Modulation of Abnormal Vasoconstriction Through 2‐Hydroxyisobutyrylation of Tropomyosin 3 Lys141: Targeting Histone Deacetylase 3 as a Key Approach

Abstract

Background

TPM3 (tropomyosin 3) is an actin‐binding protein in vascular smooth muscle cells, where posttranslational modifications critically regulate its actin affinity, influencing cardiovascular function. Emerging evidence suggests that Khib (2‐hydroxyisobutyrylation) plays a significant role in the cardiovascular system. Histone deacetylase 3 (HDAC3) serves as an “eraser” of Khib marks. However, the impact of TPM3 de‐2‐hydroxyisobutyrylation on vascular contraction remains unclear.

Methods and Results

In this study, we employed mouse models and in vitro experiments to elucidate the mechanism by which phenylephrine‐induced HDAC3 activation drives vasoconstriction via de‐2‐hydroxyisobutyrylation of TPM3. Our findings demonstrate that phenylephrine triggers HDAC3 nuclear export and promotes its interaction with TPM3, resulting in decreased Khib modification and enhanced vasoconstriction. Coimmunoprecipitation experiments confirmed that phenylephrine reduces Khib levels on TPM3 in mouse aorta. Additionally, ex vivo vascular tension assays using mouse aortic rings revealed that treatment with the Khib donor, ethyl 2‐hydroxyisobutyrate, induces endothelium‐independent vasodilation and ameliorates hypertensive vascular dysfunction. Molecular docking and kinetic simulations identified Lys141 of TPM3 as the primary site targeted by HDAC3‐mediated de‐2‐hydroxyisobutyrylation. This was further validated by adenoviral transfection of isolated blood vessels with a Lys141‐mutated TPM3 construct, which abolished the effects of HDAC3 on TPM3 Khib modification and vascular contractility.

Conclusions

These findings underscore the critical role of TPM3 de‐2‐hydroxyisobutyrylation in vasoconstriction and suggest that modulating this posttranslational modification could provide a novel therapeutic strategy for hypertensive vascular dysfunction.

Nonstandard Abbreviations and Acronyms

HDAC2

histone deacetylase 2

HDAC3

histone deacetylase 3

Khib

2‐hydroxyisobutyrylation

PTM

posttranslational modification

SAHA

suberoylanilide hydroxamic acid

TPM3

tropomyosin 3

VSMC

vascular smooth muscle cell

SOCE

store‐operated calcium entry

Research Perspective.

What Is New?

• This study identifies histone deacetylase 3 as a key regulator of the 2‐hydroxyisobutyrylation modification of TPM3 (tropomyosin 3), specifically at Lys141, in vascular smooth muscle cells.

• The findings demonstrate that selective histone deacetylase 3inhibition reduces vasoconstriction by modulating TPM3 modification, highlighting a novel epigenetic mechanism in vascular function and hypertensive disease.

• The study uncovers the role of phenylephrine in reducing TPM3 2‐hydroxyisobutyrylation, providing insights into the molecular basis of vasoconstriction.

What Question Should Be Addressed Next?

• Further studies are needed to explore the therapeutic potential of targeting TPM3 2‐hydroxyisobutyrylation in patients with hypertension and to develop localized delivery strategies that minimize systemic side effects.

• Investigating whether similar modifications are involved in other forms of vascular disease, such as atherosclerosis or aneurysms, will help determine the broader relevance of this regulatory pathway.

• Long‐term studies are essential to evaluate the safety, efficacy, and potential off‐target effects of histone deacetylase 3inhibitors in clinical settings, especially regarding their impact on nonvascular tissues.

Vascular smooth muscle cell (VSMC) contractility is a cornerstone of vascular function, playing a critical role in maintaining vascular tone and stability. Dysregulation in VSMC contractility not only contributes to hypertension and vascular remodeling but also underpins severe vascular conditions such as aortic aneurysm and dissection. ¹ , ² Abnormal calcium signaling, marked by erratic calcium influx and excessive sarcoplasmic reticulum calcium release, has been widely observed in hypertensive states. ³ , ⁴ Additionally, mutations affecting key VSMC contractile proteins, including myosin heavy chain 11, α‐actin, and protein kinase cGMP‐dependent 1, have been linked to severe vascular disorders, ⁵ , ⁶ , ⁷ , ⁸ , ⁹ , ¹⁰ emphasizing the complexity of VSMC function.

Despite advances in understanding the underlying mechanisms, the therapeutic management of abnormal vasoconstriction remains suboptimal. Calcium channel blockers, such as amlodipine and verapamil, are first‐line treatments for coronary spasm and vasomotor dysfunction and are widely used in clinical practice. However, their effectiveness varies across different patient populations. ¹¹ For instance, in Marfan syndrome, calcium channel blockers can potentially accelerate aneurysm progression and increase the risk of mortality. High doses of calcium channel blockers also raise concerns regarding blood pressure and heart rate tolerance, and they have limited efficacy in treating coronary spasm associated with myocardial bridges. ¹² Current therapies for abnormal vasoconstriction are frequently hindered by drug resistance, adverse effects, and a lack of target specificity. These limitations highlight the urgent need for alternative treatments that address the underlying molecular mechanisms driving abnormal vasoconstriction.

This study focuses on TPM3 (tropomyosin 3), a critical actin‐binding protein within VSMCs that regulates myosin‐actin interactions essential for vasoconstriction and dilation. ¹³ , ¹⁴ , ¹⁵ , ¹⁶ Although posttranslational modifications (PTMs) of TPM3, such as acetylation and phosphorylation, have been explored as potential therapeutic targets, ¹⁷ , ¹⁸ , ¹⁹ less is known about the role of more recently identified PTMs like Khib (2‐hydroxyisobutyrylation). Khib, a modification involving the addition of 2‐hydroxyisobutyryl groups to lysine residues, has emerged as a key regulator of protein stability and function in various systems. ²⁰ , ²¹ Histone deacetylase 3 (HDAC3), an eraser enzyme, specifically targets Khib marks, ²² and its dysregulation has been linked to pathological states such as glycolytic reprogramming in cancer ²³ , ²⁴ and cardiac hypertrophy. ²⁵ , ²⁶

Given these insights, our study investigates whether Khib modification of TPM3 plays a role in regulating VSMC contractility and how HDAC3‐mediated de‐2‐hydroxyisobutyrylation influences this process. We hypothesize that HDAC3 promotes abnormal vasoconstriction by selectively targeting Khib on TPM3, particularly at Lys141, and that modulating this pathway could offer a novel therapeutic strategy. By addressing the limitations of existing treatments, our work aims to provide a foundation for targeted therapies that correct epigenetic dysregulation in hypertensive vascular dysfunction.

METHODS

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Chemical and Reagents

The following chemicals were procured from Selleck Chemistry (Houston, TX): suberoylanilide hydroxamic acid (SAHA; PubChem CID: 5311), ethyl 2‐hydroxyisobutyrate (PubChem CID: 6653), verapamil (PubChem CID: 2520), N‐omega‐nitro‐L‐arginine (PubChem CID: 440005), 4‐bromo‐N′‐butylbenzohydrazide (PubChem CID: 4596836), quisinostat (PubChem CID: 11538455), N′‐butyl‐2′,3′‐difluoro‐[1,1′‐biphenyl]‐4‐carbohydrazide (PubChem CID: 118418385), CUDC‐101 (PubChem CID: 24756910), fimepinostat (CUDC‐907; PubChem CID: 54575456), sodium nitroprusside (PubChem CID: 11953895), CaCl₂ (PubChem CID: 5284359), and phenylephrine (PubChem CID: 6041). SAHA, N‐omega‐nitro‐L‐arginine, 4‐bromo‐N′‐butylbenzohydrazide, quisinostat, N′‐butyl‐2′,3′‐difluoro‐[1,1′‐biphenyl]‐4‐carbohydrazide, CUDC‐101, and CUDC‐907 were dissolved in dimethylsulfoxide to create 100 mmol/L stock solutions. Working dilutions ranging from 0.01 to 100 μmol/L were prepared in dimethylsulfoxide. The remaining reagents were dissolved in distilled water. All drug concentrations are expressed as the final molar concentration of the base in the organ bath. Dimethylsulfoxide (0.1%, v/v) was also added to the control and did not affect the tone of the aortic rings at the concentration used.

