The functional role of the circular RNA circCDR1as targeting SERCA2a in the progression of pathological cardiac hypertrophy and heart failure

Abstract

Background

Pathological cardiac hypertrophy serves as a key pathological precursor to heart failure (HF) and represents a major public health challenge worldwide. Although its clinical importance is well recognized, the underlying mechanism remains incompletely understood. Growing evidence suggests that circular RNAs (circRNAs) play significant roles in the development of cardiac pathologies. Nevertheless, the specific biological functions and regulatory mechanisms of circRNAs in cardiac hypertrophy and HF are still largely unknown. This study aimed to explore the functional role of circCDR1as in cardiac hypertrophy and elucidate its underlying molecular mechanisms.

Methods

The role of circCDR1as in myocardial hypertrophy was examined using a mouse model of pressure overload-induced hypertrophy established by transverse aortic constriction (TAC). Mass spectrometry analysis following RNA pull-down was used to identify (sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 2A) SERCA2a as an interacting protein of circCDR1as. To clarify the molecular mechanism by which circCDR1as regulates SERCA2a, protein synthesis and degradation inhibitors were used to verify the cause of SERCA2a downregulation during pathological cardiac hypertrophy.

Results

circCDR1as was upregulated in the blood of patients with cardiac hypertrophy and could be an independent risk factor for cardiac hypertrophy. Experiments in mice suggested that knockdown of circCDR1as ameliorated cardiac dysfunction and attenuated myocardial hypertrophy after TAC surgery. Conversely, the overexpression of circCDR1as mediated by the 9 adeno-associated virus (AAV9) induced hypertrophic growth and led to HF. Moreover, circCDR1as promotes an increase in the surface area of neonatal mouse cardiomyocytes (NMCMs). Mechanistically, circCDR1as promotes cardiac hypertrophy and HF by binding to SERCA2a, enhancing its NEDD4L-mediated K48-linked ubiquitination and proteasomal degradation, thereby disrupting calcium homeostasis in cardiomyocytes.

Conclusions

These findings identify circCDR1as as a potential pathological biomarker of cardiac hypertrophy and highlight its potential as a therapeutic target for treating hypertrophic cardiomyopathy. The current clinical observations are still preliminary and require validation in larger cohorts and in human myocardial tissues.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12967-026-08670-5.

Background

Pathological myocardial hypertrophy is a major independent risk factor for HF and represents a significant challenge in global public health [1]. Although the ultimate goal of clinical treatment is to curb HF, its pathological process often begins with myocardial hypertrophy. Among them, myocardial hypertrophy induced by stress overload is particularly crucial. It is not only a core precursor event for the development of HF but also an independent risk predictor [2]. Despite the promising efficacy of some novel pharmacological therapies for improving ventricular remodeling, the global prevalence of HF continues to rise [3]. Thus, a thorough elucidation of the molecular mechanisms of cardiac hypertrophy is crucial for early intervention in HF and the precise identification of therapeutic targets, which will not only effectively suppress disease progression, but also lay a foundation for the development of potential therapeutic strategies.

Accumulating evidence indicates that non-coding RNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circRNAs, play important roles in the development of cardiac hypertrophy [4–7]. CircRNAs, a recently characterized class of non-coding RNAs, are generated through back-splicing of precursor mRNAs and are abundantly expressed in eukaryotic cells [8]. Approximately 9,000 candidate circRNAs have been identified in both human patients with HF and rodent models, including neonatal and adult rats, as well as sham-operated or TAC-subjected hypertrophic mice [9]. Notably, functional studies have demonstrated that circCDYL promotes cardiomyocyte hypertrophy in vitro, underscoring the critical roles of circRNAs in cardiovascular diseases [10]. Emerging research suggests that circRNAs can interact with transcription factors or other proteins, influencing their stability and functional activity, thereby contributing to the pathogenesis of cardiac hypertrophy [11]. circCDR1as is among the highly conserved circRNAs expressed in the mammalian heart. Additionally, our previous study revealed that circCDR1as exacerbates post-infarction arrhythmias by impairing the NAMPT-NAD⁺ axis and disrupting cardiomyocyte calcium handling, making it an ideal candidate molecule for studying Ca²⁺-dependent heart diseases [12, 13]. Building on the extensive research foundation and technical expertise accumulated by our group in this field, we are continuing to delve into the potential functions of circCDR1as in the progression of pathological cardiac hypertrophy and HF.

Calcium homeostasis is essential for cardiomyocyte function and regulates the excitation-contraction coupling that underlies normal cardiac function [14]. Dysregulation of calcium handling can lead to impaired myocardial contraction and relaxation, promoting the development of pathological conditions such as cardiac hypertrophy [15]. Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a), a pivotal regulator of calcium homeostasis, facilitates the reuptake of Ca²⁺ into the sarcoplasmic reticulum (SR) to maintain the Ca²⁺ stores and cardiac rhythmicity of the SR. Aberrant SERCA2a expression or activity triggers cytosolic calcium overload, which may induce arrhythmias and cardiac damage [16–18]. Multiple studies have indicated that ncRNAs, including circRNAs, are widely involved in SERCA2a expression [19–21].

In this study, we identified circCDR1as as a critical negative regulator of SERCA2a. Specifically, circCDR1as binds directly to SERCA2a and promotes its ubiquitination and subsequent proteasomal degradation, thereby impairing its calcium handling capacity. This disruption of calcium homeostasis consequently drives the progression of cardiac hypertrophy and HF. Notably, therapeutic intervention targeting circCDR1as markedly alleviated pathological hypertrophy and improved cardiac function in vivo. These findings suggest that the inhibition of circCDR1as may represent a potential and promising therapeutic approach for treating cardiac hypertrophy-related disorders.

Methods

Blood sample analysis

All procedures in this study were performed in strict accordance with the Declaration of Helsinki. The study was approved by the ethical review committee of the Second Affiliated Hospital of Harbin Medical University (approval No. ky2020-049-01), and written informed consent was obtained from all participants before blood collection. RNA was isolated from 400 µL of each serum samples using 600 µL of TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Subsequently, RNA samples were reverse-transcribed into cDNA using a reverse transcription kit (FSQ-101; Toyobo, Japan), and circCDR1as expression were quantified via qRT-PCR with SYBR Green (Roche, Basel, Switzerland). Demographic characteristics of the human subjects from whom the blood sample were used are summarized in Table S1.

Mice

Male C57BL/6 mice (20–25 g) and C57BL/6 suckling mice of 3 days old were purchased from the Experimental Animal Center of the Second Hospital of Harbin Medical University. All experimental animals were maintained under specific conditions: a temperature of 23 ± 1 °C, humidity ranging from 55% to 60%, and a 12-hour light/12-hour dark cycle environment. All experimental protocols involving animals were carefully reviewed and approved by both the Institutional Animal Care and Use Committee and the Ethics Committee of Harbin Medical University (approval No.sydwgzr2020-153-01). Additionally, these protocols strictly complied with the NIH Guidelines for the Care and Use of Experimental Animals. To minimize potential confounding factors, all animals were randomized into experimental groups using a computer-generated random number table to ensure comparable baseline characteristics across groups. Baseline cardiac function was assessed in all animals prior to any experimental intervention. Mice with baseline LVEF < 55%, FS < 25% were excluded from the study to eliminate any pre-experimental cardiac dysfunction. Echocardiography, histological analysis and statistical analysis were conducted under a blinded condition. Specifically, all sample sizes in this study were estimated via a priori power analysis using G*Power software (version 3.1.9), with the significance level set at α = 0.05 and statistical power of 0.8. The sample size calculations were based on our pre-experiment data, combined with well-documented changes in ventricular ejection fraction (EF) observed in previous transverse aortic constriction (TAC) animal studies [22, 23]. Effect sizes were chosen based on our pilot experimental data and the characteristic effect sizes reported in laboratory animal research [24–27]. For two-group comparisons, a two-tailed independent samples t-test was applied with a predefined Cohen’s d of 1.8, which determined that a minimum of six mice per group was required to detect the anticipated phenotypic differences. For comparisons among four groups, calculations were performed under a one-way ANOVA framework with an effect size f of 0.75, which also indicated a minimum of six mice per group. This experiment strictly adhered to the 3R principles (Replacement, Reduction, Refinement). The number of experimental animals was minimized while sufficient statistical power for all measured indicators was guaranteed. From the perspective of sample size and power analysis, the current number of animals used is sufficient to detect medium to large effect sizes of cardiac function indicators, but it may not be sufficient to capture the small effects among them.

