LncRNA Sirt1-AS Protects Against Cardiac Hypertrophy by Modulating Sirt1

Abstract

Background

LncRNAs are pivotal regulators in cardiovascular diseases. Sirt1-AS, a lncRNA, has been shown to play a role in cardiovascular diseases. This study aimed to explore the role of Sirt1-AS in cardiac hypertrophy and the underlying molecular mechanism.

Methods

A mouse model of pressure-overload cardiac hypertrophy was established via transverse aortic constriction (TAC). Cardiac tissues of TAC mice and cardiomyocytes treated with angiotensin II (AngII) were examined for Sirt1-AS and Sirt1 expression levels. The effects of Sirt1-AS overexpression or knockdown on cardiomyocyte size and hypertrophic marker expression were accessed. Furthermore, the molecular interaction between Sirt1-AS and Sirt1 were investigated.

Results

Sirt1-AS expression was found to be downregulated in the hearts of TAC mice and in Ang II-treated cardiomyocytes. Overexpression of Sirt1-AS attenuated cardiac hypertrophy, while its suppression exacerbated the hypertrophic response. Mechanistic studies demonstrated that Sirt1-AS directly influenced Sirt1 mRNA and protein expression levels. Moreover, the protective effects of Sirt1-AS against cardiac hypertrophy were abolished upon Sirt1 mRNA inhibition.

Conclusion

Our findings suggest that Sirt1-AS exerts a protective effect against cardiac hypertrophy by modulating Sirt1 expression. This research provides novel insights into the role of lncRNAs in cardiac hypertrophy and highlights Sirt1-AS as a potential therapeutic target for the treatment of cardiac hypertrophy.

Introduction

Myocardial hypertrophy is a globally prevalent health issue associated with elevated morbidity and mortality, ultimately progressing to heart failure (HF). ¹ As a major public health burden, myocardial hypertrophy significantly contributes to cardiovascular disease progression. While current clinical therapies aim to alleviate symptoms and mitigate risk factors, they frequently fail to halt the transition from hypertrophy to heart failure. ² Therefore, elucidating the molecular mechanisms governing myocardial hypertrophy is imperative for developing novel therapeutic interventions and improving clinical outcomes.

Long noncoding RNAs (lncRNAs), defined as non-protein-coding RNA molecules exceeding 200 nucleotides in length, ³ have emerged as critical regulators of cardiac hypertrophy. Notable examples including CHRF, H19, and Plscr4 have been demonstrated to modulate hypertrophy progression.^4–8 Among lncRNA subtypes, natural antisense transcripts (NATs) exhibit unique regulatory potential through sequence-specific interactions with their cognate mRNAs, mediating mRNA degradation, translation inhibition, or alternative splicing.^9–14 For example, targeted interference with BDNF-AS upregulates BDNF mRNA by 2 to 7-fold, alters the chromatin configuration at the BDNF locus, increases protein levels, and induces neuronal growth and differentiation in vitro and in vivo. ¹⁵

Focusing on Sirt1-AS, the antisense transcript of the sirtuin family member Sirt1. ¹⁶ Sirt1-AS has been implicated in multiple pathological processes through its regulatory effects on Sirt1 mRNA stabilization.^17–19 Experimental evidence indicates that Sirt1-AS promotes hepatocellular carcinoma (HCC) cell proliferation by enhancing Sirt1 mRNA stability. ²⁰ Cardiac applications further underscore its clinical relevance: transgenic overexpression of Sirt1-AS enhances cardiomyocyte proliferative capacity and survival rates, significantly improving post-myocardial infarction (MI) cardiac function through mRNA stabilization mechanisms. ²¹ These findings collectively position Sirt1-AS as a promising therapeutic target for cardiac disease. Despite these advances, the functional role of Sirt1-AS in cardiac hypertrophy remains unexplored. Previous studies have shown that cardiac tissue exhibits relatively high Sirt1-AS expression levels with biological functions tightly coupled to Sirt1, ²² and Sirt1 itself demonstrates cardioprotective properties through inhibition of pathological remodeling processes. ²³ In light of these gaps in knowledge, we hypothesized that Sirt1-AS may regulate cardiac hypertrophy by influencing its sense mRNA Sirt1.

