A Mouse Model of Dilated Cardiomyopathy Produced by Isoproterenol Acute Exposure Followed by 5-Fluorouracil Administration

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  Figure 1. Echocardiography analysis reveals typical features of DCM resulting from acute Isoproterenol exposure followed by 5–Fluorouracil administration. (A–F) Cumulative and representative data of cardiac dimensions and function in the different groups and time points of the study. (A), LVEDD = left ventricular end-diastolic diameter; (B) LVESD = left ventricular end-systolic diameter; (C) EF = ejection fraction; (D), FS = fractional shortening; (E), LVPWd = left ventricular posterior wall thickness at end diastole. (F) representative M-mode tracing of long-axis left ventricle. * p < 0.001; ** p < 0.0001. (G,H) Cumulative data (G) and representative pulsed-wave (PW) Doppler mitral velocity (MV) tracing (H) assessing diastolic function. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (I–K) Cumulative data of diastolic function (E′ and E/E′ ratio, respectively I and J) and relative representative PW tissue Doppler imaging (TDI) velocity tracing (K). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. BASE = baseline (n = 26); CTRL: saline treated control animals (n = 8); ISO: isoproterenol treated mice (n = 18 at 2 days; n = 8 at 14 and 28 days); ISO + 5-FU: isoproterenol…

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  Figure 2. Strain Analysis Reveals Typical Features of DCM resulting from Isoproterenol + 5-Fluorouracil administration. (A–C) Cumulative and representative data of strain analysis in the different groups and time points of the study. (A) GLS = global longitudinal strain; (B) GCS = global circumferential strain; (C) Representative strain tracing of long-axis left ventricle. ** p < 0.01. **** p < 0.0001. BASE = baseline (n = 26); CTRL: saline treated control animals (n = 8); ISO: isoproterenol treated mice (n = 18 at 2 days; n = 8 at 14 and 28 days); ISO + 5FU: isoproterenol plus 5FU treated mice (n = 8). Data are mean ± SD. ns, no significance.

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  Figure 3. Dilated Cardiomyopathy following ISO+ 5–FU is persistent at long follow-up. (A–E) Cumulative and representative data of cardiac dimensions and function in the different groups at 56 days. (A) LVEDD = left ventricular end-diastolic diameter; (B) LVESD = left ventricular end-systolic diameter; (C) FS = fractional shortening; (D) EF = ejection fraction; (E) LVPWd = left ventricular posterior wall thickness at end diastole. ** p < 0.01; **** p < 0.0001. (F–H) Cumulative diastolic data at 56 days. *** p < 0.001; **** p < 0.0001. (I,J) Cumulative strain analysis data at 56 days. *** p < 0.001; ** p < 0.01. (K) Representative mitral and tricuspid valve color Doppler analysis. CTRL: saline treated control animals (n = 8); ISO: isoproterenol treated mice (n = 8); ISO + 5-FU: isoproterenol plus 5-FU treated mice (n = 8). Data are mean ± SD. ns, no significance.

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  Figure 4. ISO + 5–FU administration causes cardiomyocyte remodeling typical of dilated cardiomyopathy. (A,B) Bar graph with cumulative data (A) and relative representative confocal images of cardiac cross-sections (B) showing significant cardiomyocyte (CM) necrosis (revealed by myosin-MF20- in vivo antibody labeling) in ISO + 5-FU (n = 3) when compared to CTRL (n = 3) and ISO (n = 3) (MF20, green fluorescence; cTnI, red; DAPI, blue nuclei). Scale bar = 75 µm. *** p < 0.001. (C,D) Bar graph with cumulative data (C) and relative representative confocal images of cardiac cross-sections (D) showing significant CM apoptosis (assessed by TdT assay) in ISO + 5-FU (n = 5) when compared to CTRL (n = 5) and ISO (n = 5) (TdT, green; cTnI, red; DAPI, blue nuclei). Scale bar = 8 µm. **** p < 0.0001. (E,F) Bar graph with cumulative data (E) and relative representative confocal images of cardiac cross-sections (F) showing significant CM hypertrophy (assessed by WGA assay) in ISO + 5-FU (n = 5) when compared to CTRL (n = 5) and ISO (n = 5) mice. (WGA, wheat germ agglutinin, red; cTnI, green; DAPI, blue nuclei). Scale bar = 25 µm. **** p < 0.0001. (G) Bar graphs showing the expressions of…

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  Figure 5. Acute isoproterenol exposure followed by 5–FU administration causes myocardial tissue alterations typical of DCM. (A,B) Bar graph with cumulative data (A) and relative representative light microscopy images of cardiac cross-sections (B) showing significant myocardial fibrosis (revealed by Picrosirius Red staining) and cardiomyocyte disarray in ISO + 5-FU (n = 5) when compared to CTRL (n = 5) and ISO (n = 5) (cTnI, red; DAPI, blue nuclei). **** p < 0.0001. (C,D) Bar graph with cumulative data (C) and relative representative confocal images of cardiac cross-sections (D) showing exaggerated myocardial tissue ROS production (assessed by Nitrotyrosine, 3-NT) in ISO + 5-FU (n = 5) when compared to CTRL (n = 5) and ISO (n = 5) (3-NT, green; DAPI, blue nuclei). Scale bar = 25 µm. **** p < 0.0001. (E,F) Bar graph with cumulative data (E) and relative representative confocal images of cardiac cross-sections (F) showing exaggerated myocardial tissue ROS production (assessed by Dihydroethidium, DHE) in ISO + 5-FU (n = 5) when compared to CTRL (n = 5) and ISO (n = 5) mice. (DHE, red). Scale bar = 25 µm. **** p < 0.0001. ns, no significance.

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  Figure 6. ISO + 5–FU administration produces myocardial tissue inflammation associated with senescence accumulation. (A,B) Bar graph with cumulative data (A) and relative representative confocal microscopy images of cardiac cross-sections (B) showing increased inflammatory cells (revealed by leukocyte marker, CD45, staining) in ISO + 5-FU (n = 5) when compared to CTRL (n = 5) and ISO (n = 5) (CD45, red; DAPI, blue nuclei). ** p < 0.001; *** p < 0.0001. Scale bar = 25 µm. (C,D) Bar graph with cumulative data (C) and relative representative light microscopy images of cardiac cross-sections (D) showing accumulation of senescent cardiac cells (assessed by p16 antibody staining) in ISO + 5-FU (n = 5) when compared to CTRL (n = 5) and ISO (n = 5) (p16, brown DAB staining). **** p < 0.0001. Scale bar = 10 µm. (E,F) Bar graph with cumulative data (E) and relative representative light microscopy images of cardiac cross-sections (F) showing accumulation of senescent CMs (assessed by p16 antibody staining)) in ISO + 5-FU (n = 5) when compared to CTRL (n = 5) and ISO (n = 5) (p16, brown DAB staining). **** p < 0.0001. Scale bar = 10 µm. ns, no significance.