Functional analysis of a gene-edited mouse model to gain insights into the disease mechanisms of a titin missense variant

Figures

• 

  Fig. 1. Cardiac phenotype of the A178D mice. a Echocardiographic measurements: fractional shortening, end-systolic and end-diastolic dimensions are shown (Student’s t-test, n = 21/17 WT/A178D, all males, age 99 ± 1 days). For cohort characteristics and a wider set of echocardiographic parameters please refer to Table S3. b Effect of chronic adrenergic stimulation by Iso/PE infusion on cardiac performance in WT and A178D mice. Hypertrophic response to Iso/PE as indicated by LV mass is similar between both genotypes (left). Systolic function of A178D hearts was reduced upon Iso/PE treatment (right). For cohort characteristics and a wider set of echocardiographic parameters please refer to Table S5; (n = 10/9 sham/Iso/PE; Kruskal–Wallis followed by Wilcoxon rank sum test with Bonferroni correction, all males, age 116–120 days)

• 

  Fig. 2. Altered cellular characteristics in A178D hearts. a Image of isolated cardiomyocytes from WT and A178D hearts, stained with wheat germ agglutinin (WGA). b, c Quantification of cellular dimensions: b cellular area, c left—cell length, right—cell width. Values of each measured cell are shown as individual data points, colour coded by the heart they are isolated from, means are indicated by black bar. (Total of 392/369 cells from 12 independent isolations from 4 female and 8 male mice each for WT/A178D, age: 128 ± 8 days/132 ± 10 days WT/A178D; Student’s t test using hierarchical clustering see Methods). For size distribution per isolation see Fig. S7, for summary of results Table S6

• 

  Fig. 3. a Targeted assessment of transcriptional changes by qPCR for 3 month old A178D mice. All measurements are normalised to Gapdh. Significant changes are observed in the hearts of A178D mice (Mann–Whitney U test, WT: n = 10 (4M/6F), age 114.2 ± 0.7 days; A178D n = 12 (6M/6F), age 113.4 ± 0.5 days). b Targeted assessment of transcriptional changes by qPCR in 1 year old WT, Het and A178D mice. All measurements are normalised to Gapdh; Significant changes are observed in the hearts of A178D mice (Kruskal–Wallis followed by Dunn’s multiple comparison’s test, WT: n = 5, age 411 ± 3 days, HET n = 6, age 412 ± 2 days, A178D n = 6, age 423 ± 3 days, all male)

• 

  Fig. 4. a Volcano plot visualising unsupervised proteomics results (normalised by log2 transformation followed by median subtraction in Perseus, followed by Student’s t test with permutation based FDR calculation, n = 6, age WT 114 ± 1 days/A178D 113 ± 1 days, 3M/3F). Significantly downregulated proteins are telethonin and Fhl2, significantly upregulated proteins are αβ-crystallin (αβ-cryst), heat shock protein 27 (Hsp27), Dnaj homology subfamily B member (Dnajb6, another heat shock protein) and myeloid leukaemia factor 1 (Mlf1), all shown in red. b Representative Western blot demonstrating the downregulation of Fhl2 in total extracts of A178D mice. Gapdh serves as loading control. c Quantification of blot in panel B indicates downregulation of Fhl2 by approximately 50%; (mean ± SEM, n = 6, age WT 114 ± 1 days/A178D 113 ± 1 days, 3M/3F, Student’s t test)

• 

  Fig. 5. Loss of telethonin from the Z-discs of A178D hearts. a Protein destabilisation of telethonin in A178D hearts demonstrated by Western blotting. GAPDH serves as loading control. b Quantification of telethonin depletion (to < 10% of WT levels) in A178D hearts, normalised to Gapdh protein (shown as mean ± SEM, Student’s t test, n = 6, age 113 ± 1 days, all males). c Loss of telethonin from cardiac Z-disc in A178D mice. Top: Isolated cardiomyocytes from WT and A178D mice were stained with an antibody recognising titin Z1Z2 epitope and telethonin; merged images: Z1Z2 green, telethonin red. Bottom: cells were stained with a different telethonin antibody and Z-disc marker α-actinin (merged images: telethonin green, α-actinin red). Age WT: 98 days, A178D: 105 days, male)

• 

  Fig. 6. a Telethonin downregulation and re-distribution in A178D hearts: Myofilament and cytoplasmic protein fractions from WT and A178D hearts were blotted for telethonin (teleth). α-actinin served as loading control for the myofilament fraction, Gapdh as loading control for the cytoplasmic fraction, respectively. In A178D hearts, no telethonin is detected in the myofilament fraction. In contrast, the remaining telethonin protein is enriched in the cytoplasmic fraction. (n = 6, 3M/3F, age WT 114 ± 1 days, A178D 113 ± 1 days). b Quantification of telethonin accumulation in the cytoplasm in A178D hearts, normalised to Gapdh protein (shown as mean ± SEM, Student’s t test, n = 5, 2M/3F, age WT 114 ± 1 days, A178D 113 ± 1 days; the first two lanes of the blot in A were omitted for quantification)

• 

  Fig. 7. Loss of telethonin in A178D heart and partical restoration upon proteasomal inhibition. Top—Cryo-sections of hearts from WT mice, stained for telethonin and titin Z-disc epitope T12; nuclei are visualised with DAPI. Telethonin is localised at the Z-discs. Middle—same staining in A178D hearts treated with vehicle (10% dimethyl sulfoxide in 0.9% saline). Telethonin signal is lost from the Z-discs. Asterisks indicate non-specific staining of the telethonin antibody at the extracellular matrix. Bottom—A178D hearts treated with epoxomicin (0.5 mg kg−1 bodyweight per day for 7 days) show partial restoration of telethonin signal at the Z-disc (arrowheads). Scale bar represents 25 microns. All mice in this experiment are male (n = 2 per group; WT age 116 days, A178D age 115 ± 2 days)

• 

  Fig. 8. a Summary of induction of a proteo-toxic response in 1 year old A178D mice assessed by Western blotting (Mann–Whitney U test, n = 6, age WT 416 ± 2 days, A178D 423 ± 3 days, all male). b Summary of induction of a proteo-toxic response in a cohort undergoing adrenergic challenge assessed by Western blotting. Kruskal–Wallis followed by Wilcoxon rank sum test with Bonferroni correction, n = 6, age 120 ± 2 days, all male. For summary see Table 2 and for individual blots Figs. S29–34