A Titin Missense Variant Causes Atrial Fibrillation

Reviewer #1 (Public review):

Summary:

Pavel et al. analyzed a cohort of atrial fibrillation (AF) patients from the University of Illinois at Chicago, identifying TTN truncating variants (TTNtvs) and TTN missense variants (TTNmvs). They reported a rare TTN missense variant (T32756I) associated with adverse clinical outcomes in AF patients. To investigate its functional significance, the authors modeled the TTN-T32756I variant using human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs). They demonstrated that mutant cells exhibit aberrant contractility, increased activity of the cardiac potassium channel KCNQ1 (Kv7.1), and dysregulated calcium homeostasis. Interestingly, these effects occurred without compromising sarcomeric integrity. The study further identified increased binding of the titin-binding protein Four-and-a-Half Lim domains 2 (FHL2) with KCNQ1 and its modulatory subunit KCNE1 in the TTN-T32756I iPSC-aCMs.

Strengths:

This work has translational potential, suggesting that targeting KCNQ1 or FHL2 could represent a novel therapeutic strategy for improving cardiac function. The findings may also have broader implications for treating patients with rare, disease-causing variants in sarcomeric proteins and underscore the importance of integrating genomic analysis with experimental evidence to advance AF research and precision medicine.

Weaknesses:

(1) Variant Identification: It is unclear how the TTN missense variant (T32756I) was identified using REVEL, as none of the patients' parents reportedly carried the mutation or exhibited AF symptoms. Are there other TTN variants identified in the three patients carrying TTN-T32756I? Clarification on this point is necessary.

(2) Patient-Specific iPSC Lines: Since the TTN-T32756I variant was modeled using only one healthy iPSC line, it is unclear whether patient-specific iPSC-derived atrial cardiomyocytes would exhibit similar AF-related phenotypes. This limitation should be addressed.

(3) Hypertension as a Confounding Factor: The three patients carrying TTN-T32756I also have hypertension. Could the hypertension associated with this variant contribute secondarily to AF? The authors should discuss or rule out this possibility.

(4) FHL2 and KCNQ1-KCNE1 Interaction: Immunostaining data demonstrating the colocalization of FHL2 with the KCNQ1-KCNE1 (MinK) complex in TTN-T32756I iPSC-aCMs are needed to strengthen the mechanistic findings.

(5) Functional Characterization of FHL2-KCNQ1-KCNE1 Interaction: Additional functional assays are necessary to characterize the interaction between FHL2 and the KCNQ1-KCNE1 complex in TTN-T32756I iPSC-aCMs to further validate the proposed mechanism.

Reviewer #2 (Public review):

Summary:

The authors present data from a single-center cohort of African-American and Hispanic/Latinx individuals with atrial fibrillation (AF). This study provides insight into the incidences and clinical impact of missense variants in the Titin (TTN) gene in this population. In addition, the authors identified a single amino acid TTN missense variant (TTN-T32756I) that was further studied using human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs). These studies demonstrated that 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 (Iks) and is a potential mechanism for atrial fibrillation. Finally, the authors demonstrate that suppression of FHL2 could normalize the Iks current.

Strengths:

The strengths of this manuscript/study are listed below:

(1) This study includes a previously underrepresented population in the study of the genetic and mechanistic basis of AF.
(2) The authors utilize current state-of-the-art methods to investigate the pathogenicity of a specific TTN missense variant identified in this underrepresented patient population.
(3) The findings of this study identify a potential therapeutic for treating atrial fibrillation.

Weaknesses:

(1) The authors do not include a non-AF group when evaluating the incidence and clinical significance of TTN missense variants in AF patients.

(2) The authors do not provide evidence that TTN-T32756I-iPSC-aCMs are arrhythmogenic only that there is an increase in the Iks current and associated action potential changes. More specifically, the authors report "compared to the WT, TTN-T32756I-iPSC-aCMs exhibited increased arrhythmic frequency" yet is it is unclear what they are referring to by "arrhythmic frequency".

