Activation of the MET receptor attenuates doxorubicin‐induced cardiotoxicity in vivo and in vitro

FIGURE 1. The agonist mAb reduces the cachectic effect mediated by doxorubicin treatment. Adult mice were treated with placebo (n = 8), doxorubicin (DOXO; 7 mg·kg−1, once per week up to 3 weeks; n = 10), or Doxo + mAb (5 mg·kg−1 for 24 hr before each doxorubicin treatment; n = 12). For treatment protocols, see Figure S1. (a) Body weight measurements and (b) survival rates. (c) Representative images (left panels) and volume quantification (right scatterplot) of spino‐trapezius muscles calculated by magnetic resonance analysis at Day 28 after onset of doxorubicin treatment. Bars: 3 mm. *P < .05, significantly different from placebo; # P < .05, significantly different from Doxo group

FIGURE 2. MAb attenuates the doxorubicin‐induced cardiac dysfunction. (a) Representative diastolic and systolic MRI of left ventricles from placebo, Doxo, and Doxo + mAb mice analysed at Day 28 after onset of doxorubicin treatment. For mice treatments, see Figure S1. Bars: 3 mm. (b) Schematic diagram explaining the existing correlation among cardiac functional parameters evaluated by magnetic resonance analysis of left ventricles and formulas utilized to calculate the values reported in (c). The parameters reported are end diastolic volume (EDV), end systolic volume (ESV), stroke volume (SV), heart rate (HR), cardiac output (CO), and ejection fraction (EF). (c) Representation of MRI parameters changes in the three experimental groups (placebo, n = 8; Doxo, n = 10; and Doxo + mAb, n = 12). *P < .05, significantly different from placebo; # P < .05, significantly different from Doxo group

FIGURE 3. The agonist mAb exerts cardioprotective effects towards doxorubicin‐induced cardiotoxicity. (a) Representative images and quantification analysis of Masson's trichrome staining on hearts of placebo, Doxo, and Doxo + mAb mice at Day 35 after onset of doxorubicin treatment. Bars: 25 μm. (b) Western blot quantitative protein densitometry (above) and representative image (below) of α‐smooth muscle actin (α‐SMA) protein in heart tissues (n = 5 per group). (c) Gene expression analysis of connective tissue growth factor (Ctgf), collagen type I α 2 chain (Col1a2), TIMP metallopeptidase inhibitor 1 (Timp1), metallopeptidase 9 (Mmp9), α‐actin (Acta1), and atrial natriuretic peptide (Nppa) in the three cohorts of animals (n = 5 per group). Polr2a was used as reference gene for the expression data normalization. (a–c) For mice treatments, see Figure S1. (d) Western blot analysis was performed on heart lysates from mice of three treatment groups (placebo, Doxo, and Doxo + mAb; n = 5 per group). Doxorubicin (15 mg·kg−1) was injected i.p. as a single dose, and mAb (5 mg·kg−1) was administered 24 hr before doxorubicin. Mice were killed 48 hr after doxorubicin treatment. Quantitative protein…

FIGURE 4. MET activation reduces doxorubicin‐induced DNA damage in H9c2 cardiomyoblasts. (a–c) H9c2 cells were untreated (control) or treated with doxorubicin (Doxo, 25 μM), Doxo + HGF (0.5 nM), and Doxo + HGF + JNJ (500 nM). For cell treatments, see Figure S5b. (a) Protein levels of γH2AX and PARP1 were measured by western blot (left) and densitometric (right) analysis. (b) γH2AX protein levels were measured by flow cytometry. (c) γH2AX foci were analysed and counted by immunofluorescence as described in Section 2. Representative images of F‐actin (red) and γH2AX (green) co‐staining show weakly (left panels) and highly (right panels) positive nuclei. Bar: 20 μm. (d, e) H9c2 cells were pretreated with 100‐nM mAb instead of the natural ligand, HGF. The level of γH2AX protein was analysed by (d) western blot (densitometry on the top and representative image on the bottom) and (e) flow cytometry; α tubulin was used as loading control in all western blots. All experimental data were obtained from five independent experiments. *P < .05, significantly different from control; # P < .05, significantly different from Doxo; †P < .05, significantly different from Doxo + HGF

FIGURE 5. MET activation attenuates doxorubicin‐induced apoptosis in H9c2 cardiomyoblasts. (a–c) H9c2 cells were untreated (control) or treated with doxorubicin (Doxo, 25 μM), Doxo + HGF (0.5 nM), and Doxo + HGF + JNJ (500 nM). Cleaved caspase 3 apoptotic marker was quantified by (a) western blots, (b) immunofluorescence (cleaved caspase 3 green and DAPI blue; bar: 100 μm), and (c) flow cytometry. (d) H9c2 cells were pretreated with 100‐nM mAb instead of the natural ligand, HGF. The level of cleaved caspase 3 protein was determined by western blot (densitometry on the top and representative image on the bottom). Total and cleaved caspase 3 proteins (a, d) were evaluated using two different exposure times; α tubulin was used as loading control in all western blots. All experimental data were obtained from five independent experiments. *P < .05, significantly different from control; # P < .05, significantly different from Doxo; † P < .05, significantly different from Doxo + HGF

FIGURE 6. Activation of MET exerts cardioprotective effect towards doxorubicin via STAT3. (a) H9c2 cells were untreated (control) or treated with doxorubicin (DOXO, 25 μM), Doxo + HGF (0.5 nM), and Doxo + HGF + JNJ (500 nM). Protein levels of P‐STAT3 and STAT3 were analysed by western blot (densitometry above and representative image below). (b–f) H9c2 cells were untreated (control) or treated with Doxo (25 μM), Doxo + HGF (0.5 nM), and Doxo + HGF + Stattic (STAT3 inhibitor, 10 μM). Protein levels of (b) P‐STAT3 and total STAT3, (c) PARP1, and (e) total and cleaved caspase 3 were quantified by western blots (densitometric analysis (above) and representative immunoblot (below). Total and cleaved caspase 3 proteins were evaluated using two different exposure times; α tubulin was used as loading control. (d) γH2AX and (f) cleaved caspase 3 protein levels were measured by flow cytometry. All experimental data were obtained from five independent experiments. *P < .05, significantly different from control; # P < .05, significantly different from Doxo; †P < .05, significantly different from Doxo + HGF