This study aimed to investigate the correlation between the expression levels of lncRNA Sirt1-AS and the process of cardiac hypertrophy. Furthermore, we endeavor to explore the regulatory mechanisms of lncRNA Sirt1-AS in the progression of cardiac hypertrophy. Ultimately, our study provided a promising endogenous therapeutic target for cardiac hypertrophy.

Materials and Methods

Ethical Statement

All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Guizhou University (Approval No. EAE-GZU-2021-T115).

Ventricular Cardiomyocyte Isolation

In this study, C57BL/6J mice (Beijing Huafukang Biotechnology Co., Ltd) were used as experimental subjects. Neonatal mice (postnatal day 1) were euthanized using 2% isoflurane inhalation followed by cervical dislocation. Cardiac ventricles were dissected from atria, minced into tissue fragments, and subjected to sequential enzymatic digestion. Initial digestion was performed with 0.25% trypsin solution (Gibco, USA) at 4 °C for 16 h, followed by two subsequent digestions using collagenase II (Gibco) and 0.1% BSA (Solarbio, China) in PBS at 37 °C for 15 min under constant agitation. During room temperature digestion, tissue fragments were incubated in 15-min intervals with intermittent collection of supernatant containing liberated cells, which were immediately neutralized with fetal bovine serum (Bioind, Israel). After centrifugation (300 × g, 5 min), cell pellets were resuspended in DMEM/F12 medium (Gibco) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cell suspensions were plated on 100-mm culture dishes and incubated for 2 h at 37°C in a 5% CO₂ humidified chamber to facilitate differential adhesion. The supernatant enriched with cardiomyocytes was centrifuged and resuspended in complete DMEM/F12 medium. Cells were quantified using a hemocytometer and plated at appropriate densities. For angiotensin II (Ang II, Sigma-Aldrich) treatment, cells were exposed to serum-free DMEM/F12 medium containing 1 μM Ang II for 24 h.

Cell Transfection

All siRNAs and adenovirus vectors were obtained from GenePharma (Shanghai, China). Gene interference was performed using siRNA with the following sequence: 5′-CAAAGAUACAAUAAAUGGC-3′. Isolated mouse cardiomyocytes were cultured until reaching 70% confluence and maintained for 48 h pre-transfection. For adenoviral transfection, adenovirus-Sirt1 antisense lncRNA (Ad-SAS) or the adenoviral negative control (Ad-NC) was added to the cells. When transfecting with siRNA, the procedure was executed according to the guidelines provided by Lipofectamine RNA iMAX (Invitrogen). Approximately 5 µL of Lipofectamine and 50 nmol/L of siRNAs were combined in Opti-MEM medium (Gibco). Following a 6-h incubation with the transfection complex at 37 °C, the medium was replaced with fresh complete culture medium. Cells were harvested 48 h post-transfection for downstream analyses (RNA/protein extraction or immunofluorescence).

Transverse Aortic Constriction (TAC) and Injection of AAV9

TAC surgery was performed to establish a pressure-overload cardiac hypertrophy model. 8-week-old mice were randomly assigned to the TAC group or sham-operated group. After anesthesia induction with 2% isoflurane, mice were intubated with a small animal ventilator. A midline thoracotomy was then performed to expose the aortic arch. In the TAC group, the aortic arch was ligated using 6-0 silk thread, while the sham group underwent a similar surgical procedure without any occlusion of the aorta. AAV9-cTnT-SAS and AAV9-cTnT-empty viral particles were commercially obtained from Shanghai GenePharma Co., Ltd. Postoperatively, AAV9-cTnT-SAS or AAV9-cTnT-empty particles were intravenously injected through the tail vein immediately post-surgery. Postoperative analgesia was achieved by supplementing drinking water with Novalgin (200 mg/kg) for 72 h.

Tissue Collection

Mice were anesthetized with 2% isoflurane and euthanized via cervical dislocation. The cardiac tissues were carefully dissected, rinsed in 0.9% NaCl solution, weighed, and then fixed in 10% neutral buffered formalin at room temperature. Subsequently, the hearts were processed sequentially for RNA extraction, protein isolation, and paraffin embedding.

