Long noncoding RNA LncBAR enhances BRG1 protein to promote cardiomyocyte cell cycle progression and cardiac repair

Abstract

The mammalian heart retains regenerative capacity during the early postnatal period, but this ability declines as it matures. Enhancing cardiomyocyte proliferation represents a key therapeutic approach to promote heart regeneration and repair, yet the molecular mechanisms remain elusive. Here, we identified LncBAR (BAF complex-associated lncRNA) as a critical regulator of cardiac regeneration. LncBAR expression declines during heart development but is upregulated following cardiac injury. Loss of LncBAR impairs cardiomyocyte growth, suppresses cell cycle gene expression, and diminishes heart regeneration, as evidenced by reduced cytokinesis and cardiac function. Conversely, cardiac specific overexpression of LncBAR restores cardiomyocyte proliferation and enhances cardiac regeneration, especially in adult myocardial infarction model. Mechanistically, LncBAR interacts with Brg1, stabilizing BRG1 protein level and activating cell cycle progression to drive cardiomyocytes proliferation. Collectively, our study identified LncBAR as a crucial regulator for heart regeneration, highlighting the LncBAR-BRG1 axis as a promising therapeutic strategy for cardiac repair.

Introduction

Heart disease results in massive loss of cardiomyocytes (CMs) and is among the leading causes of morbidity and mortality worldwide. Due to the limited regenerative capacity of adult mammalian hearts, the infarcted heart fails to generate new CMs to replenish those lost during injury, leading to cardiac remodeling and eventually heart failure^1,2,3. In contrast, the neonatal mammalian heart possesses a transient regenerative capacity through cardiomyocyte proliferation that is limited to the first week after birth and is lost shortly thereafter^4,5,6. Growing evidence suggests that inducing adult CM re-entry into the cell cycle can remuscularize the injured myocardium and promote cardiac repair^7,8,9,10. Therefore, deciphering the regulators and molecular mechanisms that stimulate CM proliferation holds promising potential for developing therapeutic strategies to treat ischemic heart diseases in adults.

The Switch/Sucrose Non-Fermentable (SWI/SNF) complex is an ATP-dependent chromatin remodeling complex that changes the accessibility of transcription factors to DNA¹¹. Among its multiple subunits, the essential ATPase subunit Brg1 (also known as Smarca4)¹² plays critical roles in cardiac growth and differentiation under developmental and pathological conditions^13,14,15. During zebrafish heart regeneration, BRG1 interacts with DNA methyltransferase Dnmt3ab to suppress cyclin-dependent kinase inhibitors, such as Cdkn1c, thereby promoting the CM proliferation¹⁶. In mammalian heart, BRG1 expression is typically silenced in adult CMs but can be reactivated in response to cardiac stresses¹⁴. Recent studies have shown that forced expression of BRG1 protects heart from acute myocardial infarction by reducing oxidative damage and cell apoptosis¹⁵. Despite its biological significance, the regulatory mechanisms governing BRG1 expression remain largely unknown.

Long noncoding RNAs (lncRNAs) are nonprotein coding transcripts longer than 200 nucleotides¹⁷. Recent studies have demonstrated that lncRNAs play crucial roles in various cardiovascular diseases^{18,19,20,21,22,23}. LncRNAs regulates gene expression at multiple levels, including transcription, RNA splicing and protein translation to participate in the cardiac remodeling and regeneration. We recently identified an intergenic lncRNA termed LncBAR that binds with SWI/SNF complex, particularly with BRG1, to regulate neocortical development²⁴. However, the contribution of LncBAR in the regulation of cardiac regeneration and its association with BRG1 remain less understood.

In this study, we investigated the role of LncBAR in cardiomyocyte proliferation and postnatal cardiac regeneration. LncBAR deficiency blocked cardiomyocyte proliferation and impaired cardiac function recovery after apical resection (AR). In contrast, overexpressing LncBAR enhanced cardiac regeneration and post-AR heart functions. Mechanistically, LncBAR stabilized BRG1 protein levels, thereby stimulating cell cycle progression and promoting cardiomyocyte proliferation. Collectively, our results revealed that LncBAR acts as an activator of cardiomyocyte proliferation and present a promising strategy to enhance cardiac repair.

Results

LncBAR regulates murine neonatal cardiomyocyte proliferation

To investigate the potential role of LncBAR in heart regeneration, we assessed LncBAR expression levels in the hearts across different developmental stages, from embryo (embryonic day 13.5) to adulthood (12 weeks old), using quantitative reverse transcription-polymerase chain reaction (RT-qPCR). We observed that, under physiological conditions, LncBAR expression gradually decreased during cardiac development (Fig. 1a). Following apical resection (AR) in wild-type (WT) mice at day 1 (P1), LncBAR levels increased specifically in the apical zone (AZ) at both d7 and d21 post-AR (AR7 and AR21), compared to the border and remote zones (BZ and RZ) (Fig. 1b). Furthermore, following myocardial infarction (MI), LncBAR expression was markedly increased in the infarct zone (IZ) and moderately upregulated in the border zone (BZ) (Fig. 1c). These results suggested a positive correlation between LncBAR expression and cardiac regeneration, prompting us to further investigate its role in the postnatal heart.

Fig. 1: Long non-coding RNA (LncBAR) regulates cardiomyocyte proliferation.

a Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis of LncBAR expression in murine hearts at indicated time points (embryonic day (E) 13.5; postnatal days (P) 1 to P14, and 4 and 12 weeks (W)) (n = 4 mice). b Relative LncBAR expression in hearts at 7 (AR7) and 21 (AR21) days post-apical resection (AR), determined by RT-qPCR. RZ: remote zone; BZ: border zone; AZ: apex zone (n = 3 mice). c Relative LncBAR expression in murine hearts at 4 weeks post-myocardial infarction (MI) by RT-qPCR. RZ: remote zone; BZ: border zone; IZ: infarct zone. (n = 3 mice). d ICC analysis of EdU-incorporated NMCMs for cTnI (cardiomyocyte marker). Cells were transduced with shLncBAR, control shNT, or PBS and cultured with EdU for 1 day prior to staining. White arrows indicate EdU+ NMCMs. Scale bars, 50 µm. e Proportions of total (cTnI+) and proliferating (EdU+ cTnI+) NMCMs 4 days after treatment with shLncBAR, shNT, or PBS. (n = 15 fields). f Representative ICC and quantification of cTnI+ cells in EdU-incorporated NMCMs following transduction with lentivirus harboring LacZ or LncBAR, or treatment with PBS (vehicle control), after a 24 h EdU pulse. White arrows indicate EdU+ NMCMs (n = 15 fields). Scale bars, 50 µm. g Representative ICC and quantification of cTnI and Ki67 in NMCMs treated with or without lentiviral LncBAR. PBS was used as non-viral treatment control. White arrows indicate Ki67+ NMCMs (n = 15 fields). Scale bars, 50 µm. h Representative ICC and quantification of cTnI and Aurora B in NMCMs treated with or without lentiviral LncBAR. PBS was used as non-viral treatment control. White arrows indicate Aurora B+ NMCMs (n = 5 biological replicates). Scale bars, 50 µm. i, j RT-qPCR analysis showing the expression of genes positively regulating the cell cycle and proliferation upon LncBAR knockdown (i) or overexpression treatment (j) (n = 3 biological replicates). Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.

To explore this, we designed lentivirus encoding a panel of short hairpin RNA (shRNA) targeting LncBAR and the knockdown efficiency achieved approximately 80–90% (Supplementary Fig. 1a). Importantly, knocking down LncBAR had minimal effect on the expression of its two neighboring genes, Zfp207 and Psmd11 in the neonatal mouse cardiomyocytes (NMCMs) (Supplementary Fig. 1b). We then performed immunofluorescence staining (ICC) against cTnI, a marker of cardiomyocytes and 5-ethynyl-2´-deoxyuridine (EdU), a nucleotide analog that labels cells passing through the S phase, at 4 days post viral infection (Fig. 1d). ICC results indicated that LncBAR knockdown significantly reduced the number of EdU+ cardiomyocytes and total cardiomyocytes (Fig. 1e), suggesting that LncBAR may regulate NMCM proliferation. Individual shLncBAR oligonucleotides showed similar suppression effects on NMCM proliferation as pooled oligonucleotides. For the rest of the study, we used pooled shLncBARs (referred to as shLncBAR) unless otherwise indicated (Supplementary Fig. 1c).

We next performed gain-of-function experiments by delivering lentiviral LncBAR overexpression construct into NMCMs. Lentiviruses carrying LacZ fragment were used as control, and PBS was used as non-viral treatment control. Transduction efficiency, assessed via GFP, increased with MOI from 25 (~20% transduction, ~20-fold LncBAR expression) to 100 (~94% transduction, ~250-fold expression). Overexpression at MOI 50 significantly increased the number of Ki67+ cardiomyocytes, which was further enhanced at MOI 100. Therefore, MOI 100 was used in all subsequent in vitro experiments (Supplementary Fig. 1d–f).

