Clonal Hematopoiesis of Indeterminate Potential With Loss of Tet2 Enhances Risk for Atrial Fibrillation Through Nlrp3 Inflammasome Activation

BACKGROUND: Clonal hematopoiesis of indeterminate potential (CHIP), a common age-associated phenomenon, associates with increased risk of both hematological malignancy and cardiovascular disease. Although CHIP is known to increase the risk of myocardial infarction and heart failure, the influence of CHIP in cardiac arrhythmias, such as atrial fibrillation (AF), is less explored. METHODS: CHIP prevalence was determined in the UK Biobank, and incident AF analysis was stratified by CHIP status and clone size using Cox proportional hazard models. Lethally irradiated mice were transplanted with hematopoietic-specific loss of Tet2, hematopoietic-specific loss of Tet2 and Nlrp3, or wild-type control and fed a Western diet, compounded with or without NLRP3 (NLR [NACHT, LRR {leucine rich repeat}] family pyrin domain containing protein 3) inhibitor, NP3-361, for 6 to 9 weeks. Mice underwent in vivo invasive electrophysiology studies and ex vivo optical mapping. Cardiomyocytes from Ldlr−/− mice with hematopoietic-specific loss of Tet2 or wild-type control and fed a Western diet were isolated to evaluate calcium signaling dynamics and analysis. Cocultures of pluripotent stem cell–derived atrial cardiomyocytes were incubated with Tet2-deficient bone marrow–derived macrophages, wild-type control, or cytokines IL-1β (interleukin 1β) or IL-6 (interleukin 6). RESULTS: Analysis of the UK Biobank showed individuals with CHIP, in particular TET2 CHIP, have increased incident AF. Hematopoietic-specific inactivation of Tet2 increases AF propensity in atherogenic and nonatherogenic mouse models and is associated with increased Nlrp3 expression and CaMKII (Ca2+/calmodulin-dependent protein kinase II) activation, with AF susceptibility prevented by inactivation of Nlrp3. Cardiomyocytes isolated from Ldlr−/− mice with hematopoietic inactivation of Tet2 and fed a Western diet have impaired calcium release from the sarcoplasmic reticulum into the cytosol, contributing to atrial arrhythmogenesis. Abnormal sarcoplasmic reticulum calcium release was recapitulated in cocultures of cardiomyocytes with the addition of Tet2-deficient macrophages or cytokines IL-1β or IL-6. CONCLUSIONS: We identified a modest association between CHIP, particularly TET2 CHIP, and incident AF in the UK Biobank population. In a mouse model of AF resulting from hematopoietic-specific inactivation of Tet2, we propose altered calcium handling as an arrhythmogenic mechanism, dependent on Nlrp3 inflammasome activation. Our data are in keeping with previous studies of CHIP in cardiovascular disease, and further studies into the therapeutic potential of NLRP3 inhibition for individuals with TET2 CHIP may be warranted.


Lin et al
Tet2 CHIP Enhances AF Risk by Nlrp3 Activation C lonal hematopoiesis of indeterminate potential (CHIP), an age-associated phenomenon, results from somatic leukemogenic driver mutations in hematopoietic stem cells that generate clonal populations in the peripheral blood. 1,2CHIP is common, occurring in >10% of individuals older than 70 years of age, and increases the risk of myeloid malignancies.][3] Individuals with CHIP have an increased risk of myocardial infarction and ischemic stroke, independent of traditional CVD risk factors. 3Atherosclerosis-prone Ldlr −/− mice with genetic inactivation in myeloid cells of tet methylcystosine dioxygenase 2 (Tet2), a gene commonly mutated in CHIP, develop accelerated atherosclerosis with increased proinflammatory cytokine production including IL-1β (interleukin 1β) and IL-6 (interleukin 6). 3,4he NLRP3 (NLR [NACHT {NAIP (neuronal apoptosis inhibitor protein), C2TA (class 2 transcription activator of the MHC), HET-E (heterokaryon incompatibility), and TP1 (telomerase-associated protein)}, LRR {leucine rich repeat}] family pyrin domain containing protein 3) inhibi-tor MCC950 has been reported to reverse these effects, pointing to aberrant inflammation as a contributor to the mechanism of aggravated atherogenesis. 4 Additional studies continue to broaden our understanding of the role of CHIP in CVD and show that the presence of CHIP leads to accelerated and worsened outcomes in heart failure (HF) and severe aortic stenosis after transcatheter aortic valve implantation. 5,6However, the role of CHIP in arrhythmias, specifically atrial fibrillation (AF), is less understood.
Worldwide, AF is the most prevalent sustained arrhythmia and causes substantial morbidity and mortality. 7AF

