lncRNA Gm20257 alleviates pathological cardiac hypertrophy by modulating the PGC-1α–mitochondrial complex IV axis

Abstract

Pathological cardiac hypertrophy, a major contributor to heart failure, is closely linked to mitochondrial function. The roles of long noncoding RNAs (lncRNAs), which regulate mitochondrial function, remain largely unexplored in this context. Herein, a previously unknown lncRNA, Gm20257, was identified. It markedly increased under hypertrophic stress in vivo and in vitro. The suppression of Gm20257 by using small interfering RNAs significantly induced cardiomyocyte hypertrophy. Conversely, the overexpression of Gm20257 through plasmid transfection or adeno-associated viral vector-9 mitigated angiotensin II–induced hypertrophic phenotypes in neonatal mouse ventricular cells or alleviated cardiac hypertrophy in a mouse TAC model respectively, thus restoring cardiac function. Importantly, Gm20257 restored mitochondrial complex IV level and enhanced mitochondrial function. Bioinformatics prediction showed that Gm20257 had a high binding score with peroxisome proliferator–activated receptor coactivator-1 (PGC-1α), which could increase mitochondrial complex IV. Subsequently, Western blot analysis results revealed that Gm20257 substantially affected the expression of PGC-1α. Further analyses through RNA immunoprecipitation and immunoblotting following RNA pull-down indicated that PGC-1α was a direct downstream target of Gm20257. This interaction was demonstrated to rescue the reduction of mitochondrial complex IV induced by hypertrophic stress and promote the generation of mitochondrial ATP. These findings suggest that Gm20257 improves mitochondrial function through the PGC-1α–mitochondrial complex IV axis, offering a novel approach for attenuating pathological cardiac hypertrophy.

Keywords

lncRNA Gm20257 / cardiac hypertrophy / PGC-1α / mitochondrial complex IV / ATP

1 Introduction

Pathological cardiac hypertrophy results from various extrinsic or intrinsic factors, including prolonged and aberrant hemodynamic stress, chronic hypertension, valvular diseases, myocardial infarction, and genetic cardiomyopathy [1]. It is a major contributor to heart failure [2], which is one of the most serious public health concerns today [3]. Given its complicated processes, few effective treatments have been identified [4]. An improved knowledge of the molecular mechanisms behind this disease would be invaluable for developing effective diagnostic and treatment strategies for heart failure.

Long noncoding RNAs (lncRNAs), which are noncoding RNAs exceeding 200 nucleotides in length, can regulate gene expression at epigenetic, transcriptional, and posttranscriptional levels [5]. They regulate a wide range of physiological and pathological processes, including biological signaling, inflammatory responses, and cell proliferation and differentiation [6]. A growing body of evidence indicates that lncRNAs are involved in cardiac disease, particularly pathological cardiac hypertrophy [7,8]. lncRNA therapy may be a novel approach to slow or halt the progression of cardiac hypertrophy [9]. In our previous study, we found that an lncRNA, Gm20257, obviously increased under hypertrophic stress. However, the specific roles and mechanisms of lncRNA Gm20257 associated with cardiac diseases, such as cardiac hypertrophy, have not been fully explored.

Mitochondria play a critical role in cell energy generation, reactive oxygen species (ROS) production, Ca²⁺ homeostasis, cell survival, and apoptosis [10,11]. Emerging studies suggest that altered mitochondrial function is closely associated with the development of cardiac hypertrophy [12,13]. Approximately 90% of the ATP required for optimal cardiac function is produced by the mitochondrial oxidative phosphorylation system (OXPHOS) [14], which is composed of four electron transport chain (ETC) enzymes (complexes I–IV) and ATP synthase (complex V) [15]. Various changes in mitochondrial ETC complexes were observed in the hypertrophic heart [16,17], with complex IV showing a notable reduction [16]. Complex IV, the only cytochrome that transfers electrons to oxygen, is the final step in the mitochondrial ETC [18]. Peroxisome proliferator–activated receptor coactivator-1 (PGC-1α), a master regulator of mitochondrial function and biogenesis, is abundantly expressed in the heart [19,20]. PGC-1α coactivates ERRs or the NRF1 family to stimulate the expression of genes crucial for mitochondrial OXPHOS [21]. PGC-1α expression has been shown to be downregulated in response to pathological cardiac hypertrophy, which is associated with a reduction in ATP content [22,23].