Overexpression of LncBAR in NMCMs remarkably elevated EdU-positive cardiomyocytes (Fig. 1f). We used another active proliferation marker, Ki67, and cardiac troponin I (cTnI) to mark proliferating CMs. Congruently, we observed an increase in the number of both cTnI+ NMCMs and Ki67+ cTnI+ proliferating NMCMs after LncBAR overexpression (Fig. 1g). We also used marker of cytokinesis (Aurora B) and cTnI. The results showed that overexpression of LncBAR significantly increased the number of Aurora B+ CMs and total CMs (Fig. 1h). Furthermore, we conducted propidium iodide (PI) staining in LncBAR-overexpressing HL-1 cardiomyocytes, which showed a significant shift from G0/G1 to S and G2/M phases, indicating enhanced cell cycle progression (Supplementary Fig. 1 g). To assess actual cell division, we quantified NMCM numbers on day 4 after LncBAR or control lacZ overexpression, and found a significant increase in cardiomyocyte counts following LncBAR overexpression (Supplementary Fig. 1h). At the transcription level, RT-qPCR analyses showed that shLncBAR treatment significantly downregulated genes positively regulating the cell cycle and cell proliferation (Fig. 1i), while LncBAR overexpression increased the expression of these genes (Fig. 1j). Collectively, our data indicate that LncBAR plays a key role in regulating NMCM proliferation.

LncBAR depletion inhibits cardiomyocyte proliferation in postnatal hearts

To investigate the role of LncBAR in vivo, we generated LncBAR knockout (KO) mice (Fig. 2a) using a CRISPR/Cas9-mediated gene editing approach²⁴. Genotyping results confirmed successful LncBAR depletion (Fig. 2b), and RT-qPCR analysis validated deficient expression of LncBAR in KO hearts compared with controls (Fig. 2c). We assessed CM proliferation in LncBAR KO and WT hearts at postnatal days P3 and P7. LncBAR depletion remarkably inhibited the proliferation of CMs at P3 and P7, as indicated by co-staining of cTnT with mitosis markers including phospho-histone H3 (pH3) and Ki67 (Fig. 2d–g).

Fig. 2: LncBAR deficiency inhibits cardiomyocyte proliferation in postnatal hearts.

a Schematic depiction of LncBAR knockout (KO) mice and the genotyping strategy. F and R, primers designed for genotyping. b Representative genotyping results from wild-type and LncBAR KO (LncBAR^−/−) mice using PCR primer pairs shown in (a) (n = 3 mice). c Expression level of LncBAR in WT and LncBAR^−/− hearts as evaluated by RT-qPCR (n = 3 mice). Representative confocal z-stack images and quantification of cTnT+ Ki67+ (d) or cTnT+ pH3+ CMs (e) in heart sections from neonatal WT and LncBAR^−/− hearts at P3. Nuclei were labeled with DAPI. White arrows indicate Ki67+ or pH3+ CMs (n = 15 fields, 3 mice/group). Scale bars, 20 µm f, g Representative confocal z-stack images and quantification of cTnT+ Ki67+ (f) or cTnT+ pH3+ CMs (g) in heart sections from neonatal WT and LncBAR^−/− hearts at P7. Nuclei were labeled with DAPI. White arrows indicate Ki67+ or pH3+ CMs (n = 15 fields, 3 mice/group). Scale bars, 20 µm. Data are presented as mean ± SEM. Statistical significance was determined by two-tailed unpaired t test. **P < 0.01, ***P < 0.001.

To evaluate whether the effects of LncBAR are cardiomyocyte-specific, we examined its expression across cardiac cell types. RT-qPCR on sorted neonatal heart cells showed that LncBAR expression was significantly higher in NMCMs compared to cardiac fibroblasts (CFs) and endothelial cells (ECs) (Supplementary Fig. 2a). We next investigated the effects of LncBAR depletion in non-myocytes, achieving ~50% knockdown in CFs and ~85% in ECs (Supplementary Fig. 2b, c). Functional assays revealed that LncBAR knockdown did not significantly alter fibroblast proliferation or number, as assessed by vimentin and pH3 staining, nor did it affect EC proliferation or total cell counts (Supplementary Fig. 2d, e). Together, these data indicate that LncBAR is enriched in cardiomyocytes and exerts minimal effects on CFs and ECs under homeostatic conditions.

We then examined whether reduced cardiomyocyte proliferation affects heart function in LncBAR KO adult mice. Echocardiography showed minor differences in left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-diastolic volumes (LVEDV), left ventricular end-systolic volumes (LVESV), stroke volume (SV) and heart rate (HR) between LncBAR KO and WT mice (Supplementary Fig. 2f–l). However, the heart-to-body weight ratio was slightly lower in LncBAR KO mice (Supplementary Fig. 2m, n). Wheat germ agglutinin (WGA) staining showed reduced cardiomyocyte size compared to controls (Supplementary Fig. 2o). Additionally, isolated cardiomyocytes from adult LncBAR KO mice had a smaller area than those from WT mice (Supplementary Fig. 2p). These results suggest that adult LncBAR KO mice may reduce the heart size to compensate for the proliferation defects during the neonatal stage.

LncBAR depletion inhibits neonatal heart regeneration

Next, we investigated whether LncBAR deficiency undermines tissue regeneration in early postnatal hearts. We subjected LncBAR KO mice and their littermate WT counterparts to AR at P1. Heart samples were collected at AR7 and AR21 for histological and functional analyses (Fig. 3a). ICC on heart sections taken at AR7 revealed a reduced number of Ki67-positive and pH3-positive cardiomyocytes in LncBAR KO hearts compared to WT hearts (Fig. 3b, c). Additionally, Masson’s trichrome staining showed a significant increase in fibrotic area in LncBAR KO hearts at AR7 compared with WT hearts (Fig. 3d). Consistently, while neonatal WT hearts regenerated well with barely detectable fibrosis at AR21, LncBAR KO hearts exhibited significant fibrosis and more pronounced reductions in heart functions at AR21 (Fig. 3e–g), highlighting the pivotal role of LncBAR.

Fig. 3: LncBAR depletion inhibits neonatal heart regeneration.

a Schematic depiction of the experimental design in WT and LncBAR^−/− neonatal mice. b, c Representative confocal z-stack images and quantification of Ki67+ cTnT+ CMs (b), and pH3+ cTnT+ CMs (c) from WT and LncBAR^−/− heart sections in the apex zone at day 7 after AR (AR7). White arrows indicate Ki67+ or pH3+ CMs (n = 15 fields, 3 mice/group). Scale bars, 20 µm. Masson’s trichrome staining and quantification of fibrotic area (blue) in sagittal heart sections from WT and LncBAR^−/− mice at AR7(d) and AR21(e) (n = 5 mice). Scale bars, 1 mm. Echocardiography showing left ventricular ejection fraction (f) and fractional shortening (g) at AR21 of WT and LncBAR^−/− mice (n = 5 mice for sham groups, n = 8 mice for AR groups). Data are presented as mean ± SEM. Statistical significance was determined by two-tailed unpaired t test or two-way ANOVA with Tukey’s multiple comparisons test. **P < 0.01, ***P < 0.001.

To exclude potential developmental defects arising from LncBAR KO that might affect heart regeneration, we delivered shLncBAR and shNT control viruses into newborn hearts and performed AR surgery on P1. Hearts were collected at AR7 (Supplementary Fig. 3a). RT-qPCR results confirmed that viral injection decreased cardiac LncBAR expression by 50% (Supplementary Fig. 3b). ICC of hearts treated with shLncBAR demonstrated a significant reduction in Ki67+ and pH3+ proliferative cardiomyocytes (Supplementary Fig.3c, d). These findings collectively suggest that LncBAR depletion attenuates cardiomyocyte proliferation and compromises heart regeneration.

LncBAR overexpression promotes cardiomyocyte regeneration and improves cardiac functions after myocardial injury

To investigate whether increased expression of LncBAR could extend the cardiac regenerative potential, we introduced exogenous LncBAR into mice using an adeno-associated virus serotype 9 (AAV9)-mediated delivery system^25,26. Newborn mice were intramyocardially injected with AAV9-cTnT-LncBAR or AAV9-cTnT-GFP control viruses, with expression driven by cardiomyocyte-specific cTnT promoter. Saline-treated group was used as non-viral control. AR injury was performed at non-regenerative stage (P8), and heart tissues were collected at AR7 and AR21 for histological and functional analyses (Fig. 4a). We evaluated the transduction efficiency of the applied AAVs by quantifying GFP-positive cardiomyocytes, which reached approximately 83% in the AAV9-cTnT-GFP group (Fig. 4b), indicating that the majority of cardiomyocytes were successfully transduced. Overexpression of LncBAR was verified by RT-qPCR in AAV9-cTnT-LncBAR hearts (Fig. 4c). Co-immunostaining of cTnT with proliferation markers in heart sections showed that LncBAR overexpression significantly increased the number of Ki67+ and pH3+ cardiomyocytes after AR injury compared with controls (Fig. 4d, e). Masson’s trichrome staining of heart sagittal sections collected at AR7 and AR21 both showed that overexpression of LncBAR led to reduced scar size in the apical injury zone compared with control groups (Fig. 4f, g). While AR resulted in significant reduction in heart functions, LncBAR overexpression contributed to significantly improved LVEF and LVFS (Fig. 4h–j). These results suggest that elevating LncBAR expression levels can protect the heart from AR-induced injury by promoting cardiomyocyte proliferation.