Animal Experiments

All animal experiments were approved by the Animal Ethics Committee of Kunming Medical University (Approval Number: kmmu20221778) and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male C57BL/6 mice, aged 6 to 8 weeks and weighing 20 to 25 g, were obtained from the Experimental Animal Center of Kunming Medical University (Yunnan, China, License number: SCXK (滇) 2020‐0004). The mice were housed under controlled conditions (20–25 °C, 45%–60% humidity, 12‐h light/12‐h dark cycle) and provided with free access to food and water. They were acclimated for 1 week after arrival before experimentation commenced.

Tension Measurement in Isolated Blood Vessels

Vessel tension measurements were performed as previously described in our study. ²⁷ Briefly, isometric tension in mouse thoracic aorta or mesenteric artery rings was measured using a wire myograph (DMT, Aarhus, Denmark). Adult male C57BL/6 mice were euthanized by CO₂ asphyxiation. The aorta or mesenteric artery was excised and placed in ice‐cold, oxygenated Krebs buffer containing (in mmol/L) 118 NaCl, 4.7 KCl, 25 NaHCO₃, 1.2 KH₂PO₄, 1.2 MgSO₄, 2.5 CaCl₂, and 10 glucose. Vascular rings (2‐mm long) were mounted between 2 steel wires and submerged in thermostatic baths containing Krebs buffer bubbled with 95% O₂ and 5% CO₂ at 37 °C. ²⁸ The resting tension was gradually increased to 3 mN over 60 minutes. Contraction in vascular rings was induced using 60 mmol/L KCl and 1 μmol/L phenylephrine to assess contractile function. KCl was applied first to initiate contraction by depolarization, followed by the addition of phenylephrine to further evaluate the contractile response. A shrinkage percentage of ≈70% to 80% of the maximum shrinkage was considered optimal for subsequent experiments. Endothelial integrity was confirmed by relaxing phenylephrine–precontracted rings with 10 μmol/L acetylcholine. ²⁹ After washing and reequilibration, rings were precontracted with phenylephrine (1 μmol/L) and cumulative concentration–response curves were plotted for the test agents or vehicle. Vascular ring segments were washed with Ca²⁺‐free Krebs buffer (containing 0.1 mmol/L EDTA) and then treated with 1 μmol/L verapamil to block voltage‐dependent Ca²⁺ channels. After 10 minutes, 1 μmol/L phenylephrine was added to induce store‐operated calcium entry (SOCE), followed by the addition of a cumulative concentration of CaCl₂ (0.5–10 mmol/L) or a single dose of 2.5 mmol/L CaCl₂ to elicit vessel contraction. Data were recorded using LabChart software and a PowerLab data acquisition system (ADInstruments, Colorado Springs, CO). Relaxation was expressed as the percentage reduction in phenylephrine‐induced tone. Each ring served as its own control to account for between‐organ variability.

Mouse Model of Hypertension

Twenty‐four male C57BL/6 mice, weighing between 20 and 25 g and aged 6 to 8 weeks, were acclimated for 5 days. Six mice were randomly selected as normal controls. The remaining 18 mice received intraperitoneal injections of 15 mg/kg N‐omega‐nitro‐L‐arginine twice daily for 21 days to induce hypertension. A systolic blood pressure of ≥160 mm Hg was indicative of successful modeling. ³⁰ The model mice were randomly assigned to 3 groups: model group (n=6), ethyl 2‐hydroxyisobutyrate treatment group (n=6), and captopril treatment group (n=6). Captopril and ethyl 2‐hydroxyisobutyrate were dissolved in normal saline for administration. The treatment groups received captopril solution (7.615 mg/kg, corresponding to the clinical adult dose) ³¹ and ethyl 2‐hydroxyisobutyrate suspension (7.615 mg/kg, matching the dose of captopril) via oral gavage daily for 4 consecutive weeks, at a volume of 10 mL/kg. The vehicle and model groups, were gavaged with an equivalent volume of normal saline. To ensure experimental rigor, researchers who were not involved in conducting the in vivo experiments were selected to monitor systolic blood pressure, diastolic blood pressure, mean arterial pressure, and heart rate in mice on a weekly basis using tail‐cuff plethysmography. ³² Additionally, mesenteric vessels were isolated for subsequent in vitro vascular tension assessments.

Multimethod Docking Approach

In the implemented HDOCK approach, PDB files for histone deacetylase 2 (HDAC2, PDB ID: 3MAX) and HDAC3 (PDB ID: 4A69), along with the full‐length structure of TPM3 from AlphaFold (Identifier: D3TP64), were uploaded onto the HDOCK server. This server processes the structural and sequence information to perform protein–protein docking, yielding potential binding sites and calculating docking scores to reflect interaction stability and affinity. Concurrently, the PRISM server was used for its ability to predict interactions via structural matching. Structures obtained from PDB and AlphaFold were inputted into PRISM, which assesses protein interfaces and constructs models that anticipate the spatial interplay of protein complexes.

Dynamic Simulation

In the dynamic simulation study, the top 10 docking models, as determined by the highest HDOCK scores, were systematically analyzed alongside PRISM‐simulated docking models. Reference to the active sites of HDAC3 facilitated the exclusion of 3 irrelevant models from further consideration. A comprehensive compilation of all interacting amino acids was conducted, with a particular focus on lysine residues. Scores were assigned based on interaction intensities, free energy values, and the number of interaction bonds, informed by data from both HDOCK and PRISM results. Distance measurements between interacting residues and the visualization of molecular interactions were performed using PyMOL.

Transfection With siRNA

Mouse thoracic aortic vessel segments were subjected to siRNA transfection to investigate the impact of Tpm3 knockdown on cardiovascular physiology. Specifically, Tpm3 siRNA‐1, Tpm3 siRNA‐2, and Tpm3 siRNA‐3, along with a scrambled siRNA control, were employed in the transfection process as per the manufacturer's instructions (HANBIO, China). The sequences of these siRNAs were provided as follows: Tpm3 siRNA‐1 (sense strand: GCAGCUAGAGGAUGAACUA, antisense strand: UAGUUCAUCCUCUAGCUGC), Tpm3 siRNA‐2 (sense strand: GGAAAUCCAGCUAAAGGAA, antisense strand: UUCCUUUAGCUGGAUUUCC), Tpm3 siRNA‐3 (sense strand: AGACAAGUAUGAAGAAGAA, antisense strand: UUCUUCUUCAUACUUGUCU), and the scrambled siRNA (sense strand: UUCUCCGAACGUGUCACGU, antisense strand: ACGUGACACGUUCGGAGAA). The siRNA sequence includes nucleotide overhangs designed to enhance gene silencing efficiency by facilitating the unwinding of the siRNA duplex at the termini, thereby improving the accessibility and incorporation of the guide strand into the RNA‐induced silencing complex. siRNA transfections were performed using Lipofectamine 3000 (Thermo Fisher Scientific, USA). Briefly, thoracic aortic vessel segments were placed in Petri dishes containing 500 μL of complete medium (Biological Industries, Israel) supplemented with 10% fetal calf serum. The siRNA was prepared as a 20 μmol/L stock solution in diethyl pyrocarbonate‐treated water. A 100 μL transfection mixture was prepared, consisting of 50 μL of Opti‐MEM (Thermo Fisher Scientific, USA) containing 10 μL of siRNA and 50 μL of Opti‐MEM containing 2 μL of Lipofectamine 3000. This mixture was allowed to equilibrate for 20 minutes. The vessel segments were then incubated with this siRNA‐Lipofectamine mixture for 6 hours at 37 °C. After the transfection period, the mixture was replaced with complete medium, and the vessel segments were cultured for an additional 18 hours. Subsequently, tissue fragments were collected for tension measurements and Western blot experiments.

Plasmid Transfection

In the experiment, we used isolated mouse aortic rings and introduced plasmids with TPM3 constant activity mutations (Tpm3 K141R, Tpm3 K153R, Tpm3 K162R; GeneChem, China) through transfection methods. First, we diluted 4 μg of plasmids in 100 μL serum‐free Opti‐MEM. Then, we diluted 4 μL of Lipofectamine 3000 reagent in 100 μL serum‐free Opti‐MEM. We mixed the diluted plasmid solution with the diluted reagent solution and gently mixed them, followed by incubation at room temperature for 15 minutes. Next, we added the complex to Opti‐MEM culture medium without fetal bovine serum. The isolated mouse aortic ring was placed in the transfection well for transfection treatment, with a transfection time of 12 hours. After that, the transfected tissue was transferred to DMEM culture medium containing 10% fetal bovine serum for further cultivation for 24 hours.

