Obesity Enables NLRP3 Activation and Induces Myocardial Fibrosis via Hyperacetylation of HADHa

Obesity prevalence is high and is increasing worldwide. There are currently more than 650 million obese adults (BMI ≥30 kg/m²); this is triple the number in the 1970s and will keep rising (1,2). Obesity increases the risks of diabetes, hypertension, and cardiovascular diseases (2,3), resulting in heavy social burdens in both developed and developing countries. Myocardial fibrosis is an important pathological process associated with almost all heart diseases, among which ischemic heart disease accounts for the majority of deaths (4). Obesity is a known risk factor for myocardial fibrosis, but its underlying mechanism is not fully understood.

Accumulating evidence shows that both obesity and heart diseases are coupled with low-grade inflammation (5). The nucleotide-binding oligomerization-like receptor protein 3 (NLRP3) inflammasome is a cytosolic inflammasome that triggers a series of inflammatory responses (6) and contributes to cardiac fibrosis and myocardial remodeling (7,8). The NLRP3 inflammasome consists of a sensor (NLRP3), an adaptor (apoptosis-associated speck-like protein with a caspase-recruitment domain, ASC), and an effector (caspase-1) and assembles upon sensing of certain pathogen products or sterile danger signals (9,10). This inflammasome processes pro–interleukin (IL)-1β and pro–IL-18 into bioactive IL-1β and IL-18, respectively (11–13). NLRP3 and ASC deficiency in mice has been reported to protect against obesity-induced myocardial remodeling (14). Nevertheless, how the NLRP3 inflammasome is activated in this pathological process and potential therapeutic targets for obesity-induced myocardial fibrosis have not been elucidated.

Lysine acetylation (LysAc) regulates multiple cellular functions in inflammation and failing hearts (15–17). Protein hyperacetylation is usually accompanied by decreased bioavailability of nicotinamide adenine dinucleotide (Supplementary Material), a cofactor for mitochondrial deacetylases (18). Our prior study reported that hyperacetylation of cardiac mitochondrial proteins promoted NLRP3 inflammasome assembly in mice (19). However, how this process is regulated by LysAc and which lysine residue(s) play a key role remain to be explored.

In this study, we investigated the molecular mechanism for obesity-induced myocardial fibrosis. Our results demonstrate that hyperacetylation in obese mouse heart promotes the translocation of ASC to mitochondria, which facilitates NLRP3 inflammasome assembly and exacerbates myocardial fibrosis. Specifically, acetylation of K255 on hydroxyacyl-CoA dehydrogenase A (HADHa) recruits ASC to mitochondria, and reducing the acetylation level of HADHa-K255 attenuates post–ischemia/reperfusion (I/R) cardiac fibrosis in obese mice.

All animal experiments were approved by the Animal Care and Use Committee of Sichuan University (Chengdu, China). Eight-week-old male C57BL/6N mice were purchased from Beijing Charles River Laboratory Animal Inc. and housed under a 12-h light/dark cycle at 22–24°C. All animals had free access to water and food. To induce obesity, mice were fed a high-fat diet (HFD) (20% kcal protein, 20% kcal carbohydrate, and 60% kcal fat) for 12 weeks. Afterward, the mice were subjected to 30 min of ligation of the left anterior descending (LAD) coronary artery followed by 14 days of reperfusion. The mice were randomly assigned to receive PBS or nicotinamide ribose (NR) (500 mg/kg/day) for a month by oral gavage, or intraperitoneal injection of IL-1β (#BE0246; Bio X Cell) (200 µg) and IL-18 (#BE0237; Bio X Cell) (200 µg) neutralizing antibodies, or MCC950 (10 mg/kg) before and every 2 days after cardiac I/R surgery (Fig.1I). The experiments were not fully blinded, but we followed standard laboratory procedures of randomization, and the data were analyzed in a blinded manner. Information on the key resources is provided in Supplementary Table 1.