Fig. 4: LncBAR overexpression enhances cardiomyocyte proliferation after AR injury.

a Schematic depiction of AR experiments. Adeno-associated virus serotype 9 (AAV9) with LncBAR overexpression driven by cardiac specific promoter cTnT (AAV9-cTnT-LncBAR) or control virus expressing GFP (AAV9-cTnT-GFP) were intramyocardially injected into the neonatal mice hearts. The PBS group was used as a non-viral control. AR surgery was performed at P8, echo and histology samples were collected at AR7 and AR21. b Quantification analysis of the transduction efficiency of adeno-associated viruses by the percentage of GFP+ cells. Scale bars, 200 µm. c RT-qPCR analysis showing the expression level of LncBAR in hearts receiving AAV9-cTnT-LncBAR virus. AAV9-cTnT-GFP injected hearts were used as control. PBS injected hearts were used as non-viral control (n = 5 mice). Representative confocal z-stack images and quantification of Ki67+ cTnT+ CMs (d), and pH3+ cTnT+ CMs (e) in the cryosection of apex zone of hearts receiving AAV9-cTnT-LncBAR or AAV9-cTnT-GFP or PBS injection at AR7. White arrows indicate proliferating CMs (n = 15 fields, 3 mice/group). Scale bars, 20 µm. Masson’s trichrome staining and quantification of cardiac fibrosis in heart sagittal sections from AAV9-cTnT-LncBAR, AAV9-cTnT-GFP and PBS mice at AR7 (f) and AR21 (g) (n = 5 mice). Scale bars, 1 mm. h Echocardiography assessing left ventricular function at AR21 in mice treated with AAV9-cTnT-LncBAR, AAV9-cTnT-GFP, or PBS. Quantification of left ventricular ejection fraction (i) and fractional shortening (j) at 3 weeks after AR in AAV9-cTnT-LncBAR and control mice (n = 3 mice for sham groups and n = 5 mice for AR groups). Data are presented as mean ± SEM. Statistical significance was determined by one-way or two-way ANOVA with Tukey’s multiple comparisons test. **P < 0.01, ***P < 0.001.

LncBAR overexpression enhanced adult cardiac regeneration and functional recovery

We next investigated whether LncBAR plays a functional role in regulating adult heart repair. We performed both loss- and gain-of-function experiments in adult (8-week-old) mice subjected to MI via left anterior descending coronary artery (LAD) ligation. Specifically, we used LncBAR knockout (KO) and wild-type (WT) mice to examine the effects of LncBAR loss, and concurrently administered AAV9-cTnT-LncBAR or control viruses during MI surgery to evaluate the gain-of-function effects. Importantly, the gain-of-function approach also served as a rescue experiment, as LncBAR overexpression in KO mice was tested for its ability to restore impaired cardiac repair. Cardiac function was assessed longitudinally by echocardiography at weekly intervals post-MI, and hearts were collected for histological and immunohistochemical analyses at 2- and 6-week post-injury (Fig. 5a). The data demonstrated a significant increase in total cardiomyocyte number per heart at 2 weeks post-injury in LncBAR-treated mice compared to controls (Fig. 5b). Importantly, we observed an increase in the proportion of mononucleated cardiomyocytes accompanied by a corresponding decrease in binucleated and multinucleated cardiomyocytes in the LncBAR group (Fig. 5c), indicating an expanded subpopulation of cardiomyocytes undergoing cytokinesis.

Fig. 5: LncBAR promotes adult cardiomyocyte proliferation and heart repair post-MI.

a Schematic of the experiment design for evaluating the role of LncBAR in adult mice post-MI. Adeno-associated virus serotype 9 (AAV9) with LncBAR overexpression driven by cardiac specific promoter cTnT (AAV9-cTnT-LncBAR) or control virus (AAV9-cTnT-Control) were intramyocardially injected into the injured site immediately following MI. Echocardiographic assessments were performed weekly. Heart samples were collected 2 weeks or 6 weeks post-MI. b Quantification of total cardiomyocyte number from hearts 2 weeks post-MI following LncBAR or control virus treatment (n = 5 mice). Hearts were perfused using a Langendorff apparatus, and isolated cardiomyocytes were counted using a hemocytometer. c Quantification of mononucleated, binucleated, and multinucleated cardiomyocytes isolated from hearts 2 weeks post-MI following LncBAR or control virus treatment. Hearts were perfused using a Langendorff apparatus, and isolated cardiomyocytes were stained with α-actinin and DAPI for identification and nuclear counting. (n = 5 mice). Scale bars, 20 µm. Representative confocal z-stack images and quantification of Aurora B+ cTnI+ CMs (d) Ki67+ cTnI+ CMs (e), and pH3+ cTnI+ CMs (f) in the border zones of mouse hearts 2 weeks post-MI. Yellow arrows indicate proliferating CMs (n = 12 fields, 3 mice/group). Scale bars, 20 µm. Representative Masson’s trichrome staining (g) of heart cross-sections and fibrotic area quantification (h) 6 weeks post-MI (n = 5 mice). Scale bars, 1 mm. Echocardiography analysis of mouse hearts. LVEF (i): left ventricular ejection fraction; LVFS (j): left ventricular fractional shortening (n = 4 mice for sham groups, n = 8 mice for MI groups, but 2 weeks post-MI n = 5 mice for MI groups). Data are presented as mean ± SEM. Statistical significance was determined by two-tailed unpaired t test or two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.

Wheat germ agglutinin (WGA) staining revealed that cardiomyocytes in LncBAR-overexpressing hearts exhibited significantly reduced cross-sectional area, effectively reversing the hypertrophic enlargement observed in LncBAR KO mice (Supplementary Fig. 4a). Immunocytochemistry revealed an increase in Aurora B+, Ki67+ , and pH3+ cardiomyocytes following LncBAR overexpression, which rescued the proliferation deficits caused by LncBAR deletion (Fig. 5d–f). Furthermore, Masson’s trichrome staining at 6 weeks post-MI showed that LncBAR knockout significantly exacerbated fibrotic scar formation, whereas LncBAR overexpression attenuated fibrosis and rescued the detrimental effects of LncBAR loss (Fig. 5g, h). Echocardiographic assessments revealed no significant differences in cardiac function between groups under baseline conditions. However, after MI, LncBAR overexpression markedly improved cardiac function and reversed the impairment induced by LncBAR knockout. This functional benefit was sustained through 6 weeks post-MI (Fig. 5i, j and Supplementary Fig. 4b, c). Collectively, these data demonstrate that LncBAR promotes cardiomyocyte proliferation, reduces fibrosis, and improves cardiac performance following MI in adult mice.

Activation of PI3K-AKT pathway during LncBAR-induced cardiomyocyte survival and proliferation

To profile the transcriptional regulatory effects of LncBAR in cardiomyocytes, RNA sequencing (RNA-seq) was employed under conditions of LncBAR knockdown and control (Fig. 6a). We identified 1,694 differentially expressed genes (DEGs) in shLncBAR-treated NMCMs (P < 0.05, |fold change| ≥ 2) (Fig. 6b). Among them, 651 genes were upregulated and enriched in biological pathways, including positive regulating of apoptosis (e.g., Stk4 and Bak1) and mitogen-activated protein kinases (MAPK) cascade (e.g., Ntrk2 and Bcar3). The significantly downregulated 1043 DEGs in shLncBAR CMs were found to be associated with the cell cycle (e.g., Mki67, Cdc20, and Ccnd2), cell division (e.g., Cdca2, Aurka, and Kif11), and wound healing (e.g., Postn and Col3a1), consistent with our above findings (Fig. 6c).

Fig. 6: LncBAR overexpression positively regulated cardiomyocyte survival and proliferation.

a Schematic depiction of experimental design using NMCMs treated with shLncBAR or shNT for RNA-seq (n = 2 biological replicates). b Volcano plot showing differentially expressed genes (DEGs) induced by LncBAR knockdown (shLncBAR) in NMCMs compared to those treated with shNT. DEGs were defined by P < 0.05 and |fold change| ≥ 2 comparing shLncBAR-NMCMs to shNT-NMCMs. c Gene Ontology (GO) analysis of significantly enriched pathways in shLncBAR-treated NMCMs compared with shNT-treated NMCMs. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showing significantly upregulated (d) and downregulated (e) pathways enriched in shLncBAR-NMCMs compared to shNT control cells. f Heatmap showing the expression levels of representative Brg1 downstream factors involved in proliferation in shLncBAR- or shNT-NMCMs (n = 2 biological replicates). g–i Western blot showing the expression of total and phosphorylated form of PI3K and AKT in NMCMs treated with or without LncBAR overexpression. Quantification of indicated protein expression was shown in (h) and (i) (n = 3 biological replicates). Representative ICC images and quantification for cTnT+ pH3+ (j) and cTnT+ Ki67+ (k) NMCMs treated with LncBAR along with small molecule LY294002 (PI3K/AKT pathway inhibitor). Cells infected with lentiviral LacZ and treated with DMSO were used as control. White arrows indicate proliferating NMCMs (n = 15 fields). Scale bars, 100 µm. l RT-qPCR analysis showing expression of genes involved in positive regulation of cell cycle in LncBAR overexpressed NMCMs treated with or without LY294002 inhibitor (n = 3 biological replicates). Data are presented as mean ± SEM. Statistical significance was determined by two-tailed unpaired t test or two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.