Ex Vivo Adenoviral Gene Transfer in Mouse Aorta

The protocol for ex vivo infection of mouse aortic rings was based on previous studies. ³³ We used an adenovirus that carried a constitutively active mutation of TPM3 at lysine 141 (Ad‐Tpm3 K141R). An Ad‐GFP (adenovirus carrying green fluorescent protein) was used as the internal control. Aortic segments were infected with adenovirus (5×10⁹ pfu) for 12 h in fetal bovine serum‐free opti‐MEM GlutaMAX and then transferred to DMEM containing 10% fetal bovine serum for 36 hours. Transfection efficiency was verified by monitoring the green fluorescence of Ad‐GFP on the endothelium side while also preparing mouse aorta under a fluorescence microscope.

Immunofluorescence

Immunofluorescence was performed as previously described. VSMCs were cultured to 60% confluence and then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X‐100 for 10 minutes each at room temperature. Nonspecific binding sites were blocked with 5% BSA, followed by overnight incubation at 4 °C with rabbit anti‐TPM3 and mouse anti‐HDAC3 antibodies. After washing, cells were incubated with Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies for 1 hour at room temperature. DAPI was used to stain nuclei, and immunofluorescence signals were captured using a confocal microscope (A1 HD25, Nikon).

Coimmunoprecipitation

Vascular tissues were meticulously harvested, snap frozen in liquid nitrogen for 1 hour, and subsequently lysed in 200 μL of Cell lysis buffer for Western and immunoprecipitation (Beyotime, P0013). Following complete tissue homogenization, the lysate was placed on ice for 30 minutes and then centrifuged at 12 000 rpm for 15 min at 4 °C to obtain the supernatant. The resulting lysate was combined with 2 μg of specific antibodies, including anti‐TPM3 antibody (proteintech, 10 737‐1‐AP), anti‐HDAC3 antibody (Cell Signaling Technology, 3949), Flag‐tag (Abbkine, ABT2010) and anti‐2‐hydroxyisobutyllysine antibody (PTM BIO, PTM‐802). This mixture was allowed to incubate overnight at 4 °C. Subsequently, the lysate was mixed with 20 μL of protein A/G beads (APExBIO, K1305) and incubated on a shaker at 4 °C for 4 hours to facilitate the immunoprecipitation process. The coimmunoprecipitation product was resuspended in 50 μL of loading buffer and subjected to electrophoresis on a 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gel. The separated proteins were then electrotransferred onto a polyvinylidene difluoride membrane. Following the transfer, the membrane was incubated with the appropriate primary antibodies against TPM3, HDAC3, Flag‐tag, or 2‐hydroxyisobutyllysine at 4 °C overnight. Subsequently, the membrane was probed with a secondary antibody, specifically goat antirabbit IgG horseradish peroxidase‐conjugated secondary antibody (1:5000), at room temperature. Signal detection was achieved through the use of enhanced chemiluminescent substrates. The quantification of protein band densities was conducted with ImageJ software (National Institutes of Health, Bethesda, MD).

Western Blot Analysis

Endothelial‐depleted mouse thoracic aorta vascular tissues were fractionated into nucleus‐enriched and cytoplasmic samples by using a Nuclear Protein Extraction Kit (Solarbio, EX1470). Cell lysates were treated with RIPA (Beyotime, P0013B) lysis buffer, which was resolved on an 12% SDS/PAGE gel. Then, these were transferred to a polyvinylidene fluoride membrane P(0.45 μm pore; Millipore, UK). Membranes were blocked for 1 hour at room temperature in PBS containing 0.1% (v/v) Tween‐20 and 5% (w/v) semiskimmed milk. Primary antibodies against TPM3 (proteintech, 10 737‐1‐AP), HDAC3 antibody (Cell Signaling Technology, 3949), histone H3 (proteintech, 17 168‐1‐AP), 2‐hydroxyisobutyllysine antibody (PTM BIO, PTM‐801), propionyllysine antibody (PTM BIO, PTM‐201), butyryllysine antibody (PTM BIO, PTM‐301), malonyllysine antibody (PTM BIO, PTM‐901), glutaryllysine antibody (PTM BIO, PTM‐1151), succinyllysine antibody (PTM BIO, PTM‐401), crotonyllysine antibody (PTM BIO, PTM‐501), Flag‐tag (Abbkine, ABT2010), GAPDH (Abcam, ab181602), β‐tubulin (Cell Signaling Technology, 2128) and beta actin (Abcam, ab8227) were used at 1:1000 dilution, and goat antirabbit horseradish peroxidase secondary (Fisher Scientific, UK) used at 1:5000 dilution. All antibodies were diluted in blocking solution. Immunoreactivity was detected using ECL Western blotting solution (Pierce).

Statistical Analysis

Following enrollment, no animals were excluded from the study. Each experimental group included samples derived from at least 3 to 4 different mice, with n representing the number of animals used. Results are presented as the mean±SEM. For concentration‐response curves, relaxation responses to the test compounds were expressed as the percentage reduction in phenylephrine (1 μmol/L)‐induced tension. Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, La Jolla, CA). Comparisons between 2 groups were conducted using an unpaired 2‐tailed Student's t test. For multiple comparisons, 1‐way or 2‐way ANOVA followed by Sidak's post hoc test was used to assess differences between and within groups. A P value <0.05 was considered statistically significant.

RESULTS

Phenylephrine Stimulation Reduces Khib Modification of TPM3 in Blood Vessels

To investigate the impact of phenylephrine stimulation on lysine acylation modification levels of proteins in vascular tissue, we conducted Western blot analysis on mouse thoracic aorta tissue. The isolated mouse thoracic aortic vessels were subjected to phenylephrine treatment (1 μmol/L) for 30 minutes. Our results revealed that, in comparison to the control group (vehicle treatment), among the 7 lysine acylations of proteins in vascular tissue following phenylephrine treatment, only Khib exhibited a significant abundance, with a notable decrease in the level of acylation modifications (Figure 1A). Furthermore, to assess the effect of phenylephrine stimulation on the Khib modification level of TPM3 in blood vessels, coimmunoprecipitation was employed to quantify the Khib level of TPM3. Our findings demonstrated a reduction in the levels of Khib of TPM3 in blood vessels following phenylephrine treatment (Figure 1B and 1C and Figure S1). These observations provide compelling evidence that phenylephrine stimulation diminishes the level of Khib modification of TPM3 in blood vessels.

Figure 1. Phenylephrine stimulation reduces Khib modification of TPM3 in blood vessels.

A, Western blot analysis was performed on lysates of mouse thoracic aorta tissue using antibodies against 2‐hydroxyisobutyryllysine, propionyllysine, butyryllysine, malonyllysine, glutaryllysine, succinyllysine, crotonyllysine, and GAPDH (n=3 independent biological samples for each). B, Immunoprecipitation of mouse thoracic aortic vessel homogenates using anti‐TPM3 protein A/G beads following phenylephrine (1 μmol/L) treatment for 30 minutes. Immunoblotting was performed using antibodies against 2‐hydroxyisobutyllysine, TPM3, and GAPDH for detection. Representative immunoblots for each group were obtained, with n=3 independent biological samples for each. C, The effect of phenylephrine treatment on the level of Khib of TPM3 in the mouse thoracic aorta was assessed. Data are expressed as mean±SEM. *P<0.05 vs Vehicle (n=3 independent biological samples for each). IP:TPM3 indicates immunoprecipitation:tropomyosin 3; Kbu, lysine butyrylation; Kcr, lysine crotonylation; KDa, kilodaltons; Kglu, lysine glutarylation; Khib, lysine 2‐hydroxyisobutyrylation; Kmal, lysine malonylation; Kpr, lysine propionylation; Ksu, lysine succinylation; and TPM3, tropomyosin 3.