Clinical Perspective
What Is New?What Are the Clinical Implications?
• These data add to our growing understanding of AF pathogenesis, particularly the role of immune cells and the inflammasome.• Individuals with TET2-mutated CHIP may carry an elevated risk of AF compared with the general population, making CHIP a potential novel biomarker for increased risk of AF. • CHIP mutation status can allow for the identification of a patient population that may be responsive to the treatment of AF with an NLRP3 inhibitor.pathophysiology is multifactorial, and many risk factors have been identified including advancing age, atherosclerotic heart disease, valvulopathies, atrial and ventricular remodeling, hypertension, and diabetes.In addition, inflammation contributes to AF and associated increased prothrombotic risk. 8Patients with AF often have elevated circulating proinflammatory cytokines ILβ, IL-6, and TNFα (tumor necrosis factor α) and increased activation of the NLRP3 inflammasome. 8,9Mice with constitutively active Nlrp3 A350V overexpressed in cardiomyocytes recapitulate AF inducibility and susceptibility, thus establishing a role for aberrant cardiac inflammasome responses in AF. 9 We hypothesized that patients with CHIP have an increased risk for AF, resulting in part from increased inflammation, mediated by aberrant and overactive NLRP3 inflammasome activation in cardiac macrophages.

Data Availability
Additional and detailed methods are included in the Supplemental Material.Genetic sequencing data analyzed are available through the UK Biobank Data Showcase.Data supporting the findings of this study are available from the corresponding authors upon reasonable request.

Study Samples
In this study, we included 453 515 individuals with available whole-exome sequencing data in the UK Biobank from the existing >500 000 participants 40 to 70 years of age recruited between 2006 and 2010. 10,11After excluding individuals with prevalent hematological malignancies, missing covariates, and one of the third-degree relatives, 10,12 362 440 individuals remained in the cohort.Further, we excluded 4343 individuals with prevalent AF, leaving 358 097 individuals in the final analysis cohort.Using the whole-exome sequencing data, we identified CHIP, defined as the presence of somatic mutations in hematopoietic cells with a variant allele frequency (VAF) of ≥2%, as previously described. 13,14Incident AF analysis stratified by CHIP status and clone size (larger clone size defined as ≥10% VAF) was performed using Cox proportional hazard models adjusting for age, age 2 , sex (male and female), ever-smoked status (yes or no), genetic ancestry (European/non-European), body mass index (<30/≥30), prevalent type 2 diabetes, prevalent hypertension, prevalent coronary artery disease (CAD), and LDL (low-density lipoprotein; adjusted for statin use), as previously described. 15Individuals with AF, type 2 diabetes, hypertension, CAD, HF, valvular disease, and hematological malignancies were identified on the basis of the International Classification of Diseases, Ninth and 10th Revisions (ICD-9, ICD-10), and Office of Population Censuses and Surveys Classification of Interventions and Procedures-4 (OPCS-4) codes (Table S1).
The samples were followed up for up to 12 years from the time of recruitment to the UK Biobank through March 2020.The individuals diagnosed with AF ICD-9, ICD-10 codes or OPCS-4 for AF ablation were considered events, and individuals who did not experience events during the follow-up period were censored at the end of follow-up.Individuals who died before the end of the follow-up period were censored at death.For the secondary analysis, individuals with incident HF before AF diagnosis were censored at the time of HF diagnosis.

In Vivo Electrophysiology Studies
8][19] An octapolar catheter (EPR-800, Millar) was positioned in the right atrium and ventricle, with position ascertained by continuous monitoring of the intracardiac electrograms.Sinus node function was determined by measuring the sinus node recovery time after 30 s of pacing at 2 cycle lengths (100 and 80 ms).Wenckebach cycle length was determined with progressively faster atrial pacing rates.Atrial, ventricular, and atrioventricular nodal refractory periods were measured using programmed electrical stimulation with overdrive pacing trains at 100 ms followed by single extra stimuli.Provocative testing for arrhythmia induction was performed with rapid burst pacing at gradually faster rates (starting at 50 ms) to a pacing cycle length of 20 ms and lasting 3 and 6 s.AF was defined as a rapid atrial rhythm with atrial rate greater than ventricular rate and irregular ventricular response (R-R intervals).Each mouse received 36 provocative stimuli with burst pacing.The duration of AF was measured from the end of the pacing train to the end of the rapid atrial activity.Mice were considered to have AF if they had >1 episode of AF for >250 ms in duration.To measure percentage of AF inducibility, a higher threshold of 1 s was used, and the total number of AF episodes lasting >1 s for each mouse was divided by the total number of provocative maneuvers.

