Cardiomyocyte lncRNA Cpat maintains cardiac homeostasis and mitochondria function by targeting citrate synthase acetylation

Abstract

Myocardial energy metabolism disorders are essential pathophysiology in sepsis-associated myocardial injury. Yet, the underlying mechanisms involving impaired mitochondrial respiratory function upon myocardial injury remain poorly understood. Here we identify an unannotated and cardiomyocyte-enriched long non-coding RNA, Cpat (cardiac-protector-associated transcript), that plays an important role in regulating the dynamics of cardiomyocyte mitochondrial tricarboxylic acid (TCA) cycle. Cpat is essential to the mitochondrial respiratory function by targeting key metabolic enzymes and modulating TCA cycle flux. Specifically, Cpat enhances the association of TCA cycle core components malate dehydrogenase (MDH2), citrate synthase (CS), and aconitase (ACO2). Acetyltransferase general control non-repressed protein-5 (GCN5) acetylates CS and destabilizes the MDH2-CS-ACO2 complex formation. Cpat inhibits this GCN5 activity and facilitates MDH2-CS-ACO2 complex formation and TCA cycle flux. We reveal that Cpat-mediated mitochondrial metabolic homeostasis is vital in mitigating myocardial injury in sepsis-induced cardiomyopathy, positioning Cpat as a promising therapeutic target for preserving myocardial cellular metabolism and function.

Fig. 1. Identification of a cardiomyocyte lncRNA Cpat downregulated in septic mouse heart.

a Volcano map of differentially expressed genes from transcriptional analysis in hearts of CLP-induced septic mice vs sham mice. Fold change > 1.5. n = 3 biological replicates. b, c RT-qPCR analysis of 3 most significantly differentially expressed lncRNAs from CLP-induced (b) or LPS-induced (c) septic mouse hearts. Gapdh was used as the reference gene. n = 3 biological replicates. d Heart Cpat expression in mice treated with LPS (20 mg/kg, i.p.) for different time periods. n = 3 (24 H) or 5 (12 H) or 6 (0 H) or 7 (6 H) biological replicates. e Cpat expression in cardiomyocyte treated with conditioned medium (LPS 1 µg/ml stimulated Raw264.7 for 24 h) for different time periods. n = 3 biological replicates. f Cpat expression levels in different tissues by RT-qPCR. Gapdh was used as a reference gene. n = 5 biological replicates. g scRNA-Seq of hearts from 3 wild type (WT) mice showed Cpat expression in various heart cell types. h Schematic illustration of Cpat and XR004938650.1 originated from the intergenic region within Myh7 and transcribed into Cpat and XR004938650, and comparison between the sequence structures of Cpat and XR004938650. i CPC predicted the Cpat coding probability. The protein-coding mRNA Gapdh was used as a negative control. LncRNAs Neat1 and Hotair1 were used as positive controls. j Representative images of fluorescence in situ hybridization (FISH) showed localization of Cpat in HL-1 cells. Cytoplasm marker 18S and nuclear marker U6 acts were used. Scale bar =10 µm. Statistical significance was determined using the Wald test (two-sided) implemented in DESeq2, and P values were adjusted for multiple testing using the Benjamini-Hochberg method (a). Data are presented as mean ± SD, unpaired two-sided Student’s t-test (b, c) and one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (d, e). p values are indicated. Source data are provided as a Source Data file. CLP cecal ligation and puncture, LPS lipopolysaccharide; 0, 6, 12, 24 H: 0, 6, 12, 24 h followed by LPS administration.

Fig. 2. Cpat overexpression ameliorates cardiac dysfunction in mice with LPS-induced sepsis.