2‐Hydroxyisobutyrylation Donor Induces Endothelium‐Independent Vasodilation and Alleviates Hypertensive Vascular Dysfunction in Mice

Using coimmunoprecipitation techniques, we explored the role of ethyl 2‐hydroxyisobutyrate as a potential donor for Khib within mouse thoracic aorta vasculature following its administration. We found markedly increased levels of Khib across vascular proteins and particularly with TPM3, thereby substantiating the role of ethyl 2‐hydroxyisobutyrate as a donor, which notably augments Khib (Figure 2A and 2B). Furthermore, an investigation of the vasodilatory impact of ethyl 2‐hydroxyisobutyrate was facilitated using isolated mouse blood vessel rings. We observed that gradient concentration‐dependent exposures to ethyl 2‐hydroxyisobutyrate (200 μmol/L–400 μmol/L) induced a correlated vasodilation in mouse thoracic aortic rings precontracted with phenylephrine (1 μmol/L). Interestingly, this vasodilatory effect preserved its intensity even after vascular endothelium excision, indicating its endothelium‐independence. To accurately assess the vasodilatory effects of ethyl 2‐hydroxyisobutyrate, we added a control group treated with solvent (dd H₂O) to account for the natural attenuation of vasoconstriction after phenylephrine treatment. The experimental results showed that the solvent alone did not cause similar vasodilation, providing further evidence to support the direct vasodilatory effects of ethyl 2‐hydroxyisobutyrate (Figure 2C). Considering the in vivo antihypertensive effects of ethyl 2‐hydroxyisobutyrate, we developed a chronic hypertensive mouse model by elevating systolic blood pressure to ≥160 mm Hg using intraperitoneal injections of N‐omega‐nitro‐L‐arginine across 21 days. Subsequently, we administered ethyl 2‐hydroxyisobutyrate, the reference drug captopril, and normal saline for 28 days, respectively (Figure S2). Both ethyl 2‐hydroxyisobutyrate and captopril significantly decreased systolic blood pressure, diastolic blood pressure, mean arterial pressure, and heart rate compared with the model mice (Figure 2D). This cumulative evidence thus advocates the role of Khib donors in instigating endothelium‐independent vasodilation and reducing blood pressure in hypertensive mice. Hypertension is closely associated with vascular dysfunction. In this study, we employed vascular tension measurement to assess the contractile response of mesenteric arteries. As depicted in Figure 2E through 2G, the vasoconstrictor responses induced by phenylephrine, 60 mmol/L KCl, or CaCl₂ were significantly augmented in hypertensive mice compared with the vehicle group. Conversely, the administration of ethyl 2‐hydroxyisobutyrate significantly attenuated the contraction induced by these vasoconstrictor agents in hypertensive mice (Figure 2E and 2G). These findings suggest that the contractile response of mesenteric resistance arteries is potentiated in hypertensive mice, and this alteration can be reversed by ethyl 2‐hydroxyisobutyrate.

Figure 2. 2‐hydroxyisobutyrylation donor induces endothelium‐independent vasodilation and alleviates hypertensive vascular dysfunction in mice.

A, Immunoprecipitation of mouse thoracic aortic vessel homogenates using anti‐TPM3 protein A/G beads following ethyl 2‐hydroxyisobutyrate (400 μmol/L) treatment for 30 minutes. Immunoblotting was performed using antibodies against 2‐hydroxyisobutyllysine, TPM3, and GAPDH for detection. Representative immunoblots for each group were obtained, with n=3 independent biological samples for each. B, The effect of ethyl 2‐hydroxyisobutyrate treatment on the level of Khib of TPM3 in the mouse thoracic aorta was assessed. Data are expressed as mean±SEM. *P<0.05 vs Vehicle (n=3 independent biological samples for each). C, Isolated mouse aortic rings, precontracted with phenylephrine (1 μmol/L), were treated with cumulative doses of ethyl 2‐hydroxyisobutyrate (200 μmol/L–400 μmol/L). The relaxation response was measured in both endothelium‐intact (+ endo) and endothelium‐removed (− endo) rings. The resulting data are expressed as mean±SEM. *P<0.05, ***P<0.001 vs ethyl 2‐hydroxyisobutyrate (n=5 biologically independent samples each). D, The effects of ethyl 2‐hydroxyisobutyrate on SBP, DBP, MAP, and heart rate in hypertensive mice at different administration times were evaluated. Data are expressed as mean±SEM. *P<0.05, **P<0.01, ***P<0.001 vs Vehicle; ^# P<0.05, ^## P<0.01, ^### P<0.001 vs Model; n=6 biologically independent samples each. E, Phenylephrine‐induced changes in Mesenteric artery (MRA) contraction in hypertensive, vehicle, and ethyl 2‐hydroxyisobutyrate‐treated mice. Data are shown as mean±SEM; n=4. *P<0.05, **P<0.01 vs Vehicle, ^# P<0.05 vs Model; n=4 biologically independent samples each. F, 60 mmol/L KCl‐induced changes in MRA contraction in hypertensive, vehicle, and ethyl 2‐hydroxyisobutyrate‐treated mice. Data are shown as mean±SEM; *P<0.05, **P<0.01 vs Vehicle, ^# P<0.05 vs Model; n=4 biologically independent samples each. G, The vessels were placed in a Ca²⁺‐free Krebs buffer (CaCl₂ replaced with 0.1 mmol/L EDTA). Verapamil (1 μmol/L) was added to block the voltage‐dependent Ca²⁺channels in VSMCs. phenylephrine (1 μmol/L) was used to trigger SOCE. A cumulative concentration of CaCl₂ (0.5–10 mmol/L) was added to evoke vessel contraction. Data are shown as mean±SEM; ***P<0.001 vs Vehicle, ^## P<0.01 vs Model; n=4 biologically independent samples each. 2‐Hyd indicates ethyl 2‐hydroxyisobutyrate; DBP, diastolic blood pressure; IP:TPM3 indicates immunoprecipitation:tropomyosin 3; KDa, kilodaltons; Khib, lysine 2‐hydroxyisobutyrylation; MAP, mean arterial pressure; SBP, systolic blood pressure; SOCE, store‐operated calcium entry; and TPM3, tropomyosin 3.

TPM3 Plays a Key Role in the Signal Transduction Regulating Abnormal Vasoconstriction

To comprehend the functional impact of TPM3 on VSMC contraction and relaxation, we performed vascular tension measurements with or without siRNA interference. Notably, Tpm3‐specific siRNA substantially reduced TPM3 protein expression in the mouse thoracic aorta. Thus, siRNA‐3 exhibiting the most significant TPM3 protein knockdown was chosen for subsequent TPM3 knockdown experiments (Figure S3). To appreciate the role of TPM3 in vascular contraction and relaxation, tension measurements of mouse thoracic aortic rings were carried out. Here, compared with pretreatment with scrambled siRNA, Tpm3 siRNA‐3 pretreatment significantly diminished phenylephrine (Figure 3A), 60 mmol/L KCl (Figure 3B) and CaCl₂‐induced contraction of the mouse thoracic aorta (Figure 3C). Further, such pretreatment also caused a notable reduction in vasodilation induced by the HDAC inhibitor SAHA (Figure 3D) and ethyl 2‐hydroxyisobutyrate (Figure 3E). It is worth noting that TPM3, as a component of actin filaments, is instrumental in modulating actin filament polymerization and depolymerization. Hence, reduced TPM3 expression could facilitate actin filament depolymerization in VSMCs, thus lessening their sensitivity to constrictors. Such reduced TPM3 expression could affect VSMCs' response to vasodilators by potentially altering the activity of relevant signaling pathways or other mechanisms, thereby decreasing their sensitivity to the said vasodilators. In summary, our findings suggest that diminished TPM3 expression inhibits agonist‐induced vasoconstriction and vasodilator‐induced vasodilation, leading to the conclusion that TPM3 plays a significant role in VSMC contraction and relaxation signal transduction and stands out as an important target influencing abnormal vasoconstriction.

Figure 3. TPM3 plays a key role in the signal transduction regulating abnormal vasoconstriction.

