Hepatic ChREBP promotes cardiac remodeling through non‑transcriptional repression of ApoM

 

Preface

In today's rapidly advancing life sciences, precise, non‑invasive, and dynamic in vivo imaging technologies have become a core driving force for elucidating disease mechanisms and accelerating drug development. Small‑animal ultrasound, with its exceptional soft‑tissue resolution, multimodal imaging capabilities, and unique advantage of repeated longitudinal tracking, plays an indispensable role in cardiovascular, oncological, and abdominal organ research. In this issue, we highlight a pivotal study published in Circulation Research, which utilized the VINNO small‑animal ultrasound imaging system (VINNO Technology) to perform standardized, dynamic assessments of murine cardiac structure and function, thereby capturing key imaging evidence for liver‑heart crosstalk in the regulation of cardiac remodeling. Concurrently, advances in functional ultrasound (fUS) and ultrasound localization microscopy (ULM) are pushing the visualization of microvascular architecture and hemodynamics to new heights. We take this opportunity to explore with fellow researchers the broad application prospects of small‑animal ultrasound in basic and translational medicine, facilitating the production of high‑quality scientific outputs.

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I. Research Background

Pathological cardiac remodeling represents the common pathological substrate underlying the progression of various cardiovascular diseases to heart failure. Current clinical therapeutic strategies directly targeting the heart remain limited. Accumulating evidence indicates that cardiac remodeling is not a heart‑autonomous process; rather, metabolic alterations in peripheral organs such as the liver and gut also contribute to disease development and progression. However, the molecular mechanisms mediating pathological signal transmission across multiple organs remain to be further elucidated.

 

ApoM, as an important apolipoprotein of high‑density lipoprotein (HDL), is a key molecule mediating inter‑organ signal communication. Although ApoM is present only in a small subset of HDL subfractions, it is capable of binding the majority of plasma sphingosine‑1‑phosphate (S1P), which exerts significant cardiovascular protective effects. Previous studies have established the involvement of the ApoM‑S1P signaling axis in the pathogenesis of atherosclerosis and diabetic cardiomyopathy, among other conditions. Nevertheless, the activation pattern of this pathway and its upstream regulatory mechanisms under pathological conditions of increased cardiac pressure overload remain largely unclear.

 

This study was conducted collaboratively by Professor Yajun Duan from the University of Science and Technology of China, Professor Junbo Ge from Zhongshan Hospital, Fudan University, and Professor Deling Kong and Researcher Baofa Sun from Nankai University. Focusing on the liver‑derived transcription factor ChREBP as the entry point, the study employed the VINNO 6 LAB small‑animal ultrasound imaging system to achieve non‑invasive, dynamic, and quantitative assessments of murine cardiac structure and function, obtaining standardized imaging data with the aim of dissecting the molecular mechanisms underlying liver‑heart crosstalk in pathological cardiac remodeling.

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II. Main Experimental Methods

This study was conducted at the animal, organ, and molecular levels, with the primary methods outlined below:

1. 1.  Animal model construction

   • . Isoproterenol (ISO)‑treated mice: ISO administered at 10 mg/kg/day for 4 consecutive weeks.

   • . Transverse aortic constriction (TAC)‑operated mice: pressure overload‑induced cardiac remodeling.

   • . Hepatocyte‑specific ChREBP knockout mice.

   • . Global ApoM knockout mice.

   • . Cardiomyocyte/endothelial cell‑specific S1PR1 knockout mice.

2. 2. Core techniques: Echocardiography for cardiac function assessment, combined with exhaustive exercise testing, non‑invasive blood pressure measurement, hematoxylin‑eosin (HE) staining, Sirius Red staining, wheat germ agglutinin (WGA) staining, immunofluorescence, enzyme‑linked immunosorbent assay (ELISA), Western blotting, liver RNA‑sequencing (RNA‑seq), and co‑immunoprecipitation (Co‑IP).

3. 3.  Measured parameters: Ejection fraction (EF%), fractional shortening (FS%), relative wall thickness (RWT), E/A ratio, and E/e′ ratio.

4. 4.  Echocardiography: Echocardiographic images were acquired using a small‑animal ultrasound imaging system (VINNO Technology, Suzhou, China).

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III. Experimental Results

Following 4 weeks of ISO treatment (Figure 1A), left ventricular fractional shortening (LVFS) was significantly decreased, while relative wall thickness (RWT), E/A ratio, and E/e′ ratio were increased, indicating impaired systolic and diastolic function (Figures 1B–C). The ratios of heart weight to tibia length (HW/TL), heart weight to body weight (HW/BW), and lung weight to body weight (LW/BW) were elevated, suggesting cardiac hypertrophy and pulmonary congestion (Figure 1D). WGA staining revealed increased cardiomyocyte cross‑sectional area in the ISO group, confirming myocardial hypertrophy (Figure 1E).