Optical Mapping
Isolation and perfusion of the heart were performed as previously described. 17,20Excised and cannulated hearts were

CHIP Is Associated With Risk of AF
To determine the relationship between CHIP and AF, we analyzed whole-exome sequencing data and health data from the UK Biobank (n=358 097), a prospective cohort with genetic, demographic, and health-related data.Individuals with prevalent AF, valvular heart disease (mitral stenosis, mitral valve repair, replacement, or valvotomy), HF, or prevalent hematological malignancy before DNA collection were excluded (Table S1).22][23][24] 1C).Previous studies have associated HF with AF as well as CHIP. 5,26,27To investigate whether the relationship between CHIP and AF depends on HF, we performed a secondary analysis restricted to individuals with incident HF diagnosis censored at the time of HF diagnosis.We found that the expanded CHIP clones and expanded large TET2 clones were associated with increased incident AF (CHIP: HR,
Surface ECG of mice showed no differences in typical parameters, including heart rate, PR interval, and QRS duration, between Ldlr −/− mice with hematopoietic-specific inactivation of Tet2 versus WT control (Table S3).We then performed in vivo invasive electrophysiology studies to assay sinus node function, atrioventricular node function, tissue characteristics, and arrhythmia inducibility, as previously described. 17,18,20There were no differences in sinus node recovery time, atrioventricular Wenckebach cycle length,  S3).However, compared with WT controls, there was a 2-fold increase in the percentage of mice with any AF among those with hematopoietic-specific inactivation of Tet2 as well as a significant increase in AF inducibility (Figure 2A through 2D).There was also a shortening of the atrial effective refractory period (AERP; 29.8±9.3ms for Tet2KO versus 17.9±5.2ms for WT; P=0.034), which can contribute to increased electrical excitability and susceptibility for reentry, enhancing arrhythmogenesis and AF (Figure 2E). 31,32e next performed ex vivo optical mapping to determine the action potential characteristics and conduction propagation. 17,20In keeping with the in vivo changes in AERP, the right atrial action potential duration (RA APD) was abbreviated in Ldlr −/− mice with hematopoietic-specific inactivation of Tet2 fed WD (23.31±3.40ms) compared with WT controls (20±2.89ms; P=0.034; Figure 2F).Finally, we demonstrated a significant, >3-fold increase in atrial expression of Nlrp3 in Tet2-deficient marrow recipients, suggesting "priming" of the inflammasome (Figure 2G).Specific mutated genes carry variable risk for CVD and other disorders in CHIP. 3,23,25Mutations in DMNT3A were the most commonly identified CHIP mutation.We thus performed similar studies in Ldlr −/− mice with hematopoieticspecific inactivation of Dnmt3a (Dnmt3a fl/− Vav1-Cre + CD45.2, or Dnmt3aKO), fed WD for 6 to 9 weeks.In keeping with genotype-specific associations in human studies from the UK Biobank, only a minority of mice were inducible for AF, and mice with hematopoieticspecific inactivation of Dnmt3aKO did not have elevated AF inducibility (Figure S4A through S4C).These findings further support that increased AF inducibility is associated with hematopoietic loss of Tet2, and not the commonly mutated gene, Dnmt3a.
To determine the role of inflammasome activation in recipient cardiomyocytes, we assessed genetic inactivation of Nlrp3 in our model.Recipient Ldlr −/− Nlrp3 −/− mice were lethally irradiated and reconstituted with bone marrow with hematopoietic-specific inactivation of Tet2 or WT control and fed WD for 6 to 9 weeks (Figure 3A).In contrast with the recipient Ldlr −/− mice, recipient Ldlr −/− Nlrp3 −/− mice were protected from Tet2KO-mediated differences in AERP, APD, or AF propensity (Figure 3B through 3E).These results demonstrate the necessity of recipient Nrlp3 in AF inducibility because of hematopoietic-specific inactivation of Tet2 in Ldlr −/− mice.
Several small molecules, including MCC950, have been shown to effectively inhibit NLRP3. 40We examined the effect of Nlrp3 inhibition in Ldlr −/− mice with hematopoietic-specific inactivation of Tet2 using a potent and optimized small molecule antagonist of NLRP3, NP3-361, dosed in WD chow.NP3-361 binds to the NACHT domain of the NLRP3 protein and locks it in an inactive conformation, thereby preventing NLRP3 inflammasome activation in a manner analogous to other described NLRP3 inhibitors such as MCC950/cytokine release inhibitory drug (CRID) 3 41 (Figure S7A and S7B).In pharmacokinetic analyses, we demonstrated similar drug   S7B).AF occurred in the minority of mice treated with the antagonist, with reduced AF inducibility, comparable to the recipient Ldlr −/− Nlrp3 −/− model.Treatment of lethally irradiated Ldlr −/− mice with NP3-361 prevented the Tet2KO-mediated augmentation in AF susceptibility (Figure 3F through 3H).as well as associated shortening in RA APD and AERP (Figure 3I and 3J).
We next assessed the role of donor or hematopoietic Nlrp3 activity on the susceptibility to AF. Lethally irradiated Ldlr −/− mice reconstituted with hematopoieticspecific inactivation of both Tet2 and Nrlp3 (Tet2 fl/- Vav1-Cre + CD45.2,Nlrp3 −/− , or double knockout) or Nlrp3 −/− alone were fed WD or WD with NP3-361 for 6 to 9 weeks (Figure S8A).There were similar rates of AF and AF inducibility between mice receiving Nlrp3 −/− and double knockout bone marrow, without significant differences in AERP or RA APD (Figure S8B through S8E).However, treating double knockout mice with NP3-361 significantly reduced AF inducibility (Figure S8C).Overall, these studies underscore the necessity of recipient Nrlp3 activation in AF propensity, whereas the role of donor Nrlp3 warrants additional investigation.