A, After the thoracic aorta of mice was treated with disordered siRNA or TPM3 siRNA‐3, the vasoconstriction changes induced by the concentration gradient of phenylephrine administration. Data are shown as the mean±SEM; n=4. *P<0.05 vs scrambled siRNA analyzed by 2‐way ANOVAfollowed by Games‐Howell post hoc tests. B, After the thoracic aorta of mice was treated with scrambled siRNA or TPM3 siRNA‐3, the vascular contraction changes induced by 60 mmol/L KCl administration. Data are shown as mean±SEM; n=4. **P<0.01 vs scrambled siRNA analyzed by 2‐way ANOVAfollowed by Games‐Howell post hoc tests. C, After the thoracic aorta of mice was treated with scrambled siRNA or TPM3 siRNA‐3, the vascular contraction changes induced by CaCl₂ administration. The vessels were placed in a Ca²⁺‐free Krebs buffer (CaCl₂ replaced with 0.1 mmol/L EDTA). Verapamil (1 μmol/L) was added to block the voltage‐dependent Ca²⁺channels in VSMCs. phenylephrine (1 μmol/L) was used to trigger SOCE. A cumulative concentration of CaCl₂ (0.5–10 mmol/L) was added to evoke vessel contraction. Data are shown as the mean±SEM; n=4. **P<0.01 vs scrambled siRNA analyzed by 2‐way ANOVAfollowed by Games‐Howell post hoc tests. D, After the thoracic aorta of mice was treated with scrambled siRNA or TPM3 siRNA‐3, the vascular relaxation changes induced by the concentration gradient of pan‐HDAC inhibitor SAHA administration. Data are shown as mean±SEM; n=4. *P<0.05, **P<0.01 vs scrambled siRNA analyzed by 2‐way ANOVA followed by Games‐Howell post hoc tests. E, After the thoracic aorta of mice was treated with scrambled siRNA or TPM3 siRNA‐3, the vascular relaxation changes induced by the concentration gradient of ethyl 2‐hydroxyisobutyrate administration. Data are shown as mean±SEM; n=4. **P<0.01, ***P<0.001 vs scrambled siRNA analyzed by 2‐way ANOVAfollowed by Games‐Howell post hoc tests. HDAC indicates histone deacetylase; SAHA, suberoylanilide hydroxamic acid; SOCE, store‐operated calcium entry; TPM3, tropomyosin 3; and VSMC, vascular smooth muscle cell.

HDAC3 Stands Out as a Crucial Enzyme Regulating the Khib Modification of TPM3

Prevailing studies affirm the presence of histone dedihydroxyisobutyrylase activity in HDAC2 and HDAC3. Consequently, we postulated that phenylephrine possibly stimulates either HDAC2 or HDAC3 to interact with TPM3, facilitating TPM3's de‐2‐hydroxyisobutyrylation. This event might prompt the displacement of TPM3 and exposure of actin's myosin binding sites, leading to vasoconstriction through the amalgamation of actin and myosin filaments. To delve into the effects of histone deacetylases, HDAC2 and HDAC3, on the Khib modification of TPM3 and its subsequent influence on vascular contraction and relaxation dynamics, a rigorous computational strategy was undertaken employing an array of sophisticated bioinformatics tools. Using the capabilities of HDOCK and PRISM for molecular docking and kinetic simulations, we aimed to elucidate the complex interplay between these proteins and the PTMs of TPM3. Our computational analyses initiated with HDOCK, laying the groundwork for forecasting the binding affinities and interaction schematics between TPM3 and the HDAC isoforms. The docking scores derived from HDOCK advocate for a potent binding affinity of HDAC3 toward TPM3, with a mean docking score of −182.276±8.796, underscoring a pronounced interaction potential. This is emphasized by their negative docking scores, which signify a propitious binding interaction. The confidence scores further substantiate the robustness of these findings, averaging at 0.6549±0.0396 for HDAC3 & TPM3, indicating results of commendable quality, where higher scores denote superior outcomes. Conversely, the association between HDAC2 and TPM3 manifested a mean docking score of −174.092±3.284 and a confidence score of 0.6181±0.0155. These metrics accentuate the exceptional docking efficacy of HDAC3 with TPM3, suggesting a heightened probability of complex formation. The statistical analysis reinforces this conclusion, with P values of 0.0175 for docking scores and 0.0180 for confidence scores, demonstrating significant differences between the interactions of TPM3 with HDAC3 versus HDAC2. These P values, being below the conventional significance threshold of 0.05, validate the preferential affinity of TPM3 for forming complexes with HDAC3, thus highlighting its potential for a more specific and physiologically impactful interaction (Figure 4A).

Figure 4. HDAC3 stands out as a crucial enzyme regulating the Khib modification of TPM3.

A, Heatmap representation of the TPM3 full‐length protein, displaying the frequency of interaction across various amino acid residues with HDAC3. Each horizontal line represents a single amino acid residue along the TPM3 sequence, with colors indicating the level of interaction frequency from low (green) to high (red), suggesting regions of potential binding affinity or functional significance. B, Histogram of docking counts between HDAC3 and TPM3 full length proteins, illustrating the distribution of interaction frequencies across the amino acid sequence of TPM3. The x axis represents the amino acid positions within TPM3, and the y axis shows the count of docking interactions. Peaks in the histogram indicate amino acid residues with higher interaction counts, which are hypothesized to play a more critical role in the binding to HDAC3, thereby potentially influencing the Khib process. C, Immunoprecipitation of mouse thoracic aortic vessel homogenates using anti‐TPM3 or anti‐HDAC3 protein A/G beads following phenylephrine (1 μmol/L) treatment for 30 minutes. Immunoblotting was conducted using antibodies against HDAC3, TPM3, and β‐tubulin for detection. Representative immunoblots for each group were obtained, with n=3 independent biological samples for each. D, Colocalization of HDAC3 and TPM3 in VSMCs. Red fluorescence represents HDAC3; green fluorescence represents TPM3; blue fluorescence refers nucleus. Merged image indicates HDAC3 and TPM3 co‐localization. E, Isolated mouse thoracic aortas depleted of endothelium were treated with phenylephrine for 30 min, and the protein expression of HDAC3 in the cytoplasm and nucleus was measured by Western blot analysis. Left: Representative Western blot images showing the level of HDAC3. Right: The quantification of nucleus/cytoplasm HDAC3. Data are shown as mean±SEM; n=3. *P<0.05 vs phenylephrine analyzed by 2‐tailed Mann–Whitney test. F, Isolated mouse aortic rings precontracted with phenylephrine (1 μmol/L) and treated with cumulative doses of CUDC‐907. Relaxation response was measured in both endothelium‐intact and endothelium‐removed rings and the resulting data are expressed as mean±SEM. n=4 independent biological samples each. G, Summary data showing the effect of control or CUDC‐907 on 60 mmol/L KCl‐evoked vasoconstriction of mouse thoracic aortas. Data are shown as mean±SEM; n=4. Data analyzed by 2‐way ANOVA followed by the Games‐Howell post hoc test or by the 2‐tailed Mann–Whitney test. H, Summary data showing the effect of control or CUDC‐907 on SOCE‐induced vasoconstriction of mouse thoracic aortas. 2.5 mmol/L CaCl₂ alone was used to induce vasoconstriction. Data are shown as mean±SEM; n=4. **P<0.01 vs. phenylephrine analyzed by 2‐way ANOVA followed by the Games‐Howell post hoc test or by the 2‐tailed Mann–Whitney test. HDAC3 indicates histone deacetylase 3; IP:TPM3, immunoprecipitation:tropomyosin 3; Khib, lysine 2‐hydroxyisobutyrylation; ns, not significant; SOCE, store‐operated calcium entry;TPM3, tropomyosin 3; and VSMC, vascular smooth muscle cell.

Further validation and refinement of these findings were sought through PRISM, which specializes in protein–protein interaction predictions. PRISM's simulations corroborated the HDOCK results, revealing that HDAC3 consistently demonstrated a higher binding free energy when complexed with TPM3, with the lowest free energy values for HDAC3 being notably superior to those for HDAC2 (−29.12 versus −28.54) (Figure S4). This superior interaction likelihood, as indicated by the more negative free energy values, suggests a more stable complex formation compared with that with HDAC2 (Figure 4B). The converging evidence from both HDOCK and PRISM points to a distinct preference of TPM3 to engage with HDAC3 over HDAC2. This preference is indicative of a more specific and possibly more physiologically relevant interaction that could significantly influence the PTMs of TPM3. The kinetic simulation results further support this, demonstrating that the lysine residues involved in the interaction with HDAC3 are likely to be key sites of Khib, underscoring the potential physiological implications of this selective interaction in the context of vascular contraction and relaxation dynamics.

