Exploration of pathomechanisms triggered by a single-nucleotide polymorphism in titin's I-band: the cardiomyopathy-linked mutation T2580I

Missense single-nucleotide polymorphisms (mSNPs) in titin are emerging as a main causative factor of heart failure. However, distinguishing between benign and disease-causing mSNPs is a substantial challenge. Here, we research the question of whether a single mSNP in a generic domain of titin can affect heart function as a whole and, if so, how. For this, we studied the mSNP T2850I, seemingly linked to arrhythmogenic right ventricular cardiomyopathy (ARVC). We used structural biology, computational simulations and transgenic muscle in vivo methods to track the effect of the mutation from the molecular to the organismal level. The data show that the T2850I exchange is compatible with the domain three-dimensional fold, but that it strongly destabilizes it. Further, it induces a change in the conformational dynamics of the titin chain that alters its reactivity, causing the formation of aberrant interactions in the sarcomere. Echocardiography of knock-in mice indicated a mild diastolic dysfunction arising from increased myocardial stiffness. In conclusion, our data provide evidence that single mSNPs in titin's I-band can alter overall muscle behaviour. Our suggested mechanisms of disease are the development of non-native sarcomeric interactions and titin instability leading to a reduced I-band compliance. However, understanding the T2850I-induced ARVC pathology mechanistically remains a complex problem and will require a deeper understanding of the sarcomeric context of the titin region affected.

phenix.refine [6]. This structure was then used as search model in MR to phase the data from crystals grown in condition b, which contained 16 copies per asymmetric unit. Model refinement was as above, but included automatic model building in ARP/wARP [7].
The crystal structure of I9-I11 in condition d was solved by MR using the crystal structure of I9-I11 from c as search model. The position of individual Ig was adjusted by rigid body refinement in PHENIX [6] and the model refined as before.
The 3 J C' k-1 Hα k values were determined from a CO-coupled 3D(H)NCαHα experiment [10]. J-coupling values were extracted from processed spectra using Nmrview [9] and related to the main chain φ angles using the Karplus equation [

Molecular dynamics simulations
The crystal structure of I10-I11 was used as wild-type in MDS. Two versions of I10-I11 T2850I were modelled by replacement of T2850 with isoleucine, using a different rotamer in each case. The models were placed in the center of a rhombic dodecahedran water box (at 1.0 nm from the box edge) and counterions added to neutralize the system. To assess whether MDS parameters were appropriate to sample the amplitude of the interdomain motion in I10-I11, I65-I66 from titin (PDB ID 3B4B) was used as control. Simulations were performed using GROMACS package 5.0 [12] with the AMBER99SB-ILDN forcefield [13]. The TIP3P and SPC/E water models were used comparatively and showed not to influence the conclusion drawn. An initial energy minimization of all systems was performed using steepest descents algorithm. The Particle Mesh Ewald (PME) algorithm [14] was used for long-range electrostatics with non-bonded and van der Waals cut-offs of 1nm. All bond lengths were constrained using the LINCS algorithm [15]. Calculations used a constant temperature of 300K, a pressure of 1 bar, an integration time step of 2 fs and periodic boundary conditions. A final MD run of 50 ns was performed for all systems. Principal component analysis was performed on the concatenated backbone trajectories using the GROMACS modules g_convar and g_anaieg. PDB2PQR [16] and APBS [17] were used to calculate Poisson-Boltzmann electrostatic surfaces. Results were visualized in PyMol v.1.5.0.4 (Schrödinger LLC) using an APBS scale of -5 kT/e to +5 kT/e.

Transgenic muscle
For in vivo visualisation, wild-type and mutant I7-I13 fragments were cloned into the pEGFP-C1 vector, resulting in N-terminal fusion with eGFP. Transfection of GFP tagged Iband titin fragments into tibialis anterior muscles was done as previously described [18].
Mice were anesthetized with an intraperitoneal injection of Rompun (Bayer Pharma; active substance ketamine; 5mg/kg) plus Zoletil 100 (Vibrac Laboratories, active substances tiletamine and zolazepam; 30mg/kg). After hair removal, the skin and fascia overlying the tibialis anterior were incised. One electrode plate was inserted underneath the muscle, 10 µg of expression plasmid were injected into the tibialis anterior muscle and a second electrode plate was placed above the muscle. Five 25 V and 20 ms long electric pulses were applied (ECM 830; BTX Genetronics). The electrodes were removed, and the wound was closed. Ten days later, mice were euthanized by cervical dislocation and transfected muscle sections were counterstained with Texas Red®-X Phalloidin and visualized under a laser scanning confocal microscope.

