Overexpression of a pearl millet WRKY transcription factor gene, PgWRKY74, in Arabidopsis retards shoot growth under dehydration and salinity-stressed conditions.

Pearl millet (Cenchrus americanus) is a cereal crop that can tolerate high temperatures, drought, and low-fertility conditions where other crops lose productivity. However, genes regulating this ability are largely unknown. Transcription factors (TFs) regulate transcription of their target genes, regulate downstream biological processes, and thus are candidates for regulators of such tolerance of pearl millet. PgWRKY74 encodes a group IIc WRKY TF in pearl millet and is downregulated by drought. PgWRKY74 may have a role in drought tolerance. The objective of this study was to gain insights into the physiological and biochemical functions of PgWRKY74. Yeast one-hybrid and gel shift assays were performed to examine transcriptional activation potential and deoxyribonucleic acid (DNA)-binding ability, respectively. Transgenic Arabidopsis thaliana plants overexpressing PgWRKY74-green fluorescent protein (GFP) fusion gene were generated and tested for growth and stress-responsive gene expression under mannitol and NaCl-stressed conditions. A construct with PgWRKY74 enabled yeast reporter cells to survive on test media in the yeast one-hybrid assays. The electrophoretic mobility of DNA with putative WRKY TF-binding motifs was lower in the presence of a recombinant PgWRKY74 protein than its absence. The PgWRKY74-GFP-overexpressing Arabidopsis plants exhibited smaller rosette areas than did wild-type plants under mannitol-stressed and NaCl-stressed conditions, and exhibited weaker expression of RD29B, which is induced by the stress-related phytohormone abscisic acid (ABA), under the mannitol-stressed condition. PgWRKY74 have transcriptional activation potential and DNA-binding ability, and can negatively regulate plant responses to mannitol and NaCl stresses, possibly by decreasing ABA levels or ABA sensitivity.

Data of RNA sequencing of pearl millet panicles treated with a high temperature.

Pearl millet (Pennisetum glaucum) is a cereal crop that can grow and set seeds even under drought, high temperatures and nutrient-poor conditions. Panicles of two pearl millet cultivars that differ in seed-setting rates were exposed to two different high-temperature treatments at three different developmental stages with three replicates, and RNA was prepared from these panicles. The resulting RNA samples were subjected to sequencing with the Illumina NovaSeq 6000 sequencer. The obtained data were 150-base-paired-end reads and were approximately 5 Gb/sample in total. These read data were deposited as those for a project in the NCBI (National Center for Biotechnology Information) BioProject database.

Understanding genetic diversity in drought-adaptive hybrid parental lines in pearl millet.

Information on genetic diversity and population structure is helpful to strategize enhancing the genetic base of hybrid parental lines in breeding programs. The present study determined the population structure and genetic diversity of 109 pearl millet hybrid parental lines, known for their better adaptation and performance in drought-prone environments, using 16,472 single nucleotide polymorphic (SNP) markers generated from GBS (genotyping-by-sequencing) platforms. The SNPs were distributed uniformly across the pearl millet genome and showed considerable genetic diversity (0.337), expected heterozygosity (0.334), and observed heterozygosity (0.031). Most of the pairs of lines (78.36%) had Identity-by-State (IBS) based genetic distances of more than 0.3, indicating a significant amount of genetic diversity among the parental lines. Bayesian model-based population stratification, neighbor-joining phylogenetic analysis, and principal coordinate analysis (PCoA) differentiated all hybrid parental lines into two clear-cut major groups, one each for seed parents (B-lines) and pollinators (R-lines). Majority of parental lines sharing common parentages were found grouped in the same cluster. Analysis of molecular variance (AMOVA) revealed 7% of the variation among subpopulations, and 93% of the variation was attributable to within sub-populations. Chromosome 3 had the highest number of LD regions. Genomic LD decay distance was 0.69 Mb and varied across the different chromosomes. Genetic diversity based on 11 agro-morphological and grain quality traits also suggested that the majority of the B- and R-lines were grouped into two major clusters with few overlaps. In addition, the combined analysis of phenotypic and genotypic data showed similarities in the population grouping patterns. The present study revealed the uniqueness of most of the inbred lines, which can be a valuable source of new alleles and help breeders to utilize these inbred lines for the development of hybrids in drought-prone environments.