Complementarily, using coimmunoprecipitation technology, we evidenced a strengthened binding of HDAC3 and TPM3 in thoracic aortic vessels of mice after phenylephrine treatment (Figure 4C). Using immunofluorescence techniques, we investigated the colocalization of TPM3 and HDAC3 in VSMCs. Interestingly, we observed that HDAC3 was translocated from the nucleus to the cytoplasm in response to phenylephrine stimulation, leading to an enhanced colocalization with TPM3 compared with the control group (Figure 4D). To further elucidate this phenomenon, we isolated nuclear and cytoplasmic proteins from mouse thoracic aortic vascular tissues devoid of endothelium. Our findings confirmed that phenylephrine stimulation significantly reduced the expression of HDAC3 in the nucleus and increased its expression in the cytoplasm, providing evidence for the nuclear export of HDAC3 in VSMCs (Figure 4E). An assessment of the vasodilation function impacts of HDAC3 inhibitors was also carried out. The administration of multiple selective HDAC3 inhibitors (4‐bromo‐N′‐butylbenzohydrazide, CUDC‐907, quisinostat, N′‐butyl‐2′,3′‐difluoro‐[1,1′‐biphenyl]‐4‐carbohydrazide, CUDC101), each with an IC₅₀ of <10 nM for HDAC3, consistently resulted in a significant reduction in phenylephrine‐induced vasoconstriction in isolated mouse vessels. Among these, the HDAC3 inhibitor CUDC907 showcased the most potent vasodilating effect. Notably, the attenuation of vasoconstriction was more pronounced than that observed with the pan‐HDAC inhibitor SAHA. The consistent vasodilatory effect observed across structurally distinct HDAC3 inhibitors strongly indicates that the reduction in vascular tone is predominantly mediated through selective HDAC3 inhibition, rather than off‐target actions (Figure S5). Following this, an evaluation of whether the vasodilatory effect of CUDC907 is endothelium dependent was conducted where it was found not to inhibit the vasodilatory response significantly even after endothelial removal (Figure 4F). To elucidate the mechanism of CUDC‐907‐induced vasodilation, we preincubated isolated thoracic aortic rings with 10 μmol/L CUDC‐907. This treatment did not alter vasoconstriction induced by 60 mmol/L KCl (Figure 4G) but significantly attenuated contraction in response to 2.5 mmol/L CaCl₂ (Figure 4H), suggesting that CUDC‐907 may act via modulation of SOCE to induce vasodilation. These data establish HDAC3 as a key enzyme regulating TPM3 Khib and confirms that HDAC3 inhibitors can induce endothelium‐independent vasodilation by affecting SOCE.

HDAC3 Regulates the Khib Status of TPM3 at the 141st Lysine Residue, Which Is Specifically Involved in Abnormal Vasoconstriction

To elucidate the interaction between HDAC3 and TPM3, particularly focusing on lysine residues undergoing Khib modification, we used a comprehensive approach combining kinetic simulations and docking models. The amino acid residues of TPM3 showing significant interaction with HDAC3 were primarily located within the region spanning from 102 to 170 amino acids. Through kinetic simulation, we determined that the lysine residues participating in the interaction, ranked by descending probability, were K141, K153, and KK162 (Figure 5A and 5B). This finding was further supported by docking simulations, which highlighted the high frequency and proximity of interactions between these lysine residues and HDAC3. The lysine residues recommended for mutagenesis studies, based on their interaction with HDAC3 within the specified region, are at positions 129, 137, 141, 150, 153, 162, and 169. Among these, residues K141, K153, and K162 exhibited the highest interaction heats, indicating their critical roles in the HDAC3‐TPM3 interface.

Figure 5. HDAC3 regulates the Khib status of TPM3 at the 141st lysine residue, which is specifically involved in abnormal vasoconstriction.

A, Hydrogen bonding network of K141 with adjacent HDAC3 residues, showcasing distances in angstroms (Å) to illustrate the proximity and strength of interactions. B, Hydrogen bond specificity of K162 with HDAC3, detailing the significant bond distance with residue N38 and its potential influence on complex stability. C, Bar chart representation of interaction frequency for selected lysine residues, with K141, K153, and K162 highlighted for their high interaction counts, suggesting their functional importance in HDAC3 binding affinity. D, After transfection of plasmids carrying mutations at the Tpm3 K141R, L153R, and L162R sites in the thoracic aorta blood vessels of mice, the expression of Khib of TPM3 in the blood vessels was examined. Immunoprecipitation was performed on homogenates of mouse thoracic aortic vessels using anti‐Flag‐tag protein A/G beads. Immunoblotting was conducted using anti‐Flag‐tag and anti‐2‐hydroxyisobutyllysine antibodies for detection. Data were presented as mean±SEM. *P<0.05, **P<0.01 vs negative control. Representative immunoblots for each group were obtained, with n=3 independent biological samples for each. E, Plasmids transfection of the thoracic aorta of mice with mutations at Tpm3 K141R, followed by removal of the vascular endothelium and administration of a concentration gradient of phenylephrine, led to alterations in vascular contraction. Data are shown as mean±SEM; n=4. *P<0.05 vs NC, analyzed using a 2‐way ANOVAfollowed by Games‐Howell post hoc tests. F, After transfection of plasmids carrying mutations at the Tpm3 K141R site in the thoracic aorta blood vessels of mice, followed by removal of the vascular endothelium and administration of 60 mmol/L KCl, changes in vascular contraction were observed. Data are shown as mean±SEM; n=4. *P<0.05 vs NC, analyzed using a 2‐way ANOVA followed by Games‐Howell post hoc tests. G, Adenovirus transfection of the thoracic aorta of mice with mutations at Tpm3 K141R, followed by removal of the vascular endothelium and administration of a concentration gradient of phenylephrine, led to alterations in vascular contraction. Data are shown as the mean±SEM; n=4. *P<0.05 vs Adv‐NC, analyzed using a 2‐ANOVAfollowed by Games‐Howell post hoc tests. H, After adenovirus transfection of the thoracic aorta of mice with mutations at Tpm3 K141R, followed by removal of the vascular endothelium and administration of 60 mmol/L KCl, resulted in changes in vascular contraction. Data are shown as mean±SEM; n=4. **P<0.01 vs Adv‐NC, analyzed using a 2‐way ANOVAfollowed by Games‐Howell post hoc tests. I, Adenovirus transfection of the thoracic aorta of mice with mutations at Tpm3 K141R, followed by removal of the vascular endothelium and administration of a concentration gradient of SNP, led to alterations in vascular relaxation. Data are shown as mean±SEM; n=4. **P<0.01 vs Adv‐NC, analyzed using a 2‐way ANOVA followed by Games‐Howell post hoc tests. J, Adenovirus transfection of the thoracic aorta of mice with mutations at Tpm3 K141R, followed by removal of the vascular endothelium and administration of a concentration gradient of pan‐HDAC inhibitor SAHA, led to alterations in vascular relaxation. Data are shown as mean±SEM; n=4. *P<0.05 vs Adv‐NC, analyzed using a 2‐way ANOVAfollowed by Games‐Howell post hoc tests. K, Adenovirus transfection of the thoracic aorta of mice with mutations at Tpm3 K141R, followed by removal of the vascular endothelium and administration of a concentration gradient of HDAC3 specific inhibitor CUDC‐907, led to alterations in vascular relaxation. Data are shown as mean±SEM; n=4. *P<0.05 vs Adv‐NC, analyzed using a 2‐way ANOVAfollowed by Games‐Howell post hoc tests. 2‐Hyd indicates ethyl 2‐hydroxyisobutyrate; Adv‐NC, adenovirus negative control; Adv‐TPM3, adenovirus tropomyosin 3; HDAC3, histone deacetylase 3; Khib, lysine 2‐hydroxyisobutyrylation; and Mu‐TPM3, mutation of tropomyosin 3.