We further performed Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and identified the enrichment of upregulated MAPK signaling and downregulated cell cycle pathway induced by shLncBAR (Fig. 6d, e). Interestingly, pathways regulated by the SWI/SNF chromatin remodeling complex during cardiac lineage differentiation and myogenesis^27,28, including Wnt signaling pathway and EGFR tyrosine kinase inhibitor resistance pathway, were significantly upregulated in shLncBAR group (Fig. 6d). Further analysis revealed that the expression levels of several downstream factors (e.g., Cdkn1a, Bmp10, Mcm6, and Cdc6) reported to be regulated by BRG1 changed significantly after knockdown of LncBAR (Fig. 6f). These findings suggest that LncBAR may work synergistically with BRG1 to regulate cardiomyocyte proliferation and cardiac regeneration.

The PI3K-AKT signaling plays crucial roles in the regulation of CM proliferation, survival and function^29,30. Our KEGG analysis indicated that LncBAR depletion reduced PI3K-AKT signaling (Fig. 6e). Western blot analysis confirmed elevated phosphorylation of PI3K and AKT in cardiomyocytes with LncBAR overexpression (Fig. 6g–i). Furthermore, treating LncBAR overexpressed NMCMs with the AKT inhibitor LY294002 abrogated the pro-proliferative effect of LncBAR as evaluated by co-staining of cTnT with pH3 or Ki67 (Fig. 6j, k). Similarly, LY294002 treatment eliminated the effect of LncBAR on increasing the expression of cell cycle-related genes, including those involved in cell cycle initiation (Ccnd1, Ccnd2, and Cdk6), S phase and G2 phase progression (Mcm3 and Ccna2), and the transition from metaphase to anaphase and the progression of anaphase (Cdc20 and Cdca2) (Fig. 6l). Taken together, these data suggest that LncBAR exerts its functional role by activating the PI3K-AKT signaling pathway, thereby enhancing the proliferative capacity of cardiomyocytes. LncBAR promotes cardiomyocyte proliferation by stimulating PI3K-AKT pathway and subsequently enhancing the expression of downstream cell cycle genes.

Additionally, we noticed that shLncBAR increased expression of apoptosis-related genes, which were also targets of PI3K-AKT pathway. RT-qPCR analyses confirmed that LncBAR knockdown increased the expression of pro-apoptotic genes, including Bak1, Stk4, Moap1 and Pidd1 (Supplementary Fig. 5a). We then challenged LncBAR-overexpressed NMCMs and control NMCMs with oxygen glucose deprivation and reperfusion (OGD/R)-induced apoptotic conditions, and assessed apoptotic cells with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Our results showed that OGD/R treatment resulted in 20% cell death in control cells, whereas LncBAR overexpression reduced TUNEL+ NMCMs to approximately 10% (Supplementary Fig. 5b). TUNEL staining of heart sections from AR mice also demonstrated significantly fewer TUNEL+ CMs in heart injected with AAV9-cTnT-LncBAR compared to controls (Supplementary Fig. 5c). These findings indicate that overexpression of LncBAR protects CMs from apoptosis under stressed conditions. We then intramyocardially injected AAV9-cTnT-LncBAR or control viruses into P1 LncBAR KO and WT mice. AR was performed at P8, and hearts were harvested 24 h post-surgery (AR1) for histological analyses (Supplementary Fig. 5d). TUNEL staining of heart sections revealed that LncBAR KO significantly increased TUNEL+ cardiomyocytes, while AAV9-mediated LncBAR supplementation attenuated this apoptotic response (Supplementary Fig. 5e). These findings are consistent with our RNA-seq analyses, which showed enrichment of GO pathways related to positive regulation of apoptosis in LncBAR deficient cardiomyocytes (Fig. 6c)

LncBAR directly binds to BRG1 and increases its protein abundance

Our previous work demonstrated that LncBAR directly associates with the SWI/SNF complex in embryonic brain tissue²⁴, raising the possibility of a similar interaction in cardiomyocytes. Interestingly, Brg1 expression significantly increased in the apical zone at AR7 compared to the border and remote zones, mirroring LncBAR expression (Fig. 7a).

Fig. 7: LncBAR facilitates cardiomyocyte proliferation by upregulating BRG1 protein levels.

a RT-qPCR analysis of Brg1 expression in AR7 hearts. RZ remote zone, BZ border zone, AZ apex zone (n = 3 mice). b RT-qPCR analysis of Brg1 expression (relative to GAPDH) in NMCMs following LncBAR knockdown or overexpression, with shNT or LacZ as controls respectively (n = 3 biological replicates). c Western blot and quantification of BRG1 protein levels in NMCMs treated with or without shLncBAR (n = 3 biological replicates). d Western blot and quantification of BRG1, BAF155 and BAF250a in NMCMs upon LncBAR overexpression, with LacZ as control (n = 3 biological replicates). Western blot analysis (e) and quantification (f) of BRG1 expression in LncBAR-overexpressing NMCMs treated with cycloheximide (CHX, 10 µg/ml) for indicated times (n = 3 biological replicates). Western blot analysis (g) and quantification (h) of BRG1 expression in LncBAR-depleted NMCMs treated with proteasome inhibitor MG132 (10 µM), or lysosomal inhibitors chloroquine (CQ, 10 µM) and NH₄Cl (25 mM) for 6 h (n = 3 biological replicates). i RT-qPCR analysis of Brg1 expression in shBrg1-transduced NMCMs, with shNT as control (n = 3 biological replicates). Representative ICC and quantification of cTnT+ Ki67+ (j) and cTnT+ pH3+ (k) cells in NMCMs overexpressing LncBAR, treated with either shBrg1 or shNT control. White arrows indicate proliferating NMCMs (n = 15 fields). Scale bars, 100 µm. l RT-qPCR analysis of cell cycle genes in LncBAR-overexpressing NMCMs treated with shBrg1 or shNT lentiviruses (n = 3 biological replicates). ChIP-seq profiling showed the binding signal for BRG1 at the genomic loci of MCM3 (m) and AURKA (n) promoter regions in reported Nalm-6 cell line³¹. Western blot (o) and quantification (p, q) of total and phosphorylated PI3K and AKT in NMCMs with LncBAR overexpression and Brg1 knockdown (n = 3 biological replicates). Data are presented as mean ± SEM. Statistical significance was determined by two-tailed unpaired t test, one-way ANOVA, or two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.

To rigorously test whether LncBAR directly binds to BRG1 in cardiomyocytes, we performed RNA immunoprecipitation followed by quantitative real-time PCR (RIP-qPCR) in HL-1 cardiomyocytes. Using three independent primer pairs (P1, P2 and P3) targeting different regions of LncBAR, RIP-qPCR consistently detected its enrichment in BRG1 immunoprecipitates compared to IgG controls (Supplementary Fig. 6a–c). These results revealed that LncBAR physically interacts with BRG1 in cardiomyocytes.

We next examined the effect of LncBAR on Brg1 and found that neither knockdown nor overexpression of LncBAR affected Brg1 expression at mRNA level (Fig. 7b). However, at the protein level, knockdown of LncBAR reduced BRG1 expression, whereas LncBAR overexpression elevated BRG1 levels (Fig. 7c, d). Additionally, expression of other components of the SWI/SNF chromatin remodeling complex, including BAF155 and BAF250a were consistently increased by LncBAR overexpression (Fig. 7d). These results suggest that LncBAR directly binds with BRG1 to regulate the SWI/SNF chromatin remodeling complex.

We next explored how LncBAR overexpression enhances BRG1 protein stability. Western blot analysis revealed that LncBAR overexpression significantly inhibited BRG1 degradation in NMCMs following cycloheximide (CHX) treatment (Fig. 7e, f). Furthermore, the reduction in BRG1 protein levels caused by LncBAR knockdown was reversed by the proteasome inhibitor MG132, but not by the autophagosome inhibitor chloroquine (CQ) or the lysosome inhibitor NH₄Cl (Fig. 7g, h). Together, these findings indicate that LncBAR not only binds BRG1 but also stabilizes it by suppressing proteasome-mediated degradation.

