Yeeran

1. Research Background

Diabetic cardiomyopathy (DCM) is a common and specific cardiac complication of diabetes mellitus, primarily characterized by myocardial hypertrophy, interstitial fibrosis, and impaired systolic and diastolic function. It is a major cause of heart failure and death in diabetic patients. Currently, there remains a lack of specific therapeutic targets for DCM in clinical practice.

Existing studies have shown that myocardial mitochondrial dysfunction and inhibition of the AMPK signaling pathway are core pathological links in the development and progression of DCM. Protein ubiquitination and deubiquitination modifications play important regulatory roles in cardiovascular diseases; however, the role of the deubiquitinating enzyme VCPIP1 in the diabetic heart remains unclear.

This study addresses clinical issues by investigating the role and molecular mechanism of VCPIP1 in DCM, providing experimental evidence for the identification of novel therapeutic targets for DCM.

2. Main Experimental Methods

This study was conducted at three levels: animal, cellular, and molecular. The main methods are as follows:

Animal model construction:

. Type 2 diabetes mellitus (T2DM) mice: induced by high-fat diet for 16 weeks combined with low-dose STZ;

. Type 1 diabetes mellitus (T1DM) mice: induced by high-dose STZ for 3 consecutive days;

. Cardiomyocyte-specific VCPIP1 knockout mice (VCPIP1 CKO): generated by crossing VCPIP1fl/fl with Myh6-Cre mice;

. db/db mice: injected via tail vein with AAV9 vectors overexpressing AMPKγ1, AMPKγ1K234R, or VCPIP1.

Core techniques: Echocardiography for cardiac function assessment, combined with Western blot, co-immunoprecipitation, ubiquitination assays, pathological staining, mitochondrial function assays, and transmission electron microscopy.

Measured parameters: Ejection fraction (EF%), fractional shortening (FS%), E/A ratio, isovolumic relaxation time (IVRT), left ventricular end-diastolic diameter (LVIDd), interventricular septal thickness (IVSD).

Echocardiography: Echocardiograms were acquired using a multimodality small animal ultrasound imaging system (VINNO Technology, Suzhou, China).

3. Experimental Results

In type 2 diabetic mice, cardiac-specific knockout of VCPIP1 significantly improved cardiac structure and function. M-mode and pulsed-wave Doppler echocardiography showed that, compared with sham-operated mice, diabetic mice exhibited significantly enlarged left ventricular cavity, reduced ventricular wall contractile motion, decreased mitral E/A ratio, and significantly reduced EF% and FS%, indicating pronounced systolic and diastolic dysfunction. In contrast, VCPIP1 CKO significantly alleviated left ventricular remodeling, enhanced ventricular wall motion amplitude, and markedly restored E/A ratio, EF%, and FS% (Figures 1b–e).

Meanwhile, serum ANP levels (Figure 1f): elevated in T2DM mice and reduced in CKO mice; gross heart morphology and HW/BW ratio (Figures 1g–h): cardiac hypertrophy observed in T2DM mice was significantly attenuated in CKO mice; cardiomyocyte hypertrophy assessed by WGA staining (Figures 1i–j): increased cell area in T2DM mice was reduced in CKO mice; myocardial fibrosis assessed by Masson staining (Figure 1l): increased fibrosis in T2DM mice was significantly reduced in CKO mice; molecular analysis by Western blot (ANP/MyHC): hypertrophy markers were increased in T2DM mice and decreased in CKO mice. The changes in serum ANP levels, cardiac hypertrophy, cardiomyocyte hypertrophy, and interstitial fibrosis followed the same trend as the echocardiographic results, confirming that VCPIP1 knockout effectively ameliorates diabetes-induced cardiac structural and functional damage.

Figure 1:
a Schematic diagram of the experimental design. VCPIP1fl/fl mice and cardiomyocyte-specific VCPIP1 knockout mice were subjected to 16 weeks of high-fat diet (HFD) combined with intraperitoneal injection of low-dose streptozotocin (STZ, 35 mg/kg) for 3 consecutive days to establish a type 2 diabetes mellitus model. Cardiac function was assessed after 16 weeks, and blood samples and heart tissues were collected.
b Representative pulse-wave (PW) and M-mode echocardiograms of VCPIP1fl/fl mice and VCPIP1 conditional knockout (CKO) mice in each group.
c–e Echocardiographic assessment of mitral E/A ratio, ejection fraction (EF%), and left ventricular fractional shortening (FS%).
f Serum atrial natriuretic peptide (ANP) levels.
g Representative images of gross heart morphology.
h Heart weight to body weight ratio (HW/BW).
i Representative H&E staining images of cardiac cross-sections.
j Wheat germ agglutinin (WGA) staining and quantification of cardiomyocyte cross-sectional area.
k Protein expression levels and semi-quantitative analysis of ANP and MyHC in heart tissues.
l Representative images of Masson's trichrome staining and quantification of fibrotic area.
i Scale bars: 2.5 μm; j, l 50 μm. Data are presented as mean ± SEM; n = 6; one-way ANOVA with Tukey's test; P values are indicated in the figures.

