Design of the animal experiments

The animal experiments received approval from the Experimental Animal Research Committee of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China) and were performed in compliance with the Guide for the Care and Use of Laboratory Animals provided by the National Institute of Health (Bethesda, MD).

In vivo studies were performed on 8-week-old male mice on the C57BLKS (BKS) background, specifically leptin receptor-deficient (db/db) mice and wild-type (wt) mice, sourced from GemPharmatech (Nanjing, China). Mice were individually housed in transparent plastic cages, with two mice per cage, in a room that was carefully regulated to maintain a controlled temperature and a light–dark cycle of 12 h. Mice were given unlimited access to rodent chow and tap water. All mice were euthanized at 20 weeks, and their hearts were promptly dissected and rapidly frozen in liquid nitrogen for subsequent analysis.

Experimental design 1. To investigate the relationship between CAV3 and DCM, we administered an adeno-associated virus 9 vector expressing CAV3 (AAV9-CAV3) under the control of the cTnT promoter or control virus. The mice were randomly divided into four groups with equal numbers (n = 7 per group): (1) the wt + AAV9-Ctrl, (2) wt + AAV9-CAV3, (3) db/db + AAV9-Ctrl, and (4) db/db + AAV9-CAV3 groups. All mice were administered recombinant AAV9 via the tail vein at the age of 9 weeks and were subsequently euthanized at 20 weeks of age. Throughout the entire experiment, the body weight and food intake of the mice were documented on a weekly basis.

Experimental design 2. To investigate whether CAV3 alleviates diabetic cardiomyopathy by regulating NDUFA10, we administered AAV-CAV3, AAV-shNDUFA10 or control virus by tail vein injection at a planned time. Male db/db mice were randomly divided into four groups with equal numbers (n = 7 per group): (1) the db/db + AAV-Ctrl + AAV-shNC group, (2) db/db + AAV-Ctrl + AAV-shNDUFA10 group, (3) db/db + AAV-CAV3 + AAV-shNC group, and (4) db/db + AAV-CAV3 + AAV-shNDUFA10 group. At 9 weeks of age, all mice received tail vein injection of AVV9 to upregulate CAV3, and at 10 weeks of age, they were given tail vein injections of AAV9 to downregulate NDUFA10. They were euthanized at 20 weeks.

CAV3 was overexpressed in vivo with an AVV9 vector expressing CAV3, and NDUFA10 was downregulated with an AVV9 vector expressing shNDUFA10). All the vectors mentioned above were constructed by Hanbio Biotechnology Co. Ltd. (Shanghai, China). The effects of gene overexpression or interference were verified through Western blotting analysis.

Cell culture and treatment

Human cardiomyocytes (AC16) and mouse cardiomyocytes (HL-1) were purchased from iCell Bioscience (Shanghai, China). The cells were cultured in Dullbecco’s modified Eagle’s medium (DMEM) (Keygen Biotech, Nanjing, China) supplemented with 10% foetal bovine serum. Two different media were used: normal-glucose (NG) medium (5.5 mmol/l glucose) and high-glucose-high-fatty-acid (HGHF) medium (33 mmol/l glucose and 250 μmol/l palmitate). The cells were collected after incubation at 37 °C in 5% CO2 for 24 h.

Blood glucose level measurement

A small incision was made at the tip of the tail vein after sterilizing it with an alcohol-soaked sponge. The first drop of blood was discarded, and the second drop of blood was used to measure blood glucose levels using a glucose metre (Roche Diagnostics, Mannheim, Germany).

Fasting blood glucose (FBG) level measurement

The blood glucose levels of mice in each group were measured on the 1st day of the experiment and every 2 weeks afterward. Mice were fasted for 8 h, from 8:00 in the morning until 4:00 in the afternoon, every other Tuesday for a period of 12 weeks to measure their fasting blood glucose levels.

Glucose tolerance test (GTT)

GTTs were performed by injecting glucose intraperitoneally at a dose of 1 g/kg of body weight following a 16-h fasting period. Following glucose injection, blood glucose levels were measured at 0, 15, 30, 60, and 90 min.

