Translational pharmacokinetic/pharmacodynamic model for mRNA-0184, an investigational therapeutic for the treatment of heart failure.

Heart failure (HF) is a complex, progressive disorder that is associated with substantial morbidity and mortality on a global scale. Relaxin-2 is a naturally occurring hormone that may have potential therapeutic benefit for patients with HF. To investigate the therapeutic potential of relaxin in the treatment of patients with HF, mRNA-0184, a novel, investigational, lipid nanoparticle (LNP)-encapsulated mRNA therapy that encodes for human relaxin-2 fused to variable light chain kappa (Rel2-vlk) was developed. A translational semi-mechanistic population pharmacokinetic (PK)/pharmacodynamic (PD) model was developed using data from non-human primates at dose levels ranging from 0.15 to 1 mg/kg. The PK/PD model was able to describe the PK of Rel2-vlk mRNA and translated Rel2-vlk protein in non-human primates adequately with relatively precise estimates. The preclinical PK/PD model was then scaled allometrically to determine the human mRNA-0184 dose that would achieve therapeutic levels of Rel2-vlk protein expression in patients with stable HF with reduced ejection fraction. Model-based simulations derived from the scaled PK/PD model support the selection of 0.025 mg/kg as an appropriate starting human dose of mRNA-0184 to achieve average trough relaxin levels between 1 and 2.5 ng/mL, which is the potential exposure for cardioprotective action of relaxin.

Unveiling a novel memory center in human brain: neurochemical identification of the nucleus incertus, a key pontine locus implicated in stress and neuropathology.

BACKGROUND: The nucleus incertus (NI) was originally described by Streeter in 1903, as a midline region in the floor of the fourth ventricle of the human brain with an 'unknown' function. More than a century later, the neuroanatomy of the NI has been described in lower vertebrates, but not in humans. Therefore, we examined the neurochemical anatomy of the human NI using markers, including the neuropeptide, relaxin-3 (RLN3), and began to explore the distribution of the NI-related RLN3 innervation of the hippocampus. METHODS: Histochemical staining of serial, coronal sections of control human postmortem pons was conducted to reveal the presence of the NI by detection of immunoreactivity (IR) for the neuronal markers, microtubule-associated protein-2 (MAP2), glutamic acid dehydrogenase (GAD)-65/67 and corticotrophin-releasing hormone receptor 1 (CRHR1), and RLN3, which is highly expressed in NI neurons in diverse species. RLN3 and vesicular GABA transporter 1 (vGAT1) mRNA were detected by fluorescent in situ hybridization. Pons sections containing the NI from an AD case were immunostained for phosphorylated-tau, to explore potential relevance to neurodegenerative diseases. Lastly, sections of the human hippocampus were stained to detect RLN3-IR and somatostatin (SST)-IR. RESULTS: In the dorsal, anterior-medial region of the human pons, neurons containing RLN3- and MAP2-IR, and RLN3/vGAT1 mRNA-positive neurons were observed in an anatomical pattern consistent with that of the NI in other species. GAD65/67- and CRHR1-immunopositive neurons were also detected within this area. Furthermore, RLN3- and AT8-IR were co-localized within NI neurons of an AD subject. Lastly, RLN3-IR was detected in neurons within the CA1, CA2, CA3 and DG areas of the hippocampus, in the absence of RLN3 mRNA. In the DG, RLN3- and SST-IR were co-localized in a small population of neurons. CONCLUSIONS: Aspects of the anatomy of the human NI are shared across species, including a population of stress-responsive, RLN3-expressing neurons and a RLN3 innervation of the hippocampus. Accumulation of phosphorylated-tau in the NI suggests its possible involvement in AD pathology. Further characterization of the neurochemistry of the human NI will increase our understanding of its functional role in health and disease.

Acute Stress Affects the Relaxin/Insulin-Like Family Peptide Receptor 3 mRNA Expression in Brain of Pubertal Male Wistar Rats.

