The zebrafish caudal fin is comprised of bone, nerve, vasculature, connective and skin tissues, and fully regenerates within 10–14 days following amputation.14 The rate of caudal fin regrowth can be readily quantified. To identify small molecules capable of stimulating regeneration, adult fish were subjected to caudal fin amputation and then given daily intraperitoneal (IP) injections of either vehicle or candidate compounds. The length of regenerated caudal fin tissue was quantified at 4 days post-amputation (dpa). Treatment with 0.125 mg/kg MSI-1436 increased the rate of fin regeneration by ~ 3-fold relative to vehicle (Fig. 1a, b). No further stimulatory effect was observed at a 10-fold higher concentration (Supplementary Figure S1). MSI-1436 had no effect on caudal fin regeneration at 0.0125 mg/kg (Supplementary Figure S1).
MSI-1436 is a highly selective inhibitor of PTP1B.6, 7 The structurally closely related aminosterol, squalamine (Supplementary Figure S2) differs from MSI-1436 by the absence of an amine group on its polyamine tail. Importantly, squalamine had no effect on the rate of caudal fin regeneration (Fig. 1b).
Zebrafish appendage regeneration is mediated by formation of the blastema, a dedifferentiated and proliferative tissue that serves as a reservoir for proliferating cells.15, 16 We stained caudal fin blastemas with antibodies directed against phosphorylated histone 3 (H3P), a marker of mitosis.17, 18 Animals treated with MSI-1436 exhibited an ~ 2-fold increase in H3P positive cells within the blastema, suggesting that enhanced proliferation of blastemal cells could represent at least one mechanism for the increased rate of appendage regeneration (Fig. 1c, d).
Animals treated with MSI-1436 showed normal fin morphology at 21 dpa, with no signs of overgrowth or malformation (Fig. 1e, f). Moreover, zebrafish embryos and uninjured adult animals treated with daily IP injections of 1.25 mg/kg MSI-1436 for 14 and 40 days, respectively, did not exhibit obvious developmental abnormalities or altered mortality (Supplementary Fig. S3A, B). The lack of MSI-1436 toxicity in zebrafish is consistent with previous findings in mammals.19 Taken together, these studies suggest that MSI-1436 modulates the repair and regenerative responses of injured tissue and does not alter normal developmental processes or tissue homeostasis.
MSI-1436 stimulates zebrafish heart regeneration
Given the stimulatory effects of MSI-1436 on the regenerative response of the caudal fin, we also tested its effect on heart regeneration. Adult zebrafish were subjected to a partial ventricular resection, removing ~ 20% of the ventricular mass, and subsequently given daily IP injections of either vehicle or MSI-1436 at a concentration of 0.125 mg/kg for 3 days post-surgery. We first assessed CM proliferation indices as a metric for regenerative capacity. Hearts were isolated at 3 dpa, cryosectioned and stained to identify cells co-expressing the CM marker Mef2 and the proliferation marker proliferating cell nuclear antigen (PCNA) (Fig. 2a). To establish a CM proliferation index, we quantified Mef2-positive cells and PCNA-positive cells as a percentage of the total number of cells expressing only Mef2 within a defined injury area.20 Relative to vehicle control, MSI-1436 treatment increased CM proliferation ~ 2.6-fold at 3 dpa (Fig. 2a, b). Similar to appendage regeneration, squalamine had no effect on CM proliferation (Fig. 2b). Vivo-morpholino (MO) knockdown of PTP1B in adult zebrafish had a similar effect to that of MSI-1436 on CM proliferation (Supplementary Fig. S4A, B). Combinatorial treatments with PTP1B MO and MSI-1436 did not significantly elevate CM proliferation in comparison to PTP1B MO treatment alone (Supplementary Fig. S4B), suggesting MSI-1436 stimulation is mediated by repression of PTP1B activity.
Regeneration of the zebrafish heart is complete within 2 months following ventricular resection.21 To determine whether MSI-1436 increases the rate of heart regeneration, we quantified the expression of Tropomyosin, a muscle specific marker expressed in differentiated cardiac sarcomeres, by immunohistochemistry.22 MSI-1436 treatment increased Tropomyosin expression nearly 2-fold within the injury zone at 21 dpa (Fig. 2c, d). To confirm these findings, we employed the Tg(gata4:GFP) reporter strain.23 At 21 dpa, Tg(gata4:GFP) is expressed in the outer muscle layer of the heart and in newly regenerated muscle within the injury environment. Treatment of injured hearts with MSI-1436 stimulated an ~ 1.5-fold increase in Tg(gata4:GFP) expression within the injury zone in comparison to vehicle treatment (Fig. 2e, f). Thus, by two independent analyses, we demonstrate that MSI-1436 administration results in an increase in regenerated muscle. Taken together, data in Fig. 2 demonstrate that MSI-1436 is potent stimulator of zebrafish CM proliferation and new heart muscle regeneration following acute heart injury.