 

Figure 1:

A.  Schematic timeline of the in vivo experimental protocol.
B.  Representative M‑mode echocardiographic traces and Doppler flow images from mice.
C.  Quantitative analysis of left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), relative wall thickness (RWT), and E/A and E/e′ ratios.
D.  Heart weight‑to‑tibia length ratio (HW/TL), heart weight‑to‑body weight ratio (HW/BW), and lung weight‑to‑body weight ratio (LW/BW). All quantitative data are presented as mean ± standard deviation (SD). For panels C and D, P values were calculated using the Mann‑Whitney U test. A P value < 0.05 was considered statistically significant. FC, fold change.

 

Four experimental groups were established in this section: control (C), hepatocyte‑specific ChREBP knockout (KO), ISO model (ISO), and hepatocyte‑specific ChREBP knockout with ISO treatment (KISO). Mice in the C group received saline treatment; KO mice underwent hepatocyte‑specific ChREBP deletion without ISO challenge; ISO mice received isoproterenol to establish the cardiac remodeling model; KISO mice were hepatocyte‑specific ChREBP knockout mice concurrently treated with isoproterenol (Figure 2A). ChREBP knockout alone did not affect cardiac function. The ISO group exhibited decreased LVEF and LVFS, increased RWT, E/A ratio, and E/e′ ratio, reduced exercise distance, and elevated blood pressure. In contrast, the KISO group showed significant recovery of the aforementioned cardiac functional parameters to near‑control levels (Figures 2B–D). KISO reversed the ISO‑induced increases in HW/TL and HW/BW (Figure 2G) and reduced serum levels of cardiac injury enzymes, including creatine kinase (CK), lactate dehydrogenase (LDH), β‑hydroxybutyrate dehydrogenase (HBDH), and aspartate aminotransferase (AST) (Figure 2H). Hematoxylin‑eosin (HE), Sirius Red, and wheat germ agglutinin (WGA) staining demonstrated that KISO attenuated cardiomyocyte hypertrophy and myocardial fibrosis (Figures 2I–K). ELISA measurements of serum tumor necrosis factor‑α (TNF‑α) and interleukin‑1β (IL‑1β) levels, together with downregulation of hypertrophy markers—atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), β‑myosin heavy chain (β‑MHC), and α‑smooth muscle actin (α‑SMA)—and reduced inflammatory cytokines TNF‑α and IL‑1β, were observed in the KISO group (Figures 2L–M). Collectively, these results indicate that hepatocyte‑specific ChREBP knockout confers resistance to ISO‑induced pathological cardiac remodeling and preserves cardiac function.

 

Figure 2

A.  Schematic timeline of the in vivo experimental protocol.
B.  Representative M‑mode echocardiographic traces and Doppler flow images.
C.  Quantitative analysis of left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and relative wall thickness (RWT).
D.  E/A and E/e′ ratios.
E.  Running distance.
F.  Systolic blood pressure (SBP) and diastolic blood pressure (DBP) levels.
G.  Heart weight‑to‑tibia length ratio (HW/TL) and heart weight‑to‑body weight ratio (HW/BW).
H.  Serum levels of creatine kinase (CK), lactate dehydrogenase (LDH), β‑hydroxybutyrate dehydrogenase (HBDH), and aspartate aminotransferase (AST).
I.  Representative whole‑heart images and hematoxylin‑eosin (HE) staining.
J.  Sirius Red staining and wheat germ agglutinin (WGA) staining.
K.  Quantitative analysis of collagen fibers and cardiomyocyte cross‑sectional area.
L.  Serum levels of TNF‑α and IL‑1β measured by ELISA.
M.  Western blot analysis of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), β‑myosin heavy chain (β‑MHC), and α‑smooth muscle actin (α‑SMA) expression levels in mouse hearts.
*n* = 6. All quantitative data are presented as mean ± SD. P values were determined using the aligned rank transform (ART) test with Tukey's post hoc test for panels C through L. A P value < 0.05 was considered statistically significant.

