A Titin Missense Variant Causes Atrial Fibrillation

Abstract

Rare and common genetic variants contribute to the risk of atrial fibrillation (AF). Although ion channels were among the first AF candidate genes identified, rare loss-of-function variants in structural genes such as TTN have also been implicated in AF pathogenesis partly by the development of an atrial myopathy, but the underlying mechanisms are poorly understood. While TTN truncating variants (TTNtvs) have been causally linked to arrhythmia and cardiomyopathy syndromes, the role of missense variants (mvs) remains unclear. We report that rare TTNmvs are associated with adverse clinical outcomes in AF patients and we have identified a mechanism by which a TTNmv (T32756I) causes AF. Modeling the TTN-T32756I variant using human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) revealed that the mutant cells display aberrant contractility, increased activity of a cardiac potassium channel (KCNQ1, Kv7.1), and dysregulated calcium homeostasis without compromising the sarcomeric integrity of the atrial cardiomyocytes. We also show that a titin-binding protein, the Four-and-a-Half Lim domains 2 (FHL2), has increased binding with KCNQ1 and its modulatory subunit KCNE1 in the TTN-T32756I-iPSC-aCMs, enhancing the slow delayed rectifier potassium current (I _ks). Suppression of FHL2 in mutant iPSC-aCMs normalized the I _ks, supporting FHL2 as an I _ks modulator. Our findings demonstrate that a single amino acid change in titin not only affects function but also causes ion channel remodeling and AF. These findings emphasize the need for high-throughput screening to evaluate the pathogenicity of TTNmvs and establish a mechanistic link between titin, potassium ion channels, and sarcomeric proteins that may represent a novel therapeutic target.

Figure 1:. TTNmv prevalence and association with hospitalization in a multiethnic atrial fibrillation (AF) cohort.

A) Distribution of TTNmv in 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 region of the T23756I is highly conserved across vertebrate species. (E) Allele frequencies of TTN-T3265I in various ethnic groups (gnomAD).

Figure 2:. Human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) carrying TTN-T32756I variants have atypical contractility but no sarcomere disorganization.

(A) Workflow to generate the CRISPR/Cas9-mediated iPSC line carrying TTN-T32756I missense variation. (B-E) Contraction profile of wild type (black) and TTN-T32756I (Red) iPSC-aCMs showing increased beating frequency (C), Peak-to-Peak time (D), and Contraction duration (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 (H). n.s.; P>0.05; *P<0.05; **P< 0.01.

Figure 3:. Effect of T32756I on action potential (AP) and calcium-handling in iPSC-aCMs.

(A-C) Representative optical AP recordings of WT 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 (I_ks) in WT and TTN_T32756I iPSC-aCMs. control (n=7) (E-F) Comparison of Iks current density at 50 mV (mean ± SEM). N.s.; P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. (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 (B) and decreased transient durations (I) compared with the WT iPSC-aCMs.

Figure 4:. 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 5:

Inhibition of FHL2 rescues enhanced I_ks 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 I_ks TTN-T32756I iPSC-aCMs by the suppression of FHL2 (n=4–8). (D) Comparison of I_ks current density at 50 mV (mean ± SEM). I Schematic showing the TTN-T32756I results in increased FHL2 binding with the KNCQ1-KCNE1 complex and enhanced I_ks activity. *P<0.05, **P<0.01, ***P<0.001.