The current literature suggests that relaxin-3/relaxin/insulin-like family peptide receptor 3 (RLN-3/RXFP-3) system is involved in the pathophysiology of affective disorders because the results of anatomical and pharmacological studies have shown that the RLN-3 signaling pathway plays a role in modulating the stress response, anxiety, arousal, depression-like behavior, and neuroendocrine homeostasis. The risk of developing mental illnesses in adulthood is increased by exposure to stress in early periods of life. The available data indicate that puberty is especially characterized by the development of the neural system and emotionality and is a "stress-sensitive" period. The presented study assessed the short-term changes in the expression of RLN-3 and RXFP-3 mRNA in the stress-dependent brain regions in male pubertal Wistar rats that had been subjected to acute stress. Three stressors were applied from 42 to 44 postnatal days (first day: a single forced swim; second day: stress on an elevated platform that was repeated three times; third day: restraint stress three times). Anxiety (open field, elevated plus maze test) and anhedonic-like behavior (sucrose preference test) were estimated during these tests. The corticosterone (CORT) levels and blood morphology were estimated. We found that the RXFP-3 mRNA expression decreased in the brainstem, whereas it increased in the hypothalamus 72 h after acute stress. These molecular changes were accompanied by the increased levels of CORT and anxiety-like behavior detected in the open field test that had been conducted earlier, that is, 24 h after the stress procedure. These findings shed new light on the neurochemical changes that are involved in the compensatory response to adverse events in pubertal male rats and support other data that suggest a regulatory interplay between the RLN-3 pathway and the hypothalamus-pituitary-adrenal axis activity in the mechanisms of anxiety-like behavior.

Cardiomyocyte-specific RXFP1 overexpression protects against pressure overload-induced cardiac dysfunction independently of relaxin.

Heart failure (HF) prevalence is rising due to reduced early mortality and demographic change. Relaxin (RLN) mediates protective effects in the cardiovascular system through Relaxin-receptor 1 (RXFP1). Cardiac overexpression of RXFP1 with additional RLN supplementation attenuated HF in the pressure-overload transverse aortic constriction (TAC) model. Here, we hypothesized that robust transgenic RXFP1 overexpression in cardiomyocytes (CM) protects from TAC-induced HF even in the absence of RLN. Hence, transgenic mice with a CM-specific overexpression of human RXFP1 (hRXFP1tg) were generated. Receptor functionality was demonstrated by in vivo hemodynamics, where the administration of RLN induced positive inotropy strictly in hRXFP1tg. An increase in phospholamban-phosphorylation at serine 16 was identified as a molecular correlate. hRXFP1tg were protected from TAC without additional RLN administration, presenting not only less decline in systolic left ventricular (LV) function but also abrogated LV dilation and pulmonary congestion compared to WT mice. Molecularly, transgenic hearts exhibited not only a significantly attenuated fetal and fibrotic gene activation but also demonstrated less fibrotic tissue and CM hypertrophy in histological sections. These protective effects were evident in both sexes. Similar cardioprotective effects of hRXFP1tg were detectable in a RLN-knockout model, suggesting an alternative mechanism of receptor activation through intrinsic activity, alternative endogenous ligands or crosstalk with other receptors. In summary, CM-specific RXFP1 overexpression provides protection against TAC even in the absence of endogenous RLN. This suggests RXFP1 overexpression as a potential therapeutic approach for HF, offering baseline protection with optional RLN supplementation for specific activation.

Genome-wide analysis of two different regions of brain reveals the molecular changes of fertility related genes in rln3a-/- mutants in male Nile tilapia (Oreochromis niloticus).

Relaxin3 (rln3) has been associated with various emotional and cognitive processes, including stress, anxiety, learning, memory, motivational behavior, and circadian rhythm. Notably, previous report revealed that Rln3a played an indispensable role in testicular development and male fertility in Nile tilapia (Oreochromis niloticus). However, the underlying molecular mechanisms remain largely unknown. We found that Rln3a is expressed exclusively in the diencephalon* (Di*) of the brain. Deficiency of Rln3a resulted in a significant increase in serum dopamine level and an upregulation of gene expression of gnrh1 and kisspeptin2. To further elucidate the role of Rln3a in fish fertility, we collected two different regions of Di* and hypothalamus (Hyp) tissues for subsequent RNA-seq analysis of both wild-type (rln3a+/+) and rln3a-/- male tilapia. Upon the transcriptomic data, 1136 and 755 differentially expressed genes (DEGs) were identified in the Di* and Hyp tissues, respectively. In Di*, the up-regulated genes were enriched in circadian rhythm, chemical carcinogenesis, while the down-regulated genes were enriched in type II diabetes mellitus, dopaminergic synapse, and other pathways. In Hyp, the up-regulated genes were enriched in circadian rhythm, pyrimidine metabolism, while the down-regulated genes were enriched in type I diabetes mellitus, autoimmune thyroid disease, and other pathways. Subsequently, the results of both qRT-PCR and FISH assays highlighted a pronounced up-regulation of core circadian rhythm genes, cry1b and per3, whereas genes such as clocka, clockb, and arntl exhibited down-regulation. Furthermore, the genes associated with dopamine biosynthesis were significantly increased in the Hyp. In summary, the mutation of rln3a in male tilapia resulted in notable changes in circadian rhythm and disease-linked signaling pathways in the Di* and Hyp. These changes might account for the fertility defects observed in rln3a-/- male mutants in tilapia.

