Isolation and Analysis of Cardiac Myofibrils

Cardiac myofibrils were isolated from frozen mouse ventricles on the day of the experiment (Gresham et al., 2014 (link)). A piece of the frozen tissue was thawed in a fresh relaxing solution, homogenized, and the myofibrils were then skinned for 15 min with 1% Triton X-100 (Cheng et al., 2013 (link)). Skinned myofibrils were then resuspended in fresh relaxing solution containing protease and phosphatase inhibitors (PhosSTOP and cOmplete ULTRA Tablets; Roche Applied Science, Indianapolis, IN, USA) and stored on ice. To determine the cMyBP-C content and myofilament protein phosphorylation status, ventricular samples were solubilized by adding Laemmli buffer and were heated to 90°C for 5 min. For Western blot analysis, 10 μg of cardiac myofibrils were electrophoretically separated on 4–20% Tris-glycine gels (Lonza Walkersville Inc., Rockland, ME, USA) at 180 V for 60 min. Proteins were transferred to PVDF membranes and incubated overnight with a primary antibody that detects cMyBP-C (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as described previously (Cheng et al., 2013 (link)). For Pro-Q phosphoprotein analysis, 2.5 μg of solubilized cardiac myofibrils were electrophoretically separated at 180 V for 85 min then fixed and stained with Pro-Q diamond phosphoprotein stain (Invitrogen, Carlsbad, CA, USA) to assess the phosphorylation status of sarcomeric proteins. After imaging the Pro-Q stained gels, the gels were counterstained with Coomassie blue to determine if there are any changes in the isoform expression of sarcomeric proteins. Densitometric scanning of the stained gels was done using Image J software (U.S. National Institutes of Health, Bethesda, MD, USA) (Gresham et al., 2014 (link)).