In vivo gene delivery

In this experiment, the AAV9 vector with higher tropism to cardiomyocytes was used to deliver circCDR1as by intravenous injection. C57BL/6 mice were randomly assigned to four experimental groups to receive different serotype AAV9-mediated interventions. The overexpression virus AAV9-cTNT-CDR1as and control vectors AAV9-cTNT-GFP, purchased from Genechem (Shanghai, China), were injected into the mice via the tail vein. Similarly, the knockdown virus AAV9-cTNT-shCDR1as and control vectors AAV9-cTNT-GFP, sourced from HANBIO (Shanghai, China), were administered through the tail vein. Each mouse received AAV9 at a dose of 1 × 10¹¹ viral genomes (vg), and the infection was allowed to persist for at least 3 weeks. The sequence of shRNAs was listed in the Table S2.

Mouse model of TAC

TAC was performed following previously described methods [28]. Mice were anesthetized via intraperitoneal injection of tribromoethanol (200 mg/kg; Sigma; St. Louis, USA). The aortic arch was exposed by blunt dissection of the second intercostal space. A 6 − 0 polypropylene suture was used to ligate the aortic arch around a 27G needle. Subsequently, the muscle and skin were sutured layer by layer to close the thoracic cavity. Mice in the sham-operated group underwent a similar surgical procedure without aortic arch ligation. 8 weeks after the surgery, cardiac function was assessed by echocardiography in all groups, after which the mice were sacrificed and tissue samples were collected for future analysis.

Neonatal mouse primary cardiomyocyte culture

As previously stated, cardiomyocytes were isolated from C57BL/6 neonatal mice that were no more than 3 days old [28]. The excised hearts were placed in a mixture of D-Hank’s solution and 0.25% trypsin solution. The mixture was placed on a constant-temperature shaker at 4 °C for 12 h of overnight preliminary digestion. After the overnight digestion, type II collagenase solution (Gibco, Grand Island, USA) was added to the mixture, which was then placed on a shaker at 37 °C with a rotation speed of 180 rpm/min for further enzymatic digestion. The cell suspension was collected and centrifuged at 1000 r/min for 5 min. The precipitated cardiomyocytes were mixed with DMEM medium containing 10% fetal bovine serum (Sigma; St. Louis, USA) and placed in a 37 °C incubator for 50 min (a differential adhesion protocol) to separate cardiomyocytes from fibroblasts. Subsequently, the cardiomyocytes were cultured in a cell incubator with 5% CO₂ and 95% O₂ at 37 °C for 48 h for subsequent related experiments.

Cell infection with adenovirus

The adenovirus Adv - CDR1as and its negative control Adv - Null were purchased from Genechem (Shanghai, China). Subsequently, Adv with a final concentration of 1 × 10⁷ plaque forming units (PFU) mL^− 1 was added to NMCMs culture medium for 8 h, and then replaced with culture medium containing serum for 48 h for further experiments.

Cell transfection with siRNA and plasmid

siCDR1as and its negative control RNA (siNC) were synthesized by GENERL BIOL. (Anhui, China). siSERCA2a, siNEDD4L and the negative control RNA (siNC) were synthesized by genecreate.cn (Wuhan, China). The plasmid carrying SERCA2a cDNA (NM_001110140.3) with the CMV promoter and its negative control were constructed by Obio Technology (Shanghai, China). According to the manufacturer’s instructions, siRNAs with a final concentration of 50 µM or plasmid of 1 µg/mL were transfected into NMCMs with the X-tremeGENE transfection reagent (Roche, Basel, Switzerland). The next experiment could be carried out 6 h later. The sequence of siRNAs was listed in the Table S3.

Echocardiography

Mice were anesthetized with 2,2,2-tribromoethanol. Short-axis slice index and aortic flow velocity were obtained using the VisualSonics ultrasound imaging system (VisualSonics, Toronto, Canada) as described previously [29].

Angiotensin II (Ang II)-induced cardiomyocytes hypertrophy model

Cardiomyocyte hypertrophy model was constructed by stimulating NMCMs with Ang II (HY-13948, MCE, USA) following previously described methods [28]. Shortly, NMCMs were treated with Ang II (1 µM) for 48 h to finalize cardiac hypertrophy model.

Western blot

Total proteins from cardiac tissues and NMCMs were extracted using RIPA buffer (Beyotime; Shanghai, China). Equal amounts (50–70 µg) of proteins were separated by SDS-PAGE on 7.5%, 12.5% or 10% polyacrylamide gels, then transferred to nitrocellulose membranes (PALL, USA) via the wet-transfer method (300 mA constant current, 30–120 min; transfer time adjusted based on protein molecular weight). Membranes were blocked with 5% skim milk for 2 h at room temperature. After incubation with primary antibodies for atrial natriuretic peptide (ANP) (1:1000 dilution; 27426-1-AP, Proteintech; Wuhan, China), SERCA2a (1:1000, 27311-1-AP, Proteintech; Wuhan, China), β-myosin heavy chain (β-MHC) (1:5000 dilution; M-8421, Sigma; St. Louis, USA), β-actin (1:1000; AB0035; Abways; Shanghai, China), Ubiquitin (1:1000, 10201-2-AP, Proteintech; Wuhan, China), Flag (66008-4-Ig, Proteintech; Wuhan, China), Myc (16286-1-AP, Proteintech; Wuhan, China) and NEDD4L (1:1000, 13690-1-AP, Proteintech; Wuhan, China) overnight at 4 °C, membranes were probed with secondary antibodies and imaged using an Odyssey CLx infrared imaging system (LI-COR Bioscience, Lincoln, NE, USA).

Ubiquitination of SERCA2a

Prior to cell harvesting, the cells were treated with MG132 (10 µM) for 6 h. Cultured NMCMs or HEK293T cells (HaiXing Biosciences; JiangSu, China) were lysed on ice using a lysis buffer (P0013; Beyotime, Shanghai, China) containing 1% protease inhibitor for 30 min, followed by centrifugation at 12,000 g at 4 °C for 15 min. Ubiquitination assays were performed as previously described [30]. Briefly, the supernatant was diluted to a concentration of 2 mg/mL. Approximately 400 µg of protein per sample was used for immunoprecipitation. Protein A/G magnetic beads (HY-K0202, MCE, USA) for immunoprecipitation were conjugated with rabbit anti-SERCA2a antibody (BY2869, Abways; Shanghai, China; 1 µg per 400 µg protein) or anti-Flag antibody (66008-4-Ig, Proteintech; Wuhan, China) and incubated with cell lysates overnight at 4 °C with gentle rotation. The following day, beads were washed three times with cell lysis buffer containing PBST. Precipitates were resuspended in loading buffer, boiled at 100 °C for 8 min, and subjected to Western blot analysis to detect SERCA2a ubiquitination levels.

RNA isolation and qRT-PCR

Total RNA from cardiac tissues and NMCMs was isolated and purified using TRIzol reagent (Invitrogen; Carlsbad, USA). The concentration and purity of the extracted RNA samples were measured using NanoDrop-2000 spectrophotometer (Thermo Fisher Scientific; Waltham, USA). RNA samples were reverse-transcribed into cDNA using a reverse transcription kit (Cat #FSQ-101; Toyobo, Japan). The mRNA expression levels of ANP, brain natriuretic peptide (BNP), β-MHC and SERCA2a were accurately quantified by qRT-PCR with SYBR Green (Roche; Basel, Switzerland). Primers were purchased from GENERL BIOL (Anhui, China) as shown in Table S4.