H9C2 rat myoblasts, purchased from the ATCC, were cultured in DMEM containing 1 g/L glucose, 4 mmol/L glutamine, 110 mg/L sodium pyruvate, 10% (v/v) FBS, 100 units/mL penicillin, and 100 mg/mL streptomycin under a humidified atmosphere containing 5% CO₂ at 37°C.

An intraperitoneal glucose tolerance test was performed after 12 weeks of HFD feeding (20). Briefly, mice were fasted for 6 h and injected with 1 g/kg d-glucose intraperitoneally. Blood glucose levels were measured with a glucometer from the tail vein at 0, 15, 30, 60, and 90 min after injection.

The free fatty acid (FFA) content in plasma was measured with a reagent kit according to the manufacturer’s instructions. The levels of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) were detected with an automatic biochemical analyzer (Multiskan MK3; Thermo Fisher Scientific, Waltham, MA).

Transthoracic echocardiography was performed using a Vevo 3100 High-Resolution Imaging System (VisualSonics, Toronto, Canada) and a 30-mHz probe. Briefly, mice were anesthetized by 2% isoflurane inhalation and placed on a constant heated platform. M-mode echocardiography was performed at the papillary muscles of the midventricle. The heart rate, ejection fraction (EF), and fractional shortening of the left ventricle were measured from the M-mode images.

Mouse hearts were arrested in diastole by KCl (30 mmol/L), perfused, and then fixed with 10% neutral buffered formalin. All heart samples were embedded in paraffin and sectioned into 5-µm slices starting from the suture ligation site and continuing to the apex, and every 50th slice was reserved for subsequent staining. For quantification of the fibrotic area in the heart, sections were stained by Masson trichrome following the manufacturer’s instructions. Sections were observed through a microscope (AX10 imager A2/AX10 cam HRC; Zeiss), photographed using ZEN software (Zeiss) and analyzed with ImageJ (National Institutes of Health). The percentage of fibrotic area (blue stained) within the entire circular area was calculated from four to six hearts per group. A representative image for each group was selected to illustrate the mean value.

H9C2 cells were fixed with 2% paraformaldehyde and permeabilized in PBS containing 0.1% Triton X-100 at room temperature. The slides were blocked in 5% BSA for 1 h, incubated with primary antibodies (anti-ASC, Alexa Fluor-488 conjugated Tomm20, and anti-HADHa) at 4°C overnight, and then incubated with secondary antibodies for 1 h at 37°C. Images were acquired by a Zeiss LSM 900 with an Airyscan microscope (Zeiss). HADHa and ASC were computed into surfaces (green) and spots (yellow and red), respectively, using Imaris 9.9.0 software (Oxford Instrument) (21). The native distance from the center of ASC “spots” to the border of the HADHa “surface” was measured, and the percentage of colocalized ASC (distance ≤0) was calculated. Ten randomly selected fields were captured from 10 samples per group. A representative image for each group was selected to illustrate the average level of the group, based upon the histological feature.

A proximity ligation (PL) assay was performed using a Duolink In Situ Red Kit (Sigma). An in-depth description of the procedure is provided in the Supplementary Material. Images of five randomly selected fields per section were captured from six samples per group through a fluorescence microscope (IX83; Olympus), and the percentages of PL⁺ positive cells were calculated by ImageJ (National Institutes of Health).

Tissues or cultured cells were lysed in radioimmunoprecipitation assay buffer containing a protease inhibitor cocktail (Roche) for 30 min on ice. Protein (10–20 µg) was separated by SDS-PAGE. The detailed processes of Western blotting and immunoprecipitation are provided in the Supplementary Material.

Total RNA was isolated from frozen left ventricle tissue or cultured cells using an RNeasy Kit (Qiagen), and cDNA was synthesized using Omniscript reverse synthase and random hexamers according to the manufacturers’ guidelines. Quantitative real-time PCR was performed using SYBR Green (BIORAD). The primers are described in Supplementary Tables 2 and 3. Gene expression values were normalized to GAPDH levels and are reported as the fold change.

