Cardiomyocyte-specific long noncoding RNA Trdn-as induces mitochondrial calcium overload by promoting the m⁶A modification of calsequestrin 2 in diabetic cardiomyopathy

Abstract

Diabetic cardiomyopathy (DCM) is a medical condition characterized by cardiac remodeling and dysfunction in individuals with diabetes mellitus. Sarcoplasmic reticulum (SR) and mitochondrial Ca²⁺ overload in cardiomyocytes have been recognized as biological hallmarks in DCM; however, the specific factors underlying these abnormalities remain largely unknown. In this study, we aimed to investigate the role of a cardiac-specific long noncoding RNA, D830005E20Rik (Trdn-as), in DCM. Our results revealed the remarkably upregulation of Trdn-as in the hearts of the DCM mice and cardiomyocytes treated with high glucose (HG). Knocking down Trdn-as in cardiac tissues significantly improved cardiac dysfunction and remodeling in the DCM mice. Conversely, Trdn-as overexpression resulted in cardiac damage resembling that observed in the DCM mice. At the cellular level, Trdn-as induced Ca²⁺ overload in the SR and mitochondria, leading to mitochondrial dysfunction. RNA-seq and bioinformatics analyses identified calsequestrin 2 (Casq2), a primary calcium-binding protein in the junctional SR, as a potential target of Trdn-as. Further investigations revealed that Trdn-as facilitated the recruitment of METTL14 to the Casq2 mRNA, thereby enhancing the m⁶A modification of Casq2. This modification increased the stability of Casq2 mRNA and subsequently led to increased protein expression. When Casq2 was knocked down, the promoting effects of Trdn-as on Ca²⁺ overload and mitochondrial damage were mitigated. These findings provide valuable insights into the pathogenesis of DCM and suggest Trdn-as as a potential therapeutic target for this condition.

Keywords

Trdn-as / calsequestrin 2 / METTL14 / mitochondrial dysfunction / calcium overload

1 Introduction

Diabetes mellitus is a metabolic condition distinguished by elevated blood glucose levels, and its prevalence has increased in recent years. Research on the global prevalence of prediabetes suggests that the global diabetic population will reach 638 million by 2045 [1,2]. Diabetic cardiomyopathy (DCM) is the most common complication of diabetes mellitus and is characterized by myocardial hypertrophy, cardiac fibrosis, and diastolic dysfunction in the early stages, followed by progression to systolic dysfunction and ultimately resulting in heart failure [3]. Various factors, including Ca²⁺ homeostasis disruptions, mitochondrial dysfunction, and oxidative stress, contribute to DCM development [4–6]. However, the exact underlying mechanism is unclear. Thus, identifying key factors responsible for DCM progression is crucial to uncover potential therapeutic targets for its treatment.

Ca²⁺ is a crucial and second messenger within eukaryotic cells. Its elevation in the cytoplasm elicits various cellular responses, including cell proliferation, contraction, secretion, and cell death [7–9]. The sarcoplasmic reticulum (SR) is the central intracellular Ca²⁺ storehouse that regulates intracellular Ca²⁺ levels and dynamics [10,11]. The mitochondria, comprising 30% of the overall volume of mature cardiomyocytes, determine cell destiny mainly through their involvement in the dynamic control of cellular Ca²⁺ [12]. These two organelles intimately communicate as the SR transmits proper Ca²⁺ signals to the mitochondria on a dynamic membrane platform. Elevated Ca²⁺ translocation from the SR to the mitochondria leads to excessive Ca²⁺ accumulation within the mitochondria, causing disruptions in mitochondrial processes such as reduced adenosine triphosphate (ATP) synthesis, heightened reactive oxygen species (ROS) production, and apoptosis. SR and mitochondrial Ca²⁺ overload in cardiomyocytes have been recognized as biological hallmarks in experimental animals and individuals with DCM [13]. However, the specific factors contributing to these abnormalities remain largely unknown.

The calcium binding protein calsequestrin 2 (Casq2), a constituent of the ryanodine receptor 2 (RyR2) channel located within junctional SR (jSR), is essential for modulating the Ca²⁺ storage capability of the SR, enabling the accumulation of approximately 20 mmol/L Ca²⁺ within the SR. Alterations and mutations in Casq2 expression can disrupt intracellular Ca²⁺ homeostasis, leading to the onset of various diseases [14,15]. Mutations in the Casq2 gene is associated with catecholaminergic polymorphic ventricular tachycardia [16]. An elevated Ca²⁺ content in the SR was observed in transgenic mice with Casq2 overexpression, leading to the development of substantial cardiac hypertrophy [17]. Casq2 overexpression led to increases in SR Ca²⁺, RYRs’ sensitivity to Ca²⁺, and Ca²⁺ release [18]. The close location between junctional SR (jSR) and mitochondria in cardiomyocytes facilitates the transfer of Ca²⁺ from SR to mitochondria [19]. However, the precise role of Casq2 in regulating mitochondrial Ca²⁺ balance and the factors controlling Casq2 levels in DCM remain to be fully understood.

Human genome sequencing has revealed that a significant portion of the transcriptome consists of noncoding RNAs (ncRNAs) [20]. Among these ncRNAs, long noncoding RNAs (lncRNAs) exhibit diverse functional mechanisms, including the regulation of transcription, epigenetics, and posttranscriptional processes [21–24]. Our previous research uncovered the involvement of lncRNAs in the development of myocardial infarction, arrhythmia, and heart aging [25–28]. The potential role of lncRNAs in modulating cardiac function and Ca²⁺ homeostasis in DCM is a significant area of inquiry.

