Neuregulin-1 Treatment Research Articles

Abstract 1463: Neuregulin-1 Promotes Embryonic Stem Cell Differentiation into the Cardiac Lineage in vitro

Our previous studies demonstrated that transplantation of embryonic stem cell (ESCs) derived cardiomyocytes improved the cardiac function in myocardial infarction injured mice. However, significant challenges remain to be solved, such as how to produce sufficient amount and mature differentiated cardiomyocytes for the therapy. Neuregulin-1 (NRG-1) is essential for the development of the heart. Here, we tested whether NRG-1 could promote the differentiation of ESCs into cardiomyocytes. Differentiation of murine ESCs was induced by the hanging-drop method. NRG-1 was added at different points of ESC differentiation. The expression of cardiac specific genes, as well as NRG-1 and its erbB receptors were measured by quantitative RT-PCR and Western blot analysis. Our results showed that during the course of hanging-drop induced ESC differentiation, the mRNA level of cardiac-specific structure proteins cardiac troponin T (cTNT), cardiac troponin I (cTNI), myosin light chain 2a (MLC2a) and alpha-sarcomeric actin was elevated. The mRNA level of cardiac transcription factors NKX2.5 and GATA4 was also increased. In addition, beating EBs first appeared on day7 and about 90% of beating EBs was observed on day12. Further, the mRNA level of NRG-1α and NRG-1β, and both mRNA and protein levels of erbB receptors were elevated during the differentiation of ESCs. When NRG-1 was added during a certain developmental window, the mRNA level of cTNT, MLC2a and MEF2c, as well as the protein level of cTNT, CXN40, a marker for cardiac conduction system, and transcriptional factors NKX2.5, GATA4 were increased in NRG-1 treated ESCs compared to untreated cells. These results demonstrated that NRG-erbB signaling was activated during the course of hang-drop induced ESC differentiation; NRG-1 treatment further promoted ESC differentiation into the cardiac lineage in vitro. Our results suggest that NRG-1 might be used to produce more and/or more mature ESC derived cardiomyocytes for the treatment of cardiovascular diseases in the clinical setting.

Neuregulin-1 enhances differentiation of cardiomyocytes from embryonic stem cells

Neuregulin-1 (NRG-1) is a multifunctional regulator that acts through receptor tyrosine kinases of the epidermal growth factor (EGF/ErbB) receptor family in diverse tissue. ErbB receptors are expressed in developing embryoid bodies (EBs), and the importance of the NRG-1/ErbB signaling axis in heart development has been investigated, but the underlying molecular mechanism is poorly studied. NRG-1 treatment at 100 ng/ml significantly increased the number of beating EBs of differentiated murine embryonic stem cells (ESCs). Furthermore, NRG-1 up-regulated the expression of the cardiac-restricted transcription factors Nkx2.5 and GATA-4 and factors involved in differentiated cardiac cells (alpha-MHC, beta-MHC and alpha-actinin); NRG-1-induced increase of Nkx2.5 transcription was inhibited by treatment with the PI3 K inhibitor or ErbB receptor inhibitor. Western blot analysis confirmed that the expression of phospho-Akt in the beating foci was increased in the presence of NRG-1. Our results suggest that NRG-1 promotes cardiomyocyte differentiation of ESCs and the ErbB/PI3 K/Akt signaling pathway is one of the underlying molecular mechanisms.

Extended Therapeutic Window and Functional Recovery after Intraarterial Administration of Neuregulin-1 after Focal Ischemic Stroke

We have previously shown that neuregulin-1 (NRG-1) protects neurons from ischemic brain injury if administered before focal stroke. Here, we examined the therapeutic window and functional recovery after NRG-1 treatment in rats subjected to 90 mins of middle cerebral artery occlusion (MCAO) and 24 h of reperfusion. Neuregulin-1 (2.5 microg/kg [corrected] bolus, 1.25 microg/kg/min [corrected] infusion) reduced infarct volume by 89.2%+/-41.9% (mean+/-s.d.; n=8; P<0.01) if administered immediately after the onset of reperfusion. Neuroprotection was also evident if NRG-1 was administered 4 h (66.4%+/-52.6%; n=7; P<0.01) and 12 h (57.0%+/-20.8%; n=8; P<0.01) after reperfusion. Neuregulin-1 administration also resulted in a significant improvement of functional neurologic outcome compared with vehicle-treated animals (32.1%+/-5.7%; n=9; P<0.01). The neuroprotective effect of the single administration of NRG-1 was seen as long as 2 weeks after treatment. Neurons labeled with the neurodegeneration marker dye Fluoro-JadeB were observed after MCAO in the cortex, but the numbers were significantly reduced after NRG-1 treatment. These results indicate that NRG-1 is a potent neuroprotective compound with an extended therapeutic window that has practical therapeutic potential in treating individuals after ischemic brain injury.

Neuregulin-1 Induces Expression of Egr-1 and Activates Acetylcholine Receptor Transcription Through an Egr-1-binding Site

Localization of acetylcholine receptors (AChRs) to neuromuscular synapses is mediated, in part, through selective transcription of AChR genes in myofiber synaptic nuclei. Neuregulin-1 (NRG-1) and its receptors, ErbBs, are concentrated at synaptic sites, and NRG-1 activates AChR synthesis in cultured muscle cells, suggesting that NRG-1-ErbB signaling functions to activate synapse-specific transcription. Previous studies have demonstrated that NRG-1-induced transcription is conferred by cis-acting elements located within 100 bp of 5′ flanking DNA from the AChR epsilon subunit gene, and that it requires a GABP binding site within this region. To determine whether additional regulatory elements have a role in NRG-1 responsiveness, we used transcriptional reporter assays in a muscle cell line, and we identified an element that is required for NRG-1-induced transcription (neuregulin response element, NRE). Proteins from myotube extracts bind the NRE and NRG-1 treatment of the cells stimulates this binding. The ability of NRG-1 to stimulate formation of a protein–DNA complex with the NRE requires induction of protein expression. The complex contains early growth response-1 (Egr-1), a member of the Egr family of transcription factors, because proteins in the complex bind specifically to an Egr consensus site, and formation of the complex is inhibited by antibodies to Egr-1. NRG-1 induces expression of Egr-1 in myotubes, which presumably is responsible for the ability of NRG-1 to stimulate protein binding to the NRE. These results suggest that NRG-1 signaling in myotubes involves induction of Egr-1 expression, which in turn serves to activate transcription of the AChR epsilon subunit gene.