Crystal structure data of the HADHa and ASC proteins (6N1H and 2N1F) were downloaded from the Protein Data Bank, and molecular docking was performed using the online platform PATCHDOCK (https://bioinfo3d.cs.tau.ac.il/PatchDock/) (22). The 10 highest scoring solutions without applying restriction to calculation were analyzed with the biomolecular visualization tool PyMOL to highlight acetylated lysines on the HADHa and ASC complex. The putative interaction interface of HADHa was consistently observed from the 10 solutions and was accessible for ASC binding. According to the docking algorithm based on geometric fit and atomic desolvation energy, we further analyzed the top scoring docking model and obtained the binding sites on lysine on the HADHa-ASC complex. Structure images of the HADHa-ASC complex were generated in PyMOL (www.schrodinger.com/pymol).

An in-depth description of the acetylome process is provided in the Supplementary Material. The HFD/control ratio of each acetylated peptide was estimated as the average value of three hearts per group.

A detailed description of the procedure of mitochondria/cytosol fraction isolation is provided in the Supplementary Material.

The tissue NAD⁺/NADH ratio and SIRT1/SIRT3 activity were quantified using an NAD⁺/NADH Assay Kit and a SIRT1/3 Activity Fluorometric Assay Kit, respectively, following the manufacturer’s instructions.

Cardiac tissues were fixed for 24 h in 3% glutaraldehyde (pH 7.4) and then fixed with 1% osmium tetroxide for 1 h. After the samples were dehydrated in a series of ethanol solutions and embedded in epoxy resin, ultrathin sections were prepared and counterstained with 1% uranyl acetate and lead citrate. The sections were examined under a transmission electron microscope (JEM-1400PLUS; JEOL, Tokyo, Japan). For each heart, 50 mitochondria from 5–10 random fields were examined in a blind fashion to quantify the mitochondria with unruptured or ruptured membranes.

H9C2 cells were treated with BSA-conjugated palmitic acid (PA) (0.4 mmol/L) or an equivalent amount of fatty acid-free BSA for 24 h in the presence or absence of nicotinamide mononucleotide (NMN) (2 mmol/L). Then, the cells were stained with 200 nmol/L JC-1 dye for 20 min. Images of 10 randomly selected fields per section were captured from five samples per group by a Zeiss fluorescence microscope.

Adeno-associated virus 9 (AAV9) was used to express HADHa (HADHa-WT) or HADHa-K255R (VectorBuilder Inc, Guangzhou, China) and injected via the tail vein (3 × 10¹¹ genome copies per mouse): HADHa-K255R (F: 5′-TTTGGCAAAGAATTGGATCCCGCCACCATGGTGGCGTCCCGGGCGATTG-3′; R: 5′-AGTCCATGGTGGCGACCGGCTGGTAGAACTTCTTGCTAGAGTTG-3′) and HADHa-WT (F: 5′-CAGGTCGACTCTAGAGGATCCATGGTGGCGTCCCGGGCG-3′; R: 5′-TGGCGGCCATCGATTGAATTCTCACTGGTAGAACTTCTTGCTAGAGTT-3′).

Plasmids of HADHa with point mutations or truncated mutants were constructed as described in the Supplementary Material.

The human experiment was approved by the Biomedical Research Ethics Committee of West China Hospital of Sichuan University (Chengdu, China) (#2020137) and registered in the Chinese Clinical Trial Registry (ChiCTR2100047998). The inclusion and exclusion criteria and the clinical characteristics of the participants are described in Supplementary Tables 4 and 5, respectively (three males and five females in each group).

A human IL-18 ELISA kit (Elabscience) was used to detect the levels of IL-18 in human plasma. The plasma levels of other inflammatory cytokines were detected with a human high-sensitivity cytokine multiplex kit (Merck) on a Luminex system. The measurement was performed in duplicate, and the results were analyzed using MILLIPLEX Analyst 5.1 software (Merck).