Insulin tolerance test (ITT)

ITTs were performed via intraperitoneally injection of insulin (0.75 U/kg of body weight) following an 8-h fasting period. Following the injection of insulin, blood glucose levels were measured at 0, 15, 30, 60, 90, 120 and 150 min.

Transthoracic echocardiography

Transthoracic echocardiography was conducted when the mice reached the age of 20 weeks utilizing a high-resolution echocardiography system equipped with a 30-MHz high-frequency scanning head (VisualSonics Vevo770, VisualSonics, Toronto, Canada), as described previously [29]. The mice were anaesthetized with inhaled isoflurane (isoflurane, 1–1.5%) and placed supine on an imaging platform. Their paws were secured with ECG leads, and their chest fur was removed with Nair hair removal product. Ultrasonic coupling gel was then applied. M-mode and Doppler image loops of the left ventricle (LV) were obtained for 11–20 cardiac cycles, and at least three cycles per loop were averaged.

Haemodynamic analysis

We utilized the conductance catheter technique to perform haemodynamic analysis, assessing both the systolic and diastolic function of the LV. Following previously established protocols, we evaluated the haemodynamics of mice at 20 weeks of age. Before sacrificing the mice, LV catheterization was carried out under anaesthesia. The right carotid artery was carefully isolated from the surrounding tissue during the procedure. Subsequently, we inserted a Millar Mikro-Tip catheter transducer (1.0-Fr, Millar 1.4F, SPR 835, Millar Instruments, Houston, TX) into the LV cavity via the same carotid artery. This transducer was linked to a pressure transducer from Millar Instruments (Houston, TX). Haemodynamic parameters were then recorded and analysed using PowerLab and LabChart software. At least three measurements were obtained for each parameter.

Western blotting (WB) analysis

Frozen heart tissue and cultured cardiomyocytes were obtained and lysed at 4 ℃ for 30 min using radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitor cocktails (NCM Biotech, Shanghai, China). The lysates were subsequently centrifuged at 12,000 × g for 15 min, and the resultant supernatant was utilized for Western blotting analysis. The protein concentrations were determined utilizing a bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai, China). The homogenates were combined with sample loading buffer at a 1:1 ratio (v/v) and then boiled for 10 min. The protein samples were separated on a 12% SDS‒PAGE gel and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% BSA solution for 2 h at room temperature. Subsequently, the membranes were incubated with primary antibodies overnight at 4 ℃. After overnight incubation, the membranes were washed with TBS-T solution. Next, the membranes were incubated with either HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies for 2 h at room temperature. Subsequently, the membranes were washed with TBS-T solution. The protein bands were visualized using ECL Plus chemiluminescence detection reagent (Beyotime Institute of Biotechnology, Nanjing, China). The density of each protein band was quantified using ImageJ.

Antibodies for WB analysis

Anti-CAV3 (Abcam, ab289544, rabbit recombinant multiclonal, 1:1000), anti-NDUFA10 (Abcam, ab174829, rabbit monoclonal, 1:1000), anti-BAX (Abmart, M20008S, rabbit monoclonal, 1:1000), anti-Bcl2 (Abmart, T40056S, rabbit monoclonal, 1:1000), anti-cleaved caspase-3 (Cell Signalling, 9669 s, rabbit monoclonal, 1:1000), anti-SOD2 (ABclonal, A1340, rabbit monoclonal, 1:1000), anti-Cytochrome c (proteintech, 66264-1-Ig, mouse monoclonal, 1:5000), anti-VDAC1/Porin (proteintech, 55259-1-AP, rabbit polyclonal, 1:1000), anti-ATP1A1 (proteintech, 14418-1-AP, rabbit polyclonal, 1:5000), anti-Lamin B1 (proteintech, 12987-1-AP, rabbit polyclonal, 1:5000), anti-PDI (proteintech, 11245-1-AP, rabbit polyclonal, 1:500), anti-β-actin (ABclonal, AC026, rabbit monoclonal, 1:1000), anti-α-tubulin (Abcam, ab210797, mouse polyclonal, 1:1000), HRP-conjugated anti-rabbit or anti-mouse IgG (ABclonal, AS014, AS003, polyclonal, 1:5000), and HRP-conjugated anti-rabbit IgG- heavy chain or light chain (ABclonal, AS063, AS061, polyclonal, 1:5000) antibodies were used for WB analysis.

