Collagen 3

Freshly dissected mouse heart tissue from the peri-infarction border and CFs were harvested. Protein was extracted using cell lysis buffer intended for Western blotting analysis and immunoprecipitation assays (Beyotime Institute of Biotechnology, Shanghai, People’s Republic of China). Equal amounts of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Merck Millipore, Billerica, MA, USA) which were blocked in 5% nonfat milk for 1–2 hours. Then, the blocked membranes were incubated overnight with the primary antibodies against collagen I (Abcam), collagen III (Proteintech Group, Inc., Chicago, USA), TGF-β1, MMP-2, MMP-9 (all Abcam), or SIRT1 (Cell Signaling Technology, Danvers, MA, USA). After washing with Tris-buffered saline and Tween 20, the membranes were incubated with the appropriate secondary antibodies for 1–2 hours at room temperature. The protein bands were detected using an enhanced chemiluminescence detection system and the band density was determined using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The frozen LV tissue of the mice was added the tissue Lysis Buffer, then ground and centrifuged to get supernatant. The isovolumetric 2 × gel-loading buffer was added to the supernatant after protein excretion. Subsequently, the supernatant boiled for 10 min. The proteins were loaded onto 8% Sodium Dodecyl Sulfate polyacrylamide gel for separation, and transferred to nitrocellulose membrane, then marked the target protein band after the ponceau staining. The first antibody was enclosed for one min in 5% skimmed milk and then incubated for one min at room temperature, incubated overnight at 4 °C. The antibodies were then stripped by washing the membrane 3 × 10 min with 0.2% TPBS (Triton X-100/PBS). The secondary antibody was incubated for one hour at room temperature. The membrane was then rinsed 4 × 10 min in 0.2% TPBS. The primary proteins used were the first antibody of transforming growth factor-β (TGF-β) (Cell Signaling Technology, 1:1000), p-SMAD2/3 (Cell Signaling Technology, 1:1000), Collagen-I (Abcam, 1:500), Collagen-III (Proteintech, 1:500), β-actin (Abcam, 1:4000), and the secondary antibody of rabbit anti-goat IgG (1:2000), goat anti-mouse IgG (1:5000), goat anti-rabbit IgG (1:2000).

For histopathological analyses, mid-ventricular sections of the left ventricle were excised, fixed (4% paraformaldehyde), dehydrated, and embedded in paraffin. Four-micrometer-thick sections were prepared for further studies. Hematoxylin-eosin staining was used to show general ventricular wall morphology changes. Fluorescein isothiocyanate–conjugated wheat germ agglutinin (WGA; Invitrogen) was adopted for membrane staining to show the cross-sectional cardiomyocyte area. Masson staining was used to indicate collagen deposition. Sections were also incubated with primary antibodies against collagen I (Proteintech, China) and collagen III (Proteintech, China) to measure type I and III collagen deposition. For immunofluorescence (IF), sections were incubated with anti-NMRAL1 antibody (Proteintech, China) after protein blocking, antigen retrieval, and permeabilization. Subsequently, sections were stained with secondary antibody (conjugated with Alexa Fluor^® 488) and DAPI. Slides were photographed with a Pannoramic MIDI-II Digital Scanner (3DHISTECH, Budapest, Hungary), and the results were analyzed using ImageJ with the IHC Profiler plugin and the Coloc 2 plugin.

Kidney tissues were fixed in 10% buffered formalin solution for 30 min at room temperature and dehydrated in 75% ethanol overnight, followed by paraffin embedding. Immunohistochemical analysis was performed using the SP-9001 SPlink Detection kits (OriGene Technologies, Inc.), according to the manufacturers' instructions. Paraffin-embedded sections (4 µm) were deparaffinized with xylene and subsequently rehydrated in a descending series of ethanol washes (100, 90, 85 and 75%; 5 min each). The sections were treated for 15 min at 98°C with 3% H₂O₂ in methanol to inactivate the endogenous peroxidase activity and were subsequently incubated at room temperature for 1 h with primary antibodies rabbit against collagen I (cat. no. 14695-1-AP; 1:200; ProteinTech Group, Inc.), collagen III (cat. no. 22734-1-AP; 1:200; ProteinTech Group, Inc.), LC3 (cat. no. 14600-1-AP; 1:200; ProteinTech Group, Inc.) and p62 (cat. no. 55274-1-AP; 1:200; ProteinTech Group, Inc.). The secondary antibody from SPlink Detection kits (OriGene Technologies, Inc.) was incubated with the tissue for 30 min at room temperature. All sections were examined using a BX40 upright light microscope (Olympus BX43; Olympus Corporation). For each staining, 3×7 sections (6 mice) per group were analyzed and the representative images were presented. All image analyses were performed by a blinded reviewer.