Neuregulin-1 Administration Protocols Sufficient for Stimulating Cardiac Regeneration in Young Mice Do Not Induce Somatic, Organ, or Neoplastic Growth

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Fig 1. ErbB receptors are broadly expressed.

Mice were euthanized on day of life 1, 11, 35, and 65. Real time PCR showed all examined organs expressed ErbB receptor subunits. Statistical significance was tested in comparison to heart samples for each time point by analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig 2. A single weight-adjusted rNRG1-injection in neonatal mice induces similar peak rNRG1 concentrations in serum and significant increases in phospho ERK1/2 in kidney.

Mice of indicated ages received one subcutaneous injection of rNRG1 (0.1 μg/g). (A) The serum concentration of rNRG1 was determined by ELISA. (B-D) Mice either received no injection (control) or one BSA or rNRG1 injection on day of life 7 (P7) and organs were resected 1.5 hours later. Western blots showed phospho-ERK1/2 (B), and total ERK1/2 levels (C) in organs analyzed. Red rectangles indicate an example of corresponding control and treatment groups (B). Total ERK1/2 loading control is a representative example of all treatment groups (C). Quantification of phospho-ERK1/2 abundance normalized to their respective total ERK1/2 loading controls (D). Statistical analysis was tested with ANOVA followed by Bonferroni’s multiple comparison test (D). ****P<0.0001. Error bars indicate SEM. n = 2 for P1, n = 3 for P7, and P15 (A); n = 3 (B-D).

Fig 3. Diagrams of study design.

Daily injections of vehicle (BSA) and rNRG1 were begun on the day of life 0 (A, B) and day of life 5 (C) and are indicated with red boxes. Cryoinjury was performed on day of life 1 (P1, B, C). DOL, day of life.

Fig 4. Administration of rNRG1 in young mice does not alter somatic growth.

Mice underwent cryoinjury on day of life 1 (P1) and received daily BSA or rNRG1 injections (indicated by ± rNRG1) between day of life 0 and 35 (early, A-C) and day of life 5 and 35 (late, D-F). Live body weights (A, D), carcass body weights after resection of heart (B, E), and tibia length (C, F) show no significant difference (p>0.05) between BSA and rNRG1 treated mice. Statistical analysis by analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test.

Fig 5. Administration of rNRG1 has no effect on adult organ volumes.

Mice received daily BSA or rNRG1 injections between day of life 0 and 35 without heart injury (A-E), and day of life 5 and 35 and received cryoinjury on day of life 1(F-J). Lung (A, F), liver (B, G), kidney (C, H), brain (D, I), and spinal cord (E, J) volumes were determined by MRI and were not statistically different (p>0.05) between BSA and rNRG1 treated mice. Statistical analysis by Student’s t test (A, B, D-G, I, J) and analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test (C, H).

Fig 6. Administration of rNRG1 in neonatal mice does not alter organ growth.

Mice underwent cryoinjury on day of life 1 (P1) and received daily BSA or rNRG1 injections between day of life 0 and 35 (early, A-E) and day of life 5 and 35 (late, F-J). (A, F) Lung weights of rNRG1 mice were significantly lower at day of life 211 in late administration (F). (B-E, G-J) Spleen (B, G), liver (C, H), kidney (D, I), and brain (E, J) weights were not significantly different. Statistical analysis by Student’s t test (J) and analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test (A-I). *P<0.05.

Fig 7. Administration of rNRG1 during the somatic growth phase does not induce neoplastic growth at the organ level.

Mice received daily BSA or rNRG1 injections between day of life 0 and 35 (A), and, day of life 5 and 35 (B). Cryoinjury was performed on day of life 1 (B). (A-B) Representative whole body MRI scans for a BSA and rNRG1-treated mice on day of life 35 (A) and day of life 191 (B).

Fig 8. Administration of rNRG1 during the somatic growth phase does not induce neoplastic growth in the head and abdomen.

Mice received daily BSA or rNRG1 injections between day of life 0 and 35. Representative examples of sagittal (A, left panels), coronal (A, right panels) MRI scans of brain, and abdomen (B) for a BSA and rNRG1-treated mice on day of life 35 are shown.

Fig 9. Administration of rNRG1 during the somatic growth phase does not induce the formation of neoplastic foci.

Mice received daily BSA (A) or rNRG1 (B) injections between day of life 0 and 35, and organs were resected at the indicated time points. Cryoinjury was performed on day of life 1 (A, B, middle and right panels). Organs were sectioned and subjected to Hematoxylin and Eosin (H&E) staining. Representative images from each organ are shown. Scale bar, 10 μm. DOL, day of life.

Fig 10. Daily administration of rNRG1 does not increase phospho ERK1/2 and S6 kinase levels.

Mice received no injections (control) or daily BSA or rNRG1 injections between day of life 0 and 35 (A-D) and organs were resected on day of life 35 (24 hours after the last injection). In another experiment, mice received no injection (control) or a single dose of BSA or rNRG1 injection on day of life 7 and organs were resected 1.5 hours later (E). (A, B) Western blots showed no significant difference in phospho-ERK1/2 abundance between control and treatment groups (A), and total ERK1/2 levels (B). Red rectangles indicate an example of corresponding lanes in control and treatment groups (A). Total ERK1/2 loading control is a representative example of all treatment groups (B). (C) Quantification of phospho-ERK1/2 abundance normalized to their corresponding total ERK1/2 levels. (D, E) Quantification of phospho P70-S6 kinase abundance by ELISA normalized to corresponding total P70-S6 kinase levels were unaltered on day of life 35 (D) and day of life 7 (E). Statistical analysis was tested with ANOVA followed by Bonferroni’s multiple comparison test (C-E).