NRG-1β Treatment Research Articles

Erbb3 regulation of secretory differentiation in the intestinal epithelium

Background: Previous work by our lab showed that intestinal-specific deletion of the ErbB3 receptor tyrosine kinase results in increased ileal Paneth cell numbers. These cells produce host defense peptides and support stem cells with growth factors. The mechanisms by which ErbB3 regulates Paneth cells are not well understood, but a candidate mediator is the polycomb repressor complex 1 component BMI1, which marks secretory progenitor cells. Furthermore, while there are no Paneth cells in the mouse colon, deep crypt secretory (DCS) cells are a distinct Reg4+ population that have some functional overlap with both Paneth and goblet cells; we will also test whether ErbB3 regulates DCS differentiation. Overall we are testing the hypothesis that ErbB3 regulates secretory cell numbers through BMI1. Methods: We bred Villin-Cre;ErbB3flox/flox mice (ErbB3ΔIE) to delete ErbB3 from the intestinal epithelium. Colonic sections from ErbB3ΔIE and ErbB3flox/flox littermates were subjected to RNAscope in situ hybridization for Reg4. RNA from mucosal scrapings (both ileum and colon) was assessed by qPCR for Reg4, Bmi1, and Lgr5. To test signaling mechanisms, we treated HT-29 cells with NRG-1β to activate ErbB3, +/- inhibitors to PI3K (LY294002), MEK/MAPK (U0126), or BMI1 (PTC-209). Colonoids and enteroids from ErbB3ΔIE and ErbB3flox/flox mice were used to confirm responses in a non-transformed system. Results: In ErbB3ΔIE mice, the colonic epithelium showed increased Reg4 levels and more Reg4+ cells compared to littermate controls, similar to our published data for the Paneth cell marker Lyz1 in ileum. BMI1 RNA and protein levels were also increased in both small and large intestinal mucosal scrapings from ErbB3ΔIE mice vs. littermates. ErbB3ΔIE colonoids had increased Reg4 versus control colonoids, and NRG-1β reduced levels in control but not ErbB3ΔIE cultures. Ileal enteroids showed a decrease in Lyz1 expression with Bmi1 inhibition and an increase in Bmi1 expression with PI3K and MAPK blockade. In HT-29 cells, NRG-1β treatment reduced Reg4 and Bmi1 expression. Conversely, Reg4 and Bmi1 were induced by PI3K inhibition with LY294002 (p<0.03) (p<0.0005) or MEK/MAPK inhibition with U0126 (p<0.05) (p<0.0001). Conclusions: NRG1-ErbB3 signaling restricts both ileal Paneth ( Lyz1+) and colonic DCS ( Reg4+) cell numbers. Furthermore, ErbB3 regulates Bmi1 expression through PI3K/Akt and MAPK signaling and BMI1 activity promotes secretory cell development. Since loss of DCS or Paneth cells has been shown to disrupt the intestinal stem cell niche and secretory cell markers are altered in several intestinal disease states, these results may point to potential future therapeutic avenues targeting this regulatory mechanism. NIH/NIDDK award R01DK095004. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