To identify cardiac-specific lncRNAs implicated in cardiac function and DCM development, we analyzed dysregulated lncRNAs in heart tissues from DCM mice through transcriptomic sequencing. A cardiomyocyte-specific lncRNA named Trdn-as was identified and exhibited significant upregulation in the hearts of the DCM mice. Using loss- and gain-of-function methods in vitro and in vivo, we demonstrated that Trdn-as plays a detrimental role in DCM progression by promoting the m⁶A modification of Casq2 mRNA. This modification leads to SR and mitochondrial Ca²⁺ overload and subsequent mitochondrial dysfunction. Therefore, Trdn-as represents a promising novel target for therapeutic intervention in DCM.

2 Materials and methods

2.1 Animal care

Db/m mice (8-week-old, 20–25 g) and db/db mice (8-week-old, 42–47 g) were provided by the Nanjing Military Laboratory Animal Science and Technology Co., Ltd., China. and reared in a controlled animal facility with optimal conditions of 22 ± 1 °C temperature and 55% ± 5% humidity.

2.2 Construction of viral vectors for Trdn-as knockdown and overexpression

Adeno-associated viruses (AAVs.) have become widespread due to their characteristics such as highly efficient infection, low immunogenic response, nonpathogenicity, sustained gene expression over an extended period, and extensive tissue transduction. Among them, AAV9 has the highest infectivity for myocardial transduction. Trdn-as overexpression was completed by injecting an AAV9 vector carrying Trdn-as sequence (AAV-Trdn-as) with a conjugated GMV bGlobin promoter (Genechem Co., Ltd., Shanghai, China). An AAV9 vector with a short RNA fragment for knocking down Trdn-as and a conjugated U6 promoter were also designed (sh-Trdn-as) (Genechem Co., Ltd., Shanghai, China). Saline was injected into the control group of mice through the tail vein.

2.3 In vivo gene delivery

Viral solutions were injected into the mice via the tail vein using random delivery (AAV-Trdn-as, AAV-NC, sh-Trdn-as, and sh-NC constructs diluted in 100 μL of saline solution). For data collection on single-gene infection, experimental measurements were conducted 3 months after the animals were injected with either AAV-Trdn-as to overexpress Trdn-as or sh-Trdn-as to silence Trdn-as.

2.4 Establishment of DCM model and study design

The animals were randomly allocated into the following groups: db/m, db/db (21-week-old db/db mice), + sh-Trdn-as (9-week-old db/db mice infected with AAV9-sh-Trdn-as once a month for 3 months), + sh-NC (9-week-old db/db mice infected with AAV9-sh-NC once a month for 3 months), AAV-Trdn-as (9-week-old db/m mice infected with AAV9-Trdn-as once a month for 3 months), and NC (9-week-old db/m mice infected with AAV9-NC once a month for 3 months).

2.5 Echocardiographic assessment of cardiac function

The mice were anesthetized through intraperitoneal injection of 0.2 g/kg avertin (Sigma, St. Louis, Missouri, USA). Cardiac function was evaluated using the Vevo2100 high-resolution imaging system (Visualsonics, Toronto, ON, Canada). Cardiac systolic function was assessed using left ventricular (LV) ejection fraction (EF) and short-axis fractional shortening (FS), both calculated from M-mode recordings. Cardiac diastolic function was detected using the ratio of the E peak (LV diastolic maximum blood flow) to the A peak (atrial systolic maximum blood flow through the mitral valve). LV posterior wall diastole (LVPWD) and LV mass were also measured.

2.6 Histopathological and morphometric observations

Hematoxylin and eosin (HE) and Masson’s trichrome staining were applied to evaluate cardiac histopathological changes and collagen accumulation following the procedures described in our previous study [29]. Heart tissues were thoroughly fixed in 4% paraformaldehyde, embedded in paraffin, and transversely sliced into 5 μm-thick sections for HE staining using HE reagents (Solarbio, Beijing, China) or Masson’s trichrome staining with Masson’s trichrome kit (Solarbio, Beijing, China). Each section was imaged using a light microscope (Carl Zeiss Microscopy, Jena, Germany). Collagen volume fraction was measured with Image J software.

2.7 Immunohistochemistry assessment

Prepared paraffin-embedded slices of mouse heart were heated at 65 °C for 20 min, deparaffinized through xylene, and rehydrated using different alcohol concentrations (100%, 95%, 80%, and 70%). The sections were soaked in citric acid buffer at 100 °C for 10 min without boiling for antigen retrieval and treated with 3% H₂O₂ (Aladdin, Shanghai, China) at room temperature for 10 min to quench endogenous peroxidase. Goat serum (Beyotime, Shanghai, China) was used to block the slices for 1 h, followed by staining with anti-Casq2 (1:500, #ab3516; Abcam, Cambridge, UK) overnight at 4 °C, incubation with horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h, and diaminobenzidine color rendering (ZSGB-BIO, Beijing, China). Furthermore, the slices were counterstained with HE, treated with ethanol and xylene, and sealed using resin. Immunohistochemistry images were obtained using a panoramic scanning microscope (Leica, Wetzlar, Germany), and Casq2 expression was analyzed from the immunohistochemistry images with Image J software.