ORMDL3 regulates NLRP3 inflammasome activation by maintaining ER-mitochondria contacts in human macrophages and dictates ulcerative colitis patient outcome.

Genome-wide association studies in inflammatory bowel disease have identified risk loci in the orosomucoid-like protein 3/ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) gene to confer susceptibility to ulcerative colitis (UC), but the underlying functional relevance remains unexplored. Here, we found that a subpopulation of the UC patients who had higher disease activity shows enhanced expression of ORMDL3 compared to the patients with lower disease activity and the non-UC controls. We also found that the patients showing high ORMDL3 mRNA expression have elevated interleukin-1ß cytokine levels indicating positive correlation. Further, knockdown of ORMDL3 in the human monocyte-derived macrophages resulted in significantly reduced interleukin-1ß release. Mechanistically, we report for the first time that ORMDL3 contributes to a mounting inflammatory response via modulating mitochondrial morphology and activation of the NLRP3 inflammasome. Specifically, we observed an increased fragmentation of mitochondria and enhanced contacts with the endoplasmic reticulum (ER) during ORMDL3 over-expression, enabling efficient NLRP3 inflammasome activation. We show that ORMDL3 that was previously known to be localized in the ER also becomes localized to mitochondria-associated membranes and mitochondria during inflammatory conditions. Additionally, ORMDL3 interacts with mitochondrial dynamic regulating protein Fis-1 present in the mitochondria-associated membrane. Accordingly, knockdown of ORMDL3 in a dextran sodium sulfate -induced colitis mouse model showed reduced colitis severity. Taken together, we have uncovered a functional role for ORMDL3 in mounting inflammation during UC pathogenesis by modulating ER-mitochondrial contact and dynamics.

Musashi-2 causes cardiac hypertrophy and heart failure by inducing mitochondrial dysfunction through destabilizing Cluh and Smyd1 mRNA.

Regulation of RNA stability and translation by RNA-binding proteins (RBPs) is a crucial process altering gene expression. Musashi family of RBPs comprising Msi1 and Msi2 is known to control RNA stability and translation. However, despite the presence of MSI2 in the heart, its function remains largely unknown. Here, we aim to explore the cardiac functions of MSI2. We confirmed the presence of MSI2 in the adult mouse, rat heart, and neonatal rat cardiomyocytes. Furthermore, Msi2 was significantly enriched in the heart cardiomyocyte fraction. Next, using RNA-seq data and isoform-specific PCR primers, we identified Msi2 isoforms 1, 4, and 5, and two novel putative isoforms labeled as Msi2 6 and 7 to be expressed in the heart. Overexpression of Msi2 isoforms led to cardiac hypertrophy in cultured cardiomyocytes. Additionally, Msi2 exhibited a significant increase in a pressure-overload model of cardiac hypertrophy. We selected isoforms 4 and 7 to validate the hypertrophic effects due to their unique alternative splicing patterns. AAV9-mediated overexpression of Msi2 isoforms 4 and 7 in murine hearts led to cardiac hypertrophy, dilation, heart failure, and eventually early death, confirming a pathological function for Msi2. Using global proteomics, gene ontology, transmission electron microscopy, seahorse, and transmembrane potential measurement assays, increased MSI2 was found to cause mitochondrial dysfunction in the heart. Mechanistically, we identified Cluh and Smyd1 as direct downstream targets of Msi2. Overexpression of Cluh and Smyd1 inhibited Msi2-induced cardiac malfunction and mitochondrial dysfunction. Collectively, we show that Msi2 induces hypertrophy, mitochondrial dysfunction, and heart failure.

CLUH functions as a negative regulator of inflammation in human macrophages and determines ulcerative colitis pathogenesis.