ErbB3 Control of Pmp22 Regulates Intestinal Barrier Function

Background: ErbB3 is a member of the ErbB/EGFR family of receptor tyrosine kinases and is the constitutively-expressed neuregulin (NRG) receptor in the intestinal epithelium. Pathways downstream of ErbB3 have been implicated in regulating tight junctions in other systems, but a role for ErbB3 in barrier function in the intestines has not been described. Here we tested the hypothesis that ErbB3 is required for maintenance of normal barrier function in the intestine. Methods: We generated Villin-Cre;ErbB3 flox/flox mice (ErbB3-IEKO) with ErbB3 deletion in the intestinal epithelium. All comparisons are to ErbB3 flox/flox littermate controls. Mice were given a FITC-dextran 4 kDa (FD4) gavage to assess intestinal permeability. Isolated epithelial ileal samples were collected for RT-qPCR, immunohistochemistry, and bulk RNA-seq analysis. Enteroids were generated and treated with the ErbB3 ligand NRG-1β as well as PD153035 and CP-724714 to inhibit EGFR and ErbB2, respectively. Enteroids were also fixed and stained for whole mount immunofluorescence staining. Caco-2BBe cells were grown on Transwell filters, treated with NRG-1β, and assessed for transepithelial electrical resistance (TEER) and FD4 flux. Results: ErbB3-IEKO mice displayed increased intestinal barrier permeability to FD4 compared to controls (p < 0.01). RNA-seq analysis showed a decrease in the expression of the putative tight junction protein Pmp22 in ErbB3-IEKO mice, while other tight junctional components were unchanged. RT-qPCR analysis of ileal samples showed an 80% Pmp22 downregulation (p < 0.01) with ErbB3 loss compared to controls, with similar results in ErbB3-deficient enteroids. Immunohistochemical analysis confirmed near complete loss of PMP22 in ErbB3-IEKO intestinal epithelium. In vitro, treatment with NRG-1β induced Pmp22 expression (p < 0.001) in control, but not ErbB3-deficient, enteroids. Treatment with PD153035 blocked NRG-1β-driven expression of Pmp22 while treatment with CP-724714 did not, suggesting that EGFR is a critical ErbB3 heterodimer partner in regulating Pmp22. Whole mount staining of enteroids confirmed co-localization of PMP22 with the tight junction protein ZO-1 at the apical junctions. Furthermore, treatment of Caco-2BBe monolayers with NRG-1β increased TEER and reduced FD4 flux, demonstrating active regulation of permeability. Conclusions: We demonstrate that loss of ErbB3 in the intestinal epithelium leads to reduced expression of the tight junction protein PMP22 and impaired barrier function in mice. Co-localization of PMP22 with ZO-1 suggests a role for PMP22 in forming the barrier and may represent a mechanism for regulation of intestinal permeability through the ErbB3-PMP22 axis. Furthermore, increased TEER in Caco-2BBe monolayers following NRG-1β treatment suggests induction of PMP22 may represent a means to enhance barrier function and a potential target for future therapeutic interventions targeting the barrier. Supported by NIH awards R01DK095004 and F31DK131856 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The ErbB3 Receptor Restricts Bmi1 to Regulate Paneth Cells

ErbB3 is a RTK family member known to regulate proliferation and growth in the intestinal epithelium. Previous work by our lab determined that targeted Erbb3 deletion from the intestinal epithelium resulted in precocious Paneth cell development and an expanded population in mice. This was associated with increased expression of intestinal stem cell (ISC) markers. Conversely, ileal enteroids treated with the ErbB3 ligand neuregulin (NRG)‐1β had significantly reduced expression of ISC markers, including Lgr5 and Bmi1. These data suggest that ErbB3 restricts secretory cell differentiation and stemness in the intestinal crypt. However, the underlying mechanisms are largely unknown. BMI1 is a proto‐oncogene and member of the PRC complex. Bmi1+ intestinal epithelial cells represent a slow‐cycling ISC population that can regenerate the epithelium following injury and loss of Lgr5+ ISCs. Recent evidence determined that Bmi1+ ISCs are enriched in enteroendocrine markers and can give rise to goblet cells, suggesting BMI1 may be critical for differentiation of secretory lineages. We hypothesized that ErbB3 restricts secretory cell differentiation through regulation of BMI1.AimsTo define ErbB3‐dependent regulation of Bmi1 and determine if ErbB3 restriction of Paneth cell development and differentiation is through suppression of Bmi1.MethodsMucosal scrapings were collected from Vil‐Cre;Erbb3flox/flox mice and Erbb3flox/flox littermate controls for protein and gene expression analysis. Ileal enteroids and HT‐29 colorectal adenocarcinoma cells were treated with vehicle DMSO, the PI3K inhibitor LY294002 (10 μM), the MEK inhibitor U0126 (5 μM), the BMI1 inhibitor PTC‐209 (1 μM), and/or recombinant NRG‐1β (10 ng/mL). Enteroids and cell monolayers were harvested and prepared for gene expression analysis. RT‐qPCR for Bmi1 and Lyz1 was performed relative to Hprt as a loading control. Statistical analysis by one‐way ANOVA was conducted using Prism 9.ResultsConsistent with previous data, BMI1 protein and gene expression were significantly increased in small and large intestinal mucosal scrapings from Vil‐Cre;Erbb3flox/flox mice versus littermate controls. To determine if Bmi1 expression is sensitive to PI3K/Akt or MAPK signaling downstream of ErbB3, HT‐29 cells were treated with inhibitors followed by NRG‐1β after 1 hour, then harvested 24 hours later. PI3K and MAPK inhibition significantly increased Bmi1 expression. NRG‐1β treatment was able to overcome MAPK inhibition to reduce BMI1 expression. Similarly, in ileal enteroids, PI3K and MAPK blockade increased Bmi1 expression and NRG‐1β treatment reversed this effect. To determine whether BMI1 regulates expression of the Paneth cell marker LYZ1, HT‐29 cells were treated with the BMI1 inhibitor PTC‐209. BMI1 inhibition decreased LYZ1 expression.ConclusionsOur data demonstrate that ErbB3 regulates Bmi1 expression through PI3K/Akt and MAPK signaling in both human and mouse intestinal cells. Furthermore, BMI1 activity promotes LYZ1 expression. Together, these results support our hypothesis that ErbB3 regulates secretory cell differentiation through BMI1 in the intestinal epithelium.