 

In this section, ISO/TAC mouse models were established, along with inclusion of human clinical control and cardiac hypertrophy patient samples for parallel investigation (Figure 3A). The results demonstrated that serum ApoM levels were decreased in ISO/TAC model mice, whereas hepatocyte‑specific ChREBP knockout restored serum ApoM levels (Figures 3B–D). Correlation analyses in mice revealed that circulating ApoM levels were positively correlated with LVEF and negatively correlated with BNP; higher hepatic ApoM expression was associated with elevated LVEF and LVFS. Clinical sample results showed that serum ApoM levels in cardiac hypertrophy patients were significantly lower than those in controls, and the reduction in ApoM was accompanied by decreased LVEF and LVFS as well as increased NT‑proBNP (Figures 3E–H). These findings suggest that ApoM levels are positively associated with cardiac function, and that ApoM expression is downregulated during cardiac remodeling, positioning ApoM as a candidate mediator of the liver‑heart crosstalk axis.

 

Figure 3

A.  Venn diagram showing the overlap of upregulated/downregulated differentially expressed genes (DEGs, FDR < 0.05, |log₂FC| > 1) from isoproterenol (ISO) versus control and KISO versus ISO comparisons, intersecting with liver-secreted proteins and a liver-heart mediator database.
B.  Volcano plot of DEGs in KISO versus ISO: pink indicates upregulation (FDR < 0.05, FC > 2), blue indicates downregulation (FDR < 0.05, FC < 0.5), and gray indicates no significant difference.
C.  Serum ApoM levels measured by ELISA.
D.  Western blot analysis of hepatic ApoM expression in mice.
E, F.  Serum levels of BNP (brain natriuretic peptide) and ApoM.
G, H.  Spearman correlation analyses between circulating ApoM and left ventricular ejection fraction (LVEF) as well as BNP. All quantitative data are presented as mean ± SD. P values were determined as follows: for C, aligned rank transform (ART) test with Tukey's post hoc test; for E (BNP), Mann‑Whitney U test; for E (ApoM) and F (BNP), unpaired *t*‑test with Welch's correction; for F (ApoM), two‑tailed Student's *t*‑test; for G and H, Spearman correlation analysis. A P value < 0.05 was considered statistically significant.

 

Three experimental groups were established: ISO‑Ctrl (ISO model with control treatment), KISO‑Ctrl (hepatocyte‑specific ChREBP knockout combined with ISO treatment), and KISO‑shApoM (hepatocyte‑specific ChREBP knockout with AAV‑mediated ApoM knockdown and concurrent ISO treatment) (Figure 4A). The results showed that KISO‑Ctrl ameliorated ISO‑induced cardiac dysfunction, as evidenced by restored LVEF and LVFS, decreased RWT, E/A ratio, and E/e′ ratio, and recovered exercise capacity. In contrast, KISO‑shApoM completely reversed these cardioprotective effects, manifesting as reduced LVEF and LVFS again, worsened diastolic function parameters, and declined exercise tolerance (Figures 4B–E). Furthermore, KISO‑shApoM increased HW/TL, HW/BW, and serum levels of cardiac injury enzymes, exacerbated cardiomyocyte hypertrophy and fibrosis, and re‑upregulated hypertrophy markers including ANP, BNP, β‑MHC, and α‑SMA (Figures 4G–L). Collectively, these findings confirm that ApoM is an essential mediator for the cardioprotective effects of hepatocyte‑specific ChREBP knockout, and that ApoM knockdown abolishes this protective effect.

 

Figure 4

A.  Schematic timeline of the in vivo experimental protocol.
B.  Representative M‑mode echocardiographic traces and Doppler flow images.
C.  Quantitative analysis of left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and relative wall thickness (RWT).
D.  E/A and E/e′ ratios.
E.  Running distance.
F.  Systolic blood pressure (SBP) and diastolic blood pressure (DBP) levels.
G.  Heart weight‑to‑tibia length ratio (HW/TL), heart weight‑to‑body weight ratio (HW/BW), and lung weight‑to‑body weight ratio (LW/BW).
H.  Serum levels of creatine kinase (CK), lactate dehydrogenase (LDH), β‑hydroxybutyrate dehydrogenase (HBDH), and aspartate aminotransferase (AST).
I.  Sirius Red staining and wheat germ agglutinin (WGA) staining.
J.  Quantitative analysis of fibrosis and cardiomyocyte cross‑sectional area.
K.  Representative whole‑heart images and hematoxylin‑eosin (HE) staining.
L.  Western blot analysis of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), β‑myosin heavy chain (β‑MHC), and α‑smooth muscle actin (α‑SMA) expression levels in mouse hearts.
*n* = 6. All quantitative data are presented as mean ± SD. P values were determined using the Kruskal‑Wallis test followed by Dunn's multiple comparisons test for panels C through J. A P value < 0.05 was considered statistically significant.

