Recombinant neuregulin 1 does not activate cardiomyocyte DNA synthesis in normal or infarcted adult mice

Abstract

Objectives: Neuregulin 1 signaling plays an important role in cardiac trabecular development, and in sustaining functional integrity in adult hearts. Treatment with neuregulin 1 enhances adult cardiomyocyte differentiation, survival and/or function in vitro and in vivo. It has also been suggested that recombinant neuregulin 1β1 (NRG1β1) induces cardiomyocyte proliferation in normal and injured adult hearts. Here we further explore the impact of neuregulin 1 signaling on adult cardiomyocyte cell cycle activity.

Methods and results: Adult mice were subjected to 9 consecutive daily injections of recombinant NRG1β1 or vehicle, and cardiomyocyte DNA synthesis was quantitated via bromodeoxyuridine (BrdU) incorporation, which was delivered using mini-osmotic pumps over the entire duration of NRG1β1 treatment. NRG1β1 treatment inhibited baseline rates of cardiomyocyte DNA synthesis in normal mice (cardiomyocyte labelling index: 0.019±0.005% vs. 0.003±0.001%, saline vs. NRG1β1, P<0.05). Acute NRG1β1 treatment did result in activation of Erk1/2 and cardiac myosin regulatory light chain (down-stream mediators of neuregulin signalling), as well as activation of DNA synthesis in non-cardiomyocytes, validating the biological activity of the recombinant protein. In other studies, mice were subjected to permanent coronary artery occlusion, and cardiomyocyte DNA synthesis was monitored via tritiated thymidine incorporation which was delivered as a single injection 7 days post-infarction. Daily NRG1β1 treatment had no impact on cardiomyocyte DNA synthesis in the infarcted myocardium (cardiomyocyte labelling index: 0.039±0.011% vs. 0.027±0.021%, saline vs. NRG1β1, P>0.05).

Summary: These data indicate that NRG1β1 treatment does not increase cardiomyocyte DNA synthesis (and consequently does not increase the rate of cardiomyocyte renewal) in normal or infarcted adult mouse hearts. Thus, any improvement in cardiac structure and function observed following neuregulin treatment of injured hearts likely occurs independently of overt myocardial regeneration.

Figure 1. Examples of cardiomyocyte DNA synthesis assay.

(A) Use of BrdU to monitor cardiomyocyte DNA synthesis in non-injured adult mice receiving 9 consecutive daily injections of NRG1β1 (BrdU was delivered using a mini-osmotic pump). Left panel shows anti-β-galactosidase immune reactivity, middle panel shows anti-BrdU immune reactivity, and right panel shows the merged image. Arrow indicates a BrdU positive cardiomyocyte nucleus, arrowhead indicates a BrdU positive non-cardiomyocyte nucleus. Bar = 10 microns. (B) BrdU incorporation in the nuclei of the small intestine microvilli epithelial cells of an NRG1β1-treated mouse. Note the absence of BrdU signal in the muscularis mucosae zone (asterisk). Bar = 10 microns. (C) Use of ³H-Thy to monitor cardiomyocyte DNA synthesis in non-injured adult mice receiving 9 consecutive daily injections of NRG1β1 (³H-Thy was delivered as a single bolus 1 hour after the last NRG1β1 treatment). Arrow indicates a ³H-Thy positive cardiomyocyte nucleus, arrowhead indicates a ³H-Thy positive non-cardiomyocyte nucleus. Bar = 10 microns.

Figure 2. NRG1β1 elicits biological responses in the adult mouse heart.

(A) Western blot demonstrating the levels of total Erk1/2 p42/p44, P-Erk1/2[Thr²⁰²/Thy²⁰⁴] and RLC in mice treated with NRG1β1 or vehicle (hearts harvested and processed 90 minutes after treatment). Densometric quantitation revealed that NRG1β1 treatment resulted in a 987% increase in the level of ERK1 phosphorylation, a 5727% increase in the level of ERK2 phosphorylation, and a 21% increase in the level of phosphorylated RLC vs. vehicle-treated mice (p<0.01, Student’s t-test). (B) Non-cardiomyocyte ³H-Thy nuclear labeling index in non-injured adult mice following 9 consecutive daily injections of NRG1β1 (5 sections analyzed from each of 4 independent mice) or vehicle (4 sections analyzed from each of 4 independent mice). *: p<0.05 vs. vehicle treated animals, Student’s t-test.

Figure 3. Expression of the MHC-nLAC reporter transgene following 9 consecutive daily injections of NRG1β1.

Treated hearts were subjected to retrograde collagenase perfusion, and the resulting dispersed cell preparations were reacted with X-GAL and processed for cardiac alpha-actinin immune reactivity. Cardiac alpha-actinin immune reactivity (red signal) and nuclear β-galactosidase activity (inset) in a bi-nucleated (left panel) and a mono-nucleated (right panel) cardiomyocyte. Bar = 50 microns.