Altered mitochondrial function without a well-defined cause has been documented in patients with ulcerative colitis (UC). In our efforts to understand UC pathogenesis, we observed reduced expression of clustered mitochondrial homolog (CLUH) only in the active UC tissues compared with the unaffected areas from the same patient and healthy controls. Stimulation with bacterial Toll-like receptor (TLR) ligands similarly reduced CLUH expression in human primary macrophages. Further, CLUH negatively regulated secretion of proinflammatory cytokines IL-6 and TNF-α and rendered a proinflammatory niche in TLR ligand-stimulated macrophages. CLUH was further found to bind to mitochondrial fission protein dynamin related protein 1 (DRP1) and regulated DRP1 transcription in human macrophages. In the TLR ligand-stimulated macrophages, absence of CLUH led to enhanced DRP1 availability for mitochondrial fission, and a smaller dysfunctional mitochondrial pool was observed. Mechanistically, this fissioned mitochondrial pool in turn enhanced mitochondrial ROS production and reduced mitophagy and lysosomal function in CLUH-knockout macrophages. Remarkably, our studies in the mouse model of colitis with CLUH knockdown displayed exacerbated disease pathology. Taken together, this is the first report to our knowledge explaining the role of CLUH in UC pathogenesis, by means of regulating inflammation via maintaining mitochondrial-lysosomal functions in the human macrophages and intestinal mucosa.

RIPK3-MLKL signaling activates mitochondrial CaMKII and drives intrarenal extracellular matrix production during CKD.

Intrarenal extracellular matrix production or kidney fibrosis is a prevalent feature of all forms of chronic kidney disease (CKD). The transforming growth factor-beta (TGFß) is believed to be a major driver of extracellular matrix production. Nevertheless, anti-TGFß therapies have consistently failed to reduce extracellular matrix production in CKD patients indicating the need for novel therapeutic strategies. We have previously shown that necroinflammation contributes to acute kidney injury. Here, we show that chronic/persistent necroinflammation drives intrarenal extracellular matrix production during CKD. We found that renal expression of receptor-interacting protein kinase-1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL) increases with the production of intrarenal extracellular matrix and declined kidney function in both humans and mice. Furthermore, we found that TGFß exposure induces the translocation of RIPK3 and MLKL to mitochondria resulting in mitochondrial dysfunction and ROS production. Mitochondrial ROS activates the serine-threonine kinase calcium/calmodulin-dependent protein kinases-II (CaMKII) that increases phosphorylation of Smad2/3 and subsequent production of alpha-smooth muscle actin (αSMA), collagen (Col) 1α1, etc. in response to TGFß during the intrarenal extracellular matrix production. Consistent with this, deficiency or knockdown of RIPK3 or MLKL as well as pharmacological inhibition of RIPK1, RIPK3, and CaMKII prevents the intrarenal extracellular matrix production in oxalate-induced CKD and unilateral ureteral obstruction (UUO). Together, RIPK1, RIPK3, MLKL, CaMKII, and Smad2/3 are molecular targets to inhibit intrarenal extracellular matrix production and preserve kidney function during CKD.

Understanding Heterosis, Genetic Effects, and Genome Wide Associations for Forage Quantity and Quality Traits in Multi-Cut Pearl Millet.

