Titin's P-zone domains A164-167 are essential for thick filament structural arrangement.

Figures

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  Figure 1.. Genetically engineered mouse model lacking the first four domains of titin’s P-zone, A164–167. (A) Titin’s A-band is incorporated into the thick filament, with the P-zone linking the C-zone and M-band segments, spanning domains A164–170. Domains A164–167, which span 16 nm, were deleted in this mouse model. Ig domains: magenta; FnIII domains: white; titin kinase: purple; M-band M-is: blue. (B) Schematic of titin’s α and β conformations at the C-zone-to-P-zone transition region. (C) Titin exon usage in LV muscle from 3-mo-old TtnΔA164–167 mice (4 WT and 4 TtnΔA164–167 mice). (D) Titin exon usage in EDL muscle from 3-mo-old TtnΔA164–167 mice (4 WT and 4 TtnΔA164–167 mice). (E) Representative agarose protein gels of titin in LV and EDL muscles from WT (left lane in each pair) and TtnΔA164–167 mice (right lane in each pair). (F) For LV, the OD ratios of the N2BA to N2B titin isoforms (N2BA/N2B), total titin (TT, N2BA+N2B+T2) to MyHC, and T2 to total titin are plotted. (G) For EDL muscle, OD ratios of total titin to MHC (TT/MHC) and T2 to total titin (T2/TT) are plotted. Statistical significance was determined by an unpaired t test between WT and TtnΔA164–167 for each ratio. (H)…

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  Figure S1.. Generation and characterization of the Ttn ΔA164–167 mouse model. (A) CRISPR tools used to generate the TtnΔA164–167 allele by CRISPR shown relative to the WT transcript Ttn-203 (ENSMUST00000099981.10); locations of gRNAs are indicated with arrows, and the repair template (ssOSD) alignment is shown relative to the gene structure—fusing the end of exon 338 (encoding Fn3 domain A163) in frame with the start of Ig A168 in exon 341. The screening primers P1 (black) and P2 (red) were used for genotyping in combination with the WT-specific P3 (blue) primer. This GEMM removed (GRCm39) chr2:76,544,965-76,547,093. This removed the sequence encoding titin domains A164–A167 (395 amino acids, 44 kDa). (B) Pie chart indicating the percentage of each genotype of offspring born to heterozygous parents in the TtnΔA164–167 strain and the percentage of the total for that genotype. GraphPad Prism χ-squared test indicates that the observed ratios do not significantly deviate from the expected F2 Mendelian ratio (1:2:1) of offspring (P = 0.8869). (C) Growth curves tracking body weight of each sex and genotype from 1 to 12 wk after birth. (D) Skeletal muscle weights normalized to tibia length for 8…

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  Figure 2.. RNA sequencing and fiber typing results. (A) GO terms associated with the DEGs in LV tissue for CC (pink), BP (orange), and MF (peach) categories. Significantly enriched GO terms were determined by Fisher’s exact test. 4 WT and 4 TtnΔA164–167 mice were used for RNA-seq experiments. (B) MyHC ratios in LV muscle were determined by SDS-PAGE. LV lysate from 8 WT to 8 TtnΔA164–167 mice was used. Only α-myosin was detected. (C) Heatmap demonstrating the upregulation of slow fiber type–associated genes and downregulation of fast fiber type–associated genes in TtnΔA164–167 EDLs. 4 WT and TtnΔA164–167 mice were used for RNA-seq experiments. (D) MyHC ratios in EDL muscle were determined by SDS-PAGE. Statistical significance was determined by multiple t tests (WT vs. TtnΔA164–167 by fiber type). 5–6 mice of each genotype were used. CC, cellular component; BP, biological processes; MF, molecular function. ****p ≤ 0.0001. Source data are available for this figure: SourceData F2.