Prolonged Exposure to Hematopoietic-Specific Inactivation of Tet2 Promotes AF in Nonatherogenic Mice
To account for any confounding from the atherogenic Ldlr −/− model used thus far, we attempted to determine whether similar findings could be ascertained in a nonatherogenic mouse model.Ldlr WT (CD45.1)mice were lethally irradiated and transplanted with bone marrow from hematopoietic-specific inactivation of Tet2 (Tet2KO) or WT controls.As with the Ldlr −/− model, Ldlr WT mice were fed WD (Figure 4A).After 6 to 9 weeks of WD, there was no difference in AF inducibility between the 2 groups (Figure 4B), RA APD (Figure 4C), or atrial inflammasome protein expression (Figure S6B).Perhaps underlying this apparent discrepancy, Ldlr WT mice with hematopoietic Tet2 deficiency at this age expressed significantly less atrial Nlrp3 protein than their Ldlr −/− age-matched counterparts (Figure S6C).We noted increased AF susceptibility in Ldlr WT mice with hematopoietic Tet2 deficiency emerged after >40 weeks of WD (Figure 4D).As with the Ldlr −/− mice, increased AF inducibility was associated with shortening of the APD (Figure 4E).These data suggest a much slower effect of hematopoietic-specific inactivation of Tet2 in Ldlr WT mice.However, this phenotype was again dependent on Nlrp3 activation, because genetic deficiency of Nlrp3 prevented the AF inducibility caused by hematopoietic loss of Tet2 (Figure 4F through 4H).Given the timeline required to generate the AF phenotype in Ldlr WT mice, we used the Ldlr −/− mice for further downstream mechanistic analyses.