Neonatal mouse cardiomyocytes
Isolation and transfection was performed as previously described [19]. Briefly, 1-3 days old neonatal mice were used for cardiomyocyte isolation. Excised hearts were washed with calcium and magnesium free PBS supplemented with 20 mM 2,3-butanedione monoxime (BDM), and pre-digested overnight with 0.0125% trypsin in calcium and magnesium free HBSS supplemented with 20mM BDM at 4 °C. The next day, the trypsin solution was removed, and the cells were isolated from the tissue by collagenase/dispase (Roche) treatment at 37 °C, twice for 20 min. Isolated cells were collected through a cell strainer, centrifuged, re-suspended in plating medium (65% DMEM high glucose, 19% M-199, 10% horse serum, 5% fetal calf serum, 1% penicillin/streptomycin) and pre-plated into an uncoated cell-culture dish. After 2 hours incubation in a cell-incubator, non-adherent cardiomyocytes were collected and finally plated onto collagen-coated cell-culture dishes at a density of approximately 1.5 x 10 5 cells per cm 2 . After overnight incubation, cells were changed into Opti-Mem (Thermo Fisher Scientific), and transfected with either GFP tagged wildtype or mutant I7-I13 fragments using Escort III (Sigma-Aldrich) according manufacturer recommendations. 24-hours post transfection, cells were hung in maintenance medium (78% DMEM high glucose, 17% M-199, 4% horse serum, 1% penicillin/streptomycin) and cultured for another 24 hours before fixation or isoproterenol treatment. Cells undergoing isoproterenol stimulation were changed into maintenance medium supplemented with isoproterenol (1µM) or vehicle (water), and cultured for additional 6 hours before fixation.
After culturing, cells were fixed with 4% paraformaldehyde in PBS, and subsequently processed for immunofluorescence as described [20]. Cells were counterstained with an antibody against α-actinin (clone EA53, A7811, Sigma-Aldrich), filamentous actin (fluorescent phalloidin, Molecular Probes) and DNA (DAPI, Sigma-Aldrich) following standard protocols and imaged using a confocal microscope (Olympus). Images were processed using ImageJ with the Bio-Formats plugin, and Photoshop (Adobe).

Mouse model generation and phenotyping
A gene targeting strategy was employed to introduce an ACC to ATC point mutation in exon 37 of the mouse titin gene in order to mimic the T2850I human mutation. The targeting vector was built using a vector backbone carrying neomycin resistance for a positive selection. The targeting vector consists of two arms of homology. The short arm of homology was 3.3 kb in size and carries the point mutation, and the long arm of homology was 4.1 kb.
The point mutation was introduced using a site-directed mutagenesis kit (Agilent, Santa Clara, CA) and its presence was confirmed by sequencing. The targeting vector DNA was electroporated into 129S6 embryonic stem cells. The neomycin resistant clones were screened by PCR using primers outside the homologous arms and neomycin primers in order to select the clones that have taken the targeting DNA by homologous recombination. Mice carrying the mutation were genotyped by PCR across the mutation, using a forward primer on the 5' end of the mutation and a neo reverse primer. The PCR amplified bands were gel purified and sequenced to confirm the presence of the mutation. The mice used in this study were on a mixed background, 50% 129S6 and 50% C57BL/6. Statistical analysis used two-way ANOVAs with repeated measures or Tukey's multiple comparisons post-hoc test, paired t-tests, and unpaired t-tests were performed where noted. P < 0.05 is considered significant.

Section S1: Thermostability of the I10 domain and its variants
To estimate the effect of the T2850I exchange on the fold stability of I10, the thermal denaturation of wild-type I10 wt and I10 T2850I was measured by DSF. A truncated I10 missing the N-terminal β-strand A is included here. The findings reveal that the T2850I exchange is as destabilizing as the removal of a small secondary structure element.  Section S2: X-ray Crystallography

Figure S2: Electron density map of the β-turn A'B from I10
(2mFo-DFc)αc electron density map contoured at 1.5σ

Section S3: Analysis of type II β-turns composition in proteins
Using PDBeMotif (http://www.ebi.ac.uk/pdbe-site/pdbemotif/; [22]) and upon removal of redundant protein entries, we identified 62,641 β-turns of type II in protein structures deposited with the Protein Data Bank repository. The analysis of turn composition is shown below. Those exceptional cases where proline or isoleucine residues were found in position i+2 were examined manually and found to correspond to highly unusual turns inserted into hydrophobic pockets.   For western blot analysis transgenic muscle extracts were fractionated as described earlier [24] with minor modifications and probed for GFP using anti-GFP (abcam ab290) antibody as described [25]. Briefly, tissue was finely minced using scissors and washed in cold PBS. Tissue was homogenized using glass-teflon homogenizer in homogenization buffer

Figure S5a: Subcellular fractionation of transgenic muscle
Wester blot (left) and Coomasie SDS-PAGE (right) are shown.
Purified titin fragments were centrifuged at 100,000 g for 30 min immediately before the assay to sediment any insoluble protein. Titin fragments (20 µM) were added to polymerized F-actin (10 µM) in a total volume of 100 µl. Samples were incubated for 1 h at room temperature and centrifuged at 100,000 g for 30 min. Supernatant and pellet fractions were separated by SDS-PAGE.