α actinin

After the cells were treated with Ang II and transfected, they were fixed with 4% formaldehyde and permeabilized in 0.5% Triton X-100 for 20 min at room temperature. After washing with PBS, normal goat serum was added into cells for blocking for 30 min. A primary antibody (α-actinin; Abcam) was added to the cells, followed by incubation overnight at 4°C. On the next day, a secondary antibody (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594; Abcam) was added to the cells, followed by incubation for 1 h. Immunofluorescence was detected using a fluorescence microscope (Olympus, Tokyo, Japan) after the cells were cultured with DAPI for 10 min. In total, 100 cells in 3 wells were randomly selected to quantify the surface area using Image-Pro Plus 6.0.

After returning to the earth, the fixed cardiomyocytes were adopted for immunofluorescence staining against α-Actinin (Abcam, USA) and cTnT (Proteintech, China) to illustrate the cytoskeleton morphology, and the nuclei were stained with Hoechst 33342 (Molecular Probes, USA). The labeled cells were imaged by confocal microscopy (ZEISS, Germany). ImageJ software analyzed the sarcomere length by calculating the distance between intensity peaks. Five sarcomeres were measured along the long axis direction and then averaged to measure sarcomere length. A large number of sarcomeres (n > 10) were calculated to compare the sarcomere length.^{22 (link)} The ImageJ and Matlab software were used for orientation analysis of the sarcomere structure and myofilament arrangement. A higher-order cytoskeleton presents a highly centralized orientation, and any cytoskeletal remodeling and depolymerization will lead to a random orientation. To quantify the level of cTnT, we acquired a minimum of five images to analyze fluorescent intensity. In addition, we measured the cell size of cardiomyocytes after Alexa Fluor 647-WGA staining (Thermo Fisher, USA) to assess whether cardiac atrophy occurred in tail-suspension mice. All antibodies are listed in Supplementary Table 2.

The cells were plated on 20 mm coverslips and were fixed with 4% PFA for 20 minutes. Then, after washing with PBS three times for 10 minutes, the cells were successively treated with 0.2% Triton X‐100 (Sigma‐Aldrich) for 30 minutes, washed as above, and treated with 3% bovine serum albumin (BSA, Solarbio) at room temperature. Primary antibodies included OCT4 (1:100, Abcam), SSEA‐4 (1:100, Santa Cruz), NANOG (1:100, Abcam), TRA‐1‐60 (1:100, Santa Cruz), TNNT2 (1:100, Santa Cruz), α‐actinin (1:100, abcam), CX43 (1:100, Santa Cruz) and γ‐H2A.X (1:200, Abcam). Cells were washed and then incubated for 45 minutes at RT in the dark with 1:200 Alexa Fluor secondary antibodies (Life Technology). Cells were washed again as above, mounted with Fluoroshield Mounting Medium with DAPI (4, 6‐diamino‐2‐phenylindole), and imaged using a Confocal Microscope (Carl Zeiss, LSM 510 Meta).