Genetically defined nucleus incertus neurons differ in connectivity and function.

The nucleus incertus (NI), a conserved hindbrain structure implicated in the stress response, arousal, and memory, is a major site for production of the neuropeptide relaxin-3. On the basis of goosecoid homeobox 2 (gsc2) expression, we identified a neuronal cluster that lies adjacent to relaxin 3a (rln3a) neurons in the zebrafish analogue of the NI. To delineate the characteristics of the gsc2 and rln3a NI neurons, we used CRISPR/Cas9 targeted integration to drive gene expression specifically in each neuronal group, and found that they differ in their efferent and afferent connectivity, spontaneous activity, and functional properties. gsc2 and rln3a NI neurons have widely divergent projection patterns and innervate distinct subregions of the midbrain interpeduncular nucleus (IPN). Whereas gsc2 neurons are activated more robustly by electric shock, rln3a neurons exhibit spontaneous fluctuations in calcium signaling and regulate locomotor activity. Our findings define heterogeneous neurons in the NI and provide new tools to probe its diverse functions.

Identification and Quantification of Human Relaxin Proteins by Immunoaffinity-Mass Spectrometry.

The human relaxins belong to the Insulin/IGF/Relaxin superfamily of peptide hormones, and their physiological function is primarily associated with reproduction. In this study, we focused on a prostate tissue-specific relaxin RLN1 (REL1_HUMAN protein) and a broader tissue specificity RLN2 (REL2_HUMAN protein). Due to their structural similarity, REL1 and REL2 proteins were collectively named a 'human relaxin protein' in previous studies and were exclusively measured by immunoassays. We hypothesized that the highly selective and sensitive immunoaffinity-selected reaction monitoring (IA-SRM) assays would reveal the identity and abundance of the endogenous REL1 and REL2 in biological samples and facilitate the evaluation of these proteins for diagnostic applications. High levels of RLN1 and RLN2 transcripts were found in prostate and breast cancer cell lines by RT-PCR. However, no endogenous prorelaxin-1 or mature REL1 were detected by IA-SRM in cell lines, seminal plasma, or blood serum. The IA-SRM assay of REL2 demonstrated its undetectable levels (<9.4 pg/mL) in healthy control female and male sera and relatively high levels of REL2 in maternal sera across different gestational weeks (median 331 pg/mL; N = 120). IA-SRM assays uncovered potential cross-reactivity and nonspecific binding for relaxin immunoassays. The developed IA-SRM assays will facilitate the investigation of the physiological and pathological roles of REL1 and REL2 proteins.

Hypogonadotropic hypogonadism in male tilapia lacking a functional rln3b gene.

Relaxin 3 is a neuropeptide that plays a crucial role in reproductive functions of mammals. Previous studies have confirmed that rln3a plays an important role in the male reproduction of tilapia. To further understand the significance of its paralogous gene rln3b in male fertility, we generated a homozygous mutant line of rln3b in Nile tilapia. Our findings indicated that rln3b mutation delayed spermatogenesis and led to abnormal testes structure. Knocking out rln3b gene resulted in a decrease in sperm count, sperm motility and male fish fertility. TUNEL detection revealed a small amount of apoptosis in the testes of rln3b-/- male fish at 390 days after hatching (dah). RT-qPCR analysis demonstrated that mutation of rln3b gene caused a significant downregulation of steroid synthesis-related genes such as cyp17a1, cyp11b2, germ cell marker gene, Vasa, and gonadal somatic cell marker genes of amh and amhr2. Furthermore, we found a significant down-regulation of hypothalamic-pituitary-gonadal (HPG) axis-related genes, while a significantly up-regulation of the dopamine synthetase gene in the rln3b-/- male fish. Taken together, our data strongly suggested that Rln3b played a crucial role in the fertility of XY tilapia by regulating HPG axis genes.