2.8 Immunofluorescence assessment

Frozen heart sections were prepared for immunofluorescence assessment to determine Casq2 expression. The sections were washed with PBS for 10 min and then fixed with 4% paraformaldehyde. After three subsequent PBS washes, the slices were treated with goat serum containing 0.3% Triton X-100 for 1 h and incubated with anti-Casq2 (1:100) at 4 °C overnight. On the second day, a fluorescence-conjugated secondary antibody was applied to the sections at room temperature for 1 h. Finally, DPAI staining was performed for 15 min. Immunofluorescence images of the heart were captured by a confocal fluorescence microscope (Olympus, Fluoview1000, Tokyo, Japan), and the fluorescence density of Casq2 was analyzed from the immunofluorescence images with Image J software.

2.9 Transmission electron microscopy

Heart samples were fixed with 2.5% glutaraldehyde at 4 °C overnight. The slices were dehydrated and stained with uranyl acetate and lead citrate. The ultrastructural changes of the heart tissues were observed under a JEOL-1200 electron microscope produced by JEOL Company in Tokyo, Japan.

2.10 Primary neonatal mouse cardiomyocytes (NMCMs)

Cardiomyocytes were obtained from neonatal Kunming mice aged 1–3 days procured from the Experimental Animal Center of the Second Affiliated Hospital of Harbin Medical University in Harbin, China. After heart dissection, the specimens were placed in a mixed solution of D-hanks and trypsin on a shaker at 4 °C overnight. Preheated type II collagenase (#17101015; Gibco, NY, USA) was used to digest the hearts on the second day. The obtained solution containing mixed cells was collected and centrifuged (1500 rpm/min, 5 min). The supernatant was discarded, and the cells were resuspended on DMEM (#BL301A; Biosharp, China) containing 10% fetal bovine serum (#C04001; Vivacell, Shanghai, China) and 1% penicillin–streptomycin solution (×100) (#C0222; Beyotime, Shanghai, China). The cells were cultured in a CO₂ (5%) incubator with constant temperature (37 °C) and humidity. After 1.5 h, the supernatant suspension was collected, diluted, and dispersed with fresh culture solution and spread evenly in culture plates.

2.11 Transfection of Trdn-as siRNA and plasmids

For transfection, Trdn-as-overexpressing pcDNA3.1-plasmids or negative controls (IBSBIO, Shanghai, China) were transiently transfected into NMCMs using Lipofectamine 2000 (Invitrogen, USA). Trdn-as siRNA and siNC (RiboBio Co., China) were also transfected using the X-treme GENE Transfection Reagent (Roche, USA). The transfection agents were combined with Opti-MEM (Gibco, NY, USA) and DMEM without serum and subsequently added to the NMCMs. After 6 h, the medium was replaced, and the cells were treated with 33 mM glucose for 48 h and utilized for subsequent experiments.

2.12 RNA-interacting protein immunoprecipitation (RIP)

The RIP RNA binding protein immunoprecipitation kit (#Bes5101; Beisinbio, Guangzhou, China) was utilized for RIP analysis. The ReverTraAce qPCR RT Kit (#FSQ‐101; Toyobo, Osaka, Japan) was employed to reverse-transcribe RNA into cDNA. The SYBR Green PCR Master Mix kit (#4309155; Invitrogen, Carlsbad, USA) and LightCycler 96 real-time polymerase chain reaction system (Roche, Basel, Switzerland) were utilized for qRT-PCR on the specific gene of interest.

2.13 JC-1 staining for mitochondrial membrane potential (MMP)

JC-1 is a fluorescent indicator used to detect MMP and emits red or green fluorescence in cells with high or low potential. Cardiomyocytes were treated with specific interventions and exposed to a 5 μM JC-1 dye solution (#C2006; Beyotime, Shanghai, China) at 37 °C for 20 min. The resulting images were then acquired using a confocal fluorescence microscope. The fluorescence intensity of the red and green signals was quantified with Image J software.

2.14 Immuno-FISH

A Ribo^TM fluorescence in situ hybridization kit (RiboBio, Guangzhou, China) was acquired to perform the FISH fluorescence probe staining of Trdn-as in accordance with the regulations and suggestions in the kit manual. The sample was sealed in a prehybridization buffer (37 °C, 30 min), followed by a 12 h hybridization in a buffer containing 20 μM Trdn-as FISH Probe Mix at room temperature. The sample was then washed with 0.1% Tween-20 in 4 × SSC, 2 × SSC, and PBS for 10 min each. The nucleus was labeled with DAPI. Finally, the sample was mounted on a confocal fluorescence microscope for imaging.

2.15 Oxygen consumption rate (OCR)

OCR was measured using the Seahorse XF Cell Mito Stress Test Kit (#103015-100; Seahorse Bioscience, USA) and Seahorse XFe24 Extracellular Flux Analyzer following the manufacturer’s instructions. NMCMs were cultured in XFe24 plates and subjected to respective treatments. Before measurement, the cells were rinsed with an XF assay medium and incubated at 37°C without CO₂ for 1 h. Compounds oligomycin, FCCP, rotenone, and antimycin A were sequentially added to the XF probe card, and the plates were loaded onto the XFe24 analyzer for OCR detection.