(3) There seem to be discrepancies regarding the impact of the TTN-T32756I variant on mechanical function. Specifically, the authors report "both reduced contraction and abnormal relaxation in TTN-T32756I-iPSC-aCMs" yet, separately report "the contraction amplitude of the mutant was also increased . . . suggesting an increased contractile force by the TTN-T32756I-iPSC-aCMs and TTN-T32756I-iPSC-CMs exhibited similar calcium transient amplitudes as the WT."

Reviewer #3 (Public review):

Summary:

The authors describe the abnormal contractile function and cellular electrophysiology in an iPSC model of atrial myocytes with a titin missense variant. They provide contractility data by sarcomere length imaging, calcium imaging, and voltage clamp of the repolarizing current iKs. While each of the findings is separately interesting, the paper comes across as too descriptive because there is no merging of the data to support a cohesive mechanistic story/statement, especially from the electrophysiological standpoint. There is definitely not enough support for the title "A Titin Missense Variant Causes Atrial Fibrillation", since there is no strong causative evidence at all. There is some interesting clinical data regarding the variant of interest and its association with HF hospitalization, which may lead to future important discoveries regarding atrial fibrillation.

Strengths:

The manuscript is well written and there is a wide range of experimental techniques to probe this atrial fibrillation model.

Weaknesses:

(1) While the clinical data is interesting, it is extremely important to rule out heart failure with preserved EF as a confounder. HFpEF leads to AF due to increased atrial remodeling, so the fact that patients with this missense variant have increased HF hospitalizations does not necessarily directly support the variant as causative of AF. It could be that the variant is actually associated directly with HFpEF instead, and this needs to be addressed and corrected in the analyses.

(2) All of the contractility and electrophysiologic data should be done with pacing at the same rate in both control and missense variant groups, to control for the effect of cycle length on APD and calcium loading. A claim of shorter APD cannot be claimed when the firing rate of one set of cells is much faster than the other, since shorter APD is to be expected with a faster rate. Similarly, contractility is affected by diastolic interval because of the influence of SR calcium content on the myocyte power stroke. So the cells need to be paced at the same rate in the IonOptix for any direct comparison of contractility. The authors should familiarize themselves with the concept of electrical restitution.

(3) It is interesting that the firing rate of the myocytes is faster with the missense variant. This should lead to a hypothesis and investigation of abnormal automaticity or triggered activity, which may also explain the increased contractility since all these mechanisms are related to the calcium clock and calcium loading of the SR. See #2 above for suggestions on how to adequately probe calcium handling. Such an investigation into impulse initiation mechanisms would be very powerful in supporting the primary statement of the paper since these are actual mechanisms thought to cause AF.

(4) The claim of shortened APD without correcting for cycle length is problematic. However, the general concept of linking shortened APD in isolated cells alone to AF causation is more problematic. To have a setup for reentry, there must be a gradient of APD from short to long, and this can only be demonstrated at the tissue level, not really at the cellular level, so reentry should not be invoked here. If shortened APD is demonstrated with correction of the cycle length problem, restitution curves can be made showing APD shortening at different cycle lengths. If restitution is abnormal (i.e. the APD does not shorten normally in relation to the diastolic interval), this may lead to triggered activity which is an arrhythmogenic mechanism. This would also tie in well with the finding of abnormally elevated iKs current since iKs is a repolarizing current directly responsible for restitution.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

Pavel et al. analyzed a cohort of atrial fibrillation (AF) patients from the University of

Illinois at Chicago, identifying TTN truncating variants (TTNtvs) and TTN missense variants (TTNmvs). They reported a rare TTN missense variant (T32756I) associated with adverse clinical outcomes in AF patients. To investigate its functional significance, the authors modeled the TTN-T32756I variant using human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs). They demonstrated that mutant cells exhibit aberrant contractility, increased activity of the cardiac potassium channel KCNQ1 (Kv7.1), and dysregulated calcium homeostasis. Interestingly, these effects occurred without compromising sarcomeric integrity. The study further identified increased binding of the titin-binding protein Four-and-a-Half Lim domains 2 (FHL2) with KCNQ1 and its modulatory subunit KCNE1 in the TTN-T32756I iPSCaCMs.

Strengths:

This work has translational potential, suggesting that targeting KCNQ1 or FHL2 could represent a novel therapeutic strategy for improving cardiac function. The findings may also have broader implications for treating patients with rare, disease-causing variants in sarcomeric proteins and underscore the importance of integrating genomic analysis with experimental evidence to advance AF research and precision medicine.