Mice With Loss of Tet2 in Hematopoietic Lineages Have Altered Cardiomyocyte Function
3][44] Alterations in SR calcium handling can promote AF, [44][45][46] and existing literature links inflammation and cytokines such as IL-1β with NLRP3 and CaMKII-dependent RyR2 phosphorylation, resulting in enhanced spontaneous calcium release events or even inducible AF. 37,42,47 Given its central role in calcium handling, we were intrigued to find an upregulated expression of the phosphorylated, and hence, activated form of CaMKII in atrial tissue isolated from both Ldlr WT and Ldlr −/− mice with hematopoieticspecific inactivation of Tet2 (Figure 5A through 5C).
We next explored the effects of hematopoietic-specific inactivation of Tet2 on atrial cardiomyocyte calcium handling in our mouse model.Atrial cardiomyocytes isolated from lethally irradiated Ldlr −/− mice with hematopoieticspecific inactivation of Tet2 or WT controls were incubated with a fluorescent calcium sensitive indicator, Fluo-3 AM, to visualize and quantitatively characterize calcium transients (Figure 5D), as previously described. 48,49Atrial cardiomyocytes from mice with hematopoietic-specific inactivation of Tet2 had dysfunctional intracellular calcium handling resulting in a trend (P=0.055)toward a prolonged transient time to peak, and an increased frequency of spontaneous calcium release events after removal of depolarizing stimuli (Figure 5E through 5G).Release of calcium from the SR is predominantly mediated by RyR2; thus, SR calcium stores can provide an approximation of RyR2 activity.Caffeine stimulation was used to induce total calcium release and measure total SR calcium content.In the presence of 10 mM caffeine, cardiomyocytes isolated from Ldlr −/− mice with hematopoietic-specific inactivation of Tet2 showed a decrease in the SR calcium release compared with WT counterparts (Figure 5H).These results indicate that hematopoietic-specific loss of Tet2 may affect cardiomyocyte RyR2 activity resulting in abnormal calcium release from the SR into the cytosol. 49,50revious studies have demonstrated the importance of cardiac macrophages affecting electrical conduction by facilitating AF in mice via cell-cell communication and possibly paracrine effects in cardiac conduction and AF susceptibility. 9,18,20,29,30,51We tested whether Tet2-deficient macrophages can alter cardiomyocyte function and calcium signaling.In an in vitro model, human pluripotent stem cell atrial cardiomyocytes coincubated with Tet2KO bone marrow-derived macrophages showed decreased total SR calcium release amplitude in the presence of caffeine, decreased calcium transient amplitude, and increased or WT controls and fed WD.B, Hematopoietic-specific Tet2 inactivation did not increase number of mice with AF (left; number of mice with AF/total mice in the group)or AF inducibility (right) in Ldlr WT mice.C, Hematopoietic-specificTet2 deficiency did not affect RA APD90 in Ldlr WT recipient mice after 6 to 9 weeks of WD.Representative tracing to the left.D, After feeding WD >40 weeks, AF was provoked in all hematopoietic-specific inactivated Tet2 (Tet2KO) bone marrow recipients but none of the WT bone marrow recipients (left; number of mice with AF/total mice in the group), and there was a marked difference in AF inducibility (right).E, Tet2KO bone marrow recipients had a shorter RA APD90 compared with WT counterparts.Representative tracing to the left.F, Lethally irradiated Ldlr WT mice or Nlrp3 −/− mice were transplanted with bone marrow with hematopoietic-specific inactivation of Tet2 (Tet2KO) or WT controls and then fed WD >40 weeks.G, There were no significant differences in number of mice with AF (number of mice with AF/total mice in the group); however, H, there was a significant difference in AF inducibility among the 3 groups.).We postulated that in these mice, chronic inflammation and increased release of cytokines such as IL-1β and IL-6 from macrophages may alter SR calcium release.3][54] We found that human pluripotent stem cell atrial cardiomyocytes stimulated with either human IL-1β or IL-6 had decreased calcium transient amplitude compared with untreated human pluripotent stem cell atrial cardiomyocytes (Figure 6F).These findings suggest that cytokines IL-1β and IL-6, known to be released from Tet2-deficient macrophages, may contribute to the dysfunctional calcium handling in our CHIP model.

