Collagenase type b

Ventricular myocyte was isolated from the mouse hearts using a previously described method [43 (link)]. Mice were injected with heparin (50 units) and sacrificed by cervical dislocation. The heart was quickly removed from the chest, and the aorta was retrogradely perfused at 37 °C for 3 min with calcium-free Tyrode buffer (137 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 10 mM glucose, 10 mM HEPES, 10 mM 2,3-butanedione monoxime (Sigma), and 5 mM taurine (Sigma) at pH 7.4) gassed with 95% O₂ and 5% CO₂. Enzymatic digestion was initiated by adding collagenase type B (0.35 U/ml; Roche, Basel, Switzerland) and hyaluronidase (0.1 mg/ml; Sigma) to the perfusion solution. When the heart became swollen after 10 min of digestion, the left ventricle was quickly removed, cut into several chunks, and further digested in the same enzyme solution on a shaker (60–70 rpm) for 10 min at 37 °C. The supernatant containing the dispersed myocyte was filtered through a cell strainer (100 µm diameter; BD Falcon, New York, USA) and gently centrifuged at 500 rpm for 1 min. Extracellular calcium was incrementally added back to a final concentration of 1.25 mM over 30 min to avoid the calcium paradox.

EDL myofibers were isolated as previously described^{65 (link)}. Briefly, the extensor digitorum longus (EDL) muscle was dissected and digested in 0.2% Collagenase type B (catalog # 11088831001, Roche) in DMEM at 37 °C for 1 h. Single myofibers were isolated by triturating the digested EDL muscle with polished Pasteur pipettes and then fixed with PFA 4%.

Ventricular myocytes were isolated from mouse hearts using the method as described previously [11 (link)]. Male mice of 8–12 weeks of age (25–32 g) were used for the study. In brief, afterheparin (50 units) injection, animals were sacrificed by cervical dislocation. The heart was quickly removed from the chest and the aorta was retrogradely perfused at 37°C for 3 min with calcium-free Tyrode buffer (137 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, 10 mM 2, 3-butanedione monoxime (BDM; Sigma), and 5 mM taurine (Sigma) (pH 7.4)) gassed with 95% O2/5% CO2. The enzymatic digestion was initiated by adding collagenase type B (0.35 U/ml; Roche) and hyaluronidase (0.1 mg/ml; Sigma) to the perfusion solution. When the heart became swollen after 10 min of digestion, the left ventricle was quickly removed, cut into several chunks, and further digested in a shaker (60–70 rpm) for 10 min at 37°C in the same enzyme solution. The supernatant containing the dispersed myocytes was filtered through a cell strainer (100 μm, BD Falcon) and gently centrifuged at 500 rpm for 1 min. Extracellular Ca²⁺ was incrementally added back to 1.25 mM over a span of 30 min to avoid Ca²⁺ paradox. This procedure usually yielded more than 50% viable rod-shaped ventricular myocytes with clear sarcomere striations. Myocytes with obvious sarcolemmal blebs or spontaneous contraction were not used.

Con or CKO male mice (8–12 weeks of age) were anesthetized with 2,2,2‐tribromoethanol (TBE). The heart was quickly removed from the chest and retrograde perfused at a constant flow rate of 3 ml/min at 37°C for 2–3 min with a Ca²⁺‐free bicarbonate‐based buffer containing 120 mM NaCl, 5.4 mM KCl, 1.2 mM MgSO₄, 1.2 NaH₂PO₄, 5.6 mM glucose, 20 mM NaHCO₃, 10 mM 2,3‐butanedione monoxime (BDM; Sigma) and 5 mM taurine (Sigma), gassed with 95% O₂‐5%CO₂ to washout remaining Ca²⁺ in the heart. Following perfusion with the Ca²⁺‐free buffer, enzymatic digestion was initiated by perfusing with a collagenase buffer containing 0.4 mg/ml collagenase type B (Roche, Basel, Switzerland), 0.3 mg/ml collagenase type D (Roche), and 0.03 mg/ml protease type XIV (MilliporeSigma) in 50 ml of Ca²⁺‐free perfusion buffer. All solutions were filtered with a 0.2 µM filter. Hearts were perfused with the collagenase buffer for 15–20 min until the heart was fully digested. Collagenase buffer was then washed out by perfusing again with Ca²⁺‐free buffer for 2–3 min. Cardiomyocytes (CMs) were isolated by mechanically teasing the cells apart. They were then gently triturated with a plastic transfer pipette and filtered using a sterile 100 µM filter (Song et al., 2010). Myocytes were then allowed to settle by gravity. CMs were then snap‐frozen in liquid nitrogen for later use in Western blot analysis.