opposing homozygotes (OH) for paternity testing in Japanese Black cattle, using SNP genotype information of 50 sires and 3,420 fattened animals, 1,945 of which were fathered by the 50 genotyped sires. The number of OH was counted for each sire-progeny pair in 28,764 SNPs with minor allele frequencies of ≥0.05 in this population. Across all pairs of animals, the number of OH tended to increase as the pedigree-based coefficient of relationship decreased. With a threshold of 288 (1% of SNPs) for paternity testing, most sire-progeny pairs were detected as true relationships. The frequency of Mendelian inconsistencies was 2.4%, reflecting the high accuracy of pedigree information in Japanese Black cattle population. The results indicate the utility of OH for paternity testing in Japanese Black cattle.

Exploring the size of reference population for expected accuracy of genomic prediction using simulated and real data in Japanese Black cattle.

BACKGROUND: Size of reference population is a crucial factor affecting the accuracy of prediction of the genomic estimated breeding value (GEBV). There are few studies in beef cattle that have compared accuracies achieved using real data to that achieved with simulated data and deterministic predictions. Thus, extent to which traits of interest affect accuracy of genomic prediction in Japanese Black cattle remains obscure. This study aimed to explore the size of reference population for expected accuracy of genomic prediction for simulated and carcass traits in Japanese Black cattle using a large amount of samples. RESULTS: A simulation analysis showed that heritability and size of reference population substantially impacted the accuracy of GEBV, whereas the number of quantitative trait loci did not. The estimated numbers of independent chromosome segments (Me) and the related weighting factor (w) derived from simulation results and a maximum likelihood (ML) approach were 1900-3900 and 1, respectively. The expected accuracy for trait with heritability of 0.1-0.5 fitted well with empirical values when the reference population comprised > 5000 animals. The heritability for carcass traits was estimated to be 0.29-0.41 and the accuracy of GEBVs was relatively consistent with simulation results. When the reference population comprised 7000-11,000 animals, the accuracy of GEBV for carcass traits can range 0.73-0.79, which is comparable to estimated breeding value obtained in the progeny test. CONCLUSION: Our simulation analysis demonstrated that the expected accuracy of GEBV for a polygenic trait with low-to-moderate heritability could be practical in Japanese Black cattle population. For carcass traits, a total of 7000-11,000 animals can be a sufficient size of reference population for genomic prediction.

Pericyte-Specific Ninjurin1 Deletion Attenuates Vessel Maturation and Blood Flow Recovery in Hind Limb Ischemia.

Objective- Angiogenesis, entire step from endothelial cells (ECs) sprouts to vascular maturation, is a critical response to ischemia. To form functional mature vessels, interactions between ECs and pericytes are essential. Ninj1 (ninjurin1) is an adhesion molecule that contributes to the pathogenesis of neuroinflammation. We recently demonstrated that Ninj1 is expressed in pericytes during angiogenesis. However, the role of Ninj1 in angiogenesis under pathophysiological ischemic conditions has not yet been elucidated. Approach and Results- Ninj1 was detected in microvessels, and its expression was enhanced in ischemic tissues after mouse hindlimb ischemia. Knockdown of Ninj1 was performed by injection of biodegradable microspheres releasing Ninj1-small interfering RNA into muscle tissues. Alternatively, pericyte-specific Ninj1 knockout was induced by tamoxifen treatment of NG2-CreERT/Ninj1-flox mice. Ninj1 knockdown/knockout reduced the formation of blood-circulating functional vessels among total CD31+ microvessels within ischemic tissues and subsequently attenuated color Doppler-assessed blood flow recovery. Ninj1 overexpression enhanced expression of Anpt (angiopoietin) 1, whereas Ninj1 knockdown enhanced the endogenous Anpt1 antagonist, Anpt2 expression in pericytes and inhibited the association of pericytes with ECs and subsequent formation of capillary-like structure, that is, EC tube surrounded with pericytes in 3-dimensional gel culture. Conclusions- Our data demonstrate that Ninj1 is involved in the formation of functional matured vessels through the association between pericytes and ECs, resulting in blood flow recovery from ischemia. These findings further the current our understanding of vascular maturation and may support the development of therapeutics for ischemic diseases.

A carvedilol-responsive microRNA, miR-125b-5p protects the heart from acute myocardial infarction by repressing pro-apoptotic bak1 and klf13 in cardiomyocytes.