RNA Isolation and Real-Time Quantitative PCR

The total RNA was extracted from cardiomyocytes and heart tissue using the total RNA MiniPrep Kit (Axygen, CA, USA). Cytoplasmic and nuclear RNAs were isolated using the RNeasy Midi Kit (Qiagen, NRW, Germany). RNA purity was assessed through electrophoresis, while concentration and optical purity (A260/A280 ratio) were quantified using a microvolume spectrophotometer. Subsequently, cDNA was synthesized from 1 µg of total RNA by reverse transcription using the PrimeScript RT reagent kit (TaKaRa, Dalian, China). Quantitative real-time PCR (qPCR) was conducted using the TB Green Premix Ex Taq kit (TaKaRa) on the CFX96 Real-Time PCR Detection System (Bio-Rad, California, USA). The total reaction volume was 10 µL, and the thermal cycling protocol included an initial denaturation at 95 °C for 30 s, 40 cycles of denaturation (95 °C, 5sec) followed by combined annealing/extension (60 °C, 30sec) with fluorescence acquisition. After PCR amplification, a melting curve analysis was conducted (ranging from 65 °C to 95 °C with increments of 0.5 °C) to verify the specificity of the amplified fragments. GAPDH was employed as a housekeeping gene to normalize gene expression levels using the _ΔΔCt method. All primers were synthesized by Sangon Biotech Co., Ltd (Shanghai, China). The primer sequences utilized in this study are as follows: Sirt1-AS: Forward 5′-AATCCAGTCATTAAACGGTCTACA-3′, Reverse 5′-TAGGACCATTACTGCCAGAGGA-3′; Sirt1, Forward 5′-TTGGCACCGATCCTCGAAC-3′, Reverse 5′-CCCAGCTCCAGTCAGAACTAT-3′; Atrial Natriuretic Peptide (ANP): Forward 5′- GCTTCGGGGGTAGGATTGAC-3′, Reverse 5′- CGTGACACACCACAAGGGC-3′; Brain Natriuretic Peptide (BNP), Forward 5′- CCTCACAAAAGAACACCCAAAA-3′, Reverse 5′- CAACTTCAGTGCGTTACAGCC-3′; β-Myosin Heavy Chain (β-MHC), Forward 5′- GGACCAGACCCCAGGCAAG-3′, Reverse 5′- CAAAATGGATTCGGATGAATTTC-3′; GAPDH, Forward 5′- TGCTGAGTATGTCGTGGAGTCT-3′, Reverse 5′- ATGCATTGCTGACAATCTTGAG-3′.

Immunofluorescence Analysis

For in vitro cultured cardiomyocytes, glass coverslips were pre-positioned in 24-well cell culture plates. Primary mouse cardiomyocytes were seeded at a density of 1 × 10⁵ cells per well. Following treatment, 500 μL of 4% paraformaldehyde was added to fix cells. The coverslips were then removed and mounted on a wax plate. After permeabilization with 0.1% Triton X-100 (Solarbio), the cells were blocked with PBST containing 5% goat serum (Solarbio) for 30 min. Subsequently, the cells were incubated with primary antibodies, including cardiac troponin T (cTnT) (mouse; Invitrogen) and Ki67 (rabbit; Invitrogen) for 2 h at room temperature. Post-primary incubation, samples were incubated with goat anti-mouse IgG/Alexa Fluor 488 and goat anti-rabbit IgG/Alexa Fluor 594 (Bioworld, Nanjing, China) for 1 h at room temperature under light-protected conditions. Finally, Hoechst 33342 was used to stain the cell nuclei.

For paraffin-embedded tissue sections, formalin-fixed tissue slides were placed in a container filled with EDTA antigen retrieval buffer (pH 8.0) (Servicebio, Wuhan, China) using microwave irradiation. After natural cooling, the slides were washed with PBS buffer and then 100 μL iF555-WGA (Servicebio) working solution completely covering the tissue, followed by 37°C incubation in darkness for 30 min. TUNEL staining was performed strictly following the manufacturer's protocol (Beyotime, Shanghai, China). After light-protected mounting, images were acquired and analyzed using a Nikon inverted fluorescence microscope.