Ca²⁺ transient measurement

Freshly isolated cardiomyocytes were incubated with 5 µM Fluo-4 (MX4504, Maokangbio; Shanghai, China) and 0.01% F127 (ST501, Beyotime; Shanghai, China) in calcium-containing solution for 20 min to measure cellular Ca²⁺ transients. Cardiomyocytes were electrically paced at 1 Hz using a stimulator and images were acquired with an Olympus camera. Amplitude of Ca²⁺ transients was calculated as [(F − F0)/F0]. Measurements were performed at room temperature on > 20 myocytes per group from three or more mice [31].

Recording of intracellular resting calcium

The method for cell acquisition and staining was the same as above, and a confocal microscope (Zeiss Microsystems; Jena, Germany) was used to collect images following previously described methods [32].

Measurement of cardiomyocyte shortening

Isolated adult murine cardiomyocytes were electrically paced at 1 Hz under a magnetic field until reaching a steady-state contraction pattern. Once stable contractions were observed, continuous images were recorded for 5 s using HCIMageLive version 4.6 software. Systolic and diastolic lengths were measured using Image J. Cardiomyocyte shortening, an indicator of myocardial contractility, was calculated as [(diastolic length-systolic length)/diastolic length] × 100%.

Histological analysis

Cardiac tissues were first fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin. Sections of 4 μm thickness were prepared using a paraffin microtome and incubated at 37 °C for subsequent assays. Sections were stained with H&E (G1120, Solarbio; Beijing, China), Masson’s trichrome (G1340, Solarbio; Beijing, China) and WGA (L4895, Sigma; St. Louis, USA) according to the manufacturer’s instructions.

Fluorescence in situ hybridization (FISH) assay

To observe the distribution and expression levels of circCDR1as, FISH assay was carried out using the RiboTM FISH Kit (RiboBio, Guangzhou, China) according to previously established protocols [12, 32]. FISH staining was performed on NMCMs, adult mouse cardiomyocytes and cardiac tissue sections. The cell nuclei were counterstained with DAPI (C0065, Solarbio; Beijing, China). Images were acquired using fluorescence microscopy and subsequently analyzed.

Isolation of adult mouse cardiomyocytes

Adult mouse cardiomyocytes were isolated following established methods [19, 31]. Briefly, after euthanasia, hearts were rapidly excised and the aorta was cannulated for perfusion with a calcium-free buffer using a constant-flow Langendorff apparatus. The components of the solution were (in mmol/L): NaCl, KCl, NaH₂PO₄, glucose, MgCl₂, HEPES, and NaHCO₃ at 126, 5.4, 0.33, 10, 1, 10, 3 (pH 7.35–7.45 adjusted with NaOH). Digestion was performed with Tyrode solution containing 1 mg/mL Type II collagenase powder (Gibco; Grand Island, USA) and 0.75 mg/mL bovine serum albumin (Solarbio; Beijing, China). Subsequently, isolated cardiomyocytes were preserved in Tyrode solution containing 200 µmol/L CaCl₂ and 1% bovine serum albumin (Beyotime; Shanghai, China).

Immunofluorescence Staining (IF)

To evaluate the expression and localization of target proteins, cultured NMCMs were incubated overnight at 4 °C with α-actinin antibody (GTx29465, 1:300; GeneTex; San Antonio, USA), NEDD4L antibody (1:300, 13690-1-AP, Proteintech; Wuhan, China) or SERCA2a antibody (1:500, 27311-1-AP; Proteintech; Wuhan, Chian). After washing, the cells were washed and then incubated with secondary antibodies conjugated to Alexa Fluor 488(ab 150077, 1:300, Abcam; Cambridge, UK) and Alexa Fluor 594 (ab 150080, 1:300; Abcam; Cambridge, UK) for 1 h at room temperature. The cell nuclei were stained with DAPI (Solarbio, Beijing, China) for 15 min at room temperature. Following PBS washes, samples were mounted and imaged using a laser scanning confocal microscope.

RIP

RIP (RNA Binding Protein Immunoprecipitation) was performed using the Magna RIP™ RNA Binding Protein Immunoprecipitation Kit (Millipore; Germany) in according with the instructions and previously described methods followed by qRT-PCR [32]. The gene-specific primer sequences are as follows in Table S4.

Determination of the Half-life and Stability of SERCA2a

NMCMs were treated with 10 µM cycloheximide (CHX) (HY-12320, MCE; USA). The cells were processed according to a time gradient of 0, 3, 6, and 9 h to evaluate the half-life period of SERCA2a. Additionally, NMCMs were treated with 10 µM proteasome inhibitor MG132 (HY-13259; MCE; USA) to assess the stability of SERCA2a.

RNA pull-down assay

Biotin-labeled probes (Bersinbio; Guangzhou, China) were used for the following procedures. For the RNA-protein precipitation experiment, the Pierce™ Magnetic RNA-Protein Precipitation Kit (20164, Thermo Scientific, USA) was applied strictly following the manufacturer’s instructions. Proteins specifically pulled down by circCDR1as were detected by Western blotting using an anti-SERCA2a antibody (1:1000, 27311-1-AP, Proteintech; Wuhan, China) serving as the detection reagent. The sequences of the circCDR1as probes are listed in Table S5.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9.0. Data are presented as mean ± standard error of the mean (SEM). Prior to parametric testing, data normality was assessed using the Shapiro-Wilk test (P > 0.05 indicating normal distribution) and homogeneity of variances was then evaluated using the Brown-Forsythe test or F test (P > 0.05 indicating equal variances). Then, for normally distributed data, Student’s t-test (unpaired 2-tailed) was used to compare two groups; one-way ANOVA followed by Tukey post hoc multiple comparison test was used to compare differences among multiple groups. For nonnormally distributed data, the Mann-Whitney U test (unpaired 2-tailed) was used for two groups, and the Kruskal-Wallis test was used for multiple groups. A P-value < 0.05 was considered statistically significant. The cellular electrophysiological recordings from ≥ 3 mice were analysed using the nested t-test.

Results

Upregulation of circCDR1as expression in cardiac hypertrophy

To investigate the potential role of circCDR1as in cardiac hypertrophy, we firstly measured its expression in serum samples from human hypertrophic patients and non-hypertrophic controls. circCDR1as levels were significantly higher in the hypertrophic group (Fig. 1a). To further validate these findings, a mouse model of pressure overload-induced cardiac hypertrophy was established via TAC. Consistent with the human data, circCDR1as expression was markedly upregulated in TAC-induced hypertrophic hearts, along with the classical hypertrophy markers including ANP, BNP, and β-MHC (Fig. S1b, Fig. 1b). Similarly, in Ang II (1 µM)-stimulated NMCMs, both circCDR1as and hypertrophy markers were significantly induced (Fig. S2, Fig. 1c). As shown in Fig. 1d-i, FISH analysis further confirmed elevated circCDR1as expression in hypertrophic myocardial tissues. Moreover, circCDR1as was predominantly localized in the cytoplasm.

Fig. 1.

Upregulation of circCDR1as expression in TAC-induced heart tissues and Ang II-induced NMCMs. a, Serum levels of circCDR1as in human hypertrophic patients and non-hypertrophic controls. ^**P < 0.01 by Mann-Whitney U test, n = 6–8.b, qRT-PCR analysis of circCDR1as, ANP, BNP and β-MHC levels in the Sham group and the TAC group. ^* P < 0.05, ^** P < 0.01 by 2-way ANOVA with Tukey post hoc test; n = 6. c, qRT-PCR analysis of circCDR1as, ANP, BNP and β-MHC levels in DMSO or Ang II-treated NMCMs for 48 h. ^* P < 0.05, ^**P < 0.01 by 2-way ANOVA with Tukey post hoc test; n = 5. d and e, Representative FISH images showing the increase of circCDR1as expression in TAC mice. Scale bar: 50 μm; ^** P < 0.01 by Student t test; n = 6. f and g, Representative FISH assay visualizing the expression levels of circCDR1as expression in NMCMs treated with Ang II. Scale bar: 20 μm; ^** P < 0.01 by Mann-Whitney U test; n = 6. h and i, Representative images showing the increase of circCDR1as expression in single cardiomyocytes from adult TAC mice. Scale bar: 50 μm; ^** P < 0.01 by Student t test; n = 10

circCDR1as deficiency attenuates cardiac hypertrophy

To examine the functional impact of circCDR1as on pressure overload-induced cardiac hypertrophy, we knocked down circCDR1as specifically in adult mouse cardiomyocytes via tail vein injection of AAV9-shCDR1as for 3 weeks prior to TAC surgery (Fig. 2a). At 8 weeks post-TAC, the blood flow velocity at the site of aortic constriction was significantly greater in the TAC-operated mice than in Sham controls (Fig. S1a).