a Experimental design to test a role for Cpat in ameliorating cardiac dysfunction from LPS-induced sepsis in mice. WT mice at 0-2 days postnatal were infected with adeno-associated virus serovar 9 (AAV9)-Cpat (2x10¹¹v.g., i.h.). Mice at 8 weeks old were administered with LPS (i.p. 10 mg/kg for cardiac functions and 20 mg/kg for survival test) to induce sepsis (Created in BioRender. Fan, Y. (2025) https://BioRender.com/0pt6yyb). b RT-qPCR quantification of heart Cpat expression in AAV-Ctrl and AAV-Cpat mice. n = 6 biological replicates. c–g Echocardiogram representative M-mode (c), survival curves (d), EF% (e), FS% (f), LVESV (g) in AAV9-Cpat and AAV9-Ctrl mice after LPS administration (i.p. 10 mg/kg for cardiac functions and 20 mg/kg for survival test). n = 6 (echocardiographs) or 10 (survival curve) biological replicates. h-k ELISA quantification of peripheral blood myocardial injury markers CK-MB (h), cTnT (i) and cytokines IL-1β (j), TNF-α (k). n = 6 biological replicates. l Survival curves of Tg (Cpat-Cre) and Tg (Cpat) mice following CLP administration. n = 10 biological replicates. m–o Echocardiography analyses of cardiac functions EF% (m), FS% (n) and LVESV (o) between Tg (Cpat) and Tg (Cpat-Cre) mice after CLP or Sham administration. n = 6-10 biological replicates. p–s ELISA quantification of peripheral blood myocardial injury markers CK-MB (p), cTnT (q) and cytokines IL-1β (r) and TNF-α (s) from Tg (Cpat) mice and Tg (Cpat-Cre) mice after CLP or Sham administration. n = 6 biological replicates. Data are presented as mean ± SD, unpaired two-sided Student’s t-test (b), Kaplan-Meier survival curves were compared using the log-rank (Mantel-Cox) test (d, l). two-way (e–g) or one-way (h–k, m–s) ANOVA with Tukey’s multiple comparison test. p values are indicated. Source data are provided as a Source Data file. WT wild type, Tg transgenic, Cre Myh6-Cre, i.p. intraperitoneally, EF ejection fractions, FS fractional shortening, LVESV left ventricular end systolic volume, CK-MB creatine phosphokinase isozymes, cTnT troponin T, IL-1β interleukin-1β, TNF-α tumor necrosis factor-α.

Fig. 3. Cpat interacts with mitochondria-associated Pnpt1 using loops 3 and 4.

a Schematic diagram of RNA pulldown combined with mass spectrometry (MS) (Created in BioRender. Fan, Y. (2025) https://BioRender.com/gfnrubw). b KEGG pathway enrichment analysis of detected proteins from MS. c Gene Ontology (GO) analysis of detected proteins from MS. d Representative respiratory experiment of mitochondrial OXPHOS capacity by using the substrate uncoupler inhibitor titration (SUIT) protocol. AAV9-Ctrl vs AAV9-Cpat mice, with PBS or LPS administration. n = 3 biological replicates. e Representative electron microscopy images of myocardial mitochondria morphology after PBS or LPS treatment. AAV-Ctrl vs AAV-Cpat mice. n = 5 biological replicates. f Top 10 proteins involved in the metabolic pathway from Cpat pulldown with MS detection, ranked by abundance. g Representative immunoblot of Pnpt1 pulled down by Cpat. n = 3 biological replicates. h RNA immunoprecipitation (RIP) of Cpat by Pnpt1 antibody in HL-1 cells. IgG was used as a negative control. n = 3 biological replicates. i Schematic illustration of Pnpt1 domains. j, Immunoblot of different Flag-Pnpt1 mutants in HEK293T cells. k RIP assays in HL-1 cells using Flag-antibody for immunoprecipitation followed RT-qPCR to quantify Flag-Pnpt1-bound Cpat. l Schematic diagram of Cpat loops based on its secondary structure. m Anti-Flag immunoblot to detect Pnpt1 pulled down by different Cpat loop fragments. Data are presented as mean ± SD, one-way ANOVA with Tukey’s multiple comparison test (h, k). p values are indicated. Source data are provided as a Source Data file.

Fig. 4. HuR facilitates Cpat transportation from nucleus into cytoplasm.