Weaknesses

(1) Variant Identification: It is unclear how the TTN missense variant (T32756I) was identified using REVEL, as none of the patients' parents reportedly carried the mutation or exhibited AF symptoms. Are there other TTN variants identified in the three patients carrying TTN-T32756I? Clarification on this point is necessary.

We thank the reviewer for their insightful comment. Our study identified deleterious missense variants using a stringent REVEL score threshold of ≥0.7; however, variants with a REVEL score above 0.5 are generally considered potentially pathogenic (Ioannidis, Nilah M., et al., Am J Human Genetics 2016; 9.4: 877-885). The TTN-T32756I variant (REVEL Score: 0.58758, Supplementary Table 1) was prioritized due to its occurrence in multiple unrelated individuals within our clinical AF cohort, despite no reported family history of AF in affected individuals. While no parental inheritance was observed, the possibility of a de novo origin cannot be excluded. Furthermore, this variant is located within a region overlapping a deletion mutation recently shown to cause AF in a zebrafish model (Jiang et al., iScience, 2024;27(7):110395) supporting its potential pathogenicity. Notably, the affected individuals did not carry additional loss-of-function TTN variants. We will clarify these points in the revised manuscript.

(2) Patient-Specific iPSC Lines: Since the TTN-T32756I variant was modeled using only one healthy iPSC line, it is unclear whether patient-specific iPSC-derived atrial cardiomyocytes would exhibit similar AF-related phenotypes. This limitation should be addressed.

We acknowledge the reviewer’s concern that patient-specific iPSC lines could further validate our findings. However, due to the patients' unavailability of peripheral blood mononuclear cells (PBMCs), we utilized a healthy iPSC line and introduced the TTN-T32756I variant using CRISPR/Cas9 genome editing. This approach ensures an isogenic background, thereby minimizing genetic variability and providing a controlled system to study the direct effects of the mutation. We will acknowledge this limitation in the revised manuscript.

(3) Hypertension as a Confounding Factor: The three patients carrying TTN-T32756I also have hypertension. Could the hypertension associated with this variant contribute secondarily to AF? The authors should discuss or rule out this possibility.

We agree that hypertension is a common comorbidity in patients with AF and could contribute to disease progression. However, all three individuals carrying TTN-T32756I exhibited early-onset AF (onset before 66 years), with one case occurring as early as 36 years. This suggests a potential two-hit mechanism, where genetic predisposition and comorbidities influence disease risk. Importantly, our iPSC model isolates the genetic effects of TTN-T32756I from other factors, supporting a direct pathogenic role. We will explicitly discuss this in the revised manuscript.

(4) FHL2 and KCNQ1-KCNE1 Interaction: Immunostaining data demonstrating the colocalization of FHL2 with the KCNQ1-KCNE1 (MinK) complex in TTN-T32756I iPSC-aCMs are needed to strengthen the mechanistic findings.

We appreciate the reviewer’s suggestion and agree that additional immunostaining data would strengthen the evidence for FHL2 colocalization with the KCNQ1-KCNE1 complex in TTN-T32756I iPSC-aCMs. We will work on obtaining these additional data to validate our mechanistic findings further.

(5) Functional Characterization of FHL2-KCNQ1-KCNE1 Interaction: To further validate the proposed mechanism, additional functional assays are necessary to characterize the interaction between FHL2 and the KCNQ1-KCNE1 complex in TTN-T32756I iPSC-aCMs.

We agree with the reviewer that additional functional assays would further validate the proposed mechanism. We will perform contractility and electrophysiological experiments, such as multielectrode array (MEA) assays, to characterize better the interaction between FHL2 and the KCNQ1-KCNE1 complex in TTN-T32756I iPSC-aCMs.

Reviewer #2 (Public review):

Summary:

The authors present data from a single-center cohort of African-American and Hispanic/Latinx individuals with atrial fibrillation (AF). This study provides insight into the incidences and clinical impact of missense variants in this population in the Titin (TTN) gene. In addition, the authors identified a single amino acid TTN missense variant (TTN-T32756I) that was further studied using human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs). These studies demonstrated that the Four-and-a-Half Lim domains 2 (FHL2) has increased binding with KCNQ1 and its modulatory subunit KCNE1 in the TTN-T32756I-iPSCaCMs, enhancing the slow delayed rectifier potassium current (Iks) and is a potential mechanism for atrial fibrillation. Finally, the authors demonstrate that suppression of FHL2 could normalize the Iks current.