Pearl millet is an important food and fodder crop cultivated in the arid and semi-arid regions of Africa and Asia, and is now expanding to other regions for forage purpose. This study was conducted to better understand the forage quantity and quality traits to enhance the feed value of this crop. Two sets of pearl millet hybrids (80 single cross hybrids in Set-I and 50 top cross hybrids in Set-II) along with their parents evaluated multi-locationally for the forage-linked traits under multi-cut (two cuts) system revealed significant variability for the forage traits in the hybrids and parents. The mean better parent heterosis (BPH) for total dry forage yield (TDFY) was 136% across all the single cross hybrids and 57% across all the top cross hybrids. The mean BPH for in vitro organic matter digestibility (IVOMD) varied from -11 to 7% in the single cross hybrids and -13 to 11% in the top cross hybrids across cuts. The findings of TDFY and IVOMD heterosis in these sets indicated the potential of improvement of the hybrid cultivars for forage quantity and quality in forage pearl millet. The parental lines single cross parent (SCP)-L02, SCP-L06, and top cross parent (TCP)-T08 found superior in the forage quantity and quality traits can be utilized in the future breeding programs. Most of the forage traits were found to be controlled by using the non-additive gene action. A diverse panel of 105 forage-type hybrid parents (Set-III) genotyped following genotyping by sequencing (GBS) and phenotyped for crude protein (CP) and IVOMD under multi-cuts for 2 years identified one stable significant single nucleotide polymorphism (SNP) on LG4 for CP, and nine SNPs for IVOMD distributed across all the linkage groups except on LG2. The identified loci, once validated, then could be used for the forage quality traits improvement in pearl millet through marker-assisted selection.

Telomerase therapy attenuates cardiotoxic effects of doxorubicin.

Doxorubicin is one of the most potent chemotherapeutic agents. However, its clinical use is restricted due to the severe risk of cardiotoxicity, partially attributed to elevated production of reactive oxygen species (ROS). Telomerase canonically maintains telomeres during cell division but is silenced in adult hearts. In non-dividing cells such as cardiomyocytes, telomerase confers pro-survival traits, likely owing to the detoxification of ROS. Therefore, we hypothesized that pharmacological overexpression of telomerase may be used as a therapeutic strategy for the prevention of doxorubicin-induced cardiotoxicity. We used adeno-associated virus (AAV)-mediated gene therapy for long-term expression of telomerase in in vitro and in vivo models of doxorubicin-induced cardiotoxicity. Overexpression of telomerase protected the heart from doxorubicin-mediated apoptosis and rescued cardiac function, which was accompanied by preserved cardiomyocyte size. At the mechanistic level, we observed altered mitochondrial morphology and dynamics in response to telomerase expression. Complementary in vitro experiments confirmed the anti-apoptotic effects of telomerase overexpression in human induced pluripotent stem cell-derived cardiomyocytes after doxorubicin treatment. Strikingly, elevated levels of telomerase translocated to the mitochondria upon doxorubicin treatment, which helped to maintain mitochondrial function. Thus, telomerase gene therapy could be a novel preventive strategy for cardiotoxicity by chemotherapy agents such as the anthracyclines.

Comprehensive analysis of NAC transcription factor family uncovers drought and salinity stress response in pearl millet (Pennisetum glaucum).

BACKGROUND: Pearl millet (Pennisetum glaucum) is a cereal crop that possesses the ability to withstand drought, salinity and high temperature stresses. The NAC [NAM (No Apical Meristem), ATAF1 (Arabidopsis thaliana Activation Factor 1), and CUC2 (Cup-shaped Cotyledon)] transcription factor family is one of the largest transcription factor families in plants. NAC family members are known to regulate plant growth and abiotic stress response. Currently, no reports are available on the functions of the NAC family in pearl millet. RESULTS: Our genome-wide analysis found 151 NAC transcription factor genes (PgNACs) in the pearl millet genome. Thirty-eight and 76 PgNACs were found to be segmental and dispersed duplicated respectively. Phylogenetic analysis divided these NAC transcription factors into 11 groups (A-K). Three PgNACs (- 073, - 29, and - 151) were found to be membrane-associated transcription factors. Seventeen other conserved motifs were found in PgNACs. Based on the similarity of PgNACs to NAC proteins in other species, the functions of PgNACs were predicted. In total, 88 microRNA target sites were predicted in 59 PgNACs. A previously performed transcriptome analysis suggests that the expression of 30 and 42 PgNACs are affected by salinity stress and drought stress, respectively. The expression of 36 randomly selected PgNACs were examined by quantitative reverse transcription-PCR. Many of these genes showed diverse salt- and drought-responsive expression patterns in roots and leaves. These results confirm that PgNACs are potentially involved in regulating abiotic stress tolerance in pearl millet. CONCLUSION: The pearl millet genome contains 151 NAC transcription factor genes that can be classified into 11 groups. Many of these genes are either upregulated or downregulated by either salinity or drought stress and may therefore contribute to establishing stress tolerance in pearl millet.