Western Blotting

Isolated cardiomyocytes or samples of dissected mouse ventricular heart tissue were lysed in ice-cold PIPA buffer (Solarbio), supplemented with protease and phosphatase inhibitors. Protein concentrations were quantified using the BCA Protein Quantitative Analysis kit (Solarbio). Subsequently, protein samples were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, MA, USA). The membranes were blocked by incubating at room temperature for 2 h in 5% non-fat milk. Following this, the membranes were incubated overnight at 4 °C with primary antibodies against the following targets: Sirt1 (Cell Signaling Technology, MA, USA), FoxO1 (Affinity, Jiangsu, China), acetyl-FoxO1 (Affinity, Jiangsu, China), and β-actin (Bioworld, Nanjing, China). The membranes were subsequently washed three times with Tris-buffered saline with Tween 20 (TBST) and incubated with HRP-conjugated secondary antibody (Bioworld) for 1 h at room temperature. Freshly prepared ECL luminescent reagent (Servicebio, Wuhan, China) was utilized for the exposure on Tanon 5500 Chemiluminescence imager (Tanon, Shanghai, China). Relative protein densities were quantified using ImageJ software (National Institutes of Health, MD, USA), normalizing the intensity of each protein to that of β-actin.

RNA Fluorescence in Situ Hybridization (FISH)

RNA FISH was performed according to the manufacturer's protocol of FISH Kit (GenePharma, Shanghai, China). All buffers except PBS were supplied with the kit. Isolated cardiomyocytes cultured on coverslips were fixed with 4% paraformaldehyde and rinsed three times with PBS. Subsequently, cells were permeabilized with 0.2% Triton X-100 in PBS and hybridized with a pre-labeled Sirt1 antisense lncRNA probe in hybridization solution at 37 °C overnight. Post-hybridization, cells were washed sequentially with Buffer F and Buffer C, followed by incubation with a mouse anti-digoxin antibody conjugated to alkaline phosphatase (Sigma-Aldrich). Nuclei were counterstained with DAPI, and imaging was conducted using an LSM 880 confocal microscope (Carl Zeiss, Germany).

RNA Stability Assay

Primary neonatal mouse cardiomyocytes cultured in 6-well plates were transfected with either an adenoviral vector for overexpression or an empty vector. About 24 h post-transfection, the cells from the first well were trypsinized, pelleted by centrifugation (200 × g, 5 min), and immediately lysed in 1 mL TRIzol reagent (Invitrogen) for baseline RNA analysis. The remaining five wells received 2 μg/mL Actinomycin D (Sigma-Aldrich) to inhibit RNA synthesis. Cells from these treatment groups were sequentially harvested at 2 h intervals (2, 4, 6, 8, and 10 h) following initial sample collection. Total RNA was isolated using chloroform-isopropanol precipitation and quantified by spectrophotometry. Sirt1 mRNA expression dynamics were analyzed through qPCR with β-actin as endogenous control. The data were analyzed to generate time-dependent decay curves of Sirt1 mRNA half-life.

Statistical Analysis

Data analysis and statistical graphs were conducted using Graphpad Prism 8.0 software (GraphPad Software, CA, USA). All data are presented as mean ± SEM in the graphs. A two-tailed t-test was applied for comparisons between two groups, and one-way ANOVA with LSD post hoc test was used for comparisons among multiple groups. Statistical significance for each treatment was represented by a P value, with P < .05 considered statistically significant.

