Titin-truncating variants affect heart function in disease cohorts and the general population

Abstract

Titin-truncating variants (TTNtv) commonly cause dilated cardiomyopathy (DCM). TTNtv are also encountered in ∼1% of the general population, where they may be silent, perhaps reflecting allelic factors. To better understand TTNtv, we integrated TTN allelic series, cardiac imaging and genomic data in humans and studied rat models with disparate TTNtv. In patients with DCM, TTNtv throughout titin were significantly associated with DCM. Ribosomal profiling in rat showed the translational footprint of premature stop codons in Ttn, TTNtv-position-independent nonsense-mediated degradation of the mutant allele and a signature of perturbed cardiac metabolism. Heart physiology in rats with TTNtv was unremarkable at baseline but became impaired during cardiac stress. In healthy humans, machine-learning-based analysis of high-resolution cardiac imaging showed TTNtv to be associated with eccentric cardiac remodeling. These data show that TTNtv have molecular and physiological effects on the heart across species, with a continuum of expressivity in health and disease.

Fig 1. Ribosome profiling reveals the translational footprint of truncating variants in Ttn.

a Ribosome occupancy across Ttn: Average reads per kilobase per million mapped reads (RPKM) per exon for 10 BN/F344 F1 rats. F344 SNPs on the BN background (n=121) allow assessment of allele-specific translation for 2 distinct models of truncations and Ttn wild type animals. Purple indicates Z-disc, green I-band, pink A-band and blue M-line. b Ribo-seq reads (28-mers) show clear 3-nt periodicity across the genome for all replicates indicating that the datasets effectively capture actively translating ribosomes in the heart. c,d Ribosomes occupy the F344 allele in TTNtvA animals upstream of the premature stop codon in the A-band of Ttn exposing the synthesis of truncated Ttn isoforms. The premature stop releases ribosomes from the mutant Ttn message. Titin protein synthesized after TTNtvA is exclusively generated from the healthy BN allele (Median ± 25-75 percentile, Whiskers show 10-90 percentile). Allele frequencies in RNA-seq and Ribo-seq were below 50%, indicative for NMD. e F344 SNPs located after TTNtvZ are occupied by ribosomes, indicating translation after the proximal truncation in Ttn. f Ribo-seq reads located downstream of TTNtvZ do not lower 3-nt periodicity suggesting they actively translate canonical Ttn sequence at similar levels as animals with two wild type Ttn alleles. g TTNtvZ reduces ribosome density initially but translation of Ttn is gradually rescued. Translation of A-band exons, but not of I-band exons, is reduced in mutants compared to WT animals. TTNtvA efficiently reduces translation after the premature stop codon.

Fig 2. Proximal and distal truncations in titin alter isoform processing and trigger NMD.

a PSI and deltaPSI of titin exons expressed in the heart for TTNtvA, TTNtvZ and WT animals and human DCM. Exons with at least 10 inclusion reads are marked in solid colours. Truncating mutations in Ttn activate splicing in of I-band exons in the TTNtv rat models and also human DCM patients that carry TTN truncations. b RNA-seq reads assigned to either BN or F344 alleles: TTNtv selectively trigger nonsense-mediated decay of truncated Ttn transcripts. [Mean ± SD; Dunnett] c Ribo-seq expression of exons that are exclusively being synthesized in TTNtvA and TTNtvZ animals reveal that both TTNtv models generate 60% of full-length titin compared to WT [Mean ± SD; Student’s t test]. Purple indicates Z-disc, green I-band, pink A-band and blue M-line.

Fig 3. Hearts with proximal and distal truncations in titin undergo metabolic reprogramming.

a Pathway analyses based on RNA-seq and Ribo-seq data suggest perturbed metabolism, structural integrity and mechano-sensation of the TTNtv heart. This molecular signature is strikingly similar in rats with proximal and distal truncations (P value < 10^-15; Pearson Chi-square test). Significantly enriched pathways are indicated in red (corrected P<0.05). b Unsupervised clustering by k-means of cardiac acylcarnitine abundance in WT (n=5) and TTNtv (n=10) rats. –OH and –DC designate hydroxylated and dicarboxylic acid acylcarnitine species respectively. Metabolite profiles showing c branched chain amino acids (valine, leucine and isoleucine) [Mean ± SD; Student’s t test], d sum of measured glycolytic intermediates (metabolites are detailed in Table S3) [Mean ± SD; Student’s t test] and e glucose-6-phosphate (G6P) in cardiac tissue of WT (n=6) and TTNtv (n=12) rats [Mean ± SD; Student’s t test]. For individual genotypes (WT vs TTNtvA or TTNtvZ) see Fig S7.

Fig 4. TTNtv in rats and humans adversely affect cardiac geometry and function.

a SV (Stroke Volume), LVEF (Left Ventricular Ejection Fraction) and FWT (Fractional Wall Thickening) measured with CMR in 13-16 month old male WT (n=5) and TTNtv (TTNtvA, n=8; TTNtvZ, n=6) rats [Mean ± SD; Student’s t test]. For individual genotypes see Fig S12. b Measurements of ex vivo myocardial function during volume overload stress in 4 month old WT (n=9) and TTNtv (TTNtvA, n=8; TTNtvZ, n=8) rats. TTNtv hearts have mildly increased dp/dt max/min and LVDevP at baseline. Myocardial contraction rate, dp/dt max (mmHg/s); myocardial relaxation rate, dp/dt min (-mmHg/s); Left ventricle developed pressure, LVDevP (mmHg). [Mean ± SEM; Two-way ANOVA]. Human data: c Univariate analyses of left ventricular end diastolic volume (LVEDV), LEVEDV indexed to body surface area (LVEDVI), left ventricular end systolic volume (LVESV) and LVESV indexed to body surface area (LVESVI). Healthy human individuals without a TTNtv (TTNtv-) are compared to healthy humans with a TTNtv (TTNtv+) [Mean ± SD; Mann-Whitney]. d Computational modeling of cardiac geometry in healthy humans using 3D CMR. Positive standardised beta coefficients indicate where TTNtv genotype status is associated with enlargement of the LV cavity at end-diastole and end-systole. Septal and lateral en face projections are shown with an outline of the LV myocardium. The area enclosed by the yellow contour has a corrected P <0.05 [mass univariate linear regression]. e The distribution of corrected P values as a proportion of the endocardial surface [mass univariate linear regression].