DISCUSSION
CHIP independently increases the risk of incident atherosclerosis and CAD, but the role of CHIP in arrhythmias, particularly AF, has remained less defined.Our findings indicate that individuals with CHIP, specifically those with large VAF TET2 mutations, have an increased risk for incident AF.In both Ldlr WT and Ldlr −/− mice fed a WD, hematopoietic-specific inactivation of Tet2 led to increased AF inducibility.Mechanistically, our studies demonstrate the necessity of recipient Nlpr3 activity in AF propensity, through electrical, rather than structural, remodeling.
We found an association between CHIP and AF in the UK Biobank population, consistent with previous findings, and demonstrated that the risk of AF is dependent on the CHIP genotype. 55Individuals with mutations of TET2 had augmented risk of developing AF relative to those with DNMT3A or CHIP mutations with smaller VAF size clones, recapitulated in our murine models.7][58] It is notable that mice and humans with TET2-mutant CHIP have elevated IL-6 and IL-1β, which are key effectors of CHIPmediated atherosclerosis and CVD. 3,15,22,593][54] We found that heightened susceptibility to AF in our mouse model depends on the Nlrp3 inflammasome, whose expression is markedly increased by hematopoietic-specific Tet2 deficiency.In support of this finding, recipient Nlrp3 deficiency abrogated the hematopoietic-specific Tet2 deficiency-driven arrhythmogenesis in both atherogenic and nonatherogenic mouse models.We noted similar results in a study of NP3-361, a small-molecule NLRP3 inhibitor analogous to MCC950.However, because of its novelty, there is limited literature about this compound, and further work will be required before it can be considered for a translational role.
Our findings indicate that dysregulated cardiomyocyte calcium handling contributes mechanistically to the link between hematopoietic Tet2 deficiency and AF.Hematopoietic-specific Tet2 deficiency resulted in increased atrial expression of phosphorylated CaMKII, a central regulator of SR calcium flux.Atrial cardiomyocytes isolated from Ldlr −/− mice with hematopoieticspecific inactivation of Tet2 have abnormal calcium release from SR into the cytosol, demonstrating a role for a cell nonautonomous alteration in these cardiomyocytes.In vitro studies showed that Tet2-deficient macrophages coincubated with atrial cardiomyocytes can promote similar cardiomyocyte dysfunction, and this phenotype was also observed with the addition of IL-1β and IL-6 to atrial cardiomyocytes.Increased Nlrp3 inflammasome activation in cardiomyocytes is known to promote aberrant SR calcium release, promote electrical remodeling leading to shortening of atrial APD, and increase susceptibility for AF in mouse models. 8,9,30It is evident that further investigation is required to determine the molecular mechanisms that connect Nlrp3 activation, CaMKII, and SR calcium release, and how loss of Tet2 in macrophages contributes to these alterations.
Although there is a growing burden of evidence about the role of inflammation in CVD, it has yet to affect mainstream clinical care.Colchicine, which may have some NLRP3 and IL-1β inhibitory effects, has shown efficacy in secondary prevention of cardiovascular events. 60Moreover, the role of IL-6 in CVD is being explored in ongoing trials of anti-IL-6 in patients with CAD and chronic kidney disease. 61In the CANTOS trial (Canakinumab Anti-Inflammatory Thrombosis Outcomes Study), patients with known CAD and above median hsCRP (highsensitivity C-reactive protein) had decreased major adverse cardiac events and HF hospitalizations when treated with the neutralizing monoclonal anti-IL-1β canakinumab.Further post hoc analysis showed that individuals harboring TET2 CHIP mutations derived enhanced benefit on canakinumab treatment compared with those without CHIP. 62These data warrant further investigation, because they suggest a role for targeted therapies on the basis of the identification of somatic mutations.
Contemporary strategies for the treatment of AF focus on symptom reduction through either rate or rhythm control strategies that aim to restore/maintain sinus rhythm.Currently available pharmacological therapies primarily act through modulation of ion channels and impart modest efficacy. 63There is growing evidence linking inflammation and AF, and targeting aberrant inflammation may provide a novel mechanistic approach to addressing contributing pathways in AF.5][66][67] Similar to the CANTOS trial and subanalysis of individuals with CHIP, the presence of TET2-mutated CHIP could identify a more responsive patient population for the treatment of AF with specific NLRP3 inhibitors and the presence of TET2 CHIP mutations as a biomarker.Recent studies identified carriers of hematopoietic somatic mosaicism to be at greater risk for postoperative AF than those without these mutations, providing further support. 55ur understanding of the mechanistic basis of AF derives largely through the lens of the cardiomyocyte, which is critical to impulse propagation and myocardial contraction. 36However, the heart is composed of many noncardiomyocyte cell populations that cooperate intricately to impart cardiovascular function as well as pathophysiology. 68,69There is growing clinical and preclinical data about the importance of noncardiomyocytes in AF pathogenesis. 29Here, in both in vivo and in vitro models, we have demonstrated the contribution of mutated immune cells to electrical remodeling, altered calcium dynamics, and AF.Beyond somatic mutations such as those related to CHIP, common variants have been shown to explain a majority of the heritable predisposition to AF, but most existing mechanistic studies have focused on their role in cardiomyocytes.A broader look at noncardiomyocyte cell types, both within and extrinsic to the heart, may further our continued understanding of AF pathogenesis.
In summary, our analysis of a large human cohort study revealed that individuals with CHIP, and specifically TET2 CHIP, have increased risk of developing AF.In vivo and in vitro studies showed that hematopoieticspecific loss of Tet2-enhanced cardiomyocyte inflammasome activity and abnormal cardiomyocyte SR calcium handling may underlie associated AF pathogenesis.

Figure 2 .
Figure 2. Hematopoietic-specific inactivation of Tet2 augments AF inducibility in Ldlr −/− mice.A, Lethally irradiated Ldlr −/− mice transplanted with bone marrow with hematopoietic-specific inactivation of Tet2 (Tet2KO) or WT controls and fed Western diet (WD) for 6 to 9 weeks.B, Representative tracing of pacing-induced AF, showing surface ECG, right atrial (RA) and right ventricle (RV) intracardiac electrograms.C, Hematopoieticspecific inactivation of Tet2 resulted in an increased number of mice inducible for AF (number of mice with AF/total mice in the group).D, Increased AF inducibility was seen in Tet2KO mice.Percentage reflects ratio of total number of provoked AF episodes to total programmed electrical provocations.E, Atrial effective refractory period (AERP) measured at a drivetrain of 100 ms, demonstrating shortening in Tet2KO recipients.F, Optically derived right atrial action potential duration at 90% repolarization (RA APD90).Representative optical action potential tracing to right.G, Relative atrial protein expression of Nlrp3, with representative blots below, showing >3-fold increase in Nlrp3 expression.Western blot quantification relative to vinculin loading control and then normalized.Statistical testing: Fisher exact test for C and D; 2-tailed Student t test for E through G. Mean (SD) shown for E and F. AF indicates atrial fibrillation; A.U., arbitrary units; KO, knockout; LDLR, low-density lipoprotein receptor; Nlrp3, NLR (NACHT, LRR [leucine rich repeat]) family pyrin domain containing protein 3; TET2, tet methylcystosine dioxygenase 2; and WT, wild-type.