Relaxin-2 is a novel biomarker for differentiated thyroid carcinoma in humans.

Relaxin's role in differentiated thyroid cancer (DTC) has been suggested but its characterization in a large clinical sample remains limited. We performed immunohistochemistry for relaxin-2 (RLN2), CD68 (total macrophages), CD163 (M2 macrophages) on tissue microarrays from 181 subjects with non-distant metastatic DTC, and 185 subjects with benign thyroid tissue. Mean pixels/area for each marker was compared between tumor and adjacent tissue via paired-t test and between DTC and benign subjects via t-test assuming unequal variances. RNA qPCR was performed for expression of RLN2, RLN1, and RXFP1 in cell lines. Amongst 181 cases, the mean age was 46 years, 75 % were females. Tumoral tissue amongst the DTC cases demonstrated higher mean expression of RLN2 (53.04 vs. 9.79; p < 0.0001) compared to tumor-adjacent tissue. DTC tissue also demonstrated higher mean expression of CD68 (14.46 vs. 4.79; p < 0.0001), and CD163 (23.13 vs. -0.73; p < 0.0001) than benign thyroid. These markers did not differ between tumor-adjacent and benign thyroid tissue groups; and amongst cases, did not differ by demographic or clinicopathologic features. RLN1 and RXFP1 expression was detected in a minority of the cell lines, while RLN2 was expressed by 6/7 cell lines. In conclusion, widespread RLN2 expression in DTC tissue and most cell lines demonstrates that RLN2 acts in a paracrine manner, and that RLN1 and RXFP1 are probably not involved in thyroid cancer cell signaling. RLN2 is a biomarker for thyroid carcinogenesis, being associated with but not secreted by immunosuppressive macrophages. These findings will guide further investigations for therapeutic avenues against thyroid cancer.

Programmed Death-Ligand (PD-L1), Epidermal Growth Factor (EGF), Relaxin, and Matrix Metalloproteinase-3 (MMP3): Potential Biomarkers of Malignancy in Canine Mammary Neoplasia.

Gene expression has been suggested as a putative tool for prognosis and diagnosis in canine mammary neoplasia (CMNs). In the present study, 58 formalin-fixed paraffin-embedded (FFPE) paraffined canine mammary neoplasias from 27 different bitches were included. Thirty-seven tumours were classified as benign, whereas thirty-one were classified as different types of canine carcinoma. In addition, mammary samples from three healthy bitches were also included. The gene expression for vascular endothelial growth factor-α (VEGFα), CD20, progesterone receptor (PGR), hyaluronidase-1 (HYAL-1), programmed death-ligand 1 (PD-L1), epidermal growth factor (EGF), relaxin (RLN2), and matrix metalloproteinase-3 (MMP3) was assessed through RT-qPCR. All the assessed genes yielded a higher expression in neoplastic mammary tissue than in healthy tissue. All the evaluated genes were overexpressed in neoplastic mammary tissue, suggesting a role in the process of tumorigenesis. Moreover, PD-L1, EGF, relaxin, and MMP3 were significantly overexpressed in malignant CMNs compared to benign CMNs, suggesting they may be useful as malignancy biomarkers.

Specific expression and blood kinetics for relaxin 2, lipocalin 2, and tissue factor pathway inhibitor 2 at the canine placenta and pregnant bloods.

In general, humoral factors released from the placenta influence pregnancy progression, but the involvement of the canine placenta is often unidentified. We investigated specific genes in canine placentas and analyzed the blood dynamics of the translated proteins. Furthermore, RNAs are known to be released from placentas embedding in exosomes, a type of extracellular vesicles. Here, the presence of cell-free RNAs in pregnant serums was also confirmed. RNA specimens were purified from the normal healthy dog placentas and applied to RNA-Seq analysis. Expressions of frequent genes were confirmed by RT-PCR using placentas from other individuals and breeds. Relaxin (RLN) 2, lipocalin (LCN) 2, and tissue factor pathway inhibitor (TFPI) 2 were selected as high-expressed and placenta-specific genes. By western blot, the three factors were clearly detected in the pregnant serums. Quantitative analysis revealed that the amount of RLN2 increased significantly from non-pregnancy to day 41 of pregnancy. Regarding LCN2 and TFPI2, the protein serum levels elevated during pregnancy, but the statistical differences were not detected. Exosomes were found in all pregnant serums; however, the percentage was less than 6% in total extracellular vesicles. The cell-free RNA related to RLN2 was detected, but no elevation was confirmed during pregnancy. We found specific genes in the canine placenta and the transition of their translated protein into the blood. These factors may become useful tools for research on canine pregnancy and monitoring of reproductive management. Exosomes and cell-free RNA could not be found to be valid in canine reproduction.