This study aims to characterize the function of lncRNA Gm20257 in pathological cardiac hypertrophy and highlights the potential of Gm20257-regulated PGC-1α as a therapeutic target for cardiac hypertrophy.

2 Materials and methods

2.1 Animals

Male 12-week-old C57BL/6J mice (weighing 20–25 g) were obtained from the Experimental Animal Center of Harbin Medical University. The mice were housed under standard animal room conditions (temperature of 21 °C ± 1 °C and humidity of 55%–60%) with free access to food and water. All animal experiments followed the National Research Council’s Guide for the Care and Use of Laboratory Animals and received approval from the Institutional Animal Care and Use Committee of Harbin Medical University (No. HMUIRB-2020-04).

2.2 Transverse aortic constriction

For establishing the cardiac pressure overload model, 12-week-old (weighing 20–25 g) C57BL/6J mice were subjected to transverse aortic constriction (TAC) or sham surgery for 4 weeks as previously described [24]. Mice were anesthetized with 2% avertin at the dosage of 0.1 mL/10 g body weight through intraperitoneal injection (cat. T4840, Sigma–Aldrich) and subjected to open chest surgery. The arcus aortae was ligated by a 7-0 nylon suture line against a 27-gauge blunt needle. The blood flow velocity in the aortic arch was maintained at 2500–3000 mm/s. Mice were monitored for up to 4 weeks following TAC surgery.

2.3 Construction of adeno-associated virus serotype 9 carrying lncRNA Gm20257 and in vivo gene delivery

Adeno-associated virus serotype 9 (AAV9) vectors were used for in vivo gene delivery as described previously [25]. C57BL/6 mice received AAV9 injections containing the full-length lncRNA Gm20257 sequence to establish the Gm20257 overexpression model. An empty vector was administered to establish controls. The dosage was set at 1 × 10¹⁰ genome copies per mouse and administered through the tail vein 2 weeks prior to subsequent experiments. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Harbin Medical University.

2.4 Echocardiography

Mice were anesthetized with 2% avertin at the dosage of 0.1 mL/10 g body weight through intraperitoneal injection (cat. T4840, Sigma–Aldrich) and treated with a hair-removing cream on the chest. Subsequently, the mice were placed on a platform. Echocardiography was performed with a high-resolution Vevo2100 ultrasound imaging system (Visual Sonics, Toronto, ON, Canada). Cardiac contractile function and structure were evaluated by using M-mode recordings with a 10 MHz phased-array transducer as described previously [26]. Left ventricular ejection fraction and fractional shortening, diastolic left ventricular posterior wall (d) and wall thickness, LV inner diameter, and LV volume were calculated. LV dimension was derived from an average of six consecutive cardiac cycles per heart.

2.5 Cardiomyocyte culture and treatment

Cardiomyocytes were harvested from neonatal C57BL/6 mice (1–3 days) as previously described [26]. Briefly, cardiac tissues were sectioned into four parts and then digested with pancreatin (cat. C0201, Beyotime, Shanghai, China). Cells were kept in DMEM supplemented with 100 U/mL penicillin/streptomycin/amphot solution (cat. P7630, Solarbio, Beijing, China) and 10% fetal bovine serum (Biological Industries). After 2 h incubation, cardiomyocytes were separated by differential plating, and 0.1 mmol/L 5-bromo-2-deoxyuridine was introduced to restrain the growth of cardiac fibroblasts. Cells were then cultured in a 5% CO₂ and 37 °C humidified atmosphere for 48 h after plating.