2.16 Methylated RNA immunoprecipitation (MeRIP)-qPCR assays

MeRIP assay was conducted following the protocol outlined in the riboMeRIPTM m⁶A Transcriptome Profiling Kit (#C11051-1; RiboBio Co., China). RNAs were fragmented by RNA fragmentation buffer and heating and then prepared for MeRIP reaction. The m⁶A-specific antibody was incubated with magnetic beads A/G in an IP buffer at room temperature. The MeRIP reaction mixture, consisting of fragmented RNAs, was introduced to anti-m⁶A beads and incubated at 4 °C for 2 h on a rotator. Finally, the immunoprecipitated mixtures were treated with elution buffers at 4 °C for 1 h to extract the bound RNAs. The enriched RNA (Casq2) was examined by qPCR.

2.17 SR Ca²⁺ detection

SR Ca²⁺ level was detected using a specific fluorescent probe for SR Ca²⁺, Mag-Fluo-AM (GMS10267.1; GENMED SCIENTIFIC INC. USA). NMCMs were applied to cell slides, subjected to specific treatments, and exposed to a dyeing solution at room temperature for 1 h after being washed with cleaning fluid. Images were capture by a confocal microscope, and fluorescence density (green) was measured with Image J software [30].

2.18 Mitochondrial Ca²⁺ detection

A specific fluorescent probe for mitochondrial Ca²⁺, Rhod-2-AM (GMS10153.1; GENMED SCIENTIFIC INC. USA), was utilized to explore mitochondrial Ca²⁺ levels [31]. NMCMs were placed in a black 96-well plate, transfected, and treated with glucose. Cleaning fluid was added to the plate and mixed with the dyeing working fluid. The cells were placed in a 37 °C cell incubator for 30 min away from the light. The solution was substituted with a cleaning fluid, and the cells were incubated at 37 °C for an additional 45 min. Mitochondrial Ca²⁺ concentration was determined by measuring relative fluorescence units using a fluorescence microplate reader.

2.19 ROS detection

ROS level was measured using a ROS detection kit (#S0033S; Beyotime Biotechnology, China). NMCMs were rinsed with PBS three times and then exposed to a 10 μM DCFH-DA fluorescence probe at 37 °C for 20 min. Images were capture by a confocal microscope, and the fluorescence density of DCFH-DA was measured with Image J software.

2.20 Malonaldehyde (MDA) detection

MDA concentration was assessed using the CheKineTM Lipid Peroxidation (MDA) Assay Kit (#KTB1050; Abbkine, China) following the manufacturer’s guidelines. Treated NMCMs were collected and lysed with lysis buffer. After the solution was centrifugated at 13 000 g for 10 min at 4 °C, the supernatant was employed to assess the MDA level. The reaction mixture was added to the supernatant and incubated at 95 °C for 30 min. The mixed solution underwent centrifugation at 10 000 g for 10 min. The supernatant was collected and added to a 96-well plate for MDA detection. Absorbance was measured at 532 and 600 nm using a microplate reader.

2.21 Superoxide dismutase (SOD) detection

SOD level was measured by CheKine^TM Superoxide Dismutases (SOD) Activity Assay Kit (#KTB1030; Abbkine, China) following the guidelines provided by the manufacturer. NMCMs were lysed and then centrifugated at 12 000 g for 5 min. The supernatant was then added to the 96-well plate at 20 μL per well and mixed with 80 μL of working solution, which consisted of 5 µL of xanthine, 5 µL of WST-8, 1 µL of enhancer, and 74 µL of assay buffer. Finally, the mixture was incubated with 20 μL of xanthine Oxidase for 1 h at room temperature. The absorbance, indicative of the SOD level, was then measured at 450 nm using a microplate reader.

2.22 ATP content detection

The ATP levels in cardiomyocytes were determined using the ATP Assay Kit (#S0026; Beyotime, Shanghai, China). The heart tissue was lysed and centrifuged at 12 000 g for 5 min at 4 °C. ATP detection working fluid and the supernatant were sequentially added into the wells of the 96-well plate.

2.23 RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA from heart tissues and cardiomyocytes was extracted using TRIzol reagent (Invitrogen, Carlsbad, USA). Reverse transcription was carried out using the ReverTraAce qPCR RT Kit. qRT-PCR was performed using the SYBR Green PCR Master Mix kit and the LightCycler 96 real-time polymerase chain reaction system. β-actin served as the internal control. The sequences of primers used were listed below:

Trdn-as: F: 5′-GGGATGCAGAAGCAACAGAG-3′

R: 5ʹ-GCAGCAAAGGTACACAGAGG-3′

Casq2: F: 5′-GACCGAGTGGTCAGCCTTTC-3′

R: 5′-ACAGGTTCGTGGTAATAGAGACA-3′

β-actin: F: 5′-CGTTGACATCCGTAAAGACC-3′

R: 5′-AACAGTCCGCCTAGAAGCAC-3′

2.24 Western blot analysis

For protein blot detection, the total protein samples from heart tissues and cardiomyocytes were subjected to electrophoresis (60–100 μg/well) and subsequently transferred onto a nitrocellulose filter membrane (PALL, New York, USA). The membrane was blocked with 5% nonfat milk dissolved in PBS for 2 h. Primary antibodies against Casq2 and β-actin (Proteintech, Chicago, USA), diluted at 1:1000 in PBS buffer, were utilized to probe the membrane at 4 °C overnight. The membrane was incubated with a fluorescence-conjugated anti-rabbit or anti-mouse IgG secondary antibody at room temperature for 1 h after being washed in PBS with 0.05% Tween-20 three times. β-actin served as the internal control. Western blot results were quantified with Odyssey v1.2 software (LICOR Biosciences, Lincoln, NE, USA).