In an extensive structure–activity relationship study, 10 independent docking models were scrutinized, with the one exhibiting the lowest binding energy being subjected to comprehensive analysis. As depicted in Figure 5A, K141 of TPM3 is ensconced within a critical interaction domain of HDAC3, engaging extensively through a network of hydrogen bonds: K141(H) forms a bond with H17(O) at 2.6 Å, indicating a strong interaction, while also interacting with V96(O) and Y18(O) at distances of 4.2 Å and 3.3 Å respectively. Figure 5B highlights K162's involvement, where K162(H) engages N38(N) at a notably close 2.2 Å distance. These interactions, particularly the robust hydrogen bonds formed by K141 and K162, underscore their indispensable roles in the binding affinity and specificity to HDAC3 (Figure 5C). Given the high frequency and consistency of K141's interactions across multiple models, as well as its significant hydrogen bonding, K141 is posited as the key residue influencing the TPM3‐HDAC3 complex formation, thus making it the focal point for further mutagenic exploration. These interaction models, obtained through docking simulations using both HDOCK and PRISM tools, consistently identified the region between 102 to 170 amino acids as crucial for HDAC3‐TPM3 interaction. Despite the automatic alignment and docking performed by HDOCK with HDAC‐PC (PDB ID: 6Z6F), and the use of the full‐length alpha fold of HDAC3 in PRISM, the docking results were largely congruent, underscoring the reliability of these lysine residues as candidate mutagenesis sites for further exploring the interaction dynamics between HDAC3 and TPM3. This approach not only confirms the critical lysine residues involved but also provides a foundational basis for targeted mutagenesis to dissect the functional implications of their interactions in the context of Khib modification.

We engineered a plasmid introducing mutations at these specific sites of TPM3. Mouse thoracic aorta blood vessels were preemptively treated with these mutated plasmids, before their exposure to phenylephrine and ethyl 2‐hydroxyisobutyrate. Subsequently, we quantified the degree of Khib modification of TPM3 in the blood vessels. Our results revealed that neither phenylephrine treatment on vessels harboring mutated K141 site nor treatment with ethyl 2‐hydroxyisobutyrate instigated a significant elevation in the Khib modification of TPM3 (Figure S6; Figure 5D). These data accorded with our preliminary predictions. Moreover, the kinetic simulation had indicated the highest interaction likelihood for the K141 site (Figure 5C), leading us to select blood vessels harboring mutations at this particular site for tension measurement. The vessels carrying the K141 mutation displayed a markedly amplified vasoconstrictive response upon phenylephrine (Figure 5E) and 60 mmol/L KCl (Figure 5F) stimulation. Additionally, we transduced endothelium‐depleted mouse thoracic aortic vessels with an adenovirus bearing the K141 site mutation in TPM3 and verified the transfection efficiency (Figure S7). Post successful transfection, verified via fluorescence observation and Western blotting, we evaluated the vascular tension in the transduced vessels. A significant intensification in the vasoconstrictor response triggered by phenylephrine (Figure 5G) and 60 mmol/L KCl (Figure 5H) was recorded in vessels carrying the TPM3 K141 site mutation. This aligned with the outcomes following mutated plasmid transfection. However, vessels harboring the TPM3 K141R mutation exhibited a marked reduction in vasodilatory responses to sodium nitroprusside (an endothelium‐independent vasodilator) (Figure 5I), the pan‐HDAC inhibitor SAHA (Figure 5J), and the HDAC3‐specific inhibitor CUDC‐907 (Figure 5K). The broad reduction in response across these different vasodilators indicates that the mutation at Lys141 disrupts a critical regulatory pathway, implicating this site as a key target for Khib. More important, the specific decrease in the vasodilatory effect of CUDC‐907, compared with wild‐type vessels, suggests that HDAC3‐mediated regulation is dependent on the Khib of Lys141 in TPM3. The attenuated response to both SAHA and CUDC‐907 further underscores the significance of this specific PTM in maintaining vascular tone. Together, these findings highlight the specificity of the Lys141 modification and its essential role in HDAC3‐dependent regulation of vascular function.

DISCUSSION

Growing evidence suggests that PTMs play a crucial role in maintaining vascular homeostasis. Dysregulation of PTMs can contribute to the onset and progression of various cardiovascular diseases. ³⁴ Lysine acylation, a reversible PTM, influences enzyme activity, DNA binding capacity, and protein stability by altering the charge of lysine residues and modifying protein structure. Additionally, lysine acylation regulates the activity of numerous nonhistone proteins, thereby impacting cell survival, gene transcription, signal transduction, and mammalian cell metabolism. ³⁵ Recent studies have revealed significant associations between lysine acylation levels and cardiovascular diseases or related risk factors. For instance, studies have shown that obesity is linked to elevated levels of histone lysine propionylation, butylation, and crotonylation. ³⁶ , ³⁷ , ³⁸ Ischemic heart disease is associated with lysine crotonylation and 2‐hydroxyisobutylation. ³⁹ , ⁴⁰ Additionally, diabetes is related to increased levels of amino acid propionylation, ⁴¹ and myocardial ischemia–reperfusion injury is associated with high levels of lysine succinylation. ⁴² Furthermore, both succinylation and malonylation have been implicated in the pathogenesis of diabetic cardiomyopathy. ⁴³ , ⁴⁴ Key deacetylases such as SIRT1, SIRT2, and SIRT3 (which possess de‐propionylase, de‐butyrylase, and de‐crotonylase activities), SIRT5 (with de‐malonylase and de‐succinylase functions), and HDAC3 (with de‐crotonylase and de‐2‐hydroxyisobutyrylation activities) have emerged as promising therapeutic targets for the intervention of these cardiovascular risk factors. Despite this progress, research on the role of novel lysine acylation modifications, particularly in the context of abnormal vascular contraction and VSMC function, remains scarce. This study specifically focused on Khib, a modification closely linked to cellular metabolic regulation, and investigated its role in vascular smooth muscle function.

HDOCK is a hybrid docking web server that blends template‐based modeling with free docking to predict protein–protein and protein–DNA/RNA interactions, leveraging PDB data to enhance docking accuracy. It stands out for its rapid processing, capable of completing a docking run within 10 to 20 minutes. ⁴⁵ PRISM, on the other hand, predicts protein–protein interactions by matching structural similarities and conserving sequences at protein interfaces, using a database of nonredundant interface structures from the PDB. ⁴⁶ By employing HDOCK and PRISM, the study discovered that TPM3 Lys141 is a key target for HDAC3, which specifically regulates its Khib status to control vascular tone. This study proposed a novel strategy for reversing abnormal contraction of blood vessels.

Extensive research has unequivocally established the indispensability of TPM3, an actin‐binding protein expressed in VSMCs, in regulating the myosin‐driven contraction process involved in vasoconstriction and dilation. Multiple studies have reported that PTMs, such as acetylation and phosphorylation, of TPM3 play a crucial role in modulating the affinity between TPM3 and actin. Consequently, these modifications regulate sarcomeric function, as well as the overall contraction and relaxation functions. Furthermore, recent investigations in zebrafish embryos have revealed the existence of additional lysine acylation sites in TPM3. Remarkably, the sequences of TPM3 in zebrafish and humans exhibit a high degree of homology, including similarity in lysine acylation sites. ⁴⁷ This suggests that, apart from acetylation and phosphorylation, other forms of acylation may also be involved in regulating myofilament contraction and protein synthesis in zebrafish. In our study, we explored the effects of ethyl 2‐hydroxyisobutyrate on phenylephrine‐induced abnormal vasoconstriction. Our findings demonstrate a significant relaxation effect of ethyl 2‐hydroxyisobutyrate on the aorta's vasoconstriction. Additionally, co‐immunoprecipitation experiments provided evidence of Khib modification on TPM3. Furthermore, the expression of TPM3 was found to be influenced by phenylephrine, suggesting that TPM3 may be modified and regulated by 2‐hydroxyisobutyrylated proteins to modulate vascular pathophysiological processes. We also investigated the interaction between HDAC3, an eraser of Khib, and TPM3. Recent studies employing immunoaffinity purification, quantitative mass spectrometry analysis, bioinformatics, and other screening methods have identified >200 nonhistone proteins that potentially interact with the HDAC family, including TPM3. ⁴⁸ In our study, we successfully demonstrated for the first time the interaction between HDAC3 and TPM3 in vascular smooth muscle tissue using coimmunoprecipitation assays. Furthermore, we observed that this interaction is modulated by phenylephrine, which induces the nuclear export of HDAC3, thereby enhancing its binding to TPM3. HDAC3 inhibitors can induce endothelium‐independent vasodilation by affecting SOCE. These findings shed light on the intricate regulatory mechanisms involving TPM3, its modifications, and the interplay with HDAC3 in vascular smooth muscle tissue.