Results

Overexpression of lncRNA Sirt1-AS Attenuates Myocardial Hypertrophy in Cardiomyocytes

Primary cardiomyocytes were transfected with adenoviral vectors overexpressing lncRNA Sirt1-AS, which significantly elevated the expression of Sirt1-AS (Figure 1A). The experimental design comprised four groups: control (Con), Angiotensin II -induced hypertrophy group (Ang II), Ang II-induced hypertrophy with negative control vector (AngII + Ad-NC), and AngII-induced hypertrophy with Sirt1-AS overexpressing (AngII + Ad-SAS). Quantitative PCR analysis revealed that ANP, BNP, and β-MHC mRNA levels were markedly upregulated in the Ang II group compared to controls. Notably, Sirt1-AS overexpression attenuated Ang II-induced elevation of these hypertrophy markers (Figure 1B). Immunofluorescence results showed that AngII treatment increased in cell area, while overexpression of lncRNA Sirt1-AS led to a reduction in cell size and average area (Figure 1C). Given the established correlation between pathological hypertrophy and apoptosis, our results confirmed the increase in apoptotic cells with Ang II stimulation. Importantly, Sirt1-AS overexpression reduced the apoptosis level under identical treatment conditions (Figure 1D). These collective findings indicate that lncRNA Sirt1-AS exerts protective effects against AngII-mediated cardiomyocyte hypertrophy and associated apoptosis.

Figure 1.

Expression of Sirt1-AS and its Effect on Cardiomyocyte Hypertrophy. (A) Real-Time Quantitative PCR Results of lncRNA Sirt1-AS Level in Cardiomyocytes Transfected with Either the Sirt1-AS Adenovirus Overexpression Vector (Ad-SAS) or an Empty Vector (Ad-NC) (n = 3). (B) Real-Time Quantitative PCR Results Showing the Expression Levels of ANP, BNP, and β-MHC RNA in AngII-Treated Cardiomyocytes Transfected with Ad-SAS or Ad-NC (n = 3 Mice Per Group). (C) Immunofluorescence Staining for cTnT and Hoechst 33342, and Quantification of the Cardiomyocytes Area in AngII-Treated Cardiomyocytes Transfected with Ad-SAS or Ad-NC (n = 3 Mice Per Group). Scale Bar, 40 μm. (D) Detection of Apoptosis in AngII-Treated Cardiomyocytes Transfected with Ad-SAS or Ad-NC Using TUNEL Staining, and Quantitative Analysis of TUNEL-Positive CMs (n = 3 Mice Per Group). Scale Bar, 20 μm. *P < .05.

Interfere lncRNA Sirt1-AS Aggravates Myocardial Hypertrophy in Cardiomyocytes

To investigate the functional role of lncRNA Sirt1-AS, siRNA was employed to downregulate its expression (Figure 2A). In the AngII-induced cardiomyocyte hypertrophy model, transfection with lncRNA Sirt1-AS-targeting siRNA (si-SAS) or control siRNA (si-NC) revealed distinct effects. Compared to AngII treatment alone, si-SAS further elevated myocardial hypertrophy marker levels (Figure 2B). Morphologically, si-SAS-treated cardiomyocytes exhibited a more pronounced increase in cell surface area (Figure 2C). Notably, si-SAS intervention also amplified apoptosis, as evidenced by a significant rise in apoptotic cell count (Figure 2D).

Figure 2.

Effect of Sirt1-AS Knockdown on Cardiomyocyte Hypertrophy. (A) Real-Time Quantitative PCR Results of Sirt1 AS lncRNA Level in CMs Transfected with Sirt1-AS siRNA (si-SAS) or Negative Control (si-NC) (n = 3). (B) Real-Time Quantitative PCR Showing the Expression Levels of ANP, BNP, and β-MHC RNA in AngII-Treated Cardiomyocytes Transfected with si-SAS or si-NC (n = 3). (C) Immunofluorescence Staining for cTnT and Hoechst 33342, and Quantification of the CM Area in AngII-Treated Cardiomyocytes Transfected with si-SAS or si-NC (n = 3). Scale Bar, 20 μm. (D) TUNEL Assay for Detecting Apoptosis in AngII-Treated CMs Transfected with si-SAS or si-NC, and Quantitative Analysis of TUNEL-Positive CMs. (n = 3). Scale Bar, 40 μm. *P < .05.

