SDS gel electrophoresis, western blotting, and titin mobility gels

The ventral part of the diaphragm right costal were flash‐frozen in liquid nitrogen and stored at −80°C. Tissue was ground to a fine powder using Dounce homogenizers cooled in liquid nitrogen and acclimated to –20°C for 30 min before continuing. Tissue powder was resuspended in a 1:1 mixture of an 8 M urea buffer (in M; 8 urea, 2 thiourea, 0.05 Tris–HCl, 0.075 dithiothreitol, as well as 3% SDS and 0.03% bromophenol blue, pH 6.8) and 50% glycerol containing protease inhibitors (in mM; 0.04 E‐64, 0.16 leupeptin, and 0.2 PMSF). The solutions were mixed for 4 min, followed by 10 min of incubation at 60°C. Samples were centrifuged at 12.000 rpm and the supernatant was divided into smaller aliquots and flash frozen for storage at −80°C. SDS‐agarose (SDS‐AGE) 1% were run on in a Hoefer SE600X vertical gel system (Hoefer Inc; Holliston, USA) was used to electrophoretically separate titin from other proteins.24 Gels were run at 15 mA per gel for 3 h and 15 min, then stained using Neuhoff's Coomassie brilliant blue staining protocol, scanned using a commercial scanner (Epson 800, Epson Corporation, Long Beach CA). For titin size estimations in the mouse models, we essentially ran the same protocol with the addition of co‐migrating the samples with: human soleus N2A (3700 kDa), human cardiac (N2B 3000 kDa, N2BA 3300 kDa) and mouse RBM20Δ cardiac (N2BA 4000kDA, largest known titin isoform) samples, in the same lane and using imageJ to measure the migration distance of the reference samples relative to MyHC. By taking the logarithm of the migration distance of the reference samples, an exponential fit could be used to calculate the relative size of mouse wildtype, diaphragm RBM20^ΔRRM and Ttn^ΔIAjxn titin.

Titin binding and Z‐disc associated proteins: MARP1–3, CAPN3, MuRF1 & 2, FHL1 & 2, MYPN, CaM, Csrp3, CaN and Ldb3, were quantified using western blot. MyHC levels were determined from Coomassie stained initial gels to determine equalized loading for each sample. Samples were run on 10% acrylamide gels at 100 V for 1.5 h and subsequently transferred onto Immobilon‐P PVDF 0.45 μm membranes (Millipore, Billerica, USA) using a Trans‐Blot Turbo Transfer System (Bio‐Rad, Hercules, USA) for 30 min at 1.0 A. Membranes were incubated with primary antibodies (see Supporting Information, Table S1) at 4°C overnight. IR Western blots were analysed using Odyssey Infrared Imaging System (Li‐Cor Biosciences, USA). All proteins were normalized against GAPDH, and subsequently UDD samples were normalized to Sham diaphragms to determine relative changes in protein quantity.