Neuregulin‐1β promotes myoblast differentiation and is anti‐cachexic in respiratory muscles of post‐infarcted swine

Neuregulin‐1β (NRG‐1β) is a growth and differentiation factor with pleiotropic systemic effects. Because NRG‐1β has therapeutic potential for heart failure and known growth effects in skeletal muscle, we hypothesized that it might affect heart failure‐associated cachexia, a severe co‐morbidity characterized by a loss of muscle mass. We therefore assessed NRG‐1β’s effect on skeletal muscle gene expression in a swine model of heart failure using recombinant Glial Growth Factor 2 (USAN ‐ cimaglermin alfa), a version of NRG‐1β currently being tested in humans with systolic heart failure. Animals received one of two intravenous doses (0.67 or 2 mg/kg) of NRG‐1β bi‐weekly for 4 weeks, beginning one week after infarct. Skeletal muscle was collected at the time of euthanasia from the intercostal space and paired‐end RNA sequencing performed. NRG‐1β treatment altered expression of 583 transcripts including 242 transcripts altered at both doses. These included genes required for myofiber growth, maintenance and survival such as MYH3, MYHC, MYL6B, KY and HES1. Importantly, NRG‐1β altered the directionality of at least 85 genes associated with cachexia, including myostatin, which negatively regulates myoblast differentiation by down‐regulating MyoD expression. Consistent with this, MyoD was increased in NRG‐1β treated animals. In vitro experiments with myoblast cell lines confirmed that NRG‐1β induces skeletal muscle differentiation with an absolute requirement for ERBB signaling on myoblast differentiation. These findings suggest a NRG‐1β‐mediated anti‐atrophic, anti‐cachexia effect that may provide additional benefits to this potential therapy in heart failure.

Neuregulin (NRG-1β) Is Pro-Myogenic and Anti-Cachectic in Respiratory Muscles of Post-Myocardial Infarcted Swine.

Simple SummaryNeuregulin is a growth factor that has been shown to prevent adverse remodeling in the heart and may represent a therapeutic for patients with systolic heart failure. A common symptom in heart failure is shortness of breath, which has been related in part to impaired skeletal muscle function. Since neuregulin directly activates skeletal muscle, in addition to heart tissue, we hypothesized that neuregulin might directly affect intercostal muscle gene expression changes in heart disease. We tested this hypothesis by performing global gene expression analysis of intercostal muscle tissue collected from pigs treated with recombinant neuregulin after the induction of myocardial infarction, an experimental model clinically similar to a human heart attack. We found that neuregulin-treated pigs had massive changes in global gene expression consistent with new muscle cell formation, as compared to untreated pigs. These data suggest that neuregulin is an important mediator of muscle function that can potentially be used to study heart disease-associated muscle dysfunction and the development of new therapeutics aimed at muscle repair in heart failure, as well as other diseases associated with muscle dysfunction and weakness.Neuregulin-1β (NRG-1β) is a growth and differentiation factor with pleiotropic systemic effects. Because NRG-1β has therapeutic potential for heart failure and has known growth effects in skeletal muscle, we hypothesized that it might affect heart failure-associated cachexia, a severe co-morbidity characterized by a loss of muscle mass. We therefore assessed NRG-1β’s effect on intercostal skeletal muscle gene expression in a swine model of heart failure using recombinant glial growth factor 2 (USAN-cimaglermin alfa), a version of NRG-1β that has been tested in humans with systolic heart failure. Animals received one of two intravenous doses (0.67 or 2 mg/kg) of NRG-1β bi-weekly for 4 weeks, beginning one week after infarct. Based on paired-end RNA sequencing, NRG-1β treatment altered the intercostal muscle gene expression of 581 transcripts, including genes required for myofiber growth, maintenance and survival, such as MYH3, MYHC, MYL6B, KY and HES1. Importantly, NRG-1β altered the directionality of at least 85 genes associated with cachexia, including myostatin, which negatively regulates myoblast differentiation by down-regulating MyoD expression. Consistent with this, MyoD was increased in NRG-1β-treated animals. In vitro experiments with myoblast cell lines confirmed that NRG-1β induces ERBB-dependent differentiation. These findings suggest a NRG-1β-mediated anti-atrophic, anti-cachexia effect that may provide additional benefits to this potential therapy in heart failure.

Neuregulin-1β Protects the Rat Diaphragm during Sepsis against Oxidative Stress and Inflammation by Activating the PI3K/Akt Pathway.