Angiotensin II (Ang II) (cat. MB1677, Meilunbio, Dalian, China) was administered to cardiomyocytes at a concentration of 1 μmol/L for 48 h to induce cardiomyocyte hypertrophy [27].

lncRNA Gm20257 overexpression plasmids were constructed by GeneChem (Shanghai, China) by using pLVX-mCMV-ZsGreen carrying the lncRNA Gm20257 sequence. An empty pLVX-mCMV-ZsGreen vector was used as a negative control (NC). Si-Gm20257, si-PGC-1α, and their corresponding controls were produced by RIBOBIO (Guangzhou, China). si-Gm20257 and si-PGC-1α were transfected at concentrations of 50 nmol/L according to the manufacturer’s recommendations. The interference sequences were as follows: Si-Gm20257, AGCAAGACTGGAACATAAA; Si-PGC-1α, GCAATAAAGCGAAGAGCAT. Plasmids or siRNAs were transfected by using Lipo2000 (cat.11668019, Invitrogen, Carlsbad, CA, USA) by following the manufacturer’s protocol.

2.6 Western blot analysis

Mouse heart tissue and cultured cardiomyocytes were lysed in RIPA lysis standard sample buffer (cat. P0013B, Solarbio, Beijing, China) added with a protease inhibitor (cat. pi003, Biocolor, Shanghai, China). Protein concentrations were detected by a BCA protein assay kit (cat. P0010, Beyotime, Shanghai, China). Samples containing equivalent amounts of protein (60–80 μg) were subjected to SDS–PAGE and subsequently transferred to a nitrocellulose filter membrane. The primary antibodies utilized included antimyosin antibody (cat. M8421, 1:5000, mouse monoclonal; Sigma–Aldrich, St. Louis, MO, USA), total OXPHOS rodent (cat. ab110413, Abcam, Cambridge, UK), and anti-PGC-1α (cat. ab54481, 1:1000, mouse polyclonal; Abcam, Cambridge, UK). Proteins were normalized to GAPDH (cat. TA802519, 1:1000, mouse polyclonal; OriGene, USA). After incubation with the primary antibodies overnight at 4 °C, the membranes were rinsed with PBST and subsequently incubated with the secondary antibodies at room temperature for 60 min. Protein bands were visualized by using Odyssey software v1.2. ImageJ software was applied to calculate the gray value.

2.7 Real-time PCR analysis

TRIZOL reagent (cat. 15596026, Invitrogen, Carlsbad, CA, USA) was used for RNA extraction from mouse cardiac tissue or cultured cardiomyocytes. RNA was then reverse-transcribed with 5× All-InOne RT MasterMix (cat. G490, ab, Jiangsu, China). By using an ABI 7500 fast real-time PCR system (Applied Biosystems, Foster City, CA, USA) with TransStart Top Green qPCR SuperMix (cat. N20922, TransGen Biotech, Beijing, China), real-time qPCR was cycled at 95 °C/15 s, 60° C/30 s, and 72 °C/30 s for 40 cycles following an initial denaturation step at 95 °C for 10 min. This process aims to quantify the RNA levels of lncRNA Gm20257, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (β-MHC). GAPDH was set as the endogenous control. Dissociation curve software was used to check products regularly. The relative value to the control sample was analyzed through the 2^−ΔΔCT method. Primers were synthesized by Sangon Biotech (Shanghai, China) and are listed in Tab.1.

2.8 Transmission electron microscopy

At 2 weeks after AAV9 injection and 4 weeks after TAC or sham surgery, heart structure and function were detected by echocardiography. Next, mouse hearts were harvested, and left ventricles were isolated. The experimental procedure was performed as in a previous study [26]. Heart slices were fixed in 2.5% glutaraldehyde solution (containing 2% glutaraldehyde, 2% paraformaldehyde, and 0.1 mol/L cacodylate) for 12 h at 4 °C. Samples were then postfixed with 1% osmium tetroxide for 2 h at 4 °C and then rinsed three times in HEPES buffer before undergoing dehydration in graded ethanol solutions for 10 min each at 4 °C. By adhering to standard protocols for TEM sample preparation, samples were embedded in LR White resin and polymerized at 60 °C for 48 h.