Figure 3 .
Figure 3. NLRP3 inflammasome activation is required for enhanced AF inducibility stemming from hematopoietic-specific Tet2 deficiency.A, Lethally irradiated Ldlr −/− Nlrp3 −/− mice were transplanted with bone marrow with hematopoietic-specific inactivation of Tet2 (Tet2KO) or WT controls and then fed WD for a total of 6 to 9 weeks.B, Hematopoietic-specific Tet2 inactivation did not affect number of mice with AF (number of mice with AF/total mice in the group) or C, AF inducibility in Ldlr −/− Nlrp3 −/− mice.D, NLRP3 deficiency abrogated Tet2KO-related shortening in AERP as well as E, RA APD90.Representative tracing (right).F, Lethally irradiated Ldlr −/− mice were transplanted with bone marrow with hematopoietic-specific inactivation of Tet2 (Tet2KO) or WT controls and then fed WD with an incorporated NLRP3 inhibitor, NP3-361, for a total of 6 to 9 weeks.G, Hematopoietic-specific Tet2 deficiency did not increase number of mice with AF, nor H, AF inducibility in Ldlr −/− mice treated with NP3-361.I, NP3-361 treatment abrogated Tet2KO-related shortening in AERP as well as J, RA APD90.Representative tracing (right).Statistical testing: Fisher exact test for B, C, G, and H; 2-tailed Student t test and mean (SD) shown for D, E, I, and J. AERP indicates atrial effective refractory period; AF, atrial fibrillation; KO, knockout; LDLR, low-density lipoprotein receptor; NLRP3, NLR (NACHT, LRR [leucine rich repeat]) family pyrin domain containing protein 3; RA APD90, right atrial action potential duration at 90% repolarization; TET2, tet methylcystosine dioxygenase 2; and WD, Western diet; and WT, wild-type.

Figure 4 .
Figure 4. Inflammasome activation is required for Tet2-enhanced AF inducibility in nonaccelerated atherogenic models.A, Lethally irradiated Ldlr WT (CD45.1)mice were transplanted with bone marrow with hematopoietic-specific inactivation of Tet2 (Tet2KO)or WT controls and fed WD.B, Hematopoietic-specific Tet2 inactivation did not increase number of mice with AF (left; number of mice with AF/total mice in the group)or AF inducibility (right) in Ldlr WT mice.C, Hematopoietic-specificTet2 deficiency did not affect RA APD90 in Ldlr WT recipient mice after 6 to 9 weeks of WD.Representative tracing to the left.D, After feeding WD >40 weeks, AF was provoked in all hematopoietic-specific inactivated Tet2 (Tet2KO) bone marrow recipients but none of the WT bone marrow recipients (left; number of mice with AF/total mice in the group), and there was a marked difference in AF inducibility (right).E, Tet2KO bone marrow recipients had a shorter RA APD90 compared with WT counterparts.Representative tracing to the left.F, Lethally irradiated Ldlr WT mice or Nlrp3 −/− mice were transplanted with bone marrow with hematopoietic-specific inactivation of Tet2 (Tet2KO) or WT controls and then fed WD >40 weeks.G, There were no significant differences in number of mice with AF (number of mice with AF/total mice in the group); however, H, there was a significant difference in AF inducibility among the 3 groups.Statistical testing: Fisher exact test for B and D; 2-tailed Student t test and mean (SD) for C and E; χ 2 test for G and H. AF indicates atrial fibrillation; KO, knockout; LDLR, low-density lipoprotein receptor; NLRP3, NLR (NACHT, LRR [leucine rich repeat]) family pyrin domain containing protein 3; RA APD90, right atrial action potential duration at 90% repolarization; TET2, tet methylcystosine dioxygenase 2; WD, Western diet; and WT, wild-type.
Figure 4. Inflammasome activation is required for Tet2-enhanced AF inducibility in nonaccelerated atherogenic models.A, Lethally irradiated Ldlr WT (CD45.1)mice were transplanted with bone marrow with hematopoietic-specific inactivation of Tet2 (Tet2KO)or WT controls and fed WD.B, Hematopoietic-specific Tet2 inactivation did not increase number of mice with AF (left; number of mice with AF/total mice in the group)or AF inducibility (right) in Ldlr WT mice.C, Hematopoietic-specificTet2 deficiency did not affect RA APD90 in Ldlr WT recipient mice after 6 to 9 weeks of WD.Representative tracing to the left.D, After feeding WD >40 weeks, AF was provoked in all hematopoietic-specific inactivated Tet2 (Tet2KO) bone marrow recipients but none of the WT bone marrow recipients (left; number of mice with AF/total mice in the group), and there was a marked difference in AF inducibility (right).E, Tet2KO bone marrow recipients had a shorter RA APD90 compared with WT counterparts.Representative tracing to the left.F, Lethally irradiated Ldlr WT mice or Nlrp3 −/− mice were transplanted with bone marrow with hematopoietic-specific inactivation of Tet2 (Tet2KO) or WT controls and then fed WD >40 weeks.G, There were no significant differences in number of mice with AF (number of mice with AF/total mice in the group); however, H, there was a significant difference in AF inducibility among the 3 groups.Statistical testing: Fisher exact test for B and D; 2-tailed Student t test and mean (SD) for C and E; χ 2 test for G and H. AF indicates atrial fibrillation; KO, knockout; LDLR, low-density lipoprotein receptor; NLRP3, NLR (NACHT, LRR [leucine rich repeat]) family pyrin domain containing protein 3; RA APD90, right atrial action potential duration at 90% repolarization; TET2, tet methylcystosine dioxygenase 2; WD, Western diet; and WT, wild-type.