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  Figure S2.. Pulldown assays to probe for potential binding partners of the titin A164–167 segment. (A) Map of the plasmid used to produce the recombinant titin A164–167 bait protein. (B) Representative gel image from a pulldown assay in which the recombinant ALFA-tagged A164–167 protein (rA164–167-ALFA) was used to probe gastrocnemius (gast) or LV tissue lysates. The boxed areas were cut from the gel and analyzed by LC-MS/MS to identify the main protein components. (C) Table of the bands tested by mass spectrometry and the top protein hits. (D) Western blot quantification of ANT1/2 levels normalized to GAPDH in LV tissue lysate. (E) Western blot quantification of ANT1/2 levels normalized to GAPDH in EDL tissue lysate. Statistical significance for D and E was determined by an unpaired t test. Tissue lysates from 8 WT and 8 TtnΔA164–167 were used. Source data are available for this figure: SourceData FS2.

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  Figure S5.. Computational modeling of passive tension based on WLC model and intact EDL absolute force generation. (A) Sarcomere length vs. force plot for a single titin molecule in WT EDL sarcomere, a sarcomere in which only the TtnΔA164–167 EDL splicing differences are present, and a sarcomere in which the splicing differences and 86-nm shorter thick filament found in TtnΔA164–167 are present. (B) Sarcomere length vs. force plot for a single titin molecule in a WT LV sarcomere and the TtnΔA164–167 sarcomere in which the thick filament is 86 nm shorter. (C) Force–frequency relationship of intact EDL muscles expressed as absolute force values. 5–6 mice of each genotype were used. Statistical significance was determined by two-way repeated-measures ANOVA with Sidak’s multiple comparisons test.**p ≤ 0.01; ***p ≤ 0.001; **** p ≤ 0.0001.

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  Figure 3.. Single EDL fiber mechanics. (A) Peak passive stress produced by single skinned EDL fibers across a range of sarcomere lengths. (B) Elastic component of passive stress. (C) Viscous component of passive stress. Statistical significance for A–C was determined by nested ANOVA with Sidak’s multiple comparisons test (represented by asterisks) and exponential growth curve fitting with an extra sum-of-squares F test (P value listed on plot). (D) Maximal active stress produced by single skinned EDL muscle fibers. (E) CSA of EDL fibers used in single-fiber mechanical experiments. (F) Ktr of single skinned EDL fibers. (G) Average stress–calcium relationship of skinned EDL fibers. Statistical significance for D–G inset was determined by nested t test (nested by mouse). Statistical significance for curve fit in G was determined by an extra sum-of-squares F test. For panel A–F experiments, 3 WT and 3 TtnΔA164–167 mice were used, with 3–8 fibers per mouse tested. For panel G, 2 WT and 2 TtnΔA164–167 mice were used, with 5–8 fibers per mouse tested. ns, p ≥ 0.05; **p ≤ 0.01.

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  Figure 4.. Cardiomyocyte mechanics. (A) Peak stress generated by cardiomyocytes passively stretched to a range of sarcomere lengths. (B) Elastic component of passive stress. (C) Viscous component of passive stress. Statistical significance for A–C was determined by mixed-effects analysis at each sarcomere length (no significance found) and exponential growth curve fitting with an extra sum-of-squares F test (P value on plot). 6 WT and 6 TtnΔA164–167 mice were used, with 6 cells tested per animal. (D) Maximal active stress measurements from individual cardiomyocytes. Four animals per genotype were used, with six cells tested per animal. Statistical significance was determined by nested t test (nested by mouse). (E) Stress–Ca2+ relationship, with EC50 measured in individual cell inset. The stress–Ca2+ curve was fit with a Hill equation, and statistical significance of curve fit was determined by an extra sum-of-squares F test. Statistical significance of EC50 was determined by a nested t test (nested by mouse). 4 WT and 4 TtnΔA164–167 mice were used, with 3–4 cells tested per mouse.