BACKGROUND: Cardiac injury is accompanied by dynamic changes in the expression of microRNAs (miRs), small non-coding RNAs that post-transcriptionally regulate target genes. MiR-125b-5p is downregulated in patients with end-stage dilated and ischemic cardiomyopathy, and has been proposed as a biomarker of heart failure. We previously reported that the ß-blocker carvedilol promotes cardioprotection via ß-arrestin-biased agonism of ß1-adrenergic receptor while stimulating miR-125b-5p processing in the mouse heart. We hypothesize that ß1-adrenergic receptor/ß-arrestin1-responsive miR-125b-5p confers the improvement of cardiac function and structure after acute myocardial infarction. METHODS AND RESULTS: Using cultured cardiomyocyte (CM) and in vivo approaches, we show that miR-125b-5p is an ischemic stress-responsive protector against CM apoptosis. CMs lacking miR-125b-5p exhibit increased susceptibility to stress-induced apoptosis, while CMs overexpressing miR-125b-5p have increased phospho-AKT pro-survival signaling. Moreover, we demonstrate that loss-of-function of miR-125b-5p in the mouse heart causes abnormalities in cardiac structure and function after acute myocardial infarction. Mechanistically, the improvement of cardiac function and structure elicited by miR-125b-5p is in part attributed to repression of the pro-apoptotic genes Bak1 and Klf13 in CMs. CONCLUSIONS: In conclusion, these findings reveal a pivotal role for miR-125b-5p in regulating CM survival during acute myocardial infarction.

ß-arrestin-biased agonism of ß-adrenergic receptor regulates Dicer-mediated microRNA maturation to promote cardioprotective signaling.

RATIONALE: MicroRNAs (miRs) are small, non-coding RNAs that function to post-transcriptionally regulate target genes. First transcribed as primary miR transcripts (pri-miRs), they are enzymatically processed by Drosha into premature miRs (pre-miRs) and further cleaved by Dicer into mature miRs. Initially discovered to desensitize ß-adrenergic receptor (ßAR) signaling, ß-arrestins are now well-appreciated to modulate multiple pathways independent of G protein signaling, a concept known as biased signaling. Using the ß-arrestin-biased ßAR ligand carvedilol, we previously showed that ß-arrestin1 (not ß-arrestin2)-biased ß1AR (not ß2AR) cardioprotective signaling stimulates Drosha-mediated processing of six miRs by forming a multi-protein nuclear complex, which includes ß-arrestin1, the Drosha microprocessor complex and a single-stranded RNA binding protein hnRNPA1. OBJECTIVE: Here, we investigate whether ß-arrestin-mediated ßAR signaling induced by carvedilol could regulate Dicer-mediated miR maturation in the cytoplasm and whether this novel mechanism promotes cardioprotective signaling. METHODS AND RESULTS: In mouse hearts, carvedilol indeed upregulates three mature miRs, but not their pre-miRs and pri-miRs, in a ß-arrestin 1- or 2-dependent manner. Interestingly, carvedilol-mediated activation of miR-466g or miR-532-5p, and miR-674 is dependent on ß2ARs and ß1ARs, respectively. Mechanistically, ß-arrestin 1 or 2 regulates maturation of three newly identified ßAR/ß-arrestin-responsive miRs (ß-miRs) by associating with the Dicer maturation RNase III enzyme on three pre-miRs of ß-miRs. Myocardial cell approaches uncover that despite their distinct roles in different cell types, ß-miRs act as gatekeepers of cardiac cell functions by repressing deleterious targets. CONCLUSIONS: Our findings indicate a novel role for ßAR-mediated ß-arrestin signaling activated by carvedilol in Dicer-mediated miR maturation, which may be linked to its protective mechanisms.

Circular noncoding RNAs as potential therapies and circulating biomarkers for cardiovascular diseases.