Investigating the role of the relaxin-3/RXFP3 system in neuropsychiatric disorders and metabolic phenotypes: A candidate gene approach.

The relaxin-3/RXFP3 system has been implicated in the modulation of depressive- and anxiety-like behaviour in the animal literature; however, there is a lack of human studies investigating this signalling system. We seek to bridge this gap by leveraging the large UK Biobank study to retrospectively assess genetic risk variants linked with this neuropeptidergic system. Specifically, we conducted a candidate gene study in the UK Biobank to test for potential associations between a set of functional, candidate single nucleotide polymorphisms (SNPs) pertinent to relaxin-3 signalling, determined using in silico tools, and several outcomes, including depression, atypical depression, anxiety and metabolic syndrome. For each outcome, we used several rigorously defined phenotypes, culminating in subsample sizes ranging from 85,881 to 386,769 participants. Across all outcomes, there were no associations between any candidate SNP and any outcome phenotype, following corrections for multiple testing burden. Regression models comprising several SNPs per relevant candidate gene as exploratory variables further exhibited no prediction of outcome. Our findings corroborate conclusions from previous literature about the limitations of candidate gene approaches, even when based on firm biological hypotheses, in the domain of genetic research for neuropsychiatric disorders.

Integrative stemness characteristics associated with prognosis and the immune microenvironment in lung adenocarcinoma.

BACKGROUND: To comprehensively analyze the stemness characteristics related to prognosis and the immune microenvironment in lung adenocarcinoma (LUAD). METHODS: The OCLR machine learning method was used to calculate the stemness index (mRNAsi) of the LUAD samples. DEGs common between the low mRNAsi, normal, and high mRNAsi groups were screened and the immune-stemness genes were obtained. Then the PPI network was created and enrichment analyses were performed. Moreover, different subtypes based on immune-stemness genes associated with prognosis were identified, and the relationships between LUAD stemness and TIME variables were systematically analyzed, followed by TMB analysis. RESULTS: Patients in the high mRNAsi groups with poor prognosis were screened along with 144 immune-stemness genes. IL-6, FPR2, and RLN3 showed a higher degree in the PPI network. A total of 26 immune-stemness genes associated with prognosis were screened. Two clusters were obtained (cluster 1 and cluster 2). Survival analysis revealed that patients in cluster 2 had a poor prognosis. A total of 12 immune cell subpopulations exhibited significant differences between cluster 1 and cluster 2 (P < 0.05). A total of 10 immune checkpoint genes exhibited significantly higher expression in cluster 1 (P < 0.05) than in cluster 2. Further, the TMB value in cluster 2 was higher than that in cluster 1 (P < 0.05). CONCLUSION: Immune-stemness genes, including L-6, FPR2, and RLN3, might play significant roles in LUAD development via cytokine-cytokine receptor interaction, neuroactive ligand‒receptor interaction, and the JAK‒STAT pathway. Immune-stemness genes were related to tumor-infiltrating immune cells, TMB, and expression of immune checkpoint gene.

Comparative Genomic Characterization of Relaxin Peptide Family in Cattle and Buffalo.

Relaxin family peptides significantly regulate reproduction, nutrient metabolism, and immune response in mammals. The present study aimed to identify and characterize the relaxin family peptides in cattle and buffalo at the genome level. The genomic and proteomic sequences of cattle, buffalo, goat, sheep, horse, and camel were accessed through the NCBI database, and BLAST was performed. We identified four relaxin peptides genes (RLN3, INSL3, INSL5, and INSL6) in Bos taurus, whereas three relaxin genes (RLN3, INSL3, and INSL6) in Bubalus bubalis. Evolutionary analysis revealed the conserved nature of relaxin family peptides in buffalo and cattle. Physicochemical properties revealed that relaxin proteins were thermostable, hydrophilic, and basic peptides except for INSL5 which was an acidic peptide. Three nonsynonymous mutations (two in RLN3 at positions A16 > T and P29 > A, and one in INSL6 at position R32 > Q) in Bos taurus, whereas two nonsynonymous mutations (one in RLN3 at positions G105 > w and one in INSL3 at position G22 > R) in Bubalus bubalis, were identified. INSL3 had one indel (insertion) at position 55 in Bos taurus. Gene duplication analysis revealed predominantly segmental duplications (INSL5/RLN3 and INSL6/INSL3 gene pairs) that helped expand this gene family, whereas Bubalus bubalis showed primarily tandem duplication (INSL3/RLN3). Our study concluded that relaxin family peptides remained conserved during the evolution, and gene duplications might help to adapt and enrich specific functions like reproduction, nutrient metabolism, and immune response. Further, the nonsynonymous mutations identified potentially affect these functions in buffalo.