MiRNA-181a is a novel regulator of aldosterone-mineralocorticoid receptor-mediated cardiac remodelling.

AIM: The aldosterone-mineralocorticoid receptor (Aldo-MR) pathway is activated during cardiac stress, such as hypertension, myocardial infarction (MI), and heart failure. The importance of Aldo and MR in the pathogenesis of cardiac diseases is well established; however, the regulatory mechanisms behind Aldo/MR-induced cardiac remodelling remain uncertain. We here investigated potential miRNA-mediated regulation of the Aldo-MR pathway to improve mechanistic understanding. METHODS AND RESULTS: High-throughput screening of 2,555 miRNAs using an MR responsive stable cardiomyocyte cell line (MMTV-GFP-HL-1) identified miR-181a as a potential regulator of Aldo-MR pathway. MiR-181a was found to downregulate the expression of Ngal (lipocalin-2), a well-established downstream effector molecule of Aldo-MR. In addition, Aldo-induced cellular hypertrophy decreased significantly upon miR-181a overexpression. Genetic miR-181 knockout in murine MI model led to deteriorated cardiac function, cardiac remodelling, and activation of Aldo-MR pathway while AAV9-mediated miR-181a overexpression improved cardiac function and deactivated Aldo-MR pathway proving a cardio-protective role of miR-181a. Global RNA sequencing of cells under Aldo treatment with/without miR-181a overexpression identified potential miR-181a targets functionally contributing to Aldo-MR pathway. Adamts1, a direct target of miR-181a, was found to be downregulated with miR-181a overexpression and upregulated with inhibition. Similar to miR-181a overexpression, siRNA-mediated inhibition of Adamts1 inhibited Aldo-MR pathway. CONCLUSION: We here show that miR-181a is a novel regulator of the Aldo-MR pathway regulating the levels of Ngal via direct targeting of Adamts1. This new insight establishes miR-181a as a factor of immense value participating in downstream networks of Aldo-MR pathway. Our in vivo studies further confirmed miR-181a as cardio-protective under MI stress. Thus, miR-181a's involvement in Aldo-MR-mediated cardiac remodelling confers it with tremendous potential to be developed further as a new therapeutic target.

Non-coding RNAs: Regulators of valvular calcification.

There is currently a growing global burden of valvular heart diseases due to aging populations and changing lifestyles. Valvular heart diseases mainly include the malfunctioning of aortic and mitral valves and are characterized by extensive tissue remodeling, which includes calcification, endothelial dysfunction, and endothelial-mesenchymal transition. These valvular remodeling processes are known to be regulated by protein-coding genes as well as non-coding genes. Here, we have summarized studies highlighting the non-coding RNA mediated regulation of valvular tissue remodeling and their potential therapeutic benefits. Additionally, studies investigating the diagnostic capability of circulating non-coding RNA molecules in valvular diseases are also summarized. Overall, of the various candidates, several studies have highlighted miR-214 and miR-204 as central regulators of valvular calcification.

Formation of heterotic pools and understanding relationship between molecular divergence and heterosis in pearl millet [Pennisetum glaucum (L.) R. Br.].