Fig. 2.

circCDR1as deficiency prevents cardiac hypertrophy following cardiac pressure overload. a, Schematic diagram depicting the experimental strategy. 8-week-old male mice were injected with AAV9-shCDR1as and AAV9-shNC for 3 weeks and subsequently underwent Sham or TAC surgery. 8 weeks after TAC surgery, echocardiographic measurements were performed on the mice, followed by their euthanasia. b, qRT-PCR analysis of circCDR1as expression in the myocardium from mice with AAV9-shCDR1as and AAV9-shNC injection. ^*P < 0.05 by Mann-Whitney U test, n = 4–5. c and d, Representative images of echocardiography on the left ventricle and statistical data. Echocardiographic parameters: EF%, FS%, LVID; d and LVID; s. ^*P < 0.05, ^**P < 0.01; ^# P < 0.05, ^## P < 0.01 by 1-way ANOVA, followed by Tukey post hoc analysis; n = 6. e, Representative whole-heart images (Scale bar: 5 mm), HW/BW, HW/TL and evaluation of heart size via hematoxylin-eosin (HE) staining in heart sections (Scale bar: 2.5 mm). ^**P < 0.01; ^#P < 0.05, ^##P < 0.01; n = 6. Statistical significance was assessed by 1-way ANOVA, followed by Tukey post hoc analysis for HW/BW; Statistical significance was assessed by Kruskal-Wallis test with Dunn post hoc tests for HW/TL. f and g, WGA (wheat germ agglutinin) staining images and statistical results of the relative cardiomyocytes CSA. Scale bar: 50 μm; ^**P < 0.01; ^## P < 0.01 by 1-way ANOVA, followed by Tukey post hoc analysis; n = 6. h and i, Representative Masson’s trichrome-stained histological sections and statistical data of percent changes of fibrosis area. Scale bar: 50 μm; ^**P < 0.01; ^##P < 0.01; n = 6. Statistical significance was assessed by 2-way ANOVA analysis followed by Tukey post hoc multiple comparison test. j, Representative Western blotting results of ANP and β-MHC in heart tissues. ^**P < 0.01; ^## P < 0.01; n = 6. Statistical significance was assessed by 2-way ANOVA analysis followed by Tukey post hoc multiple comparison test. k, Real-time qPCR analysis of ANP, BNP and β-MHC in heart tissues. ^**P < 0.01; ^#P < 0.05, ^##P < 0.01; n = 6. Statistical significance was assessed by 2-way ANOVA analysis followed by Tukey post hoc multiple comparison test

circCDR1as knockdown significantly reduced its expression compared with that in AAV9-shNC-treated mice (Fig. 2b). Notably, compared with TAC+shNC mice, knockdown of circCDR1as markedly improved cardiac function (Fig. 2c, d). Morphometric analysis revealed that compared with AAV9-shNC-treated mice, TAC+shCDR1as mice presented significantly decreased heart weight-to-body weight (HW/BW) and heart weight-to-tibia length (HW/TL) ratios and reduced overall heart size (Fig. 2e). Histopathological evaluation showed knockdown of circCDR1as effectively prevented the increase of cardiomyocyte cross-sectional area (CSA) (Fig. 2f, g) and fibrosis (Fig. 2h, i) in TAC mice. Consistent with these findings, the protein and mRNA levels of β-MHC, ANP and BNP significantly reduced in TAC+shCDR1as mice relative to TAC+shNC mice (Fig. 2j, k). Taken together, these in vivo results demonstrate that circCDR1as knockdown attenuates TAC-induced myocardial hypertrophy and improves cardiac function, with robust and detectable effects observed in a sample size of n = 6 per group.

Overexpression of circCDR1as induces cardiac hypertrophy and adverse remodeling

Given the above findings, we further investigated whether circCDR1as overexpression alone could recapitulate features of pressure overload-induced cardiac remodeling. Using a gain-of-function approach, mice were injected with AAV9 encoding circCDR1as (AAV9-CDR1as) or AAV9-Null as a control (Fig. 3a). circCDR1as level was significantly elevated in cardiomyocytes after 8 weeks of AAV9-CDR1as treatment (Fig. 3b). Notably, even in the absence of pathological stimulation, circCDR1as overexpression induced mild myocardial injury at 4 weeks (Fig. S3) and resulted in pronounced ventricular wall thickening and cardiac dysfunction after 8 weeks (Fig. 3c, d). Furthermore, 8 weeks of circCDR1as overexpression led to increases in heart size, heart weight (Fig. 3e) and CSA (Fig. 3f). Histopathological analysis also revealed significant cardiac fibrosis in AAV9-CDR1as mice compared with that in AAV9-Null control mice (Fig. 3g, h). Consistent with these structural changes, the expression levels of cardiac hypertrophic markers, including ANP, BNP and β-MHC, were significantly upregulated following circCDR1as overexpression (Fig. 3i, j).

Fig. 3.

circCDR1as overexpression in hearts of mice causes cardiac hypertrophy and remodeling under physiological conditions. a, Schematic diagram depicting the experimental strategy. Male mice aged 8 weeks were injected with AAV9-CDR1as and AAV9-Null. After 8 weeks, an echocardiogram examination was performed on the mice, followed by their euthanasia. b, qRT-PCR analysis of circCDR1as expression in the myocardium from mice with AAV9-CDR1as and AAV9-Null injection for 8 weeks. ^**P < 0.01 by Mann-Whitney U test; n = 6. c and d, The transthoracic echocardiographic tracings from mice injected with AAV9-CDR1as and AAV9-Null and statistical data. ^**P < 0.01 by Student t test; n = 6. e, Representative whole-heart images (Scale bar: 5 mm), HW/BW, HW/TL and evaluation of heart size via HE staining in heart sections (Scale bar: 2.5 mm). ^*P < 0.05, ^**P < 0.01 by Student t test; n = 6. f, WGA staining images and statistical results of the relative cardiomyocytes CSA. Scale bar: 50 μm; ^**P < 0.01 by Student t test; n = 6. g and h, Representative images of Masson and statistical data of percent changes of fibrosis area (Scale bar: 50 μm). ^**P < 0.01 by 2-way ANOVA with Tukey post hoc test; n = 6. i, Representative Western blotting results of ANP and β-MHC in heart tissues. ^** P < 0.01 by 2-way ANOVA with Tukey post hoc test; n = 6. j, qRT-PCR analysis of ANP, BNP and β-MHC in heart tissues. ^**P < 0.01 by 2-way ANOVA with Tukey post hoc test; n = 6

circCDR1as promotes cardiomyocyte hypertrophy in vitro

To further investigate the role of circCDR1as in cardiomyocyte hypertrophy, we performed in vitro studies using NMCMs. Cells were transfected with siRNA targeting circCDR1as prior to stimulation with Ang II. Transfection with siCDR1as significantly reduced circCDR1as expression (Fig. S4a). Consistent with our hypothesis, circCDR1as knockdown attenuated Ang II-induced cardiomyocyte hypertrophy, as indicated by decreased mRNA and protein expression levels of the classic hypertrophy markers ANP, BNP, and β-MHC (Fig. S4b, c), along with a reduction in cardiomyocyte cell area (Fig. S4d).