Relaxin Affects Airway Remodeling Genes Expression through Various Signal Pathways Connected with Transcription Factors.

Fibrosis is one of the parameters of lung tissue remodeling in asthma. Relaxin has emerged as a natural suppressor of fibrosis, showing efficacy in the prevention of a multiple models of fibrosis. Therefore, the aim of this study was to analyze the aptitudes of relaxin, in the context of its immunomodulatory properties, in the development of airway remodeling. WI-38 and HFL1 fibroblasts, as well as epithelial cells (NHBE), were incubated with relaxin. Additionally, remodeling conditions were induced with two serotypes of rhinovirus (HRV). The expression of the genes contributing to airway remodeling were determined. Moreover, NF-κB, c-Myc, and STAT3 were knocked down to analyze the pathways involved in airway remodeling. Relaxin decreased the mRNA expression of collagen I and TGF-ß and increased the expression of MMP-9 (p < 0.05). Relaxin also decreased HRV-induced expression of collagen I and α-SMA (p < 0.05). Moreover, all the analyzed transcription factors­NF-κB, c-Myc, and STAT3­have shown its influence on the pathways connected with relaxin action. Though relaxin requires further study, our results suggest that this natural compound offers great potential for inhibition of the development, or even reversing, of factors related to airway remodeling. The presented contribution of the investigated transcription factors in this process additionally increases its potential possibilities through a variety of its activity pathways.

The Relaxin-3 Receptor, RXFP3, Is a Modulator of Aging-Related Disease.

During the aging process our body becomes less well equipped to deal with cellular stress, resulting in an increase in unrepaired damage. This causes varying degrees of impaired functionality and an increased risk of mortality. One of the most effective anti-aging strategies involves interventions that combine simultaneous glucometabolic support with augmented DNA damage protection/repair. Thus, it seems prudent to develop therapeutic strategies that target this combinatorial approach. Studies have shown that the ADP-ribosylation factor (ARF) GTPase activating protein GIT2 (GIT2) acts as a keystone protein in the aging process. GIT2 can control both DNA repair and glucose metabolism. Through in vivo co-regulation analyses it was found that GIT2 forms a close coexpression-based relationship with the relaxin-3 receptor (RXFP3). Cellular RXFP3 expression is directly affected by DNA damage and oxidative stress. Overexpression or stimulation of this receptor, by its endogenous ligand relaxin 3 (RLN3), can regulate the DNA damage response and repair processes. Interestingly, RLN3 is an insulin-like peptide and has been shown to control multiple disease processes linked to aging mechanisms, e.g., anxiety, depression, memory dysfunction, appetite, and anti-apoptotic mechanisms. Here we discuss the molecular mechanisms underlying the various roles of RXFP3/RLN3 signaling in aging and age-related disorders.

Changes in microRNA Expression Profiles in Diabetic Cardiomyopathy Rats Following H3 Relaxin Treatment.

ABSTRACT: MicroRNAs (miRNAs) are noncoding RNAs that play an important role in the mechanisms of diabetic cardiomyopathy (DCM); however, whether human recombinant relaxin-3 (H3 relaxin) inhibits myocardial injury in DCM rats and the underlying mechanisms involving miRNAs remain unknown. miRNA expression profiles were detected using miRNA microarray and bioinformatics analyses of myocardial tissues from control, DCM, and H3 relaxin-administered DCM groups, and the regulatory mechanisms of the miRNAs were investigated. A total of 5 miRNAs were downregulated in the myocardial tissues of DCM rats and upregulated in H3 relaxin-treated DCM rats, and 1 miRNA (miRNA let-7d-3p) was increased in the myocardial tissue of DCM rats and decreased in H3 relaxin-treated DCM rats as revealed by miRNA microarray and validated by real-time polymerase chain reaction. Important signaling pathways were found to be triggered by the differentially expressed miRNAs, including metabolism, cancer, Rap1, PI3K-Akt, and MAPK signaling pathways. The study revealed that H3 relaxin improved glucose uptake in DCM rats, potentially via the regulation of miRNA let-7d-3p.

