Liberase blendzyme 4

After ketamine sedation, hearts were rapidly removed from mice and mounted onto a temperature-controlled (37°C) Langendorff system. A modified Tyrode solution (Ca²⁺ free) is used to perfuse the hearts for 2 min, then the hearts were digested for 10–15 min with a modified Tyrode solution containing Liberase Blendzyme 4 (Hoffmann-La Roche Inc., Indianapolis, IN). The modified Tyrode solution (pH 7.4) contained the following (in mM): NaCl 135, KCl 4.0, MgCl₂ 1.0, HEPES 10, NaH₂PO₄ 0.33, glucose 10, and butanedione monoxime 10, and the solution was gassed with 5% CO₂−95% O₂. The digested hearts were then removed from the cannula and the left ventricles were cut into small pieces in the modified Tyrode solution. Tissue pieces were gently agitated and pellet of cells was resuspended. Extracellular Ca²⁺ was added incrementally back to 1.20 mM over a period of 30 min. Isolated cardiomyocytes were used for the study within 8 h of isolation. Only rod-shaped cardiomyocytes with clear sarcomere striations were selected for mechanical studies [26 (link), 27 (link)].

Cell shortening and re-lengthening were assessed using a SoftEdge MyoCam® system (IonOptix Corporation, Milton, MA, USA).^{50 (link)} Briefly, hearts from adult C57 BL/6J mice were removed and perfused after ketamine/xylazine (ketamine 80 mg/kg and xylazine 12 mg/kg, i.p.) sedation. After digestion with Liberase Blendzyme 4 (Hoffmann-La Roche Inc., Indianapolis, IN), left ventricles were removed, minced and filtered. And then cardiomyocytes were placed in a chamber mounted on the stage of an inverted microscope (IX-70, Olympus). The following indices were analyzed to assess cell mechanics: peak shortening (PS), maximal velocities of cell shortening and re-lengthening (+dL/dt, −dL/dt), time-to-PS (TPS) and time-to-90% re-lengthening (TR₉₀).

Mouse cardiomyocytes were isolated from the Nrf2^+/+ or Nrf2^-/- mice as previously indicated [88 (link), 89 (link)]. In brief, hearts from mice were mounted onto a temperature-controlled (37°C) system. After perfusion with a modified Tyrode’s solution (Sigma Aldrich), the heart was digested using a Ca²⁺-free Krebs Henseleit-bicarbonate (KHB) buffer supplemented with liberase blendzyme 4 (Roche, Norway) for 20 min. The modified Tyrode solution was gassed with 5% CO₂. Then, the digested heart was removed from the cannula and the left ventricle was cut into small pieces in the modified Tyrode’s solution. Heart tissue pieces were gently agitated, and then pellet of cells was resuspended. A yield of 65% viable rod-shaped cardiomyocytes with clear sacromere striations was acquired. Only rod-shaped myocytes with clear edges were chose for in vitro studies. Control and RIPK3-specific siRNAs were synthesized and purchased from Shanghai Generay Biotech (Shanghai, China) and transfected into cells using Lipofectamine® 3000 (Invitrogen, USA) following the manufacturer’s protocol. HO1 activator of CoPPIX, and ROS scavenger of NAC were purchased from Sigma-Aldrich. RIPK3 inhibitor of GSK872 (#HY-101872) was purchased from MedChemExpress (USA) to suppress RIPK3 expression.