Fresh fasting blood (10 mL) was needed for peripheral blood mononuclear cell (PBMC) isolation, as described in the Supplementary Material.

Human myocardial fibrosis was assessed by cardiac MRI as previously described (23,24). Images were obtained with a 3T scanner (Siemens Healthcare) with an 18-channel phased-array body coil combined with a spine coil. The native T1 value was assessed on a motion-corrected modified Look-Locker inversion recovery sequence. The short-axial T1 mapping images were acquired and the mean myocardial native T1 values were calculated using postprocessing software (Qmass 7.6; Medis).

Data are presented as mean ± SEM and were analyzed using an unpaired two-tailed Student t test to compare two groups and one-way or two-way ANOVA followed by Bonferroni post hoc analysis for >2 groups. A simple linear regression analysis was performed to estimate the correlation between two indicated factors. All statistical analyses were performed with GraphPad Prism v.9.0.1 (GraphPad, La Jolla, CA). P < 0.05 was considered to indicate statistical significance.

Information on the expanded methods and key resources is provided in the Supplementary Material. The data sets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

To study the influence of obesity on myocardial fibrosis, we treated 2-month-old mice with a normal chow diet or HFD for 12 weeks. Compared with the control mice, the HFD-fed mice exhibited physical and metabolic traits of overweight and obesity, including higher body weight, elevated level of fasting blood glucose, impaired glucose tolerance, and increased FFAs and lipid levels in the circulation (Fig.1A–E and Supplementary Fig. 1A). Although basal cardiac function was similar between the obese and control mice, Masson trichrome staining showed that obese mouse hearts exhibited pronounced interstitial fibrosis (Fig.1F and Supplementary Fig. 1B–D). Upregulation of expression of genes involved in collagen synthesis and profibrotic signaling was also observed, including Col1α1, Col1α2, Fn1, and α-SMA (Supplementary Fig. 1E). Established evidence suggests that fibrosis is a pathological feature of chronic inflammation (19,25). In our model, we found enhanced caspase-1 cleavage (20-KD subunit; P20) and downstream proinflammatory IL-1β and IL-18 release in obese mouse hearts. (Fig.1G). The graded increase in myocardial fibrosis was positively correlated with tissue IL-1β and IL-18 levels in the heart (Fig.1H and Supplementary Fig. 1F).

To understand whether obesity-evoked fibrosis and inflammation participate in pathological I/R conditions, we subjected obese mice to cardiac I/R injury through LAD ligation and analyzed them for 14 days (Fig.1I). Compared with the control mice, the obese mice exhibited increased release of cardiac enzymes, lower EF, increased inflammation, and worse fibrosis at 14 days after I/R (Fig.1J and K and Supplementary Fig. 1G–K). Importantly, IL-1β and IL-18 removal with neutralizing antibodies or NLRP3 activation inhibition with MCC950 abrogated the adverse effects of HFD on post-I/R cardiac function and fibrosis (Fig.1J and K and Supplementary Fig. 1G–K). Overall, the evidence indicates that the profibrotic effect of HFD-induced obesity is correlated with elevated NLRP3 inflammasome in the heart.

The NLRP3 inflammasome consists of a sensor (NLRP3), an adaptor (ASC), and an effector (caspase 1). Optimal spatial arrangement of ASC is required for activation of the entire NLRP3-dependent inflammasome. We found that ASC abundance was upregulated in the HFD group, with a great amount of ASC localized on mitochondria (Fig.2A and B and Supplementary Fig. 2A). Consistent with our previous finding that mitochondria-localized ASC facilitates inflammasome assembly, we observed more signals of NLRP3-ASC spatial approximation in the HFD heart by in situ PL assay (Fig.2C). Furthermore, immunoprecipitation showed that mitochondria-localized ASC was associated with a greater amount of NLRP3 (Supplementary Fig. 2B and C). These data suggest that mitochondria-localized ASC participates in NLRP3 inflammasome formation in obese mouse hearts.