Recent advancements in genome-wide analyses and RNA-sequencing technologies led to the discovery of small noncoding RNAs, such as microRNAs (miRs), as well as both linear long noncoding RNAs (lncRNAs) and circular long noncoding RNAs (circRNAs). The importance of miRs and lncRNAs in the treatment, prognosis and diagnosis of cardiovascular diseases (CVDs) has been extensively reported. We also previously reviewed their implications in therapies and as biomarkers for CVDs. More recently, circRNAs have also emerged as important regulators in CVDs. CircRNAs are circular genome products that are generated by back splicing of specific regions of pre-messenger RNAs (pre-mRNAs). Growing interest in circRNAs led to the discovery of a wide array of their pathophysiological functions. CircRNAs have been shown to be key regulators of CVDs such as myocardial infarction, atherosclerosis, cardiomyopathy and cardiac fibrosis. Accordingly, circRNAs have been recently proposed as potential therapeutic targets and biomarkers for CVDs. In this review, we summarize the current state of the literature on circRNAs, starting with their biogenesis and global mechanisms of actions. We then provide a synopsis of their involvement in various CVDs. Lastly, we emphasize the great potential of circRNAs as biomarkers for the early detection of CVDs, and discuss several patents and recent papers that highlight the utilization of circRNAs as promising biomarkers.

Ninjurin1 is a novel factor to regulate angiogenesis through the function of pericytes.

BACKGROUND: Capillary pericytes (cPCs), the mural cells of microvessels, play an important role in the formation and maintenance of microvessels; however, little is known about the mechanisms of how cPCs regulate angiogenesis. To identify factors that modulate cPC function, genes whose levels were altered in cPCs during neovessel formation were identified through a microarray screen. METHODS AND RESULTS: Ninjurin1 (nerve injury-induced protein, Ninj1) was selected as a candidate factor for angiogenesis regulation. Ninj1 was expressed in capillary cells including endothelial cells (cECs) and was expressed at a higher level in cPCs. Hypoxia induced the gene expression of Ninj1 in addition of vascular endothelial growth factor (VEGF) in cPCs. When cPCs were co-incubated with a thoracic aorta in a three-dimensional Matrigel system, the length of the EC-tubes sprouting from the aorta was increased. Small interfering RNA-mediated downregulation of Ninj1 in cPCs enhanced these cPCs-mediated angiogenic effects, whereas overexpression of Ninj1 attenuated their effects. The production of angiogenic growth factors, such as VEGF and angiopoietin 1, by cPCs was enhanced by the downregulation of Ninj1, and reduced by the overexpression of Ninj1. CONCLUSIONS: Ninj1 is a novel regulator for the angiogenic effect of PCs. Specifically, Ninj1 negatively regulates the formation of neovessels, that is, the EC-tube, by reducing the trophic effects of cPCs.

Immortalized multipotent pericytes derived from the vasa vasorum in the injured vasculature. A cellular tool for studies of vascular remodeling and regeneration.

Adventitial microvessels, vasa vasorum in the vessel walls, have an active role in the vascular remodeling, although its mechanisms are still unclear. It has been reported that microvascular pericytes (PCs) possess mesenchymal plasticity. Therefore, microvessels would serve as a systemic reservoir of stem cells and contribute to the tissues remodeling. However, most aspects of the biology of multipotent PCs (mPCs), in particular of pathological microvessels are still obscure because of the lack of appropriate methods to detect and isolate these cells. In order to examine the characteristics of mPCs, we established immortalized cells residing in adventitial capillary growing at the injured vascular walls. We recently developed in vivo angiogenesis to observe adventitial microvessels using collagen-coated tube (CCT), which also can be used as an adventitial microvessel-rich tissue. By using the CCT, CD146- or NG2-positive cells were isolated from the adventitial microvessels in the injured arteries of mice harboring a temperature-sensitive SV40 T-antigen gene. Several capillary-derived endothelial cells (cECs) and PCs (cPCs) cell lines were established. cECs and cPCs maintain a number of key endothelial and PC features. Co-incubation of cPCs with cECs formed capillary-like structure in Matrigel. Three out of six cPC lines, termed capillary mPCs demonstrated both mesenchymal stem cell- and neuronal stem cell-like phenotypes, differentiating effectively into adipocytes, osteoblasts, as well as schwann cells. mPCs differentiated to ECs and PCs, and formed capillary-like structure on their own. Transplanted DsRed-expressing mPCs were resident in the capillary and muscle fibers and promoted angiogenesis and myogenesis in damaged skeletal muscle. Adventitial mPCs possess transdifferentiation potential with unique phenotypes, including the reconstitution of capillary-like structures. Their phenotype would contribute to the pathological angiogenesis associated with vascular remodeling. These cell lines also provide a reproducible cellular tool for high-throughput studies on angiogenesis, vascular remodeling, and regeneration as well.

Nerve growth factor stimulates regeneration of perivascular nerve, and induces the maturation of microvessels around the injured artery.