The putative role of the relaxin-3/RXFP3 system in clinical depression and anxiety: A systematic literature review.

The relaxin-3/RXFP3 system is one of several neuropeptidergic systems putatively implicated in regulating the behavioural alterations that characterise clinical depression and anxiety, making it a potential target for clinical translation. Accordingly, this systematic review identified published reports on the role of relaxin-3/RXFP3 signalling in these neuropsychiatric disorders and their behavioural endophenotypes, evaluating evidence from animal and human studies to ascertain any relationship. We searched PubMed, EMBASE, PsycINFO and Google Scholar databases up to February 2021, finding 609 relevant records. After stringent screening, 51 of these studies were included in the final synthesis. There was considerable heterogeneity in study designs and some inconsistency across study outcomes. However, experimental evidence is consistent with an ability of relaxin-3/RXFP3 signalling to promote arousal and suppress depressive- and anxiety-like behaviour. Moreover, meta-analyses of six to eight articles investigating food intake revealed that acute RXFP3 activation had strong orexigenic effects in rats. This appraisal also identified the lack of high-quality clinical studies pertinent to the relaxin-3/RXFP3 system, a gap that future research should attempt to bridge.

Structural Insights into the Unique Modes of Relaxin-Binding and Tethered-Agonist Mediated Activation of RXFP1 and RXFP2.

Our poor understanding of the mechanism by which the peptide-hormone H2 relaxin activates its G protein coupled receptor, RXFP1 and the related receptor RXFP2, has hindered progress in its therapeutic development. Both receptors possess large ectodomains, which bind H2 relaxin, and contain an N-terminal LDLa module that is essential for receptor signaling and postulated to be a tethered agonist. Here, we show that a conserved motif (GDxxGWxxxF), C-terminal to the LDLa module, is critical for receptor activity. Importantly, this motif adopts different structures in RXFP1 and RXFP2, suggesting distinct activation mechanisms. For RXFP1, the motif is flexible, weakly associates with the LDLa module, and requires H2 relaxin binding to stabilize an active conformation. Conversely, the GDxxGWxxxF motif in RXFP2 is more closely associated with the LDLa module, forming an essential binding interface for H2 relaxin. These differences in the activation mechanism will aid drug development targeting these receptors.

Endurance exercise improves avoidance learning and spatial memory, through changes in genes of GABA and relaxin-3, in rats.

Different exercise patterns, neurotransmitters, and some genes have numerous effects on learning and memory. This research aims to investigate the long-term effects of submaximal aerobic exercise on spatial memory (SM), passive avoidance learning (PAL), levels of serum relaxin-3, gamma-aminobutyric acid (GABA), RLN3 gene, and glutamic acid decarboxylase (GAD65/67 genes) in the brainstem of adult male Wistar rats. Fifty male Wistar rats were randomly divided into five groups: aerobic exercise groups, performed on a treadmill running (TR), for 5 weeks (Ex5, n = 10), 10 weeks (Ex10, n = 10), involuntary running wheel group for 5 weeks (IRW5, n = 10), sham (Sh, n = 10) and control (Co, n = 10). Consequently, SM, PAL, serum relaxin-3, GABA, and GAD65/67 and RLN3 genes were measured by ELISA and PCR. Ex5, Ex10 and IRW5 improved significantly SM (p ≤ 0.05), PAL (p ≤ 0.001) and decreased significantly relaxin-3 (p ≤ 0.001). RLN3 in the brain also decreased. However, it was not significant. GABA and GAD65/GAD67 increased significantly (p ≤ 0.05) in Ex5, Ex10 compared to Sh and Co. Aerobic exercise enhanced SM and PAL in Ex compared to Co and Sh. However, duration and type of exercise affected the level of enhancement. The serum relaxin-3 and RLN3 gene displayed reverse functions compared to GABA and GAD65/67 genes in Ex. Therefore, the changes of neurotransmitters in serum relaxin-3, GABA, and their genes: RLN3 and GAD65/67 respectively, influenced learning and memory meaningfully.