Mitochondrial proteins are reportedly hyperacetylated in response to HFD feeding or obesity (16,26). In line with this, we detected increased levels of mitochondrial protein acetylation (LysAc) in the HFD group (Fig.2D and Supplementary Fig. 2D), as well as a reduced NAD⁺/NADH ratio and reduced SIRT1/SIRT3 activity (Supplementary Figure 2E–G). To explore whether mitochondrial LysAc affects the NLRP3 inflammasome in obesity, we sought to reduce LysAc via supplementation with NR, a precursor of the sirtuin cofactor NAD⁺. NAD⁺ repletion for 7 days did not alter the protein abundance of SIRT1/SIRT3 but significantly increased NAD⁺ levels and SIRT1/SIRT3 activity in the heart (Fig.2F and Supplementary Fig. 2H–J). As a result, it reduced mitochondrial acetylation, mitochondrial ASC levels, ASC-NLRP3 interaction, and the inflammatory cytokines released in HFD hearts (Fig.2G–I and Supplementary Fig. 2K). Moreover, NAD⁺ repletion reduced cardiac enzymes levels, improved cardiac systolic function, and attenuated myocardial fibrosis in obese mice after I/R injury (Fig.2J–M and Supplementary Fig. 2L and M). Collectively, the data suggest that obesity-related mitochondrial acetylation promotes NLRP3 inflammasome formation in obese mouse hearts through mitochondria-localized ASC.

To identify the molecular target(s) that mediate ASC mitochondrial localization, we performed coimmunoprecipitation with an anti-ASC antibody in isolated mitochondria from cardiac tissue. Mass spectrometry identified 48 mitochondrial proteins that associate with ASC. We next mapped the acetylome profiles of HFD hearts and revealed that eight proteins associated with ASC were hyperacetylated (Fig.3A and B and Supplementary Table 5). Immunoprecipitation confirmed that HADHa, a mitochondrial protein essential for fatty acid β-oxidation, physically interacted with ASC and was hyperacetylated in the HFD hearts (Fig.3C–E). Consistently, the PL⁺ signals of the HADHa-ASC spatial approximation showed stronger signals in the HFD hearts, indicating a strengthened association of HADHa and ASC (Fig.3F).

To better characterize the ASC-HADHa interaction under hyperacetylation, we performed superresolution structured illumination microscopy. After coincubation of H9C2 myoblasts with PA, the most abundant saturated fatty acid in plasma, the amount of ASC in close proximity to HADHa was increased (Fig.3I). Moreover, distance transformation analysis identified more colocalization of ASC and HADHa upon PA treatment (Fig.3H and I). Hyperacetylation induced an increase in outer mitochondrial membrane (OMM) permeability, as evidenced by the loss of mitochondrial membrane potential, the augmentation of cytochrome C released in PA-treated cells, and increased numbers of mitochondria with OMM rupture in HFD hearts, which may explain why ASC interacted with an inner mitochondrial membrane (IMM) protein (Fig.3J–L and Supplementary Fig. 3A). After treatment with NR or NMN, another NAD⁺ precursor, OMM permeability was preserved, and ASC was observed on the outside of mitochondria and not in close proximity to HADHa (Fig.3G–L). These data suggest that ASC localizes to mitochondria through its interaction with acetylated HADHa.

To understand how acetylated HADHa recruits ASC to mitochondria, we constructed a series of deletion mutants and examined their interaction with ASC. The HADHa mutants lacking amino acids 201–300 showed lower ASC binding capacity (Fig.3M and Supplementary Table 3), suggesting that acetylation within amino acids 201–300 on HADHa mediated the interaction. To identify the precise residue(s) mediating binding to ASC, we inputted the crystal structures of HADHa and ASC into molecular docking software and identified lysine (K) 254, K255, K259, K262, and K267 on HADHa as potential docking sites (Fig.3N, and Supplementary Fig. 3B and C). Then, we mutated each of these lysine residues to arginine (R) to prevent modification. The results showed that mutation of K255, a highly conserved lysine residue on HADHa across species, led to reduced interaction with ASC (Fig.3O and Supplementary Fig. 3D). On the other hand, mutation to glutamine (K255Q), which mimicked the modification, further enhanced the HADHa-ASC interaction (Fig.3P). Together, these results suggest that HFD-induced acetylation of K255 on HADHa recruits ASC to mitochondria.