An immature vasa vasorum in the adventitia of arteries has been implicated in induction of the formation of unstable atherosclerotic plaques. Normalization/maturation of the vasa vasorum may be an attractive therapeutic approach for arteriosclerotic diseases. Nerve growth factor (NGF) is a pleotropic molecule with angiogenic activity in addition to neural growth effects. However, whether NGF affects the formation of microvessels in addition to innervation during pathological angiogenesis is unclear. In the present study, we show a new role for NGF in neovessels around injured arterial walls using a novel in vivo angiogenesis assay. The vasa vasorum around arterial walls was induced to grow using wire-mediated mouse femoral arterial injury. When collagen-coated tube (CCT) was placed beside the injured artery for 7-14 days, microvessels grew two-dimensionally in a thin layer on the CCT (CCT-membrane) in accordance with the development of the vasa vasorum. The perivascular nerve was found at not only arterioles but also capillaries in the CCT-membrane. Biodegradable hydrogels containing VEGF and NGF were applied around the injured artery/CCT. VEGF significantly increased the total length and instability of microvessels within the CCT-membrane. In contrast, NGF induced regeneration of the peripheral nerve around the microvessels and induced the maturation and stabilization of microvessels. In an ex vivo nerve-free angiogenesis assay, although NGF potentially stimulated vascular sprouting from aorta tissues, no effects of NGF on vascular maturation were observed. These data demonstrated that NGF had potent angiogenic effects on the microvessels around the injured artery, and especially induced the maturation/stabilization of microvessels in accordance with the regeneration of perivascular nerves.

Inducible deletion of microRNA activity in kidney mesenchymal cells exacerbates renal fibrosis.

MicroRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. However, the physiological functions of these non-coding RNAs in renal interstitial mesenchymal cells remain unclear. To conclusively evaluate the role of miRNAs, we generated conditional knockout (cKO) mice with platelet-derived growth factor receptor-ß (PDGFR-ß)-specific inactivation of the key miRNA pathway gene Dicer. The cKO mice were subjected to unilateral ureteral ligation, and renal interstitial fibrosis was quantitatively evaluated using real-time polymerase chain reaction and immunofluorescence staining. Compared with control mice, cKO mice had exacerbated interstitial fibrosis exhibited by immunofluorescence staining and mRNA expression of PDGFR-ß. A microarray analysis showed decreased expressions of miR-9-5p, miR-344g-3p, and miR-7074-3p in cKO mice compared with those in control mice, suggesting an association with the increased expression of PDGFR-ß. An analysis of the signaling pathways showed that the major transcriptional changes in cKO mice were related to smooth muscle cell differentiation, regulation of DNA metabolic processes and the actin cytoskeleton, positive regulation of fibroblast proliferation and Ras protein signal transduction, and focal adhesion-PI3K/Akt/mTOR signaling pathways. Depletion of Dicer in mesenchymal cells may downregulate the signaling pathway related to miR-9-5p, miR-344g-3p, and miR-7074-3p, which can lead to the progression of chronic kidney disease. These findings highlight the possibility for future diagnostic or therapeutic developments for renal fibrosis using miR-9-5p, miR-344g-3p, and miR-7074-3p.

Apurinic/apyrimidinic endonucelase 1 maintains adhesion of endothelial progenitor cells and reduces neointima formation.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that processes DNA-repair function and controls cellular response to oxidative stress. Endothelial progenitor cells (EPCs) are recruited to oxidative stress-rich injured vascular walls and positively contribute to vascular repair and endothelialization. We hypothesized that APE1 functions for EPCs-mediated inhibition of neointima formation in injured vasculature. EPCs isolated from bone marrow cells of C57BL6 mice (12-16 wk old) were able to survive in the presence of hydrogen peroxide (H2O2; up to 1,000 µM) due to the highly expressed reactive oxygen species (ROS) scavengers. However, adhesion capacity of EPCs was significantly inhibited by H2O2 (100 µM) even though an intracellular ROS was retained at small level. An APE1-selective inhibitor or RNA interference-mediated knockdown of endogenous APE1 in EPCs aggravated the H2O2-mediated inhibition of EPCs-adhesion. In contrast, when APE1 was overexpressed in EPCs using an adenovirus harboring the APE1 gene (APE-EPCs), adhesion was significantly improved during oxidative stress. To examine in vivo effects of APE1 in EPCs, APE-EPCs were transplanted via the tail vein after wire-mediated injury of the mouse femoral artery. The number of adherent EPCs at injured vascular walls and the vascular repair effect of EPCs were enhanced in APE-EPCs compared with control EPCs. Among the cellular functions of EPCs, adhesion is especially sensitive to oxidative stress. APE1 enhances in vivo vascular repair effects of EPCs in part through the maintenance of adhesion properties of EPCs. APE1 may be a novel and useful target gene for effective cellular transplantation therapy.

