A titin missense variant drives atrial electrical remodeling and is associated with atrial fibrillation
- Division of Cardiology, Department of Medicine, University of Illinois at Chicago, United States
- Division of Genetics, Genomics, and Metabolism, Department of Pediatrics, Lurie Children’s Hospital of Chicago, United States
- Department of Pharmacology, Northwestern University, United States
- Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, United States
- Department of Pharmacology,University of Illinois at Chicago, United States
- Jesse Brown Veterans Administration Medical Center, United States
Figures
Figure 1 with 1 supplement
TTNmv prevalence and association with hospitalization in a multiethnic atrial fibrillation (AF) cohort.
(A) Distribution of TTNmvs in a multiethnic AF cohort based on amino acid position in the TTN gene, stratified by REVEL in silico score for prediction of deleterious effect, defined by REVEL ≥0.70. (B) Mean cumulative incidence of AF and heart failure (HF)-related hospitalizations in subjects with AF stratified by presence of TTNmv. Hazard ratio (HR), 95% confidence interval (CI), and p-value were obtained from univariable Cox proportional hazard modeling. (C) Diagram denoting the location of TTNmv-T23756I. (D) Sequence alignment shows that the T23756I region is highly conserved across vertebrate species. (E) Allele frequencies of TTN-T3265I in various ethnic groups (gnomAD).
Figure 1—figure supplement 1
TTN-T32756I position and distribution.
(A) Location of the T23756I at Ig139 domain in the A-band of titin. (B) Allele frequencies of T3265I between sexes. (C) Age distribution of the variant carriers. (D) Genotype quality. (E) Allele balance for heterozygotes. Source: https://gnomad.broadinstitute.org/variant/2-179404525-G-A?dataset=gnomad_r2_1.
Figure 2 with 2 supplements
Human-induced pluripotent stem-cell-derived atrial cardiomyocytes (iPSC-aCMs) carrying TTN-T32756I variants exhibit atypical contractility but no sarcomere disorganization.
(A) Workflow for generating the CRISPR/Cas9-mediated iPSC line carrying the TTN-T32756I missense variation. (B–E) Contraction profile of wild type (black) and TTN-T32756I (Red) iPSC-aCMs (B) showing increased beating frequency (C), decreased contraction duration (D), and increased contraction amplitude (E) in the mutant. (F) Representative sarcomeric organization of wild-type (WT) and TTN-T32756I iPSC-aCM by Transmission electron microscopy (TEM). (G) There is no significant change in the sarcomere length. n.s. p>0.05; *p<0.05; **p<0.01.
Figure 2—figure supplement 1
Generation of iPSC-aCMs with TTN-T32756I.
(A) TTN gene locus for the generation of isogenic iPSCs with the T32756I variation. Guide sequence (gRNA) shown in the bottom gray box was cloned into the vector to express gRNA guiding Cas9 exonuclease to the targeted protospacer adjacent motif sequence. (B) Next-generation sequencing of the confirming T32756I mutation. (C) Representative immunostaining of pluripotency markers OCT4 and SOX2 in iPSCs. The 4′,6-diamidino-2-phenylindole (DAPI) indicates the nucleus. (D) Karyotype analysis of the T32756I iPSCs.
Figure 2—figure supplement 2
Contractility and sarcomere organization of TTN-T32756I iPSC-aCMs.
(A–C) Compared to WT, TTN-T32756I (Red) iPSC-aCMs show decreased relaxation time (A) and peak-to-peak time (B), but no significant change to time-to-peak (C). (D–E) Immunostaining showing the sarcomeric organization of WT and TTN-T32756I iPSC-aCM by the pan-cardiomyocyte (CM) marker cardiac troponin T (cTnT; green) and α-actinin (orange). The DAPI staining indicates the nucleus. (F) Bar graph showing no change in the sarcomere length. n.s. p>0.05; **p<0.01.
Figure 3 with 2 supplements
Effect of T32756I on action potential (AP) and calcium handling in iPSC-aCMs.
(A–C) Representative optical AP recordings of spontaneously beating WT (A) and TTN-T32756I showing reduction of AP duration (APD) at the 50% (APD50) (B) and 90% (APD90) repolarization (C). (D) Current-voltage (I–V) curves of the slow delayed rectifier potassium current (Iks) in WT and TTN_T32756I iPSC-aCMs (n=7). (E–F) Comparison of Iks current density at 50 mV (mean ± SEM). (G) Representative tracings of spontaneous calcium transients of WT and TTN-T32756I iPSC-aCMs. (H–I) Calcium kinetics show that the TTN-T32756I iPSC-aCMs have increased frequency (H) and decreased transient durations (I) compared with the WT iPSC-aCMs. n.s. p>0.05; *p<0.05.