The present investigation was made to generate information on the heterotic pools amongst pearl millet hybrid parents. A set of 17 representative parents was selected from a diverse set of 147 hybrid parents using SSR based genetic distance (GD) and clustering pattern; 136 hybrids were developed in diallel fashion and evaluated at two locations in India. Moderate positive significant correlation (r = 0.37, p<0.01) and (r = 0.33, p<0.01) was found between GD and mid-parent heterosis (MPH) and better-parent heterosis (BPH), respectively, for grain yield for all the hybrids. Higher correlation between genetically closer individuals was observed for grain yield heterosis when the parents of B- and R- crosses had lesser genetic distance (<0.68 GD) in comparison to those parental combinations having GD higher than 0.68, indicating that the GD based predictions for grain yield are better when the parents are genetically related than when they are genetically diverse. In this study, all the pearl millet hybrid parents seems to exist in two broad-based heterotic pools; one each represented by seed and restorer parents as B × R hybrids showed highest mean heterosis for grain yield than either of B × B or R × R crosses. Further, four heterotic pools have been identified in this diverse set of hybrid parents of pearl millet, two each for seed parents (HPB1 and HPB2) and for restorer parents (HPR3 and HPR4). Among these, HPB1 × HPR3 was identified having the highest heterotic level, and could be further used to develop higher yielding pearl millet hybrids.

Long non-coding RNAs: A crucial part of the vasculature puzzle.

With the advent of empowering sequencing techniques over the past two decades, the scientific community has uncovered many underlying secrets of our genome. Non-coding transcripts covering a staggering 98% of our genome strongly suggest their involvement in diverse cellular pathways. A special class of non-coding RNAs (ncRNAs) namely long non-coding RNAs (lncRNAs) has garnered tremendous attention considering its implications in multiple developmental and pathophysiological processes. Accumulating evidence has established lncRNAs as robust regulators of pathways ranging from embryonic cell development to ultimate diseased phenotype. Here, in particular, we summarize the lncRNAs actively participating in the development of our vasculature and the ones which function as drivers or modulators of fatal vascular diseases.

Noncoding RNAs: potential regulators in cardioncology.

Cancer is the leading cause of morbidity and mortality in the United States and globally. Owing to improved early diagnosis and advances in oncological therapeutic options, the number of cancer survivors has steadily increased. Such efficient cancer therapies have also lead to alarming increase in cardiovascular complications in a significant proportion of cancer survivors, due to adverse cardiovascular effects such as cardiotoxicity, cardiac atrophy, and myocarditis. This has emerged as a notable concern in healthcare and given rise to the new field of cardioncology, which aims at understanding the processes that occur in the two distinct disorders and how they interact to influence the progression of each other. A key player in both cancer and heart failure is the genome, which is predominantly transcribed to noncoding RNAs (ncRNAs). Since the emergence of ncRNAs as master regulators of gene expression, several reports have shown the relevance of ncRNAs in cancer and cardiovascular disorders. However, the knowledge is quite limited regarding the relevance of ncRNAs in cardioncology. The objective of this review is to summarize the current knowledge of ncRNAs in the context of cardioncology. Furthermore, the therapeutic strategies as well as the prospective translational applications of these ncRNA molecules to the clinics are also discussed.

miR-212/132 Cluster Modulation Prevents Doxorubicin-Mediated Atrophy and Cardiotoxicity.

Improved therapy of cancer has significantly increased the lifespan of patients. However, cancer survivors face an increased risk of cardiovascular complications due to adverse effects of cancer therapies. The chemotherapy drug doxorubicin is well known to induce myofibril damage and cardiac atrophy. Our aim was to test potential counteracting effects of the pro-hypertrophic miR-212/132 family in doxorubicin-induced cardiotoxicity. In vitro, overexpression of the pro-hypertrophic miR-212/132 cluster in primary rodent and human iPSC-derived cardiomyocytes inhibited doxorubicin-induced toxicity. Next, a disease model of doxorubicin-induced cardiotoxicity was established in male C57BL/6N mice. Mice were administered either adeno-associated virus (AAV)9-control or AAV9-miR-212/132 to achieve myocardial overexpression of the miR-212/132 cluster. AAV9-mediated overexpression limited cardiac atrophy by increasing left ventricular mass and wall thickness, decreased doxorubicin-mediated apoptosis, and prevented myofibril damage. Based on a transcriptomic profiling we identified fat storage-inducing transmembrane protein 2 (Fitm2) as a novel target and downstream effector molecule responsible, at least in part, for the observed miR-212/132 anti-cardiotoxic effects. Overexpression of Fitm2 partially reversed the effects of miR-212/132. Overexpression of the miR-212/132 family reduces development of doxorubicin-induced cardiotoxicity and thus could be a therapeutic entry point to limit doxorubicin-mediated adverse cardiac effects.