Sepsis-induced diaphragm dysfunction (SIDD) which is mainly characterized by decrease in diaphragmatic contractility has been identified to cause great harms to patients. Therefore, there is an important and pressing need to find effective treatments for improving SIDD. In addition, acetylcholinesterase (AChE) activity is a vital property of the diaphragm, so we evaluated both diaphragmatic contractility and AChE activity. Though neuregulin-1β (NRG-1β) is known to exert organ-protective effects in some inflammatory diseases, little is known about the potential of NRG-1β therapy in the diaphragm during sepsis. Our study was aimed at exploring the effects of NRG-1β application on diaphragmatic contractility and AChE activity during sepsis. Proinflammatory cytokines, muscle injury biomarkers in serum, contractile force, AChE activity, proinflammatory cytokines, oxidative parameters, histological condition, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and expression of phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB/Akt) signaling proteins in the diaphragm were measured and compared between nonseptic and septic groups with or without NRG-1β treatment. In vitro, the effects of NRG-1β on reactive oxygen species (ROS) production in the lipopolysaccharide- (LPS-) stimulated L6 rat muscle skeletal cells with or without the Akt inhibitor MK-2206 were detected. NRG-1β inhibited proinflammatory cytokine release and muscle injury biomarkers soaring in serum and improved the sepsis-induced diaphragm dysfunction and AChE activity decrease significantly during sepsis. Meanwhile, the inflammatory response, oxidative stress, pathological impairment, and cell apoptosis in the diaphragm were mitigated by NRG-1β. And NRG-1β activated the PI3K/Akt signaling in the diaphragm of septic rats. Elevated ROS production in the LPS-stimulated L6 rat skeletal muscle cells was reduced after treatment with NRG-1β, while MK-2206 blocked these effects of NRG-1β. In conclusion, our findings underlined that NRG-1β could reduce circulating levels of proinflammatory cytokines in rats with sepsis, adjust diaphragmatic proinflammatory cytokine level, mitigate diaphragmatic oxidative injury, and lessen diaphragm cell apoptosis, thereby improving diaphragmatic function, and play a role in diaphragmatic protection by activating PI3K/Akt signaling.

Neuregulin-1β Partially Improves Cardiac Function in Volume-Overload Heart Failure Through Regulation of Abnormal Calcium Handling.

Background: Neuregulin (NRG-1), an essential stress-mediated paracrine growth factor, has a cardioprotective effect in failing heart. However, the underlying mechanism remains unclear. The role of NRG-1β in heart failure (HF) rats was examined. Methods and Results: Volume-overload HF rat model was created by aortocaval fistula surgery. The sham-operated (SO) rats received the same surgical intervention without the fistula. Thirty-five HF rats were injected with NRG-1β (NRG, 10 μg/kg·d) via the tail vein for 7 days, whereas 35 HF rats and 20 SO rats were injected with the same dose of saline. The echocardiographic findings showed left ventricular dilatation, systolic and diastolic dysfunction, and QTc interval prolongation in HF rats. The NRG-1β treatment attenuated the ventricular remodeling and shortened the QTc interval. Patch clamp recordings showed ICa-L was significantly decreased in the HF group, and NRG-1β treatment attenuated the decreased ICa-L. No significant differences in the kinetic properties of ICa-L were observed. The expressions of Cav1.2 and SERCA2a were significantly reduced, but the expression level of NCX1 was increased dramatically in the HF group. NRG-1β treatment could partially prevent the decrease of Cav1.2 and SERCA2a, and the increase of NCX1 in HF rats. Conclusions: NRG-1β could partly attenuate the heart function deterioration in the volume-overload model. Reduced function and expression of calcium transportation-related proteins might be the underlying mechanism.

Neuroprotective effect of neuregulin-1β on spinal cord ischemia reperfusion injury

Objective: This study was designed to see if neuregulin-1β (NRG-1β) plays a protective role in spinal cord ischemia and reperfusion injury (SCII). Design: Animal research. Setting: China. Participants: NA. Interventions: Forty-eight SD rats were randomly divided into control group (n = 16), SCII model group (n = 16) and NRG-1β-treated group (n = 16). In control group, the abdominal aorta was isolated but not clipped. The rats in NRG-1β-treated group were treated with 10μg/kg NRG-1β during developing SCII model. Outcome Measures: Neurological scores were evaluated. At 3, 6, 12 and 24 h after the reperfusion, rats were killed. Pathological changes of spinal cord were assessed with HE staining, and immunohistochemical staining of matrix metalloproteinases-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). MMP-9 and TIMP-1 mRNA levels were assessed using real-time PCR. Results: NRG-1β reduced the damage of SCII in the rats. The expression of MMP-9 protein and mRNA in NRG-1β treatment group was significantly lower than the model group (P < 0.05) at 6 h, 12 h and 24 h after the perfusion. The expression of TIMP-1 protein and mRNA in the treatment group was significantly higher than the model group at 12 h and 24 h after the perfusion. Conclusion: NRG-1β reduced the reperfusion damage in rat model of SCII, in which process MMP-9 and TIMP-1 were probably involved.