Strengths:

The strengths of this manuscript/study are listed below:

(1) This study includes a previously underrepresented population in the study of the genetic and mechanistic basis of AF.

(2) The authors utilize current state-of-the-art methods to investigate the pathogenicity of a specific TTN missense variant identified in this underrepresented patient population.

(3) The findings of this study identify a potential therapeutic for treating atrial fibrillation.

Weaknesses:

(1) The authors do not include a non-AF group when evaluating the incidence and clinical significance of TTN missense variants in AF patients.

We acknowledge the limitation of not including a non-AF group in our clinical analysis. Our cohort is derived from a single-center registry of individuals with AF, and we do not have a matched cohort of non-AF controls to compare the incidence of TTN missense variants. We recognize this as a limitation and will clarify that further studies are needed to define the prevalence of TTN missense variants in broader, multiethnic cohorts that include both AF and non-AF individuals.

(2) The authors do not provide evidence that TTN-T32756I-iPSC-aCMs are arrhythmogenic, only that there is an increase in the Iks current and associated action potential changes. More specifically, the authors report that "compared to the WT, TTN-T32756I-iPSC-aCMs exhibited increased arrhythmic frequency," yet it is unclear what they are referring to by "arrhythmic frequency."

We appreciate the reviewer’s request for clarification regarding "arrhythmic frequency." In our study, this term refers to the increased spontaneous beating rate and irregular action potentials observed in TTN-T32756I iPSC-aCMs compared to WT. Our findings suggest that the AF-associated TTN-T32756I variant induces ion channel remodeling and beating abnormalities, possibly contributing to an arrhythmogenic substrate for AF. We will refine our wording in the revised manuscript to enhance clarity and precision.

(3) There seem to be discrepancies regarding the impact of the TTN-T32756I variant on mechanical function. Specifically, the authors report "both reduced contraction and abnormal relaxation in TTN-T32756I-iPSC-aCMs" yet, separately report "the contraction amplitude of the mutant was also increased … suggesting an increased contractile force by the TTN-T32756IiPSC-aCMs and TTN-T32756I-iPSC-CMs exhibited similar calcium transient amplitudes as the WT."

We thank the reviewer for pointing this out and apologize for the inconsistency. We intended to report on contraction duration and relaxation rather than contraction force alone. The increased contraction amplitude reflects altered contractile force, whereas the reduced contraction duration and impaired relaxation indicate dysfunctional contractile dynamics. We will revise the text and corresponding figures to convey these findings accurately.

Reviewer #3 (Public review):

Summary:

The authors describe the abnormal contractile function and cellular electrophysiology in an iPSC model of atrial myocytes with a titin missense variant. They provide contractility data by sarcomere length imaging, calcium imaging, and voltage clamp of the repolarizing current iKs. While each of the findings is interesting, the paper comes across as too descriptive because there is no data merging to support a cohesive mechanistic story/statement, especially from the electrophysiological standpoint. There is not enough support for the title "A Titin Missense Variant Causes Atrial Fibrillation", since there is no strong causative evidence. There is some interesting clinical data regarding the variant of interest and its association with HF hospitalization, which may lead to future important discoveries regarding atrial fibrillation.

Strengths:

The manuscript is well written, and a wide range of experimental techniques are used to probe this atrial fibrillation model.

Weaknesses

(1) While the clinical data is interesting, it is essential to rule out heart failure with preserved EF as a confounder. HFpEF leads to AF due to increased atrial remodeling, so the fact that patients with this missense variant have increased HF hospitalizations does not necessarily directly support the variant as causative of AF. It could be that the variant is associated directly with HFpEF instead, and this needs to be addressed and corrected in the analyses.