Next, we generated AAV9 viruses carrying HADHa-K255R and HADHa-WT (Fig.4A) and injected them via the tail vein. At 4 weeks after injection, the transduction efficiency was confirmed according to Flag expression (Fig.4B). Compared with the wild-type controls, the HFD mice bearing the HADHa-K255R mutant had decreased mitochondrial ASC levels, reduced NLRP3-ASC interaction, and downregulated release of inflammatory cytokines in the heart (Fig.4C–G). The mutant did not change body weight, but it significantly decreased cardiac enzyme release, improved EF, and attenuated myocardial fibrosis after I/R injury (Fig.4H and I and Supplementary Fig. 4). Collectively, these data indicate that blockade of K255 acetylation on HADHa suppresses the NLRP3 inflammasome and mitigates myocardial fibrosis in obese mouse hearts.

To determine the clinical relevance of our findings, we measured plasma inflammatory cytokines in obese patients and lean participants. With higher BMI and circulating FFAs, obese patients exhibited significant elevations in the levels of multiple proinflammatory cytokines, notably IL-18 (Fig.5A–C). BMI was positively correlated with the levels of IL-18 and IL-1β (Fig.5D and E), reinforcing the connection between obesity and the NLRP3 inflammation in humans. To establish a direct connection between obesity and NLRP3 inflammation in humans, we separated PBMCs from blood samples of the participants and observed increased mRNA expression of Asc, Nlrp3, and Caspase-1 in the obese patients (Fig.5F). Furthermore, we quantified myocardial fibrosis in these patients by T1 mapping MRI. The obese patients showed higher T1 values than the lean participants (Fig.5G and H). Again, the T1 value was linearly correlated with the levels of IL-18 and IL-1β (Fig.5I and J), suggesting a potential causal role of NLRP3 inflammatory cytokines in myocardial fibrosis. These results support the conclusion that obesity increases NLRP3 inflammation and worsens myocardial fibrosis.

The current study demonstrates that obesity induces HADHa acetylation to promote ASC translocation to mitochondria and NLRP3 inflammasome assembly, which contributes to myocardial fibrosis in mice. We identified K255 on HADHa as the key LysAc site in this pathological process (Fig. 6). Therefore, our study reveals a critical role of the NLRP3 inflammasome in obesity-induced myocardial fibrosis and identifies K255 on HADHa as a potential therapeutic target.

Obesity is considered a type of low-grade inflammation (27), which is an important risk factor for diabetes and cardiovascular diseases. More than two-thirds of obesity-related mortalities are associated with heart diseases (28). Accumulating evidence indicates that the NLRP3 inflammasome plays a crucial role in myocardial injury (13,29,30). In agreement with this notion, we observed that obese mouse hearts, with increased NLRP3 inflammasome-downstream cytokines IL-18 and IL-1β levels, were susceptible to fibrosis, especially after I/R injury. The detrimental effect of the NLRP3 inflammasome on obesity was also confirmed by neutralizing antibody and NLRP3 inhibitor experiments. Further study demonstrated that increased mitochondria-localized ASC played a key role in the assembly of the NLRP3 inflammasome in obese mouse hearts. This finding has been corroborated in animal models of heart failure and atherosclerosis established by our group and other groups (19,31). Therefore, these results provide new evidence for the involvement of mitochondria-localized ASC in obesity-induced cardiac fibrosis.