a Immunoblot of HuR pulled down by sense and anti-sense Cpat in HL-1 cells. b RIP of Cpat by HuR antibody in HL-1 cells. IgG was used as negative control. n = 3 biological replicates. c HuR immunoblot analysis of isolated cytoplasm and nuclear fractions or whole cell lysate (WCL) from HL-1 cells transfected with HuR siRNA or negative control siRNA (NC). Cell fraction markers included GAPDH (cytoplasm) and lamin B (nucleus). d RT-qPCR of HuR in isolated cellular fractions or WCL in HL-1 cells after siRNA-HuR transfection (vs. siRNA-Ctrl). n = 3 biological replicates. e RT-qPCR of WCL Cpat after siRNA-HuR transfection in HL-1 cells (vs. siRNA-Ctrl). n = 3 biological replicates. f Schematic diagram of nascent RNA 4SU labelling and detection (Created in BioRender. Fan, Y. (2025) https://BioRender.com/sorgudk). g, h RT-qPCR of total and nascent Cpat in HL-1 nucleus (g) and cytoplasm (h). (siRNA-HuR vs. siRNA-Ctrl). n = 3 biological replicates. i Immunoblot of HuR in HL-1 cells transfected with siRNA-CRM1 or control siRNA. j Immunoblot of HuR in HL-1 nuclear and cytoplasm fractions after siRNA-CRM1 or control siRNA transfection. k Immunoblot of HuR and CRM1 in HL-1 cells to verify the efficiency of siRNA-HuR and siRNA-CRM1. l RT-qPCR of Cpat in HL-1 cell WCL and cytoplasm after siRNA transfection. n = 3 biological replicates. m IP of Cpat by HuR antibody in HL-1 cells measured by RT-qPCR after siRNA-CRM1 or control siRNA transfection. n = 3 biological replicates. n Immunoblot of Pnpt1 in different cellular components after siRNA-HuR or control siRNA transfection. o RT-qPCR of mitochondrial Cpat after siRNA-HuR transfection in HL-1 cells (vs. siRNA-Ctrl). n = 3 biological replicates. p Immunoblot of HuR pulled down by different fragments of Cpat in HEK293T cells. q Graphical illustration of HuR and CRM1 contribute to Cpat transportation from nucleus to cytoplasm (Created in BioRender. Fan, Y. (2025) https://BioRender.com/2cy50tf). Data are presented as mean ± SD, unpaired two-sided Student’s t-test (b, e, o), two-way ANOVA with Tukey’s multiple comparison test (d, g, h, l, m) and Unpaired Student’s t test (j). p values are indicated. Source data are provided as a Source Data file.

Fig. 5. Cpat prevents LPS-induced MDH2-CS-ACO2 complex disassembly.

a Immunoblot analysis of Pnpt1 in HL-1 cells. b, c RT-qPCR of Pnpt1 in WCL and mitochondria from HL-1 cells transfected with siRNA-Pnpt1 vs siRNA-Ctrl (b) or Pnpt1 overexpression (OE Pnpt1) vs. OE Ctrl (c). n = 3 biological replicates. d, e RT-qPCR of Cpat in WCL and mitochondria from HL-1 cells after siRNA-Pnpt1 (d) or Pnpt1 overexpression (e). n = 3 biological replicates. f Immunoblot analysis of nuclear lamin B, cytoplasm GAPDH, and mitochondria citrate synthase (CS) in purified HL-1 cellular components. g, h RT-qPCR of cytoplasm GAPDH, nuclear U6, and mitochondria Pnpt1, CS, and ATP8 in purified mitochondria from HL-1 cells (g) or Cpat in mitochondria from mouse neonatal cardiomyocytes treated with LPS or PBS (h). n = 3 biological replicates. i RNA FISH and immunofluorescence image of Cpat and mitochondria marker CS in HL-1 cells, showing their subcellular distribution. j Immunoblot analysis of CS pulled down by sense and antisense Cpat in HL-1 cells. n = 3 biological replicates. k RIP of Cpat by CS antibody in HL-1 cells IgG was used as negative control. n = 3 biological replicates. l Immunoblot analysis of Myc-CS after RIP with different Cpat loops or full-length Cpat (FL) in Myc-CS-treated HEK293T cells. m Graphical illustration of MDH2-CS-ACO2 complex in baseline condition. n Immunofluorescence co-localization of CS with ACO2 in mouse neonatal cardiomyocytes. Scale bar = 2 μm. o Immunoblot analyses using Myc or Flag antibodies following Myc antibody-mediated immunoprecipitations (IP) of HEK293T cells treated with or without Myc-CS and Flag-ACO2. p Immunofluorescence co-localization of CS with MDH2 in mouse neonatal cardiomyocytes. Scale bar = 2 μm. q Immunoblot analyses using Myc or Flag antibodies following Myc antibody-mediated IP of HEK293T cells treated with or without Myc-CS and Flag-MDH2. Data are presented as mean ± SD, two-way (b–e) or one-way (g) ANOVA with Tukey’s multiple comparison test, unpaired two-sided Student’s t-test (h, k). p values are indicated. Source data are provided as a Source Data file.

