Decellularized zebrafish cardiac extracellular matrix induces mammalian heart regeneration

Abstract

Heart attack is a global health problem that leads to significant morbidity, mortality, and health care burden. Adult human hearts have very limited regenerative capability after injury. However, evolutionarily primitive species generally have higher regenerative capacity than mammals. The extracellular matrix (ECM) may contribute to this difference. Mammalian cardiac ECM may not be optimally inductive for cardiac regeneration because of the fibrotic, instead of regenerative, responses in injured adult mammalian hearts. Given the high regenerative capacity of adult zebrafish hearts, we hypothesize that decellularized zebrafish cardiac ECM (zECM) made from normal or healing hearts can induce mammalian heart regeneration. Using zebrafish and mice as representative species of lower vertebrates and mammals, we show that a single administration of zECM, particularly the healing variety, enables cardiac functional recovery and regeneration of adult mouse heart tissues after acute myocardial infarction. zECM-treated groups exhibit proliferation of the remaining cardiomyocytes and multiple cardiac precursor cell populations and reactivation of ErbB2 expression in cardiomyocytes. Furthermore, zECM exhibits pro-proliferative and chemotactic effects on human cardiac precursor cell populations in vitro. These contribute to the structural preservation and correlate with significantly higher cardiac contractile function, notably less left ventricular dilatation, and substantially more elastic myocardium in zECM-treated hearts than control animals treated with saline or decellularized adult mouse cardiac ECM. Inhibition of ErbB2 activity abrogates beneficial effects of zECM administration, indicating the possible involvement of ErbB2 signaling in zECM-mediated regeneration. This study departs from conventional focuses on mammalian ECM and introduces a new approach for cardiac tissue regeneration.

Keywords: Extracellular matrix; cardiac repair; cardiomyogenesis; decellularization; heart regeneration; ischemic heart disease; myocardial infarction; zebrafish.

Fig. 1. Characterization of decellularized zECM.

(A) SEM images of fresh, decellularized, and ground decellularized normal and healing (3 dpa) zebrafish ventricular tissues. Scale bars, 10 μm (magnification, ×1000) and 1 μm (magnification, ×5000). (B) Particle size analysis of ground zECM by dynamic light scattering (n = 3). (C) Composition analyses of nzECM and adult mECM showing the amount of collagen, elastin, and GAGs in each group, respectively (n = 3 per group; data are means ± SD; *P < 0.05, **P < 0.01).

Fig. 2. Bioactivity of zECM on human cardiac precursor cell proliferation and migration.

(A to D) Relative proliferation rates of hCSCs and hHPs under stressed culture conditions following different cardiac ECM treatments. Addition of hzECM, nzECM, or mECM in the culture medium partially rescued the proliferation of (A) hCSCs and (B) hHPs under nutrient-deprived culture conditions. Addition of hzECM or nzECM, but not mECM, in the culture medium partially rescued the proliferation of (C) hCSCs and (D) hHPs under dual hypoxic (2.5% O₂) and nutrient-deprived culture conditions. *P ≤ 0.05, ^†P ≤ 0.01, ^§P ≤ 0.005, ^#P ≤ 0.001 compared to nutrient-deprived controls in all graphs. (E to H) Transwell chemotaxis assays with different cardiac ECM showing the migration of hCSCs and hHPs under nutrient-deprived culture conditions. hzECM and nzECM, but not mECM, induced prominent migration of (E) hCSCs and (G) hHPs (cells stained in green; scale bars, 50 μm). Significantly more (F) hCSCs and (H) hHPs migrated in hzECM- and nzECM-induced groups than in the mECM-induced group and saline control (n = 4 per group; data normalized to the respective saline control; **P < 0.01, ***P < 0.001 compared to mECM and saline; ^#P < 0.05 hzECM versus nzECM). Quantitative data represent means ± SD.

Fig. 3. Echocardiographic analyses of cardiac function.

