Ganapathy Balakrishnan, Nandhagopal Nikitha, Polizzotti Brian D, Bennett David, Asan Alparslan, Wu Yijen, Kühn Bernhard
Department of Cardiology, Boston Children's Hospital, Boston, MA 02115, United States of America.
Department of Pediatrics, University of Pittsburgh, and Richard King Mellon Institute for Pediatric Research and Division of Pediatric Cardiology, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA 15224, United States of America.
PLoS One. 2016 May 13;11(5):e0155456. doi: 10.1371/journal.pone.0155456. eCollection 2016.
We previously developed and validated a strategy for stimulating heart regeneration by administration of recombinant neuregulin (rNRG1), a growth factor, in mice. rNRG1 stimulated proliferation of heart muscle cells, cardiomyocytes, and was most effective when administration began during the neonatal period. Our results suggested the use of rNRG1 to treat pediatric patients with heart failure. However, administration in this age group may stimulate growth outside of the heart.
NRG1 and ErbB receptor expression was determined by RT-PCR. rNRG1 concentrations in serum were quantified by ELISA. Mice that received protocols of recombinant neuregulin1-β1 administration (rNRG1, 100 ng/g body weight, daily subcutaneous injection for the first month of life), previously shown to induce cardiac regeneration, were examined at pre-determined intervals. Somatic growth was quantified by weighing. Organ growth was quantified by MRI and by weighing. Neoplastic growth was examined by MRI, visual inspection, and histopathological analyses. Phospho-ERK1/2 and S6 kinase were analyzed with Western blot and ELISA, respectively.
Lung, spleen, liver, kidney, brain, and breast gland exhibited variable expression of the NRG1 receptors ErbB2, ErbB3, ErbB4, and NRG1. Body weight and tibia length were not altered in mice receiving rNRG1. MRI showed that administration of rNRG1 did not alter the volume of the lungs, liver, kidneys, brain, or spinal cord. Administration of rNRG1 did not alter the weight of the lungs, spleen, liver, kidneys, or brain. MRI, visual inspection, and histopathological analyses showed no neoplastic growth. Follow-up for 6 months showed no alteration of somatic or organ growth. rNRG1 treatment increased the levels of phospho-ERK1/2, but not phospho-S6 kinase.
Administration protocols of rNRG1 for stimulating cardiac regeneration in mice during the first month of life did not induce unwanted growth effects. Further studies may be required to determine whether this is the case in a corresponding human population.
我们之前研发并验证了一种通过给小鼠注射重组神经调节蛋白（rNRG1，一种生长因子）来刺激心脏再生的策略。rNRG1可刺激心肌细胞（即心肌纤维细胞）增殖，且在新生期开始给药时效果最佳。我们的研究结果提示可使用rNRG1治疗小儿心力衰竭患者。然而，在这个年龄组给药可能会刺激心脏以外部位的生长。
通过逆转录聚合酶链反应（RT-PCR）测定NRG1和表皮生长因子受体（ErbB）的表达。采用酶联免疫吸附测定（ELISA）法定量血清中的rNRG1浓度。对接受重组神经调节蛋白1-β1给药方案（rNRG1，100 ng/g体重，在出生后的第一个月每天皮下注射）的小鼠进行定期检查，该给药方案之前已证明可诱导心脏再生。通过称重对躯体生长进行定量。通过磁共振成像（MRI）和称重对器官生长进行定量。通过MRI、肉眼检查和组织病理学分析检查肿瘤生长情况。分别采用蛋白质免疫印迹法和ELISA法分析磷酸化细胞外信号调节激酶1/2（phospho-ERK1/2）和核糖体蛋白S6激酶（phospho-S6激酶）。
肺、脾、肝、肾、脑和乳腺均表现出NRG1受体ErbB2、ErbB3、ErbB4和NRG1的不同表达。接受rNRG1的小鼠体重和胫骨长度未发生改变。MRI显示，给予rNRG1未改变肺、肝、肾、脑或脊髓的体积。给予rNRG1未改变肺、脾、肝、肾或脑的重量。MRI、肉眼检查和组织病理学分析均未显示肿瘤生长。随访6个月显示躯体或器官生长未发生改变。rNRG1治疗可提高磷酸化ERK1/2的水平，但未提高磷酸化S6激酶的水平。
在出生后的第一个月给小鼠注射rNRG1以刺激心脏再生的给药方案未产生不良生长效应。可能需要进一步研究以确定在相应的人类群体中是否也是如此。
