After anesthetized with pentobarbital sodium (100 mg/kg, i.p.), the mice were perfused with cold PBS and hearts harvested, fixed with 4% paraformaldehyde (PFA) for 24 h, and embedded in paraffin wax or OCT. Serial sections were obtained at 6 µm intervals for paraffin embedded tissue and 8 µm intervals for OCT embedded tissue. The sections were stained with Masson's trichrome for detection of cardiac fibrosis and Alexa FluorTM 488 conjugated wheat germ agglutinin (WGA) (W11261, Invitrogen) for measurement of cardiomyocyte size in vivo 24. Images were captured by a Leica microscope (DM6000B, Leica, Germany). To quantify cardiac fibrosis, 10 fields were randomly selected from 3 cardiac sections and calculated as the percentage of Masson's trichrome positive-stained area to total myocardial area. Similar methods were used to evaluate cardiac hypertrophy by calculation of the average cardiomyocyte area in WGA staining heart sections.

Immunostaining was performed using the following antibodies: Mouse anti-α-smooth muscle actin (α-SMA) (5 µg/mL, A5228, Sigma-Aldrich), Rabbit anti-vimentin (5 µg/mL, #5741, Cell Signaling Technology), Mouse anti-PCNA (5 µg/mL, #2586, Cell Signaling Technology). After washing with PBST, the cells were incubated with Alexa Fluor 488-conjugated donkey anti-mouse secondary antibodies (2 µg/mL, A21206, Invitrogen) and Alexa Fluor 568- conjugated donkey anti-rabbit secondary antibodies (2 µg/mL, A10042, Invitrogen) for 45 min. Slides were mounted with Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, USA). Images were captured by a Leica fluorescent microscope (DM6000B, Leica, Germany). The quantification of histological and immunofluorescence studies was performed by an observer blind to the experimental groups.

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