Conversely, even without pathological stimulation, adenovirus-mediated overexpression of circCDR1as in NMCMs increased the mRNA and protein expression levels of ANP, BNP and β-MHC, along with cell enlargement (Fig. S4e-h), indicating that circCDR1as overexpression alone is sufficient to induce hypertrophic changes. Collectively, these in vitro results demonstrate that circCDR1as is not only necessary for Ang II-induced cardiomyocyte hypertrophy, but also independently drives this pathological process.

circCDR1as directly interacts with SERCA2a

To elucidate the molecular mechanisms by which circCDR1as affects myocardial hypertrophy, we performed RNA pull-down assays combined with mass spectrometry (RPD-MS) to identify circCDR1as-interacting proteins (Fig. 4a). Based on peptide enrichment scores, we listed the top 20 candidate interactors (Table S6). Notably, the biological process “cellular response to calcium ion” was highly enriched (Fig. 4b), suggesting a potential role of circCDR1as in calcium signaling. Among the candidate genes, the ATP2A2 gene encoding the SERCA2a protein (which is a key regulatory factor for calcium uptake in the sarcoplasmic reticulum) is considered a potential binding partner. We utilized RNAfold to predict the secondary structure of circCDR1as with the minimum free energy (MFE) (Fig. 4c). Additionally, we constructed a molecular docking model between circCDR1as and SERCA2a using the HADDOCK software, which indicated a stable physical interaction (Fig. 4d). In silico predictions from an RNA-protein interaction database further supported a high binding probability (Fig. 4e). The interaction was experimentally validated using independent RNA pull-down assays followed by Western blotting, which confirmed the binding of circCDR1as to SERCA2a (Fig. 4f). RNA immunoprecipitation (RIP) assays using an anti-SERCA2a antibody also revealed significant enrichment of circCDR1as (Fig. 4g). Additionally, FISH combined with IF revealed colocalization of endogenous circCDR1as and SERCA2a in the cytoplasm (Fig. 4h), providing further evidence for their direct interaction. Collectively, these findings demonstrate that circCDR1as physically associates with SERCA2a in NMCMs.

Fig. 4.

circCDR1as directly interacts with SERCA2a. a, Flowchart of identifying circCDR1as binding proteins by RNA pull-down and mass spectrometry. b, Bubble plot of GO pathway analysis for circCDR1as-binding proteins. c, The secondary structure (minimum free energy, MFE) of circCDR1as predicted by RNA fold WebServer. d, The interaction between circCDR1as and SERCA2a was predicted using HADDOCK and visualized with Discovery Studio. e, Interaction probabilities between SERCA2a and circCDR1as predicted by RPISeq (> 0.5 were considered “positive”). f, Western blotting analysis performed on independent RNA pull-down assays confirmed that SERCA2a protein exhibits a specific binding interaction with circCDR1as in cardiomyocytes. g, RIP assay verifying the binding between SERCA2a and circCDR1as. ^**P < 0.01; n = 3. h, FISH and IF assays showing the colocalization of SERCA2a and circCDR1as. Scale bar: 20 μm

circCDR1as disrupted Ca²⁺ homeostasis in cardiomyocytes

SERCA2a is essential for maintaining calcium homeostasis in cardiomyocytes [19, 20, 33]. Therefore, we investigated whether circCDR1as affects calcium handling. Adult cardiomyocytes were loaded with Fluo-4 AM for 20 min. Confocal laser scanning microscopy was employed to meticulously assess the impact of circCDR1as on the intracellular Ca²⁺ concentration within cardiomyocytes. Intracellular Ca²⁺ transients were subsequently triggered via electrical stimulation at a frequency of 1 Hz. We comprehensively evaluated the alterations in calcium handling processes and the degree of cell shortening. Our data verified that the intracellular Ca²⁺ concentration in cardiomyocytes was elevated in cardiomyocytes of the TAC group. Knockdown of circCDR1as reversed this increase, whereas overexpression of circCDR1as promoted an increase in the level of intracellular Ca²⁺ concentration (Fig. 5a, b). Calcium transient analysis revealed that the amplitude of Ca²⁺ transients in the TAC group was significantly reduced and this deficit was partially reversed by circCDR1as knockdown. More strikingly, knocking down circCDR1as significantly accelerated the slowed time course of the decay phase of Ca²⁺ transients, as evidenced by the reduction in the decay time constant (τd) (Fig. 5c-e). However, overexpression of circCDR1as promoted calcium transient disorder (Fig. 5f-h). These in vivo observations were further corroborated by in vitro experiments, strengthening the overall consistency of the results (Fig. 5i-n).

Fig. 5.

The effect of circCDR1as on the Ca²⁺ homeostasis of mouse cardiomyocytes. a, Representative images and analysis of intracellular calcium staining in mouse cardiomyocytes with circCDR1as knockdown using Fluo-4. Scale bar: 20 μm; ^**P < 0.01; ^##P < 0.01 by Kruskal-Wallis test with Dunn post hoc tests; n = 14. b, Representative images and quantitative analysis of intracellular calcium staining in circCDR1as-overexpressing mouse. Scale bars: 20 μm; ^**P < 0.01 by Mann-Whitney U test; n = 14. c through e, Effects of knockdown of circCDR1as on representative traces of calcium transient (Fig. 5c), the amplitude of calcium transient (Fig. 5d) and Tau value of the recovery phase of calcium transient (Fig. 5e) in isolated cardiomyocytes. ^**P < 0.01; ^##P < 0.01; n = 27 cells (from 3 mice per group); Statistical significance was assessed by nested 1-way test. f through h, Effects of circCDR1as overexpression on representative traces of calcium transients (Fig. 5f), the amplitude of calcium transients (Fig. 5g) and the Tau value of the recovery phase of calcium transients (Fig. 5h) in isolated cardiomyocytes from healthy adult mice. ^**P < 0.01; n = 27 cells (from 3 mice per group); Statistical significance was assessed by nested t test. i, Laser confocal microscopy was used to detect the changes of intracellular Ca²⁺ concentration in NMCMs after knockdown of circCDR1as. Scale bar: 10 μm; ^**P < 0.01; ^## P < 0.01 by Kruskal-Wallis test with Dunn post hoc tests; n = 6. j, The intracellular Ca²⁺ concentration in NMCMs following circCDR1as overexpression were monitored using a laser scanning confocal microscope. Scale bar: 10 μm; ^**P < 0.01 by Student t test; n = 6. k, Typical examples of calcium transient traces and calcium amplitude recorded in Fluo-4-loaded NMCMs with or without siCDR1as treatment exposed to Ang II. ^**P < 0.01; ^## P < 0.01 by 1-way ANOVA, followed by Tukey post hoc analysis; n = 11. l, Tau value of the recovery phase of calcium transient in NMCMs with or without siCDR1as treatment exposed to Ang II. ^**P < 0.01; ^## P < 0.01 by 1-way ANOVA, followed by Tukey post hoc analysis; n = 11. m, Typical examples of calcium transient traces and calcium amplitude recorded in Fluo-4-loaded NMCMs with Adv-CDR1as and Adv-Null. ^**P < 0.01 by Student t test; n = 11. n, Tau value of the recovery phase of calcium transient in NMCMs with Adv-CDR1as and Adv-Null. ^**P < 0.01 by Student t test; n = 11. o, Contractile function was assessed by measuring fractional shortening in single ventricular cardiomyocytes isolated from mice. Scale bar: 20 μm. p, The contraction fraction of isolated single ventricular myocytes after knocking down circCDR1as. ^**P < 0.01; ^## P < 0.01; n = 30 cells (from 3 mice per group). Statistical significance was assessed by nested 1-way test. q, Contractile fraction of isolated single ventricular myocytes after circ CDR1as overexpression. ^**P < 0.01; n = 27 cells (from 3 mice per group). Statistical significance was assessed by nested t test

Intracellular calcium cycling governs efficient myocardial contraction and relaxation. To further explore whether circCDR1as exerts a regulatory effect on cardiomyocyte contraction, we conducted additional investigations. Consistent with the intracellular Ca²⁺ concentration reduction in calcium treatment, the degree of cardiomyocyte shortening was significantly greater in circCDR1as - deficient cells (Fig. 5o-q). These findings indicate that circCDR1as disrupts calcium cycling and contractile function by destabilizing SERCA2a.