2.9 Immunofluorescence analysis of cell surface area

Cardiomyocyte cross-sectional area was determined as previously described [28]. Briefly, cells were fixed with 4% formaldehyde for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 45 min, and then stained with α-actinin primary antibodies (cat. ab68194, Abcam, Cambridge, UK) overnight at 4 °C. Cells were then incubated for 1 h at room temperature with a Daylight 594 goat antimouse antibody and subsequently stained with DAPI (cat. C0065, Solarbio, Beijing, China) for 10 min before immunofluorescence capture. A fluorescence microscope (Carl Zeiss, Germany) was utilized to detect immunofluorescence. ImageJ software was used to calculate cell surface area.

2.10 Histological analysis

Mouse heart tissue samples were obtained and fixed in 4% paraformaldehyde (cat. P1110, Solarbio, Beijing, China) for 48 h before being dried and embedded in paraffin (cat. YA012, Solarbio, Beijing, China) in accordance with normal histology methods [29]. Next, the tissues were sectioned into 5 μm–thick cross-sectional slices. For the assessment of myocyte cross-sectional areas, tissue slices were stained with fluorescein isothiocyanate–conjugated wheat germ agglutinin (cat. L4895; Sigma, St. Louis, MO, USA) as per the manufacturer’s instructions.

2.11 Mitochondrial membrane potential assessment

A JC-1 staining kit (cat. M8650, Solarbio, Beijing, China) was used to assess the mitochondrial membrane potential (MMP) of cardiomyocytes. The JC-1 solution was prepared in accordance with the manufacturer’s instructions. DMEM buffer (cat. 2232193, VivaCell Bioscence, Shanghai, China) was used to wash cardiomyocytes. A cell culture plate was immersed in 1 mL of DMEM and an equal volume of JC-1 staining solution for 30 min. Following incubation, red and green immunofluorescence images were captured by using a fluorescent microscope (Carl Zeiss, Germany). MMP was calculated on the basis of the ratio of red to green fluorescence. ImageJ software was used to calculate the intensity of fluorescence.

2.12 ROS detection

A ROS assay kit (cat. MA0219, Meilun, Dalian, China) was applied to measure ROS generation in cardiomyocytes in accordance with the manufacturer’s instructions. The sensitive probe for detecting intracellular ROS was 2,7-dichlorofluorescin diacetate (DCFH-DA). DCFH-DA is enzymatically degraded into dichlorofluorescin (DCFH) once delivered into cells. ROS could oxidize DCFH into dichlorofluorescein (DCF), which emits green fluorescence. Fluorescence intensity is positively correlated with ROS levels in cells. After treatment, cardiomyocytes were washed with PBS and then incubated for 30 min with DCFH-DA diluted in DMEM. DCFH-DA was then removed, and cardiomyocytes were washed with PBS and photographed by using a fluorescence microscope (Carl Zeiss, Germany). ImageJ software was used to calculate fluorescence intensity.

2.13 ATP content

ATP levels were determined by using an ATP assay kit (cat. S0026, Beyotime, Shanghai, China) in accordance with the manufacturer’s instructions. Lysis buffer was used to lyse cardiomyocytes. After lysis, the supernatant was collected post centrifugation. An assay tube was filled with 100 μL of ATP working solution and 20 μL of cell lysate. A luminometer (GLOMA X 20/20, Promega) was used for measurements. A standard curve was applied to calculate ATP concentrations. The unit of measurement for ATP content is expressed as nmol/mg protein.

2.14 Fluorescent in situ hybridization

Fluorescent in situ hybridization (FISH) was performed with a FISH kit (RiboBio, Guangzhou, China) in accordance with the manufacturer’s instructions. Cardiomyocytes were placed on a slide, fixed with 4% paraformaldehyde solution for 10 min, and washed three times with 1× PBS for 5 min. Cells were permeabilized with 0.4% Triton-X100 (cat. IT9100, Solarbio, Beijing, China) for 10 min, blocked with 1% BSA, and incubated in prehybridization buffer at 37 °C for 30 min. The lncRNA Gm20257 probe–containing hybridization or internal control FISH probe was then added to each slide overnight at 37 °C. Nuclei were stained with DAPI, and images were captured by using a fluorescence microscope.

