Cardiac effects of NRG-1β in a doxorubicin-therapy model – focus on early molecular events and sex-differences

Doxorubicin (dox) is one of the most potent chemotherapeutic drugs nowadays. However, dox use in clinics is limited by its high myocardial toxicity. Recent evidence suggests that dox-induced early disturbance of metabolic pathways in cardiomyocytes conditions the later development of chronic heart failure (CHF) in patients. Interestingly, circulating levels of neuregulin-1β (NRG-1) are correlated with the development of chronic heart failure in cancer patients after dox-therapy. Moreover, inhibition of the NRG-1 co-receptor ErbB2 worsens cardiac outcomes, indicating a cardioprotective role of NRG-1 in these patients. In the present study, our aim was to 1) characterize an in vivo model of dox-induced cardiac dysfunction in male and female mice, and 2) investigate whether NRG-1 protects against dox-induced cardiotoxicity by modulating acute metabolic mechanisms, especially autophagy.
C57BL/6J male and female mice were assigned to a CTL, dox or NRG/dox group. Mice were injected intraperitoneally with vehicle or dox (4 mg/kg) at day 0, 2, 5, 8, 10, 12 yielding a cumulative dose of 24 mg/kg. NRG-1 (20 μg/kg) or vehicle (PBS) were injected 30 min before each dox injection and continued every other day until sacrifice. Heart function and architecture were measured using echocardiography at multiple timepoints before sacrifice. Body weight loss was observed in males and females, indicative of systemic toxicity. These effects were stronger in males than in females. In males, dox increased cardiac ejection fractions and left ventricular wall thickness at 2 weeks after treatment-begin and NRG-1 prevented this. In contrast, in females dox did not modulate cardiac function, however, dox decreased LV wall thickness and NRG-1 did not affect this at 3 weeks. These results were associated with increased GLUT1 and β-MHC mRNA in dox-treated males, while dox-treated females only showed an increase in β-MHC mRNA levels. Yet, NRG-1 did not modify any of these molecular responses neither in males nor in females. Our data show that dox-induced cardiac remodeling is sex-dependent: males respond with stronger cardiac compensation to dox than females at the functional and molecular level.
For our mechanistic studies, additional mice were sacrificed at day 1 after the dox injection, 1 h after NRG-1 stimulation. In males, the single dox injection was enough to decrease the cardiac LC3II/I ratios. On the other hand, female mice did not show any dox-induced cardiac modulation of the LC3II/I ratios at day 1. While NRG-1 had no effects in males, in females it significantly decreased LC3II/I and enhanced the phosphorylation of ULK1 at its inhibitory S757 site, suggesting that NRG-1 reduces autophagy in dox-treated females.
To investigate underlying mechanisms, neonatal rat ventricular myocytes (NRVM) were used as a model. NRVM were pre-treated in serum-free medium with E64D/pepstatin A inhibitors to study autophagic flux, or directly stimulated with NRG-1. After NRG-1 stimulation, NRVM were treated with dox (1 μM). At 16 h the NRVM model mimicked the molecular response observed in females at 24 h. At 1 h, NRG-1 alleviated a dox-induced increase of LC3II protein, and this was associated with increased ULK1-pS757 as well as decreased FoxO1 nuclear location. Consistently, at 6 h, NRG/dox-treated NRVM showed decreased expression of the FoxO1 target genes LC3 and ULK1 in comparison to dox-treated cells. Furthermore, our preliminary data obtained with MTT and luciferin-based CELL TITER GLO assays showed that dox induced an increase in succinate synthetase activity, without changing ATP content at 3 h. Interestingly, this was associated with decreased mitochondrial/nuclear DNA ratios at 3 and 16 h, and NRG-1 alleviated it at 3 h.
In conclusion, our study showed that the NRG-1 treatment regimen that we used might not be suitable for males to alleviate dox-induced early cardiac autophagy disturbances. In females, evidence for NRG-1 inhibition of the autophagic pathway were uncovered upon dox-therapy and surprisingly, might be independent of the mTORC1 pathway. Our study highlighted the need to characterize sex-differences upon dox-therapy and NRG-1 preventive treatment to promote personalized medicine.