 

Two experimental groups were established in this section: ISO‑Ctrl and ISO‑ApoM (AAV8‑mediated liver‑specific ApoM overexpression combined with ISO treatment) (Figure 5A). The results demonstrated that mice in the ISO‑ApoM group exhibited increased LVEF and LVFS, decreased RWT, E/A ratio, and E/e′ ratio (Figures 5A–D), improved running distance (Figure 5E), reduced systolic and diastolic blood pressure (SBP and DBP) (Figure 5F), and lowered organ indices including HW/TL, HW/BW, and LW/BW, as well as decreased serum levels of cardiac injury enzymes CK, LDH, HBDH, and AST (Figures 5G–H). HE, WGA, and Sirius Red staining revealed marked attenuation of cardiomyocyte hypertrophy and fibrosis, accompanied by downregulation of hypertrophy markers ANP, BNP, β‑MHC, and α‑SMA (Figures 5I–K). Collectively, these findings confirm that liver‑specific ApoM overexpression alone is sufficient to significantly reverse ISO‑induced cardiac remodeling and confer cardioprotection.

 

Figure 5

A.  Schematic timeline of the in vivo experimental protocol.
B.  Representative M‑mode echocardiographic traces and Doppler flow images.
C.  Quantitative analysis of left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and relative wall thickness (RWT).
D.  E/A and E/e′ ratios.
E.  Running distance.
F.  Systolic blood pressure (SBP) and diastolic blood pressure (DBP) levels.
G.  Heart weight‑to‑tibia length ratio (HW/TL), heart weight‑to‑body weight ratio (HW/BW), and lung weight‑to‑body weight ratio (LW/BW).
H.  Serum levels of creatine kinase (CK), lactate dehydrogenase (LDH), β‑hydroxybutyrate dehydrogenase (HBDH), and aspartate aminotransferase (AST).
I.  Representative whole‑heart images, Sirius Red staining, and WGA staining.
J.  Quantitative analysis of fibrosis and cardiomyocyte cross‑sectional area.
K.  Western blot analysis of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), β‑myosin heavy chain (β‑MHC), α‑smooth muscle actin (α‑SMA), and ApoM expression levels in mouse hearts.
*n* = 6. All quantitative data are presented as mean ± SD. P values for panels C through J were determined using the Mann‑Whitney U test. A P value < 0.05 was considered statistically significant.

 

In this section, in vivo ISO models were employed, combined with S1PR1/2/3 inhibitors, AAV‑mediated specific knockdown of S1PR1, and S1PR1 agonists. The results showed that serum S1P levels were decreased in ISO model mice, while ChREBP knockout or ApoM overexpression elevated serum S1P levels. Among the S1P receptors, only S1PR1 protein expression was downregulated, with no significant changes observed in S1PR2 or S1PR3. In vitro experiments demonstrated that the S1PR1 inhibitor W146 abolished the protective effect of ApoM against cardiomyocyte hypertrophy, whereas S1PR2 and S1PR3 inhibitors showed no such effect. In vivo experiments revealed that following knockdown of S1PR1 in cardiomyocytes and endothelial cells, the cardioprotective effects conferred by hepatic ApoM overexpression were completely lost (Figures 6A–G), as evidenced by reduced LVEF and LVFS, worsened diastolic function, decreased exercise tolerance, and exacerbated cardiomyocyte hypertrophy and fibrosis. Conversely, the S1PR1 agonists FTY720 and ozanimod mimicked the protective effects of ApoM and ameliorated ISO‑induced cardiac dysfunction (Figures 6H–J). Collectively, these results confirm that the cardioprotective action of ApoM is entirely dependent on cardiac S1PR1, and that the S1P‑S1PR1 axis represents the critical downstream pathway.

 