2.15 RNA-binding protein immunoprecipitation

RNA-binding protein immunoprecipitation (RIP) was performed by using a Magna RIP™ RNA RIP kit (cat. 17-700, EMD Millipore, Darmstadt, Germany) in accordance with the manufacturer’s instructions. Briefly, after lysing specimens, complexes were incubated with anti-PGC-1α (cat. ab54481, 1:500, mouse polyclonal; Abcam, Cambridge, UK) and antirabbit IgG (ab172730, 1:500, mouse polyclonal; Abcam, Cambridge, UK) antibodies overnight at 4 °C. The antibody-bound complex was then incubated with protein G magnetic beads for 3 h at room temperature. RNA was then isolated, extracted, and reverse-transcribed into cDNA. The levels of lncRNA were determined through RT-qPCR and agarose gel electrophoresis.

2.16 RNA pull-down

A T7 RNA polymerase kit (cat. R7012L, Beyotime, Beijing, China) was used to transcribe plasmids, and Biotin RNA Labeling Mix (cat. 11685597910, Thermo Scientific, Carlsbad, USA) was utilized for labeling in accordance with the manufacturer’s instructions. Biotinylated RNAs were purified with RNase-free DNase I (cat. EN0521, Invitrogen, Carlsbad, USA). Cell lysates were preincubated with streptavidin beads (cat. HY-K0208, MedChemExpress, Shanghai, China) before being combined with biotinylated RNA at 4 °C for 2 h. RIP buffer was added to the beads, which were then boiled in SDS buffer, to release bound RNAs and proteins. Protein binding to lncRNA Gm20257 was visualized through immunoblotting. ImageJ software was used to calculate gray values.

2.17 Data analysis

Data were presented as mean ± SEM and analyzed by using appropriate statistical analysis methods. Shapiro–Wilk normality and the D'Agostino and Pearson tests were used to examine data distribution for statistical analysis. Experimental data were found to adhere to a normal distribution. Moreover, the F-test (for two groups) or Brown–Forsythe test (for three or more groups) was used to evaluate variance homogeneity (three or more groups). In cases of two groups, statistical significance was determined through an unpaired Student’s t-test (equal variance) or the Welch t-test (unequal variance). For comparisons among multiple groups, significance was determined by performing one-way ANOVA with Bonferroni post hoc analysis (equal variance) or the Brown–Forsythe and Welch ANOVA test with Dunnett’s T3 post hoc analysis (unequal variance). Statistical significance was defined as P < 0.05. GraphPad Prism 8.2.1 was employed for data analysis.

3 Results

3.1 Expression of lncRNA Gm20257 during cardiac hypertrophy

We examined the changes in the expression of lncRNA Gm20257 in response to hypertrophic stimuli in vivo and in vitro to understand its role in pathological cardiac hypertrophy. TAC surgery was employed to induce mouse pathological cardiac hypertrophy (Fig.1 and 1B). The cardiomyocyte hypertrophy model was established by imposing Ang II treatment for 48 h (Fig.1 and 1E). Gm20257 was overexpressed in hypertrophic hearts 4 weeks after TAC (Fig.1). The expression of Gm20257 increased in cultured neonatal cardiomyocytes after 48 h of Ang II treatment (Fig.1). Furthermore, FISH revealed that Gm20257 was predominantly located in the cytoplasm relative to the nucleus (Fig.1). The UCSC database indicated that Gm20257 is a 936 nucleotide–long intergenic lncRNA located on chromosome 1 (Fig. S1). By blasting the sequence of Gm20257 against the human genome, we found a conserved sequence motif (147 nt) in the human genome (Fig. S2A–S2C).

3.2 lncRNA Gm20257 suppressed cardiomyocyte hypertrophy in vitro

The effects of the depletion of Gm20257 on cardiomyocytes were assessed to investigate its role in pathological cardiac hypertrophy. When compared with that of NCs, the transfection of small interfering RNAs of Gm20257 obviously reduced the level of Gm20257 (Fig.2). In the absence of any treatment, Gm20257 suppression increased the cross-sectional area of cardiomyocytes by approximately 1.5-fold (Fig.2). Furthermore, the mRNA levels of the hypertrophic markers ANP, BNP, and β-MHC and protein level of β-MHC in si-Gm20257 groups were considerably elevated relative to those in NC groups (Fig.2 and 2D).