Neuregulin-1β attenuates sepsis-induced diaphragm atrophy by activating the PI3K/Akt signaling pathway.

The aim of this study was to investigate the protective effects of neuregulin-1β (NRG-1β) on sepsis-induced diaphragm atrophy and the possible underlying mechanisms. Sprague-Dawley rats were randomly divided into sham, sepsis and NRG groups. Sepsis was induced by cecal ligation and puncture (CLP). In the NRG group, rats received tail vein injections of NRG-1β (10μg/kg) every 12h for 72h after CLP. At 3days after surgery, diaphragm contractile forces were measured by determining the force-frequency curve and muscle fiber areas by hematoxylin-eosin staining. Moreover, the NRG-1 expression level in the diaphragm was detected by Western blotting. Furthermore, the proteins in the PI3K/Akt signaling pathway and its downstream Akt-mTOR and Akt-FOXO axes were detected by Western blotting analysis. In L6 myotubes treated with lipopolysaccharide (LPS) and NRG-1β, PI3K/Akt signaling pathway-related protein expression was further determined using the PI3K inhibitor LY294002. Exogenous NRG-1β could compensate for sepsis-induced diminished NRG-1 in the diaphragm and attenuate the reduction in diaphragm contractile forces and muscle fiber areas during sepsis. Moreover, NRG-1β treatment could activate the PI3K/Akt signaling pathway in the diaphragm during sepsis. The inhibition of p70S6K and 4E-BP1 on the Akt-mTOR axis and the increased expression of Murf1 on the Akt-FOXO axis were reversed after NRG-1 treatment. In addition, NRG-1β could activate the PI3K/Akt signaling pathway in L6 myotubes treated with LPS, while the PI3K inhibitor LY294002 blocked the effects of NRG-1β. NRG-1 expression in the diaphragm was reduced during sepsis, and exogenously administered recombinant human NRG-1β could attenuate sepsis-induced diaphragm atrophy by activating the PI3K/Akt signaling pathway.

Specific effects of neuregulin-1β on the communication between DRG neurons and skeletal muscle cells in vitro.

The communication between primary afferent neuron and skeletal muscle (SKM) is one of the important factors on maintaining the structure and function of SKM cells. Neuregulin-1β (NRG-1β) signaling is essential for regulating synaptic neurotransmission. Here, we established a neuromuscular coculture model of dorsal root ganglion (DRG) sensory neurons and SKM cells to explore the nerve-muscle communication in the presence of exogenous NRG-1β. The expression of three distinct subtypes (TrkA, TrkB, and TrkC) of tyrosine kinase receptors was monitored for the phenotypical alterations of the neurons. The aggregation extent of acetylcholine receptor (AChR) represents the specific changes of SKM cells after NRG-1β incubation in this neuromuscular coculture model. The results showed that NRG-1β not only enhanced neurite outgrowth of DRG neurons but also increased the length and branches of SKM cells. NRG-1β treatment not only induced expression of all the three subtypes of Trk receptors in neurons but also promoted AChR aggregation on the surface of SKM cells. The effects of NRG-1β could be blocked by administration of ERK1/2 inhibitor PD98059, PI3K inhibitor LY294002, and JAK2 inhibitor AG490, respectively. These data imply that NRG-1β is essential for the nerve-muscle communication by enhancing growth and modifying phenotypes of the two different kinds of cells. The specific effects produced by NRG-1β add novel interpretation for nerve-muscle communication between sensory neurons and SKM cells.

Modulation of the Neuregulin 1/ErbB system after skeletal muscle denervation and reinnervation

Neuregulin 1 (NRG1) is a growth factor produced by both peripheral nerves and skeletal muscle. In muscle, it regulates neuromuscular junction gene expression, acetylcholine receptor number, muscle homeostasis and satellite cell survival. NRG1 signalling is mediated by the tyrosine kinase receptors ErbB3 and ErbB4 and their co-receptors ErbB1 and ErbB2. The NRG1/ErbB system is well studied in nerve tissue after injury, but little is known about this system in skeletal muscle after denervation/reinnervation processes. Here, we performed a detailed time-course expression analysis of several NRG1 isoforms and ErbB receptors in the rat superficial digitorum flexor muscle after three types of median nerve injuries of different severities. We found that ErbB receptor expression was correlated with the innervated state of the muscle, with upregulation of ErbB2 clearly associated with the denervation state. Interestingly, the NRG1 isoforms were differently regulated depending on the nerve injury type, leading to the hypothesis that both the NRG1α and NRG1β isoforms play a key role in the muscle reaction to injury. Indeed, in vitro experiments with C2C12 atrophic myotubes revealed that both NRG1α and NRG1β treatment influences the best-known atrophic pathways, suggesting that NRG1 might play an anti-atrophic role.