circCDR1as promotes SERCA2a protein degradation via ubiquitination

Given the observed interaction between circCDR1as and SERCA2a, we sought to determine whether circCDR1as regulates SERCA2a expression. Our experimental results demonstrated that circCDR1as significantly downregulated SERCA2a protein levels, while exerting no detectable effect on the transcriptional process of SERCA2a (Fig. 6a-h). Therefore, we hypothesized that circCDR1as may promote SERCA2a protein degradation. Consistent with these findings, circCDR1as reduced the immunofluorescence intensity of the SERCA2a protein in mouse cardiomyocytes (Fig. S5) and NMCMs (Fig. S6). To assess protein stability, cycloheximide (CHX) chase assays were performed. The overexpression of circCDR1as significantly accelerated SERCA2a degradation (Fig. 6i). Because protein degradation primarily occurs via the proteasomal and lysosomal pathways [34, 35], we utilized MG132 (a proteasome inhibitor) and chloroquine (CQ, a lysosome inhibitor) to identify the route of degradation. MG132, but not CQ, fully rescues SERCA2a protein levels in circCDR1as overexpressing cells (Fig. 6j-k), indicating proteasome-dependent degradation occurred. Considering that polyubiquitination plays a central role in protein degradation, we investigated whether circCDR1as modulates SERCA2a ubiquitination. Our results confirmed that overexpression of circCDR1as promotes the ubiquitination and subsequent degradation of SERCA2a (Fig. 6l).

Fig. 6.

circCDR1as promoted SERCA2a degradation via the ubiquitin-proteasome system. a, Western blotting and its quantification of SERCA2a in the heart tissues of TAC mice with circCDR1as knockdown. ^**P < 0.01; ^#P < 0.05 by 1-way ANOVA, followed by Tukey post hoc analysis; n = 6. b, The relative mRNA levels of SERCA2a in the heart tissues of TAC mice with circCDR1as knockdown was determined by qRT-PCR assay. ^** P < 0.01 by 1-way ANOVA, followed by Tukey post hoc analysis; n = 4. c, Western blotting and its quantification of SERCA2a in heart tissues treated with AAV9-CDR1as and AAV9-Null. ^** P < 0.01 by Mann-Whitney U test; n = 6 d, The relative mRNA levels of SERCA2a in the heart tissues of mice treated with AAV9-CDR1as and AAV9-Null by qRT-PCR assay. Statistical analysis was performed using Student t test; n = 6. e, Western blotting and its quantification of SERCA2a in cardiomyocytes transfected with siCDR1as. ^**P < 0.01; ^#P < 0.05 by Kruskal-Wallis test with Dunn post hoc test; n = 6. f, The relative levels of SERCA2a in cardiomyocytes transfected with siCDR1as was determined by qRT-PCR assay. ^**P < 0.01 by 1-way ANOVA, followed by Tukey post hoc analysis; n = 7. g, Western blotting analysis of SERCA2a protein levels in cardiomyocytes treated with Adv-CDR1as and Adv-Null. ^** P < 0.01 by Student t test; n = 6. h, qRT-PCR assay of SERCA2a mRNA levels in cardiomyocytes treated with Adv-CDR1as and Adv-Null. Statistical analysis was performed using Student t test; n = 9. i, NMCMs were infected with Adv-CDR1as and Adv-Null and then treated with Cycloheximide (CHX, 10µM) for the indicated time periods. Representative Western blotting analysis of SERCA2a protein levels and quantification. Statistical analysis was performed using 2-way ANOVA followed by Tukey post-hoc test; n = 4. j, Western blotting analysis of SERCA2a protein levels regulated by circCDR1as with or without MG132 (5 µM) treatment. ^*P < 0.05 by 1-way ANOVA, followed by Tukey post hoc analysis; n = 3. k, Western blotting analysis of SERCA2a protein levels regulated by circCDR1as with or without CQ (5 µM) treatment. ^**P < 0.01; ^# P < 0.05 by Kruskal-Wallis test with Dunn post hoc tests; n = 3. l, Effect of circCDR1as on the ubiquitination level of SERCA2a. n = 3. m, Overexpression of circCDR1as in 293T cells resulted in enhanced K48-linked ubiquitination, as shown by Western blot analysis. n, Western blotting analysis and quantification of NEDD4L in cardiomyocytes treated with AAV9-Null or AAV9-CDR1as. ^*P < 0.05 by Mann-Whitney U test; n = 4. o, Immunofluorescence staining of NEDD4L in freshly isolated ventricular myocytes from the AAV9-Null or AAV9-CDR1as. n = 10; Scale bar: 50 μm. p, Western blotting analysis and quantification of NEDD4L in NMCMs treated with Adv-Null or Adv-CDR1as. ^*P < 0.05 by Mann-Whitney U test; n = 4. q, Immunofluorescence staining of NEDD4L in NMCMs from the Adv-Null or Adv-CDR1as. n = 6; Scale bar: 20 μm. r, Overexpression of circCDR1as enhances NEDD4L interaction with SERCA2a. ^**P < 0.01 by Student t test; n = 3 s, Knockdown of circCDR1as weaken the interaction between NEDD4L and SERCA2a. ^*P < 0.05 by Student t test; n = 3. t, Validation of the efficiency of NEDD4L siRNA. ^**P < 0.01 by Student t test; n = 6. u, NEDD4L knockdown attenuates circCDR1as-induced ubiquitination of SERCA2a. n = 3

In mammalian cells, K48- and K63-linked polyubiquitin chains represent the two major and most abundant forms of polyubiquitin ligations and regulate proteolytic and signaling pathways, respectively. Specifically, K48-linked polyubiquitin chains typically target proteins for proteasomal degradation, whereas K63-linked chains primarily mediate non-degradative functions such as protein stabilization, signaling and interaction modulation [36]. To further dissect the regulatory mechanism by which circCDR1as modulates SERCA2a ubiquitination, we co-transfected SERCA2a and the mutant ubiquitin plasmid, in which only the active sites of K48 and K63 were retained, into HEK293T cells with circCDR1as overexpression and a negative control group. Cells were subsequently treated with MG132 to inhibit proteasomal degradation of SERCA2a. Notably, compared with the cells in the Adv-Null group, the circCDR1as-overexpressing cells transfected with the Ub-K48 plasmid exhibited a significant increase in SERCA2a ubiquitination (Fig. 6m). These results demonstrate that circCDR1as degrades SERCA2a through the ubiquitin-proteasome pathway, which is preferentially targeted by the K48 ubiquitin chains it activates.

NEDD4L has been reported to be an E3 ubiquitin ligase for SERCA2a degradation [37]. Consistent with these findings, the UbiBrowser database also predicted that NEDD4L is a potential E3 ligase for SERCA2a (Fig. S7). Thus, we hypothesized that circCDR1as facilitates NEDD4L-mediated ubiquitination of SERCA2a. To verify this hypothesis, we first evaluated the effects of circCDR1as on the protein expression and subcellular localization of NEDD4L in mouse heart tissue and NMCMs using Western blotting and immunofluorescence staining. The results showed that overexpression of circCDR1as significantly increased the protein expression level of NEDD4L, but did not alter its subcellular distribution (Fig. 6n-q). To further confirm this regulatory effect, we conducted a co-immunoprecipitation (Co-IP) experiment. The results revealed that the overexpression of circCDR1as enhanced the interaction between SERCA2a and NEDD4L (Fig. 6r). However, the circCDR1as defect significantly impaired the interaction between NEDD4L and SERCA2a (Fig. 6s). Collectively, these results demonstrate that circCDR1as promotes K48-linked polyubiquitination and proteasomal degradation of SERCA2a by enhancing its binding to NEDD4L. Notably, the ubiquitination effect was markedly attenuated in NEDD4L-silenced cells (Fig. 6t-u), confirming that the circCDR1as-induced ubiquitination of SERCA2a and its subsequent degradation are dependent on the E3 ligase NEDD4L.