2.25 Statistical analysis

The data are presented as mean ± SEM. Statistical comparison between the two experimental groups was performed using a two-tailed unpaired Student’s t-test. The difference between groups was assessed using one-way ANOVA and multiple comparison tests with Tukey’s post hoc analysis. Statistical analyses were conducted using Prism 8.0 (GraphPad, La Jolla, CA) and SPSS (Version 25). A significance level of P < 0.05 was considered statistically significant.

3 Results

3.1 Trdn-as is upregulated in the cardiomyocytes of DCM mice

To investigate the regulatory role of lncRNAs in DCM, we screened the differentially expressed lncRNAs using the GEO data set (GSE161931) as illustrated in Fig.1. Heatmap analysis identified the top 30 dysregulated genes. Among them, the following lncRNAs with heart-specific expression patterns were identified: GM30556, GM35228, GNM31013, GM15543, GM26795, C130080G10Rik, and D830005E20Rik (Trdn-as) (Fig.1). Trdn-as exhibited the highest upregulation in the hearts of the DCM mice, indicating its potential significance. Furthermore, its expression pattern was assessed in various organs of wild-type (WT) mice, including the heart, liver, kidney, spleen, pancreas, lung, skeletal muscle, small intestine, skin, and fat. The results revealed that Trdn-as was prominently enriched in the heart (Fig. S1A). To elucidate the specific role of Trdn-as, we also analyzed its expression patterns across various cardiac cell types using the Tabula Muris database. The results revealed that Trdn-as was exclusively expressed in cardiomyocytes, indicating its potential involvement in modulating cardiomyocyte function. We also verified this conclusion in mouse aortic endothelial cells (MAEC), mouse RAW, cardiomyocytes (CM), and cardiac fibroblasts (CF) (Fig.1). qRT-PCR experiments demonstrated similar Trdn-as upregulation in NMCMs and AC16 cells exposed to high glucose, a condition that mimics cardiomyocyte damage associated with DCM (Fig.1 and 1E). To uncover its potential regulatory mechanism, we investigated the cellular localization of Trdn-as. qRT-PCR analysis of separated nuclear and cytoplasmic fractions and FISH staining revealed that Trdn-as was primarily sustained in the nucleus (Fig.1 and 1G). These findings indicated the potential regulatory role of Trdn-as within cardiomyocytes in DCM.

3.2 Trdn-as induces cardiac dysfunction and heart injury in mice

We then questioned whether the upregulation of Trdn-as in cardiomyocytes directly contributes to cardiac dysfunction. We developed an AAV9 plasmid to overexpress Trdn-as in WT C57BL/6J mice. The successful overexpression of Trdn-as in the hearts of the mice was confirmed 3 months after injection of the AAV9-Trdn-as plasmid (Fig.2 and S1B). Echocardiography was utilized to assess the cardiac function. The results suggested that Trdn-as overexpression impaired the diastolic function of mice as evidenced by the reduced E/A ratio and increased LV mass and LVPWD. A significant decrease was observed in EF and FS, suggesting a compromised systolic function. Left ventricular end diastolic (LVEDD) and systolic (LVESD) dimensions were also increased after overexpressing Trdn-as (Fig.2 and S1C). To examine the influence of Trdn-as on cardiac remodeling, we utilized HE and Masson staining to assess myocardial organization and interstitial fibrosis. The results revealed notable differences between the control mice and the mice overexpressing Trdn-as (Fig.2). In particular, the myocardial sarcomeres in the Trdn-as-overexpressing mice displayed disarray, enlarged myocardial cells and increased collagen production, indicating the presence of cardiac remodeling and interstitial fibrosis (Fig.2). To investigate the potential of Trdn-as as a therapeutic target for DCM, we performed a knockdown experiment by transfecting Trdn-as shRNA carried by an AAV plasmid in db/db mice. The knockdown efficiency of Trdn-as was confirmed using qRT-PCR and immunofluorescence staining (Fig.2 and S1D). Trdn-as knockdown significantly improved the cardiac systolic and diastolic function of db/db mice (Fig.2 and S1E). It also ameliorated the disorganized arrangement of cardiac sarcomeres, improved myocardial hypertrophy, and decreased the production of cardiac collagen (Fig.2). These results strongly indicated that Trdn-as modulates cardiac function in DCM.

3.3 Trdn-as regulates Casq2 expression in DCM

To investigate the molecular mechanism underlying the role of Trdn-as in DCM, we performed RNA-seq analysis to identify potential genes regulated by Trdn-as following its overexpression in NMCMs (Fig.3). Functional analysis of Gene Ontology and pathway enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes indicated the notable enrichment of pathways related to Ca²⁺, including Ca²⁺ ion homeostasis, cardiac muscle contraction, SR, and regulation of cation transmembrane transport (Fig.3 and 3C). Further analysis using a heatmap highlighted the enriched genes within these pathways (Fig.3), and subsequent verification identified Casq2 as a common gene (Fig.3 and 3F). CASQ2 overexpression (GSE62203) was observed in DCM model (Fig. S2A–S2C). Furthermore, the observed upregulation of Trdn-as and Casq2 in our sequencing data suggested that the model was effectively established and the analytical findings are reliable (Fig. S2D). Our data demonstrated the significant upregulation of Casq2 at the mRNA and protein levels in the hearts of db/db mice. By contrast, Trdn-as knockdown resulted in decreased Casq2 mRNA and protein levels. Trdn-as overexpression in the control mice increased Casq2 expression (Fig.3 and 3H). Furthermore, the expression and distribution of Casq2 in cardiac tissues was confirmed by immunofluorescence staining (Fig.3 and 3J). In NMCMs, Trdn-as knockdown reversed the upregulation of Casq2 induced by high glucose conditions, while overexpression of Trdn-as resulted in increased Casq2 mRNA and protein expression (Fig.3 and 3L).