Fig. 6. Cpat maintains MDH2-CS-ACO2 complex stability by reducing CS acetylation.

a-b immunoblot analysis of ACO2 and MDH2 in mouse neonatal cardiomyocytes after CS pull down. Cells were transduced with Lv-Cpat (a) or transfected with siRNA-Cpat (b) and then treated with LPS. This experiment was repeated three times. c immunoblot analysis of ACO2 and MDH2 in heart tissues from Tg(Cpat-Cre) and Tg(Cpat) transgenic mice treated with LPS or PBS, following CS pull-down. This experiment was repeated three times. d-e immunoblot analysis of CS acetylation in mouse neonatal cardiomyocytes treated with or without LPS and Lv-Cpat (d) or siRNA-Cpat (e). This experiment was repeated three times. f Immunoblot analysis of CS acetylation in heart tissues from Tg (Cpat-Cre) and Tg (Cpat) transgenic mice treated with LPS or PBS, following CS pull-down. This experiment was repeated three times. g-h immunoblot analysis of CS acetylation in HEK293T cells treated with or without LPS, Myc-CS, and transduced with Lv-Cpat (g) or transfected with siRNA-Cpat (h) as indicated. This experiment was repeated three times. i Graphical illustration of Cpat activity in maintaining MDH2-CS-ACO2 complex stability by reducing CS acetylation (Created in BioRender. Fan, Y. (2025) https://BioRender.com/yr42tnh). j-k NAM and TSA blocked the deacetylase activity. Immunoblot analysis of CS acetylation in HEK293T cells (j) and mouse neonatal cardiomyocytes (k) after different times of NAM + TSA treatment. This experiment was repeated three times. l, m IP combined with immunoblot to detect ACO2-CS (l) and CS-MDH2 (m) immunocomplexes in HEK293T cells with or without NAM and TSA treatment. This experiment was repeated three times. n, o IP combined with immunoblot to detect MDH2-CS and CS-ACO2 immunocomplexes in mouse neonatal cardiomyocytes with Cpat overexpression (Lv-Cpat, n) and knockdown (siRNA-Cpat, o) with or without NAM and TSA treatment. This experiment was repeated three times. Source data are provided as a Source Data file. MDH2 malate dehydrogenase, CS citrate synthase, ACO2 aconitase, NAM nicotinamide, TSA trichostatin A.

Fig. 7. Cpat blocks GCN5-mediated CS acetylation.

a Immunoblot to test the activities of different acetyltransferases in CS acetylation in HEK293T cells. This experiment was repeated three times. b IP combined with immunoblot to detect CS (Myc-CS) and GCN5 (HA-GCN5) immunocomplexes in HEK293T cells with and without LPS (1 µg/mL) treatment or Cpat rescue. This experiment was repeated three times. c IP combined with immunoblot to assess the impact of GCN5 in affecting MDH2-CS and CS-ACO2 immunocomplex formation in HEK293T cells. This experiment was repeated three times. d Graphical illustration of Cpat activity in preventing GCN5-mediated CS acetylation. e Schematic diagram of CS acetylation site detection by LC-MS/MS (Created in BioRender. Fan, Y. (2025) https://BioRender.com/4c4aqbr). f Acetylation sites identified by LC-MS/MS is displayed on the molecular structure of CS. CS Uniprot ID: Q9CZU6. g IP combined with immunoblot detected CS acetylation in HEK293T cells with or without LPS (1 µg/mL) treatment. 6KR, all six lysine residues were replaced by arginine. This experiment was repeated three times. h, i Representative immunoblots of Flag-ACO2 (h) and Flag-MDH2 (i) pulled down by Myc-CS and Myc-CS K52R, K366R, K370R and 6KR in HEK293T cells with or without NAM and TSA treatment. This experiment was repeated three times. j Schematic diagram summarizing the proposed mechanism by which Cpat overexpression modulates mitochondrial metabolism and cardiomyocyte injury in response to sepsis-induced stress (Created in BioRender. Fan, Y. (2025) https://BioRender.com/v28g941). Source data are provided as a Source Data file. MDH2: malate dehydrogenase; CS: citrate synthase; ACO2: aconitase; GCN5: general control non-repressed protein-5.