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  Figure 5.. Intact EDL studies. (A) Force–frequency relationship of intact EDL muscle with PCSA measurement inset. The force–frequency curves were fit with a Hill equation, and statistical significance for curve fit was determined by an extra sum-of-squares F test. Statistical significance for each frequency was determined by two-way repeated-measures ANOVA with Sidak’s multiple comparisons test (significance denoted by asterisks on plot), and for PCSA by an unpaired t test. (B) F50, or the frequency at which 50% of maximal force is produced, for EDL muscle. (C) Maximal rate of contraction (maximum dx/dt) during a twitch (1 Hz) stimulation of intact EDL muscles. (D) Maximal rate of relaxation (minimum dx/dt) during a twitch (1 Hz) stimulation of intact EDL muscles. (E) Fatigue index, a ratio of the average force produced by the last five stimulations to the average force produced by the first five stimulations of a 75-stimulation fatigue protocol on intact EDL muscles. ns, p ≥ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

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  Figure S3.. Analysis of centrally located nuclei in EDL muscle. (A) Representative images of a WT (top) and TtnΔA164–167 (bottom) EDL cross-section stained with anti-laminin antibody (cell borders) and DAPI (nuclei). The arrow in the inset of the TtnΔA164–167 panel indicates a centrally located nucleus. (B) Percentage of EDL fibers containing central nuclei. Statistical significance was determined by an unpaired t test. Three animals from each genotype were used, with 333–697 fibers analyzed per animal.

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  Figure S4.. Soleus function in the Ttn ΔA164–167 model. (A) Force–frequency relationship of intact soleus muscle with PCSA measurement inset. The force–frequency curves were fit with a Hill equation, and statistical significance for curve fit was determined by an extra sum-of-squares F test. Statistical significance for each frequency was determined by two-way ANOVA (no significance), and for PCSA by an unpaired t test. (B) F50, or the frequency at which 50% of maximal force is produced, for soleus muscle. (C) Maximal rate of contraction (maximum dx/dt) during twitch (1 Hz) stimulations of intact soleus muscles. (D) Maximal rate of relaxation (minimum dx/dt) during twitch (1 Hz) stimulations of intact soleus muscles. (E) Fatigue index, a ratio of the average force produced by the last five stimulations to the average force produced by the first five stimulations of a 75-stimulation fatigue protocol on intact soleus muscles. Statistical significance for B–E was determined by an unpaired t test. 5–6 mice of each genotype were used. (F) MyHC ratios in soleus muscle were determined by SDS-PAGE. Statistical significance was determined by multiple t tests (WT vs. TtnΔA164–167 by fiber type). 5–6…

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  Figure 6.. Cardiac function and morphology. (A) Fractional shortening, isovolumic relaxation time normalized to RR time interval (IVRT/RR interval), myocardial performance index (MPI), mitral valve E-wave deceleration time (MV E decel. time), mitral valve E/A ratio, and mitral valve A-wave, as measured by echocardiography. 9 WT and 12 TtnΔA164–16 mice were used in echocardiography studies. (B) Muscle weight-to-BW ratio for the whole heart, left ventricle, right ventricle, and combined atrial weights. 12–16 WT and 13–17 TtnΔA164–167 mice were used. Statistical significance for A and B was determined by an unpaired t test. (C) Representative images of WT (left) and TtnΔA164–167 (right) Masson’s trichrome-stained heart sections. Three mice of each genotype were used (one representative image from each genotype is shown). BW, body weight. ns, p≥0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

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  Figure 7.. Mapping titin’s A-band epitopes in Ttn ΔA164–167 cardiac muscle. (A) Schematic of titin’s A-band (M-band on left) with summary of epitopes used to map titin’s arrangement in the A-band. (B) Representative immunoelectron micrograph of WT (left) and TtnΔA164–167 (right) sarcomeres labeled with the M8–M10 antibody to mark the C terminus of titin. The plot profiles are below the sarcomeres; the Z-disks of each sarcomere are indicated, and arrows indicate the peak on the plot profile corresponding to the epitope label in the image on the right half of the sarcomere. The frequency distribution of the measured distance of the epitope from the middle of the M-band is plotted to the right, with the average epitope distance listed on the plot. N = 2 mice/genotype, with n = 132–168 sarcomeres measured. (C) Representative immunoelectron micrographs of WT (left) and TtnΔA164–167 (right) sarcomeres labeled with A168–170 antibody to mark the P-zone-to-M-band transition. The plot profiles are below the sarcomeres; the Z-disks of each sarcomere are indicated, and arrows indicate the peak on the plot profile corresponding to the epitope label in the image on the right half of the sarcomere. The…