Calcitriol Attenuates Doxorubicin-Induced Cardiac Dysfunction and Inhibits Endothelial-to-Mesenchymal Transition in Mice

Figures

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  Figure 1. Calcitriol attenuated the doxorubicin-induced impairment of cardiac diastolic function. (A) The C57BL/6 mice were divided into 3 groups (n = 10, in each group). Group 1 (control group) received the same volume of saline injection, group 2 received doxorubicin (4 mg/kg twice weekly for 4 weeks; a cumulative dose of 32 mg/kg) and group 3 received doxorubicin (same dose as group 2) with calcitriol (150 ng/kg/day for 12 weeks). Echocardiography was performed on all the mice before the first dose of doxorubicin injection and 12 weeks after the first dose of doxorubicin injection. All the mice were sacrificed 13 weeks after the first dose of doxorubicin injection. (B) Representative M-mode of long-axis echocardiographic images for measurements of the IVSd, LVPWd, LVIDd, and LVIDs. (C) Representative sample of pulsed-wave Doppler revealing diastolic function: IVRT, peak velocity of the E wave and A wave. (D) Tissue Doppler measurements of septal mitral E’and A’. (E) Calculated ejection fraction of M-mode. (F) E/E’ ratio by pulse-wave Doppler and tissue Doppler. (G) Calculated IVRT by pulsed-wave Doppler. * p < 0.05, vs. Ctrl group. (n = 10 in each group), & p < 0.05, vs. Dox group. (n =…

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  Figure 2. Effect of calcitriol on doxorubicin-induced cardiomyopathy. (A) Representative gross images of the whole heart. (B) Representative images of hematoxylin and eosin (H/E) staining of the ventricular sections. Scale bar: 1000 μm. (C) Quantification of the HW/BW ratio (n = 10 in each group). (D) Representative images of labeled WGA (green) and cardiac troponin I (red) staining of the ventricular sections. Scale bar: 20 μm. N = 3 in each group, 5 high power field (HPFs) per mouse, 15 HPFs total. (E) Quantification of the relative myocytes cross-sectional area (n = 3, 100 cells per field, 5 field in each heart). (F) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses of the mRNA levels of atrial Natriuretic Peptide (ANP) and brain natriuretic peptide (BNP). Data are normalized to the β-actin content (n = 3). Data are expressed as mean ± SEM, and n represents the number of animals. * p < 0.01, vs. Ctrl; & p < 0.05, vs. Dox. HW: heart weight; BW: body weight; WGA: wheat germ agglutinin.

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  Figure 3. Calcitriol inhibits doxorubicin-induced cardiac remodeling. (A) Representative images of Picro-Sirius Red staining of the ventricular sections. The red arrowheads indicate the positive Picro-Sirius Red staining area. Scale bar: 50 μm. (B) Quantification of the relative fibrotic area (n = 3 in each group, 3 fields in each heart). (C) Representative images of Masson’s trichrome staining of the ventricular sections. The white arrowheads indicate the myocardial fibrosis area.(n = 3 in each group, 3 field in each heart; right). Scale bar: 50 μm. (D) Quantification of the relative fibrotic area by Masson’s trichrome staining (n = 3 in each group, 3 field in each heart). (E) qRT-PCR analyses of the mRNA expression of Vimentin, Fibronectin, and TGF-β. The data are normalized to the β-actin content (n = 3 in each group). Data are expressed as mean ± SEM, * p < 0.05, vs. Ctrl; & p < 0.05, vs. Dox. (F) Representative immunoblotting analyses of fibronectin, vimentin, sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), TGF-β, and p-Smad2 in the heart tissue. (G) Quantification of the relative protein levels (n = 3). GAPDH was used as ian nternal control. Total-Smad2 as an internal…