LncRNA Sirt1-AS Regulates Sirt1 mRNA and Protein Expression

LncRNA Sirt1-AS is the natural antisense RNA of Sirt1 and exhibits partial regional complementary with Sirt1 mRNA. To assess its regulatory role, we examined Sirt1 mRNA and protein levels under experimental conditions. In AngII-induced cardiomyocyte hypertrophy, Sirt1 mRNA expression was suppressed, whereas overexpression of Sirt1-AS rescued its expression (Figure 3A). At the protein level, Sirt1-AS overexpression reversed the AngII-induced reduction in Sirt1 protein (Figure 3B). Conversely, siRNA-mediated knockdown of Sirt1-AS (si-SAS) further suppressed both Sirt1 mRNA and protein expression (Figure 3C and D). Many studies have shown that forkhead box protein O1 (FoxO1) is involved in the development of cardiac hypertrophy.^24–26 Based on the deacetylation regulated by Sirt1, we assessed the levels of FoxO1 and acetylated-FoxO1 proteins. The results indicated that in the vitro cardiac hypertrophy model, downregulation of Sirt1-AS expression led to an increase in FoxO1 expression and a decrease in acetylated-FoxO1 protein levels (Figure 3E). Subcellular localization analysis by qPCR and FISH confirmed predominant cytoplasmic localization of Sirt1-AS (Figure 3F and G). Furthermore, actinomycin D assays demonstrated that Sirt1-AS overexpression stabilizes Sirt1 mRNA by slowing its decay rate, extending its half-life from 2 to 10 h (Figure 3H).

Figure 3.

The Effect of Sirt1-aS on Sirt1 mRNA and Protein Expression. (A) Real-Time Quantitative PCR Results of Sirt1 mRNA Levels in Cardiomyocytes Treated with AngII and Transfected with an Adenoviral Vector Overexpression of Sirt1-AS (AngII + Ad-SAS) or an Empty Vector (AngII + Ad-NC) (n = 3). (B) Western Blotting and Quantification of Sirt1 Protein Expression in Cardiomyocytes Treated with AngII, AngII + Ad-NC, or AngII + Ad-SAS (n = 3). (C) Real-Time Quantitative PCR Results Showing Sirt1 mRNA Level in Cardiomyocytes Treated with AngII, AngII + si-NC, or AngII + si-SAS (n = 3). (D) Sirt1 Protein Expression in Cardiomyocytes Treated with AngII, AngII + si-NC, or AngII + si-SAS (n = 3). (E) FoxO1 and Acetyl-foxO1 Protein Expression in Cardiomyocytes Treated with AngII, AngII + si-NC, or AngII + si-SAS (n = 3). (F) Relative Expression Levels of Sirt1-AS in Cytoplasmic and Nuclear RNA; Total RNA was Extracted From the Cytoplasm and Nucleus of Normal Cardiomyocytes, with Real-Time Quantitative PCR Showing that Sirt1-AS is Predominantly Localized in the Cytoplasm (n = 3). (G) FISH Experiments Demonstrating the Cellular Localization of lncRNA Sirt1-AS in Cardiomyocytes, with DAPI Staining the Nucleus Blue and FISH Probes Labeled with iF555 Fluorescent Appearing Red. Scale Bar, 50 μm. (H) Actinomycin D Experiments to Assess the Relative Expression Levels of Sirt1 mRNA at 0, 2, 4, 6, 8, and 10 h. *P < .05.

Sirt1-AS Regulates Myocardial Hypertrophy by Modulating Sirt1 mRNA

Studies have confirmed that Sirt1 plays a crucial regulatory role in myocardial hypertrophy pathogenesis. ²⁷ As the natural antisense RNA of Sirt1, Sirt1-AS likely participates in this regulation by modulating Sirt1 mRNA expression. To elucidate the mechanism, we investigated the effects of blocking Sirt1 mRNA. In an AngII-induced hypertrophy model, transfection with the Sirt1-AS overexpression vector (AngII + Ad-SAS) significantly elevated Sirt1 mRNA levels. However, concurrent siRNA-mediated Sirt1 silencing (AngII + Ad-SAS + si-Sirt1) completely abolished this upregulation (Figure 4A). Correspondingly, Sirt1-AS overexpression enhanced Sirt1 protein expression, whereas si-Sirt1 transfection suppressed it (Figure 4C). Further functional analyses revealed that blocking Sirt1 mRNA eliminated the capacity of Sirt1-AS overexpression to counteract myocardial hypertrophy, as evidenced by unaltered levels of hypertrophy markers, cardiomyocyte area, and apoptosis rates (Figure 4B, D and E). These results demonstrate that lncRNA Sirt1-AS regulates myocardial hypertrophy through Sirt1 mRNA-dependent mechanisms.