3.4 Trdn-as induces SR and mitochondrial Ca²⁺ overload and mitochondrial damage

Considering the role of Casq2 in modulating Ca²⁺ storage within the SR, we evaluated the impact of Trdn-as on SR Ca²⁺ levels. Our findings demonstrated a substantial augmentation in SR Ca²⁺ levels upon Trdn-as overexpression (Fig. S3A). The transport of Ca²⁺ from the SR to the mitochondria is critical in regulating mitochondrial function, including mitochondrial biogenesis, respiration, and oxidative stress. Trdn-as upregulation led to a significant elevation in Ca²⁺ levels within the mitochondria (Fig. S3B). Excessive Ca²⁺ accumulation induced by Trdn-as in the mitochondria impaired mitochondrial function. JC-1 staining results indicated that Trdn-as overexpression led to a reduction in MMP (Fig. S3C). TEM results suggested that Trdn-as overexpression markedly diminished normal mitochondrial content, leading to mitochondrial damage characterized by swelling and loss of ridge structure (Fig. S3D). Subsequent analysis showed increased oxidative stress (as evidenced by ROS overload), decreased SOD levels, and increased MDA levels (Fig. S3E–S3G).

3.5 Trdn-as knockdown inhibits mitochondrial Ca²⁺ overload and mitochondrial damage caused by high glucose

We then investigated the potential of Trdn-as knockdown to mitigate mitochondrial Ca²⁺ overload and damage. High glucose treatment induced significant SR and mitochondrial Ca²⁺ overload, which were attenuated upon Trdn-as knockdown (Fig.4 and 4B). In addition, Trdn-as knockdown restored MMP and mitochondrial respiration (Fig.4 and 4D). In the hearts of db/db mice, a notable reduction in normal mitochondrial content and many damaged mitochondria were observed. However, Trdn-as knockdown attenuated mitochondrial damage in db/db mice (Fig.4). In the presence of elevated glucose levels, the ROS production and MDA content increased and the SOD levels decreased. When Trdn-as was genetically disrupted, the ROS and MDA production were inhibited and the SOD levels increased (Fig.4–4H). ATP synthesis was observed in the myocardium of mice. The ATP levels in the cardiac muscle of DCM mice were significantly reduced, and those in the myocardium of Trdn-as knockout mice were restored. Conversely, Trdn-as overexpression led to a decrease in ATP content in the myocardium of mice (Fig. S4A). We also explored the mechanism of Ca²⁺ overload in mitochondria. VDAC1 and MCU were identified as mediators of Ca²⁺ entry into the mitochondria, and knocking them down successfully mitigated the mitochondrial Ca²⁺ overload induced by Trdn-as, indicating the contribution of these two calcium channels to this process (Fig. S4B). These findings demonstrated that Trdn-as knockdown can alleviate mitochondrial Ca²⁺ overload and dysfunction, thereby protect cardiomyocytes against high glucose-induced damage.

3.6 Casq2 knockdown restores Ca²⁺ homeostasis and ameliorates mitochondrial dysfunction

We subsequently confirmed the involvement of Casq2 in the functional deterioration of cardiomyocytes caused by Trdn-as. When the calcium-sensing protein Casq2 was disrupted under typical physiologic circumstances, the Ca²⁺ within the SR and mitochondria in cardiomyocytes were reduced (Fig.5 and 5B). Our findings also suggested that the decrease in Casq2 can ameliorate the mitochondrial dysfunction induced by Trdn-as overexpression, and this ability involved the restoration of MMP, enhancement of mitochondrial respiration, mitigation of ROS and MDA generation, and an increase in SOD activity (Fig.5–5F). However, knocking down Casq2 leads to a reduction in Ca²⁺ in the SR and mitochondria, resulting in mitochondrial damage and dysfunction. The changes in SR Ca²⁺ are related to contractile function, and we also detected the changes of SERCA2a. Western blot results showed no evident change in the SERCA2a expression of db/db mice. Trdn-as knockdown did not affect SERCA2a expression, indicating that Trdn-as did not influence the function of cardiomyocytes by affecting SERCA2a (Fig. S5A). These findings highlighted that Trdn-as disrupts Ca²⁺ homeostasis in cardiomyocytes mediated by Casq2, ultimately inducing mitochondrial dysfunction.

3.7 Trdn-as enhances Casq2 mRNA stability by promoting METTL14-mediated m⁶A modification

The above findings suggested that Trdn-as could regulate the mRNA expression of Casq2. Hence, molecular mechanism was further investigated. Trdn-as downregulation led to a significant decrease in the stability of Casq2 mRNA (Fig.6). m⁶A modification promotes epigenetic reprogramming in mRNA, often leading to changes in mRNA stability. To examine the potential involvement of m⁶A modification in the modulation of Casq2 mRNA stability by Trdn-as, we analyzed the total m⁶A levels following the overexpression or knockdown of Trdn-as. Our results demonstrated that Trdn-as overexpression increased the m⁶A level in normal cardiomyocytes (Fig.6). Conversely, Trdn-as knockdown inhibited the high glucose-induced elevation of m⁶A levels (Fig.6). The regulatory potential of Trdn-as on m⁶A modification encouraged us to investigate the m⁶A modification sites on Casq2 mRNA.