LncBAR promotes cardiomyocyte proliferation through BRG1 in vitro

Next, we investigated whether LncBAR induces cardiomyocyte proliferation through BRG1. We designed shRNAs targeting Brg1, achieving knockdown efficiency over 80%, and performed epistasis experiments (Fig. 7i). Lentiviral shBrg1 was introduced into NMCMs with or without LncBAR overexpression, and the proliferation of treated NMCMs were assayed by ICC with Ki67 and pH3. We found that while LncBAR overexpression consistently increased the number of Ki67+ and pH3+ NMCMs, shBrg1 treatment significantly abrogated the LncBAR-induced NMCM expansion (Fig. 7j, k).

We then assessed the expression levels of cell cycle-related genes across different phases (Fig. 7l), including Cdk3 (G0/G1 and G1/S transition), Mcm3, Ccnd1, and Ccnd2 (S phase initiation), Cdca3 (G2/M transition), and Ccnb1, Ccnb2, and Cdc20 (M phase progression). Knockdown of Brg1 inhibited the upregulation of those cell cycle related genes in LncBAR overexpressing cardiomyocytes, suggesting that BRG1 is required for LncBAR-mediated cell cycle activation. Additionally, we analyzed published BRG1 ChIP-seq datasets³¹ and found the enrichment of BRG1 binding motifs located at the promoter regions of MCM3 and AURKA (Fig. 7m, n), indicating its direct transcriptional regulation of these genes. Furthermore, the loss of Brg1 reduced LncBAR-induced phosphorylation of PI3K and AKT, while their total protein levels remained largely unchanged (Fig. 7o–q). Collectively, these results suggest that LncBAR enhances cardiomyocyte proliferation through the upregulation of BRG1, which subsequently drives cell cycle progression.

LncBAR enhances cardiomyocyte proliferation and regeneration in vivo via BRG1

Building on our in vitro findings, we assessed BRG1 and PI3K-AKT activation in the apical zone at AR7 in LncBAR- or LacZ-treated animals. LncBAR overexpression increased BRG1 protein abundance and upregulated the expression of other SWI/SNF chromatin remodeling complex components, including BAF155 and BAF250a (Fig. 8a, c). In parallel, LncBAR enhanced PI3K and AKT phosphorylation without affecting their total protein expression (Fig. 8a–e).

Fig. 8: LncBAR enhances cardiomyocyte proliferation and cardiac regeneration by activating BRG1 and PI3K-AKT pathway.

Western blot analysis and quantification of SWI/SNF complex subunits (a, c) and PI3K-AKT pathway activation (a, b, d, e) in apical tissue post-AR injury (n = 3 biological replicates). f Schematic depiction of AR experiments. Viruses harboring LncBAR, shBrg1, or respective controls were intramyocardially injected into neonatal mouse hearts, with PBS as a non-viral control. AR surgery was performed at P8. Echocardiography and histological samples were collected at AR7 and AR21. g RT-qPCR analysis showing the expression level of LncBAR and Brg1 in hearts receiving the virus injection (n = 5 mice). Representative confocal z-stack images and quantification of Ki67+ cTnI+ CMs (h), and pH3+ cTnI+ CMs (i) in apex zone of AR7 hearts. Yellow arrows indicate Ki67+ or pH3+ CMs (n = 15 fields, 3 mice/group). Scale bars, 20 µm. j Representative Masson’s trichrome-stained sagittal heart sections with fibrotic area quantification at AR21 (n = 5 mice). Scale bars, 1 mm. Echocardiography analysis of mouse hearts at AR21. LVEF (k): left ventricular ejection fraction; LVFS (l): left ventricular fractional shortening; LVEDV (m): left ventricular end-diastolic volume; LVESV (n): left ventricular end-systolic volume (n = 6 mice). Data are presented as mean ± SEM. Statistical significance was determined by two-tailed unpaired t test, one-way ANOVA, or two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.

To determined how LncBAR-mediated BRG1 accumulation influences the cardiac regeneration, we generated four experimental groups through intramyocardial injection of saline or viral constructs for LncBAR overexpression and/or Brg1 knockdown. AR injury was performed at non-regenerative stage (P8), and heart tissues were collected at AR7 and AR21 for histological and functional analyses (Fig. 8f). RT-qPCR confirmed LncBAR overexpression and Brg1 knockdown (Fig. 8g). Immunofluorescence for cTnI with proliferation markers (Ki67 and pH3) revealed that LncBAR overexpression significantly increased proliferating cardiomyocytes at AR7 versus controls. Whereas, Brg1 knockdown markedly attenuated this effect (Fig. 8h, i). By AR21, Masson’s trichrome staining showed that LncBAR overexpression reduced fibrotic scarring, an effect abolished by Brg1 depletion (Fig. 8j). Concomitantly, echocardiography demonstrated that the functional improvements of LncBAR, as indicated by increased LVEF/LVFS and reduced LVEDV/LVESV were lost upon Brg1 knockdown (Fig. 8k–n). Together, these results confirmed that BRG1 mediates LncBAR-induced cardiomyocyte proliferation and cardiac regeneration following AR.

LncBAR overexpression promotes the proliferation and survival of human embryonic stem cell-derived cardiomyocytes

Although we did not identify a conversed sequence of LncBAR in humans²⁴, we asked whether the identified LncBAR sequence could still promote human cardiomyocyte proliferation. We used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) at day 10 of differentiation, which exhibited spontaneous beating (Supplementary Movie 1) and reached approximately 70% purity (Supplementary Fig. 7a, b). ICC analyses indicated that most cells were immature, with ~60% showing disorganized and ~10% well-aligned sarcomere structures, making them suitable for studying proliferation (Supplementary Fig. 7c).

We next determined a transduction efficiency of approximately 97% for our lentiviral vectors in hESC-CMs and RT-qPCR confirmed LncBAR overexpression (Supplementary Fig. 7d and Fig. 9a). ICC for cTnT and Ki67 or cTnT and Aurora B showed a significantly two-fold increase in the percentage of proliferating hESC-CMs with LncBAR overexpression (Fig. 9b, c). Consistently, the number of EdU+ and pH3+ hESC-CMs were doubled in LncBAR overexpressed CMs compared to controls (Supplementary Fig. 7e, f). Furthermore, to directly demonstrate cardiomyocyte proliferation, we quantified the number of hESC-CMs following LncBAR overexpression, showing a significant increase consistent with enhanced cell division (Supplementary Fig. 7g).

Fig. 9: LncBAR overexpression improves proliferation and survival of human embryonic stem cell-derived cardiomyocytes (hESC-CMs).

a RT-qPCR detecting LncBAR expression in hESC-CMs transduced with LncBAR- or control (LacZ)-encoding lentivirus (n = 3 biological replicates). b Representative ICC and quantification of cTnT+ Ki67+ hESC-CMs with LncBAR overexpression versus LacZ or PBS controls. White arrows indicate proliferating hESC-CMs (n = 15 fields). Scale bars, 25 µm. c Representative ICC and quantification of cTnT+ Aurora B+ hESC-CMs with LncBAR overexpression versus LacZ or PBS controls. White arrows indicate proliferating hESC-CMs. (n = 5 biological replicates). Scale bars, 25 µm. RT-qPCR detecting the expression of genes promoting (d) and inhibiting (e) cell cycle progression in LncBAR-overexpressing hESC-CMs vs. LacZ control (n = 3 biological replicates). f RT-qPCR analysis of BRG1 expression in hESC-CMs transduced with shBRG1 lentivirus (n = 3 biological replicates). g Representative ICC and quantification of cTnT+ pH3+ hESC-CMs treated with LncBAR-overexpressing together with shBRG1 or shNT control. White arrows indicate proliferating hESC-CMs (n = 15 fields). Scale bars, 50 µm. Western blot (h) and quantification (i, j) of total and phosphorylated PI3K and AKT in hESC-CMs with LncBAR overexpression and shBRG1 knockdown (n = 3 biological replicates). k Representative ICC and quantification of apoptotic hESC-CMs with LncBAR overexpression. hESC-CMs with or without LncBAR overexpression were subjected to α-actinin and TUNEL staining under normal or oxygen-glucose deprivation/reperfusion (OGD/R) conditions. White arrows indicate TUNEL+ hESC-CMs (n = 15 fields). Scale bars, 200 µm. l Representative ICC and quantification of hESC-CMs probed with cleaved caspase 3 (casp3) and cTnT. Cells were treated as in (k). White arrows indicate casp3+ hESC-CMs (n = 15 fields). Scale bars, 200 µm. m Western blot showing the protein level of cleaved caspase 3 in LncBAR-overexpressing hESC-CMs subjected to OGD/R treatment (n = 3 biological replicates). Data are presented as mean ± SEM. Statistical significance was determined by two-tailed unpaired t test, one-way ANOVA, or two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.

At the transcriptional level, RT-qPCR analysis showed that overexpression of LncBAR triggered the significant increase of a panel of genes positively regulating cell cycle, including control of mitosis and G1 phase progression (CDC16), initiation of S phase (e.g., CDK2, CDK4, CCND2, and CCNE2), promotion of G2 to M phase transition (CDK1 and CHEK1), and M phase progression (CCNB1 and CCNB2) (Fig. 9d). Conversely, negative regulators of the cell cycle, including p27 and p16, which switch off CDK2, 4 and 6 and arrest the cell cycle, demonstrated reduced expression in LncBAR-transduced hESC-CMs compared to control cells (Fig. 9e). These findings indicate that LncBAR enhances the hESC-CM proliferation by modulating the expression of both positive and negative cell cycle regulators.