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  Figure 4. Calcitriol attenuated doxorubicin-induced endothelial-to-mesenchymal transition (EndMT) in the mouse model. (A) Representative photomicrographs of double immunofluorescence staining for the antibodies: CD31 (red) and GFP (green). (B) Quantification of CD31+/GFP+ cells per visual field in the Ctrl, Dox and Dox–calcitriol groups. (C) Representative photomicrographs of double immunofluorescence staining for the antibodies: CD31 (red). Quantification of CD31+ cells per visual field in the Ctrl, Dox, and Dox–calcitriol groups. (D) Representative photomicrographs of immunofluorescence staining for the antibodies: GFP (green) and vimentin (red). (E) Quantification of GFP+ and Vimentin+ cells per visual field in the Ctrl, Dox, and Dox–calcitriol groups. (F) Representative photomicrographs of double immunofluorescence staining for the antibodies: vimentin (red). Quantification of vimentin+ cells per visual field in the Ctrl, Dox and Dox–calcitriol groups. n = 3 mice per group, (5 high power field (HPFs) per mouse, 15 HPFs total). Scale bar: 200 μm; Data are expressed as mean ± SEM, and n represents the number of animals. * p < 0.05 vs. Ctrl, & p < 0.05 vs. Dox.

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  Figure 5. Calcitriol-attenuated doxorubicin-induced Smad2 activation in vivo. (A) Representative photomicrographs of double immunofluorescence staining antibodies with p-Smad2 (red) and GFP (green) in different groups. (B) Enlarged area from (A, white box). The yellow arrows indicated p-Smad2+ co-localized in GFP+ cells. (C) Quantification of p-Smad2+/GFP+ cells per visual field in the Ctrl, Dox, and Dox–calcitriol groups. N = 3 mice per group (5 high power field (HPFs) per mouse, 15 HPFs total). Scale bar: 200 μm. Experiments were conducted in triplicate. Data are expressed as mean ± SEM, and n represents the number of animals. * p < 0.05 vs. Ctrl, & p < 0.05 vs. Dox.

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  Figure 6. Calcitriol-attenuated TGF-β-induced EndMT in vitro. (A) Representative immunoblotting analyses of CD31, smooth muscle 22alpha, and p-Smad2 in the human umbilical vein cells (HUVECs). Quantification of the relative protein levels (n = 3). GAPDH was used as an internal control, and total Smad2 as an internal control for p-Smad2. Data are expressed as mean ± SEM. * p < 0.05 vs. Ctrl, & p < 0.05 vs. TGF-β. Experiments were conducted in triplicate. (B) Representative photomicrographs of double immunofluorescence staining antibodies with SM22α (red) and VE-cadherin (green). Quantification of vimentin+ cells per visual field in the Ctrl, TGF-β.only, and TGF-β–calcitriol groups. Experiments were conducted in triplicate (5 high power fields (HPFs) per mouse, 15 HPFs total). Scale bar: 200 μm * p < 0.05 vs. Ctrl, & p < 0.05 vs. TGF-β.

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  Figure 7. Calcitriol-attenuated TGF-β-induced fibroblast-to-myofibroblast transition (FMT) in vitro. (A) Representative photomicrographs of the myofibroblast. (B) Quantification of myofibroblast cells per visual field in the Ctrl, TGF-β only, and TGF-β–calcitriol groups. Experiments were conducted in triplicate (5 high power fields (HPFs) per mouse, 15 HPFs total). * p < 0.05 vs. Ctrl, & p < 0.05 vs. TGF-β. (C) Representative immunoblotting analyses of fibronectin, alpha-SMA, collagen I, and p-Smad2 in the cardiac fibroblasts. (D) Quantification of the relative protein levels (n = 3). GAPDH was used as an internal control. Total Smad2 was used as an internal control for p-Smad2. Data are expressed as mean ± SEM, and n represents the number of animals. * p < 0.05 vs. Ctrl, & p < 0.05 vs. TGF-β. Experiments were conducted in triplicate.

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  Figure 8. Schematic representation of the mechanism of action of calcitriol in the protection against DoIC-associated cardiac remodeling. Doxorubicin induced damage to cardiomyocytes and endothelial cells and caused subsequent tissue remodeling by the augmentation of the TGF-β pathway. Calcitriol attenuated Doxorubicin-caused cardiac remodeling by the attenuation of TGF-β-mediated EndMT and FMT.