Characteristics of Physiological Parameters of Japanese Black Calves Relate to Carcass Weight.

This study aimed to identify the growth performance and blood factors associated with carcass weight in Japanese Black calves based on 675 performance tests and field carcass records. We measured the body weight, withers height, and chest girth at the start of fattening age (approximately 8-10 months) and analyzed eight blood factors, including vitamins and metabolites. Single- and two-trait animal models were used to estimate the heritability and genetic correlations. The heritability estimates for growth performance were moderate to high (ranging from 0.48 to 0.74), and those for blood metabolites were low to moderate (ranging from 0.19 to 0.51). Estimates for genetic correlations of carcass or body weight with body weight, withers height, and chest girth were high (ranging from 0.42 to 0.80). The body weight and withers height at 8 months of age are possibly closely related to the final carcass weight. The blood metabolites associated with body weight were vitamin E in steers (castrated males) and ß-carotene in heifers. Our findings indicate that body measurements and blood metabolites measured during the growing period could be used to determine the nutritional and physiological status of cattle as well as predict carcass weight.

SPRR1A is a key downstream effector of MiR-150 during both maladaptive cardiac remodeling in mice and human cardiac fibroblast activation.

MicroRNA-150 (miR-150) is conserved between rodents and humans, is significantly downregulated during heart failure (HF), and correlates with patient outcomes. We previously reported that miR-150 is protective during myocardial infarction (MI) in part by decreasing cardiomyocyte (CM) apoptosis and that proapoptotic small proline-rich protein 1a (Sprr1a) is a direct CM target of miR-150. We also showed that Sprr1a knockdown in mice improves cardiac dysfunction and fibrosis post-MI and that Sprr1a is upregulated in pathological mouse cardiac fibroblasts (CFs) from ischemic myocardium. However, the direct functional relationship between miR-150 and SPRR1A during both post-MI remodeling in mice and human CF (HCF) activation was not established. Here, using a novel miR-150 knockout;Sprr1a-hypomorphic (Sprr1ahypo/hypo) mouse model, we demonstrate that Sprr1a knockdown blunts adverse post-MI effects caused by miR-150 loss. Moreover, HCF studies reveal that SPRR1A is upregulated in hypoxia/reoxygenation-treated HCFs and is downregulated in HCFs exposed to the cardioprotective ß-blocker carvedilol, which is inversely associated with miR-150 expression. Significantly, we show that the protective roles of miR-150 in HCFs are directly mediated by functional repression of profibrotic SPRR1A. These findings delineate a pivotal functional interaction between miR-150 and SPRR1A as a novel regulatory mechanism pertinent to CF activation and ischemic HF.

MiR-150 blunts cardiac dysfunction in mice with cardiomyocyte loss of ß1-adrenergic receptor/ß-arrestin signaling and controls a unique transcriptome.

The ß1-adrenergic receptor (ß1AR) is found primarily in hearts (mainly in cardiomyocytes [CMs]) and ß-arrestin-mediated ß1AR signaling elicits cardioprotection through CM survival. We showed that microRNA-150 (miR-150) is upregulated by ß-arrestin-mediated ß1AR signaling and that CM miR-150 inhibits maladaptive remodeling post-myocardial infarction. Here, we investigate whether miR-150 rescues cardiac dysfunction in mice bearing CM-specific abrogation of ß-arrestin-mediated ß1AR signaling. Using CM-specific transgenic (TG) mice expressing a mutant ß1AR (G protein-coupled receptor kinase [GRK]-ß1AR that exhibits impairment in ß-arrestin-mediated ß1AR signaling), we first generate a novel double TG mouse line overexpressing miR-150. We demonstrate that miR-150 is sufficient to improve cardiac dysfunction in CM-specific GRK-ß1AR TG mice following chronic catecholamine stimulation. Our genome-wide circular RNA, long noncoding RNA (lncRNA), and mRNA profiling analyses unveil a subset of cardiac ncRNAs and genes as heretofore unrecognized mechanisms for beneficial actions of ß1AR/ß-arrestin signaling or miR-150. We further show that lncRNA Gm41664 and GDAP1L1 are direct novel upstream and downstream regulators of miR-150. Lastly, CM protective actions of miR-150 are attributed to repressing pro-apoptotic GDAP1L1 and are mitigated by pro-apoptotic Gm41664. Our findings support the idea that miR-150 contributes significantly to ß1AR/ß-arrestin-mediated cardioprotection by regulating unique ncRNA and gene signatures in CMs.