SERCA2a mediates the pro-hypertrophic effects of circCDR1as

To further investigate whether circCDR1as exerts pro-hypertrophic effects in cardiomyocytes by downregulating SERCA2a expression, we performed in vitro rescue assays. We designed siRNAs targeting SERCA2a, constructed SERCA2a overexpression plasmids, and validated their respective efficiencies (Fig. 7a, b). The results revealed that circCDR1as overexpression no longer affected the expression of hypertrophy markers when SERCA2a was overexpressed (Fig. 7c-e). Conversely, knockdown of SERCA2a attenuated the protective effects of circCDR1as silencing in cardiomyocytes (Fig. 7f-h). Taken together, these findings suggest that SERCA2a may serve as a key downstream effector circCDR1as-mediated cardiomyocyte hypertrophy.

Fig. 7.

circCDR1as promotes cardiomyocyte hypertrophy by targeting SERCA2a. a and b, Western blotting analysis of transfection efficiency of pCMV-SERCA2a and siSERCA2a. n = 6. c, Western blotting analysis and quantification assessment of the effects of pCMV-SERCA2a on the levels of ANP and β-MHC in NMCMs treated with and Adv-CDR1as for 48 h. ^**P < 0.01; ^##P < 0.01 by 2-way ANOVA analysis followed by Tukey post hoc multiple comparison test; n = 6. d, qRT-PCR assay of ANP, BNP and β-MHC levels in circCDR1as-overexpressing NMCMs treated with pCMV-SERCA2a for 48 h. ^**P < 0.01; ^#P < 0.05, ^##P < 0.01 by 2-way ANOVA analysis followed by Tukey post hoc multiple comparison test; n = 6. e, Representative α-actinin-immunostaining images of circCDR1as-overexpressing NMCMs transfected with pCMV-SERCA2a, along with corresponding statistical analysis of cell surface area. Scale bar: 20 μm; ^**P < 0.01; ^##P < 0.01 by 1-way ANOVA, followed by Tukey post hoc analysis; n = 6. f, Western blotting analysis and quantification of ANP and β-MHC levels in NMCMs treated with siSERCA2a and induced with Ang II for 48 h. ^*P < 0.05, ^**P < 0.01 by 2-way ANOVA analysis followed by Tukey post hoc multiple comparison test; n = 4. g, qRT-PCR assay of ANP, BNP and β-MHC levels in NMCMs treated with siSERCA2a and induced with Ang II for 48 h. ^*P < 0.05, ^**P < 0.01 by 2-way ANOVA analysis followed by Tukey post hoc multiple comparison test; n = 4. h, Representative α-actinin-immunostaining images of NMCMs transfected with siSERCA2a and induced with Ang II for 48 h, along with corresponding statistical analysis of cell surface area. Scale bar: 20 μm; ^**P < 0.01 by 1-way ANOVA, followed by Tukey post hoc analysis; n = 6

Therapeutic knockdown of circCDR1as attenuates cardiac hypertrophy

Multiple studies have demonstrated that packaging circRNAs in exosomes [38], loading circRNA into hydrogels [39] or making vaccines [40] can enhance the durability, safety, targeting ability, stability and controlled drug release profile of therapeutic agents. To evaluate the translational potential of targeting circCDR1as, we conducted a circCDR1as knockdown experiment 4 weeks post-TAC (Fig. 8a). The results showed a significant improvement in cardiac hypertrophy and an increase in functional parameters, supporting its therapeutic promise for treating pressure overload-induced heart disease (Fig. 8b-j).

Fig. 8.

Knocking down circCDR1as ameliorates cardiac hypertrophy following cardiac pressure overload. a, Schematic of the experimental timeline. 8-week-old male mice underwent TAC surgery. After 4 weeks, they received injections of AAV9-shCDR1as or AAV9-shNC. 4 weeks post-injection, they underwent echocardiography and were subsequently euthanized. b and c, Representative images of echocardiography on the left ventricle and statistical data. ^*P < 0.05, ^**P < 0.01; ^#P < 0.05, ^##P < 0.01; Statistical analysis was performed using Kruskal-Wallis test with Dunn post hoc tests for EF%, FS% and LVID; d; Statistical analysis was performed using 1-way ANOVA, followed by Tukey post hoc analysis for LVID; s; n = 6. d, Representative whole-heart images (Scale bar: 5 mm), HW/BW, HW/TL, and evaluation of heart size via HE staining (Scale bar, 2.5 mm). ^**P < 0.01; ^#P < 0.05 by 1-way ANOVA, followed by Tukey post hoc analysis; n = 6. e and f, WGA staining images and the quantification of cardiomyocytes CSA. Scale bar: 50 μm; ^**P < 0.01; ^# P < 0.05 by Kruskal-Wallis test with Dunn post hoc test; n = 6. g and h, Masson staining and the statistical data of percent changes of fibrosis area. Scale bar: 50 μm; ^**P < 0.01; ^##P < 0.01 by 2-way ANOVA analysis followed by Tukey post hoc multiple comparison test; n = 6. i, Western blotting analysis on ANP and β-MHC in heart tissues. ^**P < 0.01; ^#P < 0.05, ^## P < 0.01 by 2-way ANOVA analysis followed by Tukey post hoc multiple comparison test; n = 4. j, qRT-PCR analysis of ANP, BNP and β-MHC in heart tissues. ^**P < 0.01; ^##P < 0.01 by 2-way ANOVA analysis followed by Tukey post hoc multiple comparison test; n = 6

Discussion

Accumulating evidence supports the role of circRNAs in cardiac disease, but the detailed mechanism underlying this biological process remains elusive. This study demonstrated the pivotal involvement of circCDR1as-mediated SERCA2a ubiquitination in pressure overload-induced cardiac hypertrophy and elucidated the underlying molecular mechanism (Graphic abstract). In this study, we demonstrate that circCDR1as acts as a pro-hypertrophic circRNA that exacerbates cardiac dysfunction and HF. Mechanistically, circCDR1as augments NEDD4L-mediated K48-linked ubiquitination and proteasomal degradation of SERCA2a, which disturbs cardiomyocyte calcium homeostasis. These findings establish a novel mechanistic paradigm for pressure-overload cardiac remodeling: TAC→ circCDR1as↑→ SERCA2a↓→Ca²⁺↑ →cardiac contraction↓ →myocardial hypertrophy → cardiac dysfunction. Although we cannot entirely exclude the involvement of other E3 ligases in SERCA2a ubiquitination, our data strongly support a circCDR1as-dependent mechanism driving SERCA2a degradation under specific pathological conditions.

Our study provides profound insights into the cardiac effects of circCDR1as. Previous researches have suggested the involvement of circCDR1as in various cardiovascular diseases. Recent investigations have demonstrated that the activation of the ALKBH5- FOXO3- circCDR1as/Hippo signaling axis is activated in a mouse model of diabetic cardiomyopathy, indicating that targeting this pathway could be a promising therapeutic strategy for diabetic cardiomyopathy [41]. Our group previously identified the deleterious effects of circCDR1as on Na⁺, K⁺ and Ca²⁺ channels after myocardial infarction (MI) [12, 13]. Although previous observations have highlighted circCDR1as as a potential biomarker for CHF, its mechanistic role in cardiomyocyte proliferation and apoptosis via the miR-135a/b-HMOX1 axes remains to be validated in vivo [42]. Crucially, while this study characterizes circCDR1as as a microRNA sponge, the possibility of additional regulatory modes, including interactions with RNA-binding proteins (RBPs) to modulate protein stability, localization or activity, warrants further exploration. Although recent studies have demonstrated that macrophage-specific overexpression or AAV9-CMV-CDR1as can improve cardiac function after MI via anti-inflammatory effects, it is important to note that the animal models used in these studies and the promoter carried by AAV9 are differ from those used in our research [43].

