Nerve growth factor stimulates cardiac regeneration via cardiomyocyte proliferation in experimental heart failure

Abstract

Although the adult heart likely retains some regenerative capacity, heart failure (HF) typically remains a progressive disorder. We hypothesise that alterations in the local environment contribute to the failure of regeneration in HF. Previously we showed that nerve growth factor (NGF) is deficient in the failing heart and here we hypothesise that diminished NGF limits the cardiac regenerative response in HF. The capacity of NGF to augment cardiac regeneration was tested in a zebrafish model of HF. Cardiac injury with a HF phenotype was induced in zebrafish larvae at 72 hours post fertilization (hpf) by exposure to aristolochic acid (AA, 2.5 µM, 72-75 hpf). By 168 hpf, AA induced HF and death in 37.5% and 20.8% of larvae respectively (p<0.001). NGF mRNA expression was reduced by 42% (p<0.05). The addition of NGF (50 ng/ml) after exposure to AA reduced the incidence of HF by 50% (p<0.01) and death by 65% (p<0.01). Mechanistically, AA mediated HF was characterised by reduced cardiomyocyte proliferation as reflected by a 6.4 fold decrease in BrdU+ cardiomyocytes (p<0.01) together with features of apoptosis and loss of cardiomyocytes. Following AA exposure, NGF increased the abundance of BrdU+ cardiomyocytes in the heart by 4.8 fold (p<0.05), and this was accompanied by a concomitant significant increase in cardiomyocyte numbers. The proliferative effect of NGF on cardiomyocytes was not associated with an anti-apoptotic effect. Taken together the study suggests that NGF stimulates a regenerative response in the failing zebrafish heart, mediated by stimulation of cardiomyocyte proliferation.

Figure 1. Experimental heart failure model in zebrafish.

A Zebrafish (100 hpf) without HF (left panel) and with the HF phenotype (right panel). Bar graphs showing B. Ventricular fractional area change at 96 hpf, C. Incidence of AA induced HF at 168 hpf. D. Incidence of AA induced mortality at 168 hpf. ***p<0.001.

Figure 2. Aristolochic acid increases apoptosis in the heart, deplete CMs and reduces CM proliferation.

Bar graphs demonstrating A. Expression of caspase 3 mRNA in the heart at 96 hpf, B. Time dependent changes cardiomyocyte number and C. Abundance of BrdU positive cardiomyocytes at 100 hpf.

Figure 3. NGF decreases the incidence of AA induced HF and death.

Kaplan Meier Curves showing A. Effect of NGF on frequency of HF, B. Effect of NGF on frequency of death and C. Effect of the NGF trkA receptor antagonist, K-252a, on frequency of HF. D. Effect of K-252a on frequency of death. **p<0.01, ***p<0.001.

Figure 4. NGF does not attenuate AA induced apoptosis. A.

Bar graph represents absence of effect of NGF on caspase 3 mRNA expression in AA treated (72–75 hpf) zebrafish hearts at 96 hpf. B and C. Representative IMARIS images of cardiomyocyte TUNEL staining in control and AA treated zebrafish heart at 100 hpf. Scale bar = 50 µm. D. Bar graph showing quantitative analysis of TUNEL staining (n = 6 per group, * p<0.05 vs control).

Figure 5. NGF stimulates CM proliferation following AA exposure. A.

Bar graph showing the effect of NGF on cardiomyocyte proliferation. B–D. Representative IMARIS images of BrdU+ cardiomyocytes in the heart (76–100 hpf) from control (B), AA treated (C) and AA treated and post treated with NGF (D). Scale Bars  = 50 µm. *p<0.05, **p<0.01.

Figure 6. NGF restores total CM number following AA exposure.

Bar graphs showing that NGF restores total cardiomyocyte number in zebrafish model of experimental heart failure. Time dependent effects at A. 96 hpf, B. 120 hpf, and C. 144 hpf. **p<0.01.

Figure 7. NGF induced CM proliferation and total CMs.

Bar graphs show that fish exposed to egg water supplemented with NGF from 76 hpf onwards (grey bars) increases cardiomyocyte proliferation (A), and increases total cardiomyocyte number over time (B). *p<0.05.