Chronic Neuregulin-1β Treatment Mitigates the Progression of Postmyocardial Infarction Heart Failure in the Setting of Type 1 Diabetes Mellitus by Suppressing Myocardial Apoptosis, Fibrosis, and Key Oxidant-Producing Enzymes

BackgroundType 1 diabetes mellitus (DM) patients surviving myocardial infarction (MI) have substantially higher cardiovascular morbidity and mortality compared to their nondiabetic counterparts owing to the more frequent development of subsequent heart failure (HF). Neuregulin (NRG)-1β is released from cardiac microvascular endothelial cells and acts as a paracrine factor via the ErbB family of tyrosine kinase receptors expressed in cardiac myocytes to regulate cardiac development and stress responses. Because myocardial NRG-1/ErbB signaling has been documented to be impaired during HF associated with type 1 DM, we examined whether enhancement of NRG-1β signaling via exogenous administration of recombinant NRG-1β could exert beneficial effects against post-MI HF in the type 1 diabetic heart. Methods and ResultsType 1 DM was induced in male Sprague Dawley rats by a single injection of streptozotocin (STZ) (65 mg/kg). Two weeks after induction of type 1 DM, rats underwent left coronary artery ligation to induce MI. STZ-diabetic rats were treated with saline or NRG-1β (100 µg/kg) twice per week for 7 weeks, starting 2 weeks before experimental MI. Residual left ventricular function was significantly greater in the NRG-1β–treated STZ-diabetic MI group compared with the vehicle-treated STZ-diabetic MI group 5 weeks after MI as assessed by high-resolution echocardiography. NRG-1β treatment of STZ-diabetic MI rats was associated with reduced myocardial fibrosis and apoptosis as well as decreased gene expression of key oxidant–producing enzymes. ConclusionsThese results suggest that recombinant NRG-1β may be a promising therapeutic for HF post-MI in the setting of type 1 DM.

NRG-1β exerts neuroprotective effects against ischemia reperfusion-induced injury in rats through the JNK signaling pathway

BackgroundNeuregulin-1β (NRG-1β) has great potential to be developed into therapeutics for neuroprotection. The aim of the current study was to analyze the effects and possible signaling pathway of NRG-1β on brain tissues in a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R). MethodsIn order to observe the protective effect of NRG-1β on MCAO/R, the neurological deficit and infarct volume were measured using a modified neurological severity score (mNSS) test and by triphenyl tetrazolium chloride (TTC) staining. In order to detect the antagonistic effect of NRG-1β on nerve cells and the blood–brain barrier (BBB), the morphology and structure of cortical brain tissues were observed by Evans Blue (EB) staining, hematoxylin–eosin (H&E) and Nissl staining, in situ cell death detection kit, and transmission electron microscopy (TEM). In order to investigate whether NRG-1β exhibited a significant neuroprotective effect via the JNK signaling pathway, the activity of JNK and the levels of phospho-MKK4, phospho-JNK, pan-JNK and phospho-c-Jun were tested using a JNK activity screening kit, immunofluorescent labeling, and western blot analysis, respectively. ResultsIn the NRG-1β treatment group, accompanied with a decrease in JNK activity, the protein levels of phospho-JNK, phospho-MKK4 and phospho-c-Jun decreased, the ischemia-induced apoptosis decreased, the abnormal morphological structures of nerve cells were ameliorated, the integrity of the BBB was restored, and infarct volume was reduced. At the same time, neurological function was significantly recovered. ConclusionNRG-1β exerts a neuroprotective effect through the JNK signaling pathway in MCAO/R rats.

Abstract 249: Chronic Neuregulin-1β Treatment Mitigates the Progression of Post-myocardial Infarction Heart Failure in the Setting of Type 1 Diabetes Mellitus