We recognize that AF and HFpEF frequently coexist and that HFpEF-related atrial remodeling could contribute to AF development. The primary aim of our cohort analysis was to explore the potential clinical significance of TTNmv. While we acknowledge the inherent limitations of retrospective observational data in establishing causality, our subsequent in vitro experiments were designed to demonstrate that TTNmv can alter the electrophysiological substrate, potentially predisposing individuals to AF.

As HFpEF is a potential confounder, it is reasonable to consider whether TTNmv may also be associated with HFpEF. However, to our knowledge, no existing literature directly links TTNmv to HFpEF. In contrast, loss-of-function TTN variants are typically associated with heart failure with reduced ejection fraction (HFrEF) and dilated cardiomyopathy, and even their role in HFrEF remains controversial. To address potential confounding, our multivariable analysis for clinical outcomes was adjusted for reduced ejection fraction, and we conducted a sensitivity analysis excluding patients with nonischemic dilated cardiomyopathy (Supplementary Table 6). We will clarify these points in the revised manuscript.

(2) All contractility and electrophysiologic data should be done with pacing at the same rate in both control and missense variant groups, to control for the effect of cycle length on APD and calcium loading. A shorter APD cannot be claimed when the firing rate of one set of cells is much faster than the other, since shorter APD is to be expected with a quicker rate. Similarly, contractility is affected by diastolic interval because of the influence of SR calcium content on the myocyte power stroke. So the cells need to be paced at the same rate in the IonOptix for any direct comparison of contractility. The authors should familiarize themselves with the concept of electrical restitution.

We appreciate the reviewer’s technical concern. iPSC-derived cardiomyocytes (iPSC-CMs) exhibit spontaneous beating due to the presence of pacemaker-like currents and the absence of I_k1, which allows for the study of intrinsic electrophysiological properties, ion channel function, and disease modeling. In our study, we utilized this unique property of iPSCCMs to test our hypothesis that TTNmvs alter electrophysiological properties through ion channel remodeling.

While iPSC-CMs with identical backgrounds are expected to show comparable electrophysiological phenotypes under the same conditions, variability due to biological and technical factors (e.g., protein expression and culture handling) can result in differences between samples. We agree with the reviewer that pacing iPSC-CMs at the same rate for action potential duration (APD) and contractility measurements will control for cycle length effects and improve the reliability and interpretability of our findings. We will incorporate this approach into our revised experimental design.

(3) It is interesting that the firing rate of the myocytes is faster with the missense variant. This should lead to a hypothesis and investigation of abnormal automaticity or triggered activity, which may also explain the increased contractility since all these mechanisms are related to the SR's calcium clock and calcium loading. See #2 above for suggestions on how to probe calcium handling adequately. Such an investigation into impulse initiation mechanisms would be compelling in supporting the primary statement of the paper since these are actual mechanisms thought to cause AF.

We agree with the reviewer that investigating abnormal automaticity or triggered activity about the increased firing rate observed with the missense variant could provide valuable insights into the mechanisms underlying AF. As these processes are closely linked to calcium handling and the calcium clock, probing calcium cycling abnormalities could strengthen our understanding of how TTNmvs contribute to AF. We will incorporate additional experiments to investigate these mechanisms, further supporting our study's central hypothesis.

(4) The claim of shortened APD without correcting for cycle length is problematic. However, linking shortened APD in isolated cells alone to AF causation is more complicated. To have a setup for reentry, there must be a gradient of APD from short to long, and this can only be demonstrated at the tissue level, not at the cellular level, so reentry should not be invoked here. If shortened APD is demonstrated with correction of the cycle length problem, restitution curves can be made showing APD shortening at different cycle lengths. If restitution is abnormal (i.e. the APD does not shorten normally in relation to the diastolic interval), this may lead to triggered activity which is an arrhythmogenic mechanism. This would also tie in well with the finding of abnormally elevated iKs current since iKs is a repolarizing current directly responsible for restitution.

We appreciate the reviewer’s insightful comment. We recognize that isolated cell studies cannot directly demonstrate reentrant circuits, and we agree that reentry should not be invoked solely based on cellular data. Our claim of shortened APD is based on observed abnormalities in APD and beating patterns, which may contribute to conditions conducive to reentry at the tissue level. We will clarify this distinction in the revised manuscript and refrain from directly linking APD shortening to reentry without tissue-level evidence.