Figure 3—figure supplement 1
TTN-T32756I iPSC-aCMs display anomalous action potentials, potassium currents, and calcium handling.
(A) Compared to the WT, TTN-T32756I shows reduction of action potential duration at the 10% (APD10) repolarization. (B) Bar graph showing no change in the amplitude of the AP. (C) Total potassium current (IK) and voltage relationship (I-V curves) in WT and TTN-T32756I iPSC-aCMs. (D) Total IK current density at 50 mV. (E) Representative current traces at different voltages showing the isolation of the Iks current with the selective blocker HMR-1556 in both WT and TTN-T32756I iPSC-aCMs. (F–G) Bar graph showing that the TTN-T32756I iPSC-aCMs have decreased transient durations (F), but no change in the transient peak amplitudes (G) compared with the WT iPSC-aCMs. n.s. p>0.05; *p<0.05; ***p<0.001.
Figure 3—figure supplement 2
Effect of T32756I on action potential and calcium handling in paced iPSC-aCMs.
(A) Representative optical AP recordings of paced (0.5 Hz) WT and TTN-T32756I showing reduction of AP duration (APD). (B) Bar graph showing no change in the amplitude of the AP. (C) APD50 (D) APD90. (E) Representative tracings of paced calcium transients (0.5 Hz) of WT and TTN-T32756I iPSC-aCMs. (F) No change in the transient peak amplitudes. (G–H). Decreased transient durations (I) in TTN-T32756I iPSC-aCMs compared with the WT iPSC-aCMs. n.s. p>0.05; *p<0.05.
Figure 4 with 1 supplement
Transcriptomic profile and pathway enrichment analysis comparing TTN-T32756I iPSC-aCMs with the WT.
(A) Heatmaps of cardiac-related upregulated and downregulated differentially expressed genes (DEGs). (B) Top significantly enriched downregulated cardiac-related Gene-Ontology Biological process (GO-BP) pathways in the TTN-T32756I iPSC-aCMs. (C) Top significantly enriched downregulated cardiac-related Gene-Ontology Molecular Function (GO-MF) pathways in the TTN-T32756I iPSC-aCMs. (D) Top significantly enriched downregulated cardiac-related Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the TTN-T32756I iPSC-aCMs. (E) Significantly enriched upregulated and downregulated transcription factors (TFs). (F) Network diagram showing the upregulation of KCNQ1 by FHL2 predicted by the Ingenuity pathway enrichment analysis (IPA).
Figure 4—figure supplement 1
Upregulated pathways in TTN-T32756I iPSC-aCMs with the WT.
(A) Volcano plot showing spread of downregulated and upregulated differentially expressed genes (DEGs) (B) Top significantly enriched upregulated Gene-Ontology Biological process (GO-BP) pathways in the TTN-T32756I iPSC-aCMs. (C) Top significantly enriched upregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the TTN-T32756I iPSC-aCMs. (D) Top significantly enriched TTRUST transcription factors (TFs).
Figure 5 with 1 supplement
Inhibition of FHL2 rescues enhanced Iks in TTN-T32756I iPSC-aCMs.
(A) Co-immunoprecipitation revealed increased interaction between FHL2 and KCNQ1-KCNE1 (MinK) complex. (IP: KCNE1). Immunoblotting (IB) was performed with antibodies against FHL2 (32 kDa) and MinK (32 kDa) (n=3) (B) qPCR data showing the inhibition of FHL2 gene by the siRNA in the WT and TTN-T32756I iPSC-aCMs (n=7). (C) I-V curves showing the rescue of the Iks TTN-T32756I iPSC-aCMs by the suppression of FHL2 (n=4–8). (D) Comparison of Iks current density at 50 mV (mean ± SEM). (E) Schematic showing the TTN-T32756I results in increased FHL2 binding with the KNCQ1-KCNE1 complex and enhanced Iks activity. n.s. p>0.05; *p<0.05; ***p<0.001.
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Figure 5—source data 1
Source data contains raw images of the immunoblot presented at Figure 5A.
- https://cdn.elifesciences.org/articles/104719/elife-104719-fig5-data1-v1.zip
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Figure 5—source data 2
Source data contains raw images of the immunoblot presented at Figure 5A.
- https://cdn.elifesciences.org/articles/104719/elife-104719-fig5-data2-v1.zip
Figure 5—figure supplement 1
Increased FHL2-KCNE1 co-localization in TTN-T32756I iPSC-aCMs.
(A) Representative confocal immunofluorescence images showing subcellular localization of FHL2 (red) and KCNE1 (green) in WT and TTN-T32756I iPSC-aCMs. (B) Quantitative co-localization analysis, assessed by Pearson’s correlation coefficient, demonstrated a significant increase in FHL2–KCNE1 co-localization in TTN-T32756I iPSC-aCMs compared with WT. **p<0.01.