We next assessed whether LncBAR exerts its effects on hESC-CM proliferation through BRG1. Western blot analysis showed that LncBAR overexpression significantly inhibited BRG1 degradation in CHX-treated human cardiomyocytes (Supplementary Fig. 7h), consistent with its role in murine cardiomyocytes. We then designed shRNAs targeting BRG1 achieving efficient knockdown (Fig. 9f). As expected, loss of BRG1 in hESC-CMs abolished the enhanced cell proliferation induced by LncBAR, as determined by pH3 and cTnT staining (Fig. 9g). At the protein level, introducing shBRG1 into hESC-CMs with LncBAR overexpression reduced the phosphorylation of PI3K and AKT, while having minor effects on total protein expression (Fig. 9h–j). Together, these results indicate LncBAR interacts with BRG1 to activate the cell cycle progression, promoting hESC-CM proliferation.

In addition to its pro-proliferative role, we examined whether LncBAR also enhances hESC-CMs survival under stress conditions. We performed TUNEL staining with LncBAR-transduced cells upon OGD/R treatment. CMs transduced with LacZ virus were used as controls. We found that LncBAR overexpression significantly reduced apoptosis, lowering the percentage of TUNEL+ cells from approximately 30% in controls to around 12% (Fig. 9k). Congruently, ICC for cleaved caspase3 (casp3) further confirmed that LncBAR overexpression reduced the number of apoptotic cells (Fig. 9l). Additionally, western blot analysis showed that overexpression of LncBAR reduced the protein expression of casp3 (Fig. 9m).

Together, our results demonstrated that although LncBAR lacks a conserved human sequence, its expression in hESC-CMs is still capable of enhancing cardiomyocyte proliferation. This suggests that while a direct human homolog of LncBAR has not been identified, functionally analogous regulatory elements or short sequence motifs may exist in the human genome, enabling similar effects on cardiomyocyte proliferation.

Discussion

In this study, we identified LncBAR, a novel lncRNA that regulates cardiomyocyte proliferation and heart regeneration. We demonstrated that LncBAR deficiency impaired heart regeneration, whereas its overexpression promoted cardiomyocyte proliferation and improved post-AR cardiac function. Furthermore, LncBAR overexpression promoted adult cardiomyocyte proliferation and enhanced heart repair following myocardial infarction. Mechanistically, LncBAR directly interacts with BRG1 and upregulates its protein expression, which in turn activates cell cycle progression. These findings underscore the pivotal roles of LncBAR-BRG1 connection in cardiac regeneration, offering a novel strategy to enhance cardiac repair.

LncRNAs are crucial regulators of cardiac development, differentiation and diseases, with their expression tightly associated with developmental and stress conditions in the heart^32,33. Our work demonstrated that LncBAR expression declines from embryonic (E13.5) to adult stages (12 weeks old), suggesting its potential role in heart regeneration. Functional analysis confirmed that LncBAR actively stimulates cardiomyocyte proliferation and improves cardiac function after injury. These results established LncBAR as a positive regulator for cardiomyocyte renewal, supporting the notion that developmental correlated lncRNAs could be reactivated to enhance cardiac repair in the adult heart. Recent studies have shown that LncRNA CPR (cardiomyocyte proliferation regulator) represses CM proliferation, while LncRNA Snhg1 (small nucleolar RNA host gene 1) promotes it^25,34. These observations, along with our research, reinforce the critical role of LncRNAs in cardiac repair, suggesting a complex regulatory network controlling cardiomyocyte proliferation and regeneration.

Brg1, the core component of the SWI/SNF ATP-dependent chromatin-remodeling complex also known as SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 4 (Smarca4), has been recognized as a crucial regulator of cardiomyocyte proliferation. In zebrafish heart, depletion of Brg1 upregulates Cdkn1a and Cdkn1c expression after AR and inhibits myocardial regeneration¹⁶. In mammalian embryonic and postnatal heart, knockdown of Brg1 decreases expression level of Bmp10 while increased expression level of Cdkn1c^14,35, thereby inhibiting cardiomyocyte growth. Our study identified a previously unrecognized mechanism by which LncBAR regulates BRG1 at the post-transcriptional level. Specifically, LncBAR enhances BRG1 protein stability without altering its mRNA expression, thereby sustaining BRG1 activity to drive cardiomyocyte cell-cycle progression. Nonetheless, the precise molecular mechanism, such as the involvement of the ubiquitin-proteosome pathway, warrants further investigation.

Although LncBAR itself lacks overt sequence conservation in humans, it retains the capacity to promote proliferation of human cardiomyocytes. This observation raises the possibility that functionally analogous lncRNAs or conserved regulatory motifs may exist across species to mediate similar effects. Importantly, BRG1 is highly conserved between mouse and human, sharing 97.2% protein identity, and serves as a critical epigenetic regulator in both species¹⁴. Such conservation suggests that mouse LncBAR may also interact with human BRG1 and activate similar downstream pathways, offering a mechanistic basis for its cross-species activity.

Interestingly, previous studies have revealed that Brg1 mediates cardiac pressure overload-induced myocardial hypertrophy through at least two mechanisms. First, Brg1 interacts with HDAC and PARP to repress α-MHC while activating β-MHC expression¹⁴. Second, Brg1 binds with Mhrt779 to inhibit HDAC2/p-AKT/p-GSK3β pathway, thereby exerting an antihypertrophic effect³⁶. In line with these findings, our study revealed that LncBAR loss reduces BRG1 expression and cardiomyocyte size, suggesting a potential link between LncBAR, BRG1, and myocardial hypertrophy. Further investigations are needed to unravel the intricate relationship between these factors and their role in cardiac remodeling.

The PI3K-AKT pathway has been widely reported to promote cardiomyocyte proliferation during development and neonatal stages, and its reactivation in adult or injured hearts can stimulate limited CM proliferation and regeneration^34,37,38. Consistently, this pathway is commonly activated in proliferating cardiomyocytes^26,39. Upstream signaling pathways, such as Hippo-Yap signaling, cell surface tyrosine kinases ErbB2, ErbB4 and IGFR, act through PI3K-AKT to effectively stimulate CM proliferation^37,40,41. In our research, we found that LncBAR overexpression activates PI3K-AKT signaling, whereas pharmacological inhibition of this pathway blocks LncBAR-induced cardiomyocyte expansion. However, the precise mechanism by which PI3K-AKT becomes activated remains elusive. One possibility is that BRG1, stabilized by LncBAR, drives the expression of key cell cycle regulators that in turn engage PI3K-AKT signaling. Alternatively, BRG1 and PI3K-AKT may represent parallel yet converging downstream pathways of LncBAR, both contributing to the proliferative response. Future studies, particularly comprehensive proteomic analyses, will be essential to dissect the interplay between these pathways and to define the signaling hierarchy underlying LncBAR-mediated cardiac regeneration.

While our study established LncBAR as a positive regulator of cardiomyocyte proliferation and cardiac repair, several limitations should be acknowledged. In this study, we used systemic LncBAR knockout mice and intramyocardial injection of shLncBAR lentivirus to assess the effect of LncBAR deficiency on cardiac regeneration. Although our data indicated that LncBAR is preferentially expressed in cardiomyocytes and exerts minimal effects on fibroblasts and endothelial cells under homeostatic conditions, we cannot fully exclude contributions from other cell types or potential combinatory effects within the tissue microenvironment. Further work using cardiomyocyte-specific LncBAR deficient genetic models will be helpful. In addition, we observed increased fibrosis in LncBAR KO hearts following apical resection, but the underlying mechanisms by which LncBAR regulates fibrosis remain to be determined.

To evaluate CM proliferation, we applied multiple complementary approaches, including immunostaining for canonical markers (pH3, Ki67 and Aurora B kinase) and cardiomyocyte number quantification following the guideline⁴². Nonetheless, additional methods, including single-cell transcriptomic analyses to distinguish subpopulation of CMs at different cell cycle states, and advanced genetic models such as the mosaic analysis with double markers (MADM) mouse⁴³, would provide more rigorous validation and deeper mechanistic insights.

Overall, our results elucidate a critical LncBAR-BRG1 axis in promoting cardiomyocyte proliferation and efficient cardiac repair. We revealed that overexpression of LncBAR promoted cardiomyocyte proliferation in both murine and human cardiomyocytes, offering new insights into lncRNA-mediated cardiac regeneration. These findings provide an important insight for future studies exploring lncRNA-based therapeutic strategies for heart repair and regeneration.