The online tool SRAMP suggested a high confidence of m⁶A modification position in the Casq2, including 748, 1681, and 1788 locus (Fig.6 and 6E). MeRIP results revealed that relying on the 748 locus, Trdn-as contributed to a significant increase in the m⁶A modification of Casq2 mRNA (Fig.6). Furthermore, high glucose treatment could elevate the mRNA methylation level of Casq2, which was effectively inhibited by Trdn-as knockdown (Fig.6). To identify potential methylated proteins involved in regulating Casq2 methylation, we employed the RPIseq web-based tool to assess the interaction potential of established methyltransferases and readers such as METTL3, METTL14, WTAP, and YTHDF2 with Trdn-as or Casq2 mRNA. METTL14 emerged as the most prominent candidate, displaying significantly higher RF and SVM scores than other writer proteins (Fig.6). Subsequent RIP analysis revealed that METTL14 exhibited significant binding with Trdn-as (Fig.6). METTL14 expression remained unaltered, which was also confirmed through protein-level analysis (Fig. S5B and S5C). METTL14 demonstrated binding capabilities with Casq2 mRNA. Under high glucose conditions, an increased interaction between METTL14 and Casq2 mRNA was observed, and Trdn-as knockdown attenuated this interaction (Fig.6). These findings suggested that Trdn-as can regulate the m⁶A modification of Casq2 mediated by METTL14, thereby influencing the mRNA stability of Casq2 in a glucose-dependent manner.

4 Discussion

This investigation elucidates the pathophysiological role of Trdn-as and its potential mechanism in DCM. Our findings indicate a significant increase in Trdn-as levels in the myocardium of db/db mice and cardiomyocytes treated with high glucose. Trdn-as knockdown prevents cardiac dysfunction and mitochondrial damage in db/db mice, and its overexpression has the opposite effects. From a mechanistic perspective, Trdn-as influences the mRNA methylation of Casq2 mediated by METTL14, resulting in increased Casq2 protein expression in myocardial cells and an imbalance in Ca²⁺ homeostasis in the SR and mitochondria. These findings suggest the crucial role for Trdn-as in developing cardiac dysfunction.

Similar to most lncRNAs, Trdn-as exhibits limited conservation across species, with a homology of approximately 45% between humans and mice. Nevertheless, the expression patterns of Trdn-as in cardiomyocytes exhibit remarkable consistency across various species. Aligning with our results, Zhao et al. [32] suggested that Trdn-as is specifically expressed in the cardiomyocytes of human and mouse hearts. Trdn-as downregulation has been observed in individuals with arrhythmia and heart failure, suggesting its diverse regulatory effects in the progression of cardiovascular diseases [33]. Our results revealed that Trdn-as overexpression resulted in cardiac dysfunction similar to that observed in db/db mice, and its knockdown improved cardiac dysfunction in db/db mice. Meanwhile, modifications in Trdn-as expression had no impact on the blood glucose levels in mice (the data were not shown), suggesting that the beneficial effects of Trdn-as arise from its direct regulation of cardiomyocytes, rather than its influence on blood glucose. We also observed the detrimental effect of Trdn-as on the myocardial structure in db/db mice. Cardiac remodeling of db/db mice was also influenced by Trdn-as. Given the relevance of fibroblasts and endothelial cells in cardiac remodeling, the potential impact of AAV-Trdn-as or AAV-Trdn-as shRNA on these cellular components must be examined in future studies.

SR Ca²⁺ in cardiomyocytes is responsible for regulating protein synthesis, cell contraction, and relaxation and participating in cell signaling. The Ca²⁺ level must be carefully controlled to ensure normal cell function, which is achieved through a delicate balance of introducing and releasing Ca²⁺ across the SR membrane [34]. SERCA pumps Ca²⁺ from cytoplasm ([Ca²⁺ ] 100 nm) into SR ([Ca²⁺ ] 100 μm to 1 mm) to counteract Ca²⁺ concentration [35]. At the same time, SR Ca²⁺ is passively released into the cytoplasm to maintain a stable state and prevent an overload of SR Ca²⁺. Excessive Ca²⁺ accumulation in the SR triggers SR stress and results in a subsequent increase in Ca²⁺ levels in the mitochondria, a known factor in causing cell death [36–39]. In this study, RNA-seq and bioinformatics analyses revealed the impact of Trdn-as on intracellular Ca²⁺ signaling. Meanwhile, the Ca²⁺ levels within the SR and mitochondria were elevated following Trdn-as overexpression.