Gain-of-function experiments were conducted by transfecting cells with the Gm20257 overexpression plasmid, as shown in Fig.2. The overexpression of Gm20257 effectively reduced the Ang II-induced enlargement of cross-sectional area, as depicted in Fig.2. The mRNA levels of ANP, BNP, and β-MHC remarkably elevated in response to Ang II. However, compared with the vector control, the Gm20257 overexpression plasmid suppressed the increase in the mRNA levels of ANP, BNP and protein level of β-MHC, as shown in Fig.2 and 2H. Notably, Gm20257 overexpression lacked a discernible effect on cardiomyocytes under normal conditions, while only the mRNA level of BNP decreased (Fig.2–2H). These results suggest that Gm20257 acts as a cardioprotective lncRNA against hypertrophic cardiomyopathy in vitro.

3.3 lncRNA Gm20257 overexpression attenuates TAC-induced cardiac hypertrophy in the mouse heart

The effects of Gm20257 on the cardiac hypertrophy model were investigated in vivo. AAV9 containing Gm20257 was synthesized, and the Gm20257-overexpressing adenovirus was administered at a dose of 10¹⁰ titers/mouse. The effective overexpression of Gm20257 in cardiac tissue was confirmed (Fig.3). The Gm20257 overexpression plasmid diminished the increase in HW/BW, LW/BW, and HW/TL ratios caused by TAC surgery compared with these parameters of the vector control (Fig.3). Echocardiographic indicators were detected (Tab.2), with results indicating that ejection fraction (EF) and fraction shortening (FS) were remarkably higher in the Gm20257 overexpression group than in the vector control group following TAC (Fig.3 and 3D). TAC surgery substantially increased left ventricular posterior wall diastole (LVPW; d), whereas Gm20257 overexpression greatly reduced this change. TEM revealed that myofilament dissolution, mitochondrial organization loss, and structural damage occurred in the TAC group, and the Gm20257 overexpression plasmid, but not the vector, prevented these malignant changes. The Gm20257 overexpression plasmid reduced TAC-induced cardiac enlargement (wheat germ agglutinin) when compared with the vector control (Fig.3). The mRNA levels of ANP, BNP, and β-MHC, as well as the protein level of β-MHC were consistently lower in Gm20257 overexpression mice than in vector control mice after TAC surgery (Fig.3 and 3G). Gm20257 overexpression in mice did not result in observable changes in heart shape or function at the baseline (Fig.3–3G). These findings indicate that the overexpression of Gm20257 could prevent pressure overload-induced cardiac hypertrophy.

3.4 Gm20257 attenuated hypertrophic stress-induced mitochondrial injury by augmenting mitochondrial complex IV

The mechanism of Gm20257’s cardioprotective effects was then explored. Pathway enrichment analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database predicted that Gm20257 was highly linked with the mitochondrial oxidative phosphorylation pathway (Fig.4). The Gene Ontology (GO) database analysis of Gm20257’s interacting proteins revealed significant enrichment in mitochondrial electron transport, particularly the transfer of electrons from cytochromes to oxygen mediated by mitochondrial complex IV (Fig.4). Consequently, the effect of Gm20257 on mitochondrial ETC proteins was examined. Consistent with a previous study, this work found that mitochondrial complex IV, as represented by MTCO1 [30], was suppressed in TAC mice (Fig. S3). However, Gm20257 overexpression remarkably counteracted the hypertrophic stress-induced reduction in mitochondrial complex IV (Fig.4 and 4D).

Given that the blockage of mitochondrial complex IV impedes electron flow and MMP, mitochondrial functionality was detected. The inhibition of Gm20257 induced mitochondrial membrane depolarization, excessive ROS production, and ATP content reduction (Fig.4–4G). MMP depolarization, increased ROS generation, and inhibited ATP synthesis were observed following Ang II treatment. The Gm20257 overexpression plasmid, but not the vector control, reversed these adverse changes (Fig.4–4J). These results suggest that Gm20257 enhances mitochondrial complex IV to maintain mitochondrial OXPHOS and promote ATP production.

