Regulation of glucose uptake in neonatal rat cardiomyocytes by Neuregulin1β

Purpose:
Neuregulin (Nrg) signaling plays an indispensable role in cardiac development and homeostasis. Since Nrg1β was shown to have beneficial effects on the heart in animal models of cardiac disease, several clinical trials are investigating its therapeutic value. The present study examines metabolic effects and molecular mechanisms that are induced by Nrg1β in cardiomyocytes, which could help to understand the positive effects observed in disease models in vivo and in clinics.
Methods:
Isolated neonatal rat ventricular myocytes (NRVMs), isolated adult rat ventricular myocytes (ARVMs), neonatal rats and adult mice served as models for our investigations. By pharmacological inhibition or siRNA knockdown, the role of proteins of interest was analyzed. For signaling analysis, proteins were extracted and Western blotting was performed. Glucose uptake was measured by [3H]-deoxy-D-glucose incorporation, glycolysis was investigated by Seahorse assay and protein synthesis was detected by [3H]-phenylalanine incorporation. GLUT4 translocation was evaluated by expressing a c-Myc-GLUT4-mCherry construct that was transfected into NRVMs. Proliferation of NRVMs was analyzed by immunofluorescence. In addition, neonatal rats and adult streptozotocin (STZ) mice were treated with Nrg1β or insulin to assess activation of glucose uptake pathways in the heart in vivo.
Results:
Similar to insulin and IGF-I, Nrg1β caused an 80% increase in glucose uptake and elevated the level of glycolysis in NRVMs. Dose-response curves of combinations of insulin and Nrg1β showed no additive effects on glucose uptake. Nrg1β activated the PI3K/Akt, the c-Src/FAK and the MAPK/Erk1/2 pathways, whereas only the PI3K/Akt and c-Src/FAK pathways appeared implicated in the glucose uptake. Pharmacological inhibition demonstrated that Nrg1β- and insulin-induced glucose uptake require PI3K and Akt. Both Nrg1β and insulin increased phosphorylation of Akt and AS160, whereas phosphorylation of IRS-1 at Tyr612 and IRS-1/2 degradation was only induced by insulin. Interestingly, Nrg1β and insulin showed an additive effect on Akt phosphorylation, whereas this was not the case for Akt's target AS160. Knockdown of GLUT4 and an increase in GLUT4 translocation indicated its involvement in Nrg1β- and insulin-induced glucose uptake in NRVMs. Since glucose uptake is known to contribute to protein synthesis, we measured protein synthesis after Nrg1β stimulation, which was 50% increased, matching with elevated p70S6K1 and 4E-BP1 phosphorylation. Knockdown experiments revealed that the ErbB2/ErbB4 homodimer is required for the Nrg1β-induced glucose uptake as well as protein synthesis. Given the fact that glucose uptake and protein synthesis are part of proliferative responses, we analyzed proliferation of NRVMs in presence of Nrg1β. Interestingly, our preliminary data showed that Nrg1β increases proliferation of NRVMs, however only in combination with IGF-I. Furthermore, in order to compare our in vitro findings with an in vivo model, we analyzed the effect of Nrg1β on the neonatal rat heart. Western blot analysis of Nrg1β-stimulated neonatal hearts revealed comparable signaling effects as observed in vitro. In contrast to NRVMs and neonatal rat hearts, ARVMs did not increase the phosphorylation of Akt or AS160 and showed no effect on glucose uptake after Nrg1β stimulation. Moreover, while Nrg1β injection of diabetic STZ mice revealed a systemic effect on blood glucose clearance likely mediated by the liver, no activation of the PI3K/Akt/AS160 pathway was detectable in the hearts of these mice.
Conclusions:
Our major finding is that Nrg1β induces glucose uptake in NRVMs by a similar mechanism as insulin. This mechanism requires the ErbB2/ErbB4 homodimer, PI3K, Akt and AS160. Consistently, Nrg1β increases glucose uptake by GLUT4 translocation to a similar extent as insulin. Compared to insulin, Nrg1β signaling has distinct effects on IRS phosphorylation, which is not followed by IRS degradation. Altogether, our findings on glucose uptake, protein synthesis and proliferation contribute to a better understanding of the molecular mechanisms of Nrg1β in CMs, which may be applied to improve treatments of heart disease and promote heart regeneration in humans.