RNA isolation and quantitative real-time- PCR (qPCR)

Total RNA was isolated from cells using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. cDNA synthesis was performed by utilizing MultiScribe Reverse Transcriptase and total RNA (Thermo Fisher Scientific, Waltham, MA, USA). All primers were synthesized by AuGCT DNA-SYN (Wuhan, China) (Additional file 1: Table S1). The 7900HT Fast Real-Time PCR System was used for quantitative analysis of gene expression (Applied Biosystems, Foste, WA, USA). The data were analysed using the ΔΔCt method [30], and beta-actin RNA expression was utilized to normalize target mRNA expression.

Morphological and histochemical analysis

Haematoxylin and eosin staining (H&E) (Servicebio, Wuhan, China) and wheat germ agglutinin (WGA) (Servicebio, Wuhan, China) staining of mouse cardiac tissues were formed to assess morphology according to standard methods. Sirius red staining was performed to assess the presence of myocardial fibrosis in cardiac tissue. Dihydroethidium (DHE) (Beyotime, Biotechnology, Shanghai, China), an oxidative fluorescence dye, and fluorescence microscopy were employed to assess in situ O2 generation in heart tissues. The fluorescence intensity was measured and analysed as previously described [29].

Immunohistochemistry

CAV3 protein expression in mouse cardiac sections was assessed through immunohistochemical (IHC) staining using a DAB Detection Kit (Gene Tech, Shanghai, China) according to the manufacturer’s instructions (Abcam, ab289544).

Immunofluorescence staining and confocal microscopy

To assess oxidative DNA damage, AC16 Cells were incubated with an anti-DNA/RNA Damage (8-oxoG) antibody (Abcam, ab62623), as described previously [31], and images were acquired using ZEN 2.1 software (Carl Zeiss, Oberkochen, Germany). Co-localization of DNA oxidation and mitochondria was assessed by immunofluorescent analysis of Translocase Of Outer Mitochondrial Membrane (TOM20, proteintech, 11802-1-AP) and 8-oxoG (Abcam, ab62623) in AC16 cells. To explore whether the place where CAV3 interacts with NDUFA10 is located in mitochondria, AC16 cells were labelled with the MitoTracker Red CMXRos probe (Thermo Fisher Scientific, Waltham, MA, USA), an anti-CAV3 antibody (Abcam, ab289544), an anti-NDUFA10 antibody (Santa Cruz, sc-376357), and DAPI (Servicebio, Wuhan, China). The cells were also incubated with an anti-Lamp1 antibody (Abcam, ab208943). Immunofluorescence co-localization images were captured using a laser scanning confocal microscope (Leica, SP8, Germany) and processed using LAS X 3.7.4 software (Leica, SP8, Germany).

Transmission electron microscopy

Mitochondrial morphology in the LV of the heart was examined using transmission electron microscopy (TEM). Mitochondrial morphology and crista morphology were assessed using ImageJ (Madison, WI, USA), as previously described [32].

Isolation of mitochondria and measurement of mitochondrial complex I activity

Mitochondria were isolated from freshly harvested mouse hearts, and the activity of mitochondrial complex I was assessed using a kit (Abbkin Scientific, Wuhan, China) according to the manufacturer’s instructions.

Adenosine 5ʹ triphosphate (ATP) bioluminescence assay

An Enhanced ATP Assay Kit (Beyotime Biotechnology, Shanghai, China) was used to measure ATP content in cells and tissues according to the manufacturer’s instructions. ATP bioluminescence was promptly measured using a microplate luminometer (BioTek Inc., Synergy2, Vermount, USA) and standardized by each protein level.

Analysis of apoptosis cells

Cardiomyocytes were stained with Annexin V-FITC and propidium iodide (PI) according to the instructions provided by the manufacturer of the Annexin V-FITC Apoptosis Detection Kit (Keygen Biotech, Nanjing, China). Flow cytometry (Beckman Coulter EPICS XL-MCL, Brea, CA, USA) was used to analyse apoptotic cells as previously described [33]. Apoptotic cells were detected in heart tissues through TUNEL staining following the manufacturer’s protocol (The Merck Group, Darmstadt, Germany). DAPI (Servicebio, Wuhan, China) was used to stain the nuclei of apoptotic cells. Images were acquired utilizing a laser scanning confocal microscope (Leica, SP8, Germany).