3.5 Gm20257 binds to PGC-1α to regulate its expression

The molecular mechanism underlying Gm20257’s protective effect on the expression of mitochondrial complex IV was explored. Bioinformatics analysis identified high affinity between PGC-1α and Gm20257 (Fig.5, 5B, and S4A). PGC-1α is known for coactivating multiple nuclear receptors to regulate genes required for mitochondrial OXPHOS [21]. On the basis of bioinformatics data and the association between PGC-1α and mitochondrial function, Gm20257 is hypothesized to promote mitochondrial complex IV through PGC-1α. PGC-1α expression was reduced in response to hypertrophic stress (Fig.5 and 5D). The decline in PGC-1α induced by TAC and Ang II was counteracted by Gm20257 overexpression (Fig.5 and 5F). Gm20257 inhibition led to decreased PGC-1α (Fig.5).

Subsequent experiments validated the direct regulatory effect of Gm20257 on PGC-1α. The sense sequence, but not the antisense sequence, of Gm20257 substantially pulled down PGC-1α (Fig.5). Conversely, the PGC-1α antibody captured a substantial amount of Gm20257 (Fig.5).

3.6 PGC-1α mediates the regulatory effect of Gm20257 on mitochondrial and cardiac hypertrophy

Cultured cardiomyocytes were cotransfected with Gm20257 overexpression plasmids and si-PGC-1α and then treated with Ang II to explore the role of PGC-1α in mediating the function of Gm20257. Gm20257 overexpression and PGC-1α inhibition were achieved (Fig. S4B and S4C). Transfection with the Gm20257 overexpression plasmid alone protected cardiomyocytes from the Ang II-induced reduction in mitochondrial complex IV, depolarization of MMP, increase in ROS production, and restriction of ATP production. However, in contrast to that of Gm20257 overexpression + si-NC, cotransfection with si-PGC-1α diminished these protective effects, as evidenced by the decrease in mitochondrial complex IV and MMP, increase in ROS, and restriction of ATP content (Fig.6–6D).

Gm20257 overexpression alone protected cardiomyocytes from Ang II-induced hypertrophy. However, in contrast to Gm20257 overexpression + si-NC treatment, si-PGC-1α cotransfection reversed these advantages, as evidenced by the increase in cellular area; mRNA levels of ANP, BNP, and β-MHC; and protein levels of β-MHC (Fig.6–6G). These findings indicate that Gm20257 improves mitochondrial function via the PGC-1α–mitochondrial complex IV axis, thereby attenuating cardiac hypertrophy.

4 Discussion

In this study, the upregulation of lncRNA Gm20257 during cardiac hypertrophy was observed along with its protective role in safeguarding the heart by preserving mitochondrial function. Mechanistically, Gm20257 directly binds to PGC-1α. This interaction prevents the reduction in PGC-1α and subsequently increases mitochondrial complex IV. This effect leads to increased ATP generation, which in turn protects cardiomyocytes from energy deficits (Fig.7). The study is distinct due to the following aspects: (1) Gm20257 is a novel molecule that counteracts cardiac hypertrophy; (2) Gm20257 stimulates mitochondrial activity to increase ATP generation; (3) Gm20257 exerts its effects through the PGC-1α–mitochondrial complex IV axis.

Pathological cardiac hypertrophy exhibits a complicated pathogenesis that is characterized by alterations in energy metabolism [31]. Decompensated energy metabolism has been identified as a key driver of malignant myocardial remodeling in the progression of pathological cardiac hypertrophy [32]. The mitochondrion, a critical ATP-producing organelle, generates approximately 90% of cellular ATP [33]. Mitochondria produce ATP through OXPHOS, which is composed of four ETC enzymes (complexes I–IV) and ATP synthase (complex V) located in the mitochondrial inner membrane [34]. Therefore, identifying agents that can modulate mitochondrial function to enhance ATP synthesis is of paramount importance.