MiR-150 Attenuates Maladaptive Cardiac Remodeling Mediated by Long Noncoding RNA MIAT and Directly Represses Profibrotic Hoxa4.

BACKGROUND: MicroRNA-150 (miR-150) plays a protective role in heart failure (HF). Long noncoding RNA, myocardial infarction-associated transcript (MIAT) regulates miR-150 function in vitro by direct interaction. Concurrent with miR-150 downregulation, MIAT is upregulated in failing hearts, and gain-of-function single-nucleotide polymorphisms in MIAT are associated with increased risk of myocardial infarction (MI) in humans. Despite the correlative relationship between MIAT and miR-150 in HF, their in vivo functional relationship has never been established, and molecular mechanisms by which these 2 noncoding RNAs regulate cardiac protection remain elusive. METHODS: We use MIAT KO (knockout), Hoxa4 (homeobox a4) KO, MIAT TG (transgenic), and miR-150 TG mice. We also develop DTG (double TG) mice overexpressing MIAT and miR-150. We then use a mouse model of MI followed by cardiac functional, structural, and mechanistic studies by echocardiography, immunohistochemistry, transcriptome profiling, Western blotting, and quantitative real-time reverse transcription-polymerase chain reaction. Moreover, we perform expression analyses in hearts from patients with HF. Lastly, we investigate cardiac fibroblast activation using primary adult human cardiac fibroblasts and in vitro assays to define the conserved MIAT/miR-150/HOXA4 axis. RESULTS: Using novel mouse models, we demonstrate that genetic overexpression of MIAT worsens cardiac remodeling, while genetic deletion of MIAT protects hearts against MI. Importantly, miR-150 overexpression attenuates the detrimental post-MI effects caused by MIAT. Genome-wide transcriptomic analysis of MIAT null mouse hearts identifies Hoxa4 as a novel downstream target of the MIAT/miR-150 axis. Hoxa4 is upregulated in cardiac fibroblasts isolated from ischemic myocardium and subjected to hypoxia/reoxygenation. HOXA4 is also upregulated in patients with HF. Moreover, Hoxa4 deficiency in mice protects the heart from MI. Lastly, protective actions of cardiac fibroblast miR-150 are partially attributed to the direct and functional repression of profibrotic Hoxa4. CONCLUSIONS: Our findings delineate a pivotal functional interaction among MIAT, miR-150, and Hoxa4 as a novel regulatory mechanism pertinent to ischemic HF.

Interactions between noncoding RNAs as epigenetic regulatory mechanisms in cardiovascular diseases.

Cardiovascular diseases (CVDs) represent the foremost cause of mortality in the United States and worldwide. It is estimated that CVDs account for approximately 17.8 million deaths each year. Despite the advances made in understanding cellular mechanisms and gene mutations governing the pathophysiology of CVDs, they remain a significant cause of mortality and morbidity. A major segment of mammalian genomes encodes for genes that are not further translated into proteins. The roles of the majority of such noncoding ribonucleic acids (RNAs) have been puzzling for a long time. However, it is becoming increasingly clear that noncoding RNAs (ncRNAs) are dynamically expressed in different cell types and have a comprehensive selection of regulatory roles at almost every step involved in DNAs, RNAs and proteins. Indeed, ncRNAs regulate gene expression through epigenetic interactions, through direct binding to target sequences, or by acting as competing endogenous RNAs. The profusion of ncRNAs in the cardiovascular system suggests that they may modulate complex regulatory networks that govern cardiac physiology and pathology. In this review, we summarize various functions of ncRNAs and highlight the recent literature on interactions between ncRNAs with an emphasis on cardiovascular disease regulation. Furthermore, as the broad-spectrum of ncRNAs potentially establishes new avenues for therapeutic development targeting CVDs, we discuss the innovative prospects of ncRNAs as therapeutic targets for CVDs.