MSI-1436 stimulates recovery of heart function after ischemic injury in the adult mouse
The neonatal mouse heart regenerates in a manner similar to that of the adult zebrafish. However, the capacity for heart regeneration in mice is lost rapidly approximately one week after birth.24 Given the striking effect of MSI-1436 on regeneration of multiple tissue types in adult zebrafish, we examined the effect of this compound on heart injury in the adult mouse, which has limited heart regenerative capacity.25, 26 Ischemic heart injury was induced in 6–8 week old mice by permanent ligation of the left anterior descending (LAD) coronary artery.27 Twenty-four hours after ligation, we performed echocardiography to confirm the presence of heart injury. These mice were then administered MSI-1436, at either 0.125 or 1.25 mg/kg, or vehicle only, via IP injections. Based on the known pharmacokinetics of MSI-1436 in mice,19 injections were repeated every 3 days to maintain plasma concentration levels.
MSI-1436 administration increased survival at 28 days from 55% (n = 20) in vehicle-treated control animals to 70 and 80% (n = 10; 20) in mice administered 0.125 or 1.25 mg/kg MSI-1436, respectively (Fig. 3a).
To determine the effects of MSI-1436 treatment on heart function, we performed echocardiography at 4 weeks after LAD artery ligation to quantify changes in fractional shortening and ejection fraction. As expected, at 1-day post-injury (dpi) and prior to drug treatment, all animals showed significantly reduced heart function when compared to baseline values of uninjured animals (Fig. 3b, c). However, at 28 dpi, both fractional shortening and ejection fraction exhibited a ~ 2–3-fold improvement in MSI-1436 treated animals compared to vehicle controls (Fig. 3b, c; Supplementary Table S1). No difference in the degree of improvement was observed in mice administered 0.125 vs. 1.25 mg/kg MSI-1436.
MSI-1436 treatment reduces infarct size and stimulates cell cycle entry in adult mouse heart
To determine how MSI-1436 improved heart function, we performed a series of histological studies. Infarct scar size in hearts isolated 3 dpi was similar in vehicle- and MSI-1436-treated mice (Fig. 4a, b) indicating that administration of MSI-1436 did not reduce the initial amount of ischemic damage and scar formation. However, infarct scar size was reduced 53% in mice administered 0.125 mg/kg MSI-1436 for 4 weeks compared to untreated mice (Fig. 4c, d; Supplementary Fig. S5). Quantification of scar tissue by area measurements and infarct length yielded similar levels of decrease in scar tissue (Fig. 4d).
Thinning of the ventricular wall was also greatly reduced in animals administered MSI-1436 (Fig. 4c). Hearts isolated 28 dpi from mice administered vehicle or MSI-1436 showed similar increases in heart weight to body weight ratio (HW/BW) (Fig. 4e; Supplementary Table S2) indicating the presence of hypertrophy and/or new muscle formation. Previous MSI-1436 studies on mice demonstrated a dose-dependent decrease in body weight.19 However, drug treatment at 0.125 and 1.25 mg/kg did not induce significant changes to body weight at 4 weeks, suggesting the increase in HW/BW is not due to weight loss (Supplementary Table S2).
To determine whether reduced infarct scar size and increased heart weight were associated with increased cellular proliferation, we quantified EdU labeling in hearts isolated 28 days after LAD artery ligation. EdU labeling in the infarct border zone was increased 4.5-fold by MSI-1436 treatment (Fig. 5b, c). Similar results were observed using antibodies directed against H3P (Fig. 5e, f). A sub-population of the EdU labeled cells expressed sarcomeric α-actinin, a marker of mature cardiomyocytes (Fig. 5B). MSI-1436 treatment increased the number of EdU and sarcomeric α-actinin double positive cardiomyocytes 5.7-fold from an average of 1.5 cells in vehicle to 8.7 cells in drug treated hearts (Fig. 5D). EdU labeling in regions distant from the infarct border zone was nominal and no differences were observed between vehicle- and MSI-1436-treated mice (Supplementary Fig. S6).
Caudal fin and heart regeneration in adult zebrafish14, 28 occurs through a primary mechanism of dedifferentiation of spared cells within the injury zone. To determine whether MSI-1436 also acts on stem cells per se, we quantified the proliferative activity of Pax7-expressing skeletal muscle satellite cells, which are stem cells essential for muscle regeneration.29, 30
Pax7 is a well-defined marker of the proliferative activity of satellite cells and can be monitored noninvasively using mice engineered to express a Pax7-driven luciferase reporter (Pax7CreER/LuSEAP).31, 32 To determine whether MSI-1436 alters satellite cell proliferative activity, we injured tibialis anterior muscles of Pax7CreER/LuSEAP mice by BaCl2 injection. Twenty-four hours after injury, mice were administered by IP injection vehicle or 0.125 mg/kg MSI-1436. Injections were repeated every three days. MSI-1436 increased reporter activity 1.5-2-fold 7–21 days after injury was induced (Fig. 6a, b). At 21 days after injury when muscle regeneration is complete, muscle morphology was similar in vehicle-treated mice and MSI-1436-treated mice (Fig. 6c), indicating that MSI-1436 increases satellite cell proliferation without inducing aberrant tissue regeneration.