(A) Schematic representation of the work flow using zECM (0.5 mg of suspension) for mammalian heart regeneration after AMI. Cardiac contractile function is indicated by (B) fractional area change and (C) ejection fraction; LV dimension is indicated by (D) EDA and (E) ESA (n = 7 per group; data analyzed by two-way repeated-measures analysis of variance (ANOVA); *P ≤ 0.05, ^†P ≤ 0.01, ^§P ≤ 0.005, ^#P ≤ 0.001 compared to saline controls in all graphs).

Fig. 4. Ultrasonic myocardial strain analysis.

(A) Representative B-mode images showing ROI selection at end-diastole and end-systole for myocardial strain analysis. Ant., anterior; Post., posterior; lat., lateral. (B) Representative graphs showing radial (upper panels) and circumferential (lower panels) strain estimation during a cardiac cycle: Strain of the infarcted area (dark blue) is closer to the noninfarcted area (yellow, green, red, and cyan) and normal heart in hzECM-treated group than in mECM- or saline-treated groups. Quantification of (C) radial and (D) circumferential strain (n = 3 per group; ^†P ≤ 0.01, ^#P ≤ 0.001 for hzECM and nzECM compared to mECM and saline; mECM compared to saline in all graphs).

Fig. 5. Proliferation of cardiac precursor cell populations.

Dual immunofluorescence detection and quantification of (A to C) c-kit⁺/Ki67⁺ proliferating cardiac stem cells, (D to F) PDGFRβ⁺/Ki67⁺ proliferating cardiac mesenchymal stromal cells, and (G and H) Wt1⁺/Ki67⁺ proliferating EPDCs at 6 weeks after MI at the mid-infarct level of mouse left ventricles. Arrows indicate doubly positive cells. All image analyses are performed within 20 × 10–μm areas in five images of each heart (n = 4 per group). All quantitative data represent means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared to saline and mECM; ^#P < 0.05, ^##P < 0.01 for hzECM versus nzECM. Scale bars, 50 μm.

Fig. 6. Cardiomyocyte proliferation and ErbB2 expression.

(A to C) Dual immunofluorescence detection and quantification of cTnT⁺/Ki67⁺ proliferating cardiomyocytes at 3 days after MI at the mid-infarct level of mouse left ventricles. Arrows indicate doubly positive cells. (D to F) Dual immunofluorescence detection and quantification of ErbB2⁺/cTnT⁺ cardiomyocytes at 3 days after MI at the mid-infarct level suggest the involvement of NRG1 signaling in zECM-treated groups. All image analyses were performed using 20 × 10–μm areas in five images of each heart (n = 4 per group). All quantitative data represent means ± SD. ***P < 0.001 compared to mECM and saline; ^#P < 0.05 for hzECM versus nzECM. Scale bars, 50 μm.

Fig. 7. Detection of NRG1 in zECM.

(A) Immunohistochemical detection of NRG1 (brown arrowheads) in normal and healing (3 dpa) zebrafish hearts. (B) Positive immunofluorescence detection of NRG1 (green) at the ventricular apex of normal zebrafish heart (nzH) and healing zebrafish heart (hzH) but not in the adult mouse heart (mH). (C) Consistent with the in situ NRG1 staining, Western blotting showed that both nzECM and hzECM ECM contain NRG1 protein. (D) Quantification data indicate hzECM and nzECM contain approximately 6.5 and 5 times more NRG1 than normal mECM, respectively. Data represent means ± SD. ***P < 0.001 versus mECM. Scale bars, 50 μm.

Fig. 8. Inhibition of ErbB2 activity with decellularized cardiac ECM treatment.

To inhibit ErbB2 activity in vivo, the ErbB2 inhibitor AG825 was intraperitoneally injected once (5 mg/kg) immediately after the administration of decellularized cardiac ECM. Cardiac contractile function is indicated by (A) fractional area change and (B) ejection fraction; LV dimension is indicated by (C) EDA and (D) ESA. No significant difference is observed between all groups at all time points (n = 7 per group; all P > 0.05; data analyzed by two-way repeated-measures ANOVA). Dual immunofluorescence detection and quantification of (E and F) c-kit⁺/Ki67⁺ proliferating cardiac stem cells and (G and H) ErbB2⁺/cTnT⁺ cardiomyocytes. No significant difference is observed between all groups (n = 4 per group, all P > 0.05). Scale bars, 50 μm.