Figure 4.

Sirt1-AS Regulates Myocardial Hypertrophy by Modulating Sirt1 mRNA. (A) Real-Time Quantitative PCR Results of Sirt1 mRNA Level in Cardiomyocytes Treated with AngII, AngII + Ad-SAS, or AngII + Ad-SAS + si-Sirt1, with the Untreated Group Serving as a Control (n = 3). (B) Real-Time Quantitative PCR Results for the Expression of Hypertrophy Markers ANP, BNP, and β-MHC in Cardiomyocytes Transfected as Described in (A) (n = 3). (C) Western Blot Analysis of Sirt1 Protein Expression in Cardiomyocytes Treated with AngII, AngII + Ad-SAS, or AngII + Ad-SAS + si-Sirt1, with the Untreated Group as Control, and β-Actin as an Internal Reference (n = 3). (D) Fold Changes in Cardiomyocyte Area Upon Treatment with AngII, AngII + Ad-SAS, or AngII + Ad-SAS + si-Sirt1, Compared to the Control (n = 3 Mice Per Group). Scale Bar, 20 μm. (E) Quantification of TNUEL-Positive Cells in Cardiomyocytes Treated with AngII, AngII + Ad-SAS, or AngII + Ad-SAS + si-Sirt1 (n = 3 Mice Per Group), Scale Bar, 40 μm. *P < .05.

Sirt1-AS Alleviates Myocardial Hypertrophy In Vivo

TAC, a surgical procedure that modifies cardiac hemodynamics, serves as an established animal model for inducing myocardial hypertrophy. Our study demonstrated that AAV9-cTnT-mediated delivery effectively achieved cardiac-specific overexpression of Sirt1-AS (Figure 5A). This intervention significantly attenuated pathological indicators of myocardial hypertrophy (Figure 5B), reduced cardiomyocyte cross-sectional area post-TAC (Figure 5C), and suppressed TAC-induced apoptosis (Figure 5D). Molecular analyses revealed concomitant upregulation of both Sirt1 mRNA and protein expression levels in hypertrophic cardiac tissue following overexpression of Sirt1-AS (Figure 5E and F). In this in vivo model, we also assessed the levels of FoxO1 and acetylated-FoxO1 proteins. The results showed that with the overexpression of Sirt1-AS, FoxO1 expression increased, indicating an elevated level of deacetylation (Figure 5G). These in vivo findings are consistent with and extend our previous cellular experimental observations.

Figure 5.

In Vivo Model Experiment of Mouse. (A) Relative Expression Levels of lncRNA Sirt1-AS in Cardiac Tissue From the Following Groups: TAC Surgery Group, TAC Followed by Injection of the AAV9-Sirt1-AS Vector (TAC + AAV9-SAS), and Sham-Operated Group (Sham) (n = 5 Mice Per Group). (B) Real-Time Quantitative PCR Results for the Expression of Hypertrophy Markers ANP, BNP, and β-MHC in Mouse Heart Tissues From the Sham, TAC, TAC + AAV9-SAS, or TAC + AAV9-NC Groups (n = 5 Mice Per Group). (C) WGA Staining to Visualize the Membrane of Cardiomyocytes and Measure Individual Cell Areas in Paraffin Sections From Mice Treated with Sham, TAC, TAC + AAV9-SAS, or TAC + AAV9-NC (n = 5 Mice Per Group). Scale Bar, 20 μm. (D) TUNEL Assay to Detect Apoptosis in Cardiomyocytes in Paraffin Sections From the Same Groups as in (C) (n = 5 Mice Per Group). Scale Bar, 20 μm. (E-F) Changes in Sirt1 mRNA and Protein Expression Levels Following Sham Surgery, TAC, TAC + AAV9-SAS, or TAC + AAV9-NC (n = 5 Mice Per Group). *P < .05. (G) FoxO1 and Acetyl-FoxO1 Protein Expression in Tissues Treated with the Aforementioned Groups.