Mitochondrial membrane potential assay

Following the manufacturer's instructions, a JC-1 assay kit (Beyotime Biotechnology, Shanghai, China) was used to measure the mitochondrial membrane potential. The red fluorescence intensity of JC-1 aggregates and the green fluorescence intensity of JC-1 monomers were measured using flow cytometry (Beckman Coulter EPICS XL-MCL, Brea, CA, USA).

Cellular ROS assay

The levels of cellular ROS were measured using the CM-H2DCFDA dyes (MedChemExpress, New Jersey, USA) following the manufacturer's instructions, as described previously [34]. The fluorescence intensity of the cells was assessed using flow cytometry (Beckman Coulter EPICS XL-MCL, Brea, CA, USA).

Mitochondrial ROS assay

The levels of mitochondrial ROS were measured using the mitochondrial superoxide indicator MitoSOX™ red (Invitrogen, Waltham, MA, USA) following the manufacturer's instructions and as previously described [32]. The fluorescence intensity of the cells was assessed using a confocal laser scanning microscope (Leica, SP8, Germany) and flow cytometry (Beckman Coulter EPICS XL-MCL, Brea, CA, USA).

Mitochondrial oxygen consumption rate (OCR)

The OCR was measured using a Seahorse Extracellular Flux (XFe24) Analyzer (Agilent Seahorse Technologies, Shanghai, China) to monitor real-time changes in dissolved oxygen and proton concentrations in the cell culture medium, as previously described [32]. The assay was performed using the Wave software package provided with the Seahorse XF instrument following the manufacturer’s instructions. OCR measurements were normalized to protein level in each well.

Cell transfection

Cardiomyocytes were transfected with a pcDNA3.1 plasmid containing the full-length human CAV3 gene, CAV3- or NDUFA10-specific small interfering RNA (siRNA), or shNDUFA10 (Additional file 1: Table S2) using Lipofectamine™ 2000 (Invitrogen, Shanghai, China) according to the manufacturer’s protocol. Transfection was conducted when the cells reached a confluence of 80% to 90%. After 4 h, the medium was replaced with complete medium. Subsequently, the cells were cultured under HGHF (33 mmol/L glucose and 250 μmol/L saturated free fatty acid palmitate) or NG for 24 h. The cells were harvested 48 h after transfection.

Mitochondrial fractionation

Mitochondria were isolated from cardiomyocytes using a Cell Mitochondria Isolation Kit (C3601, Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions.

Coimmunoprecipitation (Co-IP)

Co-IP assays were conducted using a previously described method [35]. Cells were lysed in RIPA lysis buffer containing a protease inhibitor cocktail. Mitochondria were isolated from cardiomyocytes. Proteins extracted from whole-cell lysates and mitochondrial fractions were used for Co-IP assays. After incubation with a rabbit anti-CAV3 antibody (Abcam, ab289544), rabbit anti-NDUFA10 antibody (Abcam, ab174829), or negative control IgG antibody (ABclonal, AC005) the protein-antibody complexes were subjected to immunoprecipitation using protein A/G agarose beads (Santa Cruz, CA, USA). The immunoprecipitates were washed with IP buffer, and the appropriate amounts of RIPA buffer and loading buffer were added before proceeding with subsequent experiments.

LC‒MS/MS analysis

To identify proteins that interact with CAV3, coimmunoprecipitated proteins were electrophoresed and then visualized using Coomassie Brilliant blue R-250 staining (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s guidelines. The protein-antibody complexes, along with protein A/G agarose beads, were sent for LC‒MS/MS analysis to Spec-ally Life Technology (Wuhan, China).

Statistical analysis

The data were analysed using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA) and are presented as the mean ± standard deviation (SD). To compare normally distributed data between two groups, Student’s t test was employed. When there were more than two groups, one-way ANOVA followed by Tukey’s post hoc test for comparisons was conducted. Statistical significance was set at P < 0.05 for all analyses.