Rescue experiments in db/db diabetic mice further demonstrated by echocardiography: db/db mice alone showed significantly enlarged ventricular cavities and markedly reduced systolic function. Overexpression of wild-type AMPKγ1 significantly reduced ventricular chamber size, enhanced ventricular wall contractility, and markedly improved E/A ratio, EF%, and FS%. In contrast, overexpression of the AMPKγ1-K234R mutant failed to confer protective effects, with cardiac function and morphology showing no significant difference from those of db/db mice. Furthermore, co-overexpression of VCPIP1 reversed the cardioprotective effects of wild-type AMPKγ1 (Figures 2b–e).

In molecular and pathological assessments: ANP levels (Figure 2f): decreased by γ1 OE, unchanged by K234R; cardiac hypertrophy assessed by HW/BW (Figures 2g–h): attenuated by γ1 OE, ineffective in K234R; cardiomyocyte cross-sectional area by WGA staining (Figure 2i): reduced by γ1 OE, no difference in K234R; fibrosis assessed by Masson staining (Figure 2j): attenuated by γ1 OE, ineffective in K234R; mitochondrial complexes III/IV (Figure 2k): restored by γ1 OE, no improvement in K234R. Collectively, these data indicate that the echocardiographic results are highly consistent with changes in myocardial hypertrophy, fibrosis, and mitochondrial complex protein expression, demonstrating that lysine 234 (K234) of AMPKγ1 is a key target through which VCPIP1 regulates cardiac function in diabetes.

Figure 2:

a AAV9 vectors carrying AMPKγ1 (AMPKγ1 overexpression group), AMPKγ1-K234R (AMPKγ1-K234R mutant group), or VCPIP1 (VCPIP1 overexpression group), driven by the cardiomyocyte-specific cTNT promoter, were delivered into db/db mice via tail vein injection.
b Representative echocardiographic images of mice in each group.
c–e Echocardiographic assessment of mitral E/A ratio, ejection fraction (EF%), and left ventricular fractional shortening (FS%).
f Serum atrial natriuretic peptide (ANP) levels in each group.
g Representative images of gross heart morphology.
h Heart weight to body weight ratio (HW/BW).
i Wheat germ agglutinin (WGA) staining and quantitative analysis of heart tissues.
j Representative images of Masson's trichrome staining and quantitative analysis of heart tissues.
k Western blot detection of mitochondrial respiratory chain complex proteins (UQCRC2, MTCO1) in heart tissues.
 i, j Scale bars:  50 μm. Data are presented as mean ± SEM; n = 6; one-way ANOVA with Tukey's test; P values are indicated in the figures.

4. Research Conclusions

This study demonstrates that VCPIP1 is aberrantly upregulated in diabetic myocardium and, through deubiquitination of AMPKγ1 at lysine 234 (K234), disrupts AMPK complex assembly and activity, thereby inducing myocardial hypertrophy, fibrosis, and mitochondrial dysfunction, ultimately driving the development and progression of diabetic cardiomyopathy. Cardiomyocyte-specific knockout of VCPIP1 restores AMPK activity via a ubiquitination-dependent pathway involving AMPKγ1-K234, improves mitochondrial function, and reverses diabetes-induced cardiac remodeling and functional decline. Small animal cardiac ultrasound sensitively and accurately reflects differences in cardiac function among mouse groups and demonstrates strong consistency with histological and molecular biological findings, providing rigorous and reliable functional evidence for this study. VCPIP1 represents a promising new therapeutic target for diabetic cardiomyopathy.

Publication Information

Title: VCPIP1 drives diabetic cardiomyopathy by deubiquitinating AMPKγ1 and preventing AMPKα-γ subunit assembly in cardiomyocytes

Authors: Xue Han, Zhuqi Huang, Guoxuan Liu, Yanan Liu, Weiqi Li, Jianing Zheng, Jie Tong, Mingyang He, Yurou Wu, Ze Li, Wu Luo, Qiaojuan Shi, Huazhong Ying & Guang Liang

Journal: Signal Transduction and Targeted Therapy

Volume/Issue/Article: Volume 11, Article number: 185 (2026)

Link: https://www.nature.com/articles/s41392-026-02701-9