Figure 6

A.  Schematic illustration of the ApoM‑S1P signaling pathway.
B.  Serum S1P levels measured by ELISA.
C.  Western blot analysis of S1PR1, S1PR2, and S1PR3 expression.
D.  Phalloidin staining and quantification of cardiomyocyte surface area.
E.  Western blot analysis of S1P receptors and hypertrophy markers in mouse primary cardiomyocytes treated with isoproterenol (ISO), ApoM, and specific inhibitors of S1PR1 (W146), S1PR2 (JTE‑013), and S1PR3 (CAY10444).
F.  Schematic timeline of the in vivo experimental protocol.
G.  Representative M‑mode echocardiographic traces and Doppler flow images.
H.  Quantitative analysis of left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), relative wall thickness (RWT), E/A ratio, E/e′ ratio, running distance, systolic blood pressure (SBP), diastolic blood pressure (DBP), heart weight‑to‑tibia length ratio (HW/TL), heart weight‑to‑body weight ratio (HW/BW), lung weight‑to‑body weight ratio (LW/BW), and serum levels of creatine kinase (CK), lactate dehydrogenase (LDH), β‑hydroxybutyrate dehydrogenase (HBDH), and aspartate aminotransferase (AST).
I.  WGA staining and quantification of cardiomyocyte cross‑sectional area.
J.  Sirius Red staining and quantification of fibrotic area.
*n* = 6. All quantitative data are presented as mean ± SD. P values were determined as follows: for B, aligned rank transform (ART) test with Tukey's post hoc test; for B (S1P), Mann‑Whitney U test; for D, one‑way ANOVA followed by Tukey's multiple comparisons test; for H through J, Mann‑Whitney U test. A P value < 0.05 was considered statistically significant.

 

In this section, wild‑type (WT) and ApoM knockout (ApoM‑KO) ISO model mice were used and treated with injections of ApoM‑free HDL or ApoM‑containing HDL, respectively (Figure 7A). The results demonstrated that mice receiving ApoM‑HDL exhibited increased LVEF and LVFS, improved RWT, E/A ratio, and E/e′ ratio (Figures 7B–C), enhanced running distance, reduced blood pressure, decreased HW/TL, HW/BW, and cardiac injury enzyme levels, and significantly attenuated cardiomyocyte hypertrophy and fibrosis. In contrast, ApoM‑free HDL conferred no cardioprotective effects. Notably, even in ApoM‑KO mice, exogenous administration of ApoM‑HDL still improved cardiac function (Figures 7D–I), confirming that the cardioprotective effect of HDL is dependent on ApoM, and that ApoM‑HDL can directly ameliorate cardiac remodeling in a manner independent of endogenous ApoM.

 

Figure 7

A.  Schematic timeline of the in vivo experimental protocol.
B.  Representative M‑mode echocardiographic traces and Doppler flow images.
C.  Quantitative analysis of left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), relative wall thickness (RWT), E/A ratio, E/e′ ratio, and running distance.
D.  Systolic blood pressure (SBP) and diastolic blood pressure (DBP) levels in mice.
E.  Heart weight‑to‑tibia length ratio (HW/TL), heart weight‑to‑body weight ratio (HW/BW), and lung weight‑to‑body weight ratio (LW/BW).
F.  Serum levels of creatine kinase (CK), lactate dehydrogenase (LDH), β‑hydroxybutyrate dehydrogenase (HBDH), and aspartate aminotransferase (AST).
G.  WGA staining and quantification of cardiomyocyte cross‑sectional area.
H.  Sirius Red staining and quantification of fibrotic area.
I.  Schematic summary illustrating that liver‑derived ApoM‑rich HDL, but not ApoM‑deficient HDL, effectively inhibits the development of cardiac hypertrophy.
*n* = 6. All quantitative data are presented as mean ± SD. P values were determined using the aligned rank transform (ART) test with Tukey's post hoc test for panels C through H. A P value < 0.05 was considered statistically significant.

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IV. Research Conclusions

This study demonstrates that cardiac pressure stress promotes the accumulation of ChREBP in the cytoplasm of hepatocytes. The accumulated ChREBP binds to SURF4, thereby inhibiting ApoM secretion, which leads to reduced circulating ApoM‑S1P levels. Consequently, insufficient activation of the cardiac S1PR1 signaling pathway occurs, ultimately inducing myocardial remodeling and cardiac dysfunction. Conversely, hepatic ChREBP knockout or upregulation of ApoM/ApoM‑HDL restores S1P‑S1PR1 signaling, thereby suppressing cardiomyocyte hypertrophy and fibrosis and conferring cardioprotection.

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Publication Information

Title：

Hepatic ChREBP Drives Cardiac Remodeling via ApoM Nontranscriptional Repression

Authors: 

Shuang Zhang, Zhenzhen Zhang, Zihan Ma, Wen Wu, Lu Tang, Yanlu Han, Songning Chen, Tengteng Yan, Ye Chen, Junwu Liu, Dongdong Jian, Ji'e Yang, Likun Ma, Zequn Yin, Houzao Chen, Baofa Sun, Deling Kong, Junbo Ge, and Yajun Duan

Publisher: 

Circulation Research

26 August 2026

doi:

https://doi.org/10.1161/CIRCRESAHA.125.327537

Original Link: 

https://www.ahajournals.o rg/doi/full/10.1161/CIRCRESAHA.125.327537