The present study offers novel insights into the role of HDAC3 in regulating vascular function and blood pressure through the Khib modification of TPM3. Our findings demonstrate that phenylephrine stimulation leads to a reduction in the Khib modification of TPM3 in blood vessels, suggesting that phenylephrine, a vasoconstrictor, modulates vascular tone by influencing the Khib status of TPM3. This highlights the importance of TPM3 in mediating vasoconstriction. Furthermore, our study reveals that the administration of Khib donors induces endothelium‐independent vasodilation and lowers blood pressure in hypertensive mice, indicating that enhancing the Khib modification of TPM3 could serve as a therapeutic strategy for hypertension. These results suggest that this specific PTM directly impacts vasodilation. We further identified HDAC3 as a key enzyme involved in regulating the Khib modification of TPM3. Although HDAC3 is traditionally recognized for its role in gene expression regulation through histone deacetylation, its involvement in the Khib of TPM3 unveils a novel epigenetic mechanism governing vascular function. This study also highlights the pivotal role of TPM3 in signaling pathways that regulate VSMC contraction and relaxation, emphasizing its significance in maintaining vascular homeostasis. The Khib modification of TPM3 appears to be a central regulatory mechanism in this process. Our experiments showed that HDAC3 selectively regulates the Khib at Lys141 of TPM3, a modification implicated in vasoconstriction and relaxation. To assess the specificity of the Khib modification at Lys141 of TPM3, we employed site‐directed mutagenesis to substitute Lys141 with a nonmodifiable arginine (K141R). Functional studies using both plasmid and adenoviral constructs demonstrated that Khib at Lys141 is crucial for regulating vasoconstriction. Vessels expressing the K141R mutant exhibited significantly impaired vasodilation in response to a Khib donor, highlighting the functional importance of this specific modification. Notably, mutagenesis of other lysine residues within TPM3 did not produce similar effects, as confirmed through functional assays, underscoring the unique and essential role of Lys141 in this regulatory pathway. Further investigation revealed that this modification is dynamically regulated by HDAC3. Inhibition of HDAC3 resulted in a marked reduction in Khib at Lys141, confirming the site‐specific regulation of this modification by HDAC3. These findings collectively provide compelling evidence that Khib at Lys141 is a precisely controlled modification with significant implications for vascular function. While it is acknowledged that HDAC inhibitors possess a broad range of effects, our study addressed potential off‐target concerns through a multi‐faceted approach. First, co‐immunoprecipitation experiments were conducted to directly assess the interaction between HDAC3 and TPM3, confirming that HDAC3 specifically targets the Khib of Lys141 in TPM3. This interaction was further substantiated using bioinformatics tools such as HDOCK and PRISM, which demonstrated that HDAC3 preferentially binds to TPM3, thereby reinforcing the specificity of this modification. Moreover, the HDAC3 inhibitors used in this study were carefully selected for their high specificity, exhibiting minimal off‐target activity against other HDAC isoforms or unrelated molecular targets. This selective inhibition significantly reduces the likelihood of confounding effects in our experimental outcomes. The consistent reduction in phenylephrine‐induced vasoconstriction observed across several HDAC3 inhibitors, each with distinct chemical structures, further strengthens the conclusion that the vascular response is specifically due to HDAC3 inhibition. Additionally, the observation that selective HDAC3 inhibitors produced a more pronounced attenuation of vasoconstriction compared with the pan‐HDAC inhibitor SAHA underscores the critical role of HDAC3 in modulating vascular tone. Taken together, these findings provide robust evidence that the observed reduction in phenylephrine‐induced vasoconstriction is not the result of nonspecific actions, but rather a targeted effect of HDAC3 inhibition. This supports the therapeutic potential of selectively targeting HDAC3 to manage hypertensive vascular dysfunction. Importantly, the results highlight that Khib at Lys141 of TPM3 is a precisely controlled process, central to the regulation of vascular function (Figure 6).

Figure 6. Schematic diagram of the mechanism by which HDAC3‐induced Khib of TPM3 regulates abnormal vasoconstriction.

In VSMCs, HDAC3 is aberrantly activated by phenylephrine, leading to its translocation out of the nucleus and binding to TPM3. This binding results in the de‐2‐hydroxyisobutyrylation of TPM3, which, in turn, regulates SOCE, culminating in vascular contraction. Conversely, HDAC3‐specific inhibitors can block the de‐2‐hydroxyisobutyrylation of TPM3 by HDAC3. This inhibition affects SOCE, leading to vasodilation. Additionally, the administration of Khib donors, such as ethyl 2‐hydroxyisobutyrate, can induce endothelium‐independent vasodilation and alleviate hypertensive vascular dysfunction in mice. HDAC3 indicates histone deacetylase 3; Khib, lysine 2‐hydroxyisobutyrylation; SOCE, store‐operated calcium entry; TPM3, tropomyosin 3; and VSMC, vascular smooth muscle cell. This figure was drawn by Figdraw.

Although our study provides novel insights into the role of HDAC3 in regulating 2‐hydroxyisobutyrylation at Lys141 of TPM3 and its impact on vascular function, several limitations should be considered. First, although the use of isolated mouse aortas provides valuable mechanistic insights, the translation of these findings to clinical settings requires validation in larger animal models and, eventually, human studies. The complexity of vascular regulation in vivo involves multiple interacting pathways, and it remains uncertain whether targeting the Khib of TPM3 alone is sufficient to achieve sustained therapeutic benefits in hypertension. Additionally, although the study employed highly selective HDAC3 inhibitors, the potential for off‐target effects or compensatory mechanisms from other HDAC isoforms cannot be completely excluded, particularly under long‐term treatment conditions. Moreover, although our site‐directed mutagenesis experiments suggest that the Khib at Lys141 of TPM3 is critical for its function, the broader implications of this modification on other cellular processes and its potential interactions with other PTMs remain unexplored. Future studies could benefit from integrating multi‐omics approaches, including proteomics and metabolomics, to comprehensively map the downstream effects of Khib at this site and identify additional regulatory nodes that could be targeted in combination with HDAC3 inhibition. Furthermore, the long‐term safety and efficacy of HDAC3 inhibitors in the context of vascular function require careful evaluation, particularly given the pleiotropic roles of HDAC3 in gene expression, cellular metabolism, and chromatin remodeling. Investigating strategies to achieve localized delivery or developing tissue‐specific inhibitors may help mitigate potential systemic side effects. Exploring the therapeutic window and optimal dosing regimens in preclinical models will also be essential for guiding future clinical development. This study lays a foundation for the epigenetic regulation of vascular tone and highlights the therapeutic potential of targeting the Khib modification of TPM3 in hypertensive treatments. Further research should aim to bridge the gap between preclinical findings and clinical applications while addressing the challenges of specificity, safety, and long‐term efficacy.

CONCLUSIONS

In conclusion, our findings highlight the critical role of HDAC3‐mediated Khib modification of TPM3 in regulating abnormal vasoconstriction. The potential therapeutic implications of this mechanism are underscored by the use of HDAC3 inhibitors and ethyl 2‐hydroxyisobutyrate as potential treatments for abnormal vasoconstriction. Unraveling the underlying mechanisms of this modification holds promise for the development of novel therapeutic strategies targeting hypertension and other cardiovascular diseases. Further investigations are warranted to explore the clinical applications of targeting TPM3 Khib modification in the management of vascular disorders.

Sources of Funding

This work was supported by grants from the National Natural Science Foundation of China (81960662, 82200550, 82460105), Yunnan Fundamental Research (202301AT070270, 202201AT070183), the Open Research Foundation of Yunnan Key Laboratory of Bioactive Peptides in Yunnan Province (HXDT‐2022‐1), Yunnan Key Research and Development Program (202303AC100026, 202403AC100033), the Program Innovative Research Team in Science and Technology in Kunming Medical University (CXTD202202), Yunnan Provincial Science and Technology Department (202401AY070001‐303), Yunnan Revitalization Talent Support Program, Research Project on Undergraduate Educational and Teaching Reforms in Yunnan Province (JG2023001), First‐Class Discipline Team of Kunming Medical University (2024XKTDPY12), Biological Medicine Special Project of Yunnan to J‐.X.L. (202402AA310015).

Disclosures

None.

Supporting information

Figures S1–S7