Titin Isoform and Phosphorylation Analysis

To determine titin isoform content, 1% Agarose gels (agarose with 0.005 M tris base, 0.039 M glycine, 2% SDS, 30% glycerol) were prepared in a large-format gel system (Hoeffer SE600X, Hoefer, Inc., Holliston, MA, United States) (Lahmers et al., 2004; Chung et al., 2013; Zhu and Guo, 2017). Briefly, solubilized samples were loaded, and gel electrophoresis was run at 15 mA/gel for 3 h and 20 min. Gels were stained using Coomassie Brilliant Blue and scanned using a commercially available scanner (Epson V850, Epson America, Inc., Long Beach, CA, United States) with a calibrated Optical Density step tablet (Stouffer Industries, Mishawaka, IN, United States). The scan was normalized for Optical Density using a custom MATLAB script (MathWorks, Natick, MA, United States) and analyzed using ImageQuant TL (GE Healthcare Bio-Sciences Corp., Marlborough, MA, United States). Relative titin mobility was evaluated by loading and co-electrophoresing rat ventricular and soleus muscle reference samples. Relative mobility was calculated using ImageQuant TL as previously described using rat soleus N2A titin isoform, Nebulin, and Myosin Heavy Chain as mobility references (Granzier and Irving, 1995; Buck et al., 2010; Li et al., 2012). Relative titin isoform content (N2BA:N2B ratio) was evaluated by loading individual samples in a range of five volumes (3–9 μL) at dilutions pre-determined to be within the linear range of detection. Background subtraction was performed using the Rolling Ball function (Sternberg, 1983). Relative content was determined using the slope of the optical density versus volume loading for MHC and all titin bands (N2BA, N2B, and T2, the titin degradation band). Two minor bands representing the N2BA isoform were summed to determine the N2BA content (Freiburg et al., 2000).

To determine the total titin phosphorylation status, 2–12% gradient acrylamide gels (2–12% Acrylamide, (1:50) Bis-acrylamide, 0.1% SDS, 5% glycerol, 10× Fairbanks buffer (400 mM Tris Base, 200 mM Sodium Acetate, 20 mM EDTA, pH adjusted to 7.5 with glacial acetic acid), 0.0625% Ammonium Persulfate, and 0.0875% TEMED) were prepared in Mini-PROTEAN Cassettes (BioRad) and allowed to polymerize. Electrophoresis was pre-run with running buffer (10× Fairbanks buffer, 0.1% SDS, and 11.44 mM βME) at 60 V for 3 min without sample. Solubilized samples were then loaded and gel electrophoresis was run at 60 V for 68 min and then 90 V for 70 min. Gels were fixed overnight in 50% methanol and 10% acetic acid, washed three times using ultrapure water, and stained for total phosphate content using a rapid protocol for Pro-Q Diamond staining (Thermo Fisher Scientific, Waltham, MA, United States). Gels were destained using Pro-Q Diamond Destain Solution and washed twice with ultrapure water before scanning with a Typhoon Trio + scanner at 532-nm excitation and 560-nm emission. Gels were then stained for total protein content with Sypro Ruby Gel Stain using the rapid protocol, washed, and scanned with a Typhoon Trio + scanner at 488-nm excitation and 610-nm emission. Bands were quantified using ImageQuant TL (GE Healthcare Bio-Sciences Corp., Marlborough, MA, United States).