Sarcopenia-derived exosomal micro-RNA 16-5p disturbs cardio-repair via a pro-apoptotic mechanism in myocardial infarction in mice.

Sarcopenia is a pathophysiological malfunction induced by skeletal muscle atrophy. Several studies reported an association between sarcopenia-induced cardiac cachexia and poor prognosis in heart disease. However, due to lack of an established animal models, the underlying mechanism of disturbed cardiac repair accompanied with sarcopenia remains poorly understood. Here, we developed a novel sarcopenia-induced cardiac repair disturbance mouse model induced by tail suspension (TS) after cardiac ischemia and reperfusion (I/R). Importantly, we identified a specific exosomal-microRNA marker, miR-16-5p, in the circulating exosomes of I/R-TS mice. Of note, sarcopenia after I/R disturbed cardiac repair and raised the level of circulating-exosomal-miR-16-5p secreting from both the atrophic limbs and heart of TS mice. Likewise, miR-16-5p mimic plasmid disturbed cardiac repair in I/R mice directly. Additionally, in neonatal rat ventricular myocytes (NRVMs) cultured in vitro under hypoxic conditions in the presence of a miR-16-5p mimic, we observed increased apoptosis through p53 and Caspase3 upregulation, and also clarified that autophagosomes were decreased in NRVMs via SESN1 transcript interference-mediated mTOR activation. In conclusion, we show the pro-apoptotic effect of sarcopenia-derived miR-16-5p, which may be behind the exacerbation of myocardial infarction. Therefore, miR-16-5p can be a novel therapeutic target in the context of cardiac repair disturbances in sarcopenia-cachexia.

Cardiomyocyte microRNA-150 confers cardiac protection and directly represses proapoptotic small proline-rich protein 1A.

MicroRNA-150 (miR-150) is downregulated in patients with multiple cardiovascular diseases and in diverse mouse models of heart failure (HF). miR-150 is significantly associated with HF severity and outcome in humans. We previously reported that miR-150 is activated by ß-blocker carvedilol (Carv) and plays a protective role in the heart using a systemic miR-150 KO mouse model. However, mechanisms that regulate cell-specific miR-150 expression and function in HF are unknown. Here, we demonstrate that potentially novel conditional cardiomyocyte-specific (CM-specific) miR-150 KO (miR-150 cKO) in mice worsens maladaptive cardiac remodeling after myocardial infarction (MI). Genome-wide transcriptomic analysis in miR-150 cKO mouse hearts identifies small proline-rich protein 1a (Sprr1a) as a potentially novel target of miR-150. Our studies further reveal that Sprr1a expression is upregulated in CMs isolated from ischemic myocardium and subjected to simulated ischemia/reperfusion, while its expression is downregulated in hearts and CMs by Carv. We also show that left ventricular SPRR1A is upregulated in patients with HF and that Sprr1a knockdown in mice prevents maladaptive post-MI remodeling. Lastly, protective roles of CM miR-150 are, in part, attributed to the direct and functional repression of proapoptotic Sprr1a. Our findings suggest a crucial role for the miR-150/SPRR1A axis in regulating CM function post-MI.

Capillary-resident EphA7+ pericytes are multipotent cells with anti-ischemic effects through capillary formation.

The presence of pericytes (PCs) with multipotency and broad distribution along capillary suggests that microvasculature plays a role not only as a duct for blood fluid transport but also as a stem cell niche that contributes to tissue maintenance and regeneration. The lack of an appropriate marker for multipotent PCs still limits our understanding of their pathophysiological roles. We identified the novel marker EphA7 to detect multipotent PCs using microarray analysis of an immortalized PC library. PCs were isolated from microvessels of mouse subcutaneous adipose tissues, then EphA7+ PCs called capillary stem cells (CapSCs) were separated from EphA7- control PCs (ctPCs) using fluorescence-activated cell sorting system. CapSCs had highly multipotency that enabled them to differentiate into mesenchymal and neuronal lineages compared with ctPCs. CapSCs also differentiated into endothelial cells and PCs to form capillary-like structures by themselves. Transplantation of CapSCs into ischemic tissues significantly improved blood flow recovery in hind limb ischemia mouse model due to vascular formation compared with that of ctPCs and adipose stromal cells. These data demonstrate that EphA7 identifies a subpopulation of multipotent PCs that have high angiogenesis and regenerative potency and are an attractive target for regenerative therapies.