Substantial evidence suggests that lncRNAs are crucial regulators of pathological cardiac hypertrophy [35–37]. However, few lncRNAs have been shown to influence OXPHOS. In this study, we investigated dysregulated lncRNAs in cardiac hypertrophy and identified Gm20257 as a critical regulator. Notably, a bioinformatics assay indicated a strong association between Gm20257 and OXPHOS. Experimental findings further suggest that Gm20257 might regulate mitochondrial ATP synthesis. These findings provide considerable evidence of Gm20257’s beneficial role in heart hypertrophy.

We demonstrated that Gm20257 protects the heart from cardiac hypertrophy through a novel molecular mechanism involving mitochondrial complex IV. Complex IV, the only cytochrome that transfers electrons to oxygen [38], plays a crucial role in the mitochondrial ETC, considerably influencing ATP synthesis. Early research from 1990 reported the reduced expression and activity of mitochondrial complex IV during human ventricular hypertrophy and heart failure [39]. Numerous subsequent studies have revealed that mitochondrial COX dramatically decreases in cardiac hypertrophy [40,41]. Consistent with previous studies, our work showed that the expression of mitochondrial complex IV dramatically reduced in our hypertrophic model. Importantly, our study demonstrated that Gm20257 acts to preserve mitochondrial complex IV content, thereby mitigating its dysfunction. Gm20257 serves as a critical regulator of mitochondrial complex IV, enhancing ATP generation and, in turn, contributing to the prevention of cardiac hypertrophy.

The biological activity of lncRNAs is believed to be closely related to their subcellular location and genetic origin [42]. We found that Gm20257 mainly localizes in the cytoplasm. Mitochondrial complex IV, with subunits encoded by nuclear and mitochondrial genomes, is thus likely influenced by Gm20257 via PGC-1α, affecting both genomes. PGC-1α is highly expressed in the heart and acts as a key regulator of genes essential for mitochondrial OXPHOS in the nuclear and mitochondrial domains [21]. Consistent with previous studies, our study discovered that PGC-1α expression decreased in hypertrophic hearts and cardiomyocytes. We further demonstrated that Gm20257 bound to PGC-1α and promoted its expression in in vivo and in vitro contexts. Furthermore, we discovered that the inhibition of PGC-1α negated the protective effect of Gm20257 on mitochondrial complex IV and, consequently, on mitochondrial function. Therefore, Gm20257 enhances mitochondrial complex IV expression by directly upregulating PGC-1α during cardiac hypertrophy. Given that cardiac hypertrophy is a multifaceted process that involves various mechanisms, other factors beyond the PGC-1α–mitochondrial complex IV axis may contribute to the inhibition of cardiac hypertrophy by Gm20257. This phenomenon remains an area for future exploration.

Notably, we discovered that Gm20257 was upregulated in response to hypertrophic stress. The overexpression of Gm20257 attenuated cardiac hypertrophy in vivo and in vitro, while its inhibition promoted cardiomyocyte hypertrophy. We speculated that Gm20257 upregulation in response to hypertrophic stress is adaptive and protective. Initially, in response to hypertrophic stress, the adaptive mechanism triggers the upregulation of Gm20257, aiming to counteract harmful changes. However, at the time point of our hypertrophic model, the Gm20257 level was insufficient to resist the alterations generated by hypertrophic stress, and heart hypertrophy exacerbated. Therefore, we overexpressed Gm20257 at the beginning of cardiac hypertrophy to maintain Gm20257 content at high levels. This approach sufficiently supplemented Gm20257 content, resulting in a robust anti-cardiac hypertrophic effect. The complexity of lncRNA functions and mechanisms is evident in our study, which showed that Gm20257 is upregulated during hypertrophy, thus playing a protective role that is similar to the action of lncRNA H19 [43].

Our study identified lncRNA Gm20257 as an antihypertrophic molecule that directly regulates PGC-1α to promote mitochondrial OXPHOS function. Our findings provide a novel strategy for mitigating pathological cardiac hypertrophy by improving cardiac energy supply, potentially offering substantial benefits. They suggest that lncRNA Gm20257 could serve as a therapeutic agent in treating cardiac hypertrophy.

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