Neuregulin-1/erbB-Activation Improves Cardiac Function and Survival in Models of Ischemic, Dilated, and Viral Cardiomyopathy
Xifu Liu, PhD*,
Xinhua Gu, MD*,
Zhaoming Li, PhD*,
Xinyan Li, MD, PhD*,
Hui Li, MD*,
Jianjie Chang, MD*,
Ping Chen, MD*,
Jing Jin, BSc*,
Bing Xi, MSc*,
Denghong Chen, BSc*,
Donna Lai, PhD ,
Robert M. Graham, FAA, MD and
Mingdong Zhou, PhD*,*
* Zensun Sci & Tech Ltd., Shanghai, China
Victor Chang Cardiac Research Institute and the School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
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Figure 1 rhNRG-1 construction, synthesis, and erbB-activating activity. (A) Schematic of full-length membrane-bound form of neuregulin (NRG)-1 showing the immunoglobulin-like (IgG-L), and epidermal growth factor-like (EGF-L) regions of its extracellular domain as well as its transmembrane domain (TM) and cytoplasmic tail (CT). The construct used here (designated receptor-active 61-residue recombinant neuregulin-1 peptide [rhNRG-1]), encoding the EGF-like region of NRG-1, encompassing residues Ser177 to Glu237, followed by a stop codon (indicated) and a stroke volume 40 poly A+ tail (open box), was expressed in E. coli from a T7 promoter (arrow). (B) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (15%) fractionation of E. coli-expressed rhNRG-1 showing the resulting 6.7 kDa Coomassie-stained rhNRG-1 protein at a purity >95% (left lane) and molecular weight standards (right lane). (C) Purified rhNRG-1 stimulated erbB2 and erbB4 phosphorylation in cultured neonatal cardiomyocytes. Cells stimulated with vehicle or with full-length NRG-1 extracellular domain (ECD) (12) were used as negative and positive control subjects, respectively. (D) In vivo activity of rhNRG-1 was similarly evaluated by immunoprecipitation (IP, anti-erbB4 antisera) and immunoblotting (IB, anti–phospho-tyrosine, p-Try, antiserum [Sigma Biochemical, St. Louis, Missouri] or anti-erbB4 antiserum) of lysates prepared from the hearts of mice given 10 µg/kg rhNRG-1, IV, 10 or 20 min before being killed (upper panel). Holo-erbB4 visualized by anti-erbB4 IB is also shown (lower panel). (E) In vivo activation of extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinases was evaluated by IP and IB of lysates prepared from the hearts of mice given rhNRG-1, 10 µg/kg IV, 0, 10, 30, 90, and 270 min before being killed, with anti–phospho-ERK- (p-ERK) (upper panel; 42 and 44 kD isoforms are shown) and anti–holo-ERK- (lower panel) specific antibodies (Santa Cruz Biotechnology, Santa Cruz, California). (F) The p-ERK bands in (E) were quantitated by PhosphorImager analysis and expressed as the fold-increase (stimulation) over the basal (0 min) levels. The time-dependent increase in p-ERK44 is shown. Increased pERK1/2 mitogen-activated protein kinases (upper panels; lower panels show holo-ERK) were also observed in the lysates of the hearts of rats (G) and dogs (H) 10 min after bolus IV administration of rhNRG-1, 10 and 3 µg/kg, respectively.
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Figure 2 rhNRG-1 improves echocardiographically determined cardiac dimensions and performance after coronary artery ligation. (Upper panel) Diagrammatic schema of treatment protocols. (Lower panel) Cardiac dimensions and performance. Protocol I (panels A to D): 1 week after left anterior descending coronary artery (LAD) ligation, the rats received no treatment (sham-operated group, black bars, n = 10), vehicle (red bars, n = 12), or rhNRG-1 (10 µg/kg IV daily, blue bars, n = 13) for 5 days (Day 5 group) or 10 days (Day 10 group) with echocardiographic evaluation at the end of these treatment periods. Protocol II (panels E to H): as for Protocol I, except that vehicle (n = 15) or rhNRG-1 (n = 30) treatment was commenced 2 months after LAD ligation and continued for 5 days (Day 5 group) or 10 days (Day 10 group). The sham-operated group received no treatment (n = 20). LVEDD = left ventricular end-diastolic diameter; LVESD = left ventricular end-systolic diameter; EF = ejection fraction; FS = fraction shortening; other abbreviations as in Figure 1. *p < 0.05 and **p < 0.01 versus vehicle.