Quaking Inhibits Doxorubicin-Mediated Cardiotoxicity Through Regulation of Cardiac Circular RNA Expression.

RATIONALE: RBPs (RNA-binding proteins) have been described to be expressed and regulated in various organs including the heart. Little is known about the role of RBPs in heart failure induced by the chemotherapy drug doxorubicin and their interaction with circular RNAs. OBJECTIVE: We aimed to identify key RBPs involved in doxorubicin-mediated heart failure and to elucidate their function. METHODS AND RESULTS: Global transcriptome profiling from murine myocardium exposed to doxorubicin identified 5 differentially expressed RBPs. Expression of the RBP QKI (Quaking) in response to doxorubicin was strongly downregulated in rodent cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes in vitro and in vivo in mice. Knockdown of Qki in primary cardiomyocytes increased apoptosis and atrophy after treatment with doxorubicin, whereas lentiviral mediated overexpression of Qki5 inhibited the doxorubicin-induced apoptosis in cardiomyocytes. In vivo, AAV9 (adeno-associated virus serotype 9)-mediated cardiac overexpression of Qki5 prevented cardiac apoptosis and cardiac atrophy induced by doxorubicin and improved cardiac function. Mechanistically, by lentiviral-based overexpression and CRISPR/Cas9-mediated silencing of Qki5, we identified regulated expression of specific circular RNAs derived from Ttn (Titin), Fhod3 (Formin homology 2 domain containing 3), and Strn3 (Striatin, calmodulin-binding protein 3). Moreover, inhibition of Ttn-derived circular RNA increased the susceptibility of cardiomyocytes to doxorubicin. CONCLUSIONS: We here show that overexpression of Qki5 strongly attenuates the toxic effect of doxorubicin via regulating a set of circular RNAs. Qki5 is, thus, an interesting target molecule to combat doxorubicin-induced cardiotoxicity.

Inhibition of the Cardiac Fibroblast-Enriched lncRNA Meg3 Prevents Cardiac Fibrosis and Diastolic Dysfunction.

RATIONALE: Cardiac fibroblasts (CFs) drive extracellular matrix remodeling after pressure overload, leading to fibrosis and diastolic dysfunction. Recent studies described the role of long noncoding RNAs (lncRNAs) in cardiac pathologies. Nevertheless, detailed reports on lncRNAs regulating CF biology and describing their implication in cardiac remodeling are still missing. OBJECTIVE: Here, we aimed at characterizing lncRNA expression in murine CFs after chronic pressure overload to identify CF-enriched lncRNAs and investigate their function and contribution to cardiac fibrosis and diastolic dysfunction. METHODS AND RESULTS: Global lncRNA profiling identified several dysregulated transcripts. Among them, the lncRNA maternally expressed gene 3 (Meg3) was found to be mostly expressed by CFs and to undergo transcriptional downregulation during late cardiac remodeling. In vitro, Meg3 regulated the production of matrix metalloproteinase-2 (MMP-2). GapmeR-mediated silencing of Meg3 in CFs resulted in the downregulation of Mmp-2 transcription, which, in turn, was dependent on P53 activity both in the absence and in the presence of transforming growth factor-ß I. Chromatin immunoprecipitation showed that further induction of Mmp-2 expression by transforming growth factor-ß I was blocked by Meg3 silencing through the inhibition of P53 binding on the Mmp-2 promoter. Consistently, inhibition of Meg3 in vivo after transverse aortic constriction prevented cardiac MMP-2 induction, leading to decreased cardiac fibrosis and improved diastolic performance. CONCLUSIONS: Collectively, our findings uncover a critical role for Meg3 in the regulation of MMP-2 production by CFs in vitro and in vivo, identifying a new player in the development of cardiac fibrosis and potential new target for the prevention of cardiac remodeling.