Methods

Mouse strains and ethical approval

All mice were maintained in specific pathogen-free (SPF) facilities at the Medical Research Institute of Wuhan University. The experimental protocols followed the International Guiding Principles for Biomedical Research Involving Animals. All animal experiments were approved by the Animal Care and Ethical Committee of Medical Research Institute at Wuhan University (approval number: MRI2020-LAC68), and conform to the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals. Both male and female mice were used in this study. Wild-type C57BL/6 and CD1 mice were obtained from the Beijing VItalstar Biotechnology Co., Ltd. (Beijing, China). The LncBAR^−/− mice were generated and maintained as previously discribed²⁴. The genotyping primers were listed in Supplementary Table 1.

Plasmid construction

For gene overexpression, pLenti-LncBAR was cloned into lentiviral backbone that was originally purchased from Addgene (#17448). The construct pLenti-LacZ was used as control. All shRNAs were annealed in the pLKO.1 backbone as previously described⁴⁴. All cloning primers were listed in Supplementary Table 1, and all constructs were confirmed by sequencing.

Cell culture and viral production

HEK293T cells were purchased from ATCC and cultured in Dulbecco’s modified Minimal Essential Medium (DMEM) (Gibco, 6123110) supplemented with 10% fetal bovine serum (FBS) (Newzerum, FBS-AU500), 1% penicillin/streptomycin (Hyclone, SV30010), and 1% non-essentail amino acids (NEAA) (Gibco, 1114050). Viral package was performed as previously defined^44,45. Briefly, 293T cells were transfected with 10 µg viral constructs, 7 µg psPAX2 and 3 µg pMD2.G using Polyethylenimine (PEI) (Polysciences, 24765). The supernatant was collected 48 h and 72 h after transfection, filtered through 0.45 µm filter (Merck Millipore), and incubated with PEG6000 solution (8% final concentration) overnight at 4 °C. Then the mixture was spun down at 3800 rpm for 30 min at 4 °C, and the pelleted virus was resuspended with 100 µL of DMEM medium per each 10 cm dish. Lentivirus can be used in fresh or frozen at −80 °C.

To prepare the lentivirus for in vivo experiments, the virus-containing supernatant was filtered through 0.45 µm filter, and concentrated by ultracentrifugation at 23,000 rpm for 1 h and 45 min. The pelleted virus was resuspended with DPBS and polybrene (8 µg/mL, Merck Millipore). 10 µL of virus mixture was injected into the cardiac ventricles using an insulin syringe equipped with a 30-gauge needle.

Isolation and culture of primary neonatal mouse cardiomyocytes (NMCMs)

Neonatal mice (1–3 days old) were subjected to decapacitation and the hearts were extracted and rinsed with ice-cold PBS for three times. CMs were then dissociated using Neonatal Heart Dissociation kit (Miltenyi Biotec, 130-098-373) following manufacturer’s instructions. Briefly, the ventricles were minced into small pieces and digested with enzymes from the reagent kit at 37 °C for 15 min. Tissues were dissociated with gentleMACS™ Dissociator (Miltenyi Biotec, 130-093-235) into single-cell suspensions. The supernatant was filtered through 100 µm cell strainers (Nest, 258367) and cells were collected by centrifuge at 1000 rpm for 5 min. Red blood cells were removed using red blood cell lysis buffer (0.15 M NH4Cl, 0.1 M Na₂EDTA-2H₂O, 10 mM KHCO₃). The remaining cells were washed with MACS buffer (DPBS with 2% FBS and 0.1 mM EDTA) and incubated with antibodies provided in the Neonatal Cardiomyocyte Isolation Cocktail kit (Miltenyi Biotec, 130-100-825) for 15 min at 4 °C. After two rounds of wash with MACS buffer, cells were applied to MACS buffer-calibrated LS column (Miltenyi Biotec, 130-042-401) and the flow-through containing unlabeled CMs was collected for use.

Isolation of adult murine cardiomyocytes

WT and LncBAR KO mice hearts were perfused with Langendorff apparatus as described with minor modification⁴⁶. Briefly, mice were anesthetized by intraperitoneal injection of a mixture of tribromoethanol (320 mg/kg) and 2-butanol (160 mg/kg) and were euthanized using cervical dislocation. Hearts were extracted and retrograde perfusion was immediately performed through an aortic cannulation with continuous flow at the speed of 16–24 drops/min in a Langendorf apparatus. Both ventricles were digested with 1 mg/mL collagenase II (Worthington, LS004176) for 10–15 min, followed by further mechanical dissociation with pipetting. The cell suspension was collected and filtered through a 100 µm cell strainer. Cardiomyocytes were then collected for subsequent experiments.

Isolation of primary neonatal mouse cardiac fibroblasts (CFs)

To obtain neonatal CFs, hearts from neonatal mice (P1 to P3) were harvested and washed with PBS to remove blood cells. Then, clean hearts were minced roughly and transferred to 10 mL warm trypsin-EDTA (Sigma, T3924), and incubated at 37 °C for 10 min. The supernatant was discarded and the heart pieces were digested with 10 mL collagenase type II (Worthington, LS004176) for 8 min at 37 °C, followed by gental vortex for 1 min. The supernatant was collected and diluted with 5 mL FB medium. After five rounds of collagenase digestion and collection.

Cell suspension was obtained by passing through 40 μm cell strainer. Cell pellet was collected and CFs were sorted by magnetic activated cell sorting (MACS) as described previously⁴⁷. Cells were incubated with biotin anti-Thy1.2 antibody (Invitrogen, 13-0903-85) for 30 min at 4 °C, followed by anti-Biotin Microbeads (Miltenyi Biotec, 130090485) for 30 min at 4 °C. The cells were then washed and resuspended in MACS buffer (DPBS with 0.5% BSA and 2 mM EDTA) and applied to calibrated LS columns (Miltenyi Biotec, 130042401). Cells that bound beads were flushed out after three washes and ready to seed for experiments.

Isolation of primary neonatal mouse cardiac endothelial cells (ECs)

Neonatal mouse cardiac endothelial cells were isolated following established protocols⁴⁸. Briefly, hearts from neonatal mice (P1–P3) were harvested and washed with PBS to remove blood cells. Then hearts were cut with scalpel blade into small pieces and transferred to 5 mL heart digestion buffer (DMEM with 0.1% collagenase II and 0.25% collagenase IV and 7.5 µg/mL DNAse I) and incubated at 37 °C for 25 min. The mix were dissociated with gentleMACS™ Dissociator (Miltenyi Biotec, 130-093-235) into single-cell suspensions. The supernatant was filtered through 100 µm cell strainers (Nest, 258367) and cells were collected by centrifuge at 300 × g for 7 min. Resuspend the pellet and filter through a 40 µm cell strainer. Centrifuge at 300 × g for 5 min, remove the supernatant. Cell pellet was collected and ECs were sorted by magnetic activated cell sorting (MACS). Cells were incubated with biotin anti-CD31 antibody (Invitrogen, 13-0311-82) for 30 min at 4 °C, followed by anti-Biotin Microbeads (Miltenyi Biotec, 130090485) for 30 min at 4 °C. The cells were then washed and resuspended in MACS buffer (DPBS with 0.5% BSA and 2 mM EDTA) and applied to calibrated LS columns (Miltenyi Biotec, 130042401). Cells that bound beads were flushed out after three washes and ready to seed for experiments.

Human embryonic stem cell-derived CMs (hESC-CMs)

The human H7 ESC line was originally purchased from WiCell and was kindly gifted from Dr. Ning Sun’s lab at Fudan University⁴⁹. The cell line was maintained and differentiated to hESC-CMs using well established protocol as previously reported⁵⁰. Differentiated beating hESC-CMs were digested and reseeded onto plates coated with Matrigel (corning, 354277) for further research.

Oxygen–glucose deprivation and reperfusion (OGD/R) treatment

The OGD/R treatment was performed as previously described⁵¹. Briefly, NMCMs and hESC-CMs were incubated in glucose-free DMEM medium (Gibco, 2323743) within an anaerobic chamber (1% O₂, 5% CO₂) at 37 °C for 10 h to achieve oxygen-glucose deprivation (OGD). The cells were then transferred to normoxic conditions (95% air, 5% CO₂) and cultured with normal culture medium for 24 h at 37 °C (reperfusion, R). Control cells are treated the same medium but without OGD/R challenge.

5-ethynyl-20-deoxyuridine (EdU) incorporation assay

EdU labeling was carried out by culturing cells with 10 µM EdU for 48 h before fixation. Cells were then stained using the Cell-Light EdU Apollo567 In Vitro Kit (Ribobio, C10310-1) according to manufacturer’s instruction. Nuclei staining was performed with DAPI. Images were captured using Nikon Eclipse Ti2 fluorescence microscope.

Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) assay

Cell apoptosis was evaluated using the One-step TUNEL In Situ Apoptosis Kit (Elabscience, AK15560) following manufacturer’s instructions. Briefly, tissue sections or cultured cells were fixed in 4% PFA for 15 min and subsequently permeabilized with 0.2% Triton X-100 for 10 min at 37 °C. Samples were then incubated with the TUNEL label reaction solution for 60 min at 37 °C. Images were acquired using Leica Thunder microscope (for tissue sections) and Nikon Eclipse Ti2 fluorescence microscope (for cultured cells).