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Figure 3 rhNRG-1 improves hemodynamics after LAD ligation. Treatments with vehicle, rhNRG-1 (10 µg/kg, IV, daily for 5 days), captopril (2.5 mg/kg twice daily by gavage for the duration of the study), or the same doses of rhNRG-1 and captopril (capt.) in combination were commenced 1 week after LAD ligation, and hemodynamic studies were performed 33 weeks thereafter. Sham-operated animals (Sham) received no treatment. (A) Heart rate (HR); (B) mean arterial pressure (MAP); (C) LV pressure (LVP) at end-systole (LVESP, open bars), and end-diastole (LVEDP, filled bars); and (D) +dP/dt (open bars) and –dP/dt (filled bars), rate of contraction and relaxation, respectively (n = 10 for sham and vehicle groups; n = 20 for each of the other groups). *p < 0.05; **p < 0.01 versus vehicle; +p < 0.01 versus captopril. Other abbreviations as in Figures 1 and 2.
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Figure 4 Neuregulin-1 improves survival of rats subjected to myocardial infarction (a and b), doxorubicin-induced cardiomyopathy (c), or mice with Coxsackie B3 myocarditis (d). Treatments indicated were commenced 1 week (a; n = 51) or 2 months (b; n = 20) after LAD ligation or when doxorubicin (3.3 mg/kg, IV, once weekly for 4 weeks) (c; n = 20) or Coxsackie B3 (0.2 ml virus solution, IP, 50% tissue culture infectious dose = 10–4) (d; n = 20) was administered. The receptor-active 61-residue recombinant neuregulin-1 peptide (rhNRG-1) was given at 10 and 30 µg/kg/day IV for 5 days to rats and mice, respectively, except for the doxorubicin-treated rats that received 20 µg/kg/day for 7 days. Captopril (Capt.) was given at 2.5 mg/kg twice daily by gavage for the duration of the study. Cumulative survival curves of the sham-operated (SO) or untreated control subjects (black), rhNRG-1-treated (dark blue), rhNRG-1 + Capt.-treated (light blue), Capt.-treated (green), or vehicle-treated (red) animals, respectively, are shown. (a) rhNRG-1 versus vehicle, p < 0.028; rhNRG-1 versus Capt. p = ns; (b) rhNRG-1 or Capt. versus vehicle, p < 0.02; (c) rhNRG-1 versus vehicle, p < 0.0001; (d) rhNRG-1 versus vehicle, p < 0.02.
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Figure 5 The receptor-active 61-residue recombinant neuregulin-1 peptide (rhNRG-1) improves doxorubicin-induced and Coxsackie B3 (CV-B3)-induced myocardial pathology. Transverse sections of myocardium obtained after: no treatment (control) (a), doxorubicin (3.3 mg/kg, IV, once weekly for 4 weeks) plus vehicle (b), or doxorubicin + rhNRG-1 (20 µg/kg/day for the first 7 days) (c) treatment of rats; or no treatment (control) (d), CV-B3 infection of mice + vehicle (e), or CV-B3 + NRG-1 (30 µg/kg/day, IV, for 5 days) (f). Staining: hematoxylin and eosin; magnification: x 10; white space bar (a) = 50 µm. Arrows indicate areas of necrosis (b) and lymphocytic infiltration (e), which are much less apparent with rhNRG-1 treatment (c, f).
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Figure 6 The receptor-active 61-residue recombinant neuregulin-1 peptide (rhNRG-1) improves cardiac functions in dogs with pacing-induced heart failure. Three groups of animals were studied (n = 6/group), as detailed in Methods. The control group (sham) had a pacing lead implanted, but animals in this group were not paced. The 2 experimental groups were paced at 230 beats/min for 3 weeks and then treated with either vehicle or rhNRG-1 (3 µg/kg/day, IV, for 5 days). Pacing was continued during the treatment periods. Heart rate (HR) (A), mean arterial pressure (MAP) (B), left ventricular (LV) end-systolic (white bars) and end-diastolic pressures (black bars) (C), and rates of LV contraction (+dP/dt, white bars) and relaxation (–dP/dt, black bars) (D) were determined after the treatment periods (i.e., after 26 days of pacing). Ejection fraction (EF, percent) (E) and cardiac output (F) were determined on 3 occasions: 1) after the recovery period immediately before pacing was commenced (black bars), 2) after pacing for 3 weeks (red bars), and 3) after 5 days of vehicle or rhNRG-1 treatment (blue bars). Pacing was discontinued at the time of the hemodynamic and cardiac contractility measurements. *p < 0.05; **p < 0.01 versus vehicle.
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