Aim: Neuregulin-1β (NRG-1β), a growth factor critical for cardiac development as well as maintenance of heart function after injury has been shown to significantly improve heart function in preclinical rodent models. Importantly, number of studies are ongoing to test the efficacy of NRG-1β as a treatment for patients with chronic heart failure. However, the efficacy of recombinant NRG-1β in a typ1 diabetic model of heart failure due to myocardial infarction (MI) has not been investigated. The aim of the present study was to determine the efficacy of exogenous NRG-1β to improve residual cardiac function after MI in type1 diabetic rats. Methods and Results: Sprague Dawley rats were induced type 1 diabetes by a single injection of streptozotocin (STZ) (65 mg/kg). Two weeks after induction of type 1 diabetes, rats underwent left coronary artery ligation to induce MI. STZ-diabetic rats were treated with saline or NRG-1β (100 ug/kg) twice a week for 7 weeks, starting two weeks prior to experimental MI. Residual left ventricular (LV) function was significantly greater in the NRG-1β-treated STZ-diabetic MI group compared to the vehicle-treated STZ-diabetic MI group 5 weeks after MI as assessed by high-resolution echocardiography. Furthermore, NRG-1β treatment in STZ-diabetic MI rats reduced myocardial fibrosis and apoptosis as well as decreased gene expression of key oxidant-producing enzymes. Conclusion: This study demonstrates that augmentation of NRG-1β signaling in STZ-diabetic post-MI rats via therapy with exogenous recombinant NRG-1β will alleviate subsequent HF through improvements in residual LV function via protection against adverse remodeling and apoptosis.

Abstract 166: Anti-cachectic Role of Neuregulin-1β in Heart Failure

Neuregulin-1β (NRG-1β) is a growth and differentiation factor with pleiotropic systemic effects. Because NRG-1β has therapeutic potential for heart failure and known growth effects in skeletal muscle, we hypothesized that it might affect heart failure-associated cachexia, a severe co-morbidity characterized by a loss of muscle mass. We therefore assessed NRG-1β’s effect on skeletal muscle gene expression in a swine model of heart failure using recombinant Glial Growth Factor 2 (USAN - cimaglermin alfa), a version of NRG-1β currently being tested in humans with systolic heart failure. Animals received one of two intravenous doses (0.67 or 2 mg/kg) of NRG-1β bi-weekly for 4 weeks, beginning one week after infarct. Skeletal muscle was collected at the time of euthanasia from the intercostal space and paired-end RNA sequencing performed. NRG-1β treatment altered expression of 583 transcripts including 242 transcripts altered at both doses. These included genes required for myofiber growth, maintenance and survival such as MYH3, MYHC, MYL6B, KY and HES1 . Importantly, NRG-1β altered the directionality of at least 85 genes associated with cachexia, including myostatin, which negatively regulates myoblast differentiation by down-regulating MyoD expression. Consistent with this, MyoD was increased in NRG-1β treated animals. In vitro experiments with myoblast cell lines confirmed that NRG-1β induces skeletal muscle differentiation with an absolute requirement for ERBB signaling on myoblast differentiation. These findings suggest a NRG-1β-mediated anti-atrophic, anti-cachexia effect that may provide additional benefits to this potential therapy in heart failure.

Neuregulin1β Effects on Brain Tissue via ERK5-Dependent MAPK Pathway in a Rat Model of Cerebral Ischemia-Reperfusion Injury.

Neuregulin1β (NRG1β), a member of the excitomotor of tyrosine kinase receptor (erbB) family, was recently shown to play a neuroprotective role in cerebral ischemia-reperfusion injury. The present study analyzed the effects and its possible signaling pathway of NRG1β on brain tissues after cerebral ischemia-reperfusion injury. A focal cerebral ischemic model was established by inserting a monofilament thread to achieve middle cerebral artery occlusion, followed by an NRG1β injection via the internal carotid artery. NRG1β injection resulted in significantly improved neurobehavioral activity according to the modified neurological severity score test. Tetrazolium chloridestaining revealed a smaller cerebral infarction volume; hematoxylin-eosin staining and transmission electron microscopy showed significantly alleviated neurodegeneration in the middle cerebral artery occlusion rats. Moreover, expression of phosphorylated MEK5, phosphorylated ERK5, and phosphorylated MEK2C increased after NRG1β treatment, and the neuroprotective effect of NRG1β was attenuated by an injection of the MEK5 inhibitor, BIX02189. Results from the present study demonstrate that NRG1β provides neuroprotection following cerebral ischemia-reperfusion injury via the ERK5-dependent MAPK pathway.

Neuregulin1 alpha activates migration of neuronal progenitors expressing ErbB4.