Figure 5 .
Figure 5. Hematopoietic-specific inactivation of Tet2 leads to altered cardiomyocyte sarcoplasmic reticulum calcium release.A, Lethally irradiated Ldlr WT or Ldlr −/− mice were transplanted with bone marrow with hematopoietic-specific inactivation of Tet2 (Tet2KO) or WT controls and fed WD.B, Western blotting was performed to determine protein expression of phosphorylated calmodulin kinase II (CaMKII) in atrial tissue of Ldlr WT and C, Ldlr −/− recipient mice.D, Lethally irradiated Ldlr −/− mice transplanted with bone marrow with hematopoietic-specific inactivation of Tet2 (Tet2KO) or WT controls were fed WD for 6 to 9 weeks.Atrial cardiomyocytes (aCM) were isolated and calcium studies performed using the calcium-sensitive dye Fluo-3 AM.E, Removal of pacing stimuli leads to spontaneous calcium release into cytosol (▼).F, Spontaneous calcium release events measured per second (data obtained from 2 mice per groups, n=12 events for WT and n=24 events for Tet2KO).G, Increased calcium transient amplitude measured during pacing (data obtained from 2 mice per groups, n=14 cells for WT and n=27 cells for Tet2KO).H, Removal of pacing stimuli and the addition of 10 mM caffeine leads to SR calcium emptying into the cytosol.Tracing to left, and quantification to right, showing reduced SR calcium release in Tet2KO aCMs (data obtained from 2 mice per group, n=4 cells for WT and n=6 cells for Tet2KO).△F/F0 is the change in fluorescence over baseline.Hz indicates the external pacing frequency stimuli.All Western blot quantification relative to loading control (vinculin) and then normalized.A.U., arbitrary units.Statistical tests: 2-tailed Student t test for all comparisons; linear mixed model for F through H. aCM indicates atrial cardiomyocte; KO, knockout; LDLR, low-density lipoprotein receptor; SR, sarcoplasmic reticulum; TET2, tet methylcystosine dioxygenase 2; WD, Western diet; and WT, wild-type.

Figure 6 .
Figure 6.Incubation with of Tet2-deficient bone marrow-derived macrophages alters cardiomyocyte calcium handling.A, Human pluripotent stem cells differentiated to atrial cardiomyocytes (hPSC-aCM) were coincubated with Tet2-deficient (Tet2KO) bone marrow-derived macrophages (BMDM) or cytokines, IL-1β and IL-6.Calcium studies were performed using the calcium sensitive dye Fluo-3 AM.B, After 1 minute pacing at 1 Hz, fast perfusion of 10 mM caffeine leads to calcium emptying into the cytosol from SR. C, Tet2KO BMDMtreated hPSC-aCM showed reduced total SR Ca2 + release.△F/F0 is the change in fluorescence intensity over baseline.D, 1 Hz paced calcium transient amplitude (measured with Fluo-3 AM) is reduced in the hPSC-aCM incubated with Tet2KO BMDM compared with WT BMDM, with E, an increase in calcium transient time to peak (TTP).F, hPSC-aCM cardiomyocytes stimulated in culture with cytokines IL-1β 10 ng/mL or IL-6 10 ng/mL showed decreased SR calcium release (measured with Fluo-4 NW).Statistical tests: 2-tailed Student t test for C through E; 1-way ANOVA with Tukey multiple comparisons for F. IL-1β indicates interleukin 1β; IL-6, interleukin 6; SR, sarcoplasmic reticulum; TET2, tet methylcystosine dioxygenase 2; and WT, wild-type.