Western blots

Cells or heart tissue were lysed by using protein lysis buffer (50 mM Tis-HCl pH 7.4, 150 mM NaCl, 1% NP40, 1 mM EDTA) with a complete phosphatase and protease inhibitor cocktail. The antibodies used in this assay were listed in Supplementary Table 2. Unprocessed original scans of western blots were deposited in Supplementary Fig. 8.

Immunofluorescence staining (ICC)

For ICC, cells were washed three times with PBS and fixed by 4% paraformaldehyde (PFA) for 15 min at room temperature. Next, cells were permeabilized with 0.1% Triton X-100, and blocked with 5% BSA for 1 h. Treated cells were incubated with primary antibodies listed in Supplementary Table 2 in 1% BSA overnight. After washing with PBS three times, cells were incubated with secondary antibodies for 1 h in dark. Nuclei staining was performed with DAPI. Around 20 images were randomly taken under ×10 or ×20 magnification at the same exposure setting for quantitative analyses.

RT-qPCR

Total RNA was extracted from cells or heart tissue by Trizol regent (TaKara, 9109) and reverse-transcribed into cDNA using 5× HiScript ⅡRT SuperMix for qPCR (Vazyme, R222-01). RT-qPCR was performed using qPCR SYBR Green Master Mix (Yeasen, 11201ES08) on the Bio-Rad Real-Time PCR Detection System. Primer sequences used in present study were provided in Supplementary Table 3. The relative expression level was qualified using the 2^−ΔΔCt method.

Wheat germ agglutinin (WGA) staining

WGA (Invitrogen, W32464) staining was performed to assess cardiomyocytes hypertrophy. Briefly, heart sections were washed three times with HBSS (Gibco, C14175500BT). Then treated sections were incubated with WGA conjugated to Alexa Fluor 555 for 1 h at room temperature and then washed with HBSS. The cross-sectional cell size was measured by Image J software. At least 30 fields and 90 cells were randomly analyzed for each group.

RNA Immunoprecipitation qPCR (RIP- qPCR)

To obtain sufficient cardiomyocytes for RIP-qPCR experiments, we employed HL-1 cells, an immortalized mouse cardiomyocyte line that can be continuously expanded while retaining essential cardiomyocyte characteristics. HL-1 cells were subjected to ultraviolet (UV) crosslinking at 254 nm with an energy of 400 MJ/cm². And then cells were homogenized in ice-cold lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1:100 protease inhibitors cocktail, 1:25 RNase inhibitor). After incubating on ice for 10 min to lysis the cell, the lysate was centrifuged at 12,000 × g for 15 min at 4 °C. The 5% lysis sample was stored for “input”, and the remaining was used in immunoprecipitation reactions with an anti-Flag antibody and mouse IgG, respectively. The RNA of input and RIP was extracted using Trizol regent (TaKara, 9109). RT-qPCR was performed using qPCR SYBR Green Master Mix (Yeasen, 11201ES08) on the Bio-Rad Real-Time PCR Detection System. Primer sequences were provided in Supplementary Table 3.

Flow cytometry

Cells were harvested by trypsin digestion at 37 °C for 3 min and fixed and permeabilized by fixation buffer (Biolegend, 420801), then incubated with antibodies listed in Supplementary Table 2 at RT for 30 min at concentrations recommended by manufacturers. Cells were then suspended with 1% PFA and run with BD flow cytometer. FlowJo v10 was used to analyze the flow cytometry data.

Cell cycle analysis

To analyze the cell cycle profile, cell pellets from the HL-1 cells were fixed in 2 mL of ice-cold 70% ethanol overnight at −20 °C. Fixed cells were collected by centrifugation and stained with 200 µL of propidium iodide (PI)/RNase staining solution (Invitrogen, F10797) for 20 min at room temperature in the dark. Stained cells were then examined by BD flow cytometer and FlowJo v10 was used to analyze the cell cycle profile.

Bulk RNA sequencing (RNA-seq) and analysis

In general, 2 × 10⁵ CMs infected with shLncBAR or shNT lentiviruses were collected in Trizol (Takara) for RNA extraction. RNA quality was assessed by examining A260/A280 ratio with Nanodrop^TM OneCspectrophotometer (Thermo Fisher Scientific Inc). RNA integrity was confirmed by 1.5% agarose gel electrophoresis. RNA sequencing was performed by GENEWIZ (Suzhou, China). Raw sequencing data were filtered by Trimmomatic (version 0.36) and the clean reads were mapped to the reference genome of mouse (mm10) using STAR software (version 2.5.3a) with default parameters. Reads mapped to the exon regions were counted using featureCounts (Subread-1.5.1, Bioconductor). The resulting BAM files were sorted and indexed using SAMtools⁵².

Differential expression analysis was performed using DESeq2 (version 1.32.0). Genes with expression showing |log₂ fold change| ≥ 1 and adjust P < 0.05 were assigned as differentially expressed genes (DEGs). Gene Ontology Biological Process (GOBP) enrichment analysis and KEGG analysis were conducted using DAVID (https://david.ncifcrf.gov/). All GO and KEGG terms presented in this study have P value lower than 0.05.

Apical resection (AR) in mouse hearts

AR was carried out in P1 and P8 mice hearts as described⁵³. Briefly, mice were anesthetized by ice freezing for 2–5 min. A left parasternal thoracotomy at the fourth intercostal space was performed using blunt scissors to expose the heart. A modified 10 µL pipette tip, connected to a vacuum pump operating at 0.06 ± 0.005 MPa, was gently applied to the apex of the left ventricle for approximately 12 s. The apex was then dissected with a single incision adjacent to the pipette tip trails using iridectomy scissors. The chest and skin incision were sutured with 7-0 silk suture. Post-operative mice were placed on the 37 °C heating pad until recovery. Sham controls underwent the same procedures without apex amputation.

Animals were randomly assigned to experimental groups before surgery. All surgery operations were performed in a double-blinded manner.

Adult mouse model of myocardial infarction (MI)

MI was performed in 8-week-old C57BL/6 mice, including LncBAR^−/− mice and WT mice. Before surgery, mice were anesthetized by intraperitoneal injection of a mixture of tribromoethanol (320 mg/kg) and 2-butanol (160 mg/kg). After exposing the heart, myocardial infarction was induced by permanent ligation of the left anterior descending artery (LAD) with a 7-0 polypropylene suture. Sham-operated animals served as surgical controls and were subjected to the same procedures as the experimental animals with the exception that the LAD was not ligated. Mice were allowed to recover using a mouse ventilator and heating pad.

Animals were randomly assigned to experimental groups before surgery. All surgery operations were performed in a double-blinded manner.

Adeno-associated virus of serotype 9 (AAV9) packaging and delivery

The LncBAR sequence was cloned into the AAV9 plasmid (pcAAV-cTnT-GdGreen-WPRE), with transgene expression driven by the cTnT promoter. Viral particles were produced by OBiO Biotechnology Co. Ltd., Shanghai, China. Neonatal AAV9-mediated LncBAR overexpression was achieved through direct injection into the cardiac ventricles using an insulin syringe with a 30-gauge needle, at a dose of 5 × 10¹⁰ vector genomes per animal. For viral transduction in adult mice post-MI, 5 × 10¹¹ vector genomes per animal were intramyocardially injected into the boundary between the border and infarct zones of infarcted hearts. All viral inoculation were performed in a double-blinded manner.

Histological analysis

For immunohistochemistry, mouse hearts were fixed with 0.5% PFA in PBS supplemented with 5% sucrose overnight. After sequential dehydration with sucrose, samples were embedded in OCT (Sakura, 4583) and slowly frozen in isopropanol on ice. The hearts were vertically sectioned into 5 µm cryosections and stained with antibodies listed in Supplementary Table 2, and subsequently underwent DAPI staining. Tissue sections were imaged using a Leica confocal microscope, and measurements were conducted in a double-blinded manner.

For Masson’s trichrome staining, mouse hearts were perfused with 10 mM KCl until arrested, followed by perfusion with DPBS and 4% PFA. After embedded in paraffin, the samples were vertically sectioned into 5 µm sections, deparaffinized, and stained using the Masson’s trichrome staining kit (Baso, BA4079B) according to the manufacturer’s instructions. Fibrotic area was calculated as the percentage of total fibrotic area relative to total heart section area.

Echocardiography

Adult mice were subjected to 1–2% isoflurane as anesthesia for 3–5 min for echocardiography. Echocardiography was performed using VINNO 6 VET (VINNO Corporation). M-mode imaging was used to measure left ventricular end-systolic anterior wall thickness and to calculate the ejection fraction and fractional shortening. All echocardiography was performed in a double-blinded manner.

Statistical analysis

Data were analyzed and plotted using GraphPad Prism v8 (GraphPad Software) and FlowJo v10 (BD Biosciences). All results were expressed as mean ± SEM. The two-tailed unpaired t test was used to determine the significance of differences between two groups. One-way or two-way analysis of variance (ANOVA) was used to determine the significance of experiments containing three or more conditions. In all cases, P < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001). All data are representative of multiple repeated experiments.