Deficits in neuronal migration during development in the central nervous system may contribute to psychiatric diseases. The ligand neuregulin1 (NRG1) and its receptor ErbB4 are genes conferring susceptibility to schizophrenia, playing a key role in the control of neuronal migration both during development and adulthood. Several NRG1 and ErbB4 isoforms were identified, which deeply differ in their characteristics. Here we focused on the four ErbB4 isoforms and the two NRG1 isoforms differing in their EGF-like domain, namely α and β. We hypothesized that these isoforms, which are differently regulated in schizophrenic patients, could play different roles in neuronal migration. Our hypothesis was strengthened by the observation that both NRG1α and NRG1β and the four ErbB4 isoforms are expressed in the medial and lateral ganglionic eminences and in the cortex during development in rat. We analysed in vitro the signal transduction pathways activated by the different ErbB4 isoforms following the treatment with soluble recombinant NRG1α or NRG1β and the ability to stimulate migration. Our data show that two ErbB4 isoforms, namely JMa-cyt2 and JMb-cyt1, following NRG1α and NRG1β treatment, strongly activate AKT phosphorylation, conferring high migratory activity to neuronal progenitors, thus demonstrating that both NRG1α and NRG1β can play a role in neuronal migration.

Neuregulin-1β induces embryonic stem cell cardiomyogenesis via ErbB3/ErbB2 receptors

NRG-1β (neuregulin-1β) serves multiple functions during embryonic heart development by signalling through ErbB family receptor tyrosine kinases (ErbB2, ErbB3 and ErbB4). Previous studies reported that NRG-1β induces cardiomyogenesis of mESCs (mouse embryonic stem cells) at the later stages of differen-tiation through ErbB4 receptor activation. In the present study we systematically examined NRG-1β induction of cardiac myocytes in mESCs and identified a novel time window, the first 48 h, for NRG-1β-based cardiomyogenesis. At this time point ErbB3, but not ErbB4, is expressed. In contrast with the later differentiation of mESCs in which NRG-1β induces cardiomyogenesis via the ErbB4 receptor, we found that knocking down ErbB3 or ErbB2 with siRNA during the early differentiation inhibited NRG-1β-induced cardiomyogenesis in mESCs. Microarray analysis of RNA expression at this early time point indicated that NRG-1β treatment in mESCs resulted in gene expression changes important to differentiation including up-regulation of components of PI3K (phosphoinositide 3-kinase), a known mediator of the NRG-1β/ErbB signalling pathway, as well as activation of CREB (cAMP-response-element-binding protein). Further study demonstrated that the NRG-1β-induced phosphorylation of CREB was required for cardiomyogenesis of mESCs. In summary, we report a previously unrecognized role for NRG-1β/ErbB3/CREB signalling at the pre-mesoderm stage for stem cell cardiac differentiation.

Improvement of cardiac function and reversal of gap junction remodeling by Neuregulin-1β in volume-overloaded rats with heart failure.

ObjectiveWe performed experiments using Neuregulin-1β (NRG-1β) treatment to determine a mechanism for the protective role derived from its beneficial effects by remodeling gap junctions (GJs) during heart failure (HF).MethodsRat models of HF were established by aortocaval fistula. Forty-eight rats were divided randomly into the HF (HF, n = 16), NRG-1β treatment (NRG, n = 16), and sham operation (S, n = 16) group. The rats in the NRG group were administered NRG-1β (10 µg/kg per day) for 7 days via the tail vein, whereas the other groups were injected with the same doses of saline. Twelve weeks after operation, Connexin 43 (Cx43) expression in single myocytes obtained from the left ventricle was determined by immunocytochemistry. Total protein was extracted from frozen left ventricular tissues for immunoblotting assay, and the ultrastructure of myocytes was observed by transmission electron microscopy.ResultsCompared with the HF group, the cardiac function of rats in the NRG group was markedly improved, irregular distribution and deceased Cx43 expression were relieved. The ultrastructure of myocytes was seriously damaged in HF rats, and NRG-1β reduced these pathological damages.ConclusionsShort-term NRG-1β treatment can rescue pump failure in experimental models of volume overload-induced HF, which is related to the recovery of GJs structure and the improvement of Cx43 expression.

Palmitate alters neuregulin signaling and biology in cardiac myocytes

The saturated fatty acid palmitate alters normal cell function via disruption of cell signaling, and this effect has been implicated in the end-organ damage associated with dyslipidemia. Neuregulin-1β (NRG-1β) is a growth and survival factor in cardiac myocytes. We tested the hypothesis that palmitate alters NRG-1β signaling and biology in isolated neonatal rat cardiac myocytes. Palmitate treatment inhibited NRG-1β activation of the PI3-kinase/Akt pathway in myocytes. We found that the pro-apoptotic activity of palmitate was increased by NRG-1β treatment. The effects of palmitate on NRG-1β signaling and survival were reversed by the mono-unsaturated fatty acid oleate. Under control conditions NRG-1β decreases p53 expression in myocytes. In the presence of palmitate, NRG-1β caused an increase in p53 expression, bax multimer formation, concurrent with degradation of mdm2, a negative regulator of p53. Thus in the presence of palmitate NRG-1β activates pro-apoptotic, rather than pro-survival signaling in cardiac myocytes.