Using mass spectrometry, we detected multiple potential circCDR1as-interacting proteins in adenovirus-mediated circCDR1as-overexpressing cardiomyocytes. Among these, we focused on SERCA2a to elucidate its role in the anti-hypertrophic mechanism. Among the top 20 candidate downstream targets of circCDR1as, only ATP2A2 (encoding SERCA2a) and Mpc1 have been reported to be directly involved in TAC-induced cardiac hypertrophy [19, 44]. Mpc1 is involved in the occurrence of hypertrophy through the regulation of the pyruvate-lactate metabolic axis, whereas SERCA2a, a core regulatory protein of calcium processing in cardiomyocytes, plays a key role in hypertrophic remodeling. Notably, bioprocess enrichment analysis revealed a significant enrichment of “cellular responses to calcium” (Fig. 4b), suggesting that circCDR1as may be involved in the regulation of calcium signaling. Based on this background, our study is the first to identify a direct interaction between circCDR1as and SERCA2a and to further reveal its key regulatory role in the progression of pathological cardiac hypertrophy.

SERCA2a serves as a “molecular switch” for cardiac calcium homeostasis, with its dysfunction representing a central pathological event in diseases such as HF [45–47]. Despite its identification as a central player in HF pathophysiology over three decades ago, SERCA2a has remained refractory to conventional pharmacological interventions. This persistent therapeutic challenge underscores the urgent need to identify upstream regulatory networks governing SERCA2a expression/activity, offering mechanistic insights to enable precision targeting in HF [33, 48]. Emerging evidence indicates that ncRNAs serve as key modulators of SERCA2a. For instance, lncDACH1 directly binds to SERCA2a and promotes its ubiquitination-dependent degradation, thereby contributing to cardiac dysfunction [19]. Similarly, lncRNA-ZFAS1 acts as an endogenous inhibitor of SERCA2a, binding to the protein and reducing its intracellular levels and activity, thereby impairing cardiac contractility following MI [20]. Circ-ITCH acts as an endogenous molecular sponge for miR-330-5p, upregulating SERCA2a expression to attenuate doxorubicin-induced myocardial cell injury and dysfunction [49].

Ubiquitination represents one of the most widespread and functionally significant post-translational modifications [50, 51]. Distinct ubiquitination patterns dictate the diverse fates of substrate proteins: K48-linked polyubiquitination typically mediates proteasome-dependent degradation, while K63-linked polyubiquitination is involved in signal transduction [52]. circRNAs have recently been implicated in regulating protein stability and function through the modulation of ubiquitination [53, 54]. In this study, we report for the first time that circCDR1as interacts with SERCA2a and promotes its ubiquitination-mediated degradation via the proteasome pathway, thereby disrupting calcium processing and cardiac function. Our results provide compelling evidence that circRNAs are involved in the regulation of protein metabolism. To our knowledge, this represents the first identified circRNA that modulates the ubiquitination homeostasis of SERCA2a, a critical regulator of calcium handling in cardiomyocytes.

Our current study has several limitations. First, although our previous work demonstrated that circCDR1as is 74% conserved [13], the scarcity of human heart tissue samples has limited the availability of clinical specimens, thereby precluding further validation of these findings. In addition, the therapeutic knockdown of circCDR1as has only been verified in a mouse model induced by stress overload. Before considering clinical application, its efficacy and safety still need to be further confirmed in large animal models and human studies. In terms of clinical utility, although this study confirmed that circCDR1as is closely related to pathological myocardial hypertrophy, the clinical specimens were limited to a small sample of serum cohorts and lacked validation with human myocardial tissues. At present, there is no systematic review or meta-analysis to comprehensively quantify its diagnostic efficacy [55, 56]. To promote the clinical translation of circCDR1as, we emphasize that subsequent studies should be conducted in larger independent patient cohorts and human myocardial tissue samples to further confirm its clinical relevance and the robustness of the findings. Specifically, expanding the clinical case cohort and supplementing histological evidence will be necessary steps to deeply validate its diagnostic potential. Second, the considerable length of the circCDR1as gene sequence poses a challenge for truncating specific functional domains, which hinders more detailed mechanistic investigations. Additionally, the lysine ubiquitination sites in the SERCA2a protein have not been reported. According to GPS-Uber (http://www.www.example.com), SERCA2a contains 58 lysine residues, 19 of which are likely to be ubiquitinated. The current study does not identify which lysine residues of SERCA2a can be ubiquitinated by circCDR1as, these questions need to be further clarified by constructing specific mutants at SERCA2a lysine residue sites. Finally, we cannot rule out the possibility that other proteins regulated by circCDR1as are involved in regulating heart function after cardiac hypertrophy.

Although the data from this study show that circCDR1as has a significant therapeutic effect at 8 weeks, the long-term persistence of this effect still needs to be further clarified. The absence of relevant data after 8 weeks is mainly due to the technical challenges associated with the high mortality rate and the variability of the chronic end-stage HF model during the experiment. Filling this data gap is crucial for the clinical translation of circCDR1as. Therefore, our subsequent research will focus on developing strategies to increase the long-term survival rate of animal to conduct a comprehensive longitudinal assessment of the durability and safety characteristics of circCDR1as treatment.

In summary, our study demonstrated that circCDR1as modulates ubiquitination to regulate SERCA2a protein stability. Specifically, circCDR1as promotes the ubiquitination and subsequent degradation of SERCA2a, leading to disrupted Ca²⁺ homeostasis and impaired cardiac function. These findings extend the understanding of circRNA role in cardiac hypertrophy and indicate that reducing cardiac circCDR1as levels could be a promising therapeutic approach for cardiac hypertrophy. The development of small-molecule inhibitors targeting circCDR1as, or cardiomyocyte-specific delivery of shCDR1as, warrants further investigation for the treatment of cardiac hypertrophy.

Conclusion

The results of the current study demonstrated that circCDR1as promotes K48-linked polyubiquitination and proteasomal degradation of SERCA2a by enhancing its interaction with the E3 ligase NEDD4L. Consequently, overexpression of circCDR1as accelerates SERCA2a degradation, whereas its deletion exerts a protective effect by stabilizing the protein. The finding that circCDR1as knockdown protects against cardiac hypertrophy induced by TAC surgery underscores the therapeutic potential of circCDR1as in HF. Further studies need to be conducted on a larger scale population and in human myocardial tissues to systematically verify and clarify its clinical application prospects.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

Ang II

Angiotensin II

ANP

Atrial natriuretic peptide

AAV9

Adeno-associated virus serotype 9

BNP

Brain Natriuretic Peptide

CHX

Cycloheximide

CircRNAs

Circular RNAs

Co - IP

Co- immunoprecipitation

CQ

Chloroquine

CSA

Cross-sectional area

HE

Hematoxylin-eosin

HF

Heart Failure

HW/BW

Heart weight-to-body weight

HW/TL

Heart weight-to-tibia length

IF

Immunofluorescence

LncRNAs

Long-chain non-coding RNAs

LVEF

Left ventricular ejection fraction

LVFS

Left Ventricular Fraction Shortening

β-MHC

β-myosin heavy chain

MI

Myocardial infarction

NMCMs

Neonatal mouse cardiomyocyte

RIP

RNA immunoprecipitation

RBPs

RNA-binding proteins

SERCA2a

Sarcoplasmic reticulum calcium ATPase 2a

SR

Sarcoplasmic reticulum

TAC

Transverse aortic constriction

Author contributions

The authors thank DZM for conceiving and designing the research; WJP, LWJ, LXD, YY, ZXY, LPF, ZMX, DCL, ZY for performing the research; WJ, LCL, CZ, WZR, LHD, DHN, HXQ, HXY and ZY, for providing insights; WJP, LXD and LWJ for writing the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by grants from the Noncommunicable Chronic Diseases-National Science and Technology Major Project (2024ZD0537900 to Zhimin Du) and the National Natural Science Foundation of China (No. 82073838 and 82273917 to Zhimin Du).

Data availability

All data supporting the findings of this study are available within the article and its supplementary files.

Declarations

Ethics approval and consent to participate

All animal protocols were approved by the Institutional Animal Care and Use Committee and the Ethics Committee of Harbin Medical University (No. sydwgzr2020-153-01), complying with NIH animal care guidelines. The research on human serum samples was performed in strict accordance with the Declaration of Helsinki which has been approved by the ethical review committee of the Second Affiliated Hospital of Harbin Medical University (approval No. ky2020-049-01).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.