Ca²⁺ is transferred to the mitochondria through the IP3Rs-VDAC1 pathway, but other factors that may influence Ca²⁺ transfer must also be considered [40,41]. As the primary Ca²⁺ binding protein within the jSR of skeletal and cardiac muscles, Casq2 assumes a pivotal role in the storage and regulation of intracellular Ca²⁺ within the Ca²⁺ release unit, which is composed of the ryanodine receptor, histidine-rich Ca²⁺ binding protein, triadin, and junction [18]. Approximately 75% of the releasable Ca²⁺ within the SR is bound to Casq2 in muscle cells [42]. IP3Rs are predominantly localized at the intercalated discs of the myocardium, and RYR2 and Casq2 are abundantly found in the jSR, which is close to the mitochondria and has been implicated in the transport of Ca²⁺ to the mitochondria [43]. However, the factors mediating SR-mitochondrial Ca²⁺ transport within jSR are poorly understood. In this study, we observed the significant upregulation of Casq2 mRNA and protein levels in the heart tissues of TRDN-as-overexpressing mice. Knocking down Casq2 markedly attenuated the mitochondrial Ca²⁺ overload and dysfunction induced by Trdn-as overexpression in cardiomyocytes. These findings suggested that Casq2 plays a pivotal role in the regulation of mitochondrial Ca²⁺ overload by Trdn-as. Moreover, we found that knocking down Casq2 in cardiomyocytes reduced SR Ca²⁺ levels, consistent with the findings reported by Knollmann et al. [44]. Our study also demonstrated the potential influence of Casq2 on mitochondrial Ca²⁺ levels, leading to a notable decrease in MMP, impairments in mitochondrial respiratory function and energy production, and increased ROS production. Overall, this work provided additional compelling evidence to support the role of Casq2 as the paramount Ca²⁺ storage protein in cardiac SR. We further elucidated its crucial involvement in regulating mitochondrial Ca²⁺ levels.

TRDN, the gene responsible for encoding triadin, is a downstream target of Trdn-as. However, RNA-seq analysis did not reveal any significant changes in TRDN expression following Trdn-as overexpression. In addition, the GEO database GSE62203 supported the lack of significant alterations in TRDN levels with DCM (Fig. S6A). These pieces of evidence suggested that TRDN may not be a potential therapeutic target of Trdn-as in DCM. Finally, we further explored the regulatory mechanisms of Trdn-as. Transcription factors that potentially bind to the promoter region of Trdn-as were predicted by PROMO. We selected some transcription factors related to metabolic diseases. The results showed that the expression of transcription factor C/EBPα increased under high glucose condition (Fig. S6B), leading to an elevated Trdn-as expression upon C/EBPα overexpression (Fig. S6C). Therefore, the upregulation of C/EBPα could potentially contribute to Trdn-as upregulation in DCM.

We also discovered that Trdn-as influenced the Casq2 mRNA level, leading us to investigate its direct regulatory mechanism on Casq2 mRNA. m⁶A is the most prevalent RNA modification and can regulate mRNA stability [45]. Our results indicated that the knocking down Trdn-as significantly reduced the stability of Casq2 mRNA and the level of m⁶A, and overexpressing Trdn-as had the opposite effect. Trdn-as overexpression increased the m⁶A level of Casq2 mRNA. Other studies also discovered the regulatory effects of lncRNAs on mRNA m⁶A modification. Zhou et al. [46,47] revealed that lncRNA STEAP3-AS1 competed with YTHDF2, leading to the upregulation of mRNA stability in STEAP3 and subsequently an increase in STEAP3 protein expression. A separate investigation highlighted the role of lncRNA LINC02253 in promoting the m⁶A modification of KRT18 mRNA. This process stabilizes KRT18 mRNA by recruiting the m⁶A writer METTL3, ultimately exerting a favorable regulatory influence on the proliferation and spread of gastric cancer [48]. In our study, predictions from bioinformatics-related websites and experimental validation suggested that METTL14 may be partially involved in the regulation of Casq2 methylation. RIP experiments confirmed that METTL14 binds to Casq2 mRNA and Trdn-as. However, its expression level was not affected by Trdn-as, indicating that Trdn-as may function as a “molecular adhesive tape” to stabilize the METTL14–Casq2 mRNA complex. Moreover, METTL14 is prone to interact with lncRNA in specific circumstances. Wang et al. [49] identified a series of RGG/RG motifs in the C-terminal intrinsically disordered region (IDR) of METTL14, which are recognized as the second most common RNA binding domains in the human genome. The arginine methylation in this C-terminal IDR enhanced METTL14–RNA interaction and amplified the methyltransferase activity of METTL3/METTL14 [49]. The lncRNA UCA1 has been identified as a positive regulator of keratinocyte-driven inflammation and psoriasis. UCA1 exerts its regulatory function by binding to METTL14 and activating the HIF-1α and NF-κB signaling pathways [50]. Furthermore, the LNC942 molecule directly recruits the METTL14 protein, leading to the stabilization of downstream targets such as CXCR4 and CYP1B1 through m⁶A methylation modification in vitro and in vivo, thereby facilitating the proliferation of breast cancer cells [51]. Whether other m⁶A-related proteins mediate the regulatory effects of Trdn-as on Casq2 mRNA must be further investigated. Our study into Casq2 is currently limited to cell experiments. Further research is necessary to validate the role of Casq2 in DCM through animal experiments to provide valuable insights.

Previous studies have highlighted the RNA regulatory functions of this lncRNA. Zhang et al. [33] proposed a theoretical model suggesting that Trdn-as hinders the transcription of the skeletal muscle triadin by causing RNA polymerase II collision. Zhao et al. [32] indicated that Trdn-as is involved in mediating the alternative splicing of the triadin gene, thereby specifically controlling the levels of cardiac isoforms of triadin in cases of cardiac arrhythmias and heart failure. Although our data strongly suggested that Trdn-as regulates the m⁶A level of Casq2 mRNA, we cannot completely dismiss the possibility that it also regulates Casq2 expression in the heart through the previously proposed mechanism. Therefore, the role of Trdn-as in the regulation of transcription and alternative splicing of Casq2 must be explored in future studies. Our findings proposed that Trdn-as regulates the m⁶A modification of Casq2 mRNA to maintain cardiac function, contributing to the understanding of the RNA modulation effects of Trdn-as.

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