Discussion

Our study demonstrates that lncRNA Sirt1-AS is predominantly localized in the cytoplasm of cardiomyocytes, and its expression increases during the progression of cardiac hypertrophy. In hypertrophic models, overexpression of Sirt1-AS mitigates myocardial hypertrophy and reduces cellular apoptosis, while suppression of lncRNA Sirt1-AS expression exacerbates AngII-induced cardiomyocyte hypertrophy and accelerates apoptosis. Mechanistically, Sirt1-AS regulates the stability of Sirt1 mRNA through RNA-RNA interactions, thereby enhancing both the transcriptional and translational efficiency of Sirt1 protein. This cascade ultimately inhibits cardiomyocyte hypertrophy, establishing Sirt1-AS as a novel antisense RNA-mediated regulator in pathological cardiac remodeling.

Over the past decade, emerging research has elucidated novel molecular mechanisms that exert regulatory effects on the pathophysiological progression of cardiac hypertrophy, including epigenetic modifications, non-coding RNA networks, metabolic reprogramming, immune-inflammatory responses, translational control, and cellular proliferation dynamics. ²⁸ In the present study, we found that lncRNA Sirt1-AS exerts a protective effect against cardiac hypertrophy by alleviating hypertrophy and reducing cell apoptosis. In our previous investigation, we presented a study utilizing mouse model of myocardial infarction to examine the mechanisms underlying cardiac regeneration. This research demonstrates that Sirt1-AS stabilizes Sirt1 mRNA through specific binding domains, thereby enhancing the mitotic activity of cardiomyocytes and promoting cardiac repair processes. Integrated analysis of previous investigations and current experimental findings demonstrates that Sirt1-AS exhibits therapeutic potential in cardiovascular pathologies including myocardial infarction and heart failure.

Mechanistically, the regulatory role of Sirt1-AS is achieved by modulating the level of Sirt1 mRNA. Studies have demonstrated a regulatory relationship between sense and antisense transcripts. Natural antisense RNAs exert downstream regulatory effects by targeting interacting with their sense mRNA to form a double-stranded RNA.^11,12,29 Sirt1 is the most prominent and extensively studied member of the sirtuins, demonstrating multidimensional physiological regulatory functions and engaging in intricate molecular interaction networks. ²⁵ Its NAD ⁺ -dependent deacetylase activity plays a critical role in metabolic homeostasis. ³⁰ Notably, Sirt1 demonstrates cardioprotective potential by modulating inflammatory responses, oxidative stress, and mitochondrial function.^31,32 Within the reports on the signaling pathways, Sirt1 serves as a pivotal regulatory hub by integrating multiple signaling pathways, including NF-κB, AMPK/mTOR, p53, and FoxO networks.^24,33–35 Our study also found that the regulation of Sirt1 by Sirt1-AS is accompanied by changes in the expression of FoxO1 and acetylated FoxO1. Mechanistic evidence from this study reveals that Sirt1-AS mediates the inhibition of cardiac hypertrophy via Sirt1-dependent molecular pathways.

Our study has several limitations. It was primarily confined to the TAC-induced cardiac overload hypertrophy model, which restricts the generalizability of our findings. Future investigations should explore the protective effects of Sirt1-AS across a wider array of models, including those related to myocardial infarction and atherosclerosis. Moreover, employing tools such as spatial single-cell RNA sequencing (scRNA-seq) will be essential for a comprehensive assessment of the metabolic characteristics of hypertrophic cardiomyocytes. ³⁶

Conclusion

In summary, our study demonstrates that lncRNA Sirt1-AS stabilizes Sirt1 mRNA, thereby upregulating both Sirt1 mRNA and protein levels. This mechanism reduces cardiac hypertrophy severity and inhibits pathological progression. These findings suggest that Sirt1-AS may serve as a novel therapeutic target for cardiac hypertrophy treatment.