Protein acetylation and deacetylation control many important cellular processes. Acetylation of mitochondrial proteins is reported to be involved in the regulation of oxidative stress and inflammatory cascades (32–35). In our study, we found that downregulation of mitochondrial acetylation through NAD⁺ precursor supplementation decreased ASC mitochondrial translocation, inhibited NLRP3 inflammasome formation, reduced IL-18 and IL-1β release, and attenuated obesity-induced myocardial fibrosis. Thus, reducing mitochondrial acetylation could be a potential therapeutic strategy for obesity-related myocardial fibrosis. A limitation of this experiment is that, in addition to reducing acetylation, NAD⁺ replenishment also regulates cardiac substrate oxidation and peroxisome proliferator–activated receptor signaling pathways (36,37), which could also contribute to the antifibrotic effect.

Mitochondrial morphological destabilization and dysfunction occur during inflammation (38). In this study, we found increased OMM permeability in obese mouse hearts, as evidenced by rupture of the OMM, loss of mitochondrial membrane potential, and enhanced release of cytochrome C. Disruption of OMM integrity may facilitate the interaction between proteins at the IMM and cytoplasm (39). Here, we verified that obesity induces OMM rupture and promotes the interaction of HADHa, an IMM protein, with ASC in the cytosol, which drives ASC mitochondrial localization and increases NLRP3 inflammasome assembly. Hyperacetylation of K255 on HADHa was identified as the key governing the HADHa-ASC interaction and mitochondrial localization of ASC. Our findings suggest that impairment of OMM integrity participates in NLRP3 inflammation and that HADHa-K255 may serve as a potential therapeutic target for cardiac fibrosis in obesity. As HADHa is a metabolic enzyme in fatty acid oxidation, whether hyperacetylated HADHa also promotes NLRP3 inflammasome by regulating fatty acid metabolism requires further investigation.

Although reducing HADHa-K255 acetylation exerted antifibrotic effects similar to those of IL-1β/IL-18 blockade, these strategies were applied for different purposes and conveyed different messages. Blockade of IL-1β and IL-18 by either neutralizing antibodies or MCC950 was used to demonstrate whether NLRP3 inflammation contributes to obesity-related fibrosis, while inhibition of HADHa-K255 acetylation was used to corroborate whether mitochondrial acetylation modulates NLRP3 inflammation and thus fibrosis in obese hearts.

Circulating PBMCs are a key source of NLRP3 inflammasome, which play an important role in systemic inflammation, and have been reported to be involved in the development of myocardial fibrosis (40). To establish a direct connection between obesity and NLRP3 inflammation in humans, we separated PBMCs from human blood and observed increased mRNA expressions of Asc, Nlrp3, and Caspase-1 in the obese patients. Along with the increased IL-1β and IL-18 release, we concluded that obesity is correlated with NLRP3 inflammation. However, whether PBMCs produce excessive NLRP3 inflammasome and thus contribute to myocardial fibrosis through hyperacetylated HADHa and ASC-HADHa interaction merits further investigation.

In summary, by using an HFD-induced mouse model, we demonstrate that obese mouse hearts are susceptible to myocardial fibrosis due to mitochondrial hyperacetylation-initiated activation of the NLRP3 inflammasome.

Acknowledgments. The authors thank Metabolomics and Proteomics Technology Platform, West China Hospital of Sichuan University for the assistance with the acetyl-proteome analysis.

Funding. This study was supported by grants from the Key Research and Development Program of Sichuan Province (2022YFS0132 and 2022YFS0198), the Natural Science Foundation of Sichuan Province (2023NSFSC1633), the National Natural Science Foundation of China (81970715, 82370260, 82300916), and Yunnan Provincial Cardiovascular Disease Clinical Medical Center Project (FZX2019-06-01 and 2022YFKY078).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. Conceptualization was performed by Y.D., X.L., and T.L.; experimental investigation was performed by Mi.X., R.Z., L.J., K.T., W.Y., W.O., and Ma.X.; data collection, analysis, and interpretation were performed by Y.D. and X.L.; manuscript writing and reviewing was performed by Y.D., X.L., and T.L.; funding acquisition was performed by Y.D. and T.L.; and supervision was performed by T.L. All authors read and approved the final manuscript prior to submission. T.L. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.