Tables
Table 1
Clinical characteristics of ethnic minority subjects with AF stratified by presence of rare missense TTN variants.
*Data are missing for the following variables: eGFR (1), electrocardiogram within 3 months of AF diagnosis (11), LVEDD (19), left atrial size (6), left atrial diameter (21). Left ventricular dilatation is defined as left ventricular end diastolic diameter greater than 2 standard deviations above the normal sex-specific mean value. Variants with a REVEL score ≥0.7 were defined as predicted deleterious. Continuous data are represented as mean (standard deviation) and categorical data are represented as count (%).
| TTN Missense Absent(N=54) | TTN Missense Present(N=77) | Total(N=131) | p-value | |
|---|---|---|---|---|
| Age at AF diagnosis (years) | 64.3 (15.2) | 62.9 (12.9) | 63.5 (13.8) | 0.575 |
| Male sex | 30 (55.6%) | 40 (51.9%) | 70 (53.4%) | 0.724 |
| Race/ethnicity | 0.696 | |||
| Non-Hispanic Black | 37 (68.5%) | 56 (72.7%) | 93 (71.0%) | |
| Hispanic/Latinx | 17 (31.5%) | 21 (27.3%) | 38 (29.0%) | |
| BMI (kg/m2) | 33.7 (9.0) | 34.2 (10.1) | 34.0 (9.6) | 0.765 |
| Diabetes | 18 (33.3%) | 32 (41.6%) | 50 (38.2%) | 0.366 |
| Hypertension | 45 (83.3%) | 68 (88.3%) | 113 (86.3%) | 0.448 |
| Coronary artery disease | 13 (24.1%) | 20 (26.0%) | 33 (25.2%) | 0.841 |
| History of stroke/transient ischemic attack | 8 (14.8%) | 18 (23.4%) | 26 (19.8%) | 0.270 |
| Congestive heart failure | 21 (38.9%) | 33 (42.9%) | 54 (41.2%) | 0.720 |
| Nonischemic dilated cardiomyopathy | 4 (7.7%) | 8 (11.1%) | 12 (9.7%) | 0.760 |
| Estimated glomerular filtration rate (mg/dL) | 71.8 (24.4) | 67.2 (24.7) | 69.1 (24.6) | 0.297 |
| Ventricular rate | 92.1 (29.0) | 97.5 (29.6) | 95.3 (29.4) | 0.326 |
| QRS interval (ms) | 97.6 (23.7) | 100.2 (28.1) | 99.2 (26.3) | 0.593 |
| QTc interval (ms) | 449.6 (37.7) | 466.5 (42.3) | 459.6 (41.2) | 0.027 |
| Left ventricular ejection fraction (%) | 0.722 | |||
| Normal (≥50%) | 32 (59.3%) | 45 (58.4%) | 77 (58.8%) | |
| Mildly decreased (40–49%) | 7 (13.0%) | 7 (9.1%) | 14 (10.7%) | |
| Moderately decreased (30–39%) | 4 (7.4%) | 9 (11.7%) | 13 (9.9%) | |
| Severely decreased (20–29%) | 7 (13.0%) | 9 (11.7%) | 16 (12.2%) | |
| Very severely decreased (<20%) | 4 (7.4%) | 7 (9.1%) | 11 (8.4%) | |
| Left ventricular end diastolic diameter (mm) | 45.3 (9.2) | 47.8 (9.8) | 46.7 (9.5) | 0.180 |
| Left ventricular dilatation | 4 (8.3%) | 13 (20.3%) | 17 (15.2%) | 0.111 |
| Left atrial size | 0.675 | |||
| Normal | 17 (32.7%) | 21 (28.8%) | 38 (30.4%) | |
| Mildly dilated | 15 (28.8%) | 23 (31.5%) | 38 (30.4%) | |
| Moderately dilated | 13 (25.0%) | 17 (23.3%) | 30 (24.0%) | |
| Severely dilated | 7 (13.5%) | 12 (16.4%) | 19 (15.2%) | |
| Left atrial diameter (mm) | 39.6 (7.4) | 41.2 (7.7) | 40.5 (7.6) | 0.286 |
| Number of TTN missense variants | - | |||
| 0 | 54 (100.0%) | 0 (0.0%) | 54 (41.2%) | |
| 1 | 0 (0.0%) | 37 (48.1%) | 37 (28.2%) | |
| 2 | 0 (0.0%) | 26 (33.8%) | 26 (19.8%) | |
| >2 | 0 (0.0%) | 14 (18.2%) | 14 (10.7%) | |
| Number of predicted deleterious TTN missense variants | - | |||
| 0 | 54 (100.0%) | 34 (44.2%) | 88 (67.2%) | |
| 1 | 0 (0.0%) | 34 (44.2%) | 34 (26.0%) | |
| 2 | 0 (0.0%) | 9 (11.7%) | 9 (6.9%) |
Table 2
Clinical characteristics of the early-onset AF patients with TTN-T32756I variation.
| Case 1 | Case 2 | Case 3 | |
|---|---|---|---|
| Age at diagnosis (years) | 52 | 60 | 38 |
| Sex | Male | Female | Male |
| Race/ethnicity | Hispanic | Black | Black |
| Body mass index (kg/m2) | 42.2 | 26.9 | 31.4 |
| Type | Paroxysmal | Paroxysmal | Paroxysmal |
| Comorbidities | Hypertension Prostate cancer | Hypertension Hyperlipidemia Coronary artery disease Uterine/vulva cancer Severe mitral regurgitation | Hypertension Asthma |
| Family history | No | No | No |
| Presenting symptoms | Asymptomatic, found during preoperative evaluation | Asymptomatic, in setting of gastrointestinal bleed | Palpitations, dyspnea |
| LA size (mm) | 51 | 33 | 35 |
| LVEF (%) | 55 | 50 | 60 |
| Antiarrhythmic | No | No | No |
| Ablation | Yes | No | No |
| Cardioversion | Yes | No | Yes |
Additional files
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Supplementary file 1
List of TTN missense variants.
Age represents patient’s age at AF diagnosis in years. M=male, F=female, HL = Hispanic/Latinx, NHB = non-Hispanic Black. Variants with a blank value in the dbSNP or gnomAD columns represent variants not present in those respective databases.
- https://cdn.elifesciences.org/articles/104719/elife-104719-supp1-v1.docx
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Supplementary file 2
Clinical characteristics of ethnic minority subjects with AF stratified by presence of predicted deleterious rare missense TTN variants.
*Data are missing for the following variables: eGFR (1), electrocardiogram within 3 months of AF diagnosis (11), LVEDD (19), left atrial size (6), left atrial diameter (21). Left ventricular dilatation is defined as left ventricular end diastolic diameter greater than 2 standard deviations above the normal sex-specific mean value. Variants with a REVEL score ≥0.7 were defined as predicted deleterious. Continuous data are represented as mean (standard deviation) and categorical data are represented as count (%)
- https://cdn.elifesciences.org/articles/104719/elife-104719-supp2-v1.docx
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Supplementary file 3
TTN missense variants in subjects meeting criteria for nonischemic dilated cardiomyopathy.
Nonischemic dilated cardiomyopathy was defined by left ventricular ejection fraction <50% and left ventricular end diastolic diameter (LVEDD) greater than 2 standard deviations above the sex-specific mean, as well as coronary angiogram confirming the absence of obstructive coronary artery disease.
- https://cdn.elifesciences.org/articles/104719/elife-104719-supp3-v1.docx
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Supplementary file 4
Parameter estimates for univariable and multivariable Cox proportional hazard models of atrial fibrillation and heart failure-related hospitalizations.
A partially adjusted multivariable model contained covariates of age and sex, and the fully adjusted model additionally accounted for race-ethnicity and ejection fraction <50% closest to AF diagnosis.
- https://cdn.elifesciences.org/articles/104719/elife-104719-supp4-v1.docx
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Supplementary file 5
Cox proportional hazard models of hospitalizations related to TTN missense variants based on in silico prediction of impact.
REVEL score of ≥0.70 indicates potentially deleterious effect. A partially adjusted multivariable model contained covariates of age and sex, and the fully adjusted model additionally accounted for race-ethnicity and ejection fraction <50% closest to AF diagnosis.
- https://cdn.elifesciences.org/articles/104719/elife-104719-supp5-v1.docx
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Supplementary file 6
Cox proportional hazard models of hospitalizations excluding cases with nonischemic dilated cardiomyopathy.
A total of 12 subjects were excluded. A partially adjusted multivariable model contained covariates of age and sex, and the fully adjusted model additionally accounted for race-ethnicity and ejection fraction <50% closest to AF diagnosis.
- https://cdn.elifesciences.org/articles/104719/elife-104719-supp6-v1.docx
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Supplementary file 7
TTN-T32756I variant information.
- https://cdn.elifesciences.org/articles/104719/elife-104719-supp7-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/104719/elife-104719-mdarchecklist1-v1.pdf
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A titin missense variant drives atrial electrical remodeling and is associated with atrial fibrillation
